Transactions on GIGAKU

2012 Editorial Board
Editor-in-Chief
Koichi Niihara (President: Nagaoka Univ. Technology)
Associate Editors
Yoshiki Mikami (Nagaoka Univ. Technology)
Takayuki Komatsu (Nagaoka Univ. Technology)
Kazuo Nakamura (Nagaoka Univ. Technology)
Takaomi Kobayashi (Nagaoka Univ. Technology)
Tomoo Shiomi (Nagaoka Univ. Technology)
Masao Fukuda (Nagaoka Univ. Technology)
Shigeharu Kamado (Nagaoka Univ. Technology)
Tadachika Nakayama ((Nagaoka Univ. Technology)
Aim
What is GIGAKU? GIGAKU is a term composed of two Japanese word-roots; GI
and GAKU. The word GI〔技〕literally stands for all kinds of arts and technology, andGAKU〔学〕stands for scientific disciplines in general when used as a suffix. The term
was originally coined to describe the fundamental philosophy of education and researchof Nagaoka University of Technology (NUT) when it was established in 1976. Throughthis term the founders of NUT intended to express their recognition that all technicalchallenges in the real world require a scientific approach. And NUT has a relentlessly
pursued GIGAKU since then.
Thirty-five years have passed and all surrounding conditions have changed
dramatically during those years. We are witnessing rapidly globalizing economics andhuge scale changes in demographic, industrial and employment structures. All thosechanges seem to necessitate the further evolution of GIGAKU. In response to this, NUT
recently announced its new “Growth Plan” and a renewed definition of the term isgiven;GIGAKU is a science of technologies, which gives us an angle toanalyze and reinterpret diverse technical processes and objects
and thus helps us to advance technologies forward. By employing
a broad range of knowledge about science and engineering,
management, safety, information technology and life sciences,
GIGAKU provides us with workable solution and induces future
innovations.
Nagaoka University of Technology has decided to launch “Transactions on
GIGAKU”, which is an online journal (free access) focused on the science andtechnology related to GIGAKU. The mission of this journal is to spread out the conceptof GIGAKU and the fruits of GIGAKU to the global world and to be a strong network
for innovations in science and technology and for development of next generations of1

Transactions on GIGAKU

high-level human resources. This journal, therefore, covers education and research
activities related to GIGAKU in broad areas.
Submission and Publishing“Transactions on GIGAKU” publishes feature issues focusing on education, science,and technology related to GIGAKU. Special issues aligned with conferences arestrongly concerned, and guest editors are welcomed. Papers submitted are
peer-reviewed before publication.
Publication charge is free for all submitted papers.
Copyright
The copyright of the papers published in Transactions on GIGAKU is transferred to
Nagaoka University of Technology.

2

Transactions on GIGAKU

Transactions on GIGAKU
Volume 1, No. 1, July 2012
The 1st International GIGAKU Conference in Nagaoka (IGCN)(February 3-5, 2012, Nagaoka University of Technology, Nagaoka, Japan)
The IGCN is designed and organized to provide a cross-border, cross-sector,
cross-disciplinary forum for those researchers, educators, and industrial leaders who are
creating and practicing GIGAKU in various technology domains in various countries.
Some papers presented in the IGCN are published in this first volume of Transactions
on GIGAKU.
Table of contents
1. Pages 01001/1-6
Engineering Terminology Dictionary: Lessons Learnt from Meiji Japan and
NUT-KOSEN’s Initiative
Y. Mikami, N. L. Iizuka, K. Nagano, K. T. Nakahira, Y. Naruse, C. Yamada,Y. Kawamura
2. Pages 01002/1-5
Interdisciplinary Education Through Double Degree Programs Between
Universidad de Guanajuato and Nagaoka University of Technology
T. Kobayashi, S. Arias-Negrete, S. Sugita
3. Pages 01003/1-9
Advanced Program for Strategic Engineering Promotion with Technical College
Collaboration : Its Concept and Initial StageK. Nakamura, M. Yamazaki, Y. Jodai, Y. Shimizu, M. Nanko, Y. Fukuzawa,
M. Takeda, T. Yamaguchi
4. Pages 01004/1-4
Engineering Education Programs for International Students Toward the Integrationof Japanese Language Education and Technical Education
S. Kamimura, Y. Mikami, N.L. Iizuka, Y. Ito
5. Pages 01005/1-7
Nanoscale Deconfinement: Isolation of Nanovolume Leads to Change
R. Nowak, D. Chrobak, W.W. Gerberich, K. Niihara
6. Pages 01006/1-7
Nanomaterials As Environmentally Compatible Next Generation Green
Carbon Capture And Utilization Materials
A.R. Mohamed, S. Bhatia, K.T. Lee, C.Y. H. Foo, Z.H. Lee, N.A. Razali
7. Pages 01007/1-8
Nanomatrix Structure Formed for natural Rubber
S. Kawahara, O. Chaikumpollert
8. Pages 01008/1-4
Mullite Formation in Presence of Y2O3 by Homogeneous Precipitation
J. J. Rocha Jiménez, J. M. Martínez Rosales, K. Uematsu, S. Tanaka, S. Sugita
3

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9.

Pages 01009/1-6
Isolation and Characterization of bzaA and bzaB of Sphingobium sp. Strain SYK-6,
Which Encode Aromatic Aldehydes Dehydrogenases with Different Substrate
Preferences
Y. Yamamoto, D. Kasai, N. Kamimura, E. Masai
10. Pages 01010/1-8
Development of an Appropriate Treatment Process for Wastewater from a Natural
Rubber Processing Factory
D. Tanikawa, T. Yamashita, M. Hatamoto, M. Fukuda, M. Takahashi,
K. Syutsubo, P. K. Choeisai, T. Yamaguchi
11. Pages 01011/1-4
Biodegradation of Persistent Chlorinated Organophosphorus Flame Retardants by
Microorganisms Newly Isolated from Soil – A Short Review
Y. Kera , S. Takahashi, K. Abe
12. Pages 01012/1-6
Electrochemical Deposition of Cobalt on Mesoporous Silica Film
M. Tagaya, M. Okuda, S.W. Jones, T. Kobayashi
13. Pages 01013/1-6
The Grafting Effects of Fe(III) on Photocatalytic Activities of WO3 and BiVO4 on
Visible-Light Irradiation
M. Nishikawa, S. Hiura, Y. Mitani, Y. Nosaka
14. Pages 01014/1-8
In Vitro Targeting of Cancer Cells with Luminescent Nanoporous Silica Spheres
M. Tagaya, J. Tanaka, N. Hanagata, T. Ikoma, T. Takemura, K. Shiba,
T. Kobayashi
15. Pages 01015/1-6
Effect of Flow Channel on Specific Surface Area of Steam Activated Carbon
Material from Coffee Bean Wastes
H. Toda, I. Toda, H. Tanikawa, T. Takahata, S. Ohshio, H. Akasaka,
S. Himeno, T. Kokubu, H. Saitoh
16. Pages 01016/1-4
Hydrogen Storage Property of Activated Carbon Fabricated with Lignins
I. Toda, H. Toda, S. Ohshio, H. Akasaka, S. Himeno, M. Nakaishi,
H. Saitoh
17. Pages 01017/1-6
Recent Progress in Laser Patterning of Crystals in Glasses
T. Komatsu, F. Suzuki, K. Ogawa, Honma
18. Pages 01018/1-6
Condensation Control of Nanoscale Polymer Aggregates by Using Atomic ForceMicroscope Tip
A. Kawai
19. Pages 01019/1-6
Intensity of Singularity Analysis for Three-dimensional Two-phase Transversely
Isotropic Piezoelectric Bonded Joints
Md. Shahidul Islam, H. Koguchi

4Transactions on GIGAKU

20. Pages 01020/1-5
Evaluation of Internal Coarse Defects in Alumina Powder Compact and Ceramics
S. Tanaka, K. Uematsu
21. Pages 01021/1-6
Ultrasound Effect of Interpenetrated Hydrogels having Hydrogen Bonding
Networks in Poly(Vinylalcohl)/Poly(2-Hydroxyethyl Methacrlylate) by Using FT-IR
Spectroscopy
K. Nakasone, V.S. J. Addiel, M. Tagaya, T. Kobayashi
22. Pages 01022/1-6
Crack Self-Repairing of Nano-Metal/Al2O3 Hybrid Materials
M. Nanko, D. Maruoka, P. V. Hai, N. D. Thuy
23. Pages 01023/1-6
Development of Oxygen Sensors Exploiting Hot Spots in GdBa2Cu3O7-δ-based
Composite Ceramic Rods
T. Okamoto, Y. Kuroki, M. Takata
24. Pages 01024/1-6
TEM Observation of Carbon-Coated Li3V2(PO4)3 Glass-Ceramic Powders and TheirHigh Rate Battery Performance
K. Nagamine, K. Oh-ishi, T. Honma, T. Komatsu
25. Pages 01025/1-6
Formation of Hierarchical Structure in Liquid Crystalline Block Copolymers
H. Takeshita, S. Adachi, S. Taniguchi, K. Takenaka, M. Miya, T. Shiomi
26. Pages 01026/1-5
Alloying Effects in the Nickel-Cobalt System on Electrochemical and Adsorptive
Properties of an SOFC Anode Cermet for the Direct Oxidation of Methane
K. Sato, T. WahTzu, H. Nishiyama, T. Sawahata, H. Takayanagi
27. Pages 01027/1-5
Preparation of Filter Paper Conjugated with High Mannose-Type Glycans
K. Furukawa, T. Tadokoro, M. Sato, E. Yamada
28. Pages 01028/1-6
Development of Trichoderma reesei Strain Specialized for Cello-oligosaccharides
Production from Cellulose
Y. Shida, R. Yoshida, T. Wakayama, Y. Konda, T. Yamaguchi
29. Pages 01029/1-6
Identification of Amino Acid Residues Essential for the Activity of
3-O-Methylgallate 3,4-Dioxygenase from Sphingobium sp. Strain SYK-6
D. Kasai and E. Masai
30. Pages 01030/1-6
In Vitro Study of BphT1 Two-Component Response Regulator Protein of
Rhodococcus jostii RHA1
J. Shimodaira, Y. Miyazawa, D. Kasai, K. Miyauchi, M. Fukuda
31. Pages 01031/1-6
Overexpression of ACEII, and Transcriptional Analysis of Cellulase Genes in
Trichoderma reesei PC-3-7
T. Furukawa, N. Kitagami, Y. shida, Y. Morikawa, W. Ogasawara
5

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32. Pages 01032/1-6
Thermal Gradient Creep of Ni-based Superalloy Coatings
S. R. Gandhi, R. Nomura, M. Okazaki, S. Yamagishi
33. Pages 01033/1-6
Characteristics and Directed Evolution of Trichoderma reesei Endoglucanase III(Cel12A) – A Review
H. Okada, H. Nakazawa, W. Ogasawara, Y. Morikawa
34. Pages 01031/1-6
Electrostatic Layer-by-Layer Polymer Membranes for Heavy Metal Ion Desalination
W. M. Zhou, H. Fu, M. Tagaya, T. Kobayashi

6Transactions on GIGAKU 1(2012)01001/1-6

Engineering Terminology Dictionary:Lessons Learnt from Meiji Japan and NUT-KOSEN’s Initiative
Yoshiki Mikami*, Naoko Lee Iizuka*, Kenjiro Nagano*, Katsuko T. Nakahira*
Yoshinori Naruse**, Chikatoshi Yamada***, Yoshiko Kawamura****Nagaoka University of Technology, **Toyama National College of Technology,***Okinawa National College of Technology, ****Tokyo International University*1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: mikami@kjs.nagaokaut.ac.jp
The first part of this paper reviews historical lessons learnt from theexperience of Meiji Japan regarding the teaching medium of engineering
education. The importance of mother language use in engineering educationand of terminology development is illustrated based on findings frominvestigations on historical documents. The second part of this paper
introduces the response of Nagaoka University of Technology and National
College of Technology (KOSEN) to those lessons. The language coverageand vocabularies of the Multilingual Engineering Terminology Dictionary
developed by the Engineering Dictionary Committee of the university arepresented.

1. Introduction
In the first International GIGAKU Conference in Nagaoka (IGCN 2012), Kozo Ishizaki, theformer Vice President of Nagaoka University of Technology (NUT) and the chairman of NUT’s
Engineering Dictionary Committee, stated that “(an) important aspect (of industrial development) is
language used in development. Japanese tried to translate every European concept into Japanese forgeneral Japanese people to be blessed by the modern scientific development. It is important not todivide a society into English speaking and non-English speaking classes.”
Although engineering subjects were taught in English at the first engineering college of Japan—
the Imperial College of Engineering (Kōbu Dai-gakkō)—from the early Meiji period, serious andcontinuous efforts have been made by pioneering teachers in engineering to translate basic
engineering terminologies into Japanese language; gradually, teaching materials had been developed
based on these achievements. These sets of teaching materials were the basis of concerted efforts inengineering education and human development in Japan. Without such efforts, the quick adoption ofimported technologies, and therefore the rapid industrialization of Meiji-era Japan, might not havebeen possible.
Similarly, international students currently studying at NUT should be encouraged to propagate
their knowledge to people in their native countries using mother language terminology. This
recognition and reasoning has formed the basis of the development of a series of trilingualengineering terminology dictionaries by NUT during the last five years.
2. Language used in Engineering Education in Meiji JapanThe first engineering education program was formulated by the initiative of the Meiji
government in the 1870s, only a few years after the Meiji Restoration. The Imperial College ofEngineering, the cradle of engineering education of modern Japan, was established in Tokyo in 1873[1]. All teaching staff members were invited from Europe, with the majority of the staff hailing from
Scotland, where professional engineering education had been implemented since the early nineteenth
century and was considered to be the most advanced engineering education center in the world. The
first president of the college, Henry Dyer, came from Glasgow v the nomination by Sir WilliamThomson.
In this newly-created college, all subjects were taught in English. Applicants to the college had to1

Transactions on GIGAKU 1(2012)01001/1-6

pass through an entrance examination which included among its subjects English language ability.
The weekly calendar of the college shows that students studied English language for two hours every
day from Mondays to Fridays during their first two years in the college [2]. At the end of six years ofeducation, students had to submit theses also written in the English language.
The college was later merged with the Imperial University (renamed again to the Imperial
University of Tokyo later), when the latter was formulated as a central institute for higher educationof Meiji Japan. A lecture notebook and examination paper on “Hydraulics and Hydraulic Machinery”
written by Professors Bunji Mano and Ariya Inokuchi of the Department of Mechanical Engineeringof the Imperial University of Tokyo has been preserved, and it reveals that lectures and examinations
were conducted in English even 40 years after the beginning of engineering education in Japan(Figure 1). Professor Mano was the first president of the Japanese Society of Mechanical Engineers(JSME), and the notebook and examination paper were designated as Mechanical Engineering
Heritage Documents by the JSME in 2007.

Fig. 1 Lecture Notebook and Examination Paper on “Hydraulics and Hydraulic Machinery” byProfessors Bunji Mano and Ariya Inokuchi at the Imperial University of Tokyo, circa 1906.
Takahashi et al [3] conducted an investigation of an archived collection of 2,913 theses
submitted by students from both the Imperial College of Engineering (1879-1885) and the Imperial
University of Tokyo (1885-1934), and categorized the theses in mechanical, electrical engineeringand architecture by languages used. The number of theses according to language is shown in Figure
2 This result provides succinct evidence of the situation described above.
In the field of mechanical engineering, all theses were written in English until as late as the
mid-1910s. In electrical engineering, the vast majority of theses were written in English until as lateas the 1920s. In architecture, however, there appeared several differences with the fields ofengineering: the researchers found several theses written in Japanese early in the Meiji era, but it
was found that these papers were exceptions, and were regarding traditional Japanese temple
architecture.
60
50
40
30
20
10
0
1870

Written in English
Written in Japanese

1880

1890

1900

1910

1920

1930

1940

Fig. 2a Number of theses in selected engineering field by language (mechanical engineering)2

Transactions on GIGAKU 1(2012)01001/1-6

60
50
40
30
20
10
0
1870

Written in English
Written in Japanese

1880

1890

1900

1910

1920

1930

1940

Fig. 2b Number of theses in selected engineering field by language (electrical engineering)
40
30
20
10
0
1870

Written in English
Written in Japanese

1880

1890

1900

1910

1920

1930

1940

Fig. 2c Number of theses in selected engineering field by language (architecture)

3. The First English-Japanese Engineering Terminology Dictionary
An early Meiji-era philosopher/politician Tatsui Baba (1850-1888, Figure 3) expressed his
objection to the opinion of the then-education minister Arinori Mori, who advocated adopting
English as the official language of newly-born Japan. Baba wrote about the importance of the motherlanguage in preface of his book “Elementary Grammar of the Japanese Language” published in 1873
in London.
We must try to educate the whole mass of the people and unite them into one, in order topromote the common happiness of the community…. Mr. W.D. Whitney, in his letter toMr. Mori, says, “Even with a fully developed system of national instruction, it would takea very long time to teach a strange language to so large a part of the population as to raise
the letter, in general, to a perceptibly higher level. If the masses to be reached, it must be
mainly through their native speech.” We think, also, that it is more desirable to try toenrich and complete that which we have already.[4]Baba’s concern was exactly the same as that of Professor Ishizaki as mentioned above.
Furthermore, in order to enrich and enable a national language to be used for professional educationin various subjects, the most urgent and important challenge is to create a sufficient set of vocabulary
and terminology in various professional domain areas, such as medicine, engineering, andjurisdiction.
In the field of engineering, efforts had been made to enrich Japanese technical terminology bymany voluntary contributors since the Meiji period. The first major step in the development ofengineering terminology development was the publication of an English-Japanese Engineering
Terminology Dictionary “KOGAKU JII” (Figure 4). This dictionary was compiled by a railway
engineer named Ryutaro Nomura (1859-1943). He studied at the Imperial University and thedictionary was published by the Engineering Society of Japan in August 1886. Over 6000
engineering terms were covered by this single volume. It is interesting to observe that in thispublication, Japanese characters are printed in the traditional vertical orientation, while English
terms are in the horizontal orientation (Figure 4, right side). This dictionary was followed bycontinuous expansion and improvements of terminology development and publication of dictionaries.
This resulted in increased access to new knowledge in engineering not only for scholars inuniversities, but also to members of the general public with insufficient fluency in English,
especially technicians at workshops, as well as teachers and students at trade schools.
3Transactions on GIGAKU 1(2012)01001/1-6

Fig. 3 Photo of Tatsui
Baba, date un known

Fig. 4 The First English-Japanese Engineering Dictionary published in
1886 – Front cover (left) and the first page starting from “Alkali” (right)

Based on achievements in language enrichment and empowerment, the number of books onengineering subjects gradually increased. As shown in Figure 5, although there have been books
published in Japanese in the fields of mathematics (sūgaku) and physics (butsurigaku) since 1870,
books in mechanical engineering (kikai kōgaku) written in Japanese had been published atsignificant volume only after the turn of the century. Before the publication of the first engineering
dictionary in 1886, only four books in Japanese had been published for mechanical engineering.
While other factors may have accounted for the observed differences among the different
fields—such as the number of potential readers and the nature of the subject matter (basic versus
applied) —it is logical that without appropriate terminologies in a mother language, it becomes
impossible to publish in-depth technical books in any specialist field in that language.
The lessons learned from this historical experience of Meiji Japan may also apply to present-day
countries that are undergoing industrial development. In order to create a broad basis of humanresources for industrial development, engineering education should be conducted in each country’s
mother language. It therefore requires a sufficient set of terminologies translated into motherlanguage. The recognition of the necessity of terminology development led to the decision by NUT
to develop a series of engineering terminology dictionary into various languages in order to support
the education of engineer students from other countries.
50
40
30

Mechanics
Mathematics
Physics

20
10
0
1870

1880

1890

1900

1910

1920

1930

Fig. 5 Number of books in mechanics, mathematics and physics written in Japanese language andpublished in Japan during 1870-1930. Source: Takahashi et al.[3]4. Language-based Composition of International Students at NUT
The question that presented itself was for which language should such a dictionary be
developed? To address this question, NUT’s Engineering Dictionary Committee has set thefollowing selection criteria: (1) the population of international students by country currently enrolled
in NUT ; (2) the usage level of their mother language in engineering education in the students’ home4Transactions on GIGAKU 1(2012)01001/1-6

countries. The first criterion means that the larger the population of speakers of a particular language
at NUT campus, the higher the priority given to that language. The second criterion means that if amother language is not used extensively at home in engineering education, a lower priority is given
to that language. This second criterion was added because many countries in Asia actually use
English for higher education, including education in engineering. These countries include India, thePhilippines, Sri Lanka, and Myanmar.
In terms of the number of speakers for each language, we designated ten languages as the major
foreign languages spoken at the NUT campus (Table 1). Among these, Myanmar and Sinhalese were
dropped due to the second selection criterion, i.e., in Myanmar and Sri Lanka, higher education is
given in English. Although Korean and Mongolian have relatively few speakers in NUT at the timeof this survey, the Committee decided to add these two languages; the former was added because of
its potential importance and geographical proximity to Japan, and the latter was added because of its
large presence at Kosen (the National College of Technology).
Table 1 Top ten languages spoken at NUT campus
Language
Number of students
As of May 2011
Past maximum
Vietnamese
80
80
Chinese
54
54
Malay
45
76
Thai
30
39
Spanish29
41
Myanmar10
14
Indonesian7
21
Sinhalese4
5
Mongolian
3
6
Korean2
7Countries and regions
VietnamChina, Taiwan
Malaysia, Brunei, Singapore, etc.
Thailand
Mexico, Venezuela, etc.
MyanmarIndonesia
Sri Lanka
Mongolia, China
Korea

5. Outline of NUT Multilingual Engineering Terminology Dictionary
After employing the selection criteria, eight languages were chosen for the publication of amultilingual engineering terminology dictionary. The languages were Vietnamese, Chinese, Malay,
Thai, Spanish, Indonesian, Korean and Mongolian. Each version of the dictionary contains trilingual
entries, i.e., the target language, Japanese and English (Figure 6). Users can search for terms using
Romanized Japanese spelling arranged in alphabetical order. The Vietnamese version of this
dictionary was published in 2008 by a commercial publisher both in Japan and Vietnam [5] [6], andother language versions were printed as private publications of NUT.
The web version also contains terminologies in Lao, Myanmar, and Sinhalese (Figure7). This
web version is accessible at http://gii2.nagaokaut.ac.jp/mtd/ [7].

Fig. 6 Dictionary page layout
displaying engineering terms inJapanese, English, and thetarget language; in this case,Vietnamese.

Fig. 7a Web version (top page)5

Fig. 7b Web version(Multilingual page)

Transactions on GIGAKU 1(2012)01001/1-6

The vocabulary set available in the current dictionary is composed of basic terms from major
engineering and science subjects taught at NUT campus, namely Civil Engineering, MechanicalEngineering, Architecture, Chemical Engineering, Communication Engineering, Computer Science,Control Engineering, Electric and Electronics, Navigation, Mathematics and Physics. The number oftechnical terms categorized by subject field is shown in Table 2.
Table 2 Number of terms by field
Field of engineering
Number of terms Field of engineering
Civil Engineering
1,263 Control Engineering
Mechanical Engineering
1,186 Electric and Electronics
Architecture
1,798 Navigation
Chemical Engineering
958 Mathematics
Communication Engineering
1,410 Physics
Computer Science
1,538 Total
Note: Total number contains duplications in some terms

Number of terms
1,545
1,200
1,403
1,284
945
14,530

6. Conclusion
NUT will continue its efforts to enrich the contents of the dictionaries and improve access tothese resources by means of online access, as well as the development and commercial publicationof pocket dictionaries. Developing an online dictionary of engineering terminology is also critical forinternational students at KOSEN, because these students are required to self-learn basic specialized
subjects at their own pace to catch up with classes. Therefore, there is an urgent need to develop andprovide effective self-studying materials for e-learning, supported by the use of an online dictionaryof engineering terminology. This type of self-studying environment would enable international
students to expand their technical terms and understanding of engineering, and reduce the language
barriers between lecturer and student. International students would be better equipped to learn fromclasses conducted in Japanese, and be at less of a disadvantage when compared to their Japanesepeers. This in turn would facilitate a more effective education system, and thereby producing
better-educated and competent engineers. The authors encourage feedback and suggestions fromreaders and users of the dictionary in order to ensure that these resources are consistently improved.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT), Japan.
References[1] Anonymous, An Engineering College in Japan, Nature, April 3, 1873, p. 430 and C.W.C.,
Engineering Education in Japan, Nature, May 17, 1877, pp. 44–45.
[2] Imperial College of Engineering Tokei, Calendar, Session 1876-77, 1976, p.23.
[3] T. Takahashi, M. Matsuda, S. Kamimura, and Y. Mikami, Language of thesis in early days ofengineering education in Japan, The 4th Int’l Symposium on Technological Innovation in
Japan, National Museum of Science, Tokyo, Dec. 10, 2008.
[4] T. Baba, An Elementary Grammar of the Japanese Language with Easy Progressive Exercises,
London, Trubner and Co., 1873, pp. x-xi
[5] Japanese–Vietnamese (English) Technological Term Dictionary (Nichi-Etsu Kōgaku Yōgo
Jiten), Shumpūsha Publishing, 2008.
[6] Từ điển thuật ngữ khoa học Nhật Việt, 2008. Publication in Vietnam is arranged by Oriental
Language and Culture Department, Foreign Languages College, Hanoi National
University.
[7] O. Htun, Y, Mikami et al. Int. Conf. Computer Applications (ICCA2009), Yangon, February
26–27, 2009.
(Received: 23 May, 2012, Accepted: 12 June, 2012)6

Transaction on GIGAKU 1 (2012) 01002/1-5

Interdisciplinary Education through Double Degree Programs between
Universidad de Guanajuato and Nagaoka University of Technology
Takaomi Kobayashi1, Sergio Arias-Negrete2 and Satoshi Sugita3,
1

Department of Materials Science and Technology, Nagaoka University of Technology
1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan
2
Universidad de Guanajuato, Dirección de Cooperación Académica, Lascuráin de Retana No. 5; CP
36000, Guanajuato, Gto., México, sergio@ugto.mx; 2Universidad de Guanajuato, 3 Departamentode Química, División de Ciencias Naturales y Exactas, Col. Noria Alta, CP 36050, Guanajuato,
Gto., México;*E-mail: takaomi@nagaokaut.ac.jp
Interdisciplinary education system through Double Degree Program (DDP) between
Universidad de Guanajuato and Nagaoka University of Technology has been
established as a new educational scheme on 2011. The present paper describes
subjects on the master DDP for the innovative education system in order to enhance
globalization of students and faculty staffs in both universities. The methodology
and the goal of the program are introduced.
.

1. Introduction
Interdisciplinary education is nowadays very important to prepare future researchers in different
areas to solve problems, innovate and promote development by using diverse approaches andperspectives. The objectives of the partnership are to be a common capacity and to enhance eachother program by cooperating different areas. Thus, to reach this goal, students require training using
different methodologies, in order to achieve profound knowledge.
At present, international academic collaboration fosters joint research programs, and it requires
student mobility among partners from Higher Education Institutions (HEI). A more complex
academic collaboration is currently utilized by HEI. By offering Double Degree Program (DDP),
which is a student's working for two different university degrees in parallel, either at the same
institution or at different institutions, it has been advantage that both of the participating institutions
reduce the amount of time required to be spent at each. Usually, the HEI is from different countries
and the language of teaching is different. Therefore, there are some benefits for students pursuing auniversity academic degree, such as development of intercultural skills, proficiency in the foreign
language of the host institution; and importantly, acquiring an international perspective of researchand collaboration. Additional benefits are attained by the partner HEI, such as an increase ofvisibility and prestige, and achievement of a significant impetus in research graduate programs.

2. Higher Education Institutions in UG
The academic tradition and history of Universidad de Guanajuato (UG) date back to theeighteenth century, colonial era of the maximum blossoming of the Villa de Guanajuato afterwards acity since 1741 and its booming mines. The UG boasts an academic staff recognized for its highquality. This staff carries out essential work in the areas of instruction, research, and extension.
Because of its quality teachers and the unceasing efforts of its academic bodies, the UG carries out
more scientific research than any other institution of higher education in the state; its academic units,
institutes, and research centers develop projects with regional applications in benefit of all thecitizens of Guanajuato. The public institution in UG offering high school and university education,
leader in the context of the Mexican educational system contains in these levels, and exemplary
because its quality, coverage, equality and pertinence has been carried our with following academic
structure by four campuses, Guanajuato, Celaya-Salvatierra, Irapuato-Salamanca, and León.
It has been noted that DDP offered by HEI from different continents is becoming a common
practice as a part of their internationalization process. The UG is a comprehensive institution with astrong determination to instill an international dimension into its academic activities. This Desired1Transaction on GIGAKU 1 (2012) 01002/1-5

Scenario is indicated in its Development Plan for the 2010-2020 terms. UG is offering some DDP atundergraduate and graduate level, and new DDP for graduate studies are envisaged. The HEIs form
Japan and UG have a long and strong academic relationship based on mutual trust and friendship.

Fig.1 Pictures of UG in Guanajuato campus (left) and research view in the chemistry laboratory
at Noria Alta for the master DDP student of NUT (right).

3. Japan–Mexico–USA Consortium for Engineering Education
In 2003, Nagaoka University of Technology (NUT) and the UG signed a Letter of Intent forcollaboration. The Japan–Mexico–USA Consortium for Engineering Education was created to foster
academic collaboration and foundation of DDP, with the participation of NUT, The University ofTexas at El Paso (UTEP), The Autonomous University of Ciudad Juarez, (UACJ), Universidad
Michoacana de San Nicolás de Hidalgo (UM) and UG. Based upon strategies and concepts aimed atuniversities’ internationalization through the Academic International Collaboration/Cooperative
Support Interdisciplinary Program from 2009-2010, NUT was organized and developed engineering
education programs accelerating internationalization under the sponsorship by the Ministry of
Education, Culture, Sports, Science and Technology (MEXT). This interdisciplinary program
developed interactive educational collaboration, such as a credit transfer system and double-degree
programs with their foreign partners.
Based upon comprehensive and systematic education programs, it promoted cultivation
approaches to leading edge engineering at each university. Through such an interdisciplinary
program, we, the five universities of the consortium, could promote international contributions.
Cooperation was made as the best use of regional characteristics on enable engineers and researchers
to obtain training that included practical skills through close mutual academic exchange programs at
the universities. In this consortium, five universities promoted a new mode of international
collaboration and contribution for the development of leading engineers with practical and creative
abilities. To undertake education of engineers and researchers with practical ability through aninternational collaboration system, NUT led the international collaboration system with mutual
academic and educational cooperation under an interactively developed network (Fig. 2) for the
purpose of following; 1) Implement interdisciplinary engineering education among consortium
universities through the Master’s Double Degree Program, especially streamlining collaboration
through overseas internships and training engineers at enterprises in the areas of Mexico and theUnited States of America to achieve practical reeducation during graduate school,2) Support for enhancement of international competition in accordance with the contribution toward
human resources in overseas enterprises, especially in Japanese enterprises, and 3) Accelerateinterdisciplinary education and research globalization through practical cooperation anddevelopment of joint international industry–academic research.

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Fig. 2 Interactively developed network scheme in Japan–Mexico–USA Consortium for Engineering
Education (right) and Guanajuato reunion in the consortium meeting on 2009 January 14th (left)

4. DDP program between NUT and UG
Implement was that interdisciplinary engineering education among consortium universities was
through the Master’s DDP, especially in streamlining collaboration through overseas internships andtraining Mexican students in engineer skill and practical knowledge. Among the consortium, the DDP in
master course between NUT and UG was interdisciplinary challenge, which started on successfully to
exchange their student candidates on August 2011. In addition with credit transfer system, thecomprehensive and systematic education program also could promote cultivation approaches to leadingedge engineering at each university. Especially, streamlining collaboration through overseas internships
and enhancing exchange students between consortium members were achieved through the construction
process.

Fig. 3 Relationship scheme between NUT and consortium universities through DDP program in masterprogram (right) and picture of exchange students with cooperation professors of the department ofChemistry in Noria Alta, Guanajuato (left)

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At present, NUT and UG can offer a Master DDP, where NUT confers the Master in Engineering
Degree and UG confers the Master in Science Degree. Both NUT and UG have agreed to establish a
new DDP in their master courses. As presented in Fig. 4, the DDP is an academic program that canenable the students enroll in the graduate program at their Home University to transfer to theprogram at the Host University in order to obtain master degrees from both universities uponcompletion of their courses. Each graduate program has specific research projects, but the common
link is the Chemical Sciences. In order to be eligible for the DDP program, the student candidates
must pass the DDP screening examination held by the respective Host University and they havestudied chemistry at the master’s level. As seen in achievement scheme of DDP between NUT and
UG, the DDP has implemented according in the bilateral DDP between both universities. The periodof stay at the Host University is for a period of one year or more, but not to exceed two years.
Therefore, the students of UG can be enrolled in August each year for study lasting two and a halfyears to obtain a master's degree at both universities. On the other hand, the students of NUT canbegin their studies in April of each year for a two-year study period. It is important to constructeducation systems of DDP in both universities. So, educational and research plans firstly are
required for each DDP student, who is decided upon by prior consultation of the academic advisor(thesis supervisor) at the Home University and the Host University. Their plans are necessally forrequiring the approval of the authorized committees at both universities and the academic advisor at
the Host University ratifies the approval of such committee under its regulation. The academic
advisor at the Host University can mainly supervise the DDP students with the assistance of theacademic advisor at the Home University.

Fig. 4 Scheme of the master DDP between UG and NUT including student transfer from the host
university to the guest one.
Moreover, doctoral in chemistry at UG is accredited by Consejo Nacional de Ciencia yTecnología (CONACYT), whereas doctoral studies at NUT offer a multidisciplinary approach.
Therefore, both NUT and UG have agreed to establish a new DDP in their doctoral courses. Facultyof both universities is working coordinately to design the operating rules of the Doctoral DDP from2011, based on know-how to the interdisciplinary education system of the master program. Under
the enhanced collaboration of NUT and UG, an international reference of partnership enables to
educate leaders to successfully perform the global program in the doctoral courses.

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5. Goals and Spillover Effects
Interdisciplinary education and research through a DDP by operation of the distinctive
consortium has following effect on development of our interactive educational collaboration andconstruction of both credit transfer systems. This is because that international consortium network
can enhance education and research through close cooperation. Also, establishment of a re-education
system has been taken place for practicing professionals in Mexico. This can accelerateinterdisciplinary education and research globalization through practical cooperation anddevelopment of joint international industry-academic research. Among them, training engineers atenterprises seems to be very important in the areas of Mexico through cooperative education processin UG and can achieve practical re-education during graduate school through research. Especiallythrough the human-resource training program to re-educate engineers of Japanese industries,
students can receive lectures in engineering education and experience in Japanese style production
for one year at the NUT. Thereafter, they can receive training and do research work at a Mexican
home university to study there on site for one year, participating in their master's degreecollaborative study program. This also can support enhancement of international competition inaccordance with the contribution toward human resources in overseas enterprises, especially inJapanese enterprises. So the achievement of these goals will contribute to both the development andacceleration of internationalization in graduate education. It will be sure in the near future that the
program indirectly may support over 400 Japanese companies that are currently operating businesses
in Mexico by providing them with training for engineers and the opportunities for basic research
work to maintain and improve their position among others in North America areas.
6. Conclusion
In conclusion, the master DDP between UG and NUT developed jointly by the two universities isdiscussed in this paper. Actually, the DDP has started from 2011 April on NUT and one student
studies (Fig. 1 left) in UG till 2012 June according to the scheme of Fig. 4. These developments canbring effective changes in a culture of academic and professional collaboration, harnessing thestrengths of both organizations. These institutions are therefore working in a complementary way tomeet mutual and national interests and then to prepare the next Doctoral DDP. It is expected that
greater and deeper collaboration between both universities is still processing particularly in areas ofmaster and doctor education and their researches on the basis of enhanced globalization. Thus, thiscollaboration may also serve as a model for the future beneficial relationships between bothcountries.

Acknowledgement
This is supported by developed engineering education programs accelerating internationalization
under the sponsorship by MEXT.
(Received: 18 May, 2012, Accepted: 17 June, 2012)5

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Advanced Program for Strategic Engineering Promotion withTechnical College Collaboration : Its Concept and Initial Stage
Kazuo Nakamura1,*, Makoto Yamazaki2, Yoshifumi Jodai3, Yoshihiko Shimizu4,
Makoto Nanko1, Yasushi Fukuzawa1, Masatoshi Takeda1, Takashi Yamaguchi1
1)
Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan
2)
Nagaoka National College of Technology,888 Nishikatakai, Nagaoka 940-8532, Japan
3)
Kagawa National College of Technology,355 Chokushi-cho, Takamatsu 761-8058, Japan4)
Toyama National College of Technology, Ebie Neriya, Imizu, Toyama 933-0293, Japan*E-mail: nakamura@kjs.nagaokaut.ac.jp

Considering the essential change of global industry and economy in the
coming era, a project "Advanced Program for Strategic Engineer Promotion
with Technical College Collaboration (ADV-program) " has been established
as a new educational scheme in collaboration of Nagaoka University ofTechnology (NUT) and technical colleges (KOSENs) for Monodukuri(Craftsmanship) in the coming society. This paper introduces the conceptual
framework and the subjects on the initial stage of ADV-program. The
proposed program focuses on enhancing the fundamental abilities on"Specialties on multiple fields", "Strategic technology management" and"Global leadership." On the initial stage at KOSEN, the students of
ADV-program gain insights into global trends and consider what is importantfor elemental technology and training. Furthermore, the course forms thebasis for developing promising personnel who will be of benefit to both
Japan and the wider world through understanding of technological-systems
and creation of strategies in society.

1. Introduction
In the coming era the growth of BRICs power [1,2] with American, European and Japanese one
is going to lead the global economic and social conditions. Under the situations industrial activities
of Korea and China are rapidly expanding in the worldwide market with specific strategic
technology developments [3,4]. And the common human problems on global environment, energy/ resource conservation and coping with huge natural / artificial hazard require intellectual resolution
by advanced technology. Under such conditions the technology strategy of Japan for "Monodukuri(Craftsmanship)" is also required to be changed not only for high-level quality, function andreliability but also for "generation of unique creative technology", "flexible correspondence tovariety of needs" and "global collaboration and/or rational competition with justice."
Nagaoka University of Technology (NUT) was established thirty five years ago to aim at
providing educational programs for promoting practical and creative leading engineers accepting
graduate students from technical colleges (KOSENs). Then the role of NUT is recognized as asupplier of excellent engineers with practical technology in Japan industries. Considering theessential change of global industry and economy in the coming era, however, the scheme ofeducation program in collaboration of NUT and KOSENs should be introduced in new orientation
under the common recognition of the above mentioned requirements for Monodukuri in the coming
society. Ministry of Education, Culture, Sports, Science and Technology in Japan has supported thisorientation of NUT and KOSENs, and the project "Advanced Program for Strategic Engineer
Promotion with Technical College Collaboration (ADV -program) " has been established in 2010.
In this paper the conceptual framework and the initial stage of ADV-program are introduced. Its
principal objective designates promotion of "strategic engineer" coping with the above mentioned
requirements for Mono-Dukuri. More specifically the proposed program focuses on enhancing thefundamental abilities on "Specialties on multiple fields", "Strategic technology management" and"Global leadership" as shown in Fig. 1, and prepares several courses on specific subjects enhancing
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the abilities by collaboration of NUT and KOSENs through the 4th and 5th grades at a KOSEN tograduation from master course at NUT.
Multiple technology fusion

Interdisciplinary transfer

Specialties on
multiple fields

AdvancedcourseBounded advanced specialty
Basic specialty / Generality
Basic management knowledge

Strategic technology
management
Reading social &industrial trends

Fig. 1

Readability of English paper

Global leadership
Global teamcollaboration

Technology
assessment

Rational
competition

Conceptual image of the fundamental abilities to be enhanced in ADV-program.

On the initial stage the collaborating organization for ADV-program consists of NUT andspecific six KOSENs of various types which are managed by Institute of National Colleges ofTechnology as shown in Fig.2.
Nagaoka University of Technology （NUT）

Current
Institute of national colleges
collaborationof technology, Japan(51 national colleges all over Japan)

Futurecollaboration

Specific six collaborating colleges of technology[ Oyama‐NCT, Fukui‐NCT, Fukushima‐NCT,
Kagawa‐NCT, Nagaoka‐NCT, Toyama‐NCT ]All technical colleges in Japan(National, Public, Private)

Fig.2

Collaborating organization for ADV-program

In 2011, the second year of the program, 58 students in 4th and 5th grades joined the ADVprogram from the six model KOSENs in all over Japan. They participated in "Introduction to
Engineering Frontier", "English Oral Presentation" and "Mathematics Supporting Technology" in
Collaborating courses - Part I, and "Intensive Seminar/Laboratory Exercise", "Advanced
Technology Lecture/Practices" and "International Conference Exercise" in Preceding courses. The
detail on the initial stage of the proposed program and further discussions will be described in thesubsequent sections.
2. Collaborating Courses at Technical Colleges
2.1 Introduction to Engineering FrontierThis sub-section shows a newly introduced subject, "Introduction to Engineering Frontier", atKagawa National College of Technology (Kagawa-NCT) [5]. The subject is intended for higher
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grade students at the college and is offered in cooperation with some private enterprises as well as
Nagaoka University of Technology (NUT). The educational objectives of the subject are (1) tounderstand advanced technology in related to engineering frontier, (2) to grasp social situation indiversification and (3) to find out global human resources required in the technical innovation,corresponding to the three major talents explained in Section 1. In addition, these objectives are also
consistent with the educational objectives of Kagawa-NCT as shown in Table 1.
Table 1 Objectives of ADV-program and Kagawa-NCT
Fundamental abilities
for strategic engineer(Key words in Japanese)
Educational objectivesof "Introduction to
Engineering Frontier"
Educational objectivesof Kagawa-NCT

Specialties on
multiple fields(Fukugan-teki)
To understandadvanced technology
in related toengineering frontier
Creative designing on
complex problems

Strategic technology
management(Senryaku-teki)
To grasp social
situation indiversification
Technologicalknowledge in an everchanging world

Global leadership(Kokusai-teki)
To find out global
human resources
required in thetechnical innovation
Communication skills
for international
career paths

Table 2 shows a simplified program of the subject with affiliation of each speaker. As shown in
the table, a few topics of the closely related field are presented every day so that students of different
departments at the two campuses can easily get to the main points. At each class, the students wererequired to submit a report. Figure 3 shows a group photograph in the "Introduction to Engineering
Frontier" class. Twelve students including auditor took the course.

Day
1
2
3
4
5

Table 2 Program of "Introduction to Engineering Frontier"Class 1
Class 2
Class 3
○Multi-functional reservoir☆Global human resources□Water environment□Mechanics and○Mechanics and processing☆Ion exchangemanufacturing□Web based information☆Software development○Cloud computing
processing
for iPhone○Communication engineering (including practice)○Semiconductor device (including experiment)
Presented by: ○; Kagawa-NCT, □; NUT, ☆; Company / InstituteFig. 3

The group engaged in "Introduction to Engineering Frontier" at Kagawa-NCT

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2.2 English Oral Presentation Seminar
New subject“English Oral Presentation Seminar”has started at Toyama-NCT since April in 2011.
The Working Group of 11 professors at Toyama-NCT designed this as a subject of Collaborating
courses - Part I in ADV-program. The aim of this subject at Toyama-NCT is to stimulate students’motivation to become professional engineers or business persons with skills to manipulate English.
Toward this goal, this subject provides students with basic knowledge of English writing and with
opportunities to make speeches and discussions in English. This is one of the elective subjects in thefirst semester at Toyama-NCT. This is placed as a 90-minute regular class on Thursday on school
time table. This is a two-credit class in 15 weeks. The target students are the fourth and the fifth
graders. Toyama-NCT has two campuses. So these two campuses are connected for this subject byusing a video conference system. More than 50 students were interested in this seminar and 18
students were allowed to take this seminar in the first year.
This subject is consisted of three stages. The first stage is “Logical Thinking” stage conducted byassociate professor at NUT, Dr. Makoto Nanko. It takes students three weeks to acquire how to write
their ideas and opinions with flow charts in this stage in Japanese. This basic skill worked
successfully at the following stages in this seminar. The second stage is “150-word ParagraphWriting” organized by an instructor at Japan Society for Technical Communication (JSTC), Ms.
Yukiko Nakayama. This stage takes seven weeks. Each lecture is focused on “Technical Writing” inEnglish. There are three points as follows. Firstly, students realize what technical writing is like.
Secondly, students choose one topic from the following topics; “Hybrid car,” ”Cellular phone,”and ”Nuclear power.” Topics are basic technologies related to students’ majors. Finally, students
complete their own writing. The teaching methods such as “Peer-Learning”,“Peer-Review” aremainly adapted at this stage. Automatism is required to become better English writers. The third
stage is “English Oral Presentation and Discussion” instructed by all professors at Toyama-NCT
with the help of an instructor at JSTC, Mr. Koki Tokuda. Students’ presentations and discussions are
conducted in English. Students are supposed to be positive at this final stage. Professors at
Toyama-NCT facilitate students’ English discussion. Students experience pseudo International
Society at this stage.
Through this seminar, several surveys were conducted to evaluate students’ awareness toward
their skills. Here are 25 questions of a survey;
1) problem-finding skill, 2) skill to map out, 3) understanding and deepening knowledge,4) self-learning skill, 5) skill to collect information, 6) skill to arrange information,7) skill of information analysis, 8) expressiveness with text, 9) expressiveness without text,10) skill to create simple explanations, 11) presentation skill, 12) questioning mind,13) communication skill, 14) skill of self-assessment, 15) skill of assessment to others,16) skill of self-modification, 17) skill of follow-up, 18) skill of action,19) collaborative learning skill, 20) problem-solving skill, 21) knowledge construction skill,22) creative mind, 23) fulfillment, 24) accomplishment, 25) research mind.
In Fig. 4 the sequential blue sticks show students’ profiles before this seminar starts, and thesedata says students cannot be confident in the skill to give a presentation. This might be one of the
8
7

Average

6
5
4
3
2
1

24

25

5

23

19

18

4

6

3

17

15

13

20

22

8

16

2

21

7

14

1

10

9

12

11

0
QuestionsFig. 4Students’ profile (Surveys BEFORE[:blue] and AFTER[:red] this seminar
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reasons they took this seminar. On the other hand, their research mind and their sense ofachievement and satisfaction are quite high. That suggests that most of participants in this seminarmight get high marks at school. The sequential red sticks in Fig. 4 present students’ awarenesstoward their skills after the seminar. The comparison between blue sticks and red ones proves thatthe students’ awareness toward ALL skills is enhanced from April, “BEFORE students learn” toAugust, “AFTER students learned” in four month.
Figure 5 reveals how much students’ awareness toward all 25 skills are enhanced. This data
indicates enhancement of two things through this seminar; knowledge and skills. Firstly, students
feel that they were able to comprehend and utilize information correctly and the understanding of
their knowledge was deepened. Secondly, students realize that they were able to express theiropinions correctly in English writing and at oral presentation and in discussion.
2.5

Average

2

1.5

1

0.5

1

11

7

12

8

13

6

3

9

21

22

17

16

14

10

23

5

20

4

2

19

18

15

24

25

0
QuestionsFig. 5Enhancement of Students’ Awareness toward skills

From these two figures above, it concludes that the teaching methods and syllabus in thisseminar worked well and stimulated students’ intellectual curiosities deeply and inspired a feeling ofachievement. These data also convince us that the collaboration of Toyama-NCT and NUT
extremely empower both institutions to foster highly-motivated students. Figure 6 shows somescenes of students’ practices on the three stages.

(a) Stage1 (Logical Thinking)(b) Stage2(Technical Writing) (c) Sage3(presentation)
Fig. 6 Scenes of practices on the three stages in “English oral presentation seminar”

2.3 Overseas Dispatch Training and the Advanced Program
Nagaoka National College of Technology (Nagaoka-NCT) has been accepting international
students since 1985, and has graduated 100 international students. Up to the present, we have hadinternational students from Malaysia, Mongolia, Vietnam, Indonesia, Bangladesh, Gabon, China, etc.
At Nagaoka-NCT, we foster well-rounded students through technical school education incorporating
activities with the purpose of creating international, cross-cultural understanding. Among theactivities promoting international exchange, we have been engaged in overseas dispatch training
since 2005. This activity aims to make the most effective use of many international students learningat Nagaoka, and we dispatch Japanese students to their country so as to deepen mutual understandingof various cultures and promote international exchange among students.
Also, one part of the overseas training is designated as "Overseas Training Workshop" wheregroups of Japanese and local students carry out observations at Japanese companies andcollaborative programming practice using microcomputers and LEGO MINDSTORMS®. We
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consider that it is also important to allow students to experience other cultures at the beginning of
their engineering education. So far students have been trained on collaborative projects together withlocal students from China, Malaysia, and Vietnam on collaborative projects (Fig.7) and observed
Japanese companies (Fig.8). In addition, in order to support the cooperation of international
exchange activities, we established "Global Lab" in 2008. The Global Lab activities are devised toencourage exchange between international and Japanese students. In addition, a seminar,"International Affairs and Communications" is now being offered to promote cultural understanding.
Two projects, the practice of "Student Support Programs for New Social Needs" and "Student
Development through Regional Cooperation and Exchange with Higher Education Institutions inAsia" have also been implemented at Nagaoka-NCT.
Because the only initiatives of one KOSEN have limitations, the "ADV-program," promoted

Fig. 7 Creative work, “Monodukuri” abroadat Vietnam National University

Fig. 8

Factory visit of the Japanese company
in Vietnam

through cooperation with NUT and KOSENs, has been started as an educational program that
includes content that we understand to be major themes within engineer training. Three
perspectives—"Specialties on multiple fields", "Strategic technology management" and "Global
leadership"—are all important in the education of engineers at KOSENs. In accordance with theaims of the Cooperating Courses – Part I at KOSENs, the courses, "Introduction to Engineering
Frontier", "English Oral Presentation" and "Mathematics Supporting Technology" have been
proposed, and some of these courses are beginning to be offered at Nagaoka-NCT. Based on
experiences in these courses and on international exchange at KOSEN, students are considered to beable to continue to educate themselves through ADV-program.
3. Preceding Courses at Nagaoka University of Technology
3.1 Intensive Seminar and Laboratory Exercise
New subjects “Intensive Seminar/Laboratory Exercise” provided in the preceding courses of
ADV-Program started for giving KOSEN students the opportunity to experience laboratory activities
for 1 week. The aim of those subjects is to let them know a fun to study with laboratory colleagues
and then to enhance their motivation to be creative engineers. Intensive seminar and laboratory
exercise are held simultaneously as a subject-unit and include experimental activities, lectures andoral presentation. Figure 9 shows a picture on the scene of the research activity on development of
new materials. In the subject-unit, almost students of KOSENs have first experience to give an oral

Fig.10

Fig.9 A scene of research activity on
development of new materials
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A scene of oral presentation in
intensive laboratory exercise

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presentation based on their research activity. They can obtain not only new knowledge andexperimental skills in a particular engineering subject, but also fundamental skill for oralpresentation. Figure 10 shows a picture on oral presentation given by a KOSEN student.
In summer of 2011, 51 KOSEN students joined to Intensive Seminar/Laboratory Exercise held
in NUT from July 25 to September 16. Figure 11 shows questionnaire results from KOSEN students
on the research activity in Intensive Seminar/Laboratory Exercise.
The questionnaire results
indicate that almost KOSEN students were satisfied with the research activity in this program. To
have communication with our students in the master and doctoral programs was also useful forkeeping their motivation.

Fig.11

Questionnaire results from KOSEN students on the research activity inIntensive Seminar/Laboratory Exercise
However the schedule for this program was not enough to prepare the system for accepting
KOSEN students in our university. Establishment of the entry system for KOSEN students to thisprogram is the most important issue to be solved up to the next academic period.
Finally excellent research results have been obtained in the research activity in “Intensive
Seminar/Laboratory Exercise” and presented in some academic conferences [6,7]. These results
are given by serious efforts and of a great deal of talent of KOSEN students.
3.2 Advanced Technology Lecture and Practices
The subject “Advanced Technology Lecture and Practices” provided in the preceding courses
in ADV-program. To progress the multifaceted thinking with flexible idea and the competence forthe development of strategic advanced technology, the program students could be entry this coursefor 1 week. The aim of this subject is to learn the environment surrounding of engineers on theadvanced technological field, and the fundamental knowledge about the role of creative engineer inthe global world. Landmark of this lecture and practices is to progress the presentation ability for thepresent subject on the industry and community without regard for their special field. As shown in
Table 3, Advanced Technology Lecture was carried out by two instructors in the morning term. One
was applied on the special lecturer of Nagaoka University on the theme and background of that day.
The other was carried out by the special engineer or CEO (Chief executive officer). After lunch time,
students transferred from University to the business facility and the advanced technology practice
was carried out. They made the study tour and discussion of the special technology outside of theirresearch field. After returned to the hotel, they wrote the report of the lectures and study tour till nextmorning. On the last day of this program, the oral presentation was held on each field by several
groups. The field and the group member for the presentation were given in the evening of Thursday.
The selected filed was outside of their own region and the different college students were jointed.
The oral presentations were criticized by various reviewers, who were professor of NUT andthe lectures of the visited companies. This year, the following fields were experienced.
1) Energy ·Security ·Engineering ethics.
2) Enviroment·Civil Engineer·Tranportration
3) Biotechnology·Medical service·Provisions. 4) Management ·Information· Distribution.
In this program, 19 KOSEN students joined. According to comments for this program,
most KOSEN students were satisfied with the aim and the activities of the program.
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MondayTuesday
WednesdayThursdayFridaySubject

Table 3 Schedule of Advanced Technology Lecture and PracticeMoring 1
Moring 2
Lunch time
Study tour
Opening ceremony Lecture (business
Lunch and transfer Business facility or
and lecture(NUT)
facility)(within 1 hour,Industrial plant
By bus)Field (1),*1
Lecture(NUT)*2
ditto
ditto
Field (2) , ditto
ditto
ditto
ditto
Field (3), ditto
ditto
ditto
ditto
Field (4), ditto
Preparation of oral
Preparation of oral Lunch time
Oral presentation
presentation
presentation*3
Advanced technology
lectureAdvanced technology
lectureAdvanced technology
practice*1: Make the reports for lecture and tour, every night from Monday to Thursday.
*2: Turn in report every morning from Tuesday to Friday.
*3: The presentation field number and the group member were announced by the program bureau.
3.3 International Conference Exercise
There is no doubt that English is an important communication tool for engineers. KOSEN
students (4th and 5th graders) have been studied English for 6 or 7 years, and most of them havealready learned basic English. Learning presentation skill will improve their communication skills in
English as well. In this program, they are provided a subject “International conference exercise” asan English practice for presentation at conference.
The goal of this subject is to give a short presentation in English on the student’s own research.
This English course is composed of the following steps to achieve the goal.
1) Writing an abstract in Japanese
2) Writing the “Title” in English
3) Writing the “Objective” in English
4) Writing the “Method” in English
5) Writing the “Result & Discussion” in English 6) Writing the “Conclusion” in English
7) Preparation of 4-sheet presentation file8) Preparation of notes for presentation in English
The abstract contains “Title”, “Objective”, “Method”, “Result & Discussion”, and “Conclusion”.
The four sheets contain “Objective”, “Method”, “Result & Discussion”, and “Conclusion”,
respectively.
The course is carried out basically through e-mail and/or Web site, because it is difficult for the
KOSEN students to come to NUT every week. In the step 1), the students are requested to write an
abstract in Japanese prior to writing it in English. Contents of the abstract and its logical structure isimportant not only for the students but also for teaching staffs of NUT in order to understand eachresearch work. This is the reason why we request the students to write the abstract in Japanese. NUT
staffs make corrections on the submitted Japanese abstract, and give suggestions.
In the steps 2) - 8), the students write each topic in accordance with reference materials sentfrom NUT staff. The submitted English document for the each step is checked and corrected by NUT
staffs with the aid of foreign students of NUT as teaching assistant.
Seven KOSEN students took this English course in 2011. They gave a short presentation(Fig.12) using the 4-sheet presentation file and notes prepared in the steps 7) and 8). After giving apresentation, the student answered questions from NUT staffs and other KOSEN students.

Fig.12A scene of an oral presentation excise in English
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We believe that this English course is a good opportunity for the KOSEN students to practicepresentation in English and effective to enhance their communication skills. For further
improvement of this course, literature searching should be introduced. This is because enhancementof their vocabulary, particularly technical terms, and pick up useful expressions are necessary forbetter scientific writing and presentation.
4. Prospective Fruits
This ADV-program is a wide-reaching collaborative education between technical colleges(KOSEN) and Nagaoka University of Technology (NUT), including 4th grade KOSEN students to
NUT master’s degree students, with the aim at promoting very excellent personnel out over theworld.
There are the three pillars of ADV-program consisting “Specialty on multiple fields”, ”Strategic
technology management” and “Global leadership”. Three subjects “Mathematics to Support
Technology”, “Introduction to Engineering Frontier”, and “English Oral Presentation Seminar”
comprising Collaborating Courses - Part I corresponding to the three pillars were provided throughdiscussion between teaching staff from the six model KOSENs, the director of the College ofTechnology and teaching staff from NUT. And three subjects "Intensive Seminar/Laboratory
Exercise", "Advanced Technology Lecture/Practices" and "International Conference Exercise" aresimultaneously introduced in Preceding courses as a part of NUT program. Collaborating Courses Part I and preceding courses take introductory roles for ADV-program. At the initial stage, Stage-1,
at KOSEN, the students of ADV-program gain insights into global trends and consider what is
important for elemental technology and training. Furthermore, the course forms the basis fordeveloping promising personnel who will be of benefit to both Japan and the wider world throughunderstanding of technological-systems and creation of strategies in society.
At Stage-2 in 3rd and 4th grades in NUT, ADV-program carry out "Second laboratories" thatprovide actions on vice-specialty demanded today. With stage-3 in NUT master course, the studentsof this program will return to the laboratories of a KOSEN as an intern and carries out teaching. The
whole ADV-program brings up a global leader being aware of local industry and globalization. This
ADV-program is introduced as a model educational program with specified six KOSENs now, butthey planned to be expanded as the program for all KOSENs in the future.
Acknowledgments
The authors should express their gratitude to teaching-staffs from companies, KOSEN and
NUT for the cooperation. They will be extremely grateful for the paper readers’ positive advice andopinions regarding excellent personnel promotion on this ADV-program.
References[1] D.Wilson and R.Purushothaman, Global Economics, Paper No: 99, Goldman Sachs Global
Research Website, https://www.gs.com (2003).
[2] D.Wilson, C.Burgi, and S.Carlson, BRICs Monthly, Issue No:11/06, Goldman Sachs Global
Economics, Commodities and Strategy Research, https://360.gs.com (2011).
[3] H.J.Moon (Translated by I. Yoshiwara), Working like Samsung (in Japanese), THENAN
Publishing Co. (2010).
[4] T.Ando, M.Kawashima and K.Kan, Technology development and technology transfer in China(in Japanese), Minerva Publishing Co. (2005).
[5] Y. Jodai, Bulletin of Kagawa National College of Technology (in Japanese), 2 (2011) 22.
[6] D.Maruoka, T.Itaya, T.Misaki and M.Nanko, Abstracts on H23 Joint Meeting of the
Hokuriku-Shinetsu Branch of JIM and ISIJ, (2011) 120.
[7] D.Maruoka, T.Itaya, T.Misaki and M.Nanko, Abstracts on the Spring Meeting of JIM, (2012)
121.
(Received: 17 May, 2012, Accepted: 15 June, 2012)9

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Engineering Education Programs for International Students
toward the integration of Japanese language educationand technical education
Seiji KAMIMURA1,*, Yoshiki MIKAMI2, Naoko LEE IIZUKA3, Yoshiro ITO1
1)
Department of Mechanical Engineering, Nagaoka University of Technology,2)
Department of System Safety, Nagaoka University of Technology,
3)
Department of General Education, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: kami@nagaokaut.ac.jp

Nagaoka University of Technology (NUT) currently conducts Twinning
Programs (TPs) with six universities from three countries. . In their native
countries, students take classes in both their specialized fields and Japanese
language. However, students are initially taught in their mother tongues fortechnical classes, and therefore only exposed to technical courses taught inJapanese upon arrival in Japan. Here, we introduce refinements that wehave implemented to improve the transition process. The 2 main aspects ofthese implementations are: (1) Improving the teaching ability of technicaleducation teachers; and (2) Development of teaching materials to improve
the integration between Japanese language education and technicaleducation.
Technical lecturers from Japan are periodically sent to partner
universities to conduct intensive technical courses in Japanese. As part ofFaculty Development, a comprehensive lecturer training package wasdeveloped, comprising of pre-training of the lecturer, the actual intensive
courses, and a follow-up evaluation stage.
Time constraints for Japanese language classes make it difficult for
students to obtain sufficient language ability for further education in Japan.
To improve the integration between Japanese language education andtechnical education, we have developed curricula and teaching materials toimprove students’ Japanese language ability in their specialized engineering
fields. We are currently developing 2 such teaching materials: a textbook to
introduce mechanical engineering topics in Japanese and a workbook to
support the abovementioned intensive technical courses. These materials,
developed through collaborations between Japanese language teachers andtechnical education academic staff, should improve the quality of the TPs.

1. Introduction
A Twinning Program (TP) is a voluntary partnership program conducted between two
universities in different countries. In these programs, participating students spend the first half of
their education in one institution and complete their studies in the partner institution. Partner
institutions agree in advance to a detailed curricula, and students benefit from the experience ofstudying overseas with reduced costs. Furthermore, these programs can serve as the starting point for
further collaborations between the partner institutions, facilitating research opportunities and theexchange of internships.
In 2003, the Nagaoka University of Technology (NUT) initiated an inaugural TP with Hanoi
University of Science and Technology (HUST), the largest technical university in Vietnam. This was
the first TP established by a Japanese national university, as well as the first to be formallyrecognized by the Vietnamese government.
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In this TP, students majoring in Mechanical Engineering spend the first two and a half years at
HUST, where they are educated in engineering fundamentals as well as Japanese language. At theend of this period, candidate students undergo an examination of both their engineering knowledge
and Japanese language capabilities. Students who successfully pass this selection stage enter NUT as
third year undergraduates, attending the same classes as Japanese students. The pioneer batch of
HUST students graduated from NUT in 2007, and NUT is currently hosting the 9th batch of HUST
students.
In the subsequent years, our university has initiated and is currently administrating TPs with six
universities from three different countries, covering a range of courses. These universities include
HUST, Da Nang University and Ho Chi Minh City University in Vietnam; Universidad Autonoma deNuevo Leon and Monterrey Institute of Technology and Higher Education in Mexico in Mexico; andZhengzhou University in China. The details of these TPs are presented in Table 1.
Table 1 Current status of Twinning Programs conducted by NUT
Country
University
CourseInaugural year
Hanoi University of Science and (M)2003
Technology
Vietnam
Da Nang University
(Cv)
2006
Ho Chi Minh City University(E)
2006
Universidad Autonoma de Nuevo Leon
(Cv)
2008
MexicoUniversidad de Monterrey
(M)
2007
China
Zhengzhou University
(Cm)
2007
Legend: (M): Mechanical Engineering Course, (Cv): Civil Engineering Course, (E) electronic
Engineering Course, (Cm) Chemical Engineering Course

2. Challenges in Twinning Programs
Mastery of the Japanese language is an essential skill for TP participant students, as lectures in
NUT are conducted entirely in Japanese. Furthermore, Japanese language is necessary for daily-lifeand communicating with fellow students and lecturers. Therefore, ensuring that the students possess
adequate Japanese language ability prior to arriving in Japan is essential to the success of these
programs.
Figure 1 shows the initial components of Japanese language education within the TPs. Students
spend the first two and a half years in their native countries, during which they undergo Japanese
language classes. Additionally, NUT periodically sends technical education teachers to conduct
intensive technical courses in Japanese, in order to prepare students for the latter half of theireducation in Japan. During this latter stage, students have access to dictionaries of technical terms inJapanese and their native languages.
First 2.5 years in Native Country

Final 2 years in Japan

Japanese Language Education
Entrance Exam.
Technical Term Dictionary

Intensive Technical Courses
Technical lecturer

Fig. 1 Course design of the initial Japanese language education components in
TPs.
However, the intensive technical courses are infrequent. As such, the students essentially conduct2

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their technical training in their mother-tongue, and are only exposed to academic classes conductedentirely in Japanese upon their arrival in Japan. This sudden and intense exposure to technicalclasses in Japanese is non-conducive to a successful TP, as the students require lengthy periods oftime to adjust.
Furthermore, as students must undergo technical education in addition to Japanese language
lessons in their native countries, it is not feasible to significantly increase their language lesson
workload. While the maximum number of Japanese language lesson hours available to the students
in our TPs is 900 hours, the reality is that it is difficult for students to receive more than 700 lesson
hours.
The Japanese language education aspect of the TPs requires improvement in order to better
prepare the participant students for their academic life in Japan. However, these improvements need
to be conducted within the boundaries of the limitations as outlined above. Our solution was tomaximize the available framework by breaking away from the standardized general Japanese
language classes, and replacing it with an Integrated Technical Japanese Language Education
Program, in which Japanese language education is combined with technical education.

3. Towards A Solution
Current and impending refinements to the TP course design include the following:
Final year Japanese students from NUT are sent on internships to the partner universities forfive-month intervals, where they teach fundamental technical terminology in Japanese to candidate
TP students using a Basic Scientific Workbook. These TP students are thus exposed to technicalterminologies that they would not ordinarily learn in a basic Japanese language class.
Next, Japanese technical education lecturers undergo a comprehensive training package prior to
conducting the intensive courses in the partner universities, comprising of pre-training of the lecturer,
the actual intensive courses, and a follow-up evaluation stage. Details of the pre-training of technicaleducation lecturers from NUT prior to intensive technical courses are shown in Table 2.
Table 2 Pre-training for lecturersofDateof No.
Twinning Program
Pre-training Participants

Subject

2008.11.5

2

Hanoi, Vietnam

2009.1.30

1Mexico2009.3.13

2Danang, Vietnam

2009.8.6

2

Ho Chi Minh City, Vietnam

2009.11.5

1

Hanoi, Vietnam

2010.1.28

2

Ho Chi Minh City, Vietnam

2010.3.1
2010.5.31
2010.7.28
2010.8.32112Danang, Vietnam
Zhengzhou, China
MexicoHo Chi Minh City, Vietnam

Manufacturing
Material Science
Thermodynamics
Hydraulics
Engineering Disasters
Hydraulic Engineering
Basic Electromagnetics
Basic Quantum Mechanics
Basic Materials*
Electric materials (dielectrics)
Electric circuit
General Civil Engineering
Organic Chemistry
Civil Engineering
Electronic Engineering*Conducted for Visiting Professor from Gunma University
The aforementioned training package has the added advantage of providing Faculty
Development (FD) opportunities for technical education lecturers in NUT. These lecturers, who have
limited experience in teaching foreign students, are trained by Japanese language lecturers, technicallecturers who have experience in conducting intensive courses, as well as current foreign students
enrolled in NUT. The pre-training consists of a 20 minute mock lecture, followed by advice and3

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exchanges of opinions. FD can be obtained as these lecturers are trained to appropriately teachforeign students, as well as improving their general teaching abilities. The self-reflection andexternal advice provided in the follow-up evaluation stage may help to further improve the teachingability of the lecturers. In addition to ensuring better intensive technical courses in the partner
universities, these improvements would be advantageous when these lecturers teach foreign students
in Japan.
Refinements are also being conducted with the development of two technical Japanese language
textbooks. After obtaining a basic level of Japanese language ability, TP participant students would
continue their education in their native countries using “Intermediate Japanese Language throughMechanical Engineering”, a textbook that we are currently developing wherein students are exposed
to technical Japanese vocabulary and grammar structures. After students arrive in Japan, they would
have access to a “Plain Japanese-based Technical Book”, in which the technical topics covered intheir studies are presented in an easy-to-read format. Furthermore, the collaborative development of
the Japanese textbooks involves frequent interactions and communication between technicaleducation staff and Japanese language lecturers, thereby providing FD opportunities for all involved
parties. These refinements are summarized below in the Figure 2.
First 2.5 years in Native Country

Specialized JL Textbook

Intensive Lectures＋
Lecturer Training

Final 2 years in Japan

Entrance Exam.

Technical lecturer

PJ-based Tech. Book

Pre-training of Lecturers

Basic Scientific Workbook
Internship Students

Fig. 2 Course design of the refined Japanese language education components
in TPs.

4. Conclusion
While there are limits on the Japanese language lesson hours available to TP participant students,
the importance of mastering Japanese language emphasizes the need for refinements andimprovements to the program. In response to this need, we have implemented training of technicallecturers to effectively conduct short intensive courses in the partner universities. Furthermore, wehave integrated general Japanese language education with technical scientific education, and aredeveloping technical Japanese language textbooks to support this integration.
Acknowledgments
This research was supported by the Program for Promoting High-Quality University Educationfrom the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
(Received: 18 May, 2012, Accepted: 12 June, 2012)4

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Nanoscale Deconfinement: Isolation of Nanovolume Leads to Change
Roman Nowak1,3,*, Dariusz Chrobak1,3, William W. Gerberich2 and Koichi Niihara3
Nordic Hysitron Laboratory, Department of Materials Science & Engineering, School of Chemical
Technology, Aalto University, Espoo, FI-02015, Finland2)
Department of Chemical Engineering & Materials Science, University of Minnesota, Minneapolis,
MN 55455, USA
3)
Extreme Energy-Density Research Institute, Nagaoka University of Technology,
Nagaoka, Niigata, 940-2188 Japan*Email: roman.nowak@alto.fi

1)

Rapid expansion of nanotechnology is accompanied by growing interest in
developing nanoparticles exhibiting mechanical behavior tailored to optimize
their production routes and to satisfy the demands of their intended use aselectronic materials. Given the evidence of the dramatic impact of crystal
imperfections on the functional properties of Si nanovolumes and theirbiocompatibility, understanding the evolution of lattice defects in Si
nanoparticles is essential. Here we present atomistic calculations andsupporting experimental results revealing an unknown dislocation-driven
mechanism in Si nanoparticles. The initial dislocation activity is reversible, incontrast to the accepted definition of plastic deformation regarded as“permanent”. Molecular Dynamics simulations match the experimental data
exhibiting striking contrast to deformation of bulk Si surfaces. The observed
behavior is examined in the framework of the dilemma concerning
dislocation, or phase-transformation origin of the incipient plasticity innanoscaled semiconductors and ceramics. Our findings reinforce previous
studies proposing that the onset of plasticity in nanoscale volumes is driven
by the crystallography and geometry of the nanoscale deconfinement.

1. Introduction
Silicon, one of the most extensively studied advanced technology materials exhibits a complex
pattern of plastic deformation combining amorphization, phase transformations, and dislocations[1-4]. Recent nanoscale compression and nanoindentation observations [3] demonstrated that
deformation of Silicon nanovolumes is markedly different from behavior of the bulk material. The
enhanced hardness of Si nanospheres is consistent with the known phenomenon of yield stressincrease due to the decreasing dimensions of deformed volumes [5,6]. However, the reversible
plasticity exhibited by the compressed Si nanoparticles cannot be justified in the framework of thetheories pertinent to the bulk Si surfaces. A model of dislocation-driven onset of plasticity
accounting for the unusual behavior of Si nanoparticles has already been proposed [5,6]. Following
this idea, we clarify the origin of reversible incipient plasticity in nanosized Si particles by providing
the detailed atomistic account of the underlying previously unverified mechanisms.

2. Experiment
In order to model processes initiated in stressed Si nanospheres we performed Molecular
Dynamics (MD) simulations designed to match experimental conditions of previously reported
nanocompression tests [5,6] and experiments carried out in the present study. MD interactions
between Silicon atoms were modeled using the Stillinger-Weber (S-W) potential [7] accuratelyreflecting elastic behavior and generation of lattice defects in Silicon [8]. Simulations were
performed for Si spheres radii R1=5.16 nm (30691 atoms) and R2=3.81 nm (10041 atoms)
compressed between two rigid parallel plates (Fig. 1a). Loading was conducted via 0.25 Åsymmetrical displacement (δ) increments applied to both plates (Fig. 1a). The atomic structure of thedeformed nanospheres was relaxed during 20000 time steps (2 fs) for each displacement increment

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occurring at a temperature of zero K. Deformation history of the Si nanospheres was captured inload-strain (P - ) curves and contact pressure-strain (pc - ) relationships (parameter definition given
in Fig. 1) derived from MD-simulations. Our examination was concluded with visualization of theatomistic structural changes during MD deformation of silicon deconfined to nanoscale volumes.
The hypersonic plasma particle deposition (HPPD) technique [9] used for synthesis of Si
nanoparticles utilizes an argon-hydrogen plasma into which a vapor-phase silicon tetrachloride is
injected. Particles achieve hypersonic velocities upon expanding to low pressure through aconverging diverging nozzle and the resulting particle beam is deposited on a sapphire chip
substrate.
A PicoIndenter (PI-95, Hysitron Inc.) was used for compressing the evaluated nanospheres withradii ranging from 33 to 169 nm. The employment of a relatively rigid substrate and the diamondflat-punch diamond indenter allowed us to follow the conditions of our computer experiments.

Fig. 1 MD simulated compression of silicon nanoparticles (a) quantified in terms of load–strain
relationship (P-) obtained for the nanoparticles of two different radii, R1 = 5.16 nm and R2 = 3.81
nm (b)(c)(d). Elastic deformation follows Hertzian theory (red curves), providing an estimate of
reduced Young’s modulus (b). After the pop-in multiple singularities in the P - ε curve reflect plastic
deformation. The relationship of contact pressure, pc P/A, (A actual contact area) versus strainεR demonstrates that the hardness of the nanoparticles (~23 GPa) is nearly two times higher
than hardness of bulk silicon (~12 GPa), Unloading paths [(d) - red and green lines] calculated forthe larger Si nanosphere prove the complete particle shape recovery after unloading. Despite theprominent pop-in the initial plasticity in the investigated Si nanoparticles is reversible. Straining tohigher levels ε 0.242 red curves) produces permanent deformation.
3. Results and Discussion
Perfectly elastic behavior of Si nanoparticles during initial stages of deformation (Fig. 1b)conforms to classic continuum mechanics expressed by the 13 decades old Hertzian equation [10]. In
agreement with the results of MD simulations by Valentini, Gerberich and Dumitrica [11], our MD
calculated elastic moduli for the nanoparticles (ER1=116.8 GPa and ER2=108.9 GPa) were lower thanthe 160 GPa measured for the bulk Si [5]. The reduction of Young’s modulus with decreasing

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particle size is attributed to the high surface-to-volume ratio of nanospheres [11] consistent with therecent results by Mo et al. [12]. Our MD simulations registered pressure reaching 23 GPa (Fig. 1c)matching the experimentally observed elevated yield-point stress of the Si nanopartices [5,6]. Thisvalue is almost two times higher than the hardness of the bulk Si [5], while nearly identical to themagnitude of peak stress reached at the point of elastic-plastic transition in previous simulations [11].
The analysis of load-strain response and the magnitude of stress at the onset of plasticity
demonstrates consistency between the experimental [5,6] and MD-modeled elastic deformation
prompting the conclusion that elastic behavior in Si nanoparticles is accurately reflected by both
Tersoff [11] and S-W potentials. However, beyond the elastic limit (Fig. 1), the two potentials
predict markedly different behavior. MD results obtained with the Tersoff potential indicated that
contact pressure remained constant after reaching its peak value [11]. Such behavior was attributed
to the stress-induced transformation to a -tin phase, considerably harder than the original diamondstructure of Silicon. Consistent with the contact pressure stabilization, the load-displacement
response calculated with the Tersoff potential maintained its smoothness through thepressure-induced transition [11].
Our simulations with the S-W potential contrast the results based on the Tersoff potential [11],and reveal that the maximum contact pressure is followed by a sudden, dramatic decay of the load
(Fig. 1b) leading to a continuous drop in contact pressure (Fig. 1c). The apparent decrease of thecontact pressure under the maintained constant rate of displacement increase (Fig. 1c) is tantamountto the ‘pop-in’ observed during nanoindentation experiments [13-17]. Pop-in, alternatively referred
to as ‘the yield point’, marks the onset of plasticity in defect-free crystals. For load controlled
experiments, pop-in appears as rapid displacement increase whereas its incidence underdisplacement control is evident as a load drop [18].
Consistent with the previous experiments [5,6], contact pressure continues to gradually decrease
after the pop-in approaching hardness of bulk Si at 12 GPa. Remarkably, despite a distinguishable
pop-in event, deformation of Si nanoparticles is reversible (yields no residual deformation) for
strains up to the threshold 0.194  εc  0.242 (Fig. 1d). Only after exceeding this threshold, thenanospheres begin to exhibit conventional plastic behavior involving permanent deformation withlimited elastic recovery (Fig. 1d). In excellent agreement with our atomistic simulations (Fig. 1d),the previous experiments [5,6] showed the reversible plastic deformation in the Si nanoparticles after
the pop-in.
Clarification of the pop-in mechanism in nanoscale Si volumes needs to be viewed in retrospectwith previous research. For nearly a decade, nucleation of the first dislocations was thought to be theonly possible mechanism giving rise to the pop-ins observed during contact loading of defect-freecrystals [13,14,19]. Chrobak, Nordlund and Nowak [16] were the first to demonstrate by atomistic
modeling that pop-ins in GaAs are borne by phase transformation. Recent nanoscale electricalresistance evaluation [17] proved the validity of the earlier simulations [16] and removed thecertainty of attributing the pop-in to dislocation nucleation. These newly emerged experimental
findings clarified by Nowak et al. [17] using ab initio calculations raised a possibility that the pop-in
phenomenon observed during compression of the Si nanoparticles might also be caused by phasetransformations. This would be consistent with the previously well documented mechanisms of thecontact-induced plasticity in bulk Si invoking a combination of amorphization and a series of phasetransformations [1,2].
Curiously, discernable singularities are noticeable in the unloading segments of the MD
simulated P - ε relationships (Fig. 1d). The appearance of these singularities is reminiscent of‘pop-outs’ observed during indentation into bulk Si and determined to be a signature oftransformation from -tin phase formed during loading to Si-III and Si-XII phases [1,2] during
unloading. Pop-outs occur as a discontinuous displacement decrease during load controlled
experiments and as a discontinuous load increase during displacement controlled experiments. In
contrast to the results of our MD-simulated response of Si nanospheres (Fig. 1), the loading responseof bulk Si surfaces is generally smooth [1,2] (Fig. 2a). Therefore, the peculiar combination of pop-in(PI) and pop-out (PO) phenomena during the loading-unloading cycle (Fig. 1d) which we choose toname PI-PO effect, is unique to the nanoscale deconfinement as opposed to nanodeformation of bulk
volumes (Fig. 2a).

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Fig. 2 Mechanical response of Silicon in bulk and deconfined state shown schematically (a) andobserved from nanocompression test results (b). A combination of pop-in (PI) during loading (L)and pop-out (PO) during unloading (U) is denoted as PI-PO effect. The sequence PO→PI-PO→PI
agrees with the findings by Ge et al. for 2D nanodeconfined silicon [3] (a), and is confirmed by our
results of nanocompression tests on Si nanospheres for radii from 33 to 169 nm. The PI only
(green) response occurs consistently for all nanoparticle sizes while the combination of PI-PO (red)is only present for nanoparticles larger than a threshold size of 65 nm. This output agrees with theresults of our computer experiment shown in Figure 1d and suggests that the onset of plastic
deformation in “large” (R > 65 nm) and “small” (R < 65 nm) Si nanospheres involves essentiallydifferent mechanisms. We propose that PI only events in smaller particles and PI-PO events inlarger particles reflect two types of dislocation involvement while the PO events are indicative of
the phase transformation.
In order to verify the results of our MD calculations predicting the PI-PO effect, we performed
nanocompression experiments utilizing the PicoIndenter system. Experimental P - ε curves obtainedfor Si nanospheres reveal two distinct kinds of mechanical response, namely the PI behavior duringloading and the ‘PI-PO effect’ (Fig. 2a) during loading and unloading. The PO effect typical for bulk
Si [1,2] disappears and PI becomes more and more pronounced with the increasing degree ofnanoscale 3D deconfinement (Fig. 2a). The experimental relationship between PO stress and PI
stress (Fig. 2b) indicates the PI-PO effect systematically occurring for Si nanoparticles of radii larger
than the threshold value of 65 nm. This threshold value marks a transition from PI-PO effect to PI
only response.

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Fig. 3 Dislocations activity analyzed in terms of the slip-vector (SV) distribution and the evolutionof the stressed atomistic structure in a case of reversible (a) and irreversible (b) deformation. SV
single peak at 2.3 Å indicates plastic deformation accommodated exclusively via formation of a

stacking fault bounded by partial dislocations | b | = 1/6|[ 1 2 1 ]| = 2.228 Å (atoms marked inyellow), and after unloading the structure perfectly recovers to the undistorted defect-free
configuration (a). Inception of the permanent plastic deformation upon attaining higher strain of ε =0.242 resulted in the appearance of a new SV peak at | b | = 1/2|[ 1 0 1 ]| = 3.84 Å (atoms denoted ingreen). Unloading from strain ε = 0.242 invokes transition from partial to perfect dislocations asproved by the visualization of the strained Si nanosphere structure.
A previous study evaluating size effects in Si nanowedges under 2D deconfinement [3]concluded that pop-ins and pop-outs are generated during loading-unloading cycles when wedge size
is under 1.6 microns, while pop-in without pop-out occurs in the submicron range. TEM
observations linked the pop-outs to plasticity accommodated by a combination of phasetransformation and dislocations and attributed pop-ins to dislocation activity exclusively [3]. The
results of our atomistic modeling (Fig. 1) and nanocompression experiments (Fig. 2) shine new light5Transactions on GIGAKU 1 (2012)01005/1-7

on the previously reported effects of 2D deconfinement [3]. Our findings inspire the idea thatnanoscale 3D deconfinement causes a transition from phase transformation-dominated todislocation-driven incipient plasticity. Indeed, deformation in nanospheres of radii exceeding 65 nm
is adequately described by the approach proposed by Valentini et al. [11], while our atomistic
modeling predicts that dislocation-driven plasticity is appropriate for particles of diameters smaller
than this threshold value.
The picture of nanoscale plastic deformation in Si under nanoscale 3D deconfinement wascompleted by explicit identification of the lattice defects with slip vector (SV) method [20]. SV
analysis enabled us to detect two kinds of dislocations on the { 111 } planes, namely partial andperfect dislocations with respective Burgers vector lengths of 1/6|[ 1 2 1 ]| = 2.228 Å and1/2|[ 1 0 1 ]| = 3.84 Å. These systems were previously observed during nanoindentation loading ofSilicon crystals [3]. At strains ε < 0.242 plasticity is accommodated entirely via formation of
stacking faults bound by partial dislocations as apparent from the SV peak at 2.3 Å obtained at strain
ε = 0.194 (Fig. 3a). Complete disappearance of this peak after unloading confirms again reversible
plastic deformation. All partial dislocations introduced during loading vanish (Fig. 3a).

Fig. 4 MD simulated cross-sectional view of the highly strained Si nanosphere Atom colors
correspond to calculated SV values. The blue part of the nanoparticle is associated withhomogeneous deformation (zero slip vector value), while green bands mark inhomogeneous strain
distribution produced by dislocations. Anchoring at the surface facilitates evolution of partial
dislocations into perfect dislocations. Irreversible deformation is produced by formation of acharacteristic step at the nanoparticle surface, the unique phenomenon captured by our analysis.
In contrast, the irreversible deformation at strain ε  0.242 results in an additional SV peak at
3.8 Å due to formation of perfect dislocations (Fig. 3b) occurring after the stacking fault reaches thenanoparticle surface (Fig. 4). This event triggers the transformation of partial dislocations into
perfect dislocations previously unrecognized in nanodeconfined Si volumes. Consequently, the SV
peak at 3.8 Å becomes even more pronounced and higher during unloading (Fig. 3b). Increasingprominence of the 3.8 Å peak is justified by the observation that upon unloading, stacking fault
anchoring at the surface facilitates evolution of the fault into perfect.

4. Conclusion
The reversible dislocation-driven plasticity we witnessed (Fig. 1, Fig. 3 and Fig. 4) is in accord
with the experimental findings (Fig. 2) and in line with the reversibility of plastic deformation
apparent during ultra-low load nanoindentation of bulk single crystal surfaces reviewed by Gerberichet al. [5,6]. While a concept of reversible plastic deformation and its experimental detection werediscussed in the past [21-25], the reversible plasticity discerned in the Si nanospheres differs fromthe previously considered phenomena such as; collective motion of dislocation ensembles [22],nucleation-annihilation cycles at the boundaries of nanograins [23], evolution of stacking fault
structures [24], the “Baushinger” effect in submicron single crystal films29, and the “plastic
shakedown” model [21].
Uniqueness of the observed reversible plasticity stems from the conditions of 3D nanoscaledeconfinement leading to a shift from phase-transformation-dominated to dislocation-driven

6Transactions on GIGAKU 1 (2012)01005/1-7

incipient plasticity evident from the PI-PO effect witnessed by the authors. Given the influence ofdislocations on the functional properties of Si nanovolumes [26], controlled evolution of reversible
dislocation structures in Si nanovolumes presents unique opportunities for developing tunable Si
nanodevices with optoelectronic and biomedical applications [26,27]. Moreover, it became
increasingly evident during this study that nanoscale deconfinement will play a critical role inproduction of nanoparticles and cluster-materials already gaining a prominent role in contemporary
nanotechnology [28].
Acknowledgments
The authors gratefully acknowledge the CSC–IT Center for Science (Finland) for computation
resources and the Ceramic Society of Japan for invaluable assistance.
References[1] I. Zarudi, J. Zou, and L. C. Zhang, Appl. Phys. Lett., 82 (2003) 874.
[2] V. Domnich, Y. Gogotsi, and S. Dub, Appl. Phys. Lett., 76 (2000) 2214.
[3] D. Ge, A. M. Minor, E. A. Stach, and J. W. Morris, Philos. Mag., 86 (2006) 4069.
[4] J. E. Bradby, J. S. Williams, J. Wong-Leung, M. V. Swain, and P. Munroe, Appl. Phys. Lett., 77(2000) 3749.
[5] W. W. Gerberich, W. M. Mook, C. R. Perrey, C. B. Carter, M. I. Baskes, R. Mukherjee, A.
Gidwani, J. Heberlein, P. H. McMurry, and S. L. Girshick, J. Mech. Phys. Solids, 51 (2003)
979.
[6] W. W. Gerberich, W. M. Mook, M. J. Cordill, C. B. Carter, C. R. Perrey, J. Heberlein, and S. L.
Girshick, Int. J. Plast., 21 (2005) 2391.
[7] F. H. Stillinger and T. A. Weber, Phys. Rev. B, 31 (1985) 5262.
[8] J. Godet, L. Pizzagalli, S. Brochard, and P. Beauchamp, Phys. Rev. B, 70 (2004) 054109.
[9] N. P. Rao, N. I. Tymiak, J. Blum, A. Neuman, H. J. Lee, S. L. Girshick, P. H. McMurry, and J.
Heberlein, J Aerosol Sci., 29 (1998) 707.
[10] H. Hertz, J. Reine Angew. Math., 92 (1882) 156.
[11] P. Valentini, W. W. Gerberich, and T. Dumitrica, Phys. Rev. Lett., 99 (2007) 175701.
[12] Y. Mo, K. T. Turner, and I. Szlufarska, Nature, 457 (2009) 1116.
[13] W. W. Gerberich, J. C. Nelson, E. T. Lilleodden, P. Anderson, and J. T. Wyrobek, Acta Mater.,
44 (1996) 3585.
[14] J. Li, K. J. Van Vliet, T. Zhu, S. Yip, and S. Suresh, Nature, 418 (2002) 307.
[15] J. K. Mason, A. C. Lund, and C. A. Schuh, Phys. Rev. B, 73 (2006).
[16] D. Chrobak, K. Nordlund, and R. Nowak, Phys. Rev. Lett., 98 (2007) 045502.
[17] R. Nowak, D. Chrobak, S. Nagao, D. Vodnick, M. Berg, A. Tukianen, and M. Pessa, Nature
Nanotech., 4 (2009) 287.
[18] O. L. Warren, S. A. Downs, and T. J. Wyrobek, Z Metallkd., 95 (2004) 287.
[19] E. T. Lilleodden, J. A. Zimmerman, S. M. Foiles, and W. D. Nix, J. Mech. Phys. Solids, 51(2003).
[20] J. A. Zimmerman, C. L. Kelchner, P. A. Klein, J. C. Hamilton, and S. M. Foiles, Phys. Rev. Lett.,
87 (2001) 165507.
[21] G. L. W. Cross, A. Schirmeisen, P. Grutter, and U. Durig, Nature Materials, 5 (200) 370.
[22] M. W. Barsoum, T. Zhen, S. R. Kalidindi, M. Radovic, and A. Murugaiah, Nature Materials, 2(2003) 107.
[23] V. Yamakov, D. Wolf, M. Salazar, S. R. Phillpot, and H. S. Gleiter, Acta Mater., 49 (2001) 2713.
[24] P.-R. Cha, D. J. Srolovitz, and K. T. Vanderlick, Acta Mater., 52 (2004) 3983.
[25] R. E. Miller, L. E. Shilkrot, and W. A. Curtin, Acta Mater., 52 (2004) 271.
[26] M. H. Nayfeh, and L. Mitas, Nanosilicon (ed. Kumar, V.) 1-78 (Elsevier, Amsterdam, 2008).
[27] N. O'Farrel, A. Houlton, and B. R. Horrocks, Int. J. Nanomedicine, 1 (2006) 451.
[28] S. Kan, T. Mokari, E. Rothenberg, and U. Banin, Nature Materials, 2 (2003) 155.
(Received: 23 March, 2012, Accepted: 10 April, 2012)7

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Nanomaterials as Environmentally Compatible Next Generation Green
Carbon Capture and Utilization Materials
Abdul Rahman Mohamed*, Subhash Bhatia, Keat Teong Lee, Chee Yew Henry Foo,
Zhi Hua Lee, Nurul Aini Razali
Low Carbon Economy (LCE) Research Group, School of Chemical Engineering, Engineering
Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia.
chrahman@eng.usm.my

Researchers are striving to find a green solution towards the global warming
issue. Looking into the significant aspects of sustainability and profitability,
carbon dioxide (CO2) capture and utilization will be a sustainable andcost-effective process route. In brief, this is a process where CO2 will beselectively captured from the points of large emission sources, collected in aconcentrated form during the regeneration process, and reused as a carbon
feedstock in order to produce various valuable chemical products. The vastdevelopments in the field of nanotechnology and green chemistry should be
relatively cheap, environmental friendly and high efficiency by the CO2
capture adsorbents and CO2 conversion catalysts. Nanotechnology is apromising technology which utilizes materials at least one dimension sizedfrom 1 to 100 nanometers in nano-sized regime. With such a quantum-realm
scale, the adsorption and reaction activities will be greatly increased due tolarger surface area, unique crystal structure, and mechanical effect for better
reaction. In order to fulfill the concept of green chemistry, it is crucial to
make the synthesis method with environmental benignancy besides topractice sustainable development, industry ecology, safety and waste
minimization, improving heat and mass transfer limitation and life-cycle
analysis in addition to the new approaches.

1. Introduction
Amongst greenhouse gases, carbon dioxide (CO2) is the main contributor to global warming [1],because it has notably high emission compare to others greenhouse gases, such as methane, nitrous
oxide, and hydrofluorocarbons. The strong liberation and accumulation of CO2 in our planet iscaused by the energy supply industry, transportation sector, and manufacturing industry. Scientists
predicted that CO2 level in the atmosphere should not exceed 450 ppm to prevent global warmingfrom threatening the world [2]. However, the global CO2 level is alarming at 383 ppm with a 2.2
ppm increment per year [3]. Consequently, CO2 capture is an extremely crucial task at the moment.
It is known that CO2 has a molecular size 3.4Ǻ and is a thermodynamically stable compound [4].
This inert property makes that CO2 capture is a difficult task to be executed. Inversely, it also can bea cheap, nontoxic and abundant carbon feedstock for various valuable chemicals. Thus, development
and integration of carbon capturing and their utilization processes in chemical industry for newenergy production suggest to be one of the directions that researchers should look into the finite
solution to stop accumulation of CO2 in the atmosphere towards alleviation of global warming.
Figure 1 shows the mapping of the concept where the CO2 emitted is captured and regenerated for its
re-utilization in the chemical production industry. Many valuable chemicals including methanol [4],carbonates [5], carbamates [6], dimethyl carbonate [7], cyclic carbonates [8,9], and synthetic gas[10] as well as formic acid and its derivatives [11] could actually be synthesized from CO2.
Profitably, low carbon economy is hoped to attract more investors to join the ranks and to cope withthe environmental issue. With the target of protecting the environment, it is of paramount importancethat the CO2 capture and utilization processes are green processes. Thus, green chemistry principles
1

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should be concerned with process operations, identification and quantification of products,
conversion, selectivity and productivities, catalyst and solvent losses, heat and mass transfer
limitations, development and application of sustainable measures, incompability of safety and waste
minimization [12].
Combustion

CO2
CO2

Regeneration

captureCO2
utilization

emission

Valuablechemicals

point

Fig. 1. Concept mapping of CO2 capture and utilization
A nano-material can classify, when it possesses at least one dimension equivalent or less than 100
nm [12]. When such material has a nanoscale dimension, many addition properties appears. These
unique properties will give a plausible enhancement in many chemical processes. In the present article,
we reveal the current technology in CO2 capture and CO2 utilization to review on research works thathave been performed in these two particular fields. In addition, it is mentioned that nanomaterials give
extraordinary performance in the chemical processes and both challenges and potentials to become
candidates for environmental compatible green materials in future.
For current CO2 capture technology, recently, solvent stripping by amine solution or ammonia is themost widely used technology in capturing CO2 from its emission points. This technology can achieve
high CO2 removal even at low CO2 concentration. Absorber and stripper make up the major part of thisprocess. The stripper is used to regenerate the saturated solvent from adsorber. Schematic diagram ofthe solvent stripping CO2 capture is shown in Fig. 2. However, still many drawbacks are faced in suchhigh energy consumption, solvent loss due to its low viscosity, and handling difficulties [15]. The CO2
capture capacity (mol/kg solvent) is relatively low for large CO2 emission source like power plants.
Hence, a large absorber and stripper are needed in this condition.
Nowadays, in current CO2 utilization, CO2 is only being utilized in a few industries due to its inert
and stable properties. Two common examples are in urea and salicylic acid synthesis. However, theresearchers are looking into different perspective of CO2 utilization so that the most threatening gas ofthis century can be removed in a more economical way. Some of the research directions include
using solar energy for CO2 conversion, developing value added CO2 sequestration, using CO2 as a mildoxidant [13].
2 Nanomaterials in CO2 capture and utilization
2.1. CO2 capture and utilization
Some potential nanomaterials for CO2 capture were reported in the literature as shown in Table 1.
Many research works were tried to synthesize sorbents in various types of nanostructure [13-22], suchas nanoporous ‘molecular basket’, nanopods, and nanoparticles. Apart from that, some researches aretrying to introduce functionality of carbon nanotubes (CNTs) in CO2 adsorption [15,16]. However,such nanomaterial will have a higher cost of the production. For instance, CNT with specific structureis only available at extremely high price (US$ 5/g) compared to granular activated carbon which cost
US$ 1/kg [15].
In recent decades for CO2 utilization, the transformation of CO2 to valuable chemicals and biofuel
has attracted much attention. Researchers have been conducted on CO2 hydrogenation, CO2
cycloaddition to epoxides, CO2 reduction under photo irradiation or electrolytic conditions. Table 2
summarizes the types of CO2 utilizations with their reaction.
Various methods and catalysts have been used in order to obtain the very active catalyst with highCO2 conversion [3]. Most of the catalysts obtained low conversion of CO2 (<40%) and low selectivity
2

Transactions on GIGAKU 1(2012)01006/1-7of chemicals products (<80%). Thus, the exploration of superior design and fabrication of highperformance nanocatalyst system for conversion of CO2 to valuable chemicals is required for CO2
removal in industrial and a beneficial investment for the plant developer.

Fig. 2

Schematic diagram of solvent stripping CO2 capture technology in industry [14].
Table 1 Nanometerials synthesized for CO2 capture with the advantages

Nanosorbents
Nanoporous

MCM-41‘molecular basket’ [15,16]

Advantages
A synergetic effect on the adsorption of CO2 bypolyethylenimine (PEI)
CO2 condensed in a pore channel like a ‘basket’ form

Mesoporous MgO [17]selective to CO2 gas
thermally stableregenerable.

Multiwalled CNT [18]have higher capture capacity with same surface area with

Single-walled CNT [21]activated carbon or zeolite

CaO Nanopods [19]higher CO2 capture capacity
retaining >50% CO2 absorption capacity after 50 CO2
capture-and-release cycles

CaO-MgAl2O4 spinel

Reduce decay problem of CaO, retain more than 115

nanoparticles [20]capture-regenaration cycles

Nanocrystalline Li2ZrO3 particles [21] Improved capture of CO2 in a wide temperature range andimproved kinetics of the regeneration
2.2 Green materials
One should always aware that a material will only be claimed potential when it is produced andassembled through green and environmentally responsible methodology. Green materials are defined as3

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materials that are efficient and durable as their conventional counterparts. But, these do not pose athreat to people or the environment and provide other major benefits [22]. In this century, it is apparent
that the full potential of as-prepared nanostructures will only be realized when nanomaterials are not
only synthesized in large quantities with reproducible size, shape, structure, crystallinity, andcomposition but also prepared and assembled using green, environmentally responsible methodologies[23].
Table 2 Types of CO2 utilizations with their reaction details.
Reaction

Catalyst

Reaction Condition

Results (%)

CO2 reforming of CH4- Metal oxide

773–973K

CH4 conversion: 59-92

to synthesis gas- Metal/Zeolite

773 -1173K

CO2 conversion: 54-94

CO2 hydrogenation- Metal oxide

433-523 K, 8.0MPa

CO2 conversion: 9-54

to methanol [28-31]- Metal/Zeolite[24-27]

CH3OH selectivity: 60- 92

Synthesis of cyclic carbonate - Metal/mesoporous 293-393K,

Epoxide conversion: 6-98

fromCyclicCO2

andepoxide - Metal/Zeolite

0.5-2.0 MPa

carbonate[32-35]283- 393 K, 2-3 MPa Selectivity: 90-97

Synthesis of DMC from CO2 - Metal/zeolite

373-403K,

CH3OH conversion: 3-10

and methanol [7,36-38]

0.2-1.5 MPa

DMC selectivity: 25-86

Especially, the latter point is the focus of the present review and the social significance associatedwith developing these types of techniques cannot be overemphasized. Green chemistry can be broadly
defined as the invention of novel reactions that can maximize the desired products and minimize
by-products, designing new synthetic schemes and apparatus that can simplify operations in chemical
productions, and seeking greener solvents that are inherently environmentally and ecologically benign[39]. Specifically, many groups worldwide have worked on similar types of materials, we have made adeliberate effort to abide by the 12 principles that had been clearly defined in the publication of Green
Chemistry. Theory and Practice in 1998 [22] listed below to provide a coherent vision for the
emerging green chemistry movement. 1) Designing safer chemicals with desired function while
minimizing their toxicity, 2) Safer solvents and auxiliaries, 3) Maximize energy efficiency as if
possible to conduct synthetic process under ambient temperature and pressure, 4) Use of renewable
feedstocks, 5) Reduce reaction derivatives, 6) Make use of selective catalytic reagents, 7) Design for
degradation of chemical products so their function breakdown into innocuous degradation products anddo not persist in the environment, 8) Real-time analysis for pollution prevention as in-processmonitoring and control prior to the formation of hazardous substances and 9) Safer chemistry for
accident prevention as substances used in chemical process should be chosen to minimize the potential
for chemical accidents, including releases, explosions and fires.
For nanomaterials made of green materials the potentials can be given that green chemistry has
been employed successfully in the preparation of highly functionalized nanomaterials proposed for arange of future applications. These materials are expected to (i) exhibit new size-dependent properties
particularly in beneficial quantum realm, (ii) involving a multidisciplinary of material science
approach and innovation with associated elemental and material compositions, including organics,
inorganics and hybrid structures, and (iii) occupied a high degree of surface functionality. Apart from
that, assessment of the potential toxicological and environmental effects of nanoscale materials before
they are accepted as mature technologies presents an opportunity to minimize putative negative
consequences from the outset and ultimately lead to the design of higher performance materials.
Therefore, understanding the structure-function relationships that related specifically to nanomaterials
could lead to new “design rules” for producing benign, high-performance nanoscale substances andtoward greener synthetic methods for functionalized metal nanoparticles. Recently, a great deal ofattentions have been paid to the development of greener synthetic methods involving innovative citrate
reduction, direct synthesis of ligand stabilized nanoparticles, seeded growth and shape control of4

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nanoparticles and alternative solvents for nanoparticles synthesis.

2.3 Some examples for green nanomaterials
Since the pioneered work for by J. Turkevich et al. in 1951 [40,41], they gave nearly monodisperse,water soluble gold clusters with diameters ranging from 7-100 nm. Although the synthesis predates
green chemistry principles by several decades, it is a rather benign procedure, as the reagent pose little
hazard. The preparation did not rely on organic solvents and few (if any) undesirable side products
were generated in the course of the reaction. Revered for its simplicity, requiring only a metal salt,
trisodium citrate, and water, it remained one of the most reliable methods of creating large metal
nanoparticles [42]. For direct synthesis of ligand stabilized nanoparticles, a wide range of metal
nanoclusters or nanoparticles stabilized by polymers and organic ligands have boosted thedevelopment of nanotechnology as one of the most important building blocks to constructnano-organized systems. The direct preparation of ligand-stabilized nanoparticles provided a simple
route to functionalized materials, usually in a single-step, straight forward, imparting stability andchemical functionality to the nanoparticle products without post-synthetic modification [43].
Current research challenges are focused on selecting solvents, reaction conditions, and reagents tosynthesis a target material, but one should not overlook the opportunity to adopt greener syntheses
pathway by giving equal consideration to more benign reaction conditions (i.e., safer solvents andavoid toxic surfactants), overall yield and atom economy, and environmental issue of new
nanoproducts. Additional aspects of metal nanoparticles in organic solvent such as their fabrication,
morphology control, growth mechanism, stabilization, characterization, physical properties, andpotential applications to be covered [44].
Focusing on seeded growth approaches for the synthesis of nanoparticles leads again to manydifferent methods that have been published. On broad sense, nanoparticles having core diameters
exceeding 5 nm can be grown from smaller seed particles through the epitaxial addition of metal atoms[43]. In addition, the use of well-defined seeds is crucial to obtain products with narrow size dispersity.
Other reagents like surfactants may promote the formation of anisotroic materials or simply as surface
passivants and stabilizing agents. Nevertheless, growth of such materials from monodisperse seeds
allows the researcher to employ milder reaction conditions for the synthesis of materials, and the wide
range of weaker reducing agents capable of reducing metal ions in a growth solution offers increased
possibilities for designing greener syntheses.
Table 3 Some details of barriers faced in green nanoscience [45].
Barriers
Location in the
Stakeholder
innovation chain
There are no clear design guidelines for
Discovery phase; link Universities
academic
researchers in initial discovery phases of green between
research and industry
nanoscience
Green nanomaterials require newDevelopment
and Universities; Small and largecommercial production techniques, which
Production
phase; industry
increases the need for basic research, engineering Research phase; link
academic
research, and coordination of the two between the between
research and industry
industrial and research communities;
and Small and large industry
There is a lack of “deep bench” of scientists Development
and engineers with experience developing green production phasenanotechnology;
Toxicology and analysis protocols need to Research phase; link Universities,
National
academic Laboratories,
be developed and constantly updated to reflect between
Regulatory
research and industry Agencies, Small and large industry
advance in the science;
Regulatory uncertainty persists, and green Commercialization Regulatory Agencies, Small andphaselarge industry, Consumers
nanotechnologies often face higher regulatory
barriers than existing or conventional chemicals;
and
large
industry.
The end-market demand is unclear,
Commercialization Small
Consumers, financing mechanisms
especially since there are only a limited number phaseof commercial grade products that can becompared to conventional materials in terms of
performance5

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3. Challenges emerging with green nanoscience
The emerging field of green nanoscience faces considerable research challenges to achieve themaximum performance and benefit from nanotechnology while minimizing impact on human healthand the environment. Several general barriers to overcome were listed below together with its locationin the innovation chain and responsible stakeholder.
As the above listing of research challenges suggests, green nanosynthesis is in its early stages andfurther research is warranted to develop the approach and examine the breadth of its application. Thereare encouraging results that suggest that the green nanoscience framework can guide design,
production, and application of greener nanomaterials across the range of compositions, sizes, shapes,
and functionality. Further development and application of this framework to the design and productionof a growing number of classes of nanoparticle materials will provide research opportunities andchallenges for this community for the foreseeable future.
4. Conclusion
Fossil fuels will still the dominant source of energy for this century, in view of the immaturity ofdevelopment of other energy resources. CO2 capture in existing power plants can be classified into
three categories: pre-combustion capture, oxy-combustion capture and post-combustion capture.
Post-combustion capture has gained the major focused. It is looked as the most feasible approach while
it can be easily retrofitted into the existing conventional coal power plant compared to other method.
While other technologies are facing their limitations, CO2 adsorption with dry-based materials has
gained numerous attentions and in an evolution towards nanomaterials. To make this process become
more economically feasible, CO2 utilization in industry need to be explored and developed in a wider
scope. It is ought to mention that the route of synthesis of the adsorbents need to be green as well inorder to achieve a totally green CO2 removal process.
Acknowledgemens
The authors would like to acknowledge for the financial supports given by (1) Long Term Research
Grant (LRGS) (203 / PKT / 6723001) from Ministry of Higher Education (MOHE) Malaysia and (2)Research University Grant (1001/PJKIMIA/854001) from University Sains Malaysia (USM).
References[1] O.T. Hui, Renewable and Sustainable Energy Reviews, 14 (2010) 2697.
[2] M.S.J. Hansen, P. Kharecha, D. Beerling, and R. Berner, V. Masson-Delmotte, M. Pagani,
M. Raymo, D.L. Royer, and J. C. Zachos, Open Atmosphere Sci. J., 2 (2008) 217.
[3] P. Canadell, P. Ciais, T. Conway, C.B. Field, C.L. Quéré, R.A. Houghton, G. Marland, and M.R.
Raupach, Carbon Budget 2007; Global Carbon Project 2008.
[4] J. Toyir, R. Miloua, N.E. Elkadri, M. Nawdali, H. Toufik, and F. Milouaet, Phys. Procedia, 2(2009) 1075.
[5] J. George, Y. Patel, S.M. Pillai, and P. Munshi, J. Mol. Catal. A: Chem., 304 (2009) 1.
[6] N. McCann, D. Phan, D. Fernandes, and M. Maeder, Int. J. Greenhouse Gas Control, 5(2011) 396.
[7] J. Bian, M. Xiao, S. Wang, X. Wang, Y. Lu, and Y. Meng, Chem. Eng. J., 147 (2009) 287.
[8] H. Xie, S. Li, and S. Zhang, J. Mol. Catal. A: Chem., 250 (2006) 30.
[9] L.F.Xiao, F.W. Li, J.J. Peng, and C.G. Xia, J. Mol. Catal. A: Chem., 253 (2006) 265.
[10] K.C. Mondal, V.R. Choudhary, and U.A. Joshi, Appl. Catal. A: Gen., 316 (2007) 47.
[11] A. Behr, G. Henze, L. Johnen, and S. Reyer, J. Mol. Catal. A: Chem., 287 (2008) 95.
[12] K. Savolainen, L. Pylkkӓen, H. Norppa, G. Falck, H. Lindberg, and T. Tuomi, Safety Sci., 48(2010) 957.
[13] C. Song, Catal. Today, 115 (2006) 2.
[14] B. Metz, O. Davidson, Hd. Coninck, M. Loos, and L. Mryer, Carbon Dioxide Capture andStorage Special Report, International Panel on Climate Change (2005).
[15] X. Xu, C. Song, J.M. Andresen, B.G. Miller, and A.W. Scaroni., Micropor. Mesopor. Mater., 62(2003) 29.
6

Transactions on GIGAKU 1(2012)01006/1-7[16] M.K. Aroua, W.M.A.W. Daud, C.Y. Yin, and D. Adinata, Sep. Purif. Technol., 62 (2008) 609.
[17] M. Bhagiyalakshmi, J.Y. Lee, and H.T. Jang, Int. J. Greenhouse Gas Control, 4 (2010) 51.
[18] F. Su, C. Lu, W. Cnen, H. Bai, and J.F. Hwang, Sci. Total Environ., 407 (2009) 3017.
[19] Z. Yang M. Zhao, N.H. Florin, and A.T. Harris, Ind. Eng. Chem. Res.,48 (2009) 10765.
[20] L. Li, D.L. King, Z. Nie, X.S. Li, and C. Howard, Energy & Fuels. 24 (2010) 3698.
[21] R.B. Khomane, B.K. Sharma, S. Saha, and B.D. Kulkarni, Chem. Eng. Sci., 61 (2006) 3415.
[22] T.A. Paul and J.C. Warner, Green Chemistry: Theory and Practice: Oxford University Press,
New York; 1998.
[23] Y. Mao, T.J. Park, F. Zhang, H. Zhou, and S.S. Wong, Small, 3 ( 2007) 1122.
[24] M. Nagai, K. Nakahira, Y. Ozawa, Y. Namiki, and Y. Suzuki, Chem. Eng. Sci., 62 (2007) 4998.
[25] J. Guo, J. Gao, B. Chen, Z. Hou, J. Fei, and H. Lou, Int. J. Hydrogen Energy, 34 (2009) 8905.
[26] J. Chen, Q. Wu, J. Zhang, and J. Zhang, Fuel. 87 (2008) 2901.
[27] R. Takahashi, S. Sato, T. Sodesawa, and S. Tomiyama, Appl. Catal. A: Gen., 286 (2005) 142.
[28] X.L. Liang, X. Dong, G.D. Lin, and H.B. Zhang, Appl. Catal. B: Environ., 88 (2009) 315.
[29] X. Guo, D. Mao, S. Wang, G. Wu, and G. Lu, Catal. Commun., 10 (2009) 1661.
[30] J. Słoczyński, R. Grabowski, P. Olszewski, A. Kozłowska, J. Stoch, and M. Lachowska, Appl.
Catal. A: Gen., 310 (2006) 127.
[31] D. Lee, J.Y. Lee, and L.J. Sung, Sang-Eon Park J-SC, Kyu-Wan L, editors. Studies in
Surface Science and Catalysis: Elsevier; 2004. p. 169-72.
[32] R. Srivastava, D. Srinivas, and P. Ratnasamy, J. Catal., 233 (2005) 1.
[33] X. Zhang, N. Zhao, W. Wei, and Y. Sun, Catal. Today, 115 ( 2006) 102.
[34] S. Udayakumar, M.K. Lee, H.L. Shim, S.W. Park, and D.W. Park, Catal. Commun., 10 (2009)659.
[35] A. Ion, V. Parvulescu, P. Jacobs, D. de Vos, Appl. Catal. A: Gen., 363 (2009) 40.
[36] J. Bian, M. Xiao, S.J. Wang, Y.X. Lu, and Y.Z. Meng, Appl. Surf. Sci., 255 (2009) 7188.
[37] X.L. Wu, Y.Z. Meng, M. Xiao, and Y.X. Lu, J. Mol. Catal. A: Chem., 249 (2006) 93.
[38] Y. Zhang, D.N. Briggs, E. de Smit, and A.T. Bell, J. Catal., 251 (2007) 443.
[39] C.J. Li and B.M. Trost, Proc. Nat. Acad. Sci., 105 (2008) 13197.
[40] J. Turkevich, P.C. Stevenson, and J. Hillier, Discussions Faraday Soc., 11 (1951) 55.
[41] J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, and A. Plech, J. Phys. Chem. B, 110(2006) 15700.
[42] L. Pei, K. Mori, and M. Adachi, Langmuir, 20 (2004) 7837.
[43] N. Toshima, Macromol. Symp., 204 (2003) 219.
[44] S. Nath, S. Jana, M. Pradhan, and T. Pal, J. Colloid. Interf. Sci., 341 (2010) 333.
[45] K.J.M Matus, J.E. Hutchison, R. Peoples, S. Rung, and R.L. Tanguay, American Chemical
Society's Green Chemistry Institute ® (ACS GCI). American Chemical Society’s Green
Chemistry Institute® (ACS GCI); 2011.
(Received: 21 March, 2012, Accepted: 30 June, 2012)7

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Nanomatrix Structure Formed for Natural Rubber
Seiichi Kawahara and Oraphin Chaikumpollert
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: kawahara@mst.nagaokaut.ac.jp
Preparation of natural rubber with nanomatrix structure was made in latex
stage by graft-copolymerization of styrene onto deproteinized natural rubber(DPNR) with tert-butyl hydroperoxide / tetraethylenepentamine as an initiator.
The DPNR was prepared by enzymatic deproteinization andurea-deproteinization, respectively. Conversion and grafting efficiency ofstyrene and acrylonitrile were more than 90% under a suitable condition of
the graft-copolymerization for both enzymatic DPNR and urea-DPNR. The
morphology of the film specimens, prepared from graft-copolymers, wasobserved by transmission electron microscopy after staining the films with
OsO4. Natural rubber particle of about 0.5m in diameter was dispersed inpolystyrene matrix of less than 15 nm in thickness.

1. Introduction“Nanomatrix structure” is a novel phase separated structure for a multi component system, which
may provide outstanding mechanical, optical and electrical properties. It is defined to consist ofdispersoid of a major component and matrix of a minor component, which is distinguished fromordinary island-matrix, cylinder, gyroid and lamellar structures for block- and graft-copolymers. The
nanomatrix structure is anticipated to achieve both outstanding properties of the dispersoid andfunctional properties of the matrix.
The nanomatrix structure may be formed by covering particles with a nano-layer followed bycoagulation of the resulting nano-layer-covered particles [1]. In this case, the particles are required tochemically link to the nano-layer, in order to stabilize the nanomatrix-structure in equilibrium state.
Especially, in the case of natural rubber, the chemical linkages may be formed bygaft-copolymerization of a monomer onto rubber-particles in latex stage, since the particles in thelatex are dispersed in water. However, the graft-copolymerization was prevented by side reactions ofproteins [2-6], which covered the rubber particle [7]. Therefore, we have to remove the proteins fromnatural rubber to form the nanomatrix structure. As long as we use deproteinized natural rubber latex
as a source, the side reaction may be suppressed to attain high conversion and high graft efficiency
for the graft-copolymerization [8].
In the present work, an attempt to form nanomatrix structure is performed bygraft-copolymerization of styrene onto deproteinized natural rubber in latex stage, in which the
DPNR was prepared by enzymatic deproteinization and urea-deproteinization, respectively.
Morphology of the products is observed by transmission electron microscopy. Mechanical propertiesof the products were related to morphology, which was dependent upon styrene unit contents.

2. Experiment
Natural rubber latex used in this study was commercial high ammonia latex. The incubation ofnatural rubber latex was made with 0.1 wt% urea in the presence of 1 wt% sodium dodecyl sulphate(SDS) at 30, 60, 90oC, respectively The cream fraction was re-dispersed in 1 wt% SDS to make
30 %DRC latex and was washed twice by centrifugation. The natural rubber latex was also
deproteinized by incubation of the latex with 0.04 wt% proteolytic enzyme (Kao, KP-3939) and 1
wt% SDS for 12 h at 32oC followed by centrifugation. The cream fraction was re-dispersed in 1 wt%
SDS to make 30 %DRC latex and was washed twice by centrifugation to prepare deproteinized
natural rubber (DPNR) latex.
Graft-copolymerization of the urea-DPNR (U-DPNR) latex and enzymatic DPNR (E-DPNR)
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latex was carried out with styrene and acrylonitrile as a monomer, using tert-butyl hydroperoxidewith tetraethylene pentamine as an initiator. The latices were charged with N2 gas for 1 h at 30oC.
The initiator of 3.3X10-2 mol/kg-rubber and monomer were added to the latex, respectively. The
reaction was carried out by stirring the latex at about 400 rpm for 2 h at 30oC. The unreacted styrene
was removed by using a rotary evaporator under reduced pressure. The as-prepared graft copolymer(gross polymer) was obtained by dipping the glass tube into the reacted latex and dried under
reduced pressure at ambient temperature for more than a week. The gross polymer was extractedwith acetone/2-butanone 3:1 mixture in a Soxhlet apparatus for 24 h under nitrogen atmosphere in
the dark and dried under reduced pressure for about one week, in which the removal of almost all
free-polystyrene, isolated from natural rubber, was completed by the extraction for 24 hours. Feed ofmonomer for the graft-copolymerization is shown in Table 1.
Measurement of nitrogen content of the rubbers was made by Kjeldahl method as described in
RRIM Test Method B7. Rubber sample, mixed with catalyst mixture, i.e. potassium sulphate :copper sulphate : selenium in the weight ratio of 15:2:1, was digested with concentrated H2SO4. The
resulting solution was distilled, and distillates were titrated with 0.05 M H2SO4 using methyl red as
an indicator.
Ozonization was carried out by blowing an equimolar amount of ozone in ozonated oxygen
through a 0.4 w/v% methylene chloride solution of the extracted graft-copolymer at –30oC.
Reductive degradation of the resulting ozonide was performed by reaction with lithium aluminum
hydride (LiAlH4) in diethyl ether followed by decomposition of residual LiAlH4 with water. After
reductive degradation, grafted polystyrene, thus isolated from graft copolymer, was dissolved in asmall amount of chloroform. The chloroform solution was centrifuged, and the polymer was
precipitated with methanol.
Measurements of molecular weight and molecular weight distribution of the rubbers were madewith a TOSOH GPC, consisting a TOSOH CCPD pump, a RI-8012 Differential Refractometer andUV-8011 UV detector. The measurement was made at 30oC and the flow rate of the mobile phase,
THF, was 0.5 ml/min.
1
H-NMR measurements were carried out at 50oC by a JEOL EX-400 NMR spectrometer at thepulse repetition time of 7 sec.
The volume mean particle diameter was determined using a Coulter LS230 equipped with asmall volume module.
Table 1 Feed of monomer for the graft-copolymerization
RubberMonomer

Feed of monomer

Sample name(mol/kg-rubber)
Enzymatic

0.5

E-DPNR-PS1

Deproteinized Natural

1.0

E-DPNR-PS2

Rubber

1.5

E-DPNR-PS3

2.0

E-DPNR-PS4

0.5

U-DPNR-PS1

1.0

U-DPNR-PS2

1.5

U-DPNR-PS3

2.0

U-DPNR-PS4

1.0

U-DPNR-PAN1

2.0

U-DPNR-PAN2

3.0

U-DPNR-PAN3

4.0

U-DPNR-PAN4

5.0

U-DPNR-PAN5

Urea Deproteinized

StyreneStyreneNatural Rubber

Urea Deproteinized

Acrylonitlire

Natural Rubber

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Observation of morphology for the graft-copolymer was made with a transmission electron
microscope (TEM), Hitachi H-800 at accelerating voltage of 120 kV. The ultra thin sections of thegraft-copolymer were prepared by a Sovall Instruments MT6000 Ultra-microtome at a temperature
lower than Tg of NR. The thin sections were stained by OsO4 after annealing the blends at 80oC for
30 min.

3. Results and Discussion(1) Removal of protein
Removal of proteins from natural rubber may be essentially concerned with methods of how tocontrol interactions between the rubber and proteins in the latex stage, that is, chemical and physicalinteractions. The former is cleaved with proteolytic enzyme such as alkaline protease and the latter
varied with denaturant such as urea. Thus, we investigated a change in the amount of proteinspresent in natural rubber after deproteinization under various conditions. Table 2 shows total
nitrogen content, X, of natural rubber and rubbers coagulated from the latex after deproteinization
with alkaline protease at 32oC for a day (E-DPNR) or incubation with 0.1 wt% urea at 30oC for anhour (U-DPNR). The total nitrogen content of natural rubber was significantly reduced by both themethods to about 1/20, reflecting that not only alkaline protease but also urea were effective to
remove the proteins from the rubber. Since urea is well-known to change only conformation of theproteins but not cleave any chemical linkages, the removal of almost all proteins from natural rubberwith urea may suggest that the proteins present in natural rubber are attached just on the surface ofthe rubber particles through physical interactions without any chemical linkages.
To prove the interactions between the rubber and proteins, temperature and time necessary to
remove the proteins from natural rubber was investigated. Figure 1 shows the total nitrogen content
of natural rubber treated with urea, as a function of time, t. At 30oC, the total nitrogen content
decreased from 0.38 wt% to 0.022 wt% after incubation with urea for 10 min. Further decrease in thecontent progressed to 0.020 wt% for 60 min at 30oC. On the other hand, as the temperature of the
incubation rose, the nitrogen content of natural rubber increased to about 0.025 wt%. The little bitincrease in the nitrogen content may be attributed to less ability of urea to form hydrogen bonds
between urea and the proteins at higher temperature. This demonstrates that most proteins, attachedon the surface of the rubber particles, undergo a conformational change to detach themselves fromthe rubber particles with urea. Consequently, the temperature and time necessary to remove proteinsfrom natural rubber with urea was determined to be room temperature and an hour, respectively.

Table 2 Nitrogen content of natural rubber
Specimens

Incubation time Nitrogen content(minute)(wt%)

Natural rubber

-

0.38

U-DPNR

60

0.020

E-DPNR

720

0.017

Fig. 1 Nitrogen content of urea-treatednatural rubber at 30oC (○), 60oC (△) and90oC (□) versus time for incubation.

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Transactions on GIGAKU 1(2012)01007/1-8(2) Graft-copolymerization
StyreneGraft-copolymer was characterized through 1H-NMR spectroscopy. Figure 2 shows a typical
1
H-NMR spectrum for the graft-copolymer, i.e. U-DPNR-PS3. Signals characteristic of
cis-1,4-isoprene units appeared at 1.76, 2.10, and 5.13 ppm, which were assigned to methyl,
methylene and unsaturated methyne protons of isoprene units, respectively. Broad signals around 6 7 ppm were assigned to phenyl proton of styrene units, whose intensity was dependent upon the feedof styrene as a monomer. Thus, we estimated a content of styrene units in the graft-copolymer andconversion of styrene from a ratio of signal intensities of phenyl to methyl protons and the feed ofstyrene. The estimated content of styrene units and conversion of styrene for U-DPNR-PS are shownin Fig. 3. The conversion of styrene was dependent upon the feed of styrene, in which a maximum
was shown at styrene-feed of 1.5 mol/kg-rubber. In contrast, the content of styrene units of
U-DPNR-PS increased monotonically, as the feed of styrene increased. These may suggest that asuitable feed of styrene is 1.5 mol/kg-rubber for the graft-copolymerization of U-DPNR with
tert-butyl hydroperoxide of 3.3X10-5 mol/g-rubber with tetraethylene pentamine at 30oC. In Fig. 3
are also shown the content of styrene units and conversion of styrene for E-DPNR-PS. The content
of styrene units and conversion of styrene for E-DPNR-PS were dependent upon the feed of styrene,
as in the case of U-DPNR-PS. Thus, it is possible to expect that the graft-copolymerization for
U-DPNR may proceed in a similar way to E-DPNR.
We estimated a grafting efficiency for U-DPNR-PS and E-DPNR-PS. To estimate the grafting
efficiency, free polystyrene, which was a mixture present in the graft-copolymer, was removed byextraction with acetone/2-butanone 3:1 mixture. The grafting efficiency, ν, was estimated as follows:ν=Mole of polystyrene linked to natural rubber
Mole of polystyrene produced during graft-copolymerization

The estimated value of grafting efficiency of styrene for U-DPNR-PS and E-DPNR-PS is shownin Fig. 4. The grafting efficiency was dependent upon the feed of styrene and a maximum was shownat 1.5 mol/kg-rubber feed of styrene. The feed of styrene at the maximum was a similar to that forthe conversion of styrene. This may be attributed to a deactivation and chain transfer of the radicals
due to less and large amount of styrene, respectively. At 1.5mol/kg-rubber feed of styrene for
U-DPNR and E-DPNR, almost all polystyrene, thus produced, was proved to link up to the rubber
molecule. This demonstrates that the reactivity of U-DPNR for graft-copolymerization with
free-radical-initiator is the same as that of E-DPNR.

Fig. 2 Typical 1H-NMR spectrum for U-DPNR-PS3.
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Fig. 3

Conversion and content of styrene
for U-DPNR-PS and E-DPNR-PS.

Fig. 4

Grafting efficiency for DPNR-St.

Acrylonitlire
Content of acrylonitlire units and conversion of acrylonitlire for U-DPNR-PAN are shown in Fig.
5. The conversion of acrylonitlire was dependent upon the feed of acrylonitlire, in which a maximum
was shown at acrylonitlire-feed of 3.0 to 4.0 mol/kg-rubber. In contrast, the content of acrylonitlire
units of U-DPNR-PAN increased monotonically up to 4.0 mol/kg-rubber, as the feed of acrylonitlire
increased. These may suggest that a suitable feed of acrylonitlire is 4.0 mol/kg-rubber for thegraft-copolymerization of acrylonitrile onto U-DPNR with tert-butyl hydroperoxide of 3.3X10-5
mol/g-rubber and tetraethylenepentamine at 303K.

Fig. 5

Fig. 6 TEM photographs for E-DPNR-PS1,
E-DPNR-PS2,
E-DPNR-PS3
and
E-DPNR-PS4, respectively.

Conversion (●) and content (□)
of acrylonitlire for U-DPNR-PAN.

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Transactions on GIGAKU 1(2012)01007/1-8Morphology
TEM photographs for E-DPNR-PS1, E-DPNR-PS2, E-DPNR-PS3 and E-DPNR-PS4 are shownin Fig. 6, in which a gloomy domain is natural rubber and a bright domain is polystyrene. As for
E-DPNR-PS1, little bright domain was scattered in the gloomy matrix. As the feed of polystyrene
increased, the bright domain segregated together to cover the gloomy spheres. To characterize thegloomy spheres, we estimated an average diameter of gloomy sphere for E-DPNR-PS3 andE-DPNR-PS4, and these are tabulated in Table 3. The average diameter of gloomy sphere wassimilar to the volume mean particle diameter of the dispersoid present in natural rubber latex, which
is about 0.7 m in diameter. Since the film specimens were prepared by coagulation of the latex, thegloomy sphere, observed in the photograph, may be attributed to the dispersoid present in the latex.
The thickness of the bright domain is also shown in Table 3. The thickness was about 15nm for
E-DPNR-PS3, while it was about 25 nm for E-DPNR-PS4. This bright domain would be a matrix,
that is, a continuous phase. To confirm the fact that polystyrene was the matrix, a volume fraction ofpolystyrene for E-DPNR-PS3 was estimated by image-analysis of the photograph. The estimatedvolume fraction of polystyrene was about 10 v/v% for E-DPNR-PS3, which corresponded to 11v/v%, estimated from styrene unit content shown in Figure 3. This demonstrates that natural rubber
particle of about 0.5 m in diameter is dispersed in polystyrene matrix of about 15 nm in thickness.
Since the grafting efficiency of styrene for E-DPNR-PS3 is more than 90mol%, as shown in Fig. 4,
almost all polystyrene may link up to E-DPNR. Figure 7 shows TEM photograph for U-DPNR-PS3.
The nano-matrix structure was also found for U-DPNR-PS3, as in the case of E-DPNR-PS3. The
thickness of bright domain was about 15nm. Since the styrene content and grafting efficiency ofstyrene for U-DPNR-PS3 were about 12w/w% and 90%, respectively, the nano-matrix structure wasproved to be formed for not only E-DPNR but also U-DPNR.
Table 4 Average diameter and average thickness for E-DPNR-PS3 and E-DPNR-PS4.
Specimen
E-DPNR-PS3
E-DPNR-PS4

D (μm)
0.5
0.6

t (nm)
15
25

D: average diametert : average thickness
Morphology of U-DPNR-PAN4 was also observed by TEM. Figure 8 shows TEM photographs
for U-DPNR-PAN4, in which a gloomy domain is natural rubber and a bright domain ispolyacrylonitrile. The natural rubber particle was covered with polyacrylonitlire to form nano-matrix
structure, in which the thickness of polyacrylonitlire was less than 15 nm. This demonstrates that theFig. 7

TEM photograph for U-DPNR-PS3.

Fig.
6

8

TEM photograph for U-DPNR-PAN4.

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nano-matrix structure is formed with not only styrene as a non-polar monomer but also acrylonitrile
as a polar monomer.
Mechanical properties and oil resistance
Figure 9 shows stress-strain curves for unvulcanized graf-copolymers, that is, E-DPNR-PS1,
E-DPNR-PS2, E-DPNR-PS3 and E-DPNR-PS4. A stress at strain of 1 increased as the styrene
content increased, reflecting an increase in Young’s modulus. On the other hand, the highest stress at
break was shown for E-DPNR-PS3. This may be attributed to not only the nano-matrix structure but
also the highest grafting efficiency. As the results, the nano-matrix structure was proved to be formedfor E-DPNR-PS3.
Oil resistance of U-DPNR-PAN is shown in Fig. 10. The oil resistance for U-DPNR-PAN1,
U-DPNR-PAN2 was identical to that for U-DPNR, itself. However, as the PAN content increased, i.e.
for U-DPNR-PAN3, U-DPNR-PAN4, U-DPNR-PAN5, the oil resistance increased significantly. The
abrupt increase in the oil resistance of U-DPNR-PAN may be attributed to the formation of
nano-matrix structure. This may be distinguished from the oil resistance of U-DPNR/PAN blend,
which consists of ordinary island-matrix morphology.

Fig. 9Stress-Strain curve for unvulcanized
E-DPNR-PS:A,
E-DPNR-PS1;
B, E-DPNR-PS2; C, E-DPNR-PS3;
D, E-DPNR-PS4.

Fig. 10

Oil resistance of U-DPNR-PAN

4. Conclusion
Graft-copolymerization of deproteinized natural rubber was carried out with tert-butylhydroperoxide / tetraethylenepentamine as an initiator. The highest conversion and grafting
efficiency were achieved at styrene feed of 1.5mol/kg-rubber to be 98% and 93%, respectively. For
E-DPNR-PS3, natural rubber particle of about 0.5m in diameter was dispersed inpolystyrene-matrix of about 15 nm in thickness. The stress at break for E-DPNR-PS3 was found tobe the highest, due to not only nano-matrix of polystyrene but also the highest grafting efficiency. It
is concluded that the nano-matrix structure, which results in outstanding mechanical properties, isformed by graft-copolymerization of styrene onto DPNR in latex stage. On the other hand,
graft-copolymerization of acrylonitrile onto DPNR was also effective to increase the oil resistance atacrylonitrile feed of more than 3.0mol/kg-rubber.
Acknowledgments
This work was supported from the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, and by Program for High Reliable
Materials Design and Manufacturing in Nagaoka University of Technology.
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References[1] S. Kawahara, T. Kawazura, T. Sawada, and Y. Isono, Polymer, 44, (2003) 4527.
[2] G. A. Jeffery, Trans. Fraday Soc., 38, (1941) 384.
[3] G. F. Bloomfield, and P. M. Swift, J. Appl. Chem., 5 (1955) 605.
[4] P. W. Allen, and F. W. Merrett, J. Polym. Sci., 22 (1956) 193.
[5] E. G. Cockbain, T. D. Pendle, and D. T. Turner, J. Polym. Sci., 39 (1959) 419.
[6] H. Y. Erbil, J. Nat. Rubb. Res., 1 (1986) 234.
[7] C. C. Ho, T. Kondo, N. Muramatsu, H. Ohshima, J Coll. Interface Sci. 178 (1996) 442.
[8] Y. Fukushima, S. Kawahara, Y. Tanaka, J. Rubb. Res., 1 (1998) 154.
(Received: 16 May, 2012, Accepted: 30,June 2012)8

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Mullite Formation in Presence of Y2O3 by Homogeneous Precipitation
José de Jesús Rocha Jiménez1, J. Merced Martínez Rosales1
Keizo Uematsu2, Satoshi Tanaka2and Satoshi Sugita1
1 Departamento de Química, Universidad de Guanajuato, México
Noria alta s/n Col Noria Alta CP.36050. Guanajuato, Gto, México
2
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: stanaka@vos.nagaokaut.ac.jp
A novel route to obtain mullite ceramics (3Al2O3-2SiO2) with less energy
consuming was proposed. As have been proved in previous works, yttria as asintering additive promotes sintering of mullite phase via glassy phase. In thisstudy, an yttria-doped mullite micro-composite powder was synthesized via
the homogeneous precipitation technique and the subsequent mullitization
reaction. Dense mullite ceramics with submicron grain size was obtained by
micro-composite powder at 1450°C. Glassy phase retained on grainboundaries was reduced in mullite ceramics.

1. Introduction
Ceramic materials are extensively used around the world for wide applications. For example, atthe moment ceramics are used in around 50% of material existing buildings worldwide and fast
growing up market in technological applications. The environmental impacts associated withceramic industry have become increasingly of importance. Usually, the manufacturing of ceramicmaterials employs highly energy intensive processes consuming natural resources, materials andenergy; generating different types of environmental impacts. As the environmental concerns haveincreased, the need for environmental friendly ceramic processing is growing [1]. The special case ismullite (3Al2O3-2SiO2) which is one of the most employed ceramics, because mullite phase is the
only thermodynamically stable at high temperatures and ambient pressure. Its excellent properties athigh temperature such as good mechanical strength, low thermal expansion coefficient, and highcreep resistance make it an attractive ceramic material for high-temperature mechanical applications[2]. However, high temperatures (~1600°C) are required for the production of high density mullite
ceramic materials. The higher energy consuming process is related to the last part of the process,sintering [3,4].
On previous works, it was possible to produce fine mullite powder (~0.2 μm) from ahomogeneous precipitation technique, in which aluminum sulphate and fumed silica were used as
raw materials [5]. On sintering of mullite ceramics, the addition of yttria powder 5wt% as a sintering
aid to mullite powder reduce the sintering temperature to 1400°C [6]. However, when a large amountof yttria powder was added more, the large defects appeared as the large amounts of glassy phase
concentrated at grain boundaries. The large defects in ceramic materials play a main role on themechanical properties [7].
The objective of this study was to produce dense mullite ceramic materials with finemicrostructure at lower temperature by using small quantities of yttria (2wt%) as additive formullitization and sintering. An yttria-doped fine mullite powder was prepared from yttria-doped
mullite precursor of pseudo-bohemite coated on silica. Preparing precursors with core shell type was
supported in three facts: first, the reaction rate of mullite can be enhanced easily by reducing the
distance between phases. Second, reducing the viscous glassy phase promotes the Al mass transport
into silica rich phase [8]. Third, the use of yttria during sintering promotes densification throughglassy phase sintering mechanisms. The reduction of temperature leads to reduction in waste energy,
in other words, low impact environmental mullite ceramic materials.

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2. Experimental
The preparation of pseudo-bohemite coated on fumed silica surface with and without yttria wasmade using aluminum sulphate, fumed silica (10-40 nm), and nanometric yttria (<50 nm) as starting
materials. The entire process was divided in 2 steps. The first step, homogeneous precipitation wasused, which had been reported in detail elsewhere [5]. The technique consists of preparing awell-dispersed aqueous suspension of fumed silica and/or yttria in ultrasonic water bath.
Subsequently, by controlling pH ~4, a basic aluminum sulphate was precipitated on the fumed silica
and yttria surfaces. After the precipitation was finished, the obtained precursor was ammonia
neutralized in order to remove the sulphate ions. The neutralization was carried out at pH ~9.5, 70°C
and 15 min. The final mullite precursor was pseudo-bohemite coated silica micro-composite withand without dopants, labeled as Al-Si and Al-Si-Y respectively. After neutralization, Al-Si and
Al-Si-Y were heated dynamically at 5°C/min in a controlled electric furnace. To study the behaviorof mullite reaction from precursor, both were heated at several temperatures until 1300°C.
After heat treatment, synthesized yttria-doped or non-doped mullite powder were ball milled for
24 h using ammonia poly-acrylate as dispersant, respectively. Green compact was formed by aconventional slip casting technique. Subsequently, the green compacts were sintered at 1400°C.
SEM observations for powders and sintered materials were made, also a XRD test were made forphase analysis during heat treatment.
3. Results and Discussion
Figure 1 shows XRD patterns of the thermal evolution of precursors with and without dopant.
On the left image, it can be seen that the precursor phase is pseudo-bohemite identified for thecharacteristic peaks at 26, 37, 48, and 63°, it also can be observed at 20° the wide amorphous silica
peak. According to XRD, yttria effect on crystallinity is almost undetectably, being the doped sample
less crystalline.

Fig. 1 XRD patterns of powders evolution before and after heat treatment. Si: SiO2,
PB: Pseudo-bohemite, °: cubic spinel and *:Mullite.
During the heat treatment, mullite formation becomes perceptible. Yittria doped sample
displayed more intense signals at 1225°C. This phenomenon is related to the increased Al masstransportation into silica by reduction of viscosity of the glassy phase, which was made by theformation of a transient glassy material Y-Al-Si-O [6]. The present phases identified on XRD
coexisting during mullite formation were recognized as: Si-Al spinel, a mixture of (θ- δ) transition
alumina and broad wide signal of amorphous silica.
The formation of mullite was almost completed at 1300°C for the doped sample, the broad peakof silica and alumina peaks were almost disappeared. Sample without yttria still has some trace ofsilica. In general, yttria accelerates the reaction velocity of mullite formation.

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Al-Si- 1300°C

Al-Si

1μm

Al-Si-Y-1300°C

1μm

1μm

Fig. 2 SEM micrograph of (left) the pseudo-bohemite before heat treatment, heat treatedpowders (center) without dopant and (right) with 2wt% yttria.
Figure 2 shows SEM micrographs of morphology of precursor and their reaction to mullite
powder after heat treatment at 1300°C. The precursor morphology consists of sub-micrometricprimary nearly spherical particles. After heat treatment, the morphology of particles is retained andagglomerates consisting of submicron particles are observed. Based on observations, no noticeable
differences on size or morphology were found between yttria doped or undoped material system. The
enlarged size on particles was related to sintering.
Figure 3 presents the microstructures obtained from yttria doped and undoped material systems
after sintering at low temperature ~1450°C during 2 h. Fig.3 (a) is related to the Al-Si system; thesample did not reach high density, and still porous. It is evident that the sintering processes such asneck forming and grain growth have being started. Usually, a high temperature above 1600°C forhigh density is necessary in undoped material system.
On doped system, at 1450°C, high density was reached as shown displaying fine microstructure(less than 0.5 μm in grain size) and without abnormal grain growth using small amounts of Y2O3
(2wt%) as sintering additive. Some pore still is observed between grain boundaries. The reduction
porosity was favored by glassy phase sintering mechanisms occurring between grain boundaries. As
the image presents, no accumulated glassy phase was observed between grain boundaries.

1μm

1μm

Fig. 3 SEM photo-micrograph of sintered materials heat treated at 1450°C. Left side undoped
sample and right side doped sample.
The main difference between both microstructures was related to two different sintering
processes. On the undoped sample, higher energy was required for densification since thedensification mechanisms are governed by solid state sintering. For yttria doped mullite system, atransient viscous sintering mechanism related to the glassy phase existent between boundaries
promotes high densification.

4. ConclusionLess energy consuming mullite ceramic materials were prepared using yttria doped mullite3Transactions on GIGAKU 1(2012)01008/1-4

micro-composite powders. The employ of small amounts of yttria (2wt%) as sintering additive
provides clear benefits, such as: Sintering temperature 1450°C, being 150°C lower than normal
mullite ceramic materials. Fine homogeneous microstructure (grain size ~0.5 μm) without abnormal
grain growth related to low temperatures of sintering. Reduced defects associated to excessive glassy
phase between grain boundaries. Increased mullite reaction rate promoted by low viscosity transient
viscous phase sintering Al-Si-O-Y.
Acknowledgments
The authors thank to CONACyT for its financial assistance by providing a doctoral scholarshipto one of us (J.J.R.J.)).
References[1] N. Tikul, P. Srichandr, J. Ceram. Soc. Jpn., 118 (2010) 887.
[2] I. A. Aksay, D. M. Dabbs, M. Sarikaya, J. Am. Ceram. Soc., 74 (1991) 2343.
[3] H. Schneider, J. Scheurer, B. Hildmann, J. Eur. Ceram. Soc., 28 (2008) 329.
[4] S. Somiya, Y. Hirata Am. Ceram. Soc. Bull., 70 (1991) 1624.
[5] S. Sugita, C. Contreras, J. Eur. Ceram. Soc., 18 (1998) 1145.
[6] P. Mechnich, H. Schneider, M. Schmücker, B. Saruhan, J. Am. Ceram. Soc., 81 (1998) 1931.
[7] S. Nakamura, S. Tanaka, Z. Kato, K. Uematsu, J. Am. Ceram. Soc., 92 (2009) 688.
[8] K. Wang, M. D. Sacks, J. Am. Ceram. Soc., 79 [1] (1996) 12.
(Received: 15 May, 2012, Accepted: 29 June, 2012)4

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Isolation and Characterization of bzaA and bzaB of Sphingobium sp.
Strain SYK-6, Which Encode Aromatic Aldehydes Dehydrogenases
with Different Substrate Preferences
Yuko Yamamoto, Daisuke Kasai, Naofumi Kamimura, Eiji Masai*
Department of Bioengineering, Nagaoka University of Technology,1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan*E-mail: emasai@vos.nagaokaut.ac.jp

Conversion of vanillin and syringaldehyde to their corresponding acids is an
essential step in the production of useful intermediate metabolites such as
2-pyrone-4,6-dicarboxylic acid (PDC). We isolated the aromatic aldehydes
dehydrogenase genes, bzaA and bzaB, of Sphingobium sp. strain SYK-6,
which showed approximately 30% identity at amino acid levels with vanillin
dehydrogenase genes of Pseudomonas strains. Both genes were expressed in
Escherichia coli, and the cell extract of E. coli carrying bzaA showed theoxidation activities toward a broad range of aromatic aldehydes, including
syringaldehyde and vanillin. On the other hand, the gene product of bzaB was
highly specific for m-anisaldehyde, and the oxidation activities towardsyringaldehyde and vanillin were less than 16% of that towardm-anisaldehyde. Disruption of bzaA and bzaB in Sphingobium sp. strain
SYK-6 had little effect on the growth of the mutant on syringaldehyde,
suggesting that the contribution of these genes to the degradation ofsyringaldehyde in SYK-6 appeared to be insignificant. However, bzaA
seemed to be a useful tool for bioprocesses converting lignin-derived
aromatic aldehydes to industrially valuable intermediate metabolites.

1. Introduction
One of the practical utilizations of lignin, which is a major component of the plant cell wall, isthe production of industrially valuable chemicals through a combination of chemical decompositionof lignin and microbial conversion. Since the chemical oxidation of lignin yields large amounts ofvanillin and syringaldehyde, these lignin-derived aromatic aldehydes are thought to be effective
starting compounds for the production of useful microbial metabolites, such as
2-pyrone-4,6-dicarboxylic acid (PDC) [1, 2]. PDC is an intermediate metabolite of the
protocatechuate 4,5-cleavage pathway, and has been found to be useful in the production of
biodegradable and high-functional polymers, for instance strong adhesives [3-5]. In order to convert
vanillin and syringaldehyde to PDC, the conversion of these aldehydes to vanillate and syringate,
respectively, is essential (Fig. 1). For this purpose, the vanillin dehydrogenase gene, ligV, wasisolated from a lignin-derived aromatics degrader, Sphingobium sp. strain SYK-6 [1, 6]. The
deduced amino acid sequence of ligV showed 50-55% identity with those of the known vanillin
dehydrogenase genes of Pseudomonas strains. The gene product of ligV produced in E. coli showed
the oxidation activities toward vanillin, benzaldehyde, protocatechualdehyde, m-anisaldehyde, andp-hydroxybenzaldehyde, but the activity toward syringaldehyde was less than 5% of that toward
vanillin. Disruption of ligV in SYK-6 indicated that this gene is essential for normal growth of
SYK-6 on vanillin. However, this disruption affected, to some extent, the ability of SYK-6 to growon syringaldehyde, suggesting that another aromatic aldehydes dehydrogenase gene is responsible
for the degradation of syringaldehyde.
In this study, we isolated and characterized the two different aromatic aldehydes dehydrogenase
genes, bzaA and bzaB, from a cosmid clone pKTV3. pKTV3 was isolated as a cosmid clone, which
conferred the ability to convert vanillin to vanillate on Sphingomonas sanguinis IAM 12578 [6].
1Transactions on GIGAKU 1(2012)01009/1-6

Fig. 1. Catabolic pathway of vanillin and syringaldehyde in Sphingobium sp. strain SYK-6.
Enzymes: LigV, vanillin dehydrogenase; LigM, vanillate/3MGA O-demethylase; LigAB, PCA
4,5-dioxygenase; LigC, CHMS dehydrogenase; LigI, PDC hydrolase; LigJ, OMA hydratase; LigK,
CHA aldolase; DesA, syringate O-demethylase; DesB, gallate dioxygenase; DesZ, 3MGA
3,4-dioxygenase. Compounds: PCA, protocatechuate; CHMS, 4-carboxy-2-hydroxymuconate-6semialdehyde;
PDC,
2-pyrone-4,6-dicarboxylate;OMA,
4-oxalomesaconate;
CHA,
4-carboxy-4-hydroxy-2-oxoadipate; 3MGA, 3-O-methylgallate; CHMOD, 4-carboxy-2-hydroxy-6methoxy-6-oxohexa-2,4-dienoate.

2. ExperimentBacterial strains and culture conditions
Sphingobium sp. strain SYK-6 and its mutant derivatives were routinely grown at 30°C in
Luria-Bertani (LB) medium or W minimal salt medium [7] containing 5 mM vanillin, 5 mM
syringaldehyde, 10 mM vanillate, or 10 mM syringate. Sphingomonas sanguinis IAM 12578 [8] andthe SYK-6 mutants were grown in LB medium. If necessary, 50 mg of kanamycin/liter, 300 mg ofcarbenicillin/liter, or 12.5 mg of tetracycline/liter were added to the cultures. Escherichia coli strains
were grown in LB medium at 37°C. For cultures of cells carrying antibiotic resistance markers, themedia for E. coli transformants were supplemented with 100 mg of ampicillin/liter, 25 mg ofkanamycin/liter, or 12.5 mg tetracycline/liter.
Cloning and nucleotide sequencing of aromatic aldehydes dehydrogenase genes
A cosmid pKTV3 [6] was digested with various restriction enzymes, and each DNA fragment
was subcloned into pBluescript II KS(+) or SK(+) [9]. Cells of E. coli JM109 [10] harboring
subclones were grown in 10 ml of LB medium until the optical density at 600 nm (OD600) of theculture reached 0.5. After the addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG), theculture was further incubated for 4 h. The cells were harvested by centrifugation (5,000  g, 10 min),washed with 50 mM Tris-HCl buffer (pH 7.5), and resuspended in 1 ml of the same buffer. Cellsuspensions (500 l) were incubated with vanillin or syringaldehyde (1 mM) at 30°C for 24 h. Afterthe incubation, cells were removed by centrifugation (15,000  g, 15 min), and the supernatants wereanalysed by high-performance liquid chromatography (HPLC). Further subcloning was performed toclone the 3.1-kb BamHI-HindIII fragment carrying ORF1 (bzaA) from pKTVH8F carrying the
8.0-kb HindIII fragment in KS(+). Southern hybridization analysis of pKTV3 was done using thedigoxigenin (DIG) system (Roche Diagnostic) to identify the DNA fragment, which carried the 5
2

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region of ORF2 (bzaB). The nucleotide sequences of bzaA and bzaB were determined by the dideoxy
termination method with a CEQ 2000XL genetic analysis system (Beckman Coulter). Sequence
analysis was performed with the GeneWorks program (Intelligenetics, Inc.). Homology searches
were performed with the non-redundant protein sequence database using the BLAST program.
Pairwise alignment was done with the EMBOSS alignment tool at the homepage of the European
Bioinformatics Institute (http://www.ebi.ac.uk/Tools/psa/).
Disruption of bzaA and bzaB
The 1.0-kb BspHI fragment carrying the -lactamase gene (bla) of pUC19 [10] was blunted, andligated into the blunt-ended NdeI site of pUCV2 carrying the 3.1-kb SalI-XbaI fragment in pUC19 todisrupt bzaA. The 4.1-kb SalI-XbaI fragment of the resultant plasmid pADV2 was ligated into
pK18mobsacB [11], and pADV2B was obtained. The 3.1-kb BamHI-HindIII fragment carrying bzaB
was cloned into pK19mobsacB [11] to obtain pKBV3. The 0.8-kb StuI fragment of pKBV3 was
replaced with the 1.9-kb SmaI fragment carrying the tetracycline resistance gene (tet) of pKRP12[12], and pTDV3 was obtained. The 1.5-kb NaeI-StuI fragment of pUCV2 was replaced by the
1.0-kb BspHI fragment carrying bla to disrupt both bzaA and bzaB. The 2.6-kb SalI-XbaI fragmentof the resulting pNSD23 was cloned into pK18mobsacB to generate pKDV23B. pADV2, pTDV3,
and pKDV23 were individually introduced into SYK-6 cells by electroporation, and the candidates
for bzaA mutant (SME044), bzaB mutant (SME045), and bzaA bzaB double mutant (SME046) werescreened using the method described previously [13]. To examine the disruption in each gene,
Southern hybridization analysis was performed. The total DNAs of candidates for SME044,
SME045, and SME046 were digested with EcoRV, ApaI, and BamHI-HindIII, respectively. The
0.8-kb HincII-Eco47III fragment carrying bzaA, the 1.2-kb BspHI-EcoRV fragment carrying bzaB,
the 1.0-kb BspHI fragment carrying bla, and the 1.9-kb HindIII fragment carrying tet were labelledwith the DIG system and used as probes.
SYK-6 and its mutants were grown in LB medium (100 ml) until the OD600 of the culture
reached 1.0. Cells were harvested by centrifugation (5,000  g, 10 min), washed with 100 mM
potassium phosphate buffer (pH 7.0), and resuspended in the same buffer. The cells were sonicated,
and the cell lysate was centrifuged at 15,000  g for 15 min. The resulting supernatant was used ascell extracts. Protein concentrations were determined using the Bradford method [14]. The oxidation
activities of cell extracts for aromatic aldehydes were determined as descried below.
Expression of bzaA and bzaB in E. coli
The 1.7-kb BamHI-PstI fragment carrying bzaA and the 2.5-kb BglII-SalI fragment carrying
bzaB were cloned into SK(+) and KS(+), respectively, to obtain pSKL2 and pBSV3. Cells of E. coli
JM109 harboring pSKL2 or pBSV3 were grown in LB medium containing 100 mg ampicillin/liter at
37C. Expressions of bzaA and bzaB were induced for 6 h and 8 h, respectively, by adding 1 mM
IPTG when the OD600 of the culture reached 0.5. Cells were harvested by centrifugation andsuspended in 100 mM potassium phosphate buffer (pH 7.0). The cell extracts were preparedsimilarly to the method mentioned above. Expressions of the genes were examined using sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with
Coomasie brilliant blue.
Enzyme assay
Degradation of vanillin and syringaldehyde was periodically analysed with an Alliance 2690
separation module HPLC system equipped with a TSKgel ODS-80 column (6 by 150 mm; Tosoh).
The mobile phase was a mixture of water (74.25%), acetonitrile (24.75%), and acetic acid (1%), andthe flow rate was 1 ml/min. Vanillin and syringaldehyde were detected at 280 nm and 308 nm,
respectively. The retention times of vanillin, syringaldehyde, vanillate, and syringate were 9.0, 9.0,
6.0, 5.9 min, respectively, by a photodiode array detector (Waters 2996).
Oxidation activities for aromatic aldehydes including vanillin and syringaldehyde were
determined using a spectrophotometer (DU-7500, Beckman-Coulter). The decrease in absorbance at
364 nm derived from syringaldehyde (364 = 8,936 M-1 cm-1, pH 7.0) was monitored in a reaction
mixture containing 100 mM potassium phosphate buffer (pH 7.0), 0.5 mM NAD+, 1.2 mM pyruvate,3

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1.0 U lactate dehydrogenase, 100 M substrate, and a cell extract (100 g of protein/ml of crude
BzaA, 500 g/ml of crude BzaB, and 500 g/ml of cell extracts of SYK-6 and its mutants) at 30C.
To examine the substrate preference of BzaA and BzaB, the cell extracts were incubated with 100
M of the following substrates as described above: benzaldehyde (293 = 1,270 M-1 cm-1),
p-hydroxybenzaldehyde (331 = 7,088 M-1 cm-1), protocatechualdehyde (341 = 9,070 M-1 cm-1),
m-anisaldehyde (330 = 2,181 M-1 cm-1), veratraldehyde (330 = 3,536 M-1 cm-1), coniferyl aldehyde(410 = 3,330 M-1 cm-1), or vanillin (346 = 8,697 M-1 cm-1). One unit of enzyme activity was definedas the amount of activity that oxidized 1 mol of aromatic aldehydes per 1 min. Specific activity was
expressed in units per milligram of protein.
Nucleotide sequence accession number
The nucleotide sequence of Sphingobium sp. strain SYK-6 chromosome containing bzaA
(SLG_27910) and bzaB (SLG_27920) were deposited in the DDBJ/EMBL/GenBank databases
under accession number AP012222.

3. Results and Discussion
Isolation of aromatic aldehydes dehydrogenase genes from Sphingobium sp. strain SYK-6
Subcloning of pKTV3, which contained approximately 20-kb SalI fragments, showed that the
3.1-kb BamHI-HindIII fragment conferred the oxidation activity for syringaldehyde and vanillin on
E. coli JM109. Nucleotide sequence of the 3.1-kb BamHI-HindIII fragment revealed an open reading
frame (ORF), encoding a polypeptide of 472 amino acids (ORF1) and an incomplete ORF (ORF2),
which are oriented convergently as shown in Fig. 2. The deduced amino acid sequence of ORF1
showed 31-35% identity with those of the vanillin dehydrogenase genes of Pseudomonas putida
KT2440 [15], Pseudomonas sp. strain HR199 [16], Pseudomonas fluorescence AN103 [17], P.
putida WCS358 [18], and Sphingobium sp. strain SYK-6 [6]. Therefore, this ORF appeared to
encode aromatic aldehydes dehydrogenase and was designated bzaA. In order to obtain a full lengthof ORF2, Southern hybridization of pKTV3 was performed using the 0.5-kb BspHI-HindIII
fragment carrying a part of ORF2 as a probe. This analysis showed that ORF2 was included in the
4.2-kb ApaI fragment. Nucleotide sequence of the 2.5-kb BglII-SalI fragment in the 4.2-kb ApaI
fragment showed that ORF2 encodes a polypeptide of 489 amino acids (Fig. 2). The deduced aminoacid sequence of ORF2 showed 27-34% identity with those of the vanillin dehydrogenase genes of
Pseudomonas strains and SYK-6, and bzaA. Based on these similarities, ORF2 was designated bzaB.

Fig. 2. Restriction map of the 3.4-kb BamHI-SalI fragment carrying bzaA and bzaB. Abbreviations:
A, ApaI; B, BamHI; Bg, BglII; Bs, BspHI; E, EcoRI; H, HindIII; Hc, HincII; Ne, NaeI; P, PstI; Pv,
PvuII; RV, EcoRV; ScII, SacII; Sl, SalI; Sm, SmaI; St, StuI; X, XhoI.
Enzymatic properties of BzaA and BzaB
Each of the bzaA and bzaB genes was expressed in E. coli JM109 under the control of lac
promoter. SDS-PAGE showed the production of 45-kDa and 48-kDa proteins in cell extracts of E.
coli JM109 harboring pSKL2 and pBSV3, which carried bzaA and bzaB, respectively. These values
were close to the predicted molecular masses of the corresponding gene products calculated from theamino acid sequences.
Cell extracts (100 g of protein/ml) of E. coli harboring pSKL2 or pBSV3 were incubated with100 M vanillin in the presence of NAD+ or NADP+ (500 M) to determine the cofactor
requirement. The vanillin dehydrogenase activities were dependent on NAD+ or NADP+. However,4Transactions on GIGAKU 1(2012)01009/1-6

2.3-fold higher activity of BzaA and 19-fold fold higher activity of BzaB were observed in thepresence of NAD+ than in the presence of NADP+.
Substrate preferences of BzaA and BzaB were assessed with various aromatic aldehydes,
including vanillin, syringaldehyde, benzaldehyde, p-hydroxybenzaldehyde, protocatechualdehyde,
m-anisaldehyde, veratraldehyde, and coniferyl aldehyde. Crude BzaA enzyme showed a broad
substrate preference exhibiting specific activities of 925, 627, 511, 473, 301, 261, and 225 mU/mg
toward p-hydroxybenzaldehyde, coniferyl aldehyde, syringaldehyde, benzaldehyde, veratraldehyde,
vanillin, and protocatechualdehyde, respectively (Fig. 3). On the other hand, BzaB was essentially
specific for m-anisaldehyde. The activities of BzaB toward veratraldehyde, coniferyl aldehyde,benzaldehyde, syringaldehyde and vanillin were less than 16% of the activities of BzaB towardm-anisaldehyde (Fig. 3).

Fig. 3. (A) Chemical structures of aromatic aldehydes used in this study. Compounds: I, vanillin;II, benzaldehyde; III, p-hydroxybenzaldehyde; IV, protocatechualdehyde; V, m-anisaldehyde; VI,veratraldehyde; VII, coniferyl aldehyde; VIII, syringaldehyde. (B) Oxidation activities of BzaA
and BzaB toward aromatic aldehydes. White and gray bars indicate the activities of BzaA and
BzaB, respectively. The data are average  standard deviations of three independent experiments.
Disruption of bzaA and bzaB in Sphingobium sp. strain SYK-6
In order to examine the roles of bzaA and bzaB in the degradation of syringaldehyde in
Sphingobium sp. strain SYK-6, each of these genes in SYK-6 was disrupted by the insertion of the
bla and tet gene, respectively, using a gene replacement technique. A bzaA bzaB double mutant wasalso constructed. Disruption of each gene was confirmed by Southern hybridization analysis. The
bzaA mutant (SME044), bzaB mutant (SME045), and bzaA bzaB double mutant (SME046) grew on
syringaldehyde at rates similar to that of the wild type, suggesting that these genes are not essential
for the degradation of syringaldehyde in SYK-6. For further characterization of the mutants, theoxidation activities toward syringaldehyde and vanillin of cell extracts of SME044, SME045, andSME046 were evaluated. The activities of these three mutants for syringaldehyde and vanillin were
almost the same as those of the wild type (Table 1). These results indicated that the contributions ofboth bzaA and bzaB to the degradation of syringaldehyde are minor in SYK-6.
Table 1. Oxidation activities of SYK-6 and its mutants
toward vanillin and syringaldehyde
strains
SYK-6
DLV
SME044SME045SME046

Specific activity (mU/mg)
vanillin
syringaldehyde
20.5 ± 2.8
129 ± 6.3
22.3 ± 0.2
20.1 ± 0.1
19.3 ± 2.5
152 ± 7.4
20.7 ± 3.0
100 ± 9.7
20.7 ± 2.7
152 ± 1.6

5

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4. Conclusion
Two novel aromatic aldehydes dehydrogenase genes, bzaA and bzaB, of Sphingobium sp. strain
SYK-6 were isolated and characterized. The gene product of bzaA exhibited a broad substratepreference oxidizing syringaldehyde, vanillin, p-hydroxybenzaldehyde, and coniferyl aldehyde
efficiently. This feature of BzaA appeared to be applicable to the bioprocesses converting
syringaldehyde and vanillin to industrially valuable compounds such as PDC. Disruption of bzaAand bzaB suggested that these genes are insignificant to catabolism of syringaldehyde by
Sphingobium sp. strain SYK-6. Our results may suggest the following two possibilities: i) there is a
specific syringaldehyde dehydrogenase gene(s) in SYK-6. ii) there is no specific syringaldehyde
dehydrogenase in SYK-6, and the sum of the activities of several aromatic aldehydes
dehydrogenases with different substrate preferences enables SYK-6 to grow on syringaldehyde.
Research is currently underway to identify the aromatic aldehydes dehydrogenase genes, which are
actually involved in the syringaldehyde catabolism in SYK-6 by genome-wide analyses.
Acknowledgments
This work was supported in part by an Industrial Technology Research Grant from the New
Energy and Industrial Technology Development Organization (NEDO) of Japan.
References[1] E. Masai, Y. Katayama, and M. Fukuda, Biosci. Biotechnol. Biochem., 71 (2007) 1.
[2] Y. Otsuka, M. Nakamura, K. Shigehara, K. Sugimura, E. Masai, S. Ohara, and Y. Katayama,Appl. Microbiol. Biotechnol., 71 (2006) 608.
[3] Y. Hasegawa, K. Shikinaka, Y. Katayama, S. Kajita, E. Masai, M. Nakamura, Y. Otsuka, S.
Ohara, and K. Shigehara, Sen'i Gakkaishi, 65 (2009) 359.
[4] M. Hishida, K. Shikinaka, Y. Katayama, S. Kajlta, E. Masai, M. Nakamura, Y. Otsuka, S.
Ohara, and K. Shigehara, Polym. J., 41 (2009) 297.
[5] T. Michinobu, M. Bito, Y. Yamada, Y. Katayama, K. Noguchi, E. Masai, M. Nakamura, S.
Ohara, and K. Shigehara, Bull. Chem. Soc. Jap, 80 (2007) 2436.
[6] E. Masai, Y. Yamamoto, T. Inoue, K. Takamura, H. Hara, D. Kasai, Y. Katayama, and M.
Fukuda, Biosci. Biotechnol. Biochem., 71 (2007) 2487.
[7] X. Peng, T. Egashira, K. Hanashiro, E. Masai, S. Nishikawa, Y. Katayama, K. Kimbara, and M.
Fukuda, Appl. Environ. Microbiol., 64 (1998) 2520.
[8] E. Yabuuchi, Y. Kosako, N. Fujiwara, T. Naka, I. Matsunaga, H. Ogura, and K. Kobayashi, Int.
J. Syst. Evol. Microbiol., 52 (2002) 1485.
[9] J. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res., 16 (1988) 7583.
[10] C. Yanisch-Perron, J. Vieira, and J. Messing, Gene, 33 (1985) 103.
[11] A. Schäfer, A. Tauch, W. Jäger, J. Kalinowski, G. Thierbach, and A. Pühler, Gene, 145 (1994)
69.
[12] K. S. Reece and G. J. Phillips, Gene, 165 (1995) 141.
[13] T. Abe, E. Masai, K. Miyauchi, Y. Katayama, and M. Fukuda, J. Bacteriol., 187 (2005) 2030.
[14] M. M. Bradford, Anal. Biochem., 72 (1976) 248.
[15] R. Plaggenborg, J. Overhage, A. Steinbüchel, and H. Priefert, Appl. Microbiol. Biotechnol., 61(2003) 528.
[16] H. Priefert, J. Rabenhorst, and A. Steinbüchel, J. Bacteriol., 179 (1997) 2595.
[17] A. Narbad and M. J. Gasson, Microbiology., 144 (1998) 1397.
[18] V. Venturi, F. Zennaro, G. Degrassi, B. C. Okeke, and C. V. Bruschi, Microbiology, 144 (1998)
965.
(Received: 17 May, 2012, Accepted: 30 June, 2012)6

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Development of an Appropriate Treatment Process for Wastewaterfrom a Natural Rubber Processing Factory
Daisuke Tanikawa1)*, Takuya Yamashita1), Masashi Hatamoto1), Masao Fukuda2), Masanobu
Takahashi3), Kazuaki Syutsubo4), Pairaya Kucivilize Choeisai5), Takashi Yamaguchi1)
1)Department of Environmental System Engineering, Nagaoka University of Technology,1603-1 Kamitomioka, Nagaoka 940-2188, Japan2)
Department of Bioengineering, Nagaoka University of Technology,1603-1 Kamitomioka, Nagaoka 940-2188, Japan3)
Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Sendai
980-8579, Japan
4)
Center for Regional Environmental Research, National Institute for Environmental Studies, 16-2
Onogawa, Tsukuba 305-8506, Japan
5)
Department of Environmental Engineering, Faculty Engineering, KhonKaen University, KhonKaen
40002, Thailand*E-mail: dtanikawa@vos.nagaokaut.ac.jp
Natural-rubber latex wastewater containing a high concentration of sulfate
was treated using a combined system consisting of a two stage reactors of
up-flow anaerobic sludge blanket (UASB) and a down-flow hanging sponge(DHS) reactor for over 10 months. The system achieved a COD removal
efficiency of 97.6 ± 1.1% at an organic loading rate (OLR) of 0.91
kgCOD/(m3.d), where the influent of 10,200 ± 1,370 mgCOD/L wasmaintained at 11.1 days of the hydraulic retention time. The
methane-producing and sulfate-reducing activity tests indicated that
sulfate-reducing bacteria (SRB) was superior to methanogenis in the firststage reactor. However, methanogens were superior to SRB in the second
stage of the UASB reactor. Microbial community analysis based on a 16S
rRNA gene sequence revealed that many species of SRB, such as
Desulfomicrobium, existed in the 1st UASB reactor. However, the ratio of
SRB was decreased in the 2nd UASB reactor. Furthermore, for the treatmentof the wastewater, the consumption of power by the two stage system was
93% less than that of the conventional lagoon system.

1. Introduction
The natural rubber industry is concentrated in the South-East Asia region, especially in Thailand,
Indonesia, and Malaysia. In addition, more than 60 % of worldwide natural rubber production comesfrom these three countries [1]. Rubber latex, which is extracted from rubber trees, is used as a raw
material for three intermediate forms of the rubber products: ribbed smoked sheets (RSS), which areused as a raw material for making vehicle tires and industrial rubber parts; technically specified
rubber (TSR), which is used as a raw material for making high viscosity products such as belts; andconcentrated latex, which is used as a raw material for making dipped products such as medical
gloves. These three intermediate products are used in downstream rubber products industries [2, 3].
In a natural rubber processing factory, a large amount of wastewaters containing a highconcentration of organic matter, nitrogen (especially, ammonia) and sulfate is discharged. This isbecause that a large amount of ammonia is used for the preservation of rubber latex and sulfuric acidis required for recovering rubber particles [3]. In South-East Asia, such wastewater is generally
treated by conventional lagoon systems such as aerated lagoons, anaerobic lagoons, and facultative
lagoons [3, 4]. However, these systems need long retention times and large treatment areas. In
addition, these systems consume a large amount of power to aerate and discharge large amounts ofexcess sludge. These systems are also responsible for polluting the environment in many ways, suchas through greenhouse gases (GHG, e.g., methane, carbon dioxide), malodor emissions, and1

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groundwater pollution [3].
In a new process of wastewater treatment consisting of two stage reactors of an up-flow
anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) reactor was developed.
The UASB + DHS system exhibited high treatment performances, low excess sludge productions,
low operational costs, and energy savings [5, 6]. Moreover, when the UASB + DHS system treatedwastewater containing sulfate, the sulfide generated in the UASB reactor was promptly oxidized in
the DHS reactor.
In the present study, an on-site pilot scale experiment was conducted in the Von Bundit natural
rubber processing factory in Srat Thani, Thailand to develop an appropriate treatment process for
wastewater from natural rubber latex waste by using a combined two-stage UASB and DHS system.
The pilot plant was operated at the natural rubber processing factory for a period of more than 300
days. The performance of a conventional lagoon system and the developed system was evaluatedand compared.
2. Experiment(1) Two-stage system combined with UASB and DHS reactors
A schematic diagram of the developed system is given as Fig.1. The system consists of anacidification tank with 1180 L of working volume, the two stage UASB reactor was combined with
997 L of the first stage UASB and 597 L of the second stage UASB, and a DHS reactor with 195 L
volume of sponge media. The UASB reactor and the DHS reactor were seeded with the sludge
obtained from an anaerobic lagoon and an aerated lagoon at the factory, respectively.
Table 1 lists chemical characteristics of the concentrated latex wastewater. The concentrated
latex wastewater discharged from the factory was received in the acidification tank and was later fed
to the 1st UASB reactor as an influent. The pH of the influent was adjusted to 6.9–7.5 by addition of
2.5 N NaOH and a part of the UASB effluent obtained during the 1st UASB reactor was returned tothe influent at a recirculation ratio of 2:1. The operational period was divided into ten separate
periods (phase 1–7b) according to the organic loading rate (OLR) and the hydraulic retention time(HRT).
(2) Chemical analysis
Gas composition was measured by a gas chromatograph equipped with a thermal conductivity
detector (GC-8A, Shimadzu). The solution pH was analyzed by using a pH meter (HM-20P, DKK).
For sulfate analysis, the samples were filtered by 0.45 μm of glass fiber filter paper (GB-140,
Advantec). Sulfate, chemical oxygen demand (COD) and sulphide were measured by Hachapparatus (DR890, Hach). Analysis of biochemical oxygen demand (BOD), suspended solids (SS),volatile suspended solids (VSS) and total nitrogen was conducted according to standard method,which was published by the Japan Sewage Works Association [7]. Volatile fatty acids (VFA) was
determined by a gas chromatograph equipped with a flame ionization detector (GC-1700,
Shimadzu).

Fig. 1 Schematic diagram of the two-stage system

2Transactions on GIGAKU 1(2012)01010/1-8

Table 1 Chemical characteristics of the concentrated latex waste water
Parameter

Average±SD *

Unit

pH

5.54±0.54-

Total COD

9,710±2,600

mgCOD/L

Soluble COD

8220±2,020

mgCOD/L

Total BOD

8,670±2,750

mgBOD/L

Soluble BOD

7,310±2,320

mgBOD/L

Total VFA

4,460±1,290

mgCOD/L

Sulfate1,430±490

mgS/L

Total Nitrogen

1,370±480

mgN/L

SS

1,780±1,260

mgSS/L

VSS

1,050±760

mgVSS/L*: Standard deviation(3) Microbial activity test
The methane producing activity (MPA) and sulfate reducing activity (SRA) of the retained
sludge in the UASB reactor at 0 (as the seed), 87, 156, and 251 days were determined in duplicate.
The test sludge was obtained from each stage of the UASB reactor having, a vertical height of 0.5 m.
The sludge was disintegrated under anaerobic conditions and was used for the activity tests. Sodium
acetate (2,000 mgCOD/L), sodium propionate (1,000 mgCOD/L), and H2/CO2 (80:20, v/v, 1.4 atm)
were used as test substrates. All vials were incubated in a reciprocal shaker (radius = 4 cm) with 120
rpm at 35°C. In the SRA test, sodium sulfate (200 mgS/L) and chloroform solution (5 mg/L) wereadded as an electron acceptor and a methanogenic inhibitor, respectively. Detailed procedures for theactivity tests are described elsewhere [8, 9].
(4) Microbial community analysis
Sludge samples obtained from each stage of the UASB reactor at 196 days were used for
microbial community analysis. The DNA was extracted from the washed sludge using an ISOIL
Beads Beating kit (Nippon gene), as described in the manufacturer’s instructions. The extracted
DNA was used for amplification of bacterial and archaeal 16S rRNA gene fragments with primer
pairs of EUB8F/150r [10, 11] and Arc109f/1500r [11, 12], respectively. Bacterial and archaeal 16S
rRNA gene clone libraries were constructed using a TOPO TA cloning kit (Invitrogen).
Classification and determination of the closest species of the obtained clones were performed using aclassifier program of a ribosomal database project.
(5) Comparison between two stage system and conventional lagoon system in at the Natural
Rubber factory
In the abovementioned factory, three types of wastewater were discharged, first type ofwastewater stream was obtained from concentrated latex (latex) production, second type ofwastewater stream was obtained from RSS production, and third type of wastewater stream was
obtained from standard Thai rubber (STR) production. Fig. 2 shows the schematic diagram of theconventional lagoon system in the factory. Each wastewater stream was discharged from eachproduction line and was treated separately. The conventional lagoon system consisted of anaerobic
lagoonsfor RSS and STR wastewater treatment, aerated lagoons for latex wastewater treatment, afacultative lagoon, and a polishing lagoon for post treatment. To compare the both systems in theperformance, influent and effluent water samples obtained from each lagoon were collected and thewater quality was analyzed. Furthermore, some data concerning operational parameters such as
power consumption, wastewater discharge volume, and excess sludge volume were collected toevaluate the performance of the two stage system developed.
3

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Fig.2 Schematic diagram of the conventional lagoon system at the factory

3. Results and Discussion(1) Process performance of the developed system
Fig. 3 shows the time course of the OLR and total COD removal. In phase 1, the total COD
removal efficiency was 89.9 ± 2.3% at 7.4 days of HRT with an OLR of 0.79 ± 0.04 kgCOD/(m3.d)for the whole system (for only 1st UASB reactor, the total COD removal efficiency was 70.2 ± 4.4%
at 1.4 days HRT with an OLR of 2.27 ± 0.08 kgCOD/(m3.d)). In phase 2, the HRT was shortened to
3.7 days and the influent COD concentration was increased, which corresponded to an OLR of 10.0
± 2.19 kgCOD/(m3.d) for the 1st UASB reactor. The COD removal efficiency of the whole systemdecreased to 64.0%. Concurrently, a large amount of SS became more than 2,000 mg/L in theeffluent of the 1st UASB reactor and then was washed out from the UASB reactor to the DHS
reactor. This was caused by the overloading of the 1st UASB reactor, which inhibited the usual
treatment processes. In phases 3a–4, the HRTs of both stages of the UASB reactor returned to thesame as in phase 1, and the wastewater supply rate for the DHS reactor was adjusted in order toinvestigate the degradation of solid sulfur that had accumulated in the upper part of the DHS reactor.
For the 1st UASB reactor (in phases 3a–3c), at an OLR of 5.59 ± 0.91 kgCOD/(m3.d), the outflow ofSS from the UASB reactor decreased. Though the total COD removal efficiency in the 1st UASB
reactor was only 30.9 ± 13.7%, the total COD removal efficiency of the whole system was 91.5 ±
10.8%. This shows that the organic matter was successfully treated in the 2nd UASB reactor. In
phases 4–7b, except for phase 6, when the COD concentration of the influent suddenly increased, the
OLRs of the 1st UASB reactor were controlled to be 2.42 ± 0.41 kgCOD/(m3.d). As a result, thetotal COD removal efficiency of the 1st UASB reactor increased and stabilized to 76.2 ± 0.7% inphase 7b.
Based on the experimental results described above, the maximum acceptable OLR of the 1st
UASB reactor is approximately 5.8 kgCOD/(m3.d) at phase 3c (i.e., ca. 1.6 kgCOD/(m3.d) for thetwo-stage UASB reactor). The optimum OLR of the 1st UASB reactor is approximately 2.5
kgCOD/(m3.d) (i.e., ca. 1.0 kgCOD/(m3.d) for the two-stage UASB reactor) because a sufficient
COD removal efficiency of 72.1 ± 3.9% was achieved in the 1st UASB reactor at phase 7a. ThisOLR was lower than the OLR of the other mesophilic UASB reactors treating in industrial
wastewater [13]. This was because of the influence of the high concentration of hydrogen sulfide
generated in 1st UASB reactor. As reported by Lens et al. [14], the methane production activity of
granular sludge was inhibited by 50% with a concentration of 90–250 mgH2S/L of hydrogen sulfide[15]. In the effluent of the 1st UASB reactor, the concentration of hydrogen sulfide was 130 ± 0.8
mgH2S/L in phase 3c and 149 ± 30 mgH2S/L in phase 7a.

4

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Fig.3 Time course of (a) organic loading rate and (b) total COD removal.
(2) Microbial activity of the retained sludge of the UASB reactor
Fig.4 shows the time course of MPA and SRA of the retained sludges of the two stages of the
UASB reactor. The MPA of both retained sludges in the UASB reactor increased during reactor
operation with an exception of H2/CO2-fed MPA (MPA–H2). After 196 days, both acetate- andpropionate-fed methanogens (MPA-Pro.) in the retained sludge for the 2nd UASB reactor weregreater than that obtained in the 1st UASB reactor. Here, the ratio for the acetate was 1.61 in the 2nd
UASB/1st UASB and that of the propionate was 1.81 on 251 days. This could be resulted by the low
sulfate concentration in the influent in the 2nd UASB reactor. In regards to the SRA of the UASB
retained sludges, the H2/CO2-fed SRA (SRA–H2) was highest for both stages. Acetate- andpropionate-fed SRAs in the retained sludge of the 1st UASB reactor were minimal. However, theacetate- and propionate-fed SRAs of the retained sludge in the 2nd UASB reactor were not detected
at all.
For acetate and propionate utilization, MPA was higher than SRA for both UASB stages at
251days. On the other hand, hydrogen-utilizing SRA was higher than that for MPA. In addition, the
SRA/MPA ratio for the first and second UASB stages were 5.72 and 6.63, respectively. These
results indicated that hydrogen-utilizing sulfate-reducing bacteria (SRBs) acted as hydrogenscavengers in both stages of the UASB reactor.
(3) Microbial communities of the UASB retained sludge
Table 2 shows the microbial community compositions derived from the 16S rRNA gene baseanalysis of the retained sludges in the UASB reactor. Proteobacteria (39%) and Firmicutes (30%) in
the 1st UASB reactor, and Firmicutes (23%), Proteobacteria (21%) and Bacteroidetes (12%) in the
2nd UASB reactor were the dominant phyla of bacteria. Among Proteobacteria, a lot of clones
relating to the SRB Desulfomicrobium were detected. In addition, 17 clones of possible 95 clones
were present in the 1st UASB reactor, which could have been due to the high concentration of
sulfate.
Moreover, only Euryarchaeota were detected as archea in both UASB reactor stages. Among theclones, Methanomethylovorans and Methanosaeta dominated with more than 70% share in bothUASB reactor stages. It was reported that Methanomethylovorans species existed in the retained
sludge of methanol-fed UASB reactors and utilized methanol and dimethyl sulfide as catabolic
substrates, however, H2/CO2 and acetate were not utilized [15, 16]. The existence ofMethanomethylovorans in our reactors might be related to the high concentration of sulfate in thefeed and the utilization of dimethyl sulfide. However, further research is needed to determine therole of Methanomethylovorans in our reactors.
5Transactions on GIGAKU 1(2012)01010/1-8

Fig.4 Time course of methane-producing/sulfate-reducing activities of the retained sludges ofthe first and second stages of the UASB reactor
Table 2 Microbial community compositions from 16S rRNA gene based analysis of retained sludges
in the UASB reactor stages
No. of clone (% of total clones)

Classification

1st UASB

2nd UASB

Bacteria
3 (3)Actinobacteria

1 (1)Armatimonadetes
Bacteroidetes

5 (5)11 (12)Chloroflexi

5 (5)

2 (2)

Firmicutes

29 (31)22 (23)

Proteobacteria
Desulfuromonadaceae

4 (4)

6 (6)

Desulfobacteraceae

15 (16)10 (11)

Desulfomicrobiaceae

17 (18)other proteobacteria

4 (4)
2 (2)

Synergistetes
1 (1)Verrucomicrobia

2 (2)Uncultured bacteria

19 (20)31 (33)

Total - Bacteria

95 (100)

94 (100)

Archaea
Methanolobus

2 (4)

3 (7)

Methanomethylovorans

29 (62)22 (49)Methanosaeta

6 (13)

16 (35)1 (2)Methanosphaera
3 (6)Methanosarcinales
Uncultured archaea

7 (14)

4 (9)Total - Archaea

47 (100)

46 (100)

6

Transactions on GIGAKU 1(2012)01010/1-8(4) Comparison between the two stage system and conventional lagoon system
After evaluating the process performance for latex wastewater treatment, the two stage system
was used to treat the RSS and STR wastewaters. Table 3 shows the chemical characteristics of eachwastewater stream and the final effluent obtained from the both systems. For the purpose ofcomparison, the assumption was made that the two stage system would treat all three wastewater
streams. The first stage of the UASB reactor would treat the latex wastewater, the second stage
would be for both the RSS and STR wastewaters, and the DHS reactor would provide post-treatment.
The two stage system achieved the Thai effluent standard for the 20 mg/L of BOD, 120 mgCOD/L of
CODcr and 50 mg/L of SS and maintained a better process performance than those of theconventional lagoon system. Table 4 shows the power consumption of each treatment system. In theconventional system, the latex wastewater treatment consumed a large amount of powers because of
the need for aeration. Also a large amount of excess sludges was discharged from the aerated lagoon.
Furthermore, 20 t CO2/d of methane gas was emitted from the anaerobic lagoons. Emission of
methane gas was calculated from the COD removal in the anaerobic lagoons. On the other hand, thetwo stage system reduced 93% of the power consumption for the latex wastewater treatment,
reduced the amount of excess sludge discharged by 90%, and reduced the GHG emissions from theaerated lagoons by 95% by recovering methane as an energy source. These results prove that the twostage system was suitable for the wastewater treatment in a natural rubber processing factory.
Table 3 Chemical characteristics of wastewater and final effluent of the two stage system andconventional system
Parameter

Final effuent(Average±SD*)

Wastewater (Average±SD*)
Latex

RSS

STR

Developed

ConventionalpH

5.54±0.54

7.34±0.44

7.77±0.41

7.43±0.67

7.79±0.08

BOD

8,670±2,750435±238509±226

9±27

46±18

COD Cr9,730±2,600

1,070±421

1,080±492

113±35112±20SS

1,780±1,260

651±677261±116

13±9

50±14

Unit
mg/L

*: Standard deviation

Table 4 Power consumption and greenhouse gases emission of each wastewater treatment
systemPower consumption (kWh/m3 effluent)

GHG emission (t CO 2 /m 3 effluent)

Developed system Conventional system

Developed system Conventional system

Source of effluent

Discharge(m 3 /d)

Latex

75

0.38

5.83

0.45

14.69

RSS

500

0.06

0.00

0.03

1.70

STR

2000

0.01

0.00

0.01

9.81

Post treatment

2575

0.00

0.34

0.00

0.20

Total system

2575

0.04

0.51

0.03

8.57

4. Conclusion
In the present research, latex wastewater was treated using a two-stage UASB and DHS system,and the wastewater treatment performance was evaluated in long term process. During theexperimental period, a COD removal efficiency of 97.6% was achieved under the operating
conditions of 0.91 kgCOD/(m3.d) of OLR and 11.1 days of HRT. The 1st UASB reactor removed
organic matter while the producing methane and reducing sulfate, the 2nd UASB reactor produced
methane, and the DHS reactor removed the remaining organic matter.
In terms of the microbial community in the stages of the UASB reactor, both the dimethyl
sulfide-utilizing methanogen, Methanomethylovorans, and some SRBs such as Desulfomicrobium
7Transactions on GIGAKU 1(2012)01010/1-8

were detected because of the high concentration of sulfate. In both UASB stages, methanogenactivity was higher than SRB activity for acetate and propionate. On the other hand,hydrogen-utilizing SRB activity was higher than methanogen. These results indicate thathydrogen-utilizing SRBs acted as hydrogen scavengers under a in both stages of the UASB reactor.
The results showed that the two stage system achieved better process performance than theconventional lagoon system. Furthermore, the former system reduced the power consumption for thelatex wastewater treatment by 93%, excess sludge volume discharge from the aerated lagoon by 90%,
and GHG emissions by 95% through recovery of methane as an energy source. These results provedthat the developed system is suitable for wastewater treatment in a natural rubber processing factory.

Acknowledgments
This research was supported by the JST/JICA, SATREPS (Science and Technology ResearchPartnership for Sustainable Development), Japan. We wish to thank Von Bundit co., ltd. and theirstaff for installation and operation of the two stage system.
References[1] M. Mohammadi, H. C. Man, M. A. Hassan, and P. L. Yee, African J. Biotech., 9 (2010) 6233.
[2] P. Takasakul and S.Tekasakul, J. Aerosol Res.,21 (2006) 122.
[3] W. Jawjit, C. Kroeze, and S. Rattanapan, J. Cleaner Production, 18 (2010) 403.
[4] S. Chaiparapatand and S. Sdoodee, Resour.,Conserv.Recy., 51 (2007) 577.
[5] I. Machdar, Y. Sekiguchi, H. Sumino, A. Ohashi, and H. Harada, Wat. Sci. Tech., 42 (2000) 83.
[6] M. Tanduker, A. Ohashi, and H. Harada, Wat. Res.41 (2007) 2697.
[7] Japan Sewage Works Association, Japanese standard methods for sewage and wastewater (1997)(in Japanese).
[8] K. Syutsubo, N. Shinthurat, A. Ohashi, and H. Harada, Wat. Sci. Tech., 43 (2001) 59.
[9] T. Yamaguchi, H. Harada, and I. C. Tseng, Proc. 8th Int. Conf. Anaerobic Digestion,2 (1997)
362.
[10] L. Ovreas, L. Forney, F.L. Daae, and V. Torsvik, Appl. Environ. Microbiol., 63 (1997) 3367.
[11] D. J. Lane, 16S/23S rRNAsequencing, In E. Stackebrandt, and M. Goodfellow (ed),Nucleicacidtechniques in bacterialsystematics, JhonWileyand Sons, Inc., N. Y. (1991) 115-175.
[12] G. Regine, H. J. Peter, and L. Werner, Appl. Environ. Microbiol., 64 (2010) 960.
[13] S. V. Gali, K. Pradeep, and M. Indu, Water Res., 39 (2005) 154.
[14] P. N. L. Lens, A. Visser, A. J. Janssen, L. W. Hulshof Pol, and G. Lettinga, Critical review inEnviron. Sci. and Technol.,28 (1998) 41.
[15] B. Jiangand and N. S. Parshina, Syst. Eval. Microbiol., 55 (2005) 2465.
[16] B. P. Lomans, R. Maas, R. Luderer, H. J. M. Op den Camp, A. Pol., C. Van der Drift, and G. D.
Vogels, Appl. Environ. Microbiol., 65 (1999) 3641.
(Received: 18 May, 2012, Accepted: 4 July, 2012)8

Transactions on GIGAKU 1(2012)01011/1-4

Biodegradation of Persistent Chlorinated Organophosphorus Flame
Retardants by Microorganisms Newly Isolated from Soil – a short review
Yoshio Kera *, Shouji Takahashi, Katsumasa Abe
Department of Environmental Systems Engineering, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: yoshkera@vos.nagaokaut.ac.jp
The novel bacterial strains that can degrade trihaloalkyl phosphates,
Sphingobium sp. TCM1 and Sphingomonas sp. TDK1, were isolated from soil
by growth on persistent chlorinated organophosphorus (OP) flame retardants,tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate(TDCPP), as a sole phosphorus source, respectively. While Sphingobium sp.
TCM1 and Sphingomonas sp. TDK1 degraded TCEP and TDCPP byhydrolyzing their phosphotriester bonds, both strains were not able to degrade
2-chloroethanol and 1,3-dichloro-2-propanol, which were hydrolysis productsof TCEP and TDCPP, respectively. The two strains were able to degrade andassimilate not only the chlorinated OP flame retardants but also several otherOP flame retardants and plasticizers and the strains possessed different
substrate specificity for trialkyl phosphates.

1. Introduction
The chlorinated organophosphorus (OP) compounds, tris(2-chloroethyl) phosphate (TCEP) andtris(1,3-dichloro-2-propyl) phosphate (TDCPP), as shown in Fig. 1, are widely used as flame
retardants mainly in a range of plastic foams, resins, and latexes and in the production of liquid
unsaturated polyester resins, respectively [1]. Their widespread use has led to contamination ofvarious environments [2-7]. Particularly, a higher contamination level was observed in leachates andraw water of waste disposal sites [8]. Furthermore, they were also detected in drinking water [1],because they were hard to eliminate not only in the environment but also in sewage treatment plants[9]. Many studies reported several toxic effects of the compounds. For example, TCEP was shown tocause adverse effects on brain, liver, and kidney and on the fertility of male rats and mice [1]. In
addition, TCEP produced tumors at various organ sites [10], also was suspected to possess
carcinogenicity [11] and was shown to inhibit the expression of cell cycle regulatory proteins, DNA
synthesis, and cell numbers [12]. It was known that TDCPP exhibited genotoxicity in several in vitro
assays conducted in prokaryotic and eukaryotic cells [13] and produced some indications ofcarcinogenicity [1].
Many bacteria and fungi that can degrade OP pesticides and insecticides, such as parathion andchlorpyrifos, have been discovered, isolated, andcharacterized [14-17]. In contrast, few studies havebeen conducted on microbial degradation ofchlorinated OP flame retardants, despite their
persistence and potentially nonnegligible toxicity.
Thus far, there has been no report of the isolationof bacteria that can degrade TCEP and TDCPP.
In the present short review, we summarize ourrecent studies about the development of 2enrichment bacterial cultures that can degrade
rapidly both TCEP and TDCPP [18] and theisolation and identification of bacteria that can Fig. 1 Structures of tris(2-chloroethyl) phosphatedegrade the compounds from each enrichment (TCEP) and tris(1,3-dichloro-2-propyl)
phosphate (TDCPP).
culture [19].
1Transactions on GIGAKU 1(2012)01011/1-4

2. Enrichment and Characterization of Chlorinated Organophosphate Esters-Degrading
Mixed Bacterial Cultures [18]Chlorinated OP ester-degrading enrichment cultures were obtained using TCEP or TDCPP as thesole phosphorus source. The degradation activity of each culture was assayed by measuring thedecrease of TCEP or TDCPP in each culture.
TCEP or TDCPP in cultures was extracted with an equal volume of ethyl acetate and analyzed
using a GC-17A gas chromatograph equipped with a flame photometric detector with a phosphorus
optical filter (Shimadzu, Japan). 2-Chloroethanol and 1,3-dichloro-2-propanol in cultures wereextracted in the same manner and analyzed by a GCMS-QP2010 gas chromatograph mass
spectrometer (Shimadzu). Chloride ion concentration in cultures was determined colorimetrically[20].
In cultures with 46 environmental samples, significant TCEP and TDCPP degradation wasobserved in 10 and 3 cultures, respectively, and successive subcultivation markedly increased theirdegradation rates. Stable enrichment culture 67E and 45D, obtained with TCEP and TDCPP,
respectively, completely degraded 20 μM of the corresponding compounds within 6 h and also theother compounds, although the degradation rate of TCEP by 45D was relatively slow. We confirmed
chloride ion generation in both degradation cases and the generation of 2-chloroethanol and1,3-dichloro-2-propanol as metabolites of TCEP and TDCPP, respectively.
The 67E and 45D also showed dehalogenation ability toward 2-chloroethanol and1,3-dichloro-2-propanol, respectively. Addition of inorganic phosphate did not significantly
influence their abilities to degrade the chlorinated OP esters but markedly increased thedehalogenation ability, which was on a maximum level at 0.2 mM of inorganic phosphate anddecreased at a higher concentration.
Denaturing gradient gel electrophoresis (DGGE) analysis, followed by 16S rRNA gene sequence
analysis (135-160 bp) of prominent DGGE bands, showed that dominant bacteria in 67E wererelated to Acidovorax spp. and Sphingomonas spp. and those in 45D were Acidovorax spp.,
Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of theSphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond anddehalogenation, respectively, in both cultures.
3. Isolation and Identification of Persistent Chlorinated Organophosphorus Flame
Retardant-Degrading Bacteria [19]As described above, we demonstrated that TCEP and TDCPP were rapidly degraded in twoenrichment bacterial cultures, named 67E and 45D, obtained by using TCEP and TDCPP as sole
phosphorus sources [18]. We therefore attempted to isolate bacteria that can degrade the compounds
from each enrichment culture. A single colony isolated from the enrichment culture 67E was named
strain TCM1 and that from the 45D was named TDK1. The morphological and physiologicalcharacteristics of the both strains were similar to those of Sphingomonas spp., although the twostrains were distinct from each other in physiological characteristics.

Fig. 2 SEM micrographs of Sphingobium sp. TCM1 (A) and Sphingomonas sp. TDK1 (B).
The nearly full-length 16S rRNA gene (approximately 1,500 bp) of TCM1 and TDK1 wasanalyzed and sequence homology searches were performed against the 16S rRNA gene sequence2

Transactions on GIGAKU 1(2012)01011/1-4

database at the DDBJ using the BLAST program (http://www.ddbj.nig.ac.jp/search/top-e.html). The
similarity between the sequences of TCM1 and TDK1 was 93.4%. The strain TCM1 and TDK1 weregrouped with the members belonging to the genera Sphingobium and Sphingomonas, respectively,
showing that strains TCM1 and TDK1 are members of the respective genera. The SEM micrographsof Sphingobium sp. TCM1 and Sphingomonas sp. TDK1 are shown in Fig.2.
While Sphingobium sp. TCM1 andSphingomonas sp. TDK1 degraded TCEPandTDCPP byhydrolyzing
theirphosphotriester bonds, both strains were notable to degrade the produced 2-chloroethanoland 1,3-dichloro-2-propanol, which werehydrolysis products of TCEP and TDCPP,
respectively.
Both strains could grow on othertrihaloalkyl phosphate like tris(2,3-dibromo
propyl) phosphate (TBPP) and triaryl
phosphates like triphenyl phosphate (TPP)and tricresyl phosphate (TCP) (Fig. 3). In
addition, the strain TCM1 was able to growmoderately on tributyl phosphate (TBP) andslightly on trimethyl phosphate (TMP),
tris(2-ethylhexyl) phosphate (TEHP), triethyl
phosphate (TEP) and tris(2-butoxyethyl)
phosphate (TBXP). However, the strain
TDK1 was not able to grow on all of trialkyl
phosphates tested. These results showed thatthe strains could degrade and assimilate not Fig. 3 Substrate specificity of strains TDK1 andonly the chlorinated OP flame retardants but TCM1. White and black bars show the growth ofalso several other OP flame retardants and strains TDK1 and TCM1, respectively. Growthplasticizers. In addition, the strains showed (OD660) was measured after 96 h. Data represent
different substrate specificity for trialkyl mean ± standard deviation of three independent
experiments. The initial OD660 of cultures was 0.05.
phosphates.

Acknowledgments
This work was supported in part by Grant-in-Aid for Scientific Research (B) from the Ministryof Education, Science, Sports, and Culture of Japan, and by Project to Form a Hub of Human
Resources Development and New Industry Creation-Building a Sustainable Society through HighlyInteractive Cooperative Educational Research with Pacific Rim Countries.
References[1] World Health Organization, Environmental health criteria 209 (1998).
[2] Y. Kawagoshi, I. Fukunaga, and H. Itoh, J. Mater. Cycles Waste Manage., 1 (1999) 53.
[3] E. Fries, and W. Puttmann, J. Environ. Monit., 5 (2003) 346.
[4] J. A. Andresen, A. Grundmann, and K. Bester, Sci. Total Environ. 332 (2004) 155.
[5] A. Marklund, B. Andersson, and P. Haglund, J. Environ. Monit., 7 (2005) 814.
[6] H. M. Stapleton, S. Klosterhaus, S. Eagle, J. Fuh, J. D. Meeker, A. Blum, and T. F. Webster,
Environ. Sci. Technol., 43 (2009) 7490.
[7] J. Regnery, and W. Püttmann., Clean Soil Air Water, 37 (2009) 334.
[8] Y. Kawagoshi, S. Nakamura, and I. Fukunaga, Chemosphere, 48 (2002) 219.
[9] J. Andresen, and K. Bester, Water Res., 40 (2006) 621.
[10] U.S. National Toxicology Program, Natl. Toxicol. Program Tech. Rep. Ser. 391 (1991) 1.
[11] H. B. Matthews, S. L. Eustis, and J. Haseman, Fundam. Appl. Toxicol. 20 (1993) 477.
[12] X. Ren, Y. J. Lee, H. J. Han, and I. S. Kim, Chemosphere, 74 (2008) 84.
[13] E. J. Søderlund, E. Dybing, J. A. Holme, J. K. Hongslo, E. Rivedal, T.Sanner, and S. D. Nelson,3Transactions on GIGAKU 1(2012)01011/1-4

Acta Pharmacol.Toxicol. 56 (1985) 20.
[14] F. M. Raushel, Curr. Opin. Microbiol., 5 (2002) 288.
[15] B. K. Singh, and A. Walker, FEMS Microbiol. Rev., 30 (2006) 428.
[16] B. K. Singh, Nat. Rev. Microbiol. 7 (2009) 156.
[17] K. Abe, S. Takahashi, and Y. Kera, In: Microbial Bioremediation of Non-metals; Current
Research (Ed., A. -I. Koukkou), Caister Academic Press, Norfolk, (2011) 45.
[18] S. Takahashi, K. Kawashima, M. Kawasaki, J. Kamito, Y. Endo, K. Akatsu, S. Horino, R. H.
Yamada, and Y. Kera, J. Biosc. Bioeng. 106 (2008) 27.
[19] S. Takahashi, I. Satake, I. Konuma, K. Kawashima, M. Kawasaki, S. Mori, J. Morino, J. Mori,
H. Xu, K. Abe, R. H. Yamada, and Y. Kera, Appl. Environ. Microbiol., 76 (2010) 5292.
[20] A. M. Fauzi, D. J. Hardman, and A. T. Bull, Appl. Microbiol. Biotechnol., 46 (1996) 660.
(Received: 10 May, 2012, Accepted: 30 June, 2012)4

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Electrochemical Deposition of Cobalt on Mesoporous Silica Films
Motohiro Tagaya1,*, Mitsuhiro Okuda2, Sarah Ward Jones2, Takaomi Kobayashi1
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan2)
H.H. Wills Physics Laboratory, University of Bristol,
Tyndall Avenue, Bristol BS8 1TL, United Kingdom*E-mail: tagaya@mst.nagaokaut.ac.jp

1)

Electrochemical deposition of cobalt (Co) particle on mesoporous silica(MPS) films formed on a Si wafer was examined. The MPS films weresynthesized by a supramolecular templating method to exhibit thehighly-ordered hexagonal structure and the surface area of 435 m2·(g ofsilica)-1 with the pore size of 5.0 nm. The deposited Co particles weresuccessfully formed on the surfaces at the voltage of −1.5 (V vs. SCE); thecracks appeared on the film surface at the deposition time of 100 s andsimultaneously nanoparticles were formed in the inside surface, andsubsequently amorphous nanoparticles were homogeneously covered over the
MPS surfaces at 300 s, whereas the flower-like structure was formed on the
Si wafer. These results suggest the generation of initial Co deposition in themesopores and the subsequent deposition processes were affected by theamorphous silica frameworks of MPS.

1. Introduction
The electrochemical deposition is known as an inexpensive and simple process for the formationof metallic materials [1,2]. Although it is considered that the electrochemical deposition can produce
relatively thick metallic layers, recent developments have demonstrated that the deposition is able to
make very thin metallic films and a wide range of nanostructured materials [3,4]. Thus, theinvestigation of the deposition process on the nanostructured materials is of great importance fordesigning the dissimilar interface.
Mesoporous silicas (MPSs) were prepared by the cooperative organization of surfactant andinorganic species, the synthesis, characterization and applications of the nanostructues have been
investigated [5,6]. The MPSs prepared by supramolecular templating methods possess attractive
features such as well-defined and controllable pore size, large surface area, and reactive surfaces[7,8]. The host–guest and guest–guest interactions based on the mesopore size can be controlled, andthe host–guest complexes have been synthesized, and the possible applications have been reported so
far [9‒17]. Accordingly, the incorporation of functional species into the silica mesopore
encompasses wide variety applications. Thus, the formation of the metal−MPS composites is ofgreat importance for controlling the metal particles at the nanometer scales.
In this study, the MPS films with the pore diameter of 5.0 nm were prepared on a Si wafer by thesupramolecular templating method, and the electrochemical deposition process of cobalt (Co)particles from the aqueous solution containing Co(II) sulfate hydrate and boric acid wasinvestigated.
2. Experiment
Preparation of MPS Films
MPS films were synthesized according to the previous reports [12,18]. 2.0 g (13.1 mmol) of
tetramethoxysilane (TMOS: Shinetsu Chemical Co., Ltd.), 472 L of ultrapure water, and 100 L ofhydrochloric acid aqueous solution (HCl: Wako Co., Ltd.) (0.1 M) were vigorously stirred at 40oC
for 60 min to generate condensation polymerization. Then, 534 mg (92 mmol) of poly(ethyleneoxide)20–poly(propylene oxide)70–poly(ethylene oxide)20 (P123: Sigma-Aldrich Co., Ltd.,
1

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Mw=5800) was dissolved in 2.6 mL of ultrapure water and the solution was added into the siliceous
solution at the molar ratio of TMOS:P123=1:7×10-3, and then stirred at 40oC for 15 min. The
resulting solution was spin-coated on a glass substrate and naturally-oxidized silicon (Si) wafer(boron-doped p-type Si(100), Ryoukou Sangyou Co., Ltd.) at the rotate speed of 6000 rpm for 10 s,
and then dried at 60oC for 18 h. The template was removed by the calcination at 450 oC for 6 h under
air.
Electrochemical Deposition of Co
The solution containing 0.1 M of Co(II) sulfate hydrate (CoSO4: Aldrich Co., Ltd.) and 0.5 M ofboric acid (H3BO3: Sigma-Aldrich Co., Ltd.) using ultrapure water was prepared. 60 ml of thesolution was added into the electrochemical cell and then the dissolved oxygen molecules were
removed by nitrogen bubbling for 15 min. In the working electrode (WE), an electrical contact wasmade to the back of the Si wafer using a GaIn eutectic and a piece of Cu foil as shown in Figure 1(a). The wafer and contact were covered with Kapton tape except for a 6 mm diameter circle on the
MPS film surface, giving an area of 0.28 cm2 of the MPS film exposed to the solution. A standard
three electrode electrochemical cell set-up with a saturated calomel electrode (SCE) as a referenceelectrode (RE) and a platinum foil counter electrode (CE) was used for the electrochemical
deposition as shown in Figure 1 (b). A SP-150 potentiostat (Bio-Logic Co., Ltd.) controlled using
EC-Lab Express software was used to control the potential and record the current by completely
shielding the light reaching the electrode. The cyclic voltammetry curve was measured on thecondition (scan rate: 50 mV/s, start potential: 0 (V vs SCE), first switching potential: −1.2 (V vsSCE), second switching potential: +1 (V vs SCE), final potential: 0 (V vs SCE)). The current
measurement with the deposition time was conducted at the fixed voltage of −1.5 (V vs. SCE).

Fig. 1 Photographs of the experiment set-up for (a) WE made of the MPS on Si wafer with Cu foil
and Kapton tape and (b) the standard three electrode cells with WE, RE and CE in the solution.
Characterization
X-ray diffraction (XRD) patterns were recorded on a X’Pert Pro MPD (PNAalytical Co., Ltd.)
using monochromatized CuK radiation. Infrared spectra were recorded on a fourier transform
infrared spectrometer (FT-IR: PerkinElmer Inc., Spectrum GX) with an accumulation of 32 times
and resolution of 2.0 cm−1. Nitrogen adsorption isotherm was measured at 77K on a SA3100
instrument (BECHMAN COULTER Co., Ltd.). Prior to the measurement, the samples weredegassed under vacuum at 393K for 4 h. The surface area was evaluated by the BET surface area,
and the pore volume was calculated by the BET surface area and averaged pore size. The
morphologies of the surfaces were observed using a field emission scanning electron microscopy(FE-SEM: Hitachi Co., Ltd., S-4500N). The MPS films on the glass substrates were applied for the
XRD, FT-IR and nitrogen adsorption/desorption measurements, and those on the Si wafers were forthe electrochemical experiments.
3. Results and Discussion
Figure 2 (A) shows the XRD patterns of the MPS films before and after the calcination and theillustration of possible mesoporous hexagonal structure. The peaks are indexed as (100), (110), (200)and (220) reflections of hexagonal structure as shown in Figure 2 (B), and the d100 values are 6.3 nm
2Transactions on GIGAKU 1(2012)01012/1-6

and 5.2 nm before and after the calcinations in Figure 2 (a, b). With the calcination, the diffractions
were shifted to higher angles due to the contraction of the silica framework. The results suggest thatthe highly-ordered nanostructures are preserved with the calcination. FT-IR spectra indicated that the
bands at around 1070 and 1225 cm−1 are originated from Si–O–Si asymmetric and symmetricstretching and the bands at around 795 and 960 cm−1 are assigned to the Si–OH stretching (data notshown). The bands at around 1480, 2854, and 2925 cm−1 which can be assigned to the C–Hstretching and bending in the surfactant completely disappeared by the calcination, indicating theremoval of the P123 surfactant. Therefore, the MPS with the hexagonal structure was successfully
prepared by the supramolecular templating method.

(A)

(B)
Nanopore
(110)
(200)

Intensity / a.u.

Intensity / a.u.

(100)

(c)

d200

(110)

(100)

(200)
1

1.5

(a)

2

2.5

3

3.5

4

4.5

2(CuK) / degree

d100

(d)
5

d110

Wall

(b)
2

4

6

2(CuK) / degree

810Fig. 2 (A): XRD patterns of the MPS films (a, c) before and (b, d) after the calcination and (inset)
the magnified patterns at the lower angles. (B): Illustration of hexagonally-aligned mesoporus
structure.

5 mm

500

Pore size distribution / cm3g-1nm-1(a)Adsorbed volume / ml (S.T.P.) (g of Silica)-1

Figure 3 (a) shows the photograph of the MPS film after the calcination. The film thickness is150−200 nm and exhibits high transparency. Figure 3 (b) shows the nitrogen adsorption isotherm of
the MPS film and the pore size distribution. A hysteresis between the adsorption and desorption wasobserved, indicating the formation of mesopores. The isotherms indicated that the BET surface area,
pore size and volume are 435 m2·(g silica)−1, 5.0 nm and 0.54 ml·g-1. Taking into account the d100
value evaluated from the XRD pattern, the thickness of the silica pore wall is 0.2 nm.

(b)
400

300

200

5.0nm

3

4

5 6 7 8 9 10
Pore size / nm

100

0

0

0.2

0.4

0.6

Relative Pressure / P P0

0.8

1

-1

Fig. 3 (a) Photograph of the MPS film on a glass substrate and (b) the nitrogen adsorption isothermand (inset) pore size distribution.
3

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Figure 4 shows the CV curve of the MPS film on the Si wafer and the current changes with thedeposition time in the preparative solution. In the CV curve in Figure 4 (a), the negative currents
induce the Co deposition and hydrogen evolution, while the positive current peak at the peak of +0.4
(V vs. SCE) corresponds to the stripping (oxidation) of Co off the electrode.
When this negative potential is applied to WE, divalent metal ions of Co2+ surrounded by their
hydration layers move towards the cathode and are reduced to the metal. The movement rate of ionsin a given electric field E depends on two factors, namely, the mobility of the ions and the potential
gradient across the WE and CE,(1)
V+ = μ+ dE/dx
V− = μ− dE/dx(2)
where the V+ and V− are the movement rates of Co cations and sulphate anions, respectively, and theμ+ and μ− are the mobilities of Co and sulphate ions, respectively. Here, the components V|| and V⊥of the metal ion movement rate (V+), and the competition between them are the key parameters
determining the resultant geometry after electrodeposition. During the electrodeposition process,Co2+ in CoSO4 precursor are surrounded by a hydration shell. This hydration layer will effectively
reduce the metal ion mobility, and the components of metal ion velocity become almost equal (V|| ≈V⊥) [26]. Therefore, the rate of reduction of metal ions would become equal in both directions,
resulting in the homogeneous Co growth in the mesopores and on the film surfaces.
At the fixed voltage, the current signal changes with the time are measured as shown in Figure
4 (b), indicating the critical point at around 30 s. The saturated part of the current transient indicatesthe production of Co on the MPS film surfaces. This indicates the successful Co deposition on the
MPS film surfaces.
0

0

(a)

(b)

-20

Current / A

Current / A

2

-2
-4
-6

-40
-60
-80

-100

-8

-120

-10

-140
-1

-0.5

0

0.5

0

1

Applied voltage / (V vs. SCE)

50

100

150

200

250

300

Time / sFig. 4 CV curve of the MPS film on the Si wafer and the current changes with the deposition time at
the fixed voltage of −1.5 (V vs. SCE).
Figure 5 shows the FE-SEM surface images of the MPS films and Si wafers after the deposition.
Before the deposition, the MPS surface shows the significant flat surface at the several nanometerscales as shown in Figure 5 (a). At the deposition time of 100 s, the cracks at the width of 10−20 mwere clearly formed on the MPS surface. N2 adsorption isotherm of the MPS film decreased with thedeposition and didn’t show the mesopores. These results indicate that the cracks are pressed up by
the Co deposition into mesopores. It has been reported that the metal growth during
electrodeposition in porous materials originates from the cathode surface at the bottom edge of thepore [26]. Thus, the high surface area and presence of sites with low coordination number in the
MPS surfaces would afford energetically favorable sites for initiating metal adsorption during
electrodeposition.
At the deposition time of 300 s, the cracks and particle-covered surfaces were clearly observedon the MPS surface and the average particle size is several ten nanometers, suggesting the formationof amorphous particles on the surfaces. At the deposition time of 900 s, the cracks andparticle-aggregated surfaces were observed on the MPS surface and the aggregated particles with thediameter of several micrometer sizes were formed on the surface. From the XRD analysis at the 2
range from 40o to 80o, the deposited particles were amorphous structure. At the deposition time of
900 s on the Si wafer, the flower-like particles were homogeneously formed on the surface and themorphology is clearly different from that on the MPS surface.
4

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The electrodeposition is capable of depositing materials in extremely confined and ordered
spaces which exist in porous media called template. Possin [19] has reported the fabrication of wires
as small as 40 nm in diameter with a method involving electroplating into etched particle tracks in
mica. Williams and Giordano [20] refined this technique and used it to routinely produce wires as
small as 8.0 nm. Recent researches for the method were pioneered by Martin et al. [21−25],
introduced template synthesis for the fabrication of one dimensional nanostructures (i.e. nanowires).
This method entails using the nanoscopic pores in a host membrane as templates to preparemonodisperse nanoscopic ultra-fine structures of a desired material. The mesoporous templates canbe used as an insulating host material which encompasses cylindrical pores with diameters innanometer range. The template fabrication methods generally use prefabricated cylindrical
nanopores in an inorganic solid material as templates. By filling the desirable metals into themesopores, the functional nanocomposites with the predetermined diameter can be fabricated. In thisstudy, the highly-ordered hexagonal structure would induce the outstanding cooperative phenomenathat clearly differ from the bulk and even from their thin film counterparts. The smaller pore with thesize of 5.0 nm that was homogeneously formed on the transparent film surface effectively affects thedeposition process, and the nanocomposites will be applicable for the optical and electronic devices.
(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Fig. 5 FE-SEM surface images of the MPS films deposited for (a) 0 s, (b, c) 100 s, (d, e) 300 s, (f, g) 900
s and the (h, i) Si wafers deposited for 900 s.

4. Conclusion
Electrochemical deposition on MPS films formed on a Si wafer was investigated. The MPS films
were synthesized by the supramolecular templating method to exhibit the highly-ordered hexagonal
structure and surface area of 435 m2·(g of silica)-1. The deposited Co particles were successfully
formed on the surfaces; the cracks were generated on the film at 100 s and subsequently thenanoparticles were homogeneously covered on the surface at 300 s, whereas the plate-like structure5Transactions on GIGAKU 1(2012)01012/1-6

was formed on the Si wafer. Thus, the ordered structures induce the outstanding cooperative
phenomena that clearly differ from the bulk. These results suggest the initial deposition in themesopores and the subsequent deposition reflected by the framework amorphous silica wall surfacesof the MPS. Therefore, the deposited MPS films will be promising candidates for nanoscopic
electrodes in applied electrochemistry and for various other fundamental studies.
Acknowledgments
This study was supported by a grant from the Japan Society for the Promotion of Science(JSPS). The authors thank Prof. Dr. Walther Schwarzacher and Prof. Dr. Natasa Vasiljevic for many
helpful discussions and Dr. Stuart Bellamy and Mr. Fred Hale for providing the facilities ofNanoscience and Quantum Information (NSQI) at Bristol University.
References[1] W. Schwarzacher, J. Phys.: Condens. Matter., 16 (2004) R859.
[2] W. Schwarzacher, Electrochem. Soc. Interf., 15 (2006) 32.
[3] U. Erb, Nanostructured Mater., 6 (1995)533.
[4] S. C. Tjong, and H. Chen, Mater. Sci. Eng., R 45 (2004) 1.
[5] T. Yanagisawa, T. Shimizu, K. Kuroda, and C. Kato, Bull. Chem. Soc. Jpn., 63 (1990) 988.
[6] C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli, and J. S. Beck, Nature, 359 (1992)
710.
[7] R. I. Nooney, D. Thirunavukkarasu, Y. Chen, R. Josephs, and A.E. Ostafin, Chem. Mater., 14(2002) 4721.
[8] M. Ogawa, K. Kato, and N. Shimura, Bull. Chem. Soc. Jpn., 82 (2009) 121.
[9] K. B. Yoon, Chem. Rev., 93 (1993) 321.
[10] K. Moller, and T. Bein, Chem. Mater., 10 (1998) 2950.
[11] M. Ogawa, Ann. Rep. (Sec. C), 94 (1998) 209.
[12] M.Ogawa, H. Ishikawa, and T. Kikuchi, J. Mater. Chem., 8 (1998) 1783.
[13] A. Stein, B. J. Melde, and R. C. Schroden, Adv. Mater., 12 (2000) 1403.
[14] B.J. Scott, G. Wirnsberger, and G. D. Stucky, Chem. Mater., 13 (2001) 3140.
[15] M. Ogawa, J. Photochem. Photobiol. C, Photochem. Rev., 3 (2002) 129.
[16] M. Tagaya, and M. Ogawa, Phys. Chem. Chem. Phys., 10 (2008) 6849.
[17] M. Tagaya, and M. Ogaawa, Chem. Lett., 35 (2006) 108.
[18] D. Zhao, Q. Huo, J. Feng, B. F. Chmelka, and G. D. Stucky, J. Am. Chem. Soc., 120 (1998)
6024.
[19] G. E. Possin, Review of Scientific Instruments, 41 (1970) 772.
[20] W. D. Williams, and N. Giordano, Review of Scientific Instruments, 55 (1984) 410.
[21] R. M. Penner, and C. R. Martin, Anal. Chem., 59 (1987) 2625.
[22] C. J. Brumlik, and C. R. Martin, J. Am. Chem. Soc., 113 (1991) 3174.
[23] C. R. Martin, Science, 266 (1994) 1961.
[24] C. R. Martin, Chem. Mater., 8 (1996) 1739.
[25] S. A. Sapp, B. B. Lakshmi, and C. R. Martin, Adv. Mater., 11 (1999) 402.
[26] T. N. Narayanan, M. M. Shaijumon, P. M. Ajayan, and M. R.Anantharaman, J. Phys. Chem. C,
112 (2008) 14281.
(Received: 26 April, 2012, Accepted: 18 June, 2012)6

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Grafting Effects of Fe(III) on Photocatalytic Activities
of WO3 and BiVO4 on Visible-Light Irradiation
Masami Nishikawa, Souta Hiura, Yasufumi Mitani, Yoshio Nosaka*
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka, Nagaoka 940-2188, Japan*E-mail: nosaka@nagaokaut.ac.jp
The grafting effects of Fe(III) on the photocatalytic activities of narrow
bandgap (≤ 2.5 eV) oxide semiconductors of WO3 and BiVO4 were examined.
The photocatalytic activities measured on visible-light irradiation for bothoxides were increased by grafting Fe ions. Moreover, it was revealed, bymeans of chemiluminescence photometry, that the electrons in the valenceband transfer to the grafted Fe ions and then the energized Fe ions can reduce
O2 into H2O2. This charge transfer would suppress electron-hole
recombination, leading to the raise of the photocatalytic activities of WO3 andBiVO4.

1. Introduction
Photocatalytic materials have been used in wide application such as air purification, water
purification, anti-pollutant and anti-virus [1,2]. Photocatalytic reaction proceeds owing toholes and electrons generated in materials by absorbing light energy. The photogenerated
holes have oxidation ability and the photogenerated electrons have reduction ability. To
decompose pollutants of organic compounds by photocatalytic reaction, the oxidation
potential of hole needs to be more positive than +1 V that is redox potential of general organic
compounds. In addition, the redox potential of electrons needs to be more negative than that ofoxygen (O2/O2- or H2O2) because surplus electrons that are not consumed by reduction ofoxygen would result in electron-hole recombination that is one of the factors in decreasing
photocatalytic activity. The most suitable material for photocatalyst, which also meets these
conditions, is TiO2. The bandgaps of rutile and anatase type TiO2 are 3.0 and 3.2 eV,
respectively. The potentials of their conduction bands are negative enough to reduce oxygen
into O2- by one-electron reduction and the potentials of their valence bands are much positive
enough to decompose organic compounds. However, it is the serious problem thatphotocatalytic reaction proceeds only under ultraviolet (UV) light irradiation. The content
ratio of UV light in solar spectrum is only 5 % and moreover those in fluorescent lights andwhite light emitting diodes are much less. To raise photocatalytic activity and use
photocatalysts indoors, it is indispensable to develop their visible light responses. To achieve
the purposes, narrowing of the bandgap of TiO2 by doping elements such as N, S, and metal
ions was reported to be effective [3-5]. Among them, N-doped TiO2 showed highphotocatalytic activity under visible light irradiation. It is considered that the level of orbitals
assigned to N atoms would be formed above the level of valence band assigned to O atoms
and accordingly the oxidation ability seems to be decreased. Recently, it was reported that theTiO2 grafted by Fe (III) or Cu (II) ions showed higher photocatalytic activity under visible
light irradiation than N-doped TiO2 [6,7]. The Fe or Cu was considered to be amorphous
FeOOH or CuO clusters, respectively. When the Fe or Cu grafted TiO2 (Fe/TiO2, Cu/TiO2)
was irradiated by visible light irradiation, electrons in the valence band of TiO2 would be
excited to grafted Fe or Cu ions. Thus it has been considered that the direct electron transferfrom valence band of TiO2 to Fe or Cu ions is the origin of visible light response [8,9].
Oxide semiconductors with narrow bandgap of ≤2.5 eV have a potential for
visible-light-driven photocatalyst as well as TiO2. In general, the quantum efficiency, however,is lowered due to high probability of electron-hole recombination with narrowing the bandgap.
In the case of WO3 whose bandgap is 2.5 eV, the valence band level is almost the same as1

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TiO2, while the conduction band level is much lower than that of TiO2 and then just able toreduce oxygen to H2O2 by two-electron reduction. Since the rate of two-electron reduction ofoxygen would be low, resulting in the recombination of electron-hole [10], WO3 alone wasconsidered to show the low quantum efficiency under visible light irradiation. When Pt
nanoparticles were deposited on the surface of WO3, the photocatalytic activity was raised
because the Pt nanoparticles play a role as a promoter of the two-electron reduction process[10]. Additionally, it was reported that the photocatalytic activity of WO3 was increased bygrafting Cu (II) and Fe (III) [7,8]. Unlike Pt nanoparticles, the Fe and Cu ions are desirable inaspects of cost and resources. However, especially, the reaction mechanisms of Fe(III) graftedWO3 (Fe/WO3) was not clear. BiVO4 which is 2.4 eV bandgap in monoclinic form has also
been reported for visible-light-driven photocatalyst for O2 evolution by oxidation of H2O [11],but, the photocatalytic activity in gas phase is not clear. Moreover, the grafting effects of Cu(II) and Fe (III) on photocatalytic activity of BiVO3 have not be reported yet. Therefore, it isimportant to examine the grafting effects of Fe (III) on photocatalytic activities of WO3 andBiVO4 for practical applications.
In this paper, the grafting effects of Fe(III) on the photocatalytic activities of WO3 andBiVO4 were examined compared to TiO2. Moreover, the photocatalytic reaction mechanism of
Fe(III) grafted WO3 and BiVO4 (Fe/BiVO4) was successfully elucidated usingchemiluminescence photometry.

2. Experimental
Materials
Fe/TiO2, Fe/WO3 and Fe/BiVO4 photocatalysts were prepared by impregnation method. 10
mL of distilled water was added to a mixture of FeCl3·6H2O and 1 g of TiO2 (MT-150A,
TAYCA), WO3 (Nacalai Tesque) or BiVO4 (Alfa Aesar), respectively. FeCl3·6H2O wasweighted so that the weight fractions of Fe relative to each photocatalyst was 0.1wt%. Eachmixed solution was heated at 90°C for 1 h under stirring. Then the solution was centrifuged
and then the solid powder was sufficiently washed with distilled water. The obtained
photocatalysts were dried at 110°C for 24 h.
Photocatalytic activity
The photocatalytic activity was measured for the decomposition of acetaldehyde as follows.
Photocatalyst powder of 0.15 g was spread on a Petri dish of 4 cm diameter and then the dishwas placed in the circulated reactor with a volume of 260 mL which was filled with mixed gasof O2/N2=1:4. The acetaldehyde gas of 50 L and water of 10 L were injected into the reactor.
The dish placed in the reactor was irradiated by a set of LED (light emitting diode) of awavelength of =470 nm at the intensity of 20 mW/cm2, and the amount of generated CO2
was monitored by a gas analyzer (LI-COR, LI-840) equipped in the circulation reactor system.
Luminol chemiluminescence probe methods
The generation of O2- was observed by using a luminol chemiluminescence probe method.
[12] Photocatalyst of 15 mg was added in 0.01 M NaOH solution of 3.5 mL in a quartz cell (1cm × 1 cm) and then the suspension was irradiated by a He-Cd laser (KIMMON, IK5652R-G)
at =442 nm with 14 mW. After the irradiation, 50 L of 7 mM luminol solution was
immediately added in the suspension. The chemiluminescence intensity was measured using aPeltier-cooled photon-counter head (Hamamatsu Photonics, H7421). For the measurements of
H2O2, the photocatalyst suspension was irradiated by the LED and then was kept in dark toeliminate O2-. 50 L of 7 mM luminol solution was added in the suspension and then thesuspension was kept in dark for 10 min. 50 L of hemoglobin (Hb) solution was added in thesuspension and then the chemiluminescence intensity was measured. To convert thechemiluminescence intensities to the absolute concentration of O2- and H2O2, the apparatus
factor was calculated from the experiment where all luminol molecules are consumed for the2Transactions on GIGAKU 1(2012)01013/1-6

reaction with an excess amount of H2O2. The integrated number of photons was proportional
to the luminol concentration. From the slope, the observed chemiluminescence intensity canbe converted to H2O2 concentration as mentioned in detail in the previous report [12].
3. Results and discussion
Figure 1 shows the amounts of CO2 generated by the decomposition of gaseousacetaldehyde for TiO2, Fe/TiO2, WO3, Fe/WO3, BiVO4 and Fe/BiVO4 on the irradiation ofvisible light (470-nm LED) for 1 h. In the case of TiO2, CO2 generated by decomposition of
acetaldehyde was not detected for the bare TiO2. This was due to lack of interband excitation
because the bandgap of rutile TiO2 is 3.0 eV, corresponding to =413 nm. In contrast, theFe/TiO2 showed a decomposition ability of 80 ppm/h in CO2 generation. This visible response
was due to the direct charge transfer form valence band of TiO2 to Fe3+ grafted on the TiO2
surface as reported previously [9]. At the same time, acetaldehyde was oxidatively
decomposed by holes in the valence band of TiO2. In the case of WO3, the bare WO3 alone
showed a decomposition ability of 170 ppm/h. This decomposition ability was due tointerband excitation because the bandgap of WO3 is 2.5 eV, corresponding to =496 nm. In
the Fe/WO3, the generation amount of CO2 was increased to 209 ppm/h. In the previous report,
the photocatalytic activity of Cu(II) grated WO3 was also higher than the bare WO3 [8]. It wasconsidered that some electrons in conduction band from valence band would transfer to thegrafted Cu2+, leading to the raise of the photocatalytic activity because electron-hole
recombination was suppressed. Based on this consideration, the Fe ions in Fe/WO3 also would
play an important role for charge separation. In the case of BiVO4, the similar tendency to
WO3 was shown. The bare BiVO4 alone showed a little decomposition ability of 3 ppm/h.
Since the bandgap of BiVO4 is 2.4 eV (=516 nm), interband excitation should occur.
However, the decomposition ability was much smaller than WO3. Therefore, it was consideredthat the probability of electron-hole recombination of the used BiVO4 was high compared to
WO3. In the Fe/BiVO3, the decomposition ability was largely increased to 49 ppm/h. The
result indicates that the Fe ions in Fe/BiVO4 also would play an important role for charge
separation. Moreover, for the photocatalysts with little photocatalytic activity such as BiVO4
under visible light irradiation, it was revealed that the grafting of Fe ions is more effective toincrease their photocatalytic activities.
Fe/WO3CO2 (ppm/h)

200Bare WO3

150
100

Fe/TiO2
Fe/BiVO450
0Fig. 1

Bare BiVO4

Bare TiO2
TiO2

WO3

BiVO4

The amounts of CO2 generated for 1 h by the decomposition of
gaseous acetaldehyde for photocatalysts under visible-lightirradiation.

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To elucidate the photocatalytic reaction mechanisms of Fe/WO3 and Fe/BiVO4, the abilitiesof their excited electrons were examined for the reduction of molecular oxygen. Reduction ofoxygen is a key reaction because photocatalytic reactions cannot proceed when thephotogenerated electrons do not react with oxygen which is the only molecule to be reduced inambient atmosphere. In the Fe/TiO2, it has been already confirmed by means ofchemiluminescence photometry that the excited Fe2+ ions reduced oxygen into H2O2 by
two-electron reduction [9]. Figure 2 shows the amounts of O2- and H2O2 generated by
reduction of oxygen by excited electrons under visible light irradiation with the 442-nm
He-Cd laser. In the bare WO3, H2O2 was detected while O2- was not detected. This reason isthat the conduction level of WO3 is +0.55 V (vs. SHE at pH = 0), which is more positive than
the redox potential of (O2,H+)/ HO2, +0.46 V (vs. SHE at pH = 0) and is more negative than
the redox potential of (O2,2H+)/H2O2, +0.695 V (vs. SHE at pH = 0). In the Fe/WO3, theamount of generated H2O2 was increased. The redox potential of Fe3+/Fe2+ is +0.77 V (vs.
SHE at pH = 0), which is located under the conduction band of WO3. It is possible thatelectrons transfer to the grafted Fe ions by direct electron transfer from valence band withinterfacial charge transfer absorption or indirect charge transfer from valence band via
conduction band (valence band → conduction band → Fe ions). In the case of the TiO2,
under visible light irradiation, the direct charge transfer form valence band of TiO2 to grafted
Fe ions occur and under UV light irradiation, some electrons excited in conduction band of
TiO2 by interband excitation transfer to grafted Fe ions [9]. From this report, it was concluded
that the increase of H2O2 in Fe/WO3 was due to the electrons excited to Fe ions as well as the
Fe/TiO2. In the case of the bare BiVO4, O2- was mainly detected while H2O2 was not almost
detected. The conduction band level of BiVO4 is 0.0 V (vs. SHE at pH = 0), which is negative
enough to reduce O2 into O2-. In contrast, in the Fe/BiVO4, the amount of O2- was decreased
while that of H2O2 was increased. These results indicated that charge transfer to Fe ions would
occur because excited Fe ions have only ability of two-electron reduction. The amount of
H2O2 seems to be somewhat small. This indicates that generated H2O2 would be oxidized into
H2O by holes.
20

(a)

250

O 2- concentration (nM)

O 2- concentration (nM)

300

WO3
Fe/WO3

200
150
100
50
0
0

20

40

60

80

100

(c)

10
5
0
0

120

BiVO4
Fe/BiVO4

15

20

250
200

(b)

H2 O2 concentration (nM)

H2 O2 concentration (nM)

Irradiation time (s)300

WO3
Fe/WO3

150
100
50
0
0

Fig. 220

40
60
80 100
Irradiation time (s)

120

20

40
60
80 100
Irradiation time (s)

120

BiVO4
Fe/BiVO4

(d)
15
10
5
0
0

20

40
60
80 100
Irradiation time (s)120The amounts of (a) O2- and (b) H2O2 generated for WO3 and Fe/WO3
photocatalysts and (c) O2- and (d) H2O2 generated for BiVO4 and Fe/BiVO4photocatalysts as a function of the irradiation time at 442 nm.
4Transactions on GIGAKU 1(2012)01013/1-6

Based on the results, the plausible reaction mechanisms of Fe/WO3 and Fe/BiVO4photocatalysts were summarized in Fig. 3. Compared to WO3, BiVO4 has valence band
positioned at more negative. Therefore, the oxidation ability of BiVO4 against organic
compounds such as acetaldehyde is low as seen in Fig. 1. In this case, the remained holes invalence band, which can not react with organic compounds, might diminish by recombination
with electrons. When both photocatalysts of WO3 and BiVO4 are grafted Fe ions on theirsurface, electrons in their balance bands are photo-excited to the grafted Fe ions under visible
light irradiation. In addition, in the case of BiVO4, electrons are excited to their conduction
band and then transfer to the Fe ions. Although it is not clear which electron transfer proceed,in either electron transfer processes, electrons transfer to the grafted Fe ions. By transfer of
excited electrons to the grafted Fe ions, holes photo-generated in WO3 or BiVO4 and areseparated from electrons, leading to a low probability of electron-hole recombination.
Therefore, the photocatalytic activities of WO3 and BiVO4 were raised by grafting Fe ions.
Since the Fe ions play an important role for charge separation, for the photocatalysts with little
photocatalytic activity such as BiVO4, it was considered that the grafting of Fe ions is more
effective to increase their photocatalytic activities.

Fig. 3The photocatalytic mechanisms of (a) Fe/WO3 and (b) Fe/BiVO4.

4. Conclusions
The grafting effects of Fe(III) on the photocatalytic activities of narrow bandgap oxide
semiconductors of WO3 and BiVO4 were examined. The photocatalytic activities of bothoxides were increased by grafting Fe ions. Especially, for the BiVO4 of little photocatalytic
activity, the grafting of Fe ions was revealed to be more effective to increase its photocatalytic
activities. Moreover, the reductive ability of the excited electrons was successfully examinedby means of chemiluminescence photometry. As a result, it was revealed that electronstransfer to the grafted Fe ions by visible light irradiation and the excited Fe ions (Fe2+) have a
reduction ability of oxygen into H2O2. This charge transfer to Fe ions on the surfaces of thephotocatalysts would suppress electron-hole recombination because holes remain in thephotocatalysts, which would be leading to the raise of the photocatalytic activity of WO3 andBiVO4.

5

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References[1] A. Fujishima, X. Zhang, and D. Tryk, Surf. Sci. Rep., 63 (2008) 515.
[2] M. Anpo and P.V. Kamat, Eds. Environmentally Benign Photocatalysis- Application of
Titanium Dioxide-based Materials; Springer: New York, 2010.
[3] R. Asahi, T. Morikawa, T. Ohwaki, K. Aoki, and Y. Taga, Science, 293 (2001) 269.
[4] T. Umebayashi, T. Yamaki, H. Ito, and K. Asahi, Appl. Phys. Lett., 81 (2002) 454.
[5] Y. Murakami, B. Kasahara, and Y. Nosaka, Chem. Lett., 36 (2007) 330.
[6] H. Irie, K. Kamiya, T. Shibanuma, S. Miura, D.A. Tryk, T. Yokoyama, and K. Hashimoto,J. Phys. Chem. C, 113 (2009)10761.
[7] H. Yu, H. Irie, Y. Shimodaira, Y. Hosogi, Y. Kuroda, M. Miyauchi, and K. Hashimoto, J.
Phys. Chem. C, 114 (2010) 16481.
[8] Y. Nosaka, S. Takahashi, H. Sakamoto, and A. Nosaka, J. Phys. Chem. C, 115 (2011)
21283.
[9] M. Nishikawa, Y. Mitani, and Y. Nosaka, J. Phys. Chem. C, 2011, submitted.
[10] R. Abe, K. Sasaki, H. Kunioku, and B. Ohtani, J. Am. Chem. Soc., 130 (2008) 7780.
[11] A. Kudo, K. Omori, and H. Kato, J. Am. Chem. Soc., 121 (1999) 11459.
[12] T. Hirakawa and Nosaka, J. Phys. Chem. C, 112 (2008) 15818.
(Received: 14 May, 2012, Accepted: 19 June, 2012)6

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In Vitro Targeting of Cancer Cells
with Luminescent Nanoporous Silica Spheres
Motohiro Tagaya1,*, Junzo Tanaka2, Nobutaka Hanagata3, Toshiyuki Ikoma2,
Taro Takemura3, Kota Shiba4, Takaomi Kobayashi1
1)Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan2)
Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology,
O-okayama 2-12-1, Meguro-ku, Tokyo 152-8550, Japan
3)
Nanotechnology Innovation Station, National Institute for Materials Science (NIMS),
1-2-1 Sengen, Tsukuba, Ibaraki 305-004, Japan4)
WPI Center for Materials Nanoarchitectonics, National Institute for Materials Science (NIMS)
Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan*E-mail: tagaya@mst.nagaokaut.ac.jp
The luminescent europium(III)-doped nanoporous silica spheres (Eu:NPS)
were successfully synthesized by a supramolecular templating method. Folicacid (FA) as the targeting ligand for cancer cells was immobilized on the
Eu:NPS surface through a mediation of 3-aminopropyltriethoxysilane(APTES). The ordered nanopores were preserved with the APTES and FA
immobilization. The surface area decreased only with the APTES
immobilization, and the FA was immobilized only on the outer surface ofpores. The photoluminescence of the immobilized spheres showed thecharacteristic luminescence peak due to interactions between the FA and Eu3+,
and the luminescence can be detected by a fluorescent microscopy. The
immobilized spheres were highly dispersed stably in the cell culture medium
to bind specifically to cancer cells and exhibit the luminescence. Thus, the FA
was successfully immobilized on the luminescent Eu:NPS spheres to exhibit
the highly targeting ability to the cancer cells.

1. Introduction
The designing of superior targeting nanomaterials against specific cells is important for use in
the biomedical science field. The targeting to cells by specific ligands or utilizing antibodies depends
on the ability of the targeting reagents to bind selectively to the cell surface and to trigger
subsequently receptor-mediated endocytosis [1]. The challenges for combining the imaging withtargeting into one system are desired using a nanoparticle. However, the coupling of multifunctionalgroups in sufficient concentration on the particle surface is difficult because of the limited number ofattachment sites.
Nanoporous silica (NPS) prepared by the supramolecular templating method possesses attractive
features such as a well-defined and controllable pore size, large surface area, and reactive surface[2,3]. The large immobilization capacity for various functional molecules is one of the many
attractive features of NPS, accompanied by controlling the nanostructural properties on a nanometerscale such as pore and particle sizes and framework structures [4-11]. The preparation of silica-based
biohybrid and the NPS spheres having therapeutic or imaging properties for bio-functions have been
recently reported [12-17]. The drug delivery systems that have been synthesized to combine thetwo-functional therapeutic and imaging moieties on the surfaces have been reported using afluorescent dye covalently linked to the surface, making them visible by fluorescence microscopy[18,19]. If the external surface and silica framework in NPS are functionalized as the targeting andimaging, respectively, the internal pore surfaces can be used for therapeutic medicine. However, thecombination of the functions (therapeutic, imaging and targeting) into one particle has not yet been
investigated in detail.
1Transactions on GIGAKU 1(2012)01014/1-8

In this study, folic acid (FA) molecules, which have been known as the targeting reagents forcancer cells [20], were covalently immobilized on luminescent Eu3+-doped NPS (Eu:NPS) spheresby mediation of the 3-aminopropyltriethoxysilane (APTES) adlayer in Scheme 1 (a), and thetargeting behavior of the spheres to the cancer cells was examined as compared with that to thenormal cells (fibroblast cells) in Scheme 1 (b).
(a)

N
H

OH
N

(b)

H
N

N

Cancer cell

NH
O

FA

N
H

N
H2N

CO2H

O

OH

N

Si
HO

O

Binding
Uptake

Eu:NPS

FA‐Eu:NPS

Scheme 1 Schemes of (a) the FA immobilization on Eu:NPS spheres and (b) the targeting of theFA-immobilized Eu:NPS to cancer cells through a receptor-mediated endocytosis.
2. Experiment
Synthesis of Eu:NPS. Eu: NPS spheres were synthesized according to our previous reports [14].
1.0 g of CTAB, 225 g of ultrapure water and 3.5 mL of 2.0 M NaOH solution were stirred. Then,
5.515 mL of TEOS and 15 mL of Eu3+ aqueus solution containing 0.226 g, 0.452 g and 0.904 g of
EuCl3·6H2O (Kanto Chemical Co., Ltd.) at the molar concentration of the Eu3+ to Si (Eu/Si) of 1/40,
1/20 and 1/10 were stirred at 80oC for 2 h. The solution was filtered, and the resulting solids were
washed, vaccuumed for 4 h, and calcined at 550oC for 4 h, which were abriviated as 2.5-Eu:NPS,
5.0-Eu:NPS and 10.0-Eu:NPS for the Eu/Si of 1/40, 1/20, 1/10, respectivly.
As reference smples, Eu2O3 was synthesized by the calcination of the EuCl3·6H2O, which was
identified with ICDD-ICSD 00-034-0072. The Eu3+ in ethanol at the concentaration of 16.5 mM wasfabricated by the dissolution of EuCl3·6H2O.
Immobilization of FA. The APTES immobilization was conducted by stirring Eu:NPS (250 mg)
with HCl (12 mL), APTES (0.78 mL) and ethanol (5 mL) at 37.5oC. The FA immobilization was
conducted by stirring the APTES-modified Eu:NPS (APTES-Eu:NPS, 150 mg) with 50 mM
phosphate buffered saline (PBS: pH=7.2, 25 mL), dimethyl sulfoxide (12 mL), and the folate
N-hydroxysuccinimidyl ester (NHS-FA) synthesized at the different initial concentrations based on
our previous reports [14]. The resulting materials with the immobilized FA amounts of 39, 61, 83,
206 and 858 mol·(g of APTES-Eu:NPS)-1 were abbreviated as FA1-, FA2-, FA3-, FA4- and FA5Eu:NPS, respectively.
Characterization of Nanostructures. The doped amount of Eu3+ was determined by the
inductively coupled plasma optical emission spectrometer (ICP-OES: Leeman Labs, INC U.S.A.,
Prodigy ICP). Nitrogen adsorption and desorption isotherms were measured at 77K on a SA3100
instrument (BECHMAN COULTER Co., Ltd.). Prior to the measurement, the samples weredegassed under vacuum at 393K for 4 h. The surface area was evaluated by the BET surface area,
and the pore volume was calculated by the BET surface area and averaged pore size. X-ray
diffraction (XRD) patterns were recorded on a X’Pert Pro MPD (PNAalytical Co., Ltd.) using
monochromatized CuK radiation.
Characterization of Luminescence Properties. Luminescence properties were evaluated byphotoluminescence spectroscopy and microscopy. The excitation and luminescence spectra wererecorded on a FluoroMax-4 spectrophotometer (Horiba Co., Ltd.) with the monitored luminescenceat 611 nm and excitation wavelength at 464 nm under room temperature (atmosphere: air,excitation-slit/detection-slit: 2 nm/2 nm, measure time: 0.1 s, step width: 1.0 nm, sample weight: 150
mg, light cut filter on the detection side: cut < 515 nm). The luminescence intensities centered at the
top of 590 nm and 611 nm, which are abbreviated as I590 and I611, were calculated by the integrated
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intensity areas in the ranges of 580−600 nm (assigned to 5D0→7F1) and 600−640 nm (assigned to
5
D0→7F1) based on the Voigt-function fitting at the peak tops, and the ratio of I611 to I590 (I611/I590)
was also calculated. The luminescence intensity centered at the top of 611 nm abbreviated as I611 wasalso calculated by the integrated intensity based on the Voigt-function fitting at the peak top in therange of 600–640 nm. The microscope images were observed by a luminescence microscope (Nikon
Co., Ltd., BX-50; excitation wavelength: 380–420 nm, exposure time: 1500 ms, sensitivity: 20). The
decay kinetics, which can be explained with an exponential function, was measured on a wavelength
at 394 nm from a Xe flash lump (Horiba Co., Ltd., Fluoromax-TCSPC), and the decay of theluminescence intensity was detected on a wavelength at 611 nm. The measured decay was recorded
by a numerical fitting to an exponential function based on the Eq. (1), and was repeatedly measured
ten times.
I (t) = I(0) exp (−t / τ)(1)
where the I(t) and I(0) are the luminescence intensity at time (t) and starting point (t = 0), and the τ isluminescence life time in the decay process, which consequently provided the I (0) and correlation
factor (R2) of 20021 ± 85 cps and 0.9992 for 2.5-Eu:MPS, 101307 ± 727 cps and 0.9978 for
5.0-Eu:NPS, 7024 ± 19 cps and 0.9978 for 10.0-Eu:NPS, 19434 ± 67 cps and 0.9997 for Eu2O3 and22639 ± 125 cps and 0.9998 for Eu3+ in ethanol. The quantum efficiency was recorded on a FP-8500(Nihondenshi Co., Ltd.) with an integrating sphere (diameter: 60 mm) equipment. The efficiency isdefined as the ratio of photons absorbed from the excitation wavelength (464 nm) to photons emitted
at 611 nm.
Cell Culture and Fabrication of Cell Suspensions. Fibroblast NIH3T3 (RCB1862) and Helacancer (RCB0007) cells as cell lines provided by Riken BioResource Center were separately
cultured in a plastic cell culture flask with an area of 75 cm2 (BD Bioscience, USA) containing 15
mL of the fetal bovine serum (FBS: SAFC Bioscience Co., Ltd., product number: 12603C, lot no.
6D0975) dispersed into Dulbecco’s minimum essential medium (DMEM: Invitrogen Co., Ltd.) at 10
vol% (10% FBS/DMEM). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2,and subcultured every 7 days using 1 mL of the trypsin-EDTA. After being washed with 15 mL ofphosphate buffer saline (PBS: SEIMI Co., Ltd.) and treated with 1 mL of the trypsin-EDTA for 10
min at 37°C, the cells were dispersed in 15 mL of PBS, separated by centrifugation (2000 rpm, 2min), and dispersed in 15 mL of 10% FBS/DMEM. The centrifugation and dispersion were carriedout twice. The number of cells in the suspension was counted and adjusted at the seeding density of
2.72 × 104 cells·mL-1.
Evaluation of Cell Viability. The fibroblast cell suspension was cultured on acommercially-available 96-well cell culture plate (Greiner Bio-one Co., Ltd., bottom area: 0.34
cm2/well, product number: 655180) at the area density of 8000 cells/cm2. At the culture time of 12 h,
the cultured fibroblast cells were adhered on the plate and had fibrous morphology in Figure 1 (a),and showed no luminescence as shown in Figure 1 (b). Then, 100 L of the FA-Eu:NPS in 10%
FBS/DMEM (concentration: 100 g/mL) was added into the cell surfaces and additionally cultured
for 48 h. The cell viability was evaluated by WST-8 method [21]. 10 L of the Cell Counting Kit-8
solution (Chemical-Dojin Co., Ltd., product number: 343-07623) was added and stored for 3 h at
37°C in a humidified atmosphere of 5% CO2. The absorbance at 450 nm of the resulting solution
was measured by a photospectrometer (Power Scan ®HT, DS Pharma Biomedical Co., Ltd.). The
background absorbance as the 10% FBS/DMEM was subtracted and the average value for fivesamples (n=5) was calculated. The absorbance maximum of the samples is fitted to be 100 %.

Fig. 1(a, c) Optical and (b, d) fluorescence microscope images of (a, b) fibroblasts and (c, d) Helacells adhered on the plate at the culture time of 12 h .
3Transactions on GIGAKU 1(2012)01014/1-8

Specific Staining of the Cells. The Hela cell suspension was cultured on both thecommercially-available 96-well cell culture plate and the 35 mm tissue culture dish (BD Falcon™
Co., Ltd.) at the area density of 8000 cells/cm2. At the culture time of 12 h, the cultured fibroblast
cells were adhered on the culture plate and had round shape in Fig. 1 (c), and showed noluminescence as shown in Fig. 1 (d). Then, 100 L and 1 mL of the FA-Eu:NPS in 10%
FBS/DMEM (concentration: 100 g/mL) were added into the adhered cells on the plate and dish,respectively.
The cells on the plate were cultured for 1, 2, 3, 4, 5, and 6 days. After the culture, the cells were
washed two times with 100 L of PBS and then the luminescence intensity was measured by aphotospectrometer (Power Scan ®HT, DS Pharma Biomedical Co., Ltd.). On the other hand, the cellson the dish were culture for 2 days. The cultured cells were washed two times with 1 mL of PBS andthen the cellular nuclei was stained at room temperature with 100 L of 4’,6-diamino-2-phenylindole(DAPI: InvitrongenTM Co., Ltd.) which was diluted 15 times into PBS. The samples in the dyesolution were then incubated in the dark for 60 min, and washed three times with 1 mL of PBS. The
cells in the PBS were covered and sealed with a glass to prevent evaporation during the observationby the fluorescence microscope.
3. Results and Discussion
Nanostructure of Eu:NPS. Table 1 shows the ICP-OES analytical values of the Eu molar
concentration to Si (Eu/Si) and the nanopore properties (SBET and V) evaluated by the N2 adsorption
and desorption isotherms. The Eu and Si concentrations in the dissolved solution contains 1.93 ±0.02 ppm and 12.82 ± 0.17 ppm for 2.5-Eu:NPS, 2.84 ± 0.04 ppm and 9.72 ± 0.13 ppm for
5.0-Eu:NPS, and 5.03 ± 0.04 ppm and 9.12 ± 0.11 ppm for 10.0-Eu:NPS. It is indicated that the
Eu/Si values by the ICP-OES analysis are similar to those estimated by the initial solution
compositions.
The isotherms indicated that the BET surface area and volume are 758 m2·(g silica)−1 and 0.68
ml·g-1 for NPS, 576 m2·(g silica)−1 and 0.48 ml·g-1 for 2.5-Eu:NPS, 557 m2·(g silica)−1 and 0.55
ml·g-1 for 5.0-Eu:NPS, and 523 m2·(g silica)−1 and 0.79 ml·g-1 for 10.0-Eu:NPS, indicating the
reduced surface area with the Eu3+-doping.
The peaks of the XRD patterns are indexed as (100), (110), (200) and (220) reflections of ahexagonal structure (data not shown), suggesting that the highly-ordered arrangements are preservedwith the doping. In the wide-angles, there are no detectable diffractions of the Eu2O3 crystalline
phase. These results significantly correspond to our previous reports [14].
Table 1 ICP-OES analytical values of the Eu molar concentration to Si (Eu/Si), the nanopore
properties (SBET and V) evaluated by the N2 adsorption and desorption isotherms, the luminescence
lifetime and the quantum efficiency the Eu:NPS.
ICP-OESN 2 adsorption/desorption isotherm

Luminescence lifetime

Quantum efficiency

SampleEu/Si(mol %)

SBET(m2 (g of Eu:Silica)-1 )V(ml (g of Eu:NPS)-1 ) (ms)

 (%)

NPS

−

758

0.68

−

−

2.5-EuNPS

2.78 ± 1.23

576

0.48

1.234 ± 0.012

6.51

5.0-EuNPS

5.40 ± 2.11

557

0.55

1.052 ± 0.016

8.67

10.0-EuNPS

10.19 ± 3.54

523

0.79

0.826 ± 0.004

3.91

Luminescence Properties of Eu:NPS. Figure 2 shows the typical excitation andphotoluminescence spectra of 5.0-Eu:NPS and FA-Eu:NPS. The excitation spectra of 5.0-Eu:NPS
4

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consists of several narrow peaks, which are located at around 361, 375, 380, 394, 412, and 465 nm,
and the spectral shapes were almost same as the Eu2O3 and Eu3+ in ethanol. The peaks are attributed
to f–f transitions within the 4f6 electron configuration [22], indicating the reflection of the electronictransitions of Eu3+ irrespective of host matrix.
The luminescence spectrum showed the narrow peaks assigned to the f–f transitions of the Eu3+,
due to the shielding of the 4f orbitals by the outer 5s2 and 5p6 orbitals [22]. All the observed peaks
correspond to the transitions from the metastable orbital singlet state 5D0 to spin–orbital states of 7FJ(J = 0, 1, 2, 4) of Eu3+ [23], which indicates the characteristic transitions from the 5D0 to 7F0 at 577
nm, 7F1 at 585 nm, 590 nm and 595 nm, 7F2 at 611 nm, 7F3 at 646 nm and 7F4 at 700 nm. The spectral
shapes were clearly different from the Eu2O3 and Eu3+ in ethanol, indicating the different relaxation
process of the Eu3+ depending on the host matrix. The luminescence of the Eu3+ doped into the silica
framework is predominantly attributed to 5D0 → 7F1 and 5D0 → 7F2, and the 5D0 → 7F2 intensity
significantly depends on the site environment of Eu3+ [24]. The I611 (due to 5D0-7F2 transition) is 3.5
times larger than the I590 (due to 5D0-7F1 transition), and the I611/I590 is constant irrespective of the
Eu/Si, and corresponds to the spectral shapes of the Eu3+ located in low symmetry sites [25]. The
Eu3+ is present in an environment of a low symmetry in the silica framework structure, and thestructural environment enhanced the 5D0→7F2 transition. Therefore, the Eu3+ concentration as wellas the silica framework structure affected the efficient luminescence of Eu3+, and the highly-efficientluminescence was observed at the Eu/Si of 5.0 mol%.
In order to understand the luminescence relaxation process, the decay kinetics of the excitedstates was investigated as shown in Table 1. The decay kinetics was significantly fitted with an
exponential function. The decay kinetics of the Eu2O3 and Eu3+ in ethanol turned out to be very fast
at 0.211 ± 0.001 ms and 0.279 ± 0.003 ms [14], indicating the effective quenching through a
non-radiative phonon in the oxide and solution states. The Eu3+ doped in the silica framework ismuch longer life time as compared with that in the oxide, solution, and Eu(Phen)33+-doped in
nanoporous silica film (0.330 ms) [26], suggesting the effective host matrix of the silica framework
for the efficient luminescence and relaxation. Although the increase in the Eu3+ concentration
enhanced the cross relaxation among the neighboring Eu3+ ions to shorten the life time, the silica
framework induced the longer life time of the Eu3+. The relationship between the Eu3+ concentrationand the life time clearly reveals a negative correlation, indicating the mono-dispersion of the Eu3+
ions in the silica frameworks.
The integral luminescence intensity is in the order of 5.0-Eu:NPS > 2.5-Eu:NPS > 10.0-Eu:NPS,
suggesting the occurrence of concentration quenching of the 10.0-Eu:NPS. Furthermore, thequantum efficiency is also in the order of 5.0-Eu:NPS > 2.5-Eu:NPS > 10.0-Eu:NPS as shown in
Table 1. Thus, the 5.0-Eu:NPS shows the highly-efficient luminescence was used for the following
experiment.
FA Immobilization on Eu:NPS. The FA was effectively immobilized on the APTES-Eu:NPS togive yellow-colored products, and the immobilized amount was controlled by the initialconcentration. The XRD patterns clearly revealed the hexagonal structure with the APTES and FA
immobilizations. In the N2 adsorption and desorption isotherms, a hysteresis between the adsorption
and desorption was observed The FA immobilization with covalent bonding on the Eu:NPS was
successfully achieved, and the ordered nanopores were preserved by the immobilization [14]. Taking
into account the molecular sizes, the pore size of Eu:NPS (ca. 2.5 nm) is large enough toaccommodate the APTES, whereas the FA cannot be incorporated into the nanopore, indicating the
FA immobilization only on the external surfaces.
The excitation and luminescence spectra of FA5-Eu:NPS in Fig. 2. With the APTES and FA
immobilization, the charge-transfer band at 240 nm in the excitation spectra (detected emission at
611 nm) increased as shown in Fig. 2 (a), which was located at the short-wavelength as compared tothat of the other oxides [25], suggesting the interaction of the O atoms of APTES with thenear-surface Eu3+ ions. The luminescence spectral shapes clearly depended on the FA amount. The
peaks in the APTES-Eu:NPS correspond to the transitions from the metastable orbital singlet state of5
D0 to the spin–orbital states of 7FJ (J = 0, 1, 2, 3) [23], which indicates the transitions from the 5D0
to 7F0 at 577 nm, 7F1 at 590 nm, 7F2 at 611 nm and 7F3 at 700 nm, and the luminescence intensity is
0.2 times weaker than that of the Eu:NPS. With the increase in the FA amount, the broad
5

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luminescence peak at around 550 nm was enhanced. Taking into account no luminescence of theneat FA and NHS-FA, the spectral change would be attributed to the excitation of the conjugation
band between the FA molecules and near-surface Eu3+ ions, implying the effective charge transfer
transition. The luminescence of the Eu3+ doped in the silica framework was predominantly attributed
to 5D0→7F1 and 5D0→7F2, and the 5D0→7F2 intensity depended on the site environment of Eu3+ [24].
The ratio of the 5D0→7F2 to 5D0→7F1 luminescence intensities was almost constant with theimmobilization. The integral luminescence intensity and the I550/I611 were clearly changed, and thesevalues were maximum at the FA amount of 60.3 mol·(g of APTES-Eu:NPS)-1 (FA2-Eu:NPS) and
83.0 mol·(g of APTES-Eu:NPS)-1 (FA2-Eu:NPS), respectively. The decrease with the much higher
increase in the FA amount would be attributed to the radiationless deactivation by the interactions
among the FA molecules. Therefore, the FA immobilization effectively induced the characteristic
luminescence by the band due to the chelete complex (i.e. FA‒Eu3+), suggesting the importance ofcontrolling of the Eu3+ concentration as well as the interactions among the FA molecules.

(a)Intesnsity / a.u.

Luminescence intensity at 612 nm(b)

FA‐Eu:NPS

FA‐Eu:NPS

Eu:NPS
200

250

300

350

400

450

500

550

Eu:NPS

600

500

550

600

650

700

750

800

850

900

Wavelength / nm

Wavelength / nm

Fig. 2 (a) Excitation and (b) photoluminescence spectra of Eu:NPS and FA-Eu:NPS.
Figure 3 shows the cell viability of the fibroblasts cultured in the Eu:NPS, APTES-Eu:NPS, andFA-Eu:NPS dispersed in 10%FBS/DMEM at the additional culture time of 48 h. The cells remained
attached to the plate and maintained their normal morphology as compared with the morphology in
Fig. 1 (a). The cell viability in the APTES-Eu:NPS shows the lowest viability due to the influence ofactivated amino groups, and that of Eu:NPS also shows the lower viability due to the oxygen atom insilanol group. With increasing the FA immobilized amount, the cell viability significantly increased.
This indicated that the amino group was deactivated with the FA immobilization and the FA
molecules were functionalized on the surfaces to exhibit the good cytocompatibility, andFA5-Eu:NPS shows the highest viability.
Figure 4 (a) shows the luminescence intensity changes of the Hela cells targeted by the
FA-Eu:NPS with the different FA immobilization amount. The luminescence intensity from the Helacells gradually increase with the culture time, and was irrespective of the FA immobilization amount,suggesting that the Hela cells realize the FA ligand at the lower immobilization amount and uptake
the FA-Eu:NPS for the initial 12 h. On the other hand, no luminescence was observed from thefibroblast cells.
Figure 4 (b–e) shows the optical and fluorescence microscope images of the fibroblast and Helacells staining with DAPI and FA5-Eu:NPS at the additional culture time of 48 h. The optical andfluorescence microscope images show the blue luminescence from the cellular nuclei due to the
DAPI staining. No binding and uptake by the fibroblasts in Fig. 4 (b, c) and the significant binding
and uptake by the Hela cells in Fig. 4 (d, e) were clearly observed. The fluorescent image of the Hela6

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cells indicates the presence of luminescent particles located around the nucleu. With an increase of
the particle concentration, the number of red-emitting particles in the cells increased. Thus, theFA-Eu:NPS was found to be effectively taken up only by the Hela cells. Here, the particle size isimportant for the uptake efficiency; 100 nm particles were studied as compared to about 400 nm
particles, and a smaller particle size may lead to enhanced nonspecific cellular uptake. For
amorphous silica, toxicity tends to be inversely related to particle size, and a particle size below 100
nm has actually been found to induce cytotoxicity [27]. In this study, the particle size is 300 nm,
which would affect the efficient uptake. The detailed nanopartile design and imaging techniques will
be reported by our group soon.

Fibroblast cell viability / %

100

80

60

40

20

0

Eu:NPSAPTES‐
Eu:NPSFA1‐
FA2‐
APTES‐ APTES‐
Eu:NPS Eu:NPS

FA5‐
FA4‐
FA3‐
APTES‐ APTES‐ APTES‐
Eu:NPS Eu:NPS Eu:NPS

Luminescence intensity

Fig. 3 Cell viability of the fibroblasts cultured in the 10%FBS/DMEM containing Eu:NPS,APTES-Eu:NPS, and FA-Eu:NPS at the additional culture time of 48 h.
5.6 104

4.8 1044 10

4

3.2 104

2.4 104

1.6 104(a)

FA1-APTES-Eu:NPS
FA2-APTES-Eu:NPS
FA3-APTES-Eu:NPS
FA4-APTES-Eu:NPS
FA5-APTES-Eu:NPS

8000

(b)

(c)

(d)

(e)

0
0

1

2

3

4

5

6

7

Culture time / day

Fig. 4(a) Luminescence intensity changes of the Hela cells targeted by the FA-Eu:NPS with theculture time, and (b, d) optical and (c, e) fluorescence microscope images of (b, c) fibroblastand (d, e) Hela cells staining with DAPI and FA5-Eu:NPS at the additional culture time of
48 h.

7

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4. Conclusion
In conclusion, FA was successfully immobilized on the luminescent Eu:NPS spheres bymediation of the APTES adlayer. The ordered nanostructures were preserved by the immobilizations.
The FA–APTES–Eu:NPS showed the characteristic luminescence due to the interactions between
FA and Eu3+, suggesting the importance of controlling the interactions among the FA molecules on
the surfaces. The immobilized spheres were highly dispersed stably in the cell culture medium tobind specifically to cancer cells and exhibit the luminescence without cytotoxicity for fibroblasts.
Thus, the FA was successfully immobilized on the luminescent Eu:NPS spheres to exhibit the highly
targeting ability to the cancer cells.
Acknowledgment
This study was supported by a grant from the Japan Society for the Promotion of Science (JSPS).
References[1] L. Brandon-Peppas and J. O. Blanchette, Adv. Drug Delivery Rev., 56 (2004) 1649.
[2] T. Yanagisawa, T. Shimizu, K. Kuroda and C. Kato, Bull. Chem. Soc. Jpn., 63 (1990) 988.
[3] C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli and J. S. Beck, Nature, 359 (1992) 710.
[4] K. B. Yoon, Chem. Rev., 93 (1993) 321.
[5] K. Moller and T. Bein, T. Chem. Mater., 10 (1998) 2950.
[6] M. Ogawa, Ann Rep (Sec C), 94 (1998) 209.
[7] A. Stein, B. J. Melde and R. C. Schroden, Adv. Mater., 12 (2000) 1403.
[8] B. J. Scott, G. Wirnsberger and G. D. Stucky, Chem. Mater., 13 (2001) 3140.
[9] H. Furukawa, T. Watanabe and K. Kuroda, Chem. Comm. (2001) 2002.
[10] M. Ogawa, J. Photochem. Photobiol., C Photochem. Rev. 3 (2002) 129.
[11] (a) M. Tagaya and M. Ogaawa, Chem. Lett., 35 (2006) 108; (b) M. Tagaya and M. Ogawa, Phys.
Chem. Chem. Phys., 10 (2008) 6849.
[12] J. L. Vivero-Escoto, I. I. Slowing, C. W. Wu and V. S. Y. Lin, J. Am. Chem. Soc., 131 (2009)3462.
[13] M. Okuda, M. Takeguchi, Y. Zhu, A. Hashimoto, N. Ogawa, M. Tagaya, S. Chen, N. Hanagata
and T. Ikoma, Surf. Interf. Sci., 42 (2010) 1548.
[14] (a) M. Tagaya, T. Ikoma, T. Yoshioka, T. Motozuka, F. Minami and J. Tanaka, Mater. Lett., 65(2011) 2287; (b) M. Tagaya, T. Ikoma, T. Yoshioka, S. Motozuka, Z. Xu, J. Colloid and Interf.
Sci., 363 (2011) 456; (c) M. Tagaya, T. Ikoma, T. Yoshioka, Z. Xu and J. Tanaka, Chem.
Commun., 47 (2011) 8430.
[15] J. M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi, J. E. Eriksson, C. Sahlgren and M. Lindn,
ACS Nano, 3 (2009) 197.
[16] (a) E. Ruiz-Hitzky, P. Aranda, M. Darder and M. Ogawa, Chem. Soc. Rev., 40 (2011) 801; (b) N.
Nassif and J. Livage, Chem. Soc. Rev., 40 (2011) 849.
[17] V. Sokolova and M. Epple, Nanoscale, 3 (2011) 1957.
[18] Y. Zhu, T. Ikoma, N. Hanagata and S. Kaskel, Small, 6 (2010) 471.
[19] X. Kang, S. Huang, P. Yang, P. an Ma, D. Yanga and J. Lin, Dalton Trans., 40 (2011) 1873.
[20] J. F. Kukowska-Latallo, K. A. Candido, Z. Y. Cao, S. S. Nigavekar, I. J. Majoros, T. P. Thomas,
L. P. Balogh, M. K. Khan and J. R. Baker, Cancer Res., 65 (2005) 5317.
[21] H. Tominaga, M. Ishiyama, F. Ohseto, K. Sasamoto, T. Hmamoto, K. Suzuki and M. Watanave,
Anal. Commun., 36 (1999) 47.
[22] W. Chen, R. Sammynaiken and Y. Huang, J. Appl. Phys., 88 (2000) 1424.
[23] N. Wan, J. Xu, T. Lin, X. Zhang and L. Xu, Appl. Phys. Lett., 92 (2008) 201109.
[24] E. J. Nassar, K. J. Ciuffi, S. J. L. Ribeiro and Y. Messaddeq, Mat. Res., 6 (2003) 557.
[25] M. A. Zaitoun, T. Kim and C. T. Lin, J. Phys. Chem. B, 102 (1998) 1122.
[26] S-J. Seo, D. Zhao, K. Suh, J. H. Shin and B-S. Bae, J. Lumin., 128 (2008) 565.
[27] K. O. Yu, C. M. Grabinski, A. M. Schrand, R. C. Murdock, W. Wang, B. Gu, J. J. Schlager, S.
M. Hussain, J. Nanopart. Res., 11 (2009) 15.
(Received: 26 April, 2012, Accepted: 31 May, 2012)8

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Effect of Flow Channel on Specific Surface Area of Steam-ActivatedCarbon Material from Coffee Bean Wastes
Hiroe Toda1, Ikumi Toda1*, Hiroki Tanikawa1, Tomokazu Takahata1, Shigeo Ohshio1,
Hiroki Akasaka1, Shuji Himeno2, Toshinori Kokubu3, Hidetoshi. Saitoh1
1)
Department of Materials Science and Technology, Nagaoka University of Technology,2)
Department of Civil and Environmental Engineering, Nagaoka University of Technology
1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan3)
Department of Chemical Science and Engineering, Miyakonojo National College of Technology
473-1, Yoshio, Miyakonojo, Miyazaki 885-8567, Japan*E-mail: ikumi_toda@mst.nagaokaut.ac.jp
The effect of a flow channel on specific surface area of steam-activatedcarbons was investigated for activated carbons fabricated from coffee bean
wastes. The specific surface areas of the activated carbons fabricated atsteam-activation temperatures of 700, 775, and 825oC for 6 h were 98, 937,
and 656 m2/g, respectively. To examine relationship between specific surface
area and other properties of activated carbons materials for crystalline andspongelike morphological structures, X-ray diffraction (XRD) analysis andcross sectional scanning electron microscopy (SEM) were performed for theactivation at each temperature. The former analysis showed that the interlayer
space d002 and crystallite size Lc of the graphite structure had no change withthe activation temperature. The SEM images showed that the spongelike
structure changed with the activation temperature. These results indicatedthat controlling the flow channel was important in the attributed spongelike
structure for the fabrication of activated carbons with a high specific surfacearea.

1. Introduction
Recently, mass disposal of agricultural waste has become a seriously environmental problem. To
solve this problem, technologies for recycling agricultural waste as a biomass resource have beenstudied [1]. One of the recycling technologies is to produce activated carbon from biomass wastes asreusable products. There have been many reports on the production of activated carbons that areused as absorbent materials for removing hazardous compounds [2-4], as a canister to control
gasoline vapor [5], as electrodes in electric double-layer capacitors [6,7], and as hydrogen storage
materials [8-10]. The utilization of biomass resources has a number of advantages, such as the
production of functional materials at a low cost and the reduction of environmental impact.
Coffee bean waste is a biomass resource that is discarded in large quantities [11], Coffee is apromising raw material for the fabrication of activated carbon, because it contains plant tissues. It
was reported that coffee bean waste formed a spongelike structure consisting of micrometer-sizeordered voids during the roasting process [12]. The surface area of such structure was increased
upon chemical activation, resulting in the production of activated carbon. Boonamnuayvitaya et al.
reported the preparation of a mesoporous carbon material from coffee bean waste with a specificsurface area of 914 m2/g and a pore volume of 1.01 cm3/g by zinc chloride (ZnCl2) impregnation
coupled with activation by carbon dioxide [13]. Activated carbons fabricated by chemical activation
using hydrogen phosphate (H3PO4) were reported by Baquero et al [14]. These materials had a
specific surface area of 1402 m2/g and a pore volume of 1.25 cm3/g.
When activated carbons are used as an adsorbent to small molecules such as hydrogen, a higher
specific surface area is generally required [9]. The pores of activated carbons are classified by theirdiameter d as micro-pores (d < 2 nm), mesopores (2 < d < 50 nm), and macropores (d > 50 nm) [15].
The specific surface area changes with the development of these pores, which is related to changes
1Transactions on GIGAKU 1(2012)01015/1-6in the resulting graphite structure on activated carbons. Takahata et al. [16] reported the relationshipbetween specific surface area and the structure of graphite on activated carbon. They found that the
specific surface area increased with decreasing graphite crystalline size Lc and increasing graphenesheet interlayer spacing d002 from the analysis of patterns of activated carbons. Additionally, Toda etal. [17] reported that the change in the specific surface area of activated carbon materials was
dominated by their micro-pore structures. They calculated the micro-pore volume as a proportion of
the total pore volume for activated carbons and found that the micro-pore volume was accounted forthe majority of total pore volume. These reports suggested that the graphite structure is related to thedevelopment of the micro-pore structure. On the other hand, to utilize the micro-pores of activatedcarbons for gas adsorption, it was necessary that the micro-pores were exposed on the surface of thematerials. David et al. proposed a pore structure model for activated carbons [18]. According to its
pore structure model, the meso-pores and macro-pores in the activated carbons acted as flow
channels for the adsorbent into the micro-pores. Thus, it was considered that the gas adsorption
ability of activated carbons depended on the micro-pore volume. For the fabrication of activatedcarbons with high specific surface area from coffee bean waste, it is important to investigate therelationship between activation temperature and the graphite structure and flow channels on
activated carbons. In the present work, activated carbons with flow channels were fabricated fromwaste coffee beans in steam activation. The relationship between the specific surface area, flow
channels and the graphite crystallite structure of activated carbons was investigated.
2. Experiments
Activated carbons were fabricated by steam-activation. The coffee bean wastes were used as
raw material, which drinking contents have been removed. Before the steam-activation
process, the coffee bean waste was dried in an oven at 120oC for 24 h to remove moisture.
Steam-activation was performed by a two-step process involving carbonization and activation. In the
carbonization process, the coffee bean waste was placed into a SiC crucible, where it was heated at
200ºC for 2 h to remove moisture from coffee bean waste, then at 600ºC for 24 h with a heating rateof 5ºC/min, carbonization was carried out. After the carbonization, the carbon materials were placedin an SUS container whose bottom was composed of a mesh, which was placed in a steam
superheater (Dai-Ichi High Frequency Co., Ltd.). The steam-activation process was performed at 700,
775, or 825ºC for 6 h with a 50 g/min flow of steam.
Specific surface area of the activated carbons was estimated by the analysis of N2 adsorption
isotherm profiles on the samples at 77 K. The isotherm profiles were obtained by a Micrometrics
Gemini V2375 automatic surface area analyzer (Shimadzu Corp.). Before the N2 adsorption
measurements, absorbed gases were removed by heating at 200ºC for 1 h under vacuum pressure(10-1 Pa). After that, the balance pressure at each pressure was measured for the sample. Here, therelative pressure was defined as the ratio of the balance pressure to the pressure of the saturated
vapor at 77 K. The specific surface area SBET was calculated using the Brunauer-Emmett-Teller(BET) model [19]. The crystallite structures of the activated carbons were determined by the θ-2θ
method using XRD. An MO3XHF MXP3 diffractometer (Mac Science) with a Cu Kα (1.54 Å)
X-ray source, a current of 40 mA, and a voltage of 40 kV was used for the XRD analysis. The
detector angle and scanning speed were 0.02 °/step and 2.00 °/min, respectively. XRD patterns were
measured in the range of 2θ=4-80°. The interlayer spacing d002 and average crystallite size Lc of theactivated carbons were calculated by Bragg’s equation and Scherrer’s equation using the obtained
XRD patterns.
The flow channels in particles of the activated carbons were observed by scanning electron
microscopy (SEM) (JSM-6700F, JEOL). To obtain smooth cross-section surfaces before SEM
observations, polishing was performed by the following process. First, each activated carbon wasembedded in epoxide resin (G2, Gatan). Second, to harden the epoxide resin, the sample with the
epoxy was heated on a hot plate at 125ºC for 30 min. Finally, the cross section of the activatedcarbon was polished by an ion beam using a cross-section polisher (SM-09010, JEOL). SEM
observation was performed in the secondary image (SEI) mode at an accelerating voltage of 3 kV
and an irradiation current of 8 A.
2Transactions on GIGAKU 1(2012)01015/1-6

3. Results and Discussion
The specific surface area of the activated carbons increased from 98 to 937 m2/g upon increasing
the activation temperature from 700 to 775ºC. However, when the temperature was increased from775 to 825ºC, the specific surface area decreased from 937 to 656 m2/g. From these results, it was
confirmed that the specific surface area of the activated carbons was changed with the activation
temperature. The change in the specific surface area suggested that the activation temperaturechanged the graphite crystallite structure and flow channels of the activated carbons. The change inthe morphological structure of the activated carbons produced at the activation temperature was
evaluated from the XRD patterns. Figure 1 shows the XRD patterns of the activated carbons. The
broad peak at 2 = 24° corresponded to the (002) plane of hexagonal graphite assigned by ICDD
card No. 00-056-0159. The broader peak at 44° can be assigned to the (100) or (101) plane. This
peak was assigned to the (10) plane [20], because neither of the above planes could be clearly
discriminated. Since the peaks in the diffraction patterns were broad, the graphite clusters in the
porous carbon were small and the order of the graphene layers was short. To understand the change
in the graphite crystallite structure, the average interlayer spacing d002 and average crystallite size Lc
was calculated from the diffraction patterns, where d002 for graphite was calculated using Bragg’s
equation with a peak at 24°. The value of d002 for the activated carbons slightly decreased from 0.377
to 0.373 nm with increasing the activation temperature from 700 to 825ºC, as shown in Fig. 2. The
average crystallite diameter Lc in the 002 direction was calculated using Scherrer’s equation. The
crystallite size Lc was reduced from 1.26 to 1.16 with an increase of the activation temperature from
700 to 825ºC. These decreases in d002 and Lc were very small in compared with the change in
specific surface area. These results indicated that the graphite crystallite structure of activated carbon
was not affected by the activation temperature. Furthermore, the results suggested that the specificsurface area was not affected by the graphite crystallite structure on steam activated carbons madefrom waste coffee beans.

0.5

1.4
d002
Lc
1.3

0.4
1.2
0.3
0.2

1.1

0.1
1.0
680
720
760
800
840
Steam-activation temperature (ºC )Fig. 1 XRD patterns of activated carbons
prepared from coffee bean wastes.

Average crystallite size, Lc (nm)Interlayer spacing, d002 (nm)0.6

Fig. 2 Relationships between interlayer
spacing or average crystallite size andactivation temperature on activated carbons.

Figures 3 and 4 show cross-sectional SEM images of the activated carbon particles. The
comparatively dark areas in the SEM images corresponded to epoxy resin, and the brighter areas
were to the activated carbons having particle forms with about 100 m. From the SEM images, we
confirmed that the activated carbons have a spongelike structure consisting of cells (arrow A in Figs.
3(a), 3(b), and 3(c)) and partition walls (arrow B in Figs. 3(a), 3(b), and 3(c)). Furthermore, pores
having about 40 nm also developed inside the partition walls of the spongelike structure as shown in
Figs. 4(a) 4(b), and 4(c). The spongelike strucuture appeared to change with increasing activation
temperature. Consequently, to evaluate the change in the spongelike structure caused by the increasein activation temperature, the partition wall thickness DT and the average diameter of pores inside3Transactions on GIGAKU 1(2012)01015/1-6

the partition walls Dp were measured from the SEM images. Figure 5 shows the relationship of
between DT or Dp and steam-activation temperature on activated carbons. The values of DT
decreased from 4.8 to 2.2 m with increasing the activation temperature from 700 to 775ºC and thenincreased slightly to 2.4 m at 825ºC. For Dp the values decreased from 19.2 to 15.3 nm withincreasing steam-activation temperature from 700 to 775ºC and then drastically increased to 59.2 nm
at 825ºC. The diameter of the pores inside the partition walls decreased with increasing thesteam-activation temperature. These results suggested that the increase of the specific surface areafrom 700 to 775ºC was related to the thickness of the partition walls. In addition, they suggest thatthe decrease in the specific surface area from 775 to 825ºC was related to the increased diameter ofpores inside the partition walls.

(a)

700℃ (b)B

775℃ (c)
B

A

825℃
A

B
A
10 m

10 m

10 m

Fig. 3 Cross-sectional SEM images of spongelike structure in activated carbons after steam
activation(a)

700℃ (b)775℃ (c)825℃

100 nm

100 nm

100 nm

6

70

5

60
50

4

40
3
30
2

20
Dp
DT

1
0
680

10

0
760
800
840
720
Activation temperature (℃)

Average diameter ofpores inside partition wall, Dp (nm)Thickness of partition wall, DT (m)Fig. 4 Cross-sectional SEM images of partition walls of the spongelike structure in activated carbons
after steam activation

Fig. 5 Relationships between thickness of partition wall or average diameter of pores inside partition
wall and activation temperature on activated carbons.

4Transactions on GIGAKU 1(2012)01015/1-6

The differences in the specific surface area of the activated carbons prepared from coffee waste
seemed to be related to the formation of flow channels led to the exposure of micro-pores. To obtain
activated carbons with a high specific surface area, the development of flow channels might benecessary to allow gases to reach the micro-pores. The analysis of the crystalline structure indicatedthat the specific surface area was changed with increasing activation temperature, whereas nographite crystallite structures was changed as shown in Fig. 2. This suggested that the formation offlow channels affected the specific surface area of activated carbons. As shown in Fig. 5, DT
decreased and specific surface area increased between 700 and 775ºC. This suggested that the
exposure of micro-pores was caused by gasification of the walls of cells. Consequently, it wasconsidered that the specific surface area was increased because the cell-like macro-pores werefunctionalized by the flow channels. On the other hand, between 775 and 825ºC, few changed in thepartition wall thickness was observed with the steam-activation temperature, and the specific surface
area decreased with increasing Dp. These changes showed that the micro-pore structure wasdestroyed by linkaging pores. As a result, the specific surface area decreased with the growth of flow
channels inside the partition walls at 825ºC.

4. Conclusion
Activated carbons were fabricated from coffee bean waste through steam activation process at
different steam-activated temperatures. To investigate the relationship between steam-activation
temperatures and the properties of the activated carbons, the spongelike and graphite crystallite
structure were measured. Between 700 and 775ºC, the specific surface area increased owing to theformation of flow channels, which was attributed to the increased thickness of the partition walls. In
contrast, the specific surface area decreased above 775ºC owing to the decreased average diameter ofpores inside the partition walls. These results indicated that the formation of flow channels mainly
determined the specific surface area and that the flow channels in the activated carbons could
fabricate the spongelike sturucture depending on the steam-activation temperature.

References[1] A. Bota, K. Laszlo, L. G. Nagy, G. Subklew, H. Schlimper, and M. J. Schwuger, Adsorption, 3(1997) 81.
[2] S. Nagano, H. Tamon, T. Adzumi, K. Nakagawa, and T. Suzuki, Carbon, 38 (2000) 915.
[3] S. Karagoz, T. Tay, S. Ucar, and M. Erdem, Bioresour. Technol., 99 (2008) 6214.
[4] E. Ekinci, T. Budinova, F. Yardim, N. Petrov, M. Razvigorova, and V. Minkova, Fuel Process.
Technol., 77-78 (2002) 437.
[5] U. Mohr, Filtr. Sep., 34 (1997) 1016.
[6] M. Toyoda, Y. Tani, and Y. Soneda, Carbon, 42 (2004) 2833.
[7] K. Kinoshita, “Carbon: Electrochemical and Physicochemical Properties,” Wiley-Interscience,
New York (1988) 294.
[8] R. Strobel, L. Jorissen, T. Schliermann, V. Trapp, W. Schutz, K. Bohmhammel, G. Wolf, and J.
Garche, J. Power Sources, 84 (1999) 221.
[9] B. Panella, M. Hirsher, and S. Roth, Carbon, 43 (2005) 2209.
[10] A. Anson, J. Jagiello, J. B. Parra, M. L. Sanjun, A. M. Benito, W. K. Maser, and M. T.
Martinez, J. Phys. Chem. B, 108 (2004) 15820.
[11] V. Boonamnuayvitaya, C. Chaiya, W. Tanthapanichakoon, and S. Jarudilokkul, Sep. Purif.
Technol., 35 (2004) 11.
[12] T. Nakabayashi, and K. Suzuki, J. Food Sci. Tech., 33 (1986) 779.
[13] V. Boonamnuayvitaya, S. Sae-ung, and W. Tanthapanichakoon, Sep. Purif. Technol., 42(2005)159.
[14] M. C. Baquero, L. Giraldo, J. C. Moreno, F. Suarez-Garcia, A. Martinez-Alonso, and J. M. D.
Tascon, J. Anal. Appl. Pyrol., 70 (2003) 779.
[15] IUPAC, Manual of Symbols and Terminology (1972).
[16] T. Takahata, I. Toda, H. Ono, S. Ohshio, H. Akasaka, S. Himeno, T. Kokubu, and H. Saitoh,
5Transactions on GIGAKU 1(2012)01015/1-6

Jpn. J. Appl. Phys. 48 (2008) 117001-1.
I. Toda, H. Ono, T. Takahata, S. Ohshio, H. Akasaka, S. Himeno, T. Kokubu, and H. Saitoh, J.
Sol. Mech. Mater. Eng., 3 (2009) 1306.
[18] E. David, J. Mater. Proc. Tech., 162-163 (2005) 169.
[19] S. Brunauer, P. H. Emmett, and E. Teller, J. Am. Chem. Soc., 60 (1938) 309.
[20] B. E. Warren, J. Chem. Phys., 2 (1934) 551.
[17]

(Received: 23 March, 2012, Accepted: 30 June, 2012)6

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Hydrogen Storage Property of Activated Carbon
Fabricated with Lignin
Ikumi Toda1*, Hiroe Toda1, Shigeo Ohshio1, Hiroki Akasaka1, Shuji Himeno1,
Manami Nakaishi2, Hidetoshi Saitoh1
1
Nagaoka University of Technology, 1603-1, Kamitomioka, Nagaoka, Niigata 940-2188, Japan,
2
Harima Chemicals, 671-4, Noguchichomizuashi, Kakogawa, Hyogo 675-4, Japan*E-mail: ikumi_toda@mst.nagaokaut.ac.jp

The micro-pore structure and hydrogen storage property at 298 and 77K ofactivated carbons fabricated with lignin through KOH activation were
investigated. The specific surface area of activated carbons decreased 2290 to1140 m2/g as the carbonization temperature increased from 400 to 800oC in
the carbonization process. The micro-pore volume of activated carbons also
decreased from 1.49 to 0.48 cm3/g as the carbonization temperature increased.
The pore size of activated carbons was distributed around 0.8 and 1.1 nm in
the micro-pore range. These results suggest that the micro-pore structure ofactivated carbons is affected by carbonization temperature in the
carbonization process on KOH activation. The stored hydrogen content ofactivated carbons at 298K was 0.59wt% under 8.9 MPa. At 77K, the storedhydrogen content of activated carbons was 6.9wt% under 8.7 MPa. The
fabrication of hydrogen storage materials from lignin was achieved. The
results suggest that activated carbons fabricated from lignin have micro-pore
structures which influence the stored hydrogen content.

1. Introduction
Environmental destruction has increased the urgency to develop technologies for the recycling ofvarious materials. The disposal of biomass waste from agricultural activities is an environmental
problem. One of the solutions to this problem is to produce functional and reusable materials frombiomass resources. Activated carbons fabricated from biomass resource have been studied for use asfunctional materials. In particular, some activated carbons with nano-meter size pores, i.e.,
micropores, offer high performance because of their high specific surface areas. These activatedcarbon materials are prepared from various raw materials such as coal [1], palm shell [2], coffee
waste [3], and rice husks [4]. Such activated carbon materials are widely used because of their highadsorption properties [5]. These approaches have been applied to hydrogen storage materials [6] andadsorbent materials for gases [7,8].
From recent reports, it is apparent that various carbon materials made from carbon nanotubes [9],graphite nanofibers [10], and activated carbons are capable of storing hydrogen and have been
considered for the next generation of energy systems. For example, porous carbons with a specificsurface area of 3220 m2/g is capable of storing hydrogen of 1.3wt.% at room temperature [11]. The
reports suggest the influence of specific surface area on the hydrogen storage efficiency of the
micro-pores. In activated carbons fabricated from biomass recourses, it was reported that activatedcarbons with micro-pores in the pore structure were fabricated from lignin [12]. Lignin is product asof a residual material in the manufacture process of cellulose pulps. Some researchers have usedpotassium hydroxide (KOH) for the activation [13,14]. The micro-pore structure found in activatedcarbons fabricated with lignin following KOH activation is expected to be utilized as hydrogenstorage materials.
In this study, activated carbons were fabricated through the KOH activation method and theirmicro-pore structure and hydrogen storage property were investigated.

1

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2. Experiment
The samples of activated carbons were fabricated by the alkali activation method with KOH as
an activation agent. Lignin of raw materials was dried at 100oC for 24 h. The sample preparation was
performed by two processes of carbonization and activation. In the carbonization process, lignin was
firstly carbonized at 400, 550, and 800oC for 3 h. The carbonized sample and KOH were mixed witha ratio of 5: 1 by weight. Then, the carbonized sample was treated by a heating process at 850℃ for
2 h.
The pore characteristics, specific surface area, pore size distribution, and pore volume were
estimated by analyses of N2 adsorption isotherm profiles at 77K for activated carbons. Isotherm
profiles were obtained using Belsorp-max (BEL JAPAN Co.). Before N2 adsorption measurements,
absorbed gases were baked out at 200oC, for 10 h under vacuum pressure. The specific surface area
(SBET) and total pore volume (Vtotal) were calculated on the basis of the Brunauer-Emmett-Teller(BET) model [15]. The micro-pore size distribution and micro-pore volume (Vmicro) were analyzed
by the micro-pore (MP) –method [16] .
The stored hydrogen content in activated carbons was measured using a volumetric analysis
defined by the JIS H7201 “Method of determining the PCT relation of hydrogen absorbing alloys”.
Prior to the analysis, powder samples with a desired weight (0.2 g) were inserted into a pressure
vessel. In the pressure vessel, the sample was degassed under 10-2 torr at 423K for 1 h and wasmaintained at 77 and 298K using a cryostat in the hydrogen pressure range of 0-12 MPa.
3. Results and Discussion
Figure 1 shows N2 isotherms of carbonized samples fabricated with lignin. The N2 adsorption
isotherms of all carbonized samples are attributed to the type-III [17], indicating that the carbonizedsamples are non-porous materials. Figure 2 shows N2 isotherms of activated carbons fabricated withlignin. The N2 adsorption volumes of all activated carbons were increased considerably at P/P0 < 0.1.
At P/P0=0.99, the N2 adsorption volumes of the activated carbon decreased from 1277 to 396 cm3/g
as the carbonization temperature increased from 400 to 800oC. The adsorption isotherms of allactivated carbons indicated the type-I [17], indicating that the activated carbons fabricated withlignin has micro-pore structure. Therefore, these results suggest that the development of pore
structure in activated carbons fabricated from lignin was affected by carbonization temperature in the
carbonization process.

6

400℃
550℃
800℃

1400

(a)

Adsorption volume (cm3/g)

Adsorption volume (cm3/g)

8

4

2
0
0.0

0.2
0.4
0.6
0.8
Relative pressure P/P0

1200

(b)

1000
800
600
400
200
0
0.0

1.0

400℃
550℃
800℃

0.2
0.4
0.6
0.8
Relative pressure P/P0

1.0

Fig. 1 Nitrogen adsorption isotherms of (a) carbonized samples and (b) activated carbons.
Table 1 shows the pore-characteristics of the carbonized samples and activated carbons. The SBETof carbonized samples fabricated at 400, 550, and 800oC were 0.1, 3.0, and 0.4 m2/g, respectively.
The SBET of activated carbons decreased from 2290 to 1140 m2/g as the carbonization temperature
increased from 400 to 800oC. The Vtotal of carbonized samples fabricated at 400, 550, and 800oC
were 8.9×10-3, 7.3×10-3, and 7.8×10-3 cm3/g, respectively. The Vtotal of activated carbons decreased2

Transactions on GIGAKU 1(2012)01016/1-4

from 1.97 to 0.61 cm3/g as the carbonization temperature increased from 400 to 800oC.

5.0

6.0

(a)

400℃
550℃
800℃

dVp/d(dp) (cm3/g/nm)

dVp/d(dp) (cm3/g/nm)

6.0

4.0
3.0
2.0
1.0
0.0

400℃
550℃
800℃

5.0

(b)

4.0
3.0
2.0
1.0

0.0

0.8
1.2
1.6
0.4
Pore diameter, dp (nm)

0.0

2.0

0.0

0.4
0.8
1.2
1.6
Pore diameter, dp (nm)2.0

Fig. 2 Pore size distributions of carbonized samples (a) and activated carbon (b) in range of
micro-pore.

Table 1 Pore-characteristics of carbonized samples and activated carbons

Temperature (℃)
400
550
800

Carbonized sample
SBET (m2/g) Vtotal (cm3/g)
0.1
8.9×10-3
7.3×10-3
3.0
0.4
7.8×10-3

SBET (m2/g)
2290
1740
1140

Activated carbon
Vtotal (cm3/g) Vmicro (cm3/g)
1.49
1.97
1.10
0.99
0.61
0.48

Figure 2 shows the pore size distribution of carbonized samples and activated carbons in therange of micro-pore. The micro-pore volume of activated carbons decreased from 1.49 to 0.48 cm3/g
as the carbonization temperature increased from 400 to 800oC. The existence of micro-pores was notseen in the carbonized samples. On the other hands, it is observed on the sample of activated carbons
fabricated with the carbonized sample. The typical pore size was recognized at approximately
0.8 nm to 1.1 nm, indicating that the micro-pore structure of activated carbons was developed by
KOH activation. Therefore, these results indicate that the micro-pore structure in activated carbons
fabricated from lignin was affected by carbonization temperature in the carbonization process.
Figure 3 shows the hydrogen storage property of activated carbons which were fabricated fromthe samples carbonized at 400oC. At 298K, the stored hydrogen content of the activated carbons
increased as the equilibrium pressure of hydrogen increased. The stored hydrogen content of theactivated carbon at 298K was 0.59wt% at 8.9 MPa. At 77K, the stored hydrogen content of theactivated carbon was increased considerably as the equilibrium pressure of hydrogen increased. The
stored hydrogen content of the activated carbon at 77K was 6.9wt% at 8.7 MPa. The hysteresis ofstored contents was observed at 298 and 77K. These hystereses at 298 and 77K indicate that achemical sorption also slightly occurred by effect of KOH activation. There results suggest that the
micro-pore of activated carbons may effectively act as the hydrogen adsorption site at 77K.

3

Stored hydrogen contents (wt.%)

Transactions on GIGAKU 1(2012)01016/1-4

8.0

77 K
298 K

6.0

4.0

2.0
0.0
0

2
4
6
8
10 12
Equilibrium pressure (MPa)Fig. 3 Hydrogen storage properties at 298 and 77K of activated carbons with a specific surface areaof 2290 m2/g.

4. Conclusion
Activated carbons were fabricated from lignin by KOH activation. The micro-pore structure andhydrogen storage property of activated carbons were investigated. The pore size of activated carbons
distributed at approximately 0.8 nm to 1.1 nm. The specific surface are and micro-pore volume ofactivated carbons decreased as the carbonization temperature increased. The specific surface areaand micro-pore volume at carbonized temperature 400oC reached 2290 m2/g and 1.49 cm3/g,
respectively. The maximum stored hydrogen contents at the specific surface area of 2290 m2/g were0.59 wt% at 298K and 6.9wt% at 77K.
References[1] J. Hayashi, T. Horikawa, K. Muroyama and V.G. Gomes, Microporous and Mesoporous Mater.,55 (2002) , 63.
[2] I. A.W. Tan, A. L. Ahmad and B. H. Hameed, Bioresour. Tech., 100 (2009) 1494.
[3] J. V. Nabais, P. Carrott, M. M. L. R. Carrott, V. Luz and A. L. Ortiz, Bioresour. Tech., 99 (2008)
7224.
[4] N. Yalcin and V. Sevinc, Carbon, 38 (2000) 1943.
[5] A. Amaya, N. Medero, N. Tancredi, H. Silva and C. Deiana, Bioresour. Tech., 98 (2007) 1635.
[6] M. Conte, P. P. Prosini and S. Passerini, Mater. Sci. Eng., B108 (2004) 2.
[7] E. Diaz, S. Ordonez, A. Vega and J. Coca, Microporous and Mesoporous Mater., 82 (2005) 173.
[8] T. Qiang, Z. Zhigang, Z. Wenpei and C. Zidong, Fuel, 84 (2005) 461.
[9] H. Takagi, H. Hatori, Y. Yamada, S. Matsuo and M. Shiraishi, J. Alloys Comp., 385 (2004) 257.
[10] E. David, J. Mater. Proc. Tech., 162-163 (2005) 169.
[11] Y. Kojima, Y. Kawai, A. Koiwai, N. Suzuki, T. Haga, T. Hioki and K. Tange: J. Alloys andComp., 421 (2006) 204.
[12] J. Hayashi, A. Kazehaya, K. Muroyama, A.P. Watkinson, Carbon 38 (2000) 1873.
[13] Y. Zou, B-X. Han, Adsorp. Sci. Technol., 19 (2001) 59.
[14] L. Khezami, A. Chetouani, B. Taouk, R. Capart, Powder Technol. 157 (2005) 48.
[15] S. Brunauer, P. H. Emmett, E. Teller, J. Amer. Chem. Soc, 60 (1938) 309.
[16] R. S. Mikhail, S. Brunauer, E. E. Bodor, J. Colloid and Interface Sci., 26 (1968) 45.
[17] S. Brunauer, L.S. Deming, W.E. Deming, E. Teller, J. Amer. Chem. Soc., 62 (1940) 1723.
(Received: 23 March, 2012, Accepted: 30 June, 2012)4

Transactions on GIGAKU 1(2012)01017/1-6

Recent Progress in Laser Patterning of Crystals in Glasses
Takayuki Komatsu*, Futoshi Suzuki, Kazuki Ogawa, Tsuyoshi Honma
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: komatsu@mst.nagaokaut.ac.jp

This paper reviews shortly the laser patterning of crystals in oxide glasses, inparticular, two-dimensional planar crystals of nonlinear optical -BaB2O4 andferroelastic ’-Gd2(MoO4)3. By scanning Yb:YVO4 fiber lasers (wavelength:1080 nm) continuously with a small step (e.g., 0.5 m) between laser
irradiated areas, planar -BaB2O4 and ’-Gd2(MoO4)3 crystals are patterned
successfully on the glass surface, and a preferential growth orientation is
confirmed from linearly polarized micro-Raman scattering spectrum andsecond harmonic intensity measurements. It is found that the crystal growth
direction is perpendicular to the laser scanning direction. This relation, i.e.,
the perpendicular relation, is different from the behavior in discrete crystal
line patterning, where the crystal growth direction is consistent with the laser
scanning direction. The laser-induced crystallization has a potential for thepatterning of epitaxial crystals on the glass surface.

1. Introduction
Glass is important and key materials in advanced technologies. But, of course, it is impossible totake out all kinds of functional properties in glass itself as similar to other kinds of materials. In
particular, as glass has random structure with inversion symmetry in atomic arrangements, inprinciple, active properties arising from anisotropic atomic arrangements such as second-order
optical nonlinearity and ferroelectricity are not expected in glass. If such active functional properties
are added to glasses, new glass-related materials with various functions would be possible forpractical applications. One of the effective methods for fabrication of such active glasses is todevelop new functional glass-crystal hybrid materials, i.e., functional glass-ceramics or crystallized
glasses. Crystallization of glass proceeds through two steps of nucleation process and crystal growth
process, and usually glass-ceramics containing active crystals are fabricated by heat treatment in anelectric furnace.
Recently, laser-induced crystallization in glass has received much attention, because in which
active crystals are patterned only in spatially selected part. So far, dots and lines consisting of
crystals have been patterned in glasses by laser irradiations, i.e., zero-dimensional dot and
one-dimensional line patterning [1-11]. From a viewpoint of practical device applications forlaser-patterned crystals, it is of interest and importance to pattern two-dimensional planar crystals[12-14]. In this review, we report mainly the patterning of two-dimensional planar crystals of
-BaB2O4 (-BBO) and ’-Gd2(MoO4)3 on the glass surface.

2. Experiment
Two glasses with the compositions of 8Sm2O3-42BaO-50B2O3 (mol%) for the pattering of
-BBO and 3Sm2O3-18.25Gd2O3-63.75MoO3-15B2O3 (mol%) for the patterning of ’-Gd2(MoO4)3
were prepared using a conventional melt quenching technique. Glass transition, Tg, andcrystallization peak, Tp, temperatures were determined using differential thermal analysis (DTA) at aheating rate of 10 K/min. The quenched glasses were annealed at Tg for 30 min to release internal
stresses. Glasses were mechanically polished to a mirror finish with CeO2 powders. The glass
surface was irradiated by continuous wave (cw) Yb:YVO4 fiber laser with a wavelength of =1080
nm using objective lens (magnification: 50 times, numerical aperture: NA=0.8). The plate-shaped
glasses were put on the stage and mechanically moved during laser irradiations to construct1

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two-dimensional planar crystals. Laser-irradiated parts were observed with a polarization optical
microscope (POM) and a confocal scanning laser microscope (CSLM) (Olympus-OLS 3000). The
crystalline phase in the laser-irradiated part was examined from micro-Raman scattering spectrum
measurements (Tokyo Instruments Co., Nanofinder; Ar+ laser with = 488 nm). Second harmonic
generations (SHGs) were measured with a Maker fringe technique, in which a Q-switched Nd:YAG(yttrium aluminum garnet) laser with =1064 nm was used as the incident light and second harmonic(SH) light (=532 nm) was detected.
For the laser pattering, the rare-earth atom heat (REAH) processing has been used, in whichlasers such as cw Yb:YVO4 fiber laser are irradiated onto glasses with some amounts of Sm3+.
Irradiated lasers are absorbed by Sm3+ ions in glass through f-f transitions, and absorbed energies aretransferred to thermal energies through non-radiative relaxation process (electron-phonon couplings).
And thus surroundings of RE ions are heated locally, consequently inducing effectively structuralmodifications such as crystallization in glass [5]. In Fig. 1, the schematic model for the laser-induced
local heating mechanism in Sm3+ ion containing glasses is shown. The equipment for thelaser-induced crystallization constructed in our study is shown in Fig. 2.
6F
9/2(~1080nm)

Non-radiative
relaxation

Laser
1064nm1080nm

6H
5/2

Sm3+

Fig. 1

Fig. 2

Non-radiative relaxation process in the
f-f transition in Sm3+ ion.

Equipment for the patterning of crystals
in glass constructed in our lab.

3. Results and Discussion(1) Patterning of -BaB2O4
The values of the glass transition and the crystallization peak temperatures for
8Sm2O3-42BaO-50B2O3 glass prepared were determined to be Tg=566oC and Tp=681oC, respectively.
It was determined from an optical absorption spectrum that the optical absorption coefficient (: atroom temperature) at =1080 nm for the glass is =10.1 cm-1. The crystalline phase formed by heat
treatments in an electric furnace was identified to be only the -BaB2O4 crystalline phase (ICDD#
85-0914) [3,14-16]. In the previous paper [3], discrete lines consisting of highly oriented(single-like) -BBO crystals have been patterned on the glass surface of 10Sm2O3-40BaO-50B2O3
and 10Dy2O3-45BaO-55B2O3 by irradiations of cw Nd:YAG laser with =1064 nm. Because
phenomena taking place in the laser-irradiated region in glass depend on laser irradiation conditions(laser type, power, scanning speed) and properties of a given glass, it would be necessary to confirm
the quality of -BBO crystal lines patterned by cw Yb:YVO4 fiber laser (=1080 nm) in the present
glass. The CSLM photograph for the discrete line patterned by cw Yb:YVO4 fiber laser irradiations
with a laser power of P=0.80 W and a laser scanning speed S=4 m/s is shown in Fig. 3. The bumpwith a height of 0.4 m and a width of 5 m is observed in the laser-irradiated part. The azimuthal
dependence of SHG signals for the discrete line is shown in Fig. 4, in which the measurements werecarried out in the configuration of H-H. The notation of H-H means that the linearly polarized
electric field of the incident laser is parallel to the electric field of SH waves in the measurements.
Furthermore, the rotation angles of 0o and 180o correspond to the configuration that the polarized2

Transactions on GIGAKU 1(2012)01017/1-6

electric field of the incident laser is parallel to the line growth direction. The maximum SH
intensities are observed at the rotation angles of ~0o and ~180o, the minimum intensities are locatedat ~90o and 270o. These profiles indicate the high orientation of -BBO crystals in the discrete line
patterned by cw Yb:YVO4 fiber laser irradiations. It was also confirmed from the linearly polarized
micro-Raman scattering spectra that the growth direction of -BBO crystals along laser scanning
direction is the c-axis [14].
2.0

SH intensity (arb.units)

P=0.8 W, S=4 m/s

h=0.4 m
w=5 m

H-H

1.5

1.0

0.5

10 m
0

60

120

180

240

300

360

Rotation angle (deg.)

Fig. 3

CSLM photograph for the sample
obtained by laser irradiations. The laser
power and scanning speed were P=0.8
W and S=4 m/s, respectively.

Fig. 4

Azimuthal dependence of SH intensities
for the discrete line patterned by laser
iradiations (Fig. 3).

The idea for the patterning of planar -BBO crystals by laser irradiations is to irradiate lasers
with small steps between laser irradiated areas [12-14]. That is, a forthcoming (next) laser irradiation
is overlapped to the former (before) laser-irradiated part. The laser scanning was repeated with a stepof 0.5 m between the lines using the condition of P=0.8 W and S=8 m/s, and the CSLM
photograph for the sample obtained is shown in Fig. 5. It is found that the surface of the overlappedlaser-irradiated part is smooth, suggesting the well overlapping of laser-irradiated parts.
2.0

SH intensity (arb.units)

P=0.8 W, S=8 m/s
Step distance: 0.5 m

H-H

1.5

1.0

0.5

20 m

0

50

100

150

200

250

300

350

Rotation angle (deg.)

Fig. 5 CSLM photographs for the sample
obtained by laser irradiations. The laser
scanning direction and step direction are
shown in the figure.

Fig. 6

Azimuthal dependence of SH intensities
for the two-dimensional planar -BBO
crystals obtained by laser irradiations(Fig. 5).

The azimuthal dependence of SHG signals for the overlapped laser-irradiated part was measured,and the result is shown in Fig. 6. The measurements were carried out in the configuration of H-H as
3

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similar to the case of the discrete line shown in Fig. 3. The maximum SH intensities are observed at
the rotation angles of ~90o and ~270o, and the minimum intensities are located at 0o and ~180o.
These results are largely different from those observed for the discrete line shown in Fig. 4.
Furthermore, the appearance of the two-fold angular dependence shown in Fig. 6 demonstrates that
-BBO crystals in the overlapped laser-irradiated part with a step of 0.5 m are highly oriented [14].
The linearly polarized micro-Raman scattering spectra at room temperature for thetwo-dimensional planar -BBO crystals were measured. The results suggest that -BBO crystals in
the overlapped laser-irradiated part are highly oriented and the c-axis direction of -BBO crystals isperpendicular to the laser scanning direction or parallel to the laser step direction. In order toexamine the orientation of -BBO crystals in the overlapped laser-irradiated part more in detail,two-dimensional micro-Raman scattering spectra (imaging) were measured at room temperature.
The results are shown in Fig. 7. In this experiment, the intensity of the peak at 637 cm-1 is focused,
and the white color means the strong intensity of the Raman peak at 637 cm-1. It is seen that theintensity of the white color is small in the initial laser-irradiated part (discrete line) and also in theoverlapped (repeated) laser-irradiated part of about 10 m at the beginning (near the discrete line).
However, it is found that the intensity of the white color increases gradually and becomes almost
constant with increasing step distance. These results indicate that the orientation of -BBO crystalschanges gradually [14].
20mInterface acts as a nucleation site

Direction of c-axisof -BBO

Super‐cooled liquid
Overlappedarea

‐BaB2O4

Glass

Transitionzone

‐BaB2O4

Direction of c-axisof -BBO

Fig. 7

Fig. 8Two-dimensional micro-Raman
scattering spectra (imaging) at room
temperature. The intensity of the peak at
637 cm-1 is focused, and the white color
means the strong intensity of the Raman
peak at 637 cm-1.

Laser scanning

Schematic model for the crystal growthin the laser overlapped region for thetwo-dimensional planar -BBO crystals
obtained by laser irradiations (Fig. 7).

The melting temperature of -BBO crystals is known to be 1095oC. At this moment, thetemperature of the laser-irradiated region (Yb:YVO4 laser (=1080 nm), P=0.8 W and S=8 m/s) is
not measured, but it is obvious that the temperature is higher than the crystallization temperature(Tp=681oC) of 8Sm2O3-42BaO-50B2O3 (mol%) glass examined in this study. As can be seen in Fig.
5, any damage such as melting or ablation is not observed at the surface of the overlappedlaser-irradiated part, and thus, it is expected that the temperature would not be higher than themelting temperature (1095oC) of -BBO crystals. In the overlapped laser-irradiated part, therefore, it
is considered that initial -BBO crystals patterned by laser irradiations are not melted, but melt (i.e.,
super-cooled liquid) is created at the side (i.e., the glass part) of initial -BBO crystals. Considering
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the c-axis direction of -BBO crystals in the overlapped laser-irradiated part, it is concluded that theinterface between the initial -BBO crystals and melt might have an important role for the growthand orientation of new (forthcoming) -BBO crystals. In other words, the initial -BBO crystals
might act as nucleation sites for the growth of new -BBO crystals.
(2) Patterning of '-Gd2(MoO4)3
The patterning of two-dimensional planar ’-Gd2(MoO4)3 crystals with a high orientation has
been succeeded [13]. This crystal shows a unique morphology in the line patterned by laser
irradiations [7,13]. The POM photograph (top view) for the sample obtained by cw Yb:YVO4 fiber
laser irradiations with a laser power of P=1.2 W and a laser scanning speed of S=5 m/s is shown in
Fig. 9, indicating that a periodic structural change with different refractive indices is formed along
the laser scanning direction. The periodic structural change shown in Fig. 9 has been proposed to call“self-organized periodic domains”. Considering the width (~4 m) of laser-patterned lines, the laser
scanning was repeated with the pitch (step) of 0.7 m between the lines, and the POM photographfor such a sample is shown in Fig. 10. A unique pattern is obtained, and its morphology is largely
different from the discrete line (Fig. 9). That is, periodic domain structures with the bright and dark
regions do not appear along the laser scanning direction, but are created to the direction
perpendicular to the laser scanning direction [13].

P=1.2 W
S=5 m/s

Fig.10 POM photograph for the sample obtainedby cw Yb:YVO4 fiber laser irradiations
with a pitch of 0.7 m between lines.

Fig. 9 POM photograph (top view) for the
sample obtained by cw Yb:YVO4 fiber
laser irradiations with P=1.2 W and S=5m/s.

It was confirmed from micro-Raman scattering spectra that crystals patterned by laser
irradiations with a pitch of 0.7 m consist of the ferroelastic ’-(Sm,Gd)2(MoO4)3 crystalline phase.
In order to examine the periodic domain structure shown in Fig. 10 more in detail, two-dimensional
micro-Raman scattering spectra (imaging of the shift from the peak position of 845 cm-1) were
measured at room temperature for laser-patterned crystals as similar to the case of two-dimensional
-BBO crystals (Fig. 7). The degradation of color depending on the domain structure was clearlyobserved, indicating that the antisymmetric Mo-O stretching vibrations in (MoO4)2- tetrahedra in’-(Sm,Gd)2(MoO4)3 crystals patterned by laser irradiations change periodically depending on thedomain structure. It was also found that the SH intensity changes depending on the rotation angle in
the Azimuthal dependence curve. These results indicate that ferroelastic ’-(Sm,Gd)2(MoO4)3
crystals in the two-dimensional planar region are oriented and their crystal growth directions are5Transactions on GIGAKU 1(2012)01017/1-6

perpendicular to the laser scanning direction [13].
4. Conclusion
The laser-induced crystallization method was applied to pattern two-dimensional planar
-BaB2O4 and ’-Gd2(MoO4)3 crystals on the glass surface. By scanning Yb:YVO4 fiber lasers(wavelength: 1080 nm) continuously with a small step (e.g., 0.5 m) between laser irradiated areas,highly oriented planar crystals were patterned successfully. In both crystals, the crystal growth
direction is perpendicular to the laser scanning direction. Two-dimensional LiNbO3 crystals werealso patterned successfully on the surface of CuO-Li2O-Nb2O5-B2O3-SiO2 glasses [12]. It is required
to examine more in detail about the orientation of crystals and the possibility of the so-calledepitaxial crystal growth. The laser-induced crystallization method presented here was first developedin Nagaoka University of Technology, and from the point of view of “GIGAKU” (the policy andtarget in Nagaoka), further studies and applications of this technique are strongly required for the
progress in materials science and technology.
Acknowledgments
This work was supported from the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan (No. 23246114), and by Program forHigh Reliable Materials Design and Manufacturing in Nagaoka University of Technology.
References[1] R. Sato, Y. Benino, T. Fujiwara, and T. Komatsu, J. Non-Cryst. Solids, 289 (2001) 228.
[2] T. Honma, Y. Benino, T. Fujiwara, T. Komatsu, and R. Sato, Appl. Phys. Lett., 82 (2003) 892.
[3] T. Honma, Y. Benino, T. Fujiwara, T. Komatsu, and R. Sato, Apply. Phys. Lett., 83 (2003)
2796.
[4] T. Honma, Y. Benino, T. Fujiwara, and T. Komatsu, Apply. Phys. Lett., 88 (2006) 231105.
[5] T. Komatsu, R. Ihara, T. Honma, Y. Benino, R. Sato, H.G. Kim, and T. Fujiwara, J. Am.
Ceram. Soc., 90 (2007) 699.
[6] M. Kusatsugu, M. Kanno, T. Honma, and T. Komatsu, J. Solid State Chem., 181 (2008) 1176.
[7] Y. Tsukada, T. Honma, and T. Komatsu, Appl. Phys. Lett., 94 (2009) 059901.
[8] A. Stone, M. Sakakura, Y. Shimotsuma, G. Stone, P. Gupta, K. Miura, K. Hirao, V. Dierolf, andH. Jain, J. Non-Cryst. Solids, 356 (2010) 3059.
[9] F. Suzuki, T. Honma, and T. Komatsu, J. Solid State Chem., 183 (2010) 909.
[10] K. Kioka, T. Honma, and T. Komatsu, Opt. Mater., 33 (2011) 1203.
[11] T. Komatsu, K. Koshiba, and T. Honma, J. Solid State Chem., 184 (2011) 411.
[12] T. Honma and T. Komatsu, Opt. Express 18 (2010) 8019.
[13] F. Suzuki, T. Honma, and T. Komatsu, Mater. Chem. Phys., 125 (2011) 377.
[14] F. Suzuki, K. Ogawa, T. Honma, and T. Komatsu, J. Solid State Chem., 185 (2011) 130.
[15] H. Tanaka, T. Honma, Y. Benino, T. Fujiwara, and T. Komatsu, J. Phys. Chem. Solids, 64(2003) 1179.
[16] T. Honma, Y. Benino, T. Fujiwara, R. Sato, and T. Komatsu, J. Phys. Chem. Solids, 65 (2004)
1705.
(Received: 28 March, 2012, Accepted: 20 April, 2012)6

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Condensation Control of Nanoscale Polymer Aggregates by Using
Atomic Force Microscope Tip
Akira Kawai
Department of Electrical Engineering, Nagaoka University of Technology
1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan
E-mail, kawai@nagaokaut.ac.jp

This paper reviews shortly condensation behavior of polymer aggregate inside-wall of resist pattern characterized by using atomic force microscope(AFM). Various hardening process, thermal heating and EB irradiation, areperformed on the resist pattern after the pattern development. The
condensation of polymer aggregates in the side-wall is clearly imaged. By thehardening treatments, condensation of polymer aggregate is accelerated. Theinteraction force among polymer aggregates can be analyzed based on
Derjaguin approximation. The condensation structure of polymer aggregate
accompanying with aggregate vacancy is also discussed.

1. Introduction
In recent years, condensation control of polymer aggregate has become important in order toimprove line edge roughness (LER) in a side-wall of resist pattern [1]. The atomic force microscope(AFM) system has proven to be a versatile instrument for imaging minute structure of condensed
matter [2]. Recently, present authors have already proposed the novel principle for direct analysis
method of resist pattern adhesion, that is, direct peeling method by using AFM tip (DPAT) [3-6]. By
the DPAT method, quantitative analysis of cohesive properties of resist pattern has been conducted.
In this paper, by using the AFM, the condensation of polymer aggregate in side–wall of resist patternis characterized. Particularly, the special attention is paid to the dependency of hardening processes.
2. Experiment2.1. Resist material and line pattern fabrication
The KrF excimer laser resist, chemically amplified type, consisting of hydroxystyrene as a basepolymer was used. The resist film was coated onto a Si(100) wafer by a spinning method. The nativeoxide layer was removed by dipping into HF aqueous solution prior to spin coating of the resist film.
The pre-baking treatment was carried out at 100℃ for 1 min on a proximity hot plate. The
silane-coupling treatment with hexamethyl-disilazane (HMDS) was performed at 80℃ for 60 s. Theline patterns of 130 nm width were imaged to the resist film by using an KrF excimer laser stepper.
After the pattern exposure, the resist films were baked at 100℃ for 1 min. Then, the resist films
were developed by dipping into tetramethyl-ammoniumhydroxide (TMAH) 2.38 % aqueous solution
for 60 s. Subsequently, the resist patterns were rinsed in the deionized water for 30 s and dried by thespin method. As the hardening processes, heat treatment and EB irradiation were performed. The
heat treatment was performed at 150℃ for 5 min in the ambient condition. The charge density of
EB irradiation was approximately 5μC/cm2 with the acceleration voltage of 50 kV.
2.2. Condensation analysis with AFM
A commercially available AFM, integrated with a microtip, was used for the condensation
analysis. As a tip used for the investigation, a conical tip mounted on the cantilever apex was used１

Transaction on GIGAKU 1 (2012) 01018/1-6(Fig.1). The radius of curvature of the tip apex was approximately 8 nm. The tip was made of a
Si3N4 film. The tips with exposure to the HMDS vapor were prepared. The calibration measurementsof the spring constant correlated well with the manufacturer’s given value of k=0.44 N/m.

50nm
Fig. 1 SEM photograph of apex of AFM tip used for the investigation.

With the invention of the AFM, we have been equipped in good time with the appropriate tools
to understand the cohesive properties of condensed matter on a nanometer scale. Figure 2 shows aschematic explanation of side-wall imaging by the DPAT method. Prior to the side-wall observation,
the line resist pattern was imaged by the AFM in the non-contact mode. The tip traversed the line
pattern by moving the piezo stage under the scanning control. By directly applying load to the top
corner of the resist pattern with the AFM tip, the line pattern adhering on the substrate can becollapsed easily (Fig.2a). After the pattern collapse, the side-wall of resist pattern can be imaged
easily as shown in Fig.2b. When the adhesion strength between the resist pattern and the substrate isrelatively large, a certain residue of resist remains on the substrate. The measurement of DPAT
method is described in detail in our previous reports [3-6].
AFM tip
Imaging

Applying load

Resistpattern

Patterncollapse
Residue

Substrate(a) Pattern collapse(b) Side-wall imaging

Fig. 2 Side-wall imaging by the DPAT method.

3. Results and Discussion
3.1. Condensation structure of polymer aggregate in side-wallFigures 3a to 3c show the side-wall images of the resist pattern after the DPAT process. The
resist pattern can be collapsed easily without any damages. The square area marked in each side-wall
image is magnified in order to analyze condensation of polymer aggregates precisely.
2

Transaction on GIGAKU 1 (2012) 01018/1-6500[nm](a) Non-treatment(after pattern development)(b) Heat treatment(c) EB irradiation

Fig. 3

AFM image of side-wall of resist pattern performed with bakeand EB hardening treatment.

Figure 4 shows the side-wall images, surface topological profile and condensation structure
model for each treatment. In the AFM images, roughened surface images in side-wall due tocondensation of polymer aggregates can be clearly observed. In the case of the non-treatment(Fig.4a), the size of each polymer aggregate is approximately 20 nm. In the case of the heat
treatment (Fig.4b), the large aggregate composed with many aggregates can be observed. Moreover,polymer aggregate becomes large due to condensation acceleration by the EB irradiation and theaggregate size is approximately 60 nm (Fig.4c). The polymer aggregate smaller than 10 nm size
could not be resolved, because the limitation is caused by tip-sample convolution. The condensation
model of polymer aggregate was discussed by Bug [7]. In this study, the condensation property ofpolymer aggregate is analyzed based on Derjaguin approximation in the following section.
3

Transaction on GIGAKU 1 (2012) 01018/1-6(a) Non-treatment(after pattern development)
100[nm]20
Surface profile [nm]A

Range: 9.0nm

15
10
5
0

A’

A
0

400

0

A’

100
200
300
400
Position in side-wall [nm](b) Heat treatment
20
Surface profile [nm]B

Range: 10.3nm

15
10
5
0

B’

B
0

400

0

B’

100
200
300
400
Position in side-wall [nm](c) EB irradiation
20
Surface profile [nm]C

Range: 8.8nm

15
10
5
0

C’
400

0

(AFM image of side-wall)

C
0

B’
C’

100
200
300
400
Position in side-wall [nm](Surface topological profile)(Condensation structure model)

Fig. 4 Condensation of polymer aggregate in side-wall of resist pattern.

4Transaction on GIGAKU 1 (2012) 01018/1-6

3.2. Condensation structure of polymer aggregate
In general, the size of micro condensed matter contributes strongly to surface roughness of solidmaterial[1,8]. The size of polymer aggregate can be regarded as the representative value whichreflects the condensation properties in side-wall of resist pattern. Figure 5 shows schematic
explanation of condensation behavior of polymer aggregate based on Derjaguin approximation.

R2
D
F

R1

･
Rn

R3
･
･

Fig. 5 Interaction force model between two spheres based on
Derjaguin approximation.

As shown in Fig.5, based on Derjaguin approximation, a sum of interaction force F acting amongsome micro spheres (radius: R1, R2, …, Rn) can be expressed as follows.

F 

R1 Rn
A
,
2 
6 D n R1  Rn

(1)

Interaction force F [a. u.]where symbols A and D represent Hamaker constant and interaction length between two spheres.
The simple simulation result of interaction force is shown in Fig.6. It seems reasonable to supposethat the interaction force decreases with decreasing size of polymer aggregates. However, when theinteraction length is less than 10 nm, the interaction force increases rapidly. Therefore, it can be
explained that the small size aggregate is more likely to condense around the large size one, as
shown in Fig.4b. This theory provides a very simple but direct approach to understand condensationof polymer aggregate.
40
R1=40 nm
D=0.4 nm
35
30
0.5
25
20
15
0.7
10
1
5
2
0
1
10
100
Radius Rn [nm]41000Fig. 6 Dependency of interaction force on spherical radius.

5

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Meanwhile, the present author found out the vacancy of polymer aggregate formed in the resist
pattern [6]. In most cases, the vacancy size is approximately 60nm. It is entirely fair to say that avacancy of polymer aggregate is formed during the condensation processes. In Fig.7, thecondensation models of polymer aggregate accompanying some vacancies are illustrated. We can be
fairly certain to discuss that these condensed model are effective to study the formation mechanismsof roughened side-wall of resist pattern.

(a) Non-treatment
Substrate

Vacancy(b) Heat treatment(condensation)
Substrate(c) EB irradiation(aggregate growth)
Substrate
Fig. 7 Condensation model of polymer aggregate by hardening treatment(cross sectional view)

4. Conclusion
Condensation properties of polymer aggregate in side-wall are characterized by the DPAT
techniques. By the hardening of resist pattern, condensation structure of polymer aggregate is
drastically changed. The condensation model of polymer aggregate can be constructed based on
Derjaguin approximation.
Acknowledgements
This work was supported from the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, and by Program for High Reliable
Materials Design and Manufacturing in Nagaoka University of Technology.
References[1] T. Yoshimura, H. Shiraishi, J. Yamamoto and Shinji Okazaki, Appl. Phys. Lett., 63 (1993) 764.
[2] G. Binnig, C. F. Quate and Ch. Gerber, Phys. Rev. Lett., 56 (1986) 930.
[3] A. Kawai and Y. Kawakami, Ext. Abstr. 44th Spring Meet. Japan Society of Applied Physics andRelated Societies, Chiba, March, (1997) p.576.
[4] A. Kawai, Proc. SPIE’s 24th Inter. Symp. Microlithography, Santa Clara, 3677 (1999) p565.
[5] A. Kawai and N. Moriike, Microelectronic Engineering, 57-58 (2001) 683.
[6] A. Kawai, J. Photopolymer Sci. Technol., 15 (2002) 371.
[7] A. L. R. Bug, M. E. Cates, A. A. Safran and T. A. Witten, J. Chem. Phys., 87 (1987) 1824.
[8] J. N. Israelachvili, “Intermolecular and surface force”, 2nd Edition, Academic press (1997) p326.
(Received: 29 May, 2012, Accepted: 12 June, 2012)6

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Intensity of Singularity Analysis for Three-dimensional Two-phase
Transversely Isotropic Piezoelectric Bonded Joints
Md. Shahidul Islam1,*, Hideo KOGUCHI2
Graduate school of Nagaoka University of Technology,2)
Department of Mechanical Engineering, Nagaoka University of Technology,1603-1 Kamitomioka-machi, Nagaoka 940-2188, Japan*E-mail: shahidul@stn.nagaokaut.ac.jp
1)

Stress singularity frequently occurs at a vertex in an interface of joints due toa discontinuity of materials. The stress singularity fields are one of the main
factors responsible for debonding under mechanical or thermal loading. The
stress distribution near the vertex in the interface of joints is very important tomaintain the reliability of joints. However, the intensity of singularity for 3D
transversely isotropic piezoelectric bi-material joints has not been made clear
until now. In this paper, intensity of singularity in transversely isotropic
piezoelectric dissimilar material joints is analyzed. The stress and electric
displacement distributions on an interface and the intensity of singularity forstress and electric displacement are investigated using BEM. From thenumerical results, it is shown that the intensity of stress singularity increases
and the intensity of electric displacement singularity decreases with theincrease of material thickness in joint.

1. Introduction
In recent years, intelligent or smart structures and systems have drawn more and more attention.
Piezoelectric materials have been extensively used as transducers and sensors due to their intrinsic
direct and converge piezoelectric effects that take place between electric field and mechanicaldeformation. Piezoelectric materials are playing a key role as active components in many fields ofengineering and technology such as electronics, laser, microwave infrared, navigation and biology[1]. Mechanical stress occurs in piezoelectric material for any electric input. The stressconcentrations caused by mechanical or electric loads may lead to crack initiation and extension, andsometimes the stress concentrations may be high enough to debond the material parts. Industrial
products such as electronic devices and heat endurance parts are composed of dissimilar materials. A
mismatch of material properties causes a failure at the free edge of joints, because a stressconcentration occurs along the free edge of interface especially at the vertex of joint [2]. When twomaterials are joined, a free-edge stress singularity usually develops at the intersection of the interface
and the free surface. Stress singularity is related to debond and delamination at interface of thebonded joints. The stress distributions around the vertex were determined using a boundary element
method (BEM). Koguchi [3] determined the intensity of singularity by fitting the stress profile
obtained from BEM analysis with a least squares method. Constabel et al.[4] proposed a method tocompute their singularity exponents and the associated angular singular functions. Recently Hwu [5]and Ikeda et al.[6] proposed the solution of singular stress field and its SIFs of an interfacial cornerof a 2D dissimilar piezoelectric material joints using extended Stroh formalism. At present, no clear
picture exists of the problem of intensity of singularity for 3D two-phase transversely isotropic
piezoelectric dissimilar material joints. Therefore, the purpose of this study is to analyze the intensityof singularity in transversely isotropic piezoelectric dissimilar material joints.
2. Formula of Analysis
In the absence of body forces and free charges, the governing equations of three-dimensionalpiezoelectric materials are expressed as follows: ij, j  0
Di,i  0(1)
1

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where ij and Di are stress tensor and electric displacement vector, respectively. These equations are
the elastic equilibrium equations and Gauss’s law of electrostatics, respectively. The constitutive
relations for piezoelectric material are expressed as follows:Di  eikl kl   ik Ek(2)
 ij  cijkl kl  ekij Ekwhere kl is the strain tensor which is the mechanical field variables, Ek is electric field, cijkl is theelastic constant, ekij (eikl) and ik are the piezoelectric constant and electric permittivity (dielectric
constant), respectively.
The elastic strain-displacement and electric field-potential equations are expressed as follows:1
ij  u j,i  ui, j (3)Ei  ,i2
where ui and  are the elastic displacement and electric potential, respectively.
According to Ding et al.[1], the fundamental solutions of the governing differential equations forthe transversely isotropic piezoelectric material are shown as follows:
3
3
 
 
u  i  ov i  o
s1  s2  s3  s1 yxi1 xi1 y3w   i1
i1

i
zi

3

   i 2
i1

i
zi

(4)

where, s1, s2, and s3 are the three roots of the characteristic equation, which is related to thefollowing equation.
as 6  bs 4  cs 2  d  0(5)
In three roots of Eq. (5), s1 is assumed to be a positive real number, s2 and s3 are either positive real

number or a pair of conjugate complex roots with positive real parts.
Function i in equation (4) for an infinite piezoelectric material is given as follows:o  0andi  Ai sign(z  zo ) ln Rii  si z  zo (i  1, 2, 3)(6)

The expressions of stress and electric displacement are obtained in terms of the function i as below.
3 
2 
2
2 
 xx   c11  c12  o   i 2  c11  c12  2 ixy i1  zi
x 
3 
2 
2
2 
 yy  c11  c12  o   i 2  c11  c12  2 ixy i1  zi
y 
3
3
2
 2 
2  2i
 zz    i1 2 i
 xy  c66  2o  2 2o   2c66 
zi
x 
 zo
i1
i 1 xy xz  o12 o 3
 2 i  i1
yzi i1
xzi

 yz  o1

2  o 3 2 i  i1
xzi i1yzi

Dx  o22 o 3
 2 i  i 2yzi i1xzi

Dy   o2

2 o 3
 2 i  i 2
xzi i1
yzi

3Dz    i 2
i1

where

 2 izi2(7)

i  c13i1  e31i 2  si  c12

o1  c44 so

i1  c44 si  i1   e15i 2

i 2  e15 si  i1   11i 2

o 2  e15 so i1  c33i1  e33i2  si  c13

 i2  e33i1   33i2  si  e31c   m s  c44  s
i1  11 112

i2  11 152
3 i

m  m s s12 ic e  m4 si2  c44 e33 si4m1  m2 si2 si

4
33 i

i

Another stress and electric displacement are derived for two domain.
2

Transaction on GIGAKU 1 (2012) 01019/1-6

3. Boundary Integral Equation
Based on the Somigliana equation, the boundary integral formulation is expressed as follows:
C(d)u(d)   U* (d, x)t(x)d   T* (d, x)u(x)d


(8)



where C is the coefficient matrix, which depends on shape of the boundary , and the generaldisplacement vector u, and surface traction vector t are as follows:u  uvt  t x  ,
T

w

ty



T

tz

and two matrices U* and T* composed of fundamental solution are:
*
u11
 *
u
U*   21
*
u31
*
u41

*
u12

*
u13

*
u22

*
u23

*
u32

*
u33

*
u42

*
u43

1* 


2* 
,
 3* 
 4* 

t11*
*
t
*
T   21*
t 31*
t 41

t12*

t13*

1* 

*
t 22

*
t 23

2* 

*
t 32

*
t 33

 3* 

*
t 42

*
t 43




 4* 

where uij* and tij* (i, j = 1, 2, 3) are displacements and surface tractions, respectively at a field
point x in the j coordinate direction due to a unit load acting in the xi directions at a load point d,
u 4* j and t 4* j (j = 1, 2, 3) are displacement components and surface tractions, respectively, at a field
point x in the j coordinate direction due to a unit electric charge at d. i* and i* (i = 1, 2, 3) areelectric potentials and surface charges, respectively, at a field point x due to a unit load acting in thexi directions at a load point d, and  4* and 4* are electric potential and surface charge respectively
at a field point x due to a unit electric charge at d.
If the boundary is discretized with an eight-node isoparametric quadratic element, then theboundary integral equation is written as follows
8

C d u d    e i 1

1

1



1

1

8

T ui N i J d d    e i 1

1

1

 U t N J d d
1

1



i

i

(9)

where Ni is the shape function, J is Jacobian matrix, ui and ti represent the displacement andsurface traction at node, and C is the coefficient matrix.
4. Eigenequation
An eigenequation based on the finite element method (FEM) was used to analyze the singularity
at the singular point in a 3D dissimilar material joint. In the formulation of FEM, a sphericalcoordinate system with the origin at a singular point is introduced, and displacement within a sphereof radius ro in the singular field is expressed using the characteristic root p, which is related to theorder of singularity. The surface of the sphere is divided into mesh.
The eigenequation was formulated for determining the order of stress singularity as follows:p A p B CU  0
2

(10)

where

U  ur

u

u

 ,
T

A    k a  k sa  , B   k b  k sb  , and C   k c  k sc .
S

S

S

3

Transaction on GIGAKU 1 (2012) 01019/1-6Here, p represents the characteristic root, which is related to the order of singularity,, as
= 1-p. [A], [B] and [C] are matrices composed of material properties, and {U} represents theelastic displacement and electric potential vector.
5. Result and Discussion
Figure 1(a) represents a model for 3D piezoelectric bonded structure used in the present analysis.
The displacement and electric potential in the z-direction on the bottom in the model is fixed. The
model is subjected to a uniform tension (1 MPa) and electric displacement (1 C/m2) whose poling
direction is parallel to the z-axis. Figure 1(b) represents the geometry of a typical case where asingular stress state occurs at the point o. The region surrounding the singular point is divided into a
number of quadratic elements with a summit o, with each element being located in sphericalco-ordinates r, , and  by its nodes 1 to 8.
1 Mpaz
1 C/m2

Material 1

10 mm

Singular pointSingularity Point

h
r

5 mm

r

o


P

y


z

Material 2

x

10 mm

y

20 mm

(a) Model of analysisx(b) Element geometry and natural

co-ordinate
Fig. 1 Model of analysis for two-phase piezoelectric bonded joint
Table1 Material properties of piezoelectric materials
Elastic Constant, 1010 N/m2

Material
PZT-4
PZT-5H

c11
12.6
12.6

c12
7.78
5.50

c13
7.43
5.30

c33
11.5
11.7

c44
2.56
3.53

Piezoelectric
Constant, C/m2
e31
e33
e15
-5.20 15.1
12.7
-6.50 23.3
17.0

Dielectric Constant,
10-10C/Vm
11
33
64.63
56.22
151.0
130.0

The model of three-dimensional two-phase transversely isotropic bonded joints and the distributionof nodal displacement are shown in Fig. 2. This figure is plotted, when 1MPa tensile load is appliedon the upper material and 1 C/m2 charge. And the elastic constants of lower material are 100 times
larger than that of the upper material.

4Transaction on GIGAKU 1 (2012) 01019/1-6

Fig. 2 Two-phase piezoelectric bonded joints and distribution of nodal displacement
The order of singularity, , at the vertex for the above model is calculated by eigenanalysis
method. Solving eigenequation yields many roots p and eigenvectors are obtained. However, if theroot p is within the range of 0 < p < 1, this fact indicates that the stress field has singularity. The
values of the order of singularity at the singularity corner are 0.582, 0.315, 0.178, and 0.079.
The stress distribution at the vertex in a singular field is expressed for spherical coordinate
system as follows: ij r,  ,    K1ij f1ij ( ,  )r    K 2ij f2ij ( ,  )(11)where r is the distance from the vertex, vertex is the order of stress singularity, Kkij (k = 1, 2) is thevertexintensity of stress singularity, fkij(, ) (k = 1, 2) is the angular function for stress component, ij.
Similarly, the electric displacement distribution at the vertex in a singular field is expressed forspherical coordinate system as follows:Di r,  ,    M 1i l1i ( ,  )r    M 2i l2i ( ,  )(12)where Mki (k = 1, 2) is the intensity of electric displacement singularity, lki (, ) (k = 1, 2) is theangular function for electric displacement component, Di.
The distributions of stress and electric displacement in the singular field for the two-phasepiezoelectric bonded structure are obtained using boundary element method. Figure 3 demonstrates
the distributions of stresses, , r,  and electric displacement, D against radial distance, r, on
the interface of piezoelectric bonded joint. The plots for stresses, , r and electric displacement,D are parallel in log-log graph. The red line indicates the line for Ar-λvertex, where A is a constant,vertex (=0.582) represents the order of singularity at the vertex. The slope of all the curves is in good
agreement with the result of eigenanalysis. Therefore, the power law singularity governs the stressand electric displacement field near the vertex. All figures show that the stress and electric
displacement is larger at the vertex of the interface. Therefore, there is a possibility to debond anddelamination at the corner of the bonded joint.
vertex(degree)
10
20
30
40
45
50
60
70
80
Curve Fit

4(MPa)

2

10

8
6
4
2

(degree)
10
20
30
40
45
50
60
70
80
Curve Fit210r(MPa)

8
6

8
6
4
2

1

8
6
4

1

8
6

2

10

-4

-3

10

10

-2

10

-1

10

-4

0
-5

10
2

5

10

-2

10

-1

(degree)
10
20
30
40
45
50
60
70
80
Curve Fit

2

D(C/m )(MPa)

10

-3

4

(degree)
10
20
30
40
45
50
60
70
80
Curve Fit

15

10

r (mm)r (mm)

4
2

1

-10

8
6

8
6
4

-15
2

0.05

0.10

0.15

0.20

10

r (mm)

-4

-3

10

10

-2

10

-1

r (mm)

Fig. 3 Distributions of stress and electric displacement against radial distance, r5Transaction on GIGAKU 1 (2012) 01019/1-6

Figure 4 shows a relationship between the intensities of singularity for stress and electric
displacement and the thickness of upper material, h. The intensities of singularity are calculated byfitting the stress and electric displacement curves with the help of the result of eigenanalysis. The
boundary condition in BEM analysis as shown in Fig. 1(a) is agreed with the first value of the orderof stress singularity. Therefore, the first value of the order of stress singularity at the vertex is used inintensities of singularity calculation. The intensity of stress singularity increases with thickness ofupper material, but the intensity of singularity for electric displacement decreases with the thickness.


7) -0.5820.14

K1r(MPa.mm

K(MPa.mm-0.582

)

0.16

0.12

6

5x10

-2

0.10
2

3

4

5

6

7

8

9

1

2

10

3

4

5

Thickness, h (mm)

8

9

10



-0.582)

M(C/m .mm)-0.582

K1(MPa.mm

7

Thickness, h (mm)

9

5

6

1

8

2

4

-2

3x10

-2

2

3

4

5

1

6

7

8

7x10

9

2

10

3

4

5

1

Thickness, h (mm)

6

7

8

9

10

Thickness, h (mm)Fig. 4 Distribution of intensity of singularity against thickness, h
6. Conclusion
In this paper, the intensity of singularity that characterizes a singular field at a vertex in 3D
dissimilar material joints was investigated using a boundary element analysis. The distributions ofstress and electric displacement with respect to radial distance were calculated. The stress andelectric displacement have a larger value at the vertex. The intensities of singularity were calculated
by fitting the stress and electric displacement curves with the help of the result of eigenanalysis at
the vertex. The intensity of stress singularity increases with the thickness of upper material, but theintensity of singularity for electric displacement decreases with the thickness, because the effect ofcharge for thin material is larger than that for the thick material.
References[1] H. Ding and B. Chen, Int. J. Solids Struct., 34(23) (1997) 3041.
[2] W. Attaporn and H. Koguchi, CMES, 39(3) (2009) 237.
[3] H. Koguchi, Trans. JSME, A72(724) (2006) 2058.
[4] M. Constabel, M. Dauge, and Y. Lafranche, Comput. Meth. Appl. Mech. Eng., 190 (2001) 2111.
[5] C. Hwu, Int. J. Solids Struct., 45 (2008) 4460.
[6] T. Ikeda, H. Hirai, and M. Abe, Proceedings of the ASME, InterPACK (2011) 1.
(Received: 23 March, 2012, Accepted: 20 April, 2012)6

Transactions on GIGAKU 1(2012)01020/1-5

Evaluation of Internal Coarse Defects in Alumina Powder Compact and Ceramics
Satoshi Tanaka*, Keizo Uematsu
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: stanaka@vos.nagaokaut.ac.jp

The internal coarse defects in alumina ceramics and powder compact before
and after firing were observed by using the micro-focus x-ray computed
tomography (micro CT) and the optical microscopy. We studied theeffectiveness of micro CT by comparing with images in the powder compact
by the optical microscopy. Furthermore, the relationships between powder
compact and ceramics was examined. CT images show the large pores among
and in granules as well as images taken by the optical microscopy. However,
the external shapes of compressed granules were clearly observed by the
optical microscopy. In ceramics, lots of ark cracks were detected well in
micro CT images, whereas only shadows of coarse defects were observed in
the optical microscopy. Many large defects were derived from interstices
between granules and dimples in granules. Some large defects which were
present in green compact and survived the densification process of sintering.

1. Introduction
Coarse defects are present in ceramics, reducing the mechanical property and reliability. New
optical microscopy successfully revealed their characteristics for the first time, allowing a directcomparison between the characteristics of coarse defects and the mechanical properties of ceramics[1-11]. Very good agreement was noted among the simulated strength and its distribution, and thosedetermined by direct measurement [12-14]. Clearly, the formation mechanism of these coarse defects
must be understood in detail for drastic improvement of properties and reliability in ceramics. A
study with an optical microscope revealed that these coarse defects are formed from non-uniformity
in the powder compact of alumina powder during sintering, or densification process. Non-uniformity
over a certain degree appeared to be transformed into coarse defects in ceramics. Recently, themicro-focus x-ray computed tomography (micro CT) has made a great progress and has started to be
used to analyze internal structure in dental materials, etc [15,16]. The micro CT is a useful tool for
non-destructive visualization of bone and tooth structures, and is expected for ceramic materials. The
objective of this study is to examine the internal coarse defects in alumina powder compact before
and after firing using micro-focus x-ray computed tomography (micro CT), and compare the micro
CT images obtained with the optical microscopy.

2. Experiment
The alumina powder compact and ceramics (Alumina Referceram, AL-1) were used forobservation. The alumina powder compact was formed by dry-pressing method using spray-driedgranules (Fig.1). The relative density of alumina ceramics was ~ 98%. The powder compact andalumina ceramics were cut to a cubic 1mm x 1mm x 1mm on an each side. The samples wereobserved by micro focus x-ray computed tomography (micro CT) (SkyScan 1172) with a voltage of
40-100kV and a current 100-250 µA. It is possible to detect internal pores larger than 0.85 µm.
Obtained image data was analyzed using 2D/3D software (SkyScan CTAn ).
For observation by the optical microscopy, the samples were sliced and polished to 100 µm thick.
Powder compact was made transparent with an adequate immersion liquid. The methylene iodide
has a refractive index 1.74, which is almost the same with that of alumina. The internal
microstructure was observed with an optical microscope in transmission mode (Olympus BX51).
1

Transactions on GIGAKU 1(2012)01020/1-550µm
Fig. 1 SEM micrograph for alumina granules used in this study
After firing, thinned ceramics with 100 µm was observed directly with an optical microscope intransmission mode. Coarse defects in the ceramics were characterized in detail with SEM after the
specimen was polished down to the depth of the defects.

3. Results and Discussion(1) Internal structure of powder compacts
Figure 2 shows the internal structures of the alumina powder compact taken by the micro CT.
Figure 3 shows the image taken by the optical microscopy. Both figures show circular shape of
granules, some coarse defects between granules, and dimples inside of granules as a black spot. The
trace of granules and dimple inside granules are very clearly observed in the optical micrograph (Fig.
3), whereas the morphology of coarse defects in micro CT images is ambiguous. In micro CT, thelarge pore which has zero density, are easily detected due to difference of adsorption of x-ray
between particles and pore. However, fine pores among the primary particles and the interstices ofdeformed granules are not found, since they have similar density each other. This is a reason why theshapes of granules are confused in micro CT images.

100µm
Fig. 2 Internal microstructure of powder
compact taken by the micro CT

Fig. 3 Internal microstructure taken by the
optical microscopy2

Transactions on GIGAKU 1(2012)01020/1-5(2) Coarse defects in alumina ceramics
Figure 4 shows the internal structures of the alumina ceramics taken by the micro CT. There arelots of large defects with several 10 µm to 100 µm in length. The coarse defects distributes
homogeneously in ceramics. Shape of coarse defects is not spherical but crack type. This result
suggests that interstices among granules and dimple in granules remain even after firing.
Figure 5 shows the structure of alumina ceramics taken by the optical microscopy Fig. 5(a) andthe SEM micrograph (Fig. 5(b)) of an extremely large crack found at center of the optical
micrograph. Sample thickness is around 100 µm. Many dark spots are noted in ceramics in Fig. 5(a),which correspond to pores. The biggest one appears to be developed from dimple of a granule. This
black region was observed by SEM in detail (Fig. 5(b)). This crack is a trace of dimple left in
ceramics after sintering. This crack is also detected in micro CT. The results show that the size ofcrack is clear in the optical microscopy, but the detail of each crack is cloudy.

(a)

(b)

50m

100m
Fig. 4 Internal microstructure of alumina
ceramics taken by the micro CT

Fig. 5100m

Internal microstructure of alumina
ceramics
takenby
opticalmicroscopy
(a),
and
SEM
micrograph of a coarse defectappeared by polishing at same
position(b).

(3) Various defects in alumina ceramics
Figure 6 shows various coarse defects in alumina ceramics and each detailed images taken by
micro CT. The coarse defects with about 60 µm (Fig. 6(a)) is at triple points of granules. In Fig. 6(b),there is a circular defect with 30 µm, which is derived from dimples of granules. Finer defects with
<10 µm can be observed.

4. Conclusion
The internal coarse defects in alumina ceramics and powder compact were characterized by
micro-focus x-ray computed tomography (micro CT) and the usual optical microscopy with thinned
sample. The large pores among and/or in granules in the powder compact was observed by micro CT.
However, the external shapes of compressed granules were ambiguous compared to the optical
microscopy. In ceramics, lots of cracks were detected well in micro CT. The origins of many large
defects were discussed from each shape of coarse defects. Interstices between granules and dimples
in granules in powder compact are left in ceramics after sintering.
3

Transactions on GIGAKU 1(2012)01020/1-5

(a)

(b)

(b)

triple point of granules
(c)

(d)

100µm

30μm
(d)

(c)

Fig. 6 Various coarse defects of alumina ceramics taken by micro CT (a), (b) three points
between granules, (c) dimple of granules and (d)fine pores in alumina ceramics.

Acknowledgments
This work was supported by Program for High Reliable Materials Design and Manufacturing in
Nagaoka University of Technology. The authors thanks to Prof. Komatsu, Prof. Kamado and Prof.
Nakayama for observation of micro-focus x-ray computed tomography.
References[1] F. F. Lange, J.Am.Ceram.Soc., 72 (1989) 3.
[2] F. F. Lange, J.Am.Ceram.Soc., 66 (1983) 396.
[3] K. Uematsu, J-Y. Kim, Z. Kato, N. Uchida and K. Saito, J.Ceram.Soc.Japan, 98 (1990) 515.
[4] K. Uematsu, J-Y.Kim, M.Miyashita, N.Uchida and K. Saito, J.Am.Ceram.Soc., 73 (1990) 2555.
[5] K. Uematsu, M. Miyashita, J-Y. Kim, Z. Kato and N. Uchida, J.Am.Ceram.Soc., 74 (1991)
2170.
[6] K. Uematsu, M. Miyashita, J-Y. Kim and N. Uchida, J.Am.Ceram.Soc., 75 (1992) 1016.
[7] J-Y. Kim M. Miyashita and N. Uchida, J.Am.Ceram.Soc., 27 (1992) 6609.
[8] M. Miyashita, J-Y. Kim, Z. Kato, N. Uchida and K. Uematsu, J.Ceram.Soc.Japan., 100 (1992)
1357.
[9] K.Uematsu, Powder Technology, 88 (1996) 291.
[10] N. Shinohara, M. Okumiya, T. Hotta, K. Nakahira, M. Naito and K. Uematsu, J. Mater. Sci., 34(1999) 4271.
[11] K.Sato, S. Tanaka, N. Uchida and K. Uematsu, J. Ceram.Soc.Japan, 111 (2003) 525.
4

Transactions on GIGAKU 1(2012)01020/1-5[12] Y. Zhang, M. Inoue, N. Uchida and K. Uematsu, J. Mater. Res., 14 (1999) 3370.
[13] S. Nakamura, S. Tanaka, Z. Kato and K. Uematsu, J.Am.Ceram.Soc., 92 (2009) 688.
[14] S. Nakamura, S. Tanaka, R. Furushima, K.Sato and K. Uematsu, J.Ceram.Soc.Japan, 117 (2009)742.
[15] T. Hayakawa, H. Mishima, I. Yokota, T. Sakae, Y. Kozawa and K. Nemotoet, Dental Materials
Journal, 19 (2000) 87.
[16] T. Nakamura, K. Wakabayashi, Y. Kawamura, S. Kinuta, Y. Mutobe and H. Yatani, Dental
Materials Journal, 26 (2007) 598.
(Received: 1 June, 2012, Accepted: 12 June, 2012)5

Transactions on GIGAKU 1(2012)01021/1-6

Ultrasound Effect of Interpenetrated Hydrogels Having Hydrogen
Bonding Networks in Poly(Vinylalcohol)/Poly(2-Hydroxyethyl
Methacrylate) by Using FT-IR Spectroscopy
Kazuki Nakasone, Venegas S. Josue Addiel, Motohiro Tagaya, Takaomi Kobayashi*
Department of Material Science and Technology, Nagaoka University of Technology
1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan*E-mail: takaomi@nagaokaut.ac.jp
In hydrogels made of interpenetrated polyvinylalcohol (PVA) and poly2-hydroxyethyl methacrylate (PHEMA), ultrasound (US) influenced tochange breaking ability of hydrogen bonds between both polymers. When the
PHEMA content in the interpenetrating hydrogel was changed from 10wt%
to 20wt%, the IR band intensity of the OH stretching band at 3400 cm-1 wasdecreased by the exposure of US at the frequency of 43 kHz and output
power of 8.5 W. This result indicated that the hydrogen bonding both
PHEMA and PVA segments were broken by the US. The loose penetration in
the 10wt% hydrogels could be easily influenced by the US exposure forbreaking hydrogen bondings, which loosely formed the interpenetration
between PHEMA and PVA.

1. Introduction
Ultrasound (US) treatment is known as a non-conventional and environmentally clean
technology for some industrial uses. For mechanical engineering, cleaning industries, and healthdiagnosis, the US has been used to promote both physical and chemical processes. Therefore, US
technology has become attractive to chemists and chemical engineers for various purposes in areas
as diverse as electrochemistry, food technology, synthetic chemistry, materials science, andnanotechnology [1,2]. Therefore, most works related to US are advancing. The origin of suchsonication effects on heterogeneous processes is known. Among them, it was reported that US
influenced strongly on breaking hydrogen bonding between poly acrylic acid (PAA) and Al2O3 inaqueous slurry solution [3]. The US absorption caused a significant decrease in the slurry viscosity,
because of the evidence supported by breaking the hydrogen bond networks in the aqueous slurries
due to US stress. According to the reported researches, it was established that the US led to change
breaking ability of hydrogen bonds of aqueous polymers of acrylic acid and N-isopropyl acrylamide[4].
On the other hand, interpenetrating polymer network (IPN) is a polymer comprising two or more
networks which are interlaced on a polymer scale but not covalent bonded to each other. These
networks are entangled in such a way that they are connected and cannot be pulled apart, but notbonded to each other by any chemical bond [5,6]. It was reported that PVA with interpenetrating
PAA, Poly 2-hyroxyethyl methacrylate (PHEMA) was prepared by UV radiation method forpolymer electrolyte membrane [7,8]. These kinds of IPN type hydrogels were cross-linked byhydrogen bond. Therefore, we expected to control their hydrogen bonding networks by using US
stimuli since these kinds of work in hydrophilic polymer gels containing water were not well known
in view points of ultrasound effects. In this study, the PVA/PHEMA hydrogels were cross-linked byboth hydrogen bond and entangled polymer chains (Scheme 1), and applied for the purpose of
examination of US effect on the hydrogen bonding networks of IPN hydrogels.
2. Experiment2.1. Materials
The experimental procedure of preparation of IPN hydrogel of PVA and PHEMA is as follows.
The 87.0~89.0 mol% hydrolyzed PVA (DP=1700) was purchased from Kuraray, Ltd..
2-hydroxyethyl methacrylate (HEMA) and Ammonium persulfate (APS) were purchased from1

Transactions on GIGAKU 1(2012)01021/1-6

Nacalai Tesque, Inc. All chemicals were used without further purification.

(a) PVA(b) PHEMA

Scheme 1 Chemical structures of (a) PVA and (b) poly HEMA for IPN hydrogels
2.2. Synthesis of interpenetrating hydrogels
Interpenetrated PVA/PHEMA hydrogels were prepared as following procedure (Figure 1).
Aqueous solution of 10wt% PVA was mixed with 10 or 20wt% of HEMA having 1wt% APS used as
initiator of HEMA polymerization. The mixture solution was spread in vial (35 mm diameter and 65
mm height) and kept at 50°C for 12 h under nitrogen atmosphere. The resultant white solid (inFigure 1) was well washed with excess of distilled water in order to remove the HEMA monomerfrom the hydrogel block. Here, the polymers of PVA/PHEMA prepared from 10wt% and 20wt% of
HEMA concentration were abbreviated as PVA90PHEMA10 and PVA80PHEMA20, respectively.

Fig. 1 Preparation procedure of IPN PVA/PHEMA hydrogels (left side) and photograph of
PVA90PHEMA10 (right side).
2.3. Measurement of US effect on PVA/PHEMA hydrogels
The US equipment used in the present work was a sonoreactor (HSR-305R, Honda Electronics
Co., Ltd.) that was connected with amplifier (HSA4012, NF Corporation) and Multi function
synthesizer (WF1943B, NF Corporation) passing through matching box. As shown in Figure 2, theUS was exposed to the sample hydrogel sandwiched by CaF2 window plates (31.5 mm diameter and1.5 mm thickness) at 25°C for 10 min. The input power of the US to the transducer was 50 W at 43
kHz and the exposed power into the water bath was 8.5 W, which was evaluated with calorimetricmethod.
As shown in Figure 1, the white hydrogels were obtained after polymerization. To obtain
information of the chemical changes in the hydrogels, Fourier transform infrared (FT-IR) spectra of
the hydrogels were recorded on a FT-IR spectrophotometer (FT-IR/4100, JAS.CO) in transmission
mode in two CaF2 windows (Pier Optics Co., Ltd.). For the preparation of the sample solutions,firstly, about 1 mg of the hydrogel sliced was put on the CaF2 window and then 0.1 l of water were
slowly dropped to the gel surface. The transparent solution was left to rest for one hour and then thehydrogel was covered with another CaF2 plate. The edges of the CaF2 windows were sealed using2

Transactions on GIGAKU 1(2012)01021/1-6

teflon tape in order to prevent water penetration through the US exposure in the water bath. The
FT-IR spectra were measured in transmittance mode before and after the US exposure was done for5 min and 10 min.

Fig. 2 Scheme showing the experimental setting for the measurement of US exposure to
PVA/PHEMA hydrogel in US water bath and preparation of hydrogels for FT-IR
measurement

Storage elastic modulus G'[Pa]Loss elastic modulus G''[Pa]3. Results and Discussion
Before investigation of US effect, the viscoelastic properties of the PVA90PHEMA10 and
PVA80PHEMA20 were examined. Figure 3 shows the relationship between strain (%) and storageand loss elastic modulus (GPa) for the IPN hydrogels. The comparison in both hydrogels suggested
that the viscoelastic data of the PVA80PHEMA20 included highly entangled polymer chains, which
implied formation of rigid networks. Actually, the water contents of the PVA90PHEMA10 and
PVA80PHEMA20 were 380% and 320%, respectively. This demonstrated that higher PHEMA
content decreased hydrophilicity of the hydrogels.

1.0E+04

1.0E+03

1.0E+02

0.01

0.1

1
10
100 1000
Strain [%]G':PVA80PHEMA20 G'':PVA80PHEMA20
G':PVA90PHEMA10 G'':PVA90PHEMA10

Fig. 3 Viscoelastic behaviors (G’ and G’’) of the IPN PVA/PHEMA hydrogels
Figure 4 shows the FT-IR spectra of PVA90PHEMA10 and PVA80PHEMA10. The spectra were
measured after 5 or 10 min exposure of US for the hydrogels sandwiched with CaF2 plates. These
indicated that the band intensities of hydrogen bonded OH stretching at around 3400 cm-1 and C=O
stretching at around 1730 cm-1 were decreased after the US exposure with depending on US
irradiation time. The peak attributed to H2O at around 2150 cm-1 was shifted toward low
wavenumber. Herein, the spectral band in 2000-2300 cm-1 resulted in hydrogen bonding of water tothe hydrogels. For the PVA90PHEMA10, the hydrogen bonding band shifted from 2160 cm-1 to2100 cm-1 with the increase of the US exposure time. Additionally, the OH stretching appeared at3

Transactions on GIGAKU 1(2012)01021/1-6

3100 cm-1 was changed in the intensity. Namely, the IR intensity at 3100 cm-1 was intense when the
exposure time was increased to 10 min. These spectral changes indicated that free H2O weregenerated by US exposure. However, the PVA80PHEMA20 showed less change of the spectra when
the US was similarly exposed. Slight change of the hydrogen band of water was shifted toward 2110
cm-1 from meaning the 2120 cm-1. Also, the OH stretching band at 3100 cm-1 showed no changeeven though US was exposed. This meant that the interpenetration of the PHEMA segments highlybound with the PVA segments as the PVA80PHEMA20 hydrogel and regulated water mobility in thehydrogel.
PVA80PHEMA20

TransmittanceTransmittance

PVA90PHEMA10

US exposure for 10min
US exposure for 5minwithout US

4000

Fig. 4

3500

3000
2500
Wavenumber [cm-1]

US exposure for 10min
US exposure for 5minwithout US
4000

2000

PVA80PHEMA20

TransmittanceTransmittanceFig. 52000

FT-IR spectra of the IPN PVA/PHEMA hydrogels swelling by water at the different US
exposure times.

PVA90PHEMA10

4000

3500
3000
2500
Wavenumber [cm-1]

After 30min
After 20min
After 10min
After 5min
US exposure
3500

3000
2500
Wavenumber [cm-1]

2000

4000

After 30min
After 20min
After 10min
After 5min
US exposure
3500

3000
2500
Wavenumber [cm-1]

2000

FT-IR spectral changes of the IPN PVA/PHEMA hydrogels swelling after stopping US
exposure.

As shown in Figure 5 for the FT-IR spectra measured after the US stopping water band near 2100
cm-1 changed to show higher wavenumber shift. Here, the exposure time of the US was 10 min.
Their FT-IR spectra of the PVA90PHEMA10 after stopping US exposure showed that peak at around2150 cm-1 was shifted toward 2200 cm-1 with increasing time, but the OH band about 3400 cm-1 waschanged insignificantly (Figure 6). It was evidenced that the hydrogen bonds related with water werebroken by US exposure and recovered with stopping US exposure. On the other hands, there wasinsignificant change of the FT-IR spectra of PVA80PHEMA20 which showed rigid IPN gel networks
as indicated in Figure 3. For the viscoelastic data in PVA80PHEMA20, it was reasonable to consider
that the highly entangled polymer chains seemed to restrict the US action for breaking hydrogenbonds networks. Therefore, water release from gel microenvironment might be weaker than that of
the PVA90PHEMA10 showing loosed IPN networks.
4

Transactions on GIGAKU 1(2012)01021/1-6

2250

2250

PVA90PHEMA10

PVA80PHEMA20

2200

Wavenumber [cm-1]

Wavenumber [cm-1]

PVA90PHEMA10

2150

2100

2050

PVA80PHEMA20

2200

2150

2100

2050
0

2.5

5
Time [min]

7.5

10

0

10

20

30

Time [min]Fig. 6 FT-IR spectra of the IPN PVA/PHEMA hydrogels swelling by water.
It is very interesting to know the ultrasound effect on the change in the FAT-IR spectra of PVA
and PHEMA with water. In order to consider the spectral change, we measured FT-IR spectra for
aqueous PVA as well as the IPN hydrogels. As presented in Figure 7a, the aqueous PVA
demonstrated quietly different change in the OH band when the US was exposed. The US action
enhanced OH stretching at 3100 cm-1. This indicated the US effected on the mobility of thestretching band for the PVA polymer chains, which were not restricted by IPN networks. It seemed
to be breakage of hydrogen bonds with water to release from the polymer microenvironment. As
shown for the PHEMA-water system in Figure 7b, the IR band intensity at 3400 cm-1 was lessenhanced rather than that of the PVA-water system. This meant that similar effect was happened in
the PHEMA. It was interesting to note that the maximum wavenumber for the OH stretching
was different in PVA and PHEMA with 3400 cm-1 and 3500-3600 cm-1, respectively.
aTransmittanceTransmittance

b

4000

US exposure for 10min
US exposure for 5minwithout US

Without US
US exposure for 5min

3500

3000
2500
Wavenumber[cm-1]

4000

2000

3500

3000
2500
Wavenumber[cm-1]

2000

Fig. 7 Comparison of FT-IR spectra of (a) aqueous PVA and (b) swelled PHEMA with and without
the US exposure.
As reported by Kitano et al. [9] with determination of a Fourier self-deconvolution of the original
O-H stretching bands of water molecules, the bulk water systems of polyethylene glycol-aqueous
solutions showed that the characteristics of water were determined by the analysis of the band for awater cluster. For example, the O-H stretching of pure water into five Gaussian components wereassigned at 3620, 3500, 3390, and 3215 cm-1 to a free water, water molecules whose hydrogen bonds
are partly broken, fully hydrogen-bonded water molecules uncorrelated (not in-phase), and fullyhydrogen-bonded water molecules correlated (in-phase, both donation of the protons and acceptanceof the protons with both of the electron lone pairs). Therefore the 3400 cm-1 in PVA assigned withfully hydrogen-bonded water molecules and the 3600 cm-1 band with free water molecules. Thus the5Transactions on GIGAKU 1(2012)01021/1-6

US exposure made partially free water from the polymer microenvironment of IPN networks.
4. Conclusion
The IPN hydrogels made of PVA and PHEMA were used for investigation of the US effect. As
influenced to change breaking ability of hydrogen bonds between both polymers, the results clearly
showed in FT-IR spectra that interpenetrated hydrogen bonds in PVA/PHEMA were broken by the
exposure of the US at 43 kHz and 8.5 W. In the case of PVA90PHEMA10, FT-IR spectra forhydrogen bonds around 3400 cm-1 were changed for the intensity of the OH stretching band by US
exposure. This meant that the US broke the hydrogen bands of the OH groups of the interpenetrated
PHEMA and PVA, and water molecules interacted with the hydrogel. The less change in the
PVA80PHEMA20 was observed by the US exposure. The tight interpenetration of the PHEMA andPVA segments caused that low mobility of the hydrogel responded to the less effect of the ultrasoundfor the water penetration into the matrix. These evidences were shown that the tendency depended
on the component of interpenetration of PVA and PHEMA for the formation of free water molecules
left from the IPN hydrogels.
References[1] K.S. Suslick and G.J. Price, Annu. Rev. Mater. Sci. 29 (1999) 295.
[2] T.J. Mason, Sonochemistry: The Uses of Ultrasound in Chemistry, The Royal Society ofChemistry, 1990, p. 132.
[3] N.L. Ngoc and T. Kobayashi, Ultrason. Sonochem., 17 (2010) 186.
[4] N.L. Ngoc and T. Kobayashi, Ultrason. Sonochem., 18 (2011) 1185.
[5] L.H. Sperling, Interpenetrating Polymer Networks, American Chemical Society, 1994, p.3–38[6] Y. Osada and K. Kajiwara, Gels Handbook, NTS, 1997, p. 63[7] J.M. Yang, H.Z. Wang, and C.C. Yang, J. Membr. Sci., 322 (2008) 74.
[8] J.M. Yang, C.Y. Chiang, H.Z. Wang, and C.C. Yang, J. Membr. Sci., 341 (2009) 186.
[9] H. Kitano, K. Ichikawa, M. Ide, M. Fukuda, and W. Mizuno, Langmuir, 17 (2001) 1889.
(Received: 18 April, 2012, Accepted: 18 June, 2012)6

Transactions on GIGAKU 1 (2012)01022/1-6

Crack Self-Repairing of Nano-Metal/Al2O3 Hybrid Materials
Makoto Nanko1, Daisuke Maruoka2, Pham Vu Hai3 and Nguyen Dang Thuy3
1

Nagaoka University of Technology,
Graduate School of Engineering, Nagaoka University of Technology,(Kamitomioka, Nagaoka, Niigata, 940-2188, Japan, Email: nanko@mech.nagaokaut.ac.jp)
3
Hanoi University of Science and Technology(1 Dai Co Viet Road, Hanoi, Vietnam)
2This paper reviews shortly the crack-repairing of Al2O3 based hybridmaterials dispersed with nano-sized metallic particles. Al2O3 dispersed with
nano-Ni particles and nano-Co particles shows crack-repairing function.
The crack-repairing of these systems is caused by the formation of the oxide
product formed by the outward diffusion of cations. Doping of Si and Y into
Al2O3 matrix is discussed on crack-repairing and high-temperature oxidationof Al2O3 dispersed with nano-Ni particles. On the other hand, no
crack-repairing was observed in Al2O3 dispersed with Mo.

1. Introduction
Al2O3-based ceramics have been used in structural applications. However they have somedisadvantages such as low fracture toughness, surface crack sensitivity for decreasing fracture
strength. Al2O3 based composites with nano-sized metallic particles (referred as to metal/Al2O3) are
expected as structural materials with high mechanical strength, high hardness and excellent thermaland chemical stability [1-19]. As well some of metal/Al2O3 have any other useful and uniquefunctions, which can be adapted in structural components. For example, dispersion of nano-Fe, Co or
Ni results in magnetism into Al2O3 based-ceramics.
Ando and his colleges have discovered the crack-repairing in some ceramic composites dispersedwith SiC particles or whiskers by a thermal oxidation [20-25]. In high-temperature applications of
those structural ceramics, crack-repairing takes place autonomously. Crack-repairing via heat
treatment was observed in monolithic Al2O3 [26, 27], crack-repairing of those composites isavailable for larger cracks and by treatment at lower temperature than the monolithic Al2O3. The
crack- repairing of those composites is caused by filling surface cracks with the oxidation product byhigh-temperature oxidation of the dispersoid. Crack-filling by the oxidation product decreases stressconcentration to surface crack. Surface cracks do not behave as the fracture origin. The mechanicalstrength will be recovered up to the level of the virgin materials without surface cracks. The similar
phenomenon of crack- repairing was observed in NiAl/Al2O3 [28], but not in TiC/Al2O3 [29].
Our research group has discovered newly crack-repairing of nano-Ni/Al2O3 [30, 31]. The
systematic studies of crack-repairing of the Al2O3-based composites are necessary for understanding
the mechanism of the crack-repairing of the Al2O3-based composites and to design the composites
with faster crack-repairing and better mechanical properties. In the present paper, crack-repairing of
those composites is discussed as well as high-temperature oxidation in order to understand the
kinetic mechanism of the crack-repairing.

2. Experiments
Al2O3-based materials dispersed with 5 vol% Ni, Co or Mo were prepared in the present study. A
slurry consisting of a commercial Al2O3 (average particle size: 0.4 μm, purity: 99.99%) and nickel or
cobalt nitrate for nano-Ni or Co/Al2O3 was produced with distilled water. In the case ofnano-Mo/Al2O3, molybdenum trioxide powder (average particle size: 0.7 μm) was mixed with the
Al2O3 powder by ball-milling. Those bulk specimens were sintered by using pulsed electric current
sintering at 1400°C under 40 MPa in uni-axial pressure during holding time of 5 min in vacuum.
1Transactions on GIGAKU 1 (2012)01022/1-6

Vickers hardness was measured with loading in 1.9 N. Fracture toughness was evaluated by using
the IF method by using Niihara’s equation [32].
Three Vickers indentations with loading at 49 and 490 N for 10 s were conducted into a sample
to possess 12 cracks. The cracks introduced were semi-circular with approximately 350 μm in depth
and 200 μm in length. Samples introduced cracks were heat-treated at temperature ranging from 600
to 1300°C for 1-48 h in air. Nano-Ni/Al2O3 were cut 3×4×26 mm and polished until mirror finish.
The 3-point bending test at room temperature was conducted to as-cracked samples and ones
oxidized at 1200°C for 6 h in air. The crosshead speed was 0.5 mm/min. Samples were evaluated byusing scanning electron microscopy (SEM) for microstructure and X-ray diffraction for phase
identification.

3. Results and Discussion(1) Mechanical properties of nano-metal/ceramic hybrid materials
Table 1 lists mechanical properties of various nano-metal/Al2O3. Nano-metal/Al2O3 has excellent
mechanical properties as high as or higher than 500 MPa and 5 MPam0.5.
Table.1 Mechanical properties of various nano-metal/ceramic hybrid materials
nano-Ni/Al2O3

nano-Co/Al2O3

nano-Mo/Al2O3

Grain Size:
Al2O3/ Metal [μm]1.1/ 0.3

1.1/ 0.3

1.1/ 0.3

Vickers Hardness [GPa]

17.1

16.1

19.9

Fracture Toughness[MPam0.5]

5.83

5.81

4.64

Bending Strength [MPa]

497

710

490

Sample(2) Crack-Repairing of nano-metal/ceramic hybrid materials
Figure 1 shows the SEM images on the surface of various Al2O3-based hybrid materials withsurface cracks before/after a thermal oxidation process. Figure 2 shows bending strength of various
Al2O3-based hybrid materials. Surface cracks on nano-Ni/Al2O3 and nano-Co/Al2O3 disappear after
heat treatment at 1200ºC for 6 h in air. The values of bending strength in nano-Ni/Al2O3 andnano-Co/Al2O3 were recovered by thermal oxidation process. XRD reveals that MAl2O4 spinel (M =Ni or Co) was formed on the surface during high-temperature oxidation. Removing surface Ni
particles by nitrate acid, NiAl2O4 was developed on the surface of nano-Ni/Al2O3. Those facts meanthat the spinel phase was formed by the oxidation of Ni/Co dispersoid with Al2O3 matrix andpartially by outward diffusion of Ni/Co ions during heat treatment. In the case of nano-Mo/Al2O3,
the surface cracks disappear via heat treatment at 700ºC for 6 h in air. However, the value of bending
strength of nano-Mo/Al2O3 with surface cracks was not increased after heat treatment. In thismaterials system, oxidation products, Al2(MoO4)3 covered only the surface of nano-Mo/Al2O3, not in
the inside of the cracks.

2Transactions on GIGAKU 1 (2012)01022/1-6

10 m

10 m
(a) nano-Ni/Al2O3(b) nano-Co/Al2O3(c) nano-Mo/Al2O3
Fig. 1 SEM images on the surface of various Al2O3-based hybrid materials with surface cracks
before/after a thermal oxidation process. (a) nano-Ni/Al2O3 (heat treatment at 1200C for 6
h in air), (b) nano-Co/Al2O3 (heat treatment at 1200C for 6 h in air), and (c)nano-Mo/Al2O3 (heat treatment at 700C for 6 h in air). The left and right ones show
as-cracked and as-heat treated ones, respectively.

3

0

Ni/Al 2O3

ere
d

Co/Al2O3

As
-c

rac
ked
70
6 h 0℃
,A
ir

As
-s

int

12
6 h 00℃
,A
ir

As
-s

int
e

red

200

As
-cr
ack
ed

400

As
-cr
ack
ed

600

12
6 h 00℃
,A
ir

ere
d
int

800

As
-s

Bending strength, / MPa

Transactions on GIGAKU 1 (2012)01022/1-6

Mo/Al2O3

Fig.2 Bending strength of various Al2O3-based hybrid materials.
(3) Relationship between high-temperature oxidation and crack-repairing of nano-metal/ceramic
hybrid materials
Some nano-metal/Al2O3 hybrid materials show the crack-repairing function to recover themechanical strength degraded by surface cracks. Because the crack-repairing process was anoxidation process at high-temperatures, degradation of the nano-metal/Al2O3 hybrid materials shouldtake place. High-temperature oxidation of nano-metal/Al2O3 hybrid materials is an oxidation of
nano-metallic particles in the Al2O3 matrix from the surface of the hybrid materials. Thickness of theoxidized zone was increased in the parabolic manner in the cases of nano-Ni/Al2O3 andnano-Co/Al2O3. Comparison between oxidation rate and crack-repairing rate is useful forunderstanding life time of nano-metal/Al2O3 hybrid materials.
Figure 3 shows thickness of the oxidized zone and ratio of crack-disappearance as a function ofoxidation time in nano-Ni/Al2O3 at 1200ºC. Crack-repairing of nano-Ni/Al2O3 occurs after thegrowth of oxidized zone. In fact, recovery of bending strength is completed for 1 h at 1200ºC in
nano-Ni/Al2O3. Degradation of nano-Ni/Al2O3 by heat treatment for crack-repairing is negligible.
As well, doping Y and Si into nano-Ni/Al2O3 can decrease growth rate of the oxidized zone andgives no significant change in ratio of crack-disappearance by thermal oxidation process [33, 34].
As shown in Fig. 3, thickness of the oxidized zone on Y-doped one is half of the un-doped one. On
the other hand, crack-disappearance of Y-doped one is comparable with un-doped one.

4

Transactions on GIGAKU 1 (2012)01022/1-6

Fig.3 Thickness of the oxidized zone and ratio of crack-disappearance as a function of oxidation
time in nano-Ni/Al2O3 at 1200ºC
4. Conclusion
Crack-repairing in nano-metal/Al2O3 hybrid materials was reported in the present paper. With
respect to nano-Ni/Al2O3 and nano-Co/Al2O3, surface semi-circular cracks with approximately 350
μm in length and 200 μm in depth can be repaired via heat treatment at 1200ºC for 1 h in air as thebending strength was recovered as high as the virgin samples. The surface cracks were filled with theoxidation product, MAl2O4 (M = Ni or Co), which is formed by both of oxidation of surface Ni
particles and reaction of Al2O3 with Ni ions diffused from the inside of the sample. During the heat
treatment for crack-repairing, nano-Ni/Al2O3 was oxidized from the surface. The oxidation depth
was much smaller than the crack size. However, nano-Mo/Al2O3 does not have crack-repairing
function via a thermal oxidation. Surface of nano-Mo/Al2O3 was covered by the oxidation product,
Al2(MoO4)3, but the cracks was not filled by the product.
Acknowledgments
This work was supported partially from the Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports, Culture and Technology, Japan by Program for High
Reliable Materials Design and Manufacturing in Nagaoka University of Technology.
References[1] H. Awaji: Strength of Ceramic Materials, (Corona Pub., 2001) pp. 6-8.
[2] K. Niihara: J. Ceram. Soc. Jpn., 99 (2000) 974-982.
[3] M. Nawa, T. Sekino and K. Niihara: J. Jpn. Soc. Powder Powder Metall., 39 (1992)
484-487.
[4] T. Sekino, T. Nakajima and K. Niihara: J. Am. Ceram. Soc., 80 (1997) 1139-1148.
[5] T.Sekino, A. Nakahira, M. Nawa and K. Niihara: J. Jpn. Soc. Powder. Powder. Metall., 38(1991) 14-18.
[6] T. Matsunaga, U. Leela-Adisorn, Y. Kobayashi, S. M. Choi and H. Awaji: J. Ceram. Soc.
Jpn., 113 (2005) 123-125.
[7] T. Isobe, K. Daimon, T. Sato, T. Matsubara, Y. Hikichi and T. Ota: Ceram. Int., 34 (2008)
213-217.
[8] X. Yao, Z. Huang, L. Chen, D. Jiang, S. Tan, D. Michel, G. Wang, L. Mazerolles and J. L.
Pastol: Matt. Lett., 59 (2005) 2314-2318.
5

Transactions on GIGAKU 1 (2012)01022/1-6[9] B. S. Kim, T. Sekino, T. Nakayama, M. Wada, J. S. Lee and K. Niihara: Mat. Res. Innovat.,
7 (2003) 57-61.
[10] T. Sekino and K. Niihara: J. Mater. Sci., 32 (1997) 3943-3949.
[11] M. Nawa, T. Sekino and K. Niihara: J. Mater. Sci., 29 (1994) 3185-3192.
[12] L. A. Diaz, A. F. Valdes, A. M. Espino and R. Torrecillas; J. Euro. Ceram. Soc., 23 (2003)
2829-2834,.
[13] W. P. Tai and T. Watanabe: J. Mater. Sci., 33 (1998) 5795-5801.
[14] W. P. Tai, Y. S. Kim and J. G. Kim: Mater. Chem. Phys., 82 (2003) 396-400.
[15] J. Li, X. Ni and G. Wang: J. Alloy. Comp. 440 (2007) 349-356.
[16] X. Shi, Y. Pan and J. Guo: Ceram. Intl., 33 (2007) 1509-1513.
[17] S. N. Hosseini, F. Karimzadeh and M. H. Enayati: Adv. powder Tech., (2011) in press.
[18] Z. Jiang and W. E. Rhine: Chem. Mater., 6 (1994) 1080-1086.
[19] A. Nakahira and K. Niihara: J. Ceram. Soc. Jpn., 100 (1992) 448-453.
[20] M. C. Chu, S. Sato, Y. Kobayashi and K. Ando: Fatig. Fract. Eng. Mater. Struct., 18 (1995)
1019-1029.
[21] K. Houjou, K. Ando, S. P. Liu and S. Sato: J. Euro. Ceram. Soc.. 24 (2004) 2329-2338.
[22] S. K. Lee, M. Ono, W. Nakao, K. Takahashi and K. Ando:, J. Euro. Ceram. Soc., 25 (2005)
3495-3502.
[23] W. Nakao, K. Takahashi and K. Ando, Self-healing of surface crack in structural ceramics,
in: S. K. Ghosh(Eds.), Self-healing Materials, Fundamentals, Design Strategies, andApplications, WILEY-VCH, Weinheim, 2009, pp. 183-217.
[24] K. Takahashi, M. Yokouchi, S. K. Lee and K. Ando: J. Am. Ceram. Soc., 86 (2003)
2143-2147.
[25] T. Osada, W. Nakao, K. Takahashi, K. Ando and S. Saito: J. Euro. Ceram. Soc., 27 (2007)
3261-3267.
[26] F. F. Lange and T. K. Gurta, J. Am. Ceram. Soc., 53 (1970) 420-421.
[27] Y. Matsuo, T. Ogasawara, S. Kimura, S. Sato and E. Yasuda: J. Ceram. Soc. Jpn., 99 (1991)
384-389.
[28] O. Abe, Y. Ohwa and Y. Kuranobu, J. Euro. Ceram. Soc., 26 (2005) 689-695.
[29] L. Jun, Z. X. Zheng, H. F. Ding, Z. H. Jin, Fatigue Fract Engng Mater Struct., 27 (2004) 89-97.
[30] A. L. Salas-Villasenor, J. Lemus-Ruiz, M. Nanko and D. Maruoka:,Adv. Mat. Res., 68(2009) 34-43.
[31] D. Maruoka and M. Nanko: Adv. Mat. Res., 89-91 (2010) 365-370.
[32] K. Niihara: Ceramics Japan, 20 (1985) 12-18.
[32] D. Maruoka and M. Nanko, Mater. Trans.,51 (2010) 1570-1573.
[33] M. Nanko, D. Maruoka and Y. Sato, Intl. J. Appl. Ceram. Technol., 9 (2012) 172-177.
(Received: 16 May, 2012, Accepted: 5 June, 2012)6

Transactions on GIGAKU 1(2012)01023/1-6

Development of Oxygen Sensors Exploiting Hot Spots in
GdBa2Cu3O7-δ-based Composite Ceramic Rods
Tomoichiro Okamoto, Yuichiro Kuroki, Masasuke Takata*
Department of Electrical Engineering, Nagaoka University of Technology,1603-1 Kamitomioka, Nagaoka 940-2188, Japan*E-mail: Takata@vos.nagaokaut.ac.jp
A hot spot, which is a local area glowing orange, appears in a LnBa2Cu3O7-δ
(Ln: rare earth element) ceramic rod when a voltage exceeding a certain
value is applied to the rod at room temperature. The electrical current running
through the hot spot depends strongly on the oxygen partial pressure inambient atmosphere, which acts as an oxygen sensor without the need for any
heating system. The GdBa2Cu3O7-δ rod tends to melt and break by a sustained
presence of the hot spot in a high Po2 (~100 kPa). The composite rods
containing dispersed high melting point materials such as BaAl2O4, BaZrO3
and Gd2BaCuO5 show a remarkable high durability in the high Po2. In a low
Po2 (< 0.02 kPa), the current decreases to almost zero and the hot spot
disappears, resulting in an insensitive rod to oxygen. The composite rods withdispersed CuO detects oxygen even in Po2 < 0.002 kPa. The response time isimproved by forming Gd2BaCuO5 core in GdBa2Cu3O7-δ rod. The results
suggest that the performance of hot spot oxygen sensors is improved by using
composite materials.

1. Introduction
LnBa2Cu3O7-δ (Ln: rare earth element), which is well known as the high-Tc superconductor, is atypical nonstoichiometric oxide. Oxygen deficiency δ increases with increasing temperature above
400oC [1]. The carrier density decreases with increasing δ, which results in a steep increase in theresistivity [2-4]. In other words, the material shows a positive temperature coefficient of resistivity(PTCR).
The present authors observed the phenomenon that a local area of a LnBa2Cu3O7-δ ceramic rod
glows orange once a voltage exceeding a certain value is applied at room temperature as shown in
Fig. 1 [5]. The visible glowing area was named a hot spot. The hot spot moves to the negativeelectrode with the velocity of a few mm/min. The appearance of the hot spot is related to the PTCR
characteristic [5-7]. The movement of the hot spot is considered to be caused by the diffusion ofoxide ions in the electric field over the hot spot [5-6]. The temperature of the hot spot in air is about
900oC which is almost the same as its
sintering temperature [6,8]. The heat
treatment using the hot spot can be used as
dc 10 V GdBa2Cu3O7-
an improvement technique for its transport
critical current density [8].
The current running through the rod
decreases abruptly when the hot spot
appears, and remains constant withincreasing voltage [6-9]. This is possible
10 mmbecause the size of the hot spot with highresistivity
increases
linearlywithincreasing voltage [5-7]. The rod with theFig. 1. Photograph of a hot spot appearing on a
hot spot can be used as a constant-current
GdBa2Cu3O7-δ ceramic rod. A dc voltage of 10 V
generator without any active component.
was applied to the rod at room temperature in air.
The current is sensitive to the gas flow1Transactions on GIGAKU 1(2012)01023/1-6

around the rod, and the rod can be used as a high sensitive gas flow sensor [10]. Since the current
vibrates in the form of damped sinusoidal oscillation under low oxygen partial pressure, the rod canbe used as a new type of sine wave oscillator [11].
The current after the appearance of the hot spot depends on the oxygen partial pressure inambient atmosphere, which acts as an oxygen sensor without the need for any heating system [6,10].
This paper reviews shortly the progress in the development of the oxygen sensors exploiting the hot
spots appearing in GdBa2Cu3O7-δ (Gd-123) -based composite ceramic rods.
2. General Features of Hot Spot Oxygen Sensor
Figure 2 shows current-voltage characteristics in various Po2 for Gd-123 ceramic rod preparedby conventional solid state reaction [6]. The current increases linearly with increasing voltage in low
voltage region. When the voltage reaches a certain value, the current abruptly decreases and a hot
spot appears in the rod. After that the current remains constant with increasing voltage and depends
strongly on Po2. This result means that the oxygen concentration in environment can be evaluatedfrom the current value, which is the principle of the hot spot oxygen sensor [6,10].
Figure 3 shows the oxygen partial pressure dependence of current through the rod with the hot
spot. The relationship between the logarithmic current and the logarithmic Po2 well agrees with adotted straight line with a slope of 1/6.
When the oxygen content (7-δ) of LnBa2Cu3O7-δ is higher than 6.2, the sample behaves like anoxygen excess (metal deficit) oxide [12]. If the LnBa2Cu3O7-δ is kept at high temperature and the Po2
is raised, the sample absorbs oxygen that dissociates into holes and oxide ions. The overall reaction
is expressed as

O2 = 2 Oi’’+ 4 h·(1)where Oi’’ is an interstitial divalent oxide ion and h· is a hole. Therefore, the LnBa2Cu3O7-δ
essentially has a p -type conduction since the excess oxygen plays the role of acceptor. The massaction law for equation (1) isK

[Oi'' ] 2 [h  ]4
[O2 ]

(2)

where K is the equilibrium constant, and [Oi’’], [h·] and [O2] represent the concentrations of
GdBa2Cu3O7-δ
1.0

PO / kPa

0.4

100
30
10
5
3
1

0.3
0.2

GdBa2Cu3O7-δ

0.7

2

Current I / A

Current / A

0.5

0.4

I ∝ Po21/6

0.2

0.1
0
0

5

10

15

Voltage / V

Fig. 2. Current-voltage
various Po2 [6].

characteristics

0.1

20

1

10

100

Po 2 / kPa
Fig. 3. Oxygen partial pressure dependenceof current through the rod with the hot spot.

in2

Transactions on GIGAKU 1(2012)01023/1-6

interstitial oxide ion, hole and oxygen (=Po2), respectively. When the formation of defects
represented by equation (2) is predominant, the insertion of the relation 2[Oi’’] = [h·] into equation(2) yields[h·] ∝ Po2 1/6(3)

Since the conductivity σ is proportional to [h·], the current is proportional to Po2 1/6.
3. Improvement of durability in high Po2
To enable the practical use of the hot spot oxygen sensor, the durability of the sensing rods isimportant. The Gd-123 rod tends to be molten and broken by a sustained presence of the hot spot ina high Po2. The authors have found that the Gd-123-based composite rods with dispersed BaAl2O4[13], BaZrO3 [14] and Gd2BaCuO5 (Gd-211) [15] shows remarkable improvement in the durability.
Figure 4 shows the withstanding electric power in air for the various composite rods. For therod with BaAl2O4, with increasing BaAl2O4 content, the power increases significantly until thecontent increases to 20 vol%, slightly increases at the contents between 20 and 40 vol%, anddecreases gradually when the content exceeds 40 vol%. The results suggest the appropriate amountof BaAl2O4 is around 20 vol%. From the same way of the evaluation, suitable amount for BaZrO3
and Gd-211 are found to be 30 and 7 vol%, respectively.
To evaluate the durability in high Po2, the presence of hot spot has been sustained under O2
atmosphere where the condition is the most stressful for the rod because the temperature of the hot
spot increases with increasing Po2 [6]. The currents through the various composite rods with hot
spots are shown in Fig. 5. For Gd-123 rod, the current increases irregularly. After approximately 1 h,
the rod breaks with melting at the hot spot. Local parts in the hot spot, particularly the grainboundaries, may become partially melting due to local deviation from the ideal composition [16].
The presence of partial melt is considered to be the cause of the breaking at the hot spot. As seen in
Fig. 5, the currents through the composite rods increase gradually and the rods do not easily break,reflecting a high durability in the high Po2. Compared with the BaAl2O4-dispersed and

GdBa2Cu3O7-δ -based composite

2.0

1.2

Gd-211

BaAl2O4

Normalized Current

Withstand Power / kW cm

-3

1.4

1.0
0.8

BaZrO3

0.6
0.4
0.2
0.0

0

10

20

30

40

50

Content / vol%in O2

with 20 vol% BaAl2 O4d.c. 11V

1.5

with 30 vol% BaZrO3d.c. 15V

Gd-123d.c. 6 V

1.0
with 7 vol% Gd-211
d.c. 7 V

0.5

0.0 -1
1060

GdBa2Cu3O7-δ -based composite

10

0

10

1

10

2

10

3

10

4

10

Time / minFig. 4. Withstand electric power for various
GdBa2Cu3O7-δ –based composite ceramic rods
with dispersed BaAl2O4, BaZrO3 and Gd-211.

Fig. 5. Change in the current in O2 atmospherefor various GdBa2Cu3O7-δ –based composite
ceramic rods with dispersed BaAl2O4, BaZrO3
and Gd-211.
3

5

Transactions on GIGAKU 1(2012)01023/1-6

BaZrO3-dispersed composites, the Gd-211-dispersed one shows higher durability, suggesting apromising material for the application in the high Po2.
4. Improvement of durability in low Po2
Figure 6 shows oxygen sensing characteristics of (a) Gd-123 rod and (b) the composite rods
containing 15 vol% of dispersed CuO [17]. For the Gd-123 rod, the current running through therod with the hot spot decreases quickly when the atmosphere is changed from air to low Po2 in
the range of 1–0.03 kPa and subsequently increases gradually, showing overshoot of current.
When the atmosphere is changed to air, the current increases abruptly, shows a steep peak, andthen return to the initial value. When the atmosphere is changed to Po2=0.02 kPa, the current
becomes almost zero and the hot spot disappears in the rod. Then the current does not return tothe initial value when the atmosphere is returned to air.
For the composite rod containing 15 vol% CuO, the current shows overshoot when theatmosphere is changed from air to Po2=1–0.2 kPa. In contrast, the overshoot is not observed in
Po2=0.02–0.002 kPa. The rod keeps the current of tens of milliamperes even in Po2=0.002 kPa,
and the current returns to the initial value when the atmosphere is returned to air.
Figure 7 summarizes the oxygen partial pressure dependence of current for the rods
containing various amount of CuO. For the rods containing CuO≤11 vol%, the currents become
too low to maintain the hot spot in the rod at ~0.02 kPa. On the other hand, the rods containing
15 -52 vol% CuO have a sensitivity to oxygen even in Po2<0.002 kPa.
The temperature of the hot spot decreases with increasing content of CuO [17]. With decreasing
temperature, the diffusion of oxygen in the hot spot decreases and the overshoot of current issuppressed. As a consequence, the hot spot is retained in the rod and the sensing oxygen in the very
low Po2 is achieved.

N2 / O2 gas mixture

GdBa2Cu3O7-δ -based composite

Air

1000

PO2 / kPa

1.0

1
0.2

0.5

0.03

(a)
0.0
0

Air

GdBa2Cu3O7‐ DC 9.9 V

0.02

100

Current / mA

Normalized Current

Air

200mixture
N2 / O2 gas

300

400
Air

PO2 / kPa

1.0

1
0.2

100

CuO / vol%
0
5
11
15
52

10

0.02

0.5

1

0.002

(b)
0.0
0

GdBa2Cu3O7‐– 15 vol% CuO DC 19 V

100

200

300

10

400

-3

-2

-1

10

10

0

10

PO / kPa

Time / sec

2

Fig. 7. Oxygen partial pressure dependencesof currents for the composite rods containing
various amount of CuO.

Fig. 6. Oxygen sensing characteristics for (a)GdBa2Cu3O7-δ rod, and (b) the composite
rods containing 15 vol% CuO.

5. Improvement of response in low Po2
The response time of hot spot oxygen sensor has been improved by forming Gd-211 core in
Gd-123 rod [18]. The composite rod with the core is prepared by dipping a Gd-211 rod into the
Gd-123 slurry followed by sintering at 950oC. Figure 8 shows SEM images and EDS elemental mapsof Gd for the cross section of the rods. In the elemental maps, the white area indicates a highconcentration of Gd. The Gd is uniformly distributed in the rods without the core (b). In the rod withthe core (d), the concentration of Gd in the core is higher than that in the coat, indicating that the4Transactions on GIGAKU 1(2012)01023/1-6

core is Gd-211 and the coat is Gd-123. The
average thickness of the Gd-123 coat is 21μm.
The response time (t0.9) is defined as
the time taken for the ratio (i - i0) / (i∞ - i0)
to reach 0.9 after the change of theatmosphere from air to N2+O2 gas mixture
as shown in Fig. 9, where i0 is the initial
current, and i ∞ is the final current
measured after 300 s from the change inatmosphere. The response times as afunction of Po2 for various rods are shownin Fig. 10. It is found that, response time ofoxygen sensor for the rod with the core isapproximately 4 s shorter than those
without the core at Po2 of 0.2 kPa. The
Gd-123 as a medium for oxygen sensing
exists uniformly in all part of the rods
without the core. On the other hand, thepresence of Gd-123 is confined in the coaton the Gd-211 core. Therefore, length ofthe oxygen diffusion, which strongly
affects the response, is assumed to be
shorter in the rod with the core than those
without the core [19].

(a)

(b)

(c)

(d)

200 μmFig. 8. SEM images and EDS elemental maps of
Gd for the cross section of the rods: (a, b) Gd-123
with dispersed 10 mol% Gd-211 and (c, d) Gd-123
with Gd-211 core.

Gd-123 / Sintered at 910℃ / DC 3.9 V
N2+O2 mixed gas

with dispersed 10 mol% Gd-211

5Po2=0.2 kPa

t0.9

i0
0.5

6

0.9

t0.9 / sec

Current, i / A

Air

EDS map of Gd

Gd-211 core

Dispersed
10 mol% Gd-211

SEM

1

i∞

4

Gd-123

3
2
1

0.0
-10

0

10

20

30

40

50

60

70

80

0
0.1

90 1 280 290 300

Time / sec

with Gd-211 core1

10

Po2 / kPa

100

Fig. 10. Response time (t0.9) for rods withvarious structures as a function of the Po2.

Fig. 9. Oxygen sensing characteristics for
GdBa2Cu3O7-δ rod.

6. Conclusion
The progress in the development of the hot spot oxygen sensor was reviewed. Though the
GdBa2Cu3O7-δ rod tends to melt and break by a sustained presence of the hot spot in a high Po2, theGdBa2Cu3O7-δ composite rods with dispersed high melting point materials, especially Gd2BaCuO5,
show higher durability. In a low Po2 GdBa2Cu3O7-δ rod becomes insensitive to oxygen in Po2<0.02
kPa, while the composite rods containing appropriate amount of CuO can detect oxygen even in
Po2<0.002 kPa. The response time of the oxygen sensor using a composite GdBa2Cu3O7-δ rod with
Gd2BaCuO5 core is shorter than that without the core. The results suggest that the performance of
hot spot oxygen sensors is improved by using composite materials.
5Transactions on GIGAKU 1(2012)01023/1-6

Acknowledgments
This work was supported from the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, and by Program for High Reliable
Materials Design and Manufacturing in Nagaoka University of Technology..
References[1] K. Kishio, J. Shimoyama, T. Hasegawa, K. Kitazawa and K. Fueki, Jpn. J. Appl. Phys., 26(1987) L1228.
[2] V.A.M. Brabers, W.J.M.de Jonge, L.A. Bosch, C.v.d. Steen, A.M.W.de Groote, A.A. Verheyen
and C.W.H.M. Vennix, , Mater. Res. Bull., 23 (1988) 197.
[3] A.T. Fiory, M. Gurvitch, R.J. Cava and G.P. Espinosa, Phys. Rev. B, 36 (1987) 7262.
[4] T.K. Chaki and M. Rubinstein, Phys. Rev. B, 36 (1987) 7259.
[5] T. Okamoto, B. Huybrechts and M. Takata, Jpn. J. Appl. Phys., 33 (1994) L1212.
[6] M. Takata, Y. Noguchi, Y. Kurihara, T. Okamoto and B. Huybrechts, Bull. Mater. Sci., 22(1999) 593.
[7] H. Sunatori, T. Okamoto and M. Takata, J. Ceram. Soc. Japan, 111 (2003) 217.
[8] Y. Kurihara, Y. Noguchi and M. Takata, Key Eng. Mater., 157-158 (1999) 127.
[9] Y. Kurihara, T. Okamoto, B. Huybrechts and M. Takata, J. Mater. Res., 11 (1996) 549.
[10] K. Kikuchi, M. Kanzaki, T. Neda, T. Kondo and M. Takata, Jpn. J. Appl. Phys., 34 (1995)
L1311.
[11] Y. Ibaraki, T. Okamoto and M. Takata, Trans. Mater. Res. Soc. Jpn., 26 (2001) 23.
[12] K. Kakinuma and K. Fueki, Phys. Rev. B., 56 (1997) 3494.
[13] T. Okamoto and M. Takata, J. Ceram. Soc. Japan, 112 (2004) S567.
[14] Y. Tsutai, T. Okamoto, A. Kawamoto and M. Takata, J. Ceram. Soc. Japan, 112 (2004) S599.
[15] T. Okamoto, K. Iihama and M. Takata, Adv. Mater. Res., 11-12, (2006) 137.
[16] R.S. Roth, K.L. Davis and J.R. Dennis, Adv. Ceram. Mater., 2 (1987) 295.
[17] K. Iihama, Y. Kuroki, T. Okamoto and M. Takata, Current Appl. Phys., 9 (2009) S167.
[18] S. Fujihara, Y. Kuroki, T. Okamoto and M. Takata, Key Eng. Mate., 445 (2010) 213.
[19] S. Fujihara, Y. Kuroki, T. Okamoto, and M. Takata, Inst. Phys. Conf. Series: Mater. Sci. andEng., 18 (2011) 212004.
(Received: 31 May, 2012, Accepted: 7 June, 2012)6

Transactions on GIGAKU 1(2012)01024/1-6

TEM Observation of Carbon-Coated Li3V2(PO4)3 Glass-Ceramic
Powders and Their High Rate Battery Performance
Kenta Nagamine, Keiichiro Oh-ishi, Tsuyoshi Honma, Takayuki Komatsu*
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: komatsu@mst.nagaokaut.ac.jp

The morphology of carbon-coated Li3V2(PO4)3 crystals synthesized via
glass-ceramic
processing
(the
base
glass
composition:
37.5Li2O-25V2O5-37.5P2O5 (mol%)) was clarified from high resolution
transmission electron microscope (HRTEM) observations and their high rate
battery performance was examined. Glucose was added to the precursor glass
powders as a reducing agent of vanadium ions (V4+, V5+ →V3+) and a sourceof carbon coating, and two different techniques of hand mixing in a mortar
and ball-milling mixing were applied. HRTEM observations indicate that asintering of glass powders prepared by a ball-milling mixing is prevented bydispersed carbons on the surface of the primary glass particles, although thecoating state of carbon on the surface of glass-ceramic powders is nothomogeneous. The glass-ceramics with a carbon coating (a ball-milling
mixing) synthesized by a heat treatment at 700oC for 30 min in 7%H2/Ar
atmosphere showed a discharged capacity of 109 mAhg-1 in the range of
3.0-4.3 V and a good cycle performance at a high rate of 5C. The present
study suggests that the glass-ceramic processing is a novel method for thesynthesis of Li3V2(PO4)3 crystals with good lithium ion battery performance.

1. Introduction
Lithium ion secondary batteries have been widely used as energy sources of laptop-type personal
computers, mobile phones, and so on, because of advantages such as portability and high energy
densities. LiCoO2, which is a usual cathode material in commercially available lithium ion
secondary batteries, has critical disadvantages such as an extraction of oxygen in the charged state,high cost of cobalt, and its toxicity. These problems prohibit its use in large-scale devices such as
electronic vehicles. Instead, lithium transition metal phosphates such as LiFePO4 [1], Li3V2(PO4)3 [2],and LiVOPO4 [3] have been proposed as potential cathode materials for lithium ion batteries over a
decade. A framework consisting of phosphate network has effects that prevent the extraction ofoxygen and that improve a cycle performance as a battery. Especially monoclinic Li3V2(PO4)3
crystals exhibit excellent electrochemical performances such as the highest theoretical capacity of
197 mAhg-1 in phosphate cathodes reported and high-potentials of 3.60, 3.65, 4.10 V (for V4+/V3+)
and 4.55 V (for V5+/V4+). These features ensure that Li3V2(PO4)3 can replace LiCoO2 as practicalbattery cathode. On the other hand, phosphate materials have poor electronic conductivities, which
limit charge-discharge rates for lithium ion batteries. This problem has been considered to be solved
by carbon coating, particle size reduction, control of interface, and so on.
Recently, our group has reported the glass-ceramics processing as a synthesizing method forcathode materials of lithium ion secondary batteries and succeeded in synthesizing LiFePO4 [4,5],
LiMnxFe1-xPO4 [6], and LiVOPO4 [3]. The glass-ceramic technique has some advantages such as
simple fabrication process, high-speed synthesis, no requirement of high cost reagent, and so on.
These advantages are preferable to practical applications in lithium ion secondary batteries. In
addition, Nagakane et al. [7] reported that LiFePO4 prepared via glass-ceramic processing showed
better performance due to the presence of an amorphous phase than commercial LiFePO4 ceramic
powder. More recently, we reported the fast synthesis of Li3V2(PO4)3 crystals via glass-ceramics
processing from 37.5Li2O-25V2O5-37.5P2O5 precursor glass [8]. In order to improve battery1Transactions on GIGAKU 1(2012)01024/1-6

performance of Li3V2(PO4)3 crystals prepared by glass-ceramic processing such as high discharge
capacity at high discharging rates, it would be necessary to design well coating of carbon (i.e., highelectronic conductivity) on Li3V2(PO4)3 crystal particles and to clarify the contribution of amorphous
phase (i.e., maybe high Li+ ion conductivity) on the battery performance. In this paper, Li3V2(PO4)3
crystals were synthesized using a glass-ceramic processing and two different carbon coating
techniques of hand mixing in a mortar and ball-milling mixing were applied. The morphology ofcarbon coated Li3V2(PO4)3 crystals and the state of amorphous phase were clarified from highresolution transmission electron microscope (HRTEM) observations and their high rate battery
performance was examined.

2. Experiment
A glass with the composition of 37.5Li2O-25V2O5-37.5P2O5 (mol%) corresponding to thestoichiometric composition of Li3V2(PO4)3 crystal was prepared in air by using a conventional
melt-quenching method. The detailed glass preparation process has been reported elsewhere [8], inwhich raw materials of simple oxide compounds of LiPO3 and V2O5 are used. In this study, theprecursor glass prepared is described as LVP323 glass. The LVP323 glass was pulverized by using aplanetary ball-milling method. Glucose as a reducing agent was mixed with the glass powders by
two different ways: 1) 10wt% glucose was mixed with ethanol in an agate mortar by hand, and 2)
10-20wt% glucose was mixed by using a planetary ball-milling (Fritsch premium line P-7). Mixtures
were heat-treated at 700ºC for 0.5-12 h under 7%H2/Ar atmosphere to synthesize Li3V2(PO4)3
crystals. The crystalline phases present in the crystallized samples were identified by X-ray
diffraction (XRD; Cu K radiation; RIGAKU ULTIMA IV) analyses at room temperature. The
surface and inner morphologies of LVP323 glass ceramics were studied by scanning electron
microscopy (SEM; JIB-4500) and high resolution transmission electron microscopy (HRTEM;
JEM-2100F). Electrical conductivities of carbon coated glass-ceramics with a disk shape were
measured using a four-terminal method, in which silver paste was painted onto the surface of thesample as blocking electrodes. Battery performances were evaluated in testing cells. Cathodecomposites were prepared by mixing 80wt% glass–ceramics (for active material), 15wt% acetylene
black (for electronic conductor), and 5wt% polyvinylidene difluoride (PVDF) (for binder). Mixtures
were then pressed onto aluminum thin sheet, and circular disks were prepared by cutting the sheet.
Stainless test cells were constructed using a lithium metal as an anode and an electrolyte of 1M
LiPF6 consisting of a 1:1 solution of ethylene carbonate and diethyl carbonate. The lithium ion
battery performance at room temperature was evaluated from charge/discharge measurements(Hokuto denko Co., HJ-1001). The cell potential was swept in the voltage range of 3.0–4.3 V, andthe charge/discharge rate was 0.01–5 C.
3. Results and Discussion
A SEM image for the precursor LVP323 glass powders is shown in Fig. 1. The agglomerated
particles having the diameter of >5 m are present. The surface of particles is rough, implying thatthey consist of sparsely-connected primary glass particles. Such agglomerates might be produced
during the pulverizing process of plate-shaped melt-quenched glass and the following drying process.
The precursor LVP323 glass powders with the morphology shown in Fig. 1 were used in thefollowing two experiments: the precursor LVP323 glass powders were mixed with glucose (10 or
20wt%) by using a hand mixing method or a ball-milling mixing method and then mixtures were
heat-treated at 700ºC for 0.5-12 h under 7%H2/Ar atmosphere. For simplicity, the sample preparedusing a hand mixing method is called as “hand mixing sample”, and the sample prepared using aball-milling mixing method is called as “ball-milling mixing sample” in this paper.
The XRD patterns for the ball-milling mixing samples (heat-treated at 700ºC for 30 min) are
shown in Fig. 2. Only Li3V2(PO4)3 crystals are formed in the samples, indicating that Li3V2(PO4)3 is
successfully synthesized by the crystallization of LVP323 glass powders with glucose under
7%H2/Ar atmosphere. In the previous paper [8], it has been reported that the vanadium ions of V4+
and V5+ in the precursor LVP glass are reduced largely to V3+ ions during heat treatments withglucose in N2 or 7%H2/Ar atmosphere. The XRD pattern for the ball-milling mixing samples wasrefined by using Rietveld method, and the lattice parameters for Li3V2(PO4)3 crystals synthesized2

Transactions on GIGAKU 1(2012)01024/1-6

were calculated to be a=1.2047 nm, b=0.8607 nm, c=0.8609 nm, and =90.504º. The size of
crystallites was estimated from the Lorentz parameter X which relates to the full width of half
maximum (FWHM) of XRD peaks. The Scherrer’s equation is expressed as p = K/X, where p, Kand  are the size of crystallites (nm), Scherrer’s constant (0.9), and the wavelength (0.1540562 nm)of X-ray, respectively. The sizes of Li3V2(PO4)3 crystallites in the hand mixing sample and in theball-milling mixing sample with 20wt% glucose were estimated to be 122 and 72 nm. It is, therefore,
concluded that the ball-milling mixing provides more fine Li3V2(PO4)3 crystals compared with thehand mixing in the glass-ceramic processing.

Intensity / arb. units

700°C, 0.5h

BMM glucose 20wt%

BMM glucose 10wt%

Li3V2(PO4)315

Fig. 1

SEM image for LVP323 glass powders
pulverized by using a planetaryball-milling.

Fig. 220

25
30
2 / deg.

35

40

XRD patterns for the ball-milling
mixing (BMM) samples prepared byheat-treatments of LVP323 glass
powers with glucose in 7%H2/Ar
atmosphere.

From SEM observations for the hand mixing samples (heat-treated at 700ºC for 30 min), it was
found that the secondary particles having the diameter of >20 m are present and their surface issmooth unlike the precursor glass particles with a rough surface (Fig. 1). Such a smooth surface
suggests that the surfaces of the glass-ceramic particles are covered with the carbon formed by apyrolysis of glucose. It should be pointed out that the glucose added transforms into liquid phase(melting temperature is ~146ºC) on the heating (up to 700oC) for the crystallization of glass powders.
From a SEM image for the cross-section of a glass-ceramic particle obtained by a focused ion beam
(FIB) method, it was found that the agglomerated particle has many fine pores or hollows in its
inside. In order to clarify the morphology of the inside of glass-ceramic particle more in detail,
HRTEM observations were carried out. Many hollows having the size of several dozen nanometers
are again confirmed from the high angle annular dark field (STEM-HAADF) image for the
cross-section. They look like open pores. These hollows might enable liquid electrolytes to penetrate
into the glass-ceramics particles. It was confirmed from energy dispersed spectrum (EDS)
experiments that the composition of the cross-sectioned particle corresponds to the composition of
Li3V2(PO4)3 and any other phases with different compositions such as metal phosphides are notdetected. The bright field image and electron diffraction pattern for the inside of the glass-ceramics
are shown in Fig. 3. The bright field image indicates that the carbon formed by the pyrolysis of
glucose is not present in the hollows, indicating that carbon is attached only on the surface of thesecondary particles. It is confirmed from the electron diffraction pattern that Li3V2(PO4)3 crystals areformed in the inside (the region marked with a dotted line). The results on HRTEM observations
demonstrate that the glass-ceramics prepared by the hand mixing with glucose and by theheat-treatment at 700ºC for 30 min are well crystallized and consist of Li3V2(PO4)3.
In the SEM image for the ball-milling mixing samples (heat-treated at 700ºC for 30 min), the3

Transactions on GIGAKU 1(2012)01024/1-6

agglomeration of particles is observed as well as the precursor LVP323 glass powders (Fig. 1).
However, it is noted that the surface of each particle is not smooth unlike the glass-ceramic powders
prepared by the hand mixing method. The bright field and HRTEM images for the ball-milling
mixing samples are shown in Fig. 4 (a-c). Pores or hollows are not found, which are seen in the handmixing samples, and the structure of each particle is clear, suggesting that a sintering of the
precursor LVP323 glass powders is prevented by dispersed carbons on the surface of the primary
glass particles. The HRTEM images showed that some particles are not coated by carbon (Fig. 4 (b)),
but some particles are coated by carbon (Fig. 4 (c)), indicating that the dispersion state of carbon, i.e.,the coating state of carbon, on the surface of glass-ceramic powders is not homogeneous orinsufficient.

Fig. 3Fig. 4 Bright field image (a) and high resolution
TEM images for the ball milling mixing
samples prepared by a heat treatment(700oC, 30 min) of LVP323 glass powers
with glucose (20wt%) in 7%H2/Ar
atmosphere. (b): the surface region
without the coating of glucose, (c): thesurface with a carbon layer.

Bright field image and electron
diffraction pattern for the hand mixing
samples prepared by a heat treatment(700oC, 30 min) of LVP323 glass
powers with glucose (10wt%) in
7%H2/Ar atmosphere.

The electrical conductivities () at room temperature for the hand mixing samples (heat-treated
at 700ºC for 0.5-12 h, 10wt% glucose) were measured using a direct current four-terminal method. It
was found that the electrical conductivity increases with increasing heat treatment time. The
glass-ceramics prepared in the heat treatments of 30 min and 12 h show the values of =4x10-3
S/cm-1 and =3x10-2 S/cm-1, respectively. In the previous paper [8], it has been reported that theamount of carbon remained in the glass-ceramics after heat treatments decreases with increasing
heat-treatment time. It is, therefore, considered that the quality of carbon coating is improved asincreasing heat-treatment time. The electrochemical performance of the hand mixing samples was
examined, and the discharge capacities cycled between 3.0-4.3V at the rate of 0.01-1C were obtained.
At the slow rate of 0.01C, the discharged capacities are 117~126 mAhg-1. On the other hand, thedischarged capacities at the rate of 1C for the glass-ceramics obtained in the heat treatments of 0.5, 3,
6, and 12 h are 86, 89, 91, and 96 mAhg-1, respectively. These values mean that lithium ionscorresponding to the fraction of x=1.3 (for 30 min) and x=1.45 (for 12 h) in Li3-xV2(PO4)3 areinserted at the rate of 1C. It is seen that the decrease in the discharged capacity for the glass-ceramics
obtained by long time heat-treatments such as 12 h is slightly smaller than that for the glass-ceramicobtained by a short time heat-treatment of 30 min. Such an improvement of the high rate discharge
4Transactions on GIGAKU 1(2012)01024/1-6

performance would be closely related to the coating state of carbon, i.e., the electronic conductivityof coated carbons.

2

125
100

1.5
0.1C

75

0.5C

1

1C

50 0.01C

2C
5C

700°C 0.5h2500x in Li3-xV2(PO4)3

Discharged capacity / mAhg -1

150

0.5

hand mixing
bmm glu. 10
bmm glu. 20

5

10
15
20
Number of cycles / times

250Fig. 5 Discharged capacities at the rate of 0.01-5C between 3.0-4.3 V for the ball-milling mixing(BMM) samples prepared by a heat treatment (700oC, 30 min) of LVP323 glass powers
with glucose 10-20 wt% in 7%H2/Ar atmosphere.
The discharge capacities at the rate of 0.01-5C between 3.0-4.3V for the ball-milling mixing
samples (heat-treated at 700ºC for 0.5 h, 10-20wt% glucose) are shown in Fig. 5. The data for thehand mixing sample (heat-treated at 700ºC for 30min, 10wt% glucose) are also shown in Fig. 5 forcomparison. At the slow rate of 0.01C, the values of discharge capacities are 124-133 mAhg-1.
Especially, the discharge capacities of the glass-ceramics with 20wt% glucose at the rates of
0.01-0.5C correspond to almost 100% of the theoretical capacity of Li3-xV2(PO4)3 (x=2). At the rateof 1C, the values of discharged capacities are 124-128 mAhg-1. In addition, even at the high rate of
5C the discharged capacities of 92 (for 10wt% glucose) and 109 (for 20wt% glucose) are obtained.
As shown in Fig. 5, the battery performance of LVP323 glass-ceramics is drastically improved byapplying the ball-milling mixing method. In high rate discharging process, electrical conductivity
and contact between carbon and active materials are more and more important. In the ball-milling
mixing method applied in this study, finer Li3V2(PO4)3 crystals are formed in the glass-ceramics
compared with the hand mixing method. Therefore, the improvement of the battery performance
shown in Fig. 5 might result from a good contact of fine Li3V2(PO4)3 crystals with carbon coatings.
However, even in the ball-milling mixing method, still Li3V2(PO4)3 crystals having no carbon
coatings are present as shown in HRTEM observations (Fig. 4). In this point of view, there would bea possibility to improve more the battery performance of glass-ceramics.
There have been several reports on the synthesis of Li3V2(PO4)3 crystals and their Li+ ion battery
performances, in which Li3V2(PO4)3 crystals have been fabricated using various techniques such as
conventional solid state reaction, hydrothermal method, sol-gel method, carbothermal reduction
method, and soft chemistry route [9-12]. In those studies [9-12], a well carbon coating andnano-scaled uniform particle size have been established, and consequently Li3V2(PO4)3 crystals
synthesized show good Li+ ion battery performances. The present study demonstrates that theglass-ceramic processing also provides Li3V2(PO4)3 crystals showing a moderate performance as
cathode materials for lithium ion secondary battery. In particular, it should be emphasized that raw
materials for the fabrication of Li3V2(PO4)3 crystals are simple oxide compounds of LiPO3 and V2O5,
suggesting that cost for their fabrication would be low.

5Transactions on GIGAKU 1(2012)01024/1-6

4. Conclusion
Carbon-coated Li3V2(PO4)3 crystals were synthesized using glass-ceramic processing, in which
the composition of the precursor glass was 37.5Li2O-25V2O5-37.5P2O5 (mol%) and simple oxide
compounds of LiPO3 and V2O5 were used as raw materials. Their morphology of carbon-coated
Li3V2(PO4)3 crystals synthesized was clarified from HRTEM observations, and their high rate
battery performance was examined. Two different techniques of hand mixing in a mortar andball-milling mixing were applied. The glass-ceramics with a carbon coating (a ball-milling mixing)
synthesized by a heat treatment at 700oC for 30 min showed a discharged capacity of 109 mAhg-1
and a good cycle performance at a high rate of 5C. The present study suggests that the glass-ceramic
processing is a simple and fast method for the synthesis of Li3V2(PO4)3 crystals with a good lithium
ion battery performance.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture, and Technology, Japan (No. 23246114), and by Program forHigh Reliable Materials Design and Manufacturing in Nagaoka University of Technology. One (K.
Nagamine) of the authors thanks for JSPS Research Fellowships for Young Scientists.
References[1] A. Yamada and S.C. Chung, J. Electrochem. Soc., 148 (2001) A960.
[2] H. Huang, S.C. Yin, T. Kerr, N. Taylor, and L.F. Nazar, Adv. Mater., 14 (2002) 1525.
[3] K. Nagamine, T. Honma, and T. Komatsu, J. Am. Ceram. Soc., 91 (2008) 3920.
[4] T. Honma, K. Hirose, T. Komatsu, T. sato, and S. Marukane, J. Non-Cryst. Solids, 356 (2010)3032.
[5] K. Nagamine, S. Reinsch, R. Mueller, T. Honma, and T. Komatsu, J. Am. Ceram. Soc., 94(2011) 2890.
[6] T. Honma, K. Nagamine, and T. Komatsu, Ceram. Int., 36 (2010) 1137.
[7] T. Nagakane, H. Yamauchi, K. Yuki, M. Ohji, A. Sakamoto, T. Komatsu, T. Honma, M. Zou, G.
Park, and T. Sakai, Solid State Ionics, 206 (2012) 78.
[8] K. Nagamine, T. Honma, and T. Komatsu, J. Power Sources, 196 (2011) 9618.
[9] Y.H. Nien, J.R. Carey, and J.S. Chen, J. Power Sources, 193 (2009) 822.
[10] L. Wang, X. Zhou, and Y, Guo, J. Power Sources, 195 (2010) 2844.
[11] Y.Q. Qiao, J.P. Tu, J.Y. Xiang, X.L. Wang, Y.J. Mai, D. Zhang, and W.L. Liu, Electrochim. Acta,
56 (2011) 4139.
[12] W. Yuan, J. Yan, Z. Tang, O. Sha, J. Wang, W. Mao, and L. Ma, J. Power Sources, 201 (2012)
301.
(Received: 17 May, 2012, Accepted: 24 May, 2012)6

Transactions on GIGAKU 1(2012)01025/1-6

Formation of Hierarchical Structure
in Liquid Crystalline Block Copolymers
Hiroki Takeshita, Shunsuke Adachi, Shin-ichi Taniguchi, Katsuhiko Takenaka,
Masamitsu Miya, Tomoo Shiomi*
Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: shiomi@vos.nagaokaut.ac.jp

This paper reviews the hierarchical structure and its formation process forliquid crystalline block copolymers. Block copolymers exhibit microphaseseparation with various kinds of morphologies such as sphere, cylinder andlamella in the order of several ten nanometers, while the liquid crystalline
polymers form mainly smectic or nematic structure with several
nanometer-sizes. For the superimposed structure formed in block copolymers
composed of liquid crystalline and amorphous blocks, the followings werefocused on: the correlation between the microphase separation structure andliquid crystalline structure, the orientation of mesogen axis, and the processof the structure formation. Photoresponsiveness of the microphase separation
structure to the liquid crystalline/isotropic transition caused by thephotoisomerization of the mesogen group was also presented.

1. Introduction
Polymer molecules can contain two or more kinds of monomers, which are combined in various
sequence manners. Block copolymers are composed of linear arrangement of different blocks
comprising the respective constitutional monomers, like -A-A-A-A-A-A-B-B-B-B-B-B- for a block
copolymer consisting of monomers A and B. When the block A is immiscible with the block B, theblock A and block B segregate from each other to form so-called microphase separation (MS)structure. Various kinds of morphologies occur in the MS structure: sphere, cylinder, gyroid, lamella,etc., depending on the volume fraction of blocks, even in the block copolymer composed of only two
blocks. The domain size of the MS structure is parallel to that of polymer molecules; usually several
ten nm for polymers with common molecular weights. Such morphologies and their formation
mechanism in the amorphous or molten state have been extensively investigated [1].
When one or more constitutional blocks are crystalline or liquid crystalline, the structure isformed by combination or competition between their phase transitions and microphase separation.
There are various combinations among crystalline, liquid crystalline and amorphous blocks in block
copolymers. For crystalline block copolymers, their phase structure and crystallization behavior havestatically and dynamically studied so far [1-11].
In this paper, we will focus on liquid crystalline block copolymers containing a side-group liquid
crystalline (LC) block. The liquid crystalline block copolymer has a potential to exhibit ahierarchical phase structure which consists of several ten nanometers of MS structure and a few
nanometers of LC structure. Nematic (Nm) or smectic (Sm) liquid crystalline structure exists in the
MS domain with various morphologies described above. In order to clarify such superimposed
structure, it is required to reveal the interplay between microphase separation and liquid
crystallization; the correlation between the MS structure and LC structure, the orientation of
mesogenic groups to the interface of the microdomain, and in addition, the process of the structureformation on liquid crystallization from microphase-separated states.
In the liquid crystalline block copolymers, the binary combinations with amorphous[12-15,18,19], liquid crystalline [16] and crystalline [17] blocks have been investigated. In this paper,
we review the hierarchical structure and its formation process for block copolymers consisting of
liquid-crystalline and amorphous block chains.
1Transactions on GIGAKU 1(2012)01025/1-6

2. Experimental Methods
In order to realize a well-controlled MS structure, we must use block copolymers having anarrow molecular-weight distribution. Although such block copolymers have usually beensynthesized by living anionic polymerization, this polymerization method is not suitable forpreparation of liquid crystalline polymers having a polar side group. Therefore, liquid crystalline
block copolymers have been prepared by atomic transfer radical polymerization (ATRP) that is akind of so-called living radical polymerizations. The liquid crystalline copolymers described here
consist of a liquid crystalline block (PLC) with a cyanobiphenyl-type or azobenzene-type mesogengroups and an amorphous block of poly(n-butyl acrylate) (PBA) or polystyrene (PS). The chemical
structure of PBA-PLC block copolymer is shown in Figure 1 as an example. The spacer length
between a main chain and mesogen group was controlled by the number x of methylene groups.
Microphase separation structure and Sm layer can beevaluated by a small-angle X-ray scattering (SAXS)
technique. Here, the scattering vector q is defined asq=(4/)sin(/2), where  is the scattering angle. The
time-resolved SAXS measurements using a very strong X-ray
source from synchrotron radiation give a useful informationon the structure formation process in the liquid crystallization.
The SAXS results described here were obtained at the Photon
Factory in the Institute of Materials Structure Science, HighEnergy Accelerator Research Organization in Tsukuba, Japan:
for 1-dimensional and 2-diensional measurements at beam
Fig. 1 An example of liquidlines of BL-10C and BL-15A, respectively. Polarized opticalcrystalline-amorphous
block
microscopy (POM) and differential scanning calorimetry
copolymer.
(DSC) are also used for observation of a texture of liquid
crystals and a transition temperature, respectively.

Log Intensity /a.u.

3. Results and Discussion(1) Liquid crystalline structure of homopolymer
The liquid crystalline/isotropic transition temperature Tiso increases with increasing spacer lengthx [14,15]. Also, the cyanobiphenyl-type homopolymer with a short spacer x=6 does not exhibit Sm
structure but only Nm structure, while other ones with a longer spacer can form Sm structure[14,15].
For example, Figure 2 shows SAXS profiles for cyanobiphenyl-type PLC with x=11, in which
two peaks can be observed below Tiso [12]. The ratio of these peak positions is 1:2, which means
layer structure, that is,
Sm structure.
Thelayer spacing l is**estimated from thenOfirst-orderpeakO
position around q=1.4
nm-1 to be about 4.5
50.6 °C
nm. This value of the
1.64 nm102.5 °C
layer spacing is about
4.5 nmtwice the length of theO
107.4 °Cside group with themesogen and trans112.6 °C
0.89 nmzigzag conformation of
138.1 °C
C N
which
(CH2)11,suggests that the Sm
0.0
1.0
2.0
3.0
-1
q /nm
structure is a bilayer
structure consisting ofFig. 2 SAXS profiles and liquid crystalline structure of cyanobiphenyltwo mesogenic layers
type polymer with x=11. (Reproduced from ref. 12.)2

Transactions on GIGAKU 1(2012)01025/1-6

Fig. 3 SAXS profiles for PBA-PLC (a) with LC=0.44 and (b) with LC=0.20. (Reproduced
from ref. 12)

as shown in Figure 2. A monolayer structure
was also found for the azobenzene-type liquid
crystalline polymers [18,19].
(2) Hierarchical structure of liquid
crystalline/amorphous block copolymers
In order to elucidate the hierarchicalstructure, it is required to estimate the MS
structure, LC structure and the orientation of
the mesogen axis to the interface of the
microdomain.
Figure 3a shows SAXS profiles at eachtemperature for PBA-PLC with LC=0.44
weight fraction [12]. In both isotropic(127.7oC) and liquid crystalline states, a peak
due to MS structure is observed at q=0.17nm-1.
Although any higher-order peaks are notclearly observed, the MS structure isconsidered to be lamellar by taking account of
the copolymer composition. A peak due toFig. 4 SAXS profiles for PS-PLC at isotropic
Sm layers also appears around q=1.4nm-1(140oC) and liquid crystalline (25oC) states.
below about 100oC. This means that Sm(Reproduced from ref. 13)
structure is formed in the lamellar MS
structure. On the other hand, as shown in
Figure 3b [12], SAXS profiles for PBA-PLC with LC=0.20 exhibits a hexagonal packing, that is,cylindrical MS structure, but no peak due to Sm structure is found. In this case, since the formation3

Transactions on GIGAKU 1(2012)01025/1-6of liquid crystalline structure isconfirmed by POM, it isconcluded that the Nm structure
is formed within the cylindrical
MS domain.
The same result can beobtained for a series of PS-PLC
block copolymers as shown in
Figure 4 [13]. Sm structure is notformed in the cylindrical domain(PSt09PCB04; 2=0.22), while
Sm can be formed in bothlamellar and matrix domains(PSt14PCB09 with 2=0.38 and2=0.59,
PSt09PCB13
with

Fig. 5 2-D SAXS images for PBA-PLC (lamella) andfor PS-PLC (PS, cylinder). (Reproduced from ref. 12 andref. 13.)respectively),
where
for
PSt09PCB13, PS is located in thecylindrical domain.
The orientation of themesogen axis can be determinedusing a 2-dimensional CCD
X-ray detector. Figure 5 shows
2-D SAXS images for PBA-PLC
Fig. 6 Schematic pictures of superimposed structure(lamellar) [12] and PS-PLC
composed of LC and MS structures. (Reproduced from ref.
(PS-cylindrical) [13] observed
12 and ref. 13.)
after applying oscillating shear
flow (1 Hz). The scattering peaks
from the Sm layer are located perpendicularly to those from the MS structure for both samples,
which means that the Sm layer aligns perpendicularly to the microdmain interface, that is, thedirection of the mesogen axis is parallel to the interface.
From the above results, we can draw schematic images of hierarchical structure as shown in
Figure 6 [12,13]. Since the orientation of the Nm structure cannot be observed by the SAXS
technique, it was estimated using both 2D-SAXS and POM which give orientations of the
microdomain interface and of the mesogen axis, respectively [14,15].
(3) Process of MS structural change on liquid crystallization
Morphology or spacing of MS structure is expected to be changed by liquid crystallization in thesame way as crystallization of crystalline block copolymers [5]. Figure 7(left) shows timedevelopment of SAXS profiles in the liquid crystallization process from the lamellar MS structure

Fig. 7 The development of SAXS profiles (left) and the position and intensity of SAXS peak4
(right) in liquid-crystallization process from lamellar
MS structure for PBA-PLC. (Reproduced
from ref. 12)

Transactions on GIGAKU 1(2012)01025/1-6

for PBA-PLC [12]. The position and intensity of the peak due to the MS structure are plotted against
the temperature in Figure 7(right) [12]. As a peak from the Sm layer appears at q=1.4 nm-1, theintensity of a preexisting peak around q=0.17 nm-1 (indicated by ‘A’ in Figure 7) above Tiso decreases
and the peak is succeeded by peak ‘B’. Furthermore, at almost the same time, a new peak (‘C’)
appears at a smaller angular side and develops. The peak B disappears with developing the peak C.
The peak C comes from the MS structure containing Sm LC layers, because the peak from the Sm
layer exists in the whole temperature range below Tiso. The peak B might be assigned to the Nmstructure by the POM observation.
From the above behavior of the SAXS profiles, we can conclude the structural change in theliquid crystallization process as follows: (1) the Sm layer structure is evolved through a transient Nm
structure and (2) the MS structure is reorganized by the formation of the Sm structure because thepeak position changes discretely. Such change of the structure is schematically shown in Figure8(left). In the liquid crystallization from the cylindrical structure, on the other hand, the peak
position changed continuously probably because of the Nm formation. In addition, (3) it should be
noted that the MS peak position changes toward a low-angle side in liquid crystallization, whichmeans that the spacing of the MS structure is larger in the liquid crystalline state as shown in Figure8(left). This may be related to the alignment of the mesogen groups. Especially in the Sm structure,
as shown in Figure 8(right), the perpendicular alignment of the Sm layer expands the main chain
toward the microdomaininterface,andsimultaneously the main
chain of the amorphous
block is also expanded
because the interface
area per block chain mustmatch between liquid
crystalline
andFig. 8 (left) Formation of Sm structure via. Nm structure
amorphous block chains.
accompanied by reorganization of lamellar MS structure (A, B and C
Themorphology
correspond to those of Fig. 7), and (right) the predicted main-chain
change such as lamellar
conformation in Sm structure.
to cylindrical has also
been found for other liquid crystalline block copolymers [18,19]. This morphology change may becaused by the requirement that the entropic deficit coming from excessive expansion of the main
chain should be avoided.
(4) Responsiveness of MS structure to photosensitive liquid crystalline/isotropic transition
Mesogen groups containing azobenzene derivatives exhibit a transition from liquid crystalline toisotropic state due to trans to cis photoisomerization triggered by UV irradiation.
Photoresponsiveness of the MS structure has been investigated for the block copolymer consisting of
PBA amorphous and azobenzene-type liquid crystalline blocks [18,19]. The spacing of the MS
structure was shorter in the isotropic state caused by UV irradiation in the same way as the case in
the thermal transition. The change of the spacing was reversible in the repeat of UV-On and –Off,and the responsive time was 20-30 sec. If block copolymers containing a photosensitive liquid
crystalline block are employed, it is expected that the spacing or morphology of the MS structure canbe changed rapidly and easily by the photoexposure, compared with the change by the thermaltransition, which is a better way for practical application of the structural change.
4. Conclusion
The hierarchical structure containing liquid crystalline or crystalline phases has been exploredrecently for bock copolymers. Especially the formation process of the MS structure for both
crystalline and liquid crystalline block copolymers was first revealed by us [5,12]. In polymericsystems, there can coexist crystalline, liquid crystalline and amorphous phases. The overall phasestructure of such systems is formed by interplay between these phase transitions and phaseseparation, and the structure is determined dynamically as well as thermodynamically because the5Transactions on GIGAKU 1(2012)01025/1-6

diffusion of polymer chains is not so fast. Therefore, in order to clarify the complicated structure
systematically, the investigations on the formation process as well as the resulting structure are
required.
Acknowledgments
Our works referred to in this paper were supported by the Grant-in-Aid for Scientific Researchon Basic Area (B) and for Challenging Exploratory Research from Japan Society for the
Promotion of Science and by the 21st Century COE Program for Scientific Research from theministry of Education, Culture, Sports, Science and Technology. The SAXS experiments usingsynchrotron radiation were performed under the approval of the Photon Factory Program
Advisory Committee (Proposal Nos. 2004G090, 2006G087 and 2007G090 and 2008G066).
References[1] I.W. Hamley, The Physics of Block Copolymers. New York: Oxford; 1998.
[2] A.J. Müller, V. Balsamo, and M.L. Arnal, Advances in Polymer Science, Volume 190, 1-63;
2005.
[3] S. Nojima, K. Kato, S. Yamamoto, and T. Ashida, Macromolecules, 25 (1992) 2237.
[4] T. Shiomi, H. Tsukada, K. Takeshita, K. Takenaka, and Y. Tezuka, Polymer, 42 (2001) 4997.
[5] T. Shiomi, H. Takeshita, H. Kawaguchi, M. Nagai, K. Takenaka, and M. Miya, Macromolecules,
35 (2002) 8056.
[6] H. Takeshita, N. Ishii, C. Araki, M. Miya, K. Takenaka, and T. Shiomi, J Polym Sci Part B
Polym Phys, 42 (2004) 4199.
[7] H. Takeshita, Y.J. Gao, T. Natsui, E. Rodriguez, M. Miya, K. Takenaka, and T. Shiomi., Polymer,
48 (2007) 7660.
[8] Y.J. Gao, H. Takeshita, Y. Takata, K. Takenaka, and T. Shiomi, e-J Soft Mater, 4 (2008) 12.
[9] H. Takeshita, Y.J. Gao, Y. Takata, K. Takenaka, T. Shiomi, and C. Wu, Polymer, 51 (2010) 799.
[10] F. Arai, H. Takeshita, M. Dobashi, K. Takenaka, M. Miya, and T. Shiomi, Polymer, 53 (2012)851.
[11] H. Takeshita, K. Fukumoto, T. Ohnishi, T. Ohkubo, M. Miya, K. Takenaka, and T. Shiomi,
Polymer, 47 (2006) 8210.
[12] S. Taniguchi, H. Takeshita, M. Arimoto, M. Miya, K. Takenaka, and T. Shiomi, Polymer, 49(2008) 4889.
[13] H. Takeshita, S. Taniguchi, M. Arimoto, M. Miya, K. Takenaka, and T. Shiomi, Polymer, 50(2009) 271.
[14] H. Takeshita, S. Adachi, H. Taniguchi, M. Miya, K. Takenaka, and T. Shiomi, Polymer Prepr,
Jpn, 58 (2009) 3543.
[15] T. Shiomi, H. Takeshita, S. Adachi, H. Taniguchi, and K. Takenaka, European Polymer
Congress, Book of Abst, 8 (2011) 1233.
[16] T. Shiomi, H. Takeshita, H. Taniguchi, S. Adachi, M. Miya, and K. Takenaka, Polymer Prepr,
Jpn, 58 (2009) 3541.
[17] H. Takeshita, Y. Sakima, M. Miya, K. Takenaka, and T. Shiomi, Polymer Prepr, Jpn, 59 (2010)3397.
[18] H. Takeshita, T. Fjise, K. Haginoya, M. Miya, K. Takenaka, and T. Shiomi, Polymer Prepr, Jpn,
60 (2011) 4489.
[19] K. Haginoya, R. Kudo, T. Fujise, H. Takeshita, M. Miya, K. Takenaka, and T. Shiomi, Polymer
Prepr, Jpn, 61 (2012) 739.
(Received: 18 May, 2012, Accepted: 30 June, 2012)6

Transactions on GIGAKU 1(2012)01026/1-5

Alloying Effects in the Nickel-Cobalt System on Electrochemicaland Adsorptive Properties of an SOFC Anode Cermet
for the Direct Oxidation of Methane
Kazunori Sato*, Teoh WahTzu, Hiroshi Nishiyamaa), Tatsuya Sawahata, Hirotaka Takayanagi
Department of Environmental Engineering, Nagaoka University of Technologya) Department of Materials Science and Technology, Nagaoka University of Technology
Nagaoka, Niigata 940-2188, Japan*E-mail: sato@analysis.nagaokaut.ac.jp

Substitution of Co atoms for Ni atoms in the Ni-based cermet anodeenhanced the electrochemical performance of a solid oxide fuel cell (SOFC)
for the direct oxidation of methane. This effect was confirmed for the cells
using an yttria-stabilized zirconia (YSZ) electrolyte. The result ofelectrochemical measurements has revealed that the enhancement of thedirect oxidation of methane and hydrogen is caused by an improvement ofadsorptive properties for the reactants and products due to thesolid-solutioning effect in the Ni-Co binary alloy system. We found that
adsorptive abilities of Ni0.5Co0.5-YSZ and Ni0.5Co0.5-CeO2 cermet particles forCH4 and CO are affected by the Co atoms substituted for the Ni atoms.

1. Introduction
The development of proper metal catalysts combined with a high oxide-ion conductor is required
for the electrochemical oxidation of hydrocarbon fuels [1-6]. We have investigated the performanceof an anode, which consists of Ni-Co solid-solution alloy particles dispersed on the zirconia-based
electrolytes or ceria-based electrolytes [7,8]. However, the effect of the Ni1-xCox composition on themethane (CH4) adsorption has not been investigated so far, and the reaction mechanism remained
uncertain. The electrochemical reaction occurring at the electrode-electrolyte interface is important toimprove the power generation performance of SOFCs. Both CO and graphite produced at the anodecan degrade the cell performance. The strong adsorption of CO on the electrode surface results in a
carbon deposition and poisoning as well as the thermal decomposition of CH4 into graphite. To
understand the reaction mechanism the adsorptive ability of cermet materials must be characterized.
The objective of this research is to characterize the adsorptive properties of Ni1-xCox-YSZ and
Ni1-xCox-CeO2 cermet particles by Temperature-Programmed Desorption (TPD) analysis. The results
were discussed in terms of the metal-support interaction for the direct oxidation of CH4.
2. Experimental
The Ni1-XCoXO solid solution was prepared by heating a mixture of reagent-grade NiO andCo3O4 powders at 1273 K for 10 h in air. Phase identification of the prepared powders was made by
X-ray diffraction (XRD). Pulverized Ni1-XCoXO powders were mixed with 8 mol%Y2O3-ZrO2 (YSZ)
powder or 10 mol%Sc2O3-1 mol%CeO2-ZrO2 (ScSZ) powder in a mass ratio of 30% to form a slurryusing glycerol. The slurry was painted as the anode on one face of the electrolyte disk
(Shinko-Denki). The disk was 15 mm in diameter and 0.3 mm thick. The painted disk was heated at1373 K for 10 h. (La0.85Sr0.15)0.9Mn0.1O3 (LSM) was used as the cathode. LSM powders were mixed
with glycerol to form slurry. This slurry was painted on the other face of the electrolyte disk, and thedisk was heated at 1523 K for 1 h. The thickness of the anode and cathode was approximately 50 m.
Two 0.3 mm diameter platinum lead wires were attached to each electrode with a platinum mesh(#100 mesh) 3 mm x 3 mm in size. Following this fabrication procedure, the cell was fired at 1323 K
for 1 h. The anode was reduced in a H2 atmosphere at 1073 K to obtain a Ni1-XCoX-YSZ (or ScSZ)
cermet before the cell measurement. The fuel cell test assembly with the two-electrode configuration1

Transactions on GIGAKU 1(2012)01026/1-5

was used to evaluate the current-voltage (I-V) characteristics. The anodic overvoltage measurement
was conducted by the current interruption method. The reference electrode was bonded as a small
circle on the anode face separated by a 2-3 mm gap from the circular working electrode. Glass ringgaskets were used to fix and seal the peripheral areas of the anode and cathode faces of the cell to theends of alumina tubes. O2 gas was supplied to the cathode at a flow rate of 2 x 10-5 m3/min. CH4 wasalso used as the fuel. CH4 diluted with He in a volume ratio of 1:9 was used as the fuel gas at a total
flow rate of 2 x 10-5 m3/min. I-V curves of the cells were measured at 973-1073 K. Reproducibilityof these I-V curves on increasing and decreasing the temperature was confirmed for each experiment.
The outlet gas from the anode was analyzed by gas chromatography. Impedance spectra were
measured with an impedance analyzer (HP-4192A).
As for the TPD analysis Ni1-xCox-YSZ and Ni1-xCox-CeO2 cermet samples were prepared by theconventional impregnation method. The starting materials were Ni(NO3)2 6H2O (Wako Pure
Chemical), Co(NO3)2 6H2O (Nacalai Tesque) and YSZ powder (Tosoh, 8 mol%Y2O3-ZrO2: TZ-8Y),
and CeO2 powder (Nacalai Tesque). The metal nitrates were mixed in an appropriate amount, and themass ratio of the Ni1-xCox to the oxides was adjusted to be 1:5. The impregnated samples were dried at
373 K for 1 h and subsequently calcined at 773 K for 1 h in air. The calcined samples were placed in analumina boat and reduced at 773 K for 1 h in a 20%H2-He atmosphere. The TPD experiment was
carried out with a ChemBET-3000 (YUASA-IONICS). The prepared cermet samples were placed in aquartz vessel for TPD and kept at 623 K for 1 h in a N2 atmosphere, and subsequently reduced at 623 K
for 1 h in a 5%H2-He atmosphere before the TPD experiment. The cermet samples were exposed in anadsorbed gaseous species at room temperature. Desorption was performed by increasing thetemperature from the room temperature to 973 K at different heating rates (β). Desorbed species weredetected with a thermal conductivity detector (TCD).
3. Results and Discussion
Figs. 1a and 1b show the current-voltage (I-V) and current-power curves (I-P) for the cell using a
Ni-YSZ cermet (cell 1) and one using a Ni0.5Co0.5-YSZ cermet (cell 2) as the anode both combinedwith the YSZ electrolyte. These results have revealed that the enhancement of the cell performanceby substituting Co atoms for Ni atoms in the Ni-based cermet is significant for CH4. The maximum
power densities were compared among the cells, whose alloy compositions in the Ni1-XCoX system
1.4

0.3

1.4

(b)

(a)
1.2

0.3

0.2

0.8
0.6
0.1

0.4

1

0.2

0.8
0.6
0.1

0.4

0.2

Power density, P / W・cm-

1

Power density, P / W・
cm-

1.2

0.2

0

0
0

0.2

0.4

0

0.6

0
0

0.2

0.4

0.6

Current density, I / A・cm-

Current density , I / A・cm-

Fig. 1(a) I-V and I-P curves of cells using the
Ni-YSZ (○) and Ni0.5Co0.5-YSZ (●)
anodes for H2 at 1073 K.

Fig. 1(b) I-V and I-P curves of cells using the
Ni-YSZ (○) and Ni0.5Co0.5-YSZ (●)
anodes for CH4 at 1073 K.

were from x = 0 through x=1, as shown in Fig. 2. We confirmed that increasing amount of Co in theNi1-XCoX system results in an increase in the cell performance up to approximately 0.8. However, the2

Transactions on GIGAKU 1(2012)01026/1-5

Co-YSZ cermet showed the lowest cell performance both for CH4 and H2. This result agrees with acomparison of the anodic polarization curves at 1273 K for the M-YSZ (M = Mn, Fe, Co, Ni, Ru, Rh,
Pd, Pt, and Au) cermet for H2 [5].
T/℃
700

800

100

Q = 100 kJ・mol-1

-0.5

80
60

-1

40

-1.5

○H2
CH4

20

0

0.2

Q = 110 kJ・mol-1-2
Q = 140 kJ・mol-10Fig. 2

600

0

0.4
0.6
X Co/(Co+Ni)

0.8Comparison of the maximum
power density among the cells using
Ni1-X CoX-YSZ cermet at 1073 K.

-2.5
0.9

1

1

1.1

1.2

T-1 / 10-3・K-

Fig. 3Arrhenius plots of Rp-1 for theelectrochemical oxidation of CH4 using the
Ni-YSZ (○) and Ni0.5Co0.5-YSZ (●) anodes.

Fig. 3 shows the reciprocal polarization resistance, Rp-1, determined from the slope of the line
showing a linear relationship between the anodic overvoltage and current density at low current
densities less than approximately 50 mA/cm2. The larger values in Rp-1 mean a high activity of theanode for the electrochemical oxidation of CH4; the anodic activation overvoltage, act = Rp  I, iscaused by the charge transfer process at the interface between the anode and electrolyte.
Replacement of half of the Ni atoms with Co atoms increased the activity of the anode for theelectrochemical oxidation of CH4 by about two times. However, the slope of the Arrhenius plots,shown in Fig. 3, remained almost constant above approximately 700 degrees Celsius, indicating thatthe solid solutioning of Co does not change the apparent activation energy for the anode reaction but
increases the amount of reactants at the active sites of the anodic oxidation of CH4. The value of Rp-1
for cell 1 below approximately 700 degrees Celsius showed a somewhat high value. Since in the gas
phase CH4 is thermally very stable and only begins to decompose into the elements, H2 and C, at 785
degrees Celsius [9], the increase in Rp-1 for cell 1 appears to be caused by the difference in theadsorptive property for CH4 between the Ni-YSZ and Ni0.5Co0.5-YSZ cermets. The strong adsorptive
ability of Ni for CH4 can account for this result. We have also reported an increase in the cell
performance for CH4 using the Ni0.5Co0.5-CeO2 cermet anode combined with a samaria-doped ceria(SDC) electrolyte [7]. These results indicate that both the alloying effect in the Ni-Co system and anoxide component in the cermet affect the adsorptive property both for CH4 and reaction products inthe electrochemical oxidation of CH4.
Figs. 4a and 4b show TPD curves of the Ni-CeO2 and Ni-YSZ cermet samples. The TPD curve of
the Ni-YSZ sample for CH4 showed one main peak at ca. 750 K, whereas that of the Ni-CeO2 sample
exhibited two main peaks at ca. 480 K and ca. 800 K. These TPD curves of the Ni-YSZ and Ni-CeO2
samples for CO showed almost the same peak shape. The amount of desorbed CO from the Ni-CeO2
sample was larger than that from the Ni-YSZ sample. Quantitative TPD analysis for these samples wasdifficult due to the low TCD signals; however, the adsorptive property of Ni metal particles was foundto be dependent on the oxide support in the cermet anode materials. Fig. 5 shows the Arrhenius plot
for the prepared Ni1-xCox-YSZ samples using the peak-method calculation based on the peaktemperature, Tp, and the heating rate, , during the TPD experiment. The slope of these Arrhenius
plots appears to be decreased with increasing the Co content up to x = 0.5. The Polanyi-Wigner
equation was used to determine the activation energy for desorption, Ed [11]. Although this equation
is inapplicable to accurately describe the kinetics of thermal desorption, which is caused by the3

Transactions on GIGAKU 1(2012)01026/1-5

interaction between the adsorbed species and the heterogeneity of adsorption sites, the value of Ed
for CH4 apparently decreased by the partial substitution of Co atoms for Ni atoms in the fcc lattice.
Since Ni atoms have a strong affinity for CH4 and CO molecules, this causes carbon deposition and
CO poisoning at SOFC operating temperatures. We have shown that the partial substitution of Co
atoms for Ni atoms in the cermet anode enhances the activity for the electrochemical oxidation of CH4,
which resulted in an increase in the cell performance due to a decrease in the anodic overvoltage.
This enhancement is most likely to be explained by the decrease in the activation energy fordesorption as well as the increase in adsorption sites for CH4 molecules, which results in the
suppression of carbon deposition and CO poisoning. By comparison, the adsorption of H2 molecules
was not affected the composition in Ni1-xCox. This adsorptive property of the Ni1-xCox-YSZ sample
for the TPD analysis shows an agreement with the result of the cell performance, as shown in Figs.
1a and 1b.

Fig. 4(b) TPD curves of the Ni-CeO2 andNi-YSZ samples for CO at  = 10 K-1.

Fig. 4(a) TPD curves of the Ni-CeO2 andNi-YSZ samples for CH4 at  = 10 K-1.

Fig. 5 Arrhenius plots for thermal desorption of the adsorbed CH4 in the Ni1-xCox-YSZ samples.
4. Conclusion
Substitution of Co atoms for Ni atoms in the Ni-based cermet anode enhanced theelectrochemical oxidation of CH4. This effect was confirmed for the cells using an YSZ electrolyte.
The cell using the Ni1-XCoX-YSZ cermet anode showed an increase in the cell performance withincreasing amount of Co content up to approximately 0.8. The decrease in the anodic overvoltage
4Transactions on GIGAKU 1(2012)01026/1-5

contributes to an enhancement of the cell performance. The TPD results showed that the adsorptive
ability of Ni1-xCox-YSZ and Ni1-xCox-CeO2 cermet particles for CH4 and CO can well account for thecell performance of these anode materials on the direct oxidation of CH4 by the O2- ions supplied
through the oxide electrolyte. The adsorptive property of Ni1-xCox particles was affected by the oxide
component of the cermet materials. The partial substitution of Co for Ni in the fcc lattice increases theamount of adsorbed species, and decreases the desorption activation energy of desorption for CO andCH4.
Acknowledgment
A part of this work was supported by a Grant-in-Aid of Scientific Research (B) (#24360304)
from Japan Society for the Promotion of Science.
References[1] A. McEvoy, “High Temperature Solid Oxide Fuel Cells”, S. C. Singhal and K. Kendall, Editors,
pp.149-169, Elsevier, UK (2003).
[2] A. Hammou and J. Guindet, “The CRC Handbook of Solid State Electrochemistry”, P. J.
Gellings and H. J. M. Bouwmeester, Editors, pp. 419-422, CRC Press, Boca Raton, Florida(1997).
[3] H. Uchida, T. Osuga, and M. Watanabe, J. Electrochem. Soc., 146 (1999) 1677.
[4] M. Watanabe, H. Uchida, and M. Yoshida, J. Electrochem. Soc., 144 (1997) 1739.
[5] T. Setoguchi, K. Okamoto, K. Eguchi, and H. Arai, J. Electrochem. Soc., 139 (1992) 2875.
[6] H. Yoshida and T. Inagaki, J. Alloys Compounds, 408/412 (2006) 632.
[7] H. Tsuchiya, W. Weerapakkaroon, T. Kanjanaboonmalert, T. Wahtzu, and K. Sato, ECS Trans.,
25 (2009) 2169.
[8] T. Sawahata, H. Takayanagi, T. WahTzu, and K. Sato, ECS Trans., 35 (2011) 1545.
[9] W. A. Bone and H. F. Coward, J. Chem. Soc., 93 (1908) 1197.
[10] J. Zhang, H. Wang, and A.K. Dalai, Appl. Catalysis A, 339 (2008) 121.
[11] J. L. Falconer and J. A. Schwarz, Catal. Rev. Sci. Eng., 25 (1983) 141.
(Received: 21 May, 2012, Accepted: 5 June, 2012)5

Transactions on GIGAKU 1(2012)01027/1-5

Preparation of Filter Paper Conjugated with High Mannose-Type
Glycans
Kiyoshi Furukawa*, Tomomi Tadokoro, Mayumi Sato, Eriko Yamada
Department of Bioengineering, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: furukawa@vos.nagaokaut.ac.jp

This paper describes the method for conjugating N-glycans from animal
sources to filter paper. High mannose-type glycans were released frombovine RNase B by hydrazinolysis, and then they were treated with succinic
dihydrazide. Hydrazide-derivatives were incubated with filter paper which
was pretreated with 100 mM sodium periodate for 60 min to generate
aldehyde groups on cellulose backbones, and then they were treated withdimethylamine borane in acetate buffer (pH3.9). The resulting glycanconjugated filter paper was incubated with peroxidase-conjugated Con A, andsubjected to color development in the presence of a chromogenic substrate,showing that color intensities correlate with amounts of high mannose-typeglycans conjugated to filter paper. The results from analyses of chemicallymodified filter paper suggest that glycan-conjugated filter paper could be
useful for trapping microorganisms which express glycan-binding proteins,
lectins.

1. Introduction
Many microorganisms recognize cell surface glycans as receptors for cell attachment and tissuecolonization. This is because viruses, bacteria and protozoa express a large number of glycan-binding proteins so called lectins (reviewed in 1). For examples, human influenza viruses recognize theSia2→6Gal group by their hemagglutinins expressed on virus surfaces, and Helicobacter pylori
binds Fuc1→2Gal→3(Fuc1→4)GlcNAc1→R group (Leb antigen) by its adhesin BabA
expressed on flagella (2, 3). In fact, such glycans are expressed on cell surfaces of humanrespiratory tract and stomach tissues, respectively. Furthermore, E. coli binds high mannose-typeglycans rich in intestinal mucosa and glycans with the Gal1→4Galgroup in urinary tract
depending on the strains, and pathogenic strains often cause enterohemorrhagy with verotoxin
produced, hemolytic uremic syndrome, acute encephalopathy, pyelonephritis, cystitis and urithritis
for human beings (1, 4, 5). Therefore, we are always facing the crisis of infection with pathogenic
microorganisms that recognize our glycans. However, this indicates that glycans can protect us
against a variety of microorganisms. Using daily necessaries and foods conjugated with glycans
with respective structures, they could trap microorganisms floating in air, living in water, attached on
foods and so on, and remove them resided in our air tract, and digestive and excretory organs.
Since some filters and foods made from plant polysaccharides such as cellulose, mannan, andcarageenan, such plant polysaccharides can be oxidized with sodium periodate to generate aldehydegroups which react quickly with hydrazide glycans, thus producing filters and foods with glycans
containing respective structures. In the present paper, we report a basic method to prepare filter
paper conjugated with high mannose-type glycans from animal glycoproteins.

2. Materials and Methods
Chemicals - Anhydrous hydrazine, dimethylamine boran, blue dextran, bovine RNase B, bovine
serum albumin (fraction V), and 4-chloro-1-naphthol were purchased from Nacalai Tesque, Inc.
(Kyoto) and Sigma Chemicals Co. (St. Loius, MO), respectively. Horseradish peroxidase-conjugated Con A was from Seikagaku Kogyo Co. (Tokyo). Whatman 3MM filter paper was from GE
Healthcare Japan (Tokyo). Shoji paper containing 15% of a mixture of rayon, vinylon and binder
was from Fujita Paper Mills Co. (Misato, Saitama), and white colored paste made of konnyaku flour1Transactions on GIGAKU 1(2012)01027/1-5

was from a local food store.
Liberation of N-glycans from glycoproteins - RNase B, which has been shown to contain highmannose-type glycans (6), dried thoroughly over P205 in vacuo, was subjected to hydrazinolysis for
10 h as described previously (7). After N-acetylation, liberated glycans were separated by paper
chromatography using 1-butanol-ethanol-water (4:1:1 v/v) as a solvent for 2 days, and extracted
from paper by the method described previously (8).
Conjugation of glycans with filter paper, shoji paper and konnyaku paste – Glycans wereincubated with succinic dihydrazide and dimethylamine borane in a mild acidic condition to prepare
hydrazide glycans. The derivatized glycans (50-100 nmoles/sample for blue dextran and 20-40
nmoles/sample for N-glycans) were then incubated for 8 hrs at 40°C with filter paper, shoji paper
and konnyaku paste (surface areas of 24-40 mm2) oxidized with sodium periodate under theconditions examined in the present study. The reaction mixture was cooled down and incubatedfurther with dimethylamine borane in acetate buffer (pH4.0) for 36 hrs at 4°C according to themethod described previously (9). A chemical reaction for conjugation of glycans to the above
materials is illustrated in Fig. 1.

Fig. 1A schematic illustration of the chemical reaction for conjugation.

Detection of N-glycans conjugated to filter paper, shoji paper and konnyaku paste – N-Glycanconjugated materials were blocked with 1% bovine serum albumin in 10 mM phosphatebuffered-saline (pH7.4) for 2 h, and incubated with 10 µg/ml Con A conjugated with horseradish
peroxidase for 1 h at 4°C followed by 500 µg/ml of the chromogenic peroxidase-substrate,
4-chloro-1-naphthol, for 10 min according to the method described previously (10). Colorintensities developed were determined by NIH image J analysis.

3. Results and Discussion
Preparation of oxidized filter paper
Optimal conditions for derivatization of N-glycans with succinic dihydrazide were establishedpreviously (9). In the present study, optimal conditions for oxidizing filter paper with sodium
periodate were determined. Pieces of filter paper (40 mm2) were treated with 10, 50 and 100 mM
sodium periodate in 50 mM acetate buffer (pH 4.5) for up to 60 min at room temperature. Then,they were conjugated with hydrazide blue dextran (100 nmols/sample) and amounts of blue dextran
conjugated to filter paper were determined by measuring intensities of blue color on filters by theimage analysis. The results showed that not so much blue dextran is conjugated to filters treatedwith 10 and 50 mM sodium periodate for 60 min, but significant amounts of blue dextran isconjugated to filters treated with 100 mM sodium periodate for 60 min at room temperature (data notshown). In order to determine an optimal incubation time period of filter paper with 100 mM
sodium periodate, pieces of filter paper were treated with 100 mM sodium periodate for 15, 30, 45
and 60 min, and then conjugated with hydrazide blue dextran. The results showed that more blue
dextran is conjugated to filter paper treated for a longer time period with 100 mM sodium periodate(Figs. 2 and 3). However, treatment of filter paper with 100 mM sodium periodate for more than
60 min, particularly over 90 min resulted in decomposition of filter paper (unpublished data),
indicating that an optimal incubation time period for oxidizing filter paper is 60 min. Hereafter,
filter paper was treated with 100 mM sodium periodate for 60 min at room temperature. When2

Transactions on GIGAKU 1(2012)01027/1-5

pieces of filter paper (40 mm2) treated with 100 mM sodium periodate for 60 min were incubatedwith different amounts of hydrazide blue dextran, 10-20 nmols of blue dextran were conjugated to
the filter paper effectively (Fig. 4).

Fig. 2 Blue dextran-conjugated filters. Filters were treated with 100 mM sodium periodate forthe time periods indicated, and then conjugated with blue dextran.

Fig. 3 Incubation time with sodium periodatedependent conjugation of blue dextran on
filters. Filters were incubated with 100 mM
sodium periodate for the time periods indicated, and then conjugated with blue dextran.
Blue color was monitored by the image
analysis.

Fig. 4 Concentration-dependent conjugation ofblue dextran on filters. Oxidized filters wereincubated with different amounts of hydrazide
blue dextran. Blue color was monitored by theimage analysis.

Conjugation of hydrazide N-glycans to oxidized filter paper
Hydrazide high mannose-type glycans were prepared and conjugated to oxidized filter paper by
the method described above. Conjugation of the N-glycans was monitored by binding of Con A
which interacts preferentially with high mannose-type ones (11). By measuring color intensities
indicating amounts of Con A which interacted with the glycans conjugated to filter paper (Fig. 5), it
was shown that about 5-10 nmoles of high mannose-type glycans bind to a piece of filter paper witha surface area of 40 mm2.

Fig. 5 High mannose-type glycan-conjugated filter paper. Oxidized filters were incubated withdifferent amounts of hydrazide glycans, and glycans bound were detected with Con A-binding as
described in the text.
Preparation of oxidized shoji paper and paste made from konnyaku flour
Since an oxidizing condition for filter paper was established, we then examined those for shoji
paper and konnyaku paste. Pieces of shoji paper (36 mm2) and of konnyaku paste (about 36 mm2
3

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surface area and 5 mm thickness) were similarly treated with 10, 50 and 100 mM sodium periodatefor 60 min at room temperature, and oxidized materials were conjugated with hydrazide highmannose-type glycans (40 nmoles/sample) followed by visualization with peroxidase-conjugated
Con A as described above. The results showed that toptimal oxidizing conditions for shoji paper
and konnyaku paste are 10 mM sodium periodate for 60 min, and that other conditions bring about
denature or decomposition of the materials treated (data not shown). Shoji paper and konnyaku
paste oxidized with 10 mM sodium periodate for 60 min were incubated with different amounts ofhydrazide high mannose-type glycans and visualized with Con A-binding as describe above (Figs. 6
and 7, respectively).

Fig. 6 Glycan concentration-dependent conjugation to shoji paper. A, B, C and D indicate 0, 2.5, 5 and 10 nmoles of glycans/sample.

Fig. 7 Glycan concentration-dependent conjugation to konnyaku paste. A, B, C and D indicate 0, 2.5, 5 and 10 nmoles of glycans/ sample.

In the present study, blue dextran and high mannose-type glycans were used to conjugate to filter
paper. However, it appeared that more blue dextran chains are conjugated to filter paper than highmannose-type glycan chains. This could be due to differences in the branching structures between
them. Further examination remains to be elucidated. Since oxidized materials used in the present
study were strips, glycans were conjugated on both sides of them. This indicates that even though
microorganisms fail to be trapped at the front side of filter paper, they could be trapped at thebackside when they pass through the filter.
Since microorganisms including pathogenic viruses and bacteria bind to glycans with respective
structures expressed on cell surfaces of our organs and tissues, it is possible that glycan-conjugateddaily necessaries and foods protect us against a variety of infectious microorganisms. A major
strain of E. coli in nature contains bacterial adhesin FimH at the tip of type 1 fimbria (12), whichbinds to -mannose-terminated glycans (4). Therefore, high mannose-type glycan-conjugated filter
paper prepared in the present study can trap E. coli in nature, particularly in well water to drink andeven in tap water when water supply facilities are destroyed by disasters by soaking it or filtering
through it. In some underdeveloped countries, inhabitants are always scared with infection by
microorganisms through drinking water. Introduction of a large scale-filtering system using
glycans in waterwork of such countries will supply microorganism-free drinking water to inhabitants.
In other aspects of their application, ventilating fan filters with glycans having the Sia2→6Gal
structures (2) can trap human influenza viruses floating in air under a humidified condition, andreduce absolute numbers of the viruses in living spaces, thus protecting us against infection with theviruses. Konnyaku paste, an indigestible food, with glycans having the Fuc1→2Gal→3(Fuc1→4)GlcNAc structure can trap H. pylori in stomach and take it out from us if we have been
infected with the bacterium. Such ideal trapping of H. pylori with the glycan-conjugated foods will
reduce the possibility of development and progression of stomach ulcer and eventually of stomach
cancer caused by H. pylori (13).
The present method can extend to any types of glycans to be conjugated with polysaccharide-based materials depending on which microorganism(s) to be removed from our living
spaces and from our bodies. In Japan, we have least chance to appreciate blessing of drinking
water since it is free and safe. However, once Japan is hit by serious natural disasters such as
earthquakes and flooding, we will be immediately placed into low quality of living environments
particularly by shortage of water for daily life and secondary by spreading of infectious diseases due
to collapse of sanitary condition. The glycan-conjugated daily necessaries and foods might be ofhelp for us in those circumstances, too.
4

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4. Conclusion
The present study showed that optimal conditions for oxidizing filter paper and for shoji paper
and konnyaku paste are 100 mM and 10 mM of sodium periodate for 60 min at room temperature,
respectively. The oxidized materials were conjugated with hydrazide high mannose-type glycans.
The glycans attached to filter paper, shoji paper and konnyaku paste were visualized by staining withCon A that binds high mannose-type glycans, and quantified by NIH image analysis. These results
indicate that not only high mannose-type glycans but also complex-type and hybrid-type glycans,and disaccharides to polysaccharides could be conjugated with these materials. Since microorganisms including pathogenic viruses and bacteria bind glycans with respective structures
expressed on our cells (reviewed in 1), it is possible to protect us against infection with such
pathogenic microorganisms and/or to elimination of such pathogenic microorganisms alreadyinfected us by dairy necessaries and foods conjugated with glycans having structures specific toindividual microorganisms as discussed above. The method presented here was first developed in
Nagaoka University of Technology, and from the point of view of “GIGAKU” (the policy and target
in Nagaoka), further studies and applications of this method are strongly required for the progress inbiological science and glycotechnology.
Acknowledgments
This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, by the Research Promotion Fund fromthe Japan Science and Technology Agency, and by Program for the Pacific Rim Green Innovation
Hub Project in Nagaoka University of Technology.
References[1] A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart, andM. E. Etzler, In “Essentials of Glycobiology” (2nd ed.), Cold Spring Harbor Lab. Press, New
York, 2008.
[2] J. N. Couceiro, J. C. Paulson, and L. G. Baum, Virus Res., 29 (1993) 155-165.
[3] D. Ilver, A. Amrnqvist, J. Ogren, I. M. Frick, D. Kersulyte, E. T. Incecik, D. E. Berg, A. Covacci,
L. Engstrand, and T. Boren, Science, 279 (1998) 373-377.
[4] I. Ofek, and E. H. Beachey, Infect. Immun., 22 (1978) 247-254.
[5] K. Bock, M. E. Breimer, A. Bringnole, G. C. Hansson, K.-A. Karlsson, G. Larson, H. Leffler, B.
E. Samuelsson, N. Stromberg, and C. S. Eden, J. Biol. Chem., 260 (1985) 8545-8551.
[6] C.-J. Liang, K. Yamashita, and A. Kobata, J. Biochem., 88 (1980) 51-58.
[7] S. Takasaki, T. Mizuochi, and A. Kobata, Methods Enzymol., 83 (1982) 263-268.
[8] T. Hironaka, K. Furukawa, P. C. Esmon, M. A. Fournel, S. Sawada, M. Kato, T. Minaga, and A.
Kobata., J. Biol. Chem., 267 (1992) 8012-8020.
[9] N. Yoshitani, and S. Takasaki, Anal. Biochem., 277 (2000) 127-134.
[10] T. Sato, K. Furukawa, D. E. Greenwalt, and A. Kobata, J. Biochem. 114 (1993) 890-900.
[11] S. Ogata, T. Muramatsu, and A. Kobata, J. Biochem., 78 (1975) 687-696.
[12] E. Hahn, P. Wild, U. Hermanne, P. Sebbel, R. Glockshuber, M. Haner, N. Taschner, P.
Burkhard, U. Aebi, and S. A. Muller, J. Mol. Biol., 323 (2002) 845-857.
[13] F. J. Hardin, and R. A. Wright, Hospital Physician, May (2002) 23-31.
(Received: 17 May, 2012, Accepted: 31 May, 2012)5

Transactions on GIGAKU 1(2012)01028/1-6

Development of Trichoderma reesei Strain Specialized for
Cello-oligosaccharides Production from Cellulose
Yosuke Shida1*, Rina Yoshida1, Tatsuki Wakayama2, Yoshiro Konda2, Takashi Yamaguchi1,
and Wataru Ogasawara1
1
Department of Bioengineering, Nagaoka University of Technology
1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188,
2
INPEX Corporation
Akasaka Biz Tower, 5-3-1 Akasaka, Minato-ku, Tokyo 107-6332, Japan*E-mail: yshida@vos.nagaokaut.ac.jp

The filamentous fungus Trichoderma reesei is a well-known cellulolyticorganism that has been widely used in industrial applications such as
saccharification of cellulosic biomass. T. reesei produces a complete set ofcellulases to degrade cellulose to glucose. However, it is difficult to control the
enzyme reaction to obtain cello-oligosaccharides, which are the intermediatesof cellulose degradation. In this study, we created a T. reesei strain in which thegene encoding ß-glucosidase I of cellulases was disrupted. The resultant bgl1
disruptant exhibited higher accumulation of cellobiose compared to the parent
PC-3-7 strain in cellulose hydrolysis experiments.

1. Introduction
Cellulose is the most abundant biomass on the earth as the major component of plant cell wall.
It is a linear polymer composed of ß-D-glucose units, linked by ß-1,4-glycosidic bonds. Glucose
derived from cellulosic biomass can serve not only as food, but also as raw material for chemicals
and fuels [1]. In addition to monosaccharides, cello-oligosaccharides (COSs), which areintermediates of cellulose degradation, have the potential to be converted into functional foods orother raw materials [2, 3]. However, a major impediment to this process is the high refractory natureof cellulose to hydrolysis, which is due to its crystalline structure. In nature, cellulose is degraded bya variety of cellulolytic microorganisms.
The filamentous fungus Trichoderma reesei is a potent producer of cellulase and produces three
types of cellulases, which synergistically degrade cellulose to glucose [4]. The cellobiohydrolase(CBH, EC 3.2.1.91) releases cellobiose from the end of the cellulose chain. The endoglucanase (EG,EC 3.2.1.4) is an end-acting enzyme that randomly cleaves the cellulose chain in an amorphous
region. Finally, the ß-glucosidase (BGL, EC 3.2.1.21) hydrolyzes cellobiose to glucose. The weakest
link of T. reesei cellulase preparation is the comparatively low activity of BGL, which results in theinhibition of cellobiohydrolase by accumulated cellobiose. Attempts have been made to constructgenetically modified derivative of T. reesei to enhance ß-glucosidase activity for effectivesaccharification of cellulosic biomass [5]. On the other hand, production of cellobiose can beachieved by disrupting the gene encoding ß-glucosidase, and production of longer COSs can beachieved by disruption of cellobiohydrolases and ß-glucosidase genes. COSs derived from cellulosic
biomass are estimated to be the most widely available nondigestible oligosacchalides, which areresistant to digestion by human gastric and pancreatic enzymes. Hence, COSs can be the potential
sources for prebiotic functional foods by stimulating bacteria in the colon [6]. A low-cost efficient
production system of COSs from cellulosic biomass and a subsequent separation system of specific
COS are expected to interpret the physiological functions of COSs and to establish wide industrial
use of COSs. In this study, we present the effect of BGL deletion in T. reesei on the production of
COSs from cellulosic biomass.

1

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2. Experiments(1) Fungal strains and culture conditions
T. reesei strain PC-3-7 is a cellulase-hyperproducing mutant with enhanced ability in cellulase
induction by L-sorbose [7] and was obtained from Kyowa Hakko, Japan. Strains were maintained on
potato dextrose agar (PDA; Difco) slants.
For production of cellulase enzymes, conidia obtained from T. reesei strains grown on PDA
plates were inoculated in a medium composed of 50 mM Na-tartrate buffer (pH4.0) containing
0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·H2O, 0.03% MgSO4·7H2O, 0.05% yeast extract,
0.1% Polypeptone, 0.1% Tween 80, 0.1% trace element solution (0.005 mg/L FeSO4, 0.002 mg/L
MnSO4H2O, 0.001 mg/L CuSO45H2O, 0.002 mg/L ZnSO4·7H2O, 0.001 mg/L (NH4)6Mo7O24·4H2O,
0.002 mg/L CoCl2·6H2O and 0.001 mg/L NiCl2·6H2O), which was supplemented with 1% Avicel orcellobiose as a carbon source. The cultures were then incubated at 28°C on rotary shaker at 220 rpm
for 5 days.
(2) Construction of bgl1 disruption cassette
The chromosomal DNA of T. reesei PC-3-7 was prepared as described previously [8]. DNA
fragment of bgl1 upstream region and downstream region were amplified by polymerase chain
reaction (PCR). The amdS gene from Aspergillus nidulans encoding acetamidase was used as aselection marker of T. reesei transformant was prepared from the plasmid p3SR2. Three DNA
fragments were connected in the order of bgl1 upstream region first, followed by amdS and then
bgl1 downstream region. The resultant bgl1 disruption cassette was used for transformation of T.
reesei strains.
(3) Transformation of T. reesei
T. reesei strain PC-3–7 was transformed using a protoplast-polyethylene-glycol procedure [9].
Prior to transformation, the bgl1 disruption cassette was subjected to restriction enzyme digestion toremove the vector backbone. Candidate transformants were streaked twice on selective plates
containing acetamide as the sole nitrogen source, and were transferred to PDA plates to form
conidia.
(4) Enzyme assays and protein analysis
Carboxymethyl–cellulase (CMCase) activity was determined by measuring reducing sugarusing Somogyi-Nelson method [10]. CMCase activity was determined with a final concentration of
1.0% carboxymethylcellulose (low viscosity, 50-200 cps, Sigma Chemical) in 50 mM sodium
acetate buffer (pH 5.0) at 50˚C for 15 min. One unit of activity was defined as the amount of enzymethat produced 1 µmol of reducing sugar per minute in glucose equivalents. BGL activity wasdetermined by measuring the amount of pNP-ß-D-glucopyranoside (pNP-G, Sigma Chemical)
released in 50 mM sodium acetate buffer (pH 5.0) at 50˚C. One unit of activity was defined as theamount of enzyme that produced 1 µmol of pNP per minute. Protein concentration was determinedusing the Bradford protein assay [11] (Bradford, 1976) with bovine gamma globulin as a standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 12.5%
polyacrylamide gel slabs as described [12]. Proteins were stained with Coomassie brilliant blue
R-250. The molecular-mass markers used were the Precision Plus Dual Standard Marker kit for
SDS-PAGE (Bio-Rad).
(5) Cellobiose production
The substrates used for cellobiose production were Avicel™, microcrystalline cellulose andphosphoric-acid swollen cellulose, which was prepared as described by Walseth [13]. The reaction
was performed in an 1.5-mL microtube containing 5% (w/w) dry substrate in 100 mM sodium
acetate buffer, pH 5.0 with an enzyme at a loading of 5 mg protein/g of dry weight substrate. The
reaction was performed at 50˚C with vigorous shaking for the period of 6, 12, or 24 h. Samples wereboiled for 5 min to inactivate enzymes, and products were measured using high performance liquid
chromatography (HPLC) equipped with Prominence reducing sugar analysis system (Simadzu).

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3. Results and Discussion(1) Construction of bgl1 disruptant of T. reesei PC-3-7
Among the five genes in T. reesei encoding extracellular BGL, the bgl1 product is mainly
responsible for BGL activity. Therefore, disruption of the bgl1 gene was performed. We constructed
the bgl1 disrupting cassette, in which bgl1 was replaced by amdS as a selectable marker (Fig. 1A).
After transformation of T. reesei, we successfully obtained two candidates that utilize acetamide as asole nitrogen source. They were grown on Avicel to produce cellulase enzymes and then the culture
filtrates were assayed for pNPase activity. The pNPase activities of candidates were significantly
lower than that of the parental strain, PC-3-7 (Fig. 1B). In addition, the 82-kD protein bandcorresponding to BGL1 was not detected in the culture filtrates in SDS-PAGE analysis (Fig 1C).
Thus, we concluded that bgl1 disruptants were successfully constructed. Out of the bgl1 disruptants
obtained, ∆bgl1-2 was used as the representative ∆bgl1 for subsequent experiments.

Fig. 1 Construction of bgl1 disruption mutant by gene targeting technique. A: Schematic
representation of gene replacement of bgl1. B: Comparison of BGL activity of parental strain PC-3-7
and two bgl1 disruptants, ∆bgl1-1 and ∆bgl1-2. C: SDS-PAGE analysis of enzyme preparations frombgl1 disruptants. Samples subjected to electrophoresis were prepared from culture filtrates grown on
Avicel for 6 days.
(2) Cellulase production by T. reesei ∆bgl1 strain
When T. reesei was grown on cellulose as a sole carbon source, it produces a set of cellulases[14]. Although its direct assimilation of cellulose is hard to grasp due to the highly crystalline andinsoluble nature of cellulose, it is generally believed that soluble cello-oligosaccharides released by
the action of constitutively expressed cellulases served as inducers of inducible cellulase production[15-18]. As the major soluble product from cellulose cellobiose can be the natural inducer ofcellulase. However, growth on cellobiose inhibited cellulase induction in T. reesei QM9414 [19] due
to its rapid hydrolysis to glucose, which suppressed cellulase formation by carbon catabolite
repression controlled by a transcription regulator, Cre1 [20]. Among inducers reported so far
sophorose exerted the strongest cellulase induction, and sophorose has been considered as thenatural inducer of cellulases and was estimated to be formed by transglycosylation by ß-glucosidase.
Therefore, it has been assumed that BGL is the key enzyme for cellulase induction in T. reesei.
Although the bgl1 gene disruption causing reduction of cellulase formation has been reported in T.
reesei QM9414 [21], the effect of bgl1 disruption has not been examined in PC-3-7, a mutant strain

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obtained after six generations from QM9414 by UV irradiation and chemical mutagenesis that
produces large amount of cellulose. The time course measurement of CMCase activity was
performed during the growth on Avicel and cellobiose and the results were compared between
PC-3-7 and ∆bgl1. When PC-3-7 and ∆bgl1 were grown on Avicel, CMCase activity of ∆bgl1 wasslightly higher than that of PC-3-7 (Fig. 2A). However, when they were grown on cellobiose,
significantly higher CMCase activity was observed in ∆bgl1 compared to PC-3-7 strain (Fig. 2B).
These results are dramatically opposite to those previously reported for QM9414. Recently, it wasreported that cellulase production of PC-3-7 was highly responsive to cellobiose in the liquidmedium and that a mutation of the gene encoding a transcription regulator in PC-3-7 altered bothcellobiose utilization and cellulase production [22]. These phenotypes were due to some mutation in
the PC-3-7 genome, which has been demonstrated previously [22]. The results obtained in this study
suggest that the accumulation of cellobiose during the growth period caused higher cellulase
induction in ∆bgl1 strain, and the mutation in PC-3-7 is also involved in the phenotypes observe.

Fig. 2. Time course of CMCase activity
produced by PC-3-7 and ∆bgl1. These strains
were grown on Avicel (A) and cellobiose (B)as a sole carbon source. The CMCase activity
in the culture supernatant was assayed. Opencircles and filled squares represent ∆bgl1 andPC-3-7, respectively.

Fig. 3. HPLC analysis of hydrolytic products ofcellulosic material by cellulase preparation.
Avicel, micro crystalline cellulose (A) and PSC,
phosphoric-acid swollen cellulose (B) were usedas substrates.

(3) Cellobiose production from cellulosic material
To assess cellobiose accumulation by enzyme preparations, two kinds of celluloses, Avicel and
PSC, were used as substrates, and cellobiose and glucose accumulation were compared between∆bgl1 and PC-3-7 strains. When Avicel was added to the enzyme preparation of ∆bgl1, the amountof cellobiose increased and glucose remained at a significantly lower level over the course of the4Transactions on GIGAKU 1(2012)01028/1-6

reaction time. In contrast, cellobiose level stayed constant and glucose increased during the reaction
period in the PC-3-7 enzyme preparation (Fig. 3A). Similar results were obtained when PSC wasused as a substrate. The enzyme preparation of ∆bgl1 exhibited higher cellobiose accumulation andreduced glucose accumulation compared to the PC-3-7 enzyme preparation (Fig. 3B). Because PSC
is susceptible to degradation by endoglucanases owing to the swollen nature of its cellulose, we
expected to observe longer cello-oligosaccharides from PSC, but no other COSs than cellobiose was
detected.

4. Conclusions
A novel mutant strain of T. reesei PC-3-7 with the disruption of bgl1 gene was constructedusing gene replacement technique. The resulting mutant showed significant reduction of
ß-glucosidase activity. In addition, bgl1 disruption brought increased production of cellulase
compared to the parent PC-3-7 strain. When Avicel and PSC were treated with the enzyme
preparation of ∆bgl1, accumulation of cellobiose was observed. Mass production of cellobiose from
cellulosic materials is expected using this ∆bgl1 strain and development of a strain with thecellobiohydrolase gene disruption on the background of ∆bgl1 is also expected to produce longer
cello-oligosaccharides. Exploitation of a more practical purification method of COSs from thereaction mixture is required to achieve mass production of these intermediary products of cellulose
degradation.

Acknowledgement
This work was mainly supported by INPEX Corporation. This work was partially supported by
Establishment of Carbon-Cycle-System with Natural Rubber program of Science and Technology
Research Partnership for Sustainable Development in Japan science and technology agency (JST)and Japan International Cooperation Agency (JICA).

References[1] B. Kamm, and M. Kamm, Adv. Biochem. Eng. Biotechnol., 105 (2007) 175–204[2] M. Satouchi, T. Watanabe, S. Wakabayashi, T. Ohokuma, T. Koshijima, and M. Kuwahara, J Jpn
soc nutr food sci 49 (1996) 143– 148. (in Japanese).
[3] S. R. Hall, S. C. Wimbush, Y. Shida, and W. Ogasawara, Chem. phys. lett., 507 (2011) 144-150(2011)[4] A. Schuster, and M. Schmoll, Appl. Microbiol. Biotechnol 87(2010) 787–79[5] H. Nakazawa, T. Kawai, N. Ida, Y. Shida, Y. Kobayashi, H. Okada, S. Tani, J. Sumitani, T.
Kawaguchi, Y. Morikawa, and W. Ogasawara, Biotechnol. Bioeng. 109 (2012) 92-99[6] G.R. Gibson, and M. B. Roberfroid, J. Nutr. 125 (1995) 1401-1412.
[7] M. Kawamori, Y. Morikawa, and S. Takasawa, Appl. Microbiol. Biotechnol. 24(2986) 449–453.
[8] B. Seiboth, L. Hartl, M. Pail, E. Fekete, L. Karaffa, and C.P. Kubicek, Mol. Microbiol. 51(2004)
1015–1025[9] M. Penttilä, H. Nevalainen, M. Rättö, E. Salminen, and J.K.C. Knowels, Gene 61(1987) 155–164[10] M. Somogyi, J Biol Chem 195(1952) 19-23[11] M. M. Bradford, Anal Biochem 72(1976) 248–254[12] U. K. Laemmli, Nature 227 (1970) 680–685[13] C. S. Walseth, Am Tech Assoc Pulp Paper Ind 35(1952) 228-233[14] Nogawa M, Goto M, Okada H, Morikawa Y (2001) L-Sorbose induces cellulase genetranscription in the cellulolytic fungus Trichoderma reesei. Curr Genet 38:329–334[15] M. Gritzali, and R. D. Brown, Adv. Chem. Ser. 181(1960) 816-826[16] M. Mandels, F. W. Parrish, and E. T. Reese, J. Bacteriol 83(1962) 400-408[17] S. El-Gogary, A. Leite, O. Crivellaro, D. E. Eveleigh, and H. El-Dorry, Proc. Natl. Acad. Sci.
USA 86(1989) 6138-6141

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Transactions on GIGAKU 1(2012)01028/1-6[18] J. C. Carle-Urioste, J. Escobar-Vera, S. El-Gogary, F. Henrique-Silva, E. Torigoi, O. Crivellaro,
A. Herrera-Estrella, and H. El-Dorry, J. Biol. Chem. 272(1989) 10169-10174[19] C. Fritscher, R. Messner, and C. P. Kubicek, Exp. Mycol. 14(1990) 451-461[20] J. Strauss, R. L. Mach, S. Zeilinger, G. Hartler, G. Stöffler, M. Wolschek, and C. P. Kubicek,
FEBS Lett. 376(1995) 103-107[21] R. L. Mach, B. Seiboth, A. Myasnikov, R. Gonzalez, J. Strauss, A. M. Harkki, and C. P.
Kubicek, Mol Microbiol 16(1995) 687-697[22] M. Nitta, T. Furukawa, Y. Shida, K. Mori, S. Kuhara, Y. Morikawa, and W. Ogasawara, Fungal
Genet Biol 49(2012) 388-397(Received: 30 May, 2012, Accepted: 18 June, 2012)6

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Identification of Amino Acid Residues Essential for the Activity of
3-O-Methylgallate 3,4-Dioxygenase from Sphingobium sp. Strain
SYK-6
Daisuke Kasai* and Eiji Masai
Department of Bioengineering, Nagaoka University of Technology,
Nagaoka, Niigata 940-2188, Japan*E-mail: dkasai1@vos.nagaokaut.ac.jp
The 3-O-methylgallate 3,4-dioxygenase gene, desZ, is involved in thesyringate catabolism of the lignin-derived aromatic degrader Sphingobium
sp. strain SYK-6. Its product, DesZ is classified as a type II extradiol
dioxygenase, which usually contains two histidines and one glutamate tocoordinate active site non-heme Fe(II) and another histidine to deprotonate thehydroxyl group of a substrate. Based on the amino acid sequence alignment of
DesZ with the related type II extradiol dioxygenases His250 is predicted to be
involved in deprotonation of the substrate hydroxyl group. His12 and Glu287
are estimated to coordinate Fe(II) and the other histidine is deduced to be
replaced by Glu100 in DesZ. Each of DesZ mutants with His12Ala,
Glu100Ala, His250Ala, or Glu287Ala replacements lost its catalytic activity
completely. These results indicate that these four amino acid residues are
essential for the DesZ activity and sauggest that His12, Glu100, and Glu242
coordinate Fe(II) in DesZ.

1. Introduction
Extradiol dioxygenases play a critical role in the aerobic bacterial degradation of aromaticcompounds. These enzymes catalyze the cleavage of the aromatic ring of catechol derivatives, suchas catechol, protocatechuate (PCA), and homoprotocatechuate (HPCA), through the incorporation of
two oxygen atoms at a position adjacent to the hydroxyl groups. It has been reported that a large
number of extradiol dioxygenases contain Fe(II) at the active site. According to the similarity of
their amino acid sequences, the extradiol dioxygenases have been classified into three different
evolutionarily independent families. Type I extradiol dioxygenases belong to the vicinal oxygenchelate superfamily, including 2,3-dihydroxybiphenyl 1,2-dioxygenase of Burkholderia xenovorans
LB400 and Acidovorax sp. strain KKS102 and catechol 2,3-dioxygenase of Pseudomonas putida
mt-2. Type II extradiol dioxygenases comprise enzymes consisting of one or two different subunits.
For example, 2,3-dihydroxyphenylpropionate 1,2-dioxygenase from Escherichia coli K-12 andHPCA 2,3-dioxygenase of E. coli W are homooligomers of a single subunit, whereas PCA
4,5-dioxygenase (4,5-PCD) of Sphingobium, Pseudomonas, and Comamonas strains and2-aminophenol 1,6-dioxygenase of Pseudomonas and Comamonas strains have  and  subunits.
Type III dioxygenases belong to the cupin superfamily, which includes such enzymes as gentisate
dioxygenase, homogentisate dioxygenase, and 1-hydroxy-2-naphthoate dioxygenase. Despite theirdifferent primary structures, these three types of enzymes have similar active sites and usuallypossess the same iron ligands, two histidines and one glutamate called 2-His-1-carboxylate structural
motif. In addition to these residues, another histidine in the active site, which is involved in the
deprotonation of the hydroxyl group of the substrate, is conserved among the extradiol dioxygenases.
Sphingobium sp. strain SYK-6 is able to grow on a wide variety of lignin-derived aromaticcompounds, including 5,5'-dehydrodivanillate, vanillate, and syringate, as the sole source of carbon
and energy [2, 3]. The catabolic pathway for syringate has been characterized and is shown in Fig. 1.
3-O-Methylgallate (3MGA), a metabolite of syringate via the reaction catalyzed by syringate
O-demethylase (DesA), is degraded through three different pathways in which LigAB, 3MGA
3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) and gallate dioxygenase (DesB)
participate [4-7]. DesZ catalyzes the ring cleavage between C3 and C4 of 3MGA to generate1Transactions on GIGAKU 1(2012)01029/1-6

Fig. 1. Catabolic pathways of syringate by Sphingobium sp. strain SYK-6. Enzymes: DesA,
syringate O-demethylase; DesZ, 3MGA 3,4-dioxygenase; LigA and -B, small and large subunits,
respectively, of 4,5-PCD; LigM, vanillate/3MGA O-demethylase; DesB, gallate dioxygenase;
LigI, PDC hydrolase; LigJ, OMA hydratase; LigK, CHA aldolase/oxaloacetate decarboxylase.
Compounds: 3MGA, 3-O-methylgallate; PDC, 2-pyrone-4,6-dicarboxylate; CHMOD,
4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate; OMA, 4-oxalomesaconate; CHA,
4-carboxy-4-hydroxy-2-oxoadipate.
4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate and 2-pyrone-4,6-dicarboxylate [4].
According to the amino acid sequence similarity, DesZ appeared to belong to the type II extradiol
dioxygenase family. However, the second histidine, which is the constituents of the
2-His-1-carboxylate structural motif, is not found in DesZ. In the present study, we investigated theamino acid residues involved in the activity of DesZ using site-directed mutagenesis.
2. Experiment
Strains and plasmids
The Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37C or 30C. For
culturing the cells carrying an antibiotic resistance marker, the media for the E. coli transformants
were supplemented with 100 mg of ampicillin (Ap) per liter.
DNA manipulations, site-directed mutagenesis, and nucleotide sequencing
The DNA manipulations were performed essentially as previously described [8, 9]. The
site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit(Stratagene, San Diego, CA) following the manufacturer’s instructions. The pTZNE6 and pTZEN3
recombinant plasmids carrying the 0.7-kb NdeI-EcoRI fragment and 0.3-kb EcoRI-NdeI fragment,
respectively, were used as the templates for the amino acid changes H12A, E100A, H250A, andE287A in DesZ, together with the appropriate primers (Primer sequences are available upon request),
to obtain pT6, pT8, pT11, and pT12. The mutated desZ genes were constructed by inserting the
0.3-kb EcoRI fragment from pBH700 into pT6 and pT8 and by inserting the 0.7-kb EcoRI fragmentfrom pBE660 into pT11 and pT12. The 1.0-kb NdeI fragments from the resulting plasmids, pSD6,
pSD8, pSD11, and pSD12, were cloned into pET21a(+) to obtain pETZM6 (H12A), pETZM8(E100A), pETZM11 (H250A), and pETZM12 (E287A), respectively. Mutagenesis of eachnucleotide base was verified by sequencing both strands of the cloned insert using the dideoxy
termination method [10] with a CEQ 2000XL genetic analysis system (Beckman Coulter, Inc.,
Fullerton, CA). A Sanger reaction was performed using a CEQ Dye terminator cycle
sequencing-quick start kit (Beckman Coulter, Inc.). The analysis of the nucleotide sequence was2Transactions on GIGAKU 1(2012)01029/1-6

performed as described in a previous study [6]. The multiple sequence alignment was produced
using the Clustal W program, and the phylogenetic tree was inferred from the alignments using theneighbor-joining method [11]. The graphics for the phylogenetic trees were produced using the
TreeView program [12]. The DDBJ, EMBL, and GenBank databases were used for searching forhomologous proteins.
Expression of desZ and its mutants in E. coli and preparation of crude enzymes
E. coli BL21(DE3) cells carrying either the wild-type or mutant desZ gene were grown in 100
ml of LB medium containing Ap at 30C. The expression of the genes was induced for 4 h by adding
1 mM isopropyl--D-thiogalactopyranoside when the absorbance of the culture at 600 nm reached0.5. The cells were harvested and resuspended in FE2 buffer consisting of 50 mM Tris-HCl buffer(pH 7.0), 10% glycerol, 0.1 mM ferrous ammonium sulfate, and 2 mM L-cysteine hydrochloride.
The suspended cells were sonicated, and the cell lysate was centrifuged at 15,000  g for 15 min.
The resulting supernatant was then used as the crude enzyme fraction. The protein concentration wasdetermined by the method of Bradford [13]. The expression of the gene was confirmed by sodium
dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE).
Enzyme assays
The dioxygenase activities of DesZ and its mutants were assayed by measuring thesubstrate-dependent oxygen consumption rate. The 2-ml assay mixture contained 50 mM GTA
buffer (pH 7.0) consisting of 50 mM 3,3-dimethylglutarate, 50 mM Tris, 50 mM
2-amino-2-methyl-1,3-propanediol, the crude enzyme (500 g of protein), and 1 mM of 3MGA. The
reaction was performed at 30C, and the oxygen consumption rate was measured using an oxygen
electrode (B-505; Iijima Electronics Manufacturing Co., Ltd.). The enzyme activity (one unit) was
defined as the amount that consumed 1 mol of O2 per min at 30C. The specific activity was
expressed as units per milligram of protein.

3. Results
Amino acid sequence alignment of DesZ with type II extradiol dioxygenases
Based on the three dimensional structure of LigB, His12, His61, and Glu242 in LigB are
estimated to coordinate non-heme iron and constitute an active center and His195 is predicted to act
as the catalytic base [14]. Alignment of the deduced amino acid sequence of the desZ gene with thatof LigB indicated that DesZ conserved residues His12, His250, and Glu287, which correspond to
His12, His195, and Glu242 of LigB, respectively (Fig. 2). The alignment also revealed that theFig. 2. Alignment of the deduced amino acid sequences of desZ, ligZ, and ligB. Identical aminoacids are indicated with asterisks. The conserved residues among the type II extradiol
dioxygenases that are thought to be active site residues in LigAB are shaded in black. The
arrows show the positions subjected to mutagenesis.

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residue corresponding to His61 of LigB was not conserved in DesZ and 2,2,3-trihydroxy-3methoxy-5,5-dicarboxybiphenyl dioxygenase (LigZ) of SYK-6 [4, 15]. Because Glu242 coordinates
non-heme iron along with His12 and His61 in LigB, Glu100 of DesZ appeared to coordinate
non-heme iron along with His12 and Glu287. In LigZ Glu99 is conserved at the corresponding
position of Glu100 in DesZ.
Site-directed mutagenesis of the putative active site residues of DesZ
To investigate whether His12, His250, Glu287, and Glu100 are involved in the activity of DesZ,
each of the residues was replaced with an Ala residue as indicated in Fig. 2 using site-directedmutagenesis. Each desZ mutant gene was inserted in pET21a(+) and expressed in E. coli BL21(DE3)under the control of the T7 promoter. The SDS-PAGE analysis revealed that there was no differencein the production of DesZ protein between the wild-type desZ (E. coli harboring pEDZA) and themutant desZ genes (Fig. 3), suggesting that the sequence alterations in mutant DesZ genes did notaffect the expression of mutant genes in E. coli.
3MGA 3,4-dioxygenase activities of DesZ and its mutants
The crude extracts of E. coli carrying the desZ gene or its mutants (250 g of protein/ml) wereincubated with 1 mM 3MGA at 30°C to determine the dioxygenase activity on 3MGA. No
dioxygenase activities were detected with any of the four mutants, whereas wild-type DesZ
exhibited a specific activity of 1.87 ± 0.15 U/mg. These results suggest that His12, Glu100, His250,
and Glu287 were essential for the 3MGA 3,4-dioxygenase activity of DesZ.

4. Discussion
The substitutions of His12, Glu100, His250, and Glu287 in DesZ with an Ala residue resulted in
the complete loss of the dioxygenase activity on 3MGA. These results indicate that all these residues
are essential for the dioxygenase activity, and suggest that Glu100 as well as His12 and Glu287 are
involved in the coordination of non-heme iron, Fe(II) and His250 acts as the catalytic base.
A sequence database search revealed that theamino-acid sequences of DesZ and LigZ are closely
related to those of the putative extradiol dioxygenase
genes of Ralstonia eutropha JMP134, Mycobacterium sp.
strains KMS and JLS as illustrated in the phylogenetic
tree of the type II extradiol dioxygenases in Fig. 4. The
deduced amino acid sequences of putative dioxygenase
genes YP_299962 and YP_298976 of JMP134,
ZP_01286210 of KMS, and ZP_01280861 of JLS
showed 57%, 37%, 23%, and 23% identities with that ofdesZ and 38%, 33%, 26%, and 24% identities with that
Fig. 3. SDS-PAGE analysis ofof ligZ, respectively. These enzymes constitute a kind of
extracts of the E. coli cell expressing
subfamily in the phylogenetic tree of the type II extradiol
mutant DesZ enzymes. Proteins (10dioxygenases (Fig. 4). Interestingly, the Glu residues
g) were separated on a 12%
corresponding to Glu100 of DesZ are completely
SDS-PAGE and stained withconserved among these enzymes as presented in Fig. 5,
Coomassie brilliant blue. Lanes: 1,
suggesting that Glu residue of these enzymes aremolecular weight markers; 2 to 6,
involved in the coordination of non-heme iron.
cell extracts of E. coli BL21(DE3)
Employment of one His and two Glu for Fe(II) ligands
harboring pEDZA (DesZ), pETZM6
instead of two His and one Glu appears to common(H12A),
pETZM8(E100A),
feature of this subfamily and may be extended to otherpETZM11(H250A),andpETZM12
enzymes surrounding this subfamily.
(E287A), respectively. The molecularmasses are given on the left.

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Fig. 4. Phylogenetic tree of type II extradiol dioxygenases. The scale corresponds to a genetic
distance of 0.1 substitution per position (10% difference). Enzymes: LigB_SYK-6,  subunit of
4,5-PCD of SYK-6 (BAA97118); FldU_LB126,  subunit of putative 4,5-PCD of Sphingomonas
sp. LB126 (CAB87561); PcmA_12B, 4,5-PCD of Arthrobacter keyseri 12B (AAK16524);
PmdB_BR6020,  subunit of 4,5-PCD of Comamonas testosteroni BR6020 (AAM09637);
ProOb_NGJ1,  subunit of 4,5-PCD of Pseudomonas ochraceae NGJ1; DesB_SYK-6, gallate
dioxygenase of SYK-6 (BAD80871); GalA_KT2440, gallate dioxygenase of P. putida KT2440(AE016783); MhpB_K12, 2,3-dihydroxyphenylpropionate 1,2-dioxygenase of E. coli K-12
(CAA70748); MpcI_JMP222, catechol 2,3-dioxygenase I of Cupriavidus necator JMP222(CAA36665); MhpB_TA441, 3-(2,3-dihydroxyphenyl)propionate 1,2-dioxygenase of C.
testosteroni TA441 (BAA82879); OhpD_V49, 2,3-dihydroxyphenylpropionate 1,2-dioxygenaseof Rhodococcus sp. V49 (AAF81826); HppB_PWD1, 2,3-dihydroxyphenylpropionate
1,2-dioxygenase of R. globerulus PWD1 (AAB81314); EdoD_I1, extradiol dioxygenase of
Rhodococcus sp. I1 (CAA06875); CarBb_CA10, catalytic subunit of 2'-aminobiphenyl-2,3-diol
1,2-dioxygenaseofP.
resinovorans
CA10(BAA21731);
LigZ_SYK-6,
2,3,2'-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl dioxygenase of SYK-6 (BAA75884);
YP298976_JMP134, extradiol dioxygenase of Ralstonia eutropha JMP134 (YP_298976);
DesZ_SYK-6, 3MGA 3,4-dioxygenase of SYK-6 (BAC79261); YP299962_JMP134, extradiol
dioxygenase of R. eutropha JMP134 (YP_299962); ZP01286210_KMS, extradiol dioxygenase of
Mycobacterium sp. KMS (ZP_01286210); ZP01280861_JLS, extradiol dioxygenase of
Mycobacterium sp. JLS (ZP_01280861); PhnC_RP007, extradiol dioxygenase of Burkholderia
sp. RP007 (AAD09870); HpaD_W, HPCA 2,3-dioxygenase of E. coli W (CAA86042); HpcB_C,
HPCA 2,3-dioxygenase of E. coli C (CAA38985); AmnA_CNB-1 and AmnB_CNB-1,  and 
subunits of 2-aminophenol 1,6-dioxygenase of Comamonas sp. CNB-1 (AAT35227 andAAT35226); AmnA_JS45 and AmnB_JS45,  and  subunits of 2-aminophenol 1,6-dioxygenaseof P. pseudoalcaligenes JS45 (AAB71525 and AAB71524); and AmnA_AP3 and AmnB_AP3, and  subunits of 2-aminophenol 1,6-dioxygenase of Pseudomonas sp. AP-3 (BAB03532 andBAB03531).
Acknowledgments
This work was supported in part by research fellowship 5468 from the Japan Society for the
Promotion of Science for Young Scientists.

5

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References[1] C. C. Tseng, F. H. Vaillancourt, S. D. Bruner, C.
T. Walsh, Chem. Biol., 11 (2004) 1195.
[2] Y. Katayama, S. Nishikawa, M. Nakamura, K.
Yano, M. Yamasaki, N. Morohoshi, T.
Haraguchi, Mokuzai Gakkaishi, 33 (1987) 77.
[3] Y. Katayama, S. Nishikawa, A. Murayama, M.
Yamasaki, N. Morohoshi, T. Haraguchi, FEBS
Lett., 233 (1988) 129.
[4] D. Kasai, E. Masai, K. Miyauchi, Y. Katayama,
M. Fukuda, J. Bacteriol., 186 (2004) 4951.
[5] E. Masai, M. Sasaki, Y. Minakawa, T. Abe, T.
Sonoki, K. Miyauchi, Y. Katayama, M. Fukuda,
J. Bacteriol., 186 (2004) 2757.
[6] T. Abe, E. Masai, K. Miyauchi, Y. Katayama,
M. Fukuda, J. Bacteriol., 187 (2005) 2030.
[7] D. Kasai, E. Masai, K. Miyauchi, Y. Katayama,
M. Fukuda, J. Bacteriol., 187 (2005) 5067.
[8] F. M. Ausubel, R. Brent, R. E. Kingston, D. D.
Moore, J. G. Seidman, J. A. Smith, K. Struhl,
John Wiley & Sons, Inc., New York, N.Y.
(1990).
[9] J. Sambrook, E. F. Fritsch, T. Maniatis, Cold
Spring Harbor Laboratory Press, Cold Spring
Fig. 5. Alignment of the deduced amino
Harbor, N.Y. (1989).
acid sequences of the DesZ-LigZ subfamily[10] F. Sanger, S. Nicklen, C. A. R., Proc. Natl.
of enzymes and LigB. Identical amino acids
Acad. Sci. USA, 74 (1977) 5463.
are indicated with asterisks. The conserved[11] N. Saitou, M. Nei, Mol. Biol. Evol., 4 (1987)
active site residues are shaded in black.
406.
[12] R. D. M. Page, Appl. Biosci., 12 (1996) 357.
[13] M. M. Bradford, Anal. Biochem., 72 (1976) 248.
[14] K. Sugimoto, T. Senda, H. Aoshima, E. Masai, M. Fukuda, Y. Mitsui, Structure, 7 (1999) 953.
[15] X. Peng, T. Egashira, K. Hanashiro, E. Masai, S. Nishikawa, Y. Katayama, K. Kimbara, M.
Fukuda, Appl. Environ. Microbiol., 64 (1998) 2520.
(Received: 19 May, 2012, Accepted: 22 June, 2012)6

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In vitro study of BphT1 two-component response regulator protein of
Rhodococcus jostii RHA1
Jun Shimodaira*,1 Yuta Miyazawa,1 Daisuke Kasai,1 Keisuke Miyauchi,2 Masao Fukuda1
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka-cho,
Nagaoka 940-2188, Japan; and 2Department of Civil and Environmental Engineering, Tohoku
Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537*E-mail: jshimo@stn.nagaokaut.ac.jp

1

BphT1 is a response regulator (RR) protein of the BphS1-BphT1
two-component system regulating the transcription of gene clusters for
degradation of biphenyl/polychlorinated biphenyls (PCBs) in Rhodococcus
jostii RHA1. BphT1 is estimated to bind to the degradation-gene promoters
including the bphAa promoter (bphAap) to activate their transcription
because its C-terminal domain contains a helix-turn-helix DNA binding motif.
In this study, BphT1 was overexpressed in E. coli and purified as N-terminal
histidine-tagged protein (ht-BphT1). Purified ht-BphT1 did not bind to thebphAap DNA fragment in the gel shift assays. The ht-bphT1 genecomplemented transcriptional activation activity of the original bphT1 genein bphT1/T2 double disruption mutant, TDR12, and restored the growth on
biphenyl. Because DNA-binding activity of RR usually depends on its
phosphorylation status, in vitro phosphorylation of ht-BphT1 by
low-molecular-weight phosphate donors, carbamoyl phosphate and acetyl
phosphate was examined using phos-tag acrylamide SDS-PAGE. However,phosphorylation of ht-BphT1 was not detected. These results suggested that
ht-BphT1 purified from E. coli is dephosphorylated and hard to bephosphorylated in vitro. ht-BphT1 appears to require phosphorylation to
exhibit its binding activity to the target promoter sequence.

1. Introduction
Polychlorinated biphenyls (PCBs) had been widely used for industrial purposes because of
their excellent stability, insulation properties, and resistance to combustion. The production and useof PCBs have been banned because of their toxicity and their contamination and persistence in theenvironment. However, PCBs still remain widely in the environmental and cause environmental
problems. Microbial degradation of PCBs has been regarded as a promising procedure for theirremoval from the environment. A variety of bacteria, which aerobically degrade PCBs, have been
isolated, and their degradation enzymes and genes have been characterized [1, 6, 14, 20]. Most ofthese bacteria cometabolize PCBs through the metabolic pathway of biphenyl. A PCB degrader,
Rhodococcus jostii RHA1, was isolated from the -hexachlorocyclohexane-contaminated upland soil
and has a great capacity to degrade mono- to octa-chlorobiphenyls by cometabolism with biphenyl[23, 24].
The biphenyl/PCB degradation enzyme genes of the upper biphenyl degradation pathway of
RHA1 are distributed among the gene clusters including bphAaAbAcAdC1B1, etbD1,
etbAa1Ab1CbphD1, etbAa2Ab2AcD2, and etbAdbphB2. The transcription of these gene clusters
from the promoters of bphAa, etbAa1, etbAa2, etbAd, and etbD1 (bphAap, etbAa1p, etbAa2p, etbAdp,
and etbD1p, respectively) are simultaneously induced in the presence of aromatic compounds suchas biphenyl and ethylbenzene [27]. These five promoters are under the control of dual
two-component systems coded by bphS1T1 and bphS2T2 [28, 29]. Two-component systems are
employed by a number of bacteria and certain higher organisms [26]. The typical two-component
system consists of a sensor protein-histidine kinase (HK) and a cognate response regulator (RR) [16,18]. HK perceives an environmental stimulus via its N-terminal input domain and1

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autophosphorylates the conserved histidine residue in its C-terminal transmitter domain. This
phosphoryl group is subsequently transferred to the conserved aspartic acid residue in the receiverdomain of RR. This phosphorylation causes a conformational change of RR and activation of its
output domain which usually retains DNA-binding property [16]. The receiver and output domains
usually occupy N- and C-termini of RR.
The bphS1 and bphS2 products, BphS1 and BphS2, and the bphT1 and bphT2 products,
BphT1 and BphT2, are estimated to be the HK and RR of a two component system, respectively.
BphS1 and BphS2 are 92% identical between their amino acid sequences. BphT1 and BphT2 are
97% identical. The BphS1T1 and BphS2T2 (BphSTs) systems share the same broad inducing
substrate spectrum containing benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, andchlorinated benzenes [28, 29]. The exception is biphenyl, which is an inducing substrate only for the
BphS1T1 system. The C-terminal domains of the BphT1 and BphT2 (BphTs) contain ahelix-turn-helix (HTH) DNA-binding motif. Thus, BphTs are deduced to bind directly to somesequence in the promoter to induce the transcription. In the previous study, the 24-bp consensus
sequence conserved among five BphSTs-dependent promoters was identified and the significance ofthe 24-bp consensus sequence in transcriptional regulation by BphS1T1 was demonstrated by thehybrid promoter analysis [25]. This 24-bp consensus sequence appears to be the BphTs binding
sequence.
In this study, BphT1 was overexpressed in E. coli, purified, and subjected to the in vitro
phosphorylation to examine its binding to the 24-bp consensus sequence.

2. ExperimentBacterial strains, plasmids, and culture conditions.
The strains and plasmids used in this study are listed in Table 1. Rhodococcus jostii TDR12
was grown at 30°C in 1/5 LB (2 g/l Bacto-tryptone, 1 g/l yeast extract, and 5 g/l NaCl) and on a W
minimal salt medium plate [2]. A host strain, Escherichia coli JM109 was grown at 37°C in LB (10g/l Bacto-tryptone, 5 g/l yeast extract, and 5 g/l NaCl). If necessary, antibiotics were used at thefollowing concentrations: ampicillin, 100 g/ml and kanamycin, 50 g/ml.
DNA manipulations and analysis.
DNA manipulations, including electrotransformation (electroporation), were performed aspreviously described [17]. Nucleotide sequences were determined by the dideoxy termination
method using a CEQ 8000 genetic analysis system (Beckman Coulter, Inc., Fullerton, CA). A Sanger
reaction [22] was performed using the CEQ Dye Terminator Cycle Sequencing Quick Start kit(Beckman Coulter, Inc.). Sequence analysis was performed using BioEdit [7] and in silico
MolecularCloning (in silico biology, Inc., Yokohama, Japan).
Construction of overexpression and complementation plasmids.
The bphT1 gene was amplified by PCR using pG1013F as a template and the primers,
BglII-T1F and Pst-EcoRI-T1R, which had the BglII and PstI sites at their 5’ termini, respectively.
The PCR product was subcloned into pT7Blue T-Vector to verify its nucleotide sequence. The 0.6-kb
BglII-PstI fragment containing bphT1 of the resulting plasmid, pT7T1BEP was cloned into
BamHI-PstI site of an expression vector, pColdI to obtain pColdIT1. The ompR gene was amplified
from the total DNA of E. coli JM109 using the primers, ompR-F and ompR-R, which had the BamHI
and NdeI sites at their 5’ termini, respectively. The PCR product was subcloned into a pT7Blue
T-Vector to verify its nucleotide sequence. The resulting plasmid was named pT7ompR. The 0.8-kb
BamHI-SalI fragment of pT7ompR containing the ompR gene was cloned into the BamHI-SalI siteof the pColdI to obtain pColdIompRF. The histidine-tagged bphT1 (ht-bphT1) gene fragment was
amplified by PCR using pColdIT1 as a template and the primers, EcoRI-SDpColdIF and
EcoRI-T1AR. These primers had EcoRI sites at their 5’ termini. The PCR product was subcloned
into pT7Blue T-Vector to verify its nucleotide sequence. The 0.8-kb EcoRI fragment containing
ht-bphT1 of the resulting plasmid, pT7SDColdIT1 was cloned into the EcoRI site of a vector,
pKPA1 to obtain pKPA1SDColdIT1. The nucleotide sequences of primers used are listed in Table 1.
2

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Table 1. Strains plasmids, and primers used in this study
Relevant characteristic(s)aStrain, plasmid, or
primers
Strains
R. jostii
RHA1
TDR12

PCB degrader, BPH+, ETB+, Nalr
RHA1 derivative, BPH-, ETB-, Nalr; bphT1, bphT2[23]
This study

Escherichia coli
JM109

endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ (lac-proAB)
e14- hsdR17(rK- mK+) [F' traD36 proAB+ lacIqZΔM15][32]

Cloning vector, Apr
Cold shock-based expression vector, Apr
pColdI with 0.6-kb BglII-PstI bphT1 fragment, Apr
pColdI with 0.8-kb BamHI-SalI ompR fragment, Apr
pBluescript II KS(+) with an 8.8-kb HindIII fragment
containing bphS1T1 in the forward orientation to lacZ gene, Apr
Rhodococcus-E. coli shuttle vector, Kmr
pK4 with a insertion of bphAap in the reverse orientation tokanamycin resistance gene, Kmr
pKPA1 with 0.8-kb EcoRI ht-bphT1 fragment, Kmr
TA Cloning vector
pT7Blue T-vector with 0.6-kb BglII-PstI bphT1 fragment, Apr
pT7Blue T-vector with 0.8-kb BamHI-NdeI ompR fragment, Apr
pT7Blue T-vector with 0.8-kb EcoRI ht-bphT1 fragment, Apr

Stratagene
TakaraThis study
This study[29]

Plasmids
pBluescript II KS(+)
pColdI
pColdIT1
pColdIompR
pG1013F
pK4
pKPA1
pKPA1SDColdIT1
pT7Blue T-vector
pT7BEP
pT7ompR
pT7SDColdIT1

Source orreference[8][10]This study
Novagene
This study
This study
This study

Primers
BglII-T1F
5'-AGATCTATCAGCGTTGTGGTGTGTGACG-3'
This study
Pst-EcoRI-T1R
5'-CTGCAGGAATTCCTAGTCCAAAAGACCTGCG-3'
This study
ompRF
5'-GGATCCATGCAAGAGAACTACAAGATTC-3'
This study
ompRR
5'-CATATGTCATGCTTTAGAGCCGTC-3'
This study
EcoRI-SDpColdIF
5'-GAATTCTTAAGAGGTAATACACC-3'
This study
EcoRI-T1AR
5'-GAATTCCCTAGTCCAAAAGACC-3'
This studyaBPH+, growth on biphenyl; BPH-, no growth on biphenyl; ETB+, growth on ethylbenzene; ETB-, nogrowth on ethylbenzene; Apr, ampicillin resistance; Kmr, kanamycin resistance, Nalr, nalidixic acidresistance.

Purification of the histidine-tagged recombinant proteins.
For the overexpression of histidine-tagged BphT1 (ht-BphT1) and OmpR (ht-OmpR), E. coli
JM109 harboring each expression plasmid was grown at 37°C in LB medium containing ampicillin.
When the optical density at 600 nm reached 0.6, cells were kept at 15°C for 30 min and incubated at
15°C for a further 24 h after adding 0.5 mM isopropyl--D-thiogalactopyranoside. Cells were
harvested by centrifugation, resuspended in the elution buffer [50 mM Tris-HCl, 500 mM NaCl;
pH7.4] containing 100 mM imidazole, and disrupted by sonication. A crude cell lysate was preparedby the removal of cell debris by centrifugation at 18,800 × g for 20 min at 4ºC. Proteins were
purified by using a Ni Sepharose Fast Flow column (GE Healthcare UK Ltd., Buckinghamshire,
England). ht-BphT1 and ht-OmpR were eluted with the elution buffer containing 500 mM and 300
mM imidazole, respectively. The purified proteins were desalted and concentrated with the elution
buffer or phosphorylation buffer [50 mM Tris-HCl, 50 mM KCl, pH 7.4] using an Amicon Ultra-15
Ultracel-10 k (Millipore Corporation, Billerica, MA,).
In vitro phosphorylation of ht-BphT1.
Treatment of purified ht-BphT1 (12.9 M) with 50 mM carbamoyl phosphate was carried out
for 120 min at 30°C or 37°C in the phosphorylation buffer containing either 10 mM or 20 mM
MgCl2. The resultant solution was mixed with 2 × SDS-PAGE loading buffer [220 mM Tris-HCl,3

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8.9% sodium dodecyl sulfate (SDS), 10% glycerol, 100 mM dithiothreitol, 0.1% bromophenol blue,
pH 6.8], incubated at room temperature for 2 min, and applied onto a 10% polyacrylamide gel
containing 75 M Phos-tag acrylamide (NARD Institute Ltd., Amagasaki, Japan) and 150 M
MnCl2. After electrophoresis, the gel was stained with Coomassie brilliant blue.

3. Results and Discussion
Overexpression and purification of ht-BphT1
The BphT1 protein was overexpressed as an
N-terminally histidine-tagged protein (ht-BphT1) in
E. coli JM109 harboring pColdIT1. ht-BphT1 was
purified to near homogeneity by Ni2+- nitrilotriacetic
acid (Ni2+-NTA) affinity chromatography andsubjected to SDS-PAGE analysis (Fig. 1). ht-BphT1
migrated with apparent molecular mass ofapproximately 28 kDa, which is close to thecalculated molecular mass of ht-BphT1 (25,734 Da).

Fig. 1. SDS-PAGE analysis of purified
ht-BphT1. Proteins were separated on an
SDS-15% polyacrylamide gel and stainedwith Coomassie brilliant blue. Lanes: 1,
molecular weight markers; 2 and 3,
insoluble and soluble fractions of E. coli
JM109 harboring pColdIT1, respectively; 4,elute from Ni sepharose column
chromatography. The molecular weights(kDa) of the marker proteins are indicatedon the left.

Fig. 2. Complementation of the bphT1T2
double disruption mutant, TDR12 by
ht-bphT1 gene. Four transformants of
pKPA1SDColdIT1 containing ht-bphT1(upper half) and those with a vector pKPA1(lower half) were streaked and grown on
biphenyl on a W minimal medium plate.

Binding of the ht-BphT1 to bphAap
To investigate the ability of BphT1 to bind tobphAap, the gel shift assays using ht-BphT1 and thebphAap fragment containing the 24-bp consensus
sequence was performed. No shifted band wasobserved at any tested concentration (data notshown). It was reported that the histidine-tag
sometimes affected the activity of recombinant
protein [9, 12, 21]. To confirm whether ht-BphT1
maintain its activity in vivo, pPA1SDColdIT1
containing the ht-bphT1 gene was constructed andintroduced into the mutant strain TDR12 with double
disruption of bphT1 and bphT2, which could not
grow on biphenyl as a sole souse of carbon. The
resulting transformant grew well on biphenyl (Fig. 2).
These results indicate that ht-BphT1 maintain its
in-vivo activity in transcriptional regulation.
Phosphorylation of the ht-BphT1 in vitro
The DNA-binding activity of RR usuallydepends on its phosphorylation status. In vitro
phosphorylation of RR proteins have been performed
using low-molecular-weight phosphate donors, suchas acetyl phosphate and carbamoyl phosphate [4, 31].
To examine in vitro phosphorylation of ht-BphT1,
ht-BphT1 was incubated with carbamoyl phosphate,
and was subjected to phos-tag acrylamide
SDS-PAGE, which specifically retards the migrationof phosphoproteins [3, 15]. histidine-tagged OmpR
(ht-OmpR) protein of E. coli was used as a positive
control. OmpR has been shown to be efficiently
phosphorylated by carbamoyl phosphate in vitro [13].
ht-OmpR was overexpressed in E. coli JM109
harboring pColdIompR and purified to near
homogeneity by Ni2+-NTA affinity chromatography.
As shown in Fig. 3, the retarded band of4

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phosphorylated ht-OmpR was detected after incubation with carbamoyl phosphate in the presence of
Mg2+, which is required for phosphorylation [13, 19]. The retarded band was not observed after
incubation with carbamoyl phosphate in the absence of Mg2+. No retarded band of phosphorylated
ht-BphT1 was detected regardless of the concentrations of Mg2+ and the incubation temperatures. We
also incubated ht-BphT1 with acetyl phosphate, but phosphorylated ht-BphT1 was not detected(data not shown). These results suggest that carbamoyl phosphate and acetyl phosphate are notsuitable for in vitro phosphorylation of ht-BphT1.

Fig. 3. In vitro phosphorylation of ht-BphT1 with carbamoyl phosphate. ht-OmpR(Lanes 1 and 2) and ht-BphT1 (Lanes 3-7) were incubated with carbamoyl phosphate for
120 min at 30°C (Lanes 4 and 6) or 37°C (Lanes 1, 2, 3, 5, and 7), and analyzed byphos-tag acrylamid gel SDS-PAGE. The reaction solutions contained 10 mM (Lanes 2, 4,
and 5) or 20 mM (Lanes 6 and 7) MgCl2. In lanes 1 and 3, MgCl2 was omitted from thereaction solutions to prevent phosphorylation. The expected positions of ht-OmpR and
ht-BphT1 and phosphorylated ht-OmpR (ht-OmpR-P) are indicated on the left.
Dephosphorylation of RRs was reported to eliminate their binding activities completely [5, 11,30]. These reports imply that phosphorylation is required for binding of RR to the target DNA. The
results of this study suggest that ht-BphT1 purified from E. coli is not phosphorylated and that
ht-BphT1 in the mutant strain TDR12, which exhibited complete activity in transcriptional
regulation, is phosphorylated in vivo. Because the low-molecular-weight phosphate donors
examined in this study did not work in phosphorylation of ht-BphT1, in vivo phosphorylation in theoriginal Rhodococcus strain, RHA1 is expected to be useful for phosphorylation of ht-BphT1.
Because BphT2, which is mostly identical to BphT1, governs transcription of the same fivepromoters as BphT1 and cross-communicate with BphS1 [28], the results obtained in this study
appear to be extended to BphT2.
References[1] D. Ahmad, R. Massé, M. Sylvestre, Gene, 86 (1990) 53.
[2] N. Araki, T. Suzuki, K. Miyauchi, D. Kasai, E. Masai, M. Fukuda, J. Mol. Microbiol.
Biotechnol., 20 (2011) 125.
[3] C. M. Barbieri, A. M. Stock, Anal. Biochem., 376 (2008) 73.
[4] R. B. Bourret, Curr. Opin. Microbiol., 13 (2010) 142.
[5] L. E. Cybulski, G. del Solar, P. O. Craig, M. Espinosa, D. de Mendoza, J. Biol. Chem., 279(2004) 39340.
[6] K. Furukawa, A. M. Chakrabarty, Appl. Environ. Microbiol., 44 (1982) 619.
[7]
T. A. Hall, Nucleic Acids Symp. Ser., 41 (1999) 95.
[8]
Y. Hashimoto, M. Nishiyama, F. Yu, I. Watanabe, S. Horinouchi, T. Beppu, J. Gen. Microbiol.,
138 (1992) 1003.
[9]
H. Horchani, S. Ouertani, Y. Gargouri, A. Sayari, Journal of Molecular Catalysis B:
Enzymatic, 61 (2009) 194.
[10] T. Iwasaki, H. Takeda, K. Miyauchi, T. Yamada, E. Masai, M. Fukuda, Biosci. Biotechnol.
5Transactions on GIGAKU 1(2012)01030/1-6

[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]

Biochem., 71 (2007) 993.
Y. Jeon, Y. S. Lee, J. S. Han, J. B. Kim, D. S. Hwang, J. Biol. Chem., 276 (2001) 40873.
L. Jessop, T. Bankhead, D. Wong, A. M. Segall, J.. Bacteriol., 182 (2000) 1024.
L. J. Kenney, M. D. Bauer, T. J. Silhavy, Proc. Natl. Acad. Sci. USA, 92 (1995) 8866.
K. Kimbara, T. Hashimoto, M. Fukuda, T. Koana, M. Takagi, M. Oishi, K. Yano, J. Bacteriol.,
171 (1989) 2740.
E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, S. Yamada, H. Nakamura, Y. Shiro, Y. Aoki,
K. Okita, T. Koike, Proteomics, 8 (2008) 2994.
M. T. Laub, M. Goulian, Annu Rev Genet, 41 (2007) 121.
E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, K. Yano, Appl. Environ.
Microbiol., 61 (1995) 2079.
T. Mascher, J. D. Helmann, G. Unden, Microbiol. Mol. Biol. Rev., 70 (2006) 910-38.
W. R. McCleary, J. B. Stock, J. Biol. Chem., 269 (1994) 31567.
F. J. Mondello, J. Bacteriol., 171 (1989) 1725.
C. L. Richard-Fogal, E. R. Frawley, R. G. Kranz, J. Bacteriol., 190 (2008) 3489.
F. Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. U. S. A., 74 (1977) 5463.
M. Seto, K. Kimbara, M. Shimura, T. Hatta, M. Fukuda, K. Yano, Appl. Environ. Microbiol.,61 (1995) 3353.
M. Seto, E. Masai, M. Ida, T. Hatta, K. Kimbara, M. Fukuda, K. Yano, Appl. Environ.
Microbiol., 61 (1995) 4510.
J. Shimodaira, Y. Furusawa, Y. Miyazawa, D. Kasai, K. Miyauchi, E. Masai, M. Fukuda, J.
Biosci. Bioeng., 113 (2012) 279.
A. M. Stock, V. L. Robinson, P. N. Goudreau, Annu. Rev. Biochem., 69 (2000) 183.
H. Takeda, N. Hara, M. Sakai, A. Yamada, K. Miyauchi, E. Masai, M. Fukuda, Biosci.
Biotechnol. Biochem., 68 (2004) 1249.
H. Takeda, J. Shimodaira, K. Yukawa, N. Hara, D. Kasai, K. Miyauchi, E. Masai, M. Fukuda,
J. Bacteriol., 192 (2010) 4741.
H. Takeda, A. Yamada, K. Miyauchi, E. Masai, M. Fukuda, J. Bacteriol., 186 (2004) 2134.
J. R. Wei, Y. H. Tsai, P. C. Soo, Y. T. Horng, S. C. Hsieh, S. W. Ho, H. C. Lai, J. Bacteriol.,
187 (2005) 5683.
A. J. Wolfe, Microbiol. Mol. Biol. Rev., 69 (2005) 12.
C. Yanisch-Perron, J. Vieira, J. Messing, Gene, 33 (1985) 103.

(Received: 18 May, 2012, Accepted: 18 June, 2012)6

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Overexpression of ACEII, and transcriptional analysis of cellulase
genes in Trichoderma reesei PC-3-7
Takanori Furukawa, Naoki Kitagami, Yosuke shida, Yasushi Morikawa, Wataru Ogasawara*
Department of Bioengineering, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: owataru@vos.nagaokaut.ac.jp

The transcription factor ACEII (Activator of cellulase 2) of Trichoderma

reesei is the first identified transactivator for cellulase and xylanase genes.
However, relatively little information is available about the regulatory
function of ACEII. In this study, strains expressing tagged-ACEII under
the control of the cbh1 and the xyn3 promoters were developed toinvestigate the effect of overexpression of ACEII. The resultant strains
expressed markedly higher amount of ace2 transcript in thecellulase-inducing conditions, and the maximum level of the transcript
induction was estimated to be 570-fold for the cbh1 construct and 20-foldfor the xyn3 construct. Analysis of production and expression profiles ofcellulase revealed the possible involvement of ACEII in the regulation ofcellulase and xylanase gene expressions in T. reesei. Our study provides
insights into the effect of overexpresion of ACEII in T. reesei.

1. Introduction
Plant biomass, which primarily consists of lignocellulosic material, is the most abundant
renewable resource in nature. The major components of lignocellulose are polysaccharides, such as
cellulose and hemicellulose, which can potentially be converted into biofuels by hydrolysis into
fermentable sugars followed by downstream fermentation. Many microorganisms secrete a wide
variety of hydrolases that can degrade plant polysaccharides, and their potential application in thebiorefinery area has received considerable attention in the last decades.
The filamentous fungus Trichoderma reesei is one of the most efficient producers of cellulolytic
and xylanolytic enzymes, and its hydrolytic enzyme system has been subjected to extensive
biochemical and genetic analyses. T. reesei produces at least two cellobiohydrolases (CBHI andCBHII), eight endoglucanases (EGLI to EGLVIII), and two ß-glucosidases (BGLI and BGLII) thatact synergistically to degrade crystalline cellulose to glucose. The xylanolytic enzymes produced by
T. reesei include at least four endoxylanases (XYNI to XYNIII), and one ß-xylosidase (BXL1) [1].
In T. reesei, the production of cellulolytic and xylanolytic enzymes is regulated at thetranscriptional level depending on the carbon source available. Transcription of cellulase genes is
induced by cellulose, its derivatives, lactose, and a monosaccharide L-sorbose, and is subject tocarbon catabolite repression [2]. It has been reported that major cellulase genes are co-ordinately
expressed under various inducing conditions, suggesting the presence of common regulatory
mechanisms. However, the independent gene exprepression of xylanases from cellulases suggest that
expression of cellulase and xylanase genes is modulated by a complex regulatory network [3].
Even though significant progress has been made in the molecular biology of the cellulase andxylanase system of T. reesei, our knowledge about the regulatory mechanisms of production of these
enzymes is rather fragmentary. So far, four transcriptional activators, Xyr1, ACEII, BglR, and theHAP 2/3/5 complex, as well as two repressors including ACEI and the carbon catabolite repressor
CREI were identified [14-17]. Xyr1 is the main activator of the cellulolytic and xylolytic genes,
controlling the enzyme production regardless of the inducer and the mode of expression. Deletion of
xyr1 is reported to eliminate the induction of the hydrolase and hemicellulase genes involved inarabinan degradation [4]. The Xyr1-binding consensus, 5’-GGC(A/T)4-3’ have been identified [5].
1Transactions on GIGAKU 1(2012)01031/1-6

ACEII belongs to the zinc binuclear cluster protein family. The ace2 gene was originally isolated
from T. reesei by applying a yeast expression screening system using T. reesei cbh1 promoter [6].
ACEII has been shown to bind to a 5’-GGCTAATAA-3’ motif in the cbh1 promoter and
5’-GGGTAA-3’ sequences in the cbh2 and xyn2 promoters [2]. Deletion of ace2 is reported toreduce the expression of the major cellulase genes particularly at the early stage of induction bycellulose but not by sophorose [6]. Hence ACEII appears to play a role in the regulation of thehydrolase genes as a narrow domain activator by modulating Xyr1 activation. However, relatively
little information is available about the transcriptional targets and true function of ACEII.
In this study, we investigated the effect of overexpression and deletion of ace2 on transcriptional
regulation of the major cellulase and xylanase genes under different inducing conditions.

2. Experiments
Microbial strains and culture conditions Trichoderma reesei PC-3-7, which is a hypercellulolytic
mutant with enhanced cellulase and xylanase inducibility by L-sorbose [7], was used throughout thisstudy. For induction experiments, PC-3-7 and its transformants were grown for 48 h at 28°C on arotary shaker (220 rpm) in a glucose medium as described [7]. Mycelia collected by filtration were
washed twice with saline, and transferred to a basal medium [7] containing 0.5 mg/mL L-sorbose as
an inducer. As a control of inducer glucose was added at 1.0 mg/mL. Induction was performed for 6
h at 28°C on a rotary shaker (220 rpm). After induction, mycelia harvested by filtration and washedtwice with saline were used for RNA extraction.
Construction of an ace2-knockout strain of T. reesei To construct the ace2-disruption cassette, a
4.2-kbp fragment containing the ace2 structural gene with 1.6-kbp of 5’- and 3’-flanking regions wasamplified from the PC-3-7 genomic DNA by PCR using the primers, ace2-5’Fw and ace2-3’Rv(Table 1). The PCR product was cloned into the HincII site of pUC118 to yield pUace2FFL. A
3.1-kbp fragment containing the selective marker, A. nidulans amdS was excised from p3SR2 [8] by
SpeI and XbaI digestion and blunted using a blunting kit (TaKaRa Bio). The blunted fragment wasinserted into theTable 1. Primers used in this study
blunted-BspEI site
PrimerSequence
located in the ace2
ace2-5’Fw
5’-CGCTCCTTGAGCTCTTCCTTGGTACCTTCCTTTGG-3’
coding region oface2-3’Rv
5’-GGGAGGTACCTGCGTCTGCCAAAGAGGGGT-3’
pUace2FFL
toace2-MatFragFw 5’-CCCTTCTCTGAAAGCTTATGGACCTCCGGC-3’
obtainp∆ace2-amdS. The ace2-MatFragRv 5’-CCATATGGGTACCTCACTTCAGCAGTCTGGC-3’
5’-ATGGCTCACAACCACCGTCACAAACACG-3’
disruption cassette ace2N-tagFw
releasedfrom ace2pPICZCFw 5’-CCCTTCGAATTCAAAATGGACCTCCGGC-3’
p∆ace2-amdS by ace2pPICZCRv 5’-CCATATGAGATACGTACTTCAGCAGTCTGGC-3’
KpnI
digestion ace2CterFw
5’-ATGGACCTCCGGCAAGCATGTG-3’
wasused
for ace2CterRv
5’-CATTGAGTTTGTAGCCTTAGGTACCACTGTTCC-3’transformation.
Construction of an ace2 over-expressing strains of T. reesei To construct the N-terminal tagged
ace2 over-expression cassette, the ace2 coding region was amplified from the PC-3-7 genomic DNA
by PCR using the primers, ace2-MatFragFw and ace2-MatFragRv (Table 1). The amplified fragment
was digested with HindIII and KpnI and cloned into the HindIII/KpnI site of pT7-MAT-FLAG1 toyield pT7MFace2. N-terminal tagged ace2 was amplified from pT7MFace2 by PCR using theprimers, ace2N-tagFw and ace2-MatFragRv (Table 1). The resultant fragment was cloned in-frame
into an inverse PCR product of pcbh1-gus [9] harboring cbh1 expression signals and the amdS toyield pcbh1-N-tagace2, and inserted into an inverse PCR product of pBxyn3D0-ag [10], containing
xyn3 expression signals and the amdS to yield pBD0N-tagace2. The ace2 overexpression cassettes
were excised from pcbh1-N-tagace2 and pBD0N-tagace2 with KpnI, and used for transformation.
To construct the C-terminal tagged ace2 over-expression cassette, the coding region of ace2 wasamplified from the PC-3-7 genomic DNA by PCR using the primers, ace2pPICZCFw andace2pPICZCRv (Table 1). The PCR product was digested with EcoRI and SnaBI and cloned into the
EcoRI/SnaBI site of pPICZC to yield pPICace2. C-terminal tagged ace2 gene was amplified from2

Transactions on GIGAKU 1(2012)01031/1-6

pPICace2 by PCR using the primers ace2CterFw and ace2CterRv (Table 1). The amplified fragment
was cloned in-frame into the inverse PCR product of pcbh1-gus and pBxyn3D0-ag to yield
pcbh1-C-tagace2 and pBD0C-tagace2, respectively. The C-terminal tagged ace2 expression cassettes
were excised with KpnI and used for transformation.
Determination of gene copy number and integration locous in the genome of the transformants
Chromosomal DNA of transformant was extracted as described previously [10]. Southernhybridization was done using an AlkPhos Direct kit (GE Healthcare Bio Science). Standard methods
were used for electrophoresis, blotting, and hybridization. Chromosomal DNA of the ace2-disrupted
mutant was digested by XhoI and hybridized with an alkaline-phosphatase-labeled ace2 gene probe.
Integration of the ace2 overexpression cassette was verified by hybridization with the ace2 probe.
Enzyme assay Cellulase activity was determined at 50°C for 15 min in 50 mM sodium acetate
buffer (pH 5.0) using 1% carboxy methyl cellulose (CMC) as a substrate. Released reducing sugar
was measured by the Somogyi-Nelson method. One unit of activity (U) was defined as the amountof enzyme that released 1 μmol of glucose-equivalent reducing sugar per minute. Protein
concentration was determined by the Lowry method using bovine serum albumin as standard.
RNA extraction, cDNA synthesis and qRT-PCR Total RNA was extracted from frozen mycelia bya modified hot-phenol method using TRIZOL LS reagent, and purified with an RNAspin Mini RNA
isolation kit (GE Healthcare Bio Science). Synthesis of cDNA from total RNA was carried out usinga Transcriptor First-Strand cDNA Synthesis kit (Roche Diagnostics) with an Anchored-oligo (dT)18
and random hexamer primers.
qRT-PCR was performed in a final volume of 20 µL containing 0.5 µM each of forward andreverse primers and 2 µL of 100-fold diluted cDNA using a LightCycler 480 System andLightCycler 480 SYBR Green I Master kit (Roche Diagnostics). Primers used in real-time PCR were
described previously [5]. Conditions for thermal cycling are as follows: 5 min at 95°C followed by
45 cycles of 10 s at 95°C, 10 s at 60°C and 10 s at 72°C. All assays were performed in triplicate witha no-amplification control. The specificity of amplification was documented by melting curve
analysis. The actin gene (act1) was used as a reference for quantification.

3. Results and Discussion(1) Deletion and overexpression of the ace2 gene in T. reesei PC-3-7.
To investigate the roles of ACEII in the transcriptional regulation of cellulase and xylanase genes,
an ace2-knockout mutant was constructed by inserting the A. midulans amdS into the coding regionof ace2 in PC-3-7 background. We also developed strains expressing N-terminal or C-terminal
tagged ACEII under the control of either the cbh1 promoter or the xyn3 promoter. The cbh1
promoter is the strongest inducible promoter in T. reesei. Expression level of xyn3 was estimated to
be about 6–9% of that of cbh1[9], which is still high and 100-fold stronger than that of thephosphoglycerate kinase 1 gene (pgk1). The transformation cassettes were introduced into
chromosomal DNA of PC-3-7 and the resulting transformants were screened by Southern blot
analysis (data not shown). Both the homologously and heterologously integrated transformants wereselected for the tagged-ACEII expressing strains.
(2) Screening of ace2 overexpessing strains.
After pre-cultivation, the tagged-ACEII expressing strains and the wild-type (WT) weretransferred into basal media containing L-sorbose as an inducer and were incubated for 6 h. The
levels of the ace2 transcripts were analyzed by quantitative real-time PCR (qRT-PCR). As shown in
Table 1, ace2 expression levels in the transformants of the cbh1 construct were markedly higher thanthose in the WT, and the maximum elevation of the expression from the WT was estimated to be
570-fold in the transformant CN6. The maximum elevation of the ace2 expression in thetransformants of the xyn3 consyruct was as low as 20-fold (data not shown). This is in good
agreement with the previous results on the relative strength of these promoters, which was obtainedusing a heterologous Escherichia coli ß-glucuronidase gene, gus as a reporter [9]. It was reported
earlier that calf chymosin, when expressed heterologously using the T. reesei cbh1 promoter, showed
reduced levels of expressed mRNA (only 1-5% compared to the native cbh1 mRNA) [9]. This3

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difference could be attributed to the origin of the gene, because our previous study also suggested
that expression of the heterologous gene in T. reesei is limited at the level of tranasctiption [9]. The
results of the qRT-PCR analysis indicate that the transformants could successfully overexpress thetagged-ACEII gene constructs in the cellulase-inducing condition. Disruption of cbh1 has beenreported to decrease total cellulase activity that could affect cellulase production profiles of thetransformants [9]. Thus, the CC2 stain harboring the Pcbh1: C-terminal tagged-ACEII construct at
Table 2. Relative expression levels of ace2
in the cbh1 construct transformants.
Transformant

Relative expression (×act1)
ace2
cbh1
WT
0.1
54
Homologous strains
CN5
47
0.0
CN6
57
0.0
CC5
29
0.0
Heterologous strain
CC2
46
120

Fig. 1. SDS-PAGE analysis of the enzymes
secreted by the transformants.

an ectopic locus (which shows about 460-fold overexpression), was chosen to perform further
analysis.
(3) Effect of overexpression of ace2 on cellulase production.
To determine whether increased levels of ace2 transcripts affect cellulase formation of thetransformant, we evaluated production levels of the enzyme in WT, CC2, and ∆ace2 in Avicel
culture over a period of 6 days. As shown in Fig. 2, no significant differences were observed in the
production profiles of CMCase between WT and ∆ace2 in the time-course experiment. In contrast,
CC2 strain produced significantly lower levels of CMCase after 3 days of cultivation, and it reachedonly 25% of WT after 6 days. At the earliest time point (day 2), CC2 produced almost the samelevels of CMCase as WT, but it only slightly increased by day 6. Because we used the cbh1
promoter for expression of ace2, overproduction of ACEII was expected to start after 2 days ofcultivation. It was previously reported that
disruption of ace2 in a hyper-celluolytic
mutant T. reesei ALKO2221 led to 70% and
40% reduction of CMCase production after
3 days and 6 days of cultivation on Avicel,
respectively [6]. However, the ∆ace2
mutation in PC3-7 background did not result
in apparent reduction of the production of
CMCase. Enhancement of the xylanolytic
and cellulolytic enzyme production by theoverexpression of the trasncavtivator XlnR
was reported in Aspergillus niger and A.
oryzae [11,12], but that by the ACEII
overexpression was not detected in theFig. 2 Cellulase production profiles of the ace2
enzyme assay and SDS-PAGE analysis ofoverexpressing and deletion strain.
the ∆ace2 strain.
(4) Effect of overexpression of ace2 on cellulase and xylanase gene transcription.
To investigate the effect of ace2 overexpression and deletion at the transcriptional level, theexpression profiles of the major cellulases (cbh1, cbh2, egl1 to egl3, and bgl1 to bgl2) were analyzed
by qRT-PCR every 24 hours for up to 120 hours (Figures 3 and 4). Consistent with the CMCase
production profiles, WT expressed cellulase and xylanase genes in a coordinate manner from 24 h,and the maximum expression was attained at 72 h of cultivation. In contrast, the gene expression in4Transactions on GIGAKU 1(2012)01031/1-6

the CC2 strain reached the maximum at 24 h, decreased to approximately 50% of the maximum after
48 h, and was maintained at the same level for the rest of the experiment. Cellulase expression in the∆ace2 strain followed the similar patter as that of WT except for a precipitous drop in the gene
expression at 96 h. In ALKO2221 strain a substantial reduction in transcript levels (from 30 to 70%)
has been reported at the early stagesof inductionn, as a result of
disruption of ace2 [6]. This may be
due to differences in the genetic
backgrounds of the isogenic mutans
PC3-7 and ALKO2221.
Bearing in mind the proposed
activating function of ACEII [6, 13],
it can be assumed that high-levels ofcellulase gene expression observed
at 24 h in the CC2 strain resultsfrom the overexpression of ACEII.
Fig. 3. qRT-PCR expression analysis of the major cellulase
There are quite a few studies thatgenes in the ace2 overexpressing and deletion strain
described the binding properties ofthese transcription factors. The heterologously expressed DNA-binding domain of ACEII has beenshown to bind in vitro to the 5’-GGCTAATAA-3’ and 5’-GGGTAA-3’ sequences [6]. A similarconsensus binding sequence, 5’-GGC(A/T)4-3’[5], has also been determined for the other maintranscriptional activator, Xyr1. It was recently reported that not only the in vitro translation productof full-length ACEII but also Xyr1 bound to an inverted repeat consisting of a 5’-GGCTAA-3’ and a
5’-GGCTGG-3’ motifs within the xyn2 promoter region [13]. It was proposed that there may be a
regulatory interplay between Xyr1 and ACEII on the promoter region. Hence the results obtained inthis study suggest that ACEII can act as an antagonist of Xyr1 at the early stage of induction.

Fig. 4. qRT-PCR analysis of the egl and bgl genes in the ace2 overexpressing and deletion strains.
Since the CC2 strain expressed about 470-fold ace2 transcripts, the reduced gene expression
detected in the later stage of induction (48 to 96h) is deduced to be a result of competition for thecis-acting elements between Xyr1 and ACEII. Overexpressed ACEII might occupy many of thebinding sites for Xyr1, causing transcriptional downregulation of the Xyr1-target genes. On the other
hand we observed that overexpression of ACEII did not evoke any enhancement of the transcript
formation. This suggests that the transcriptional activation by ACEII needs co-activating protein(s)for its overall function. Taken together, our results suggest a possible involvement of ACEII in theregulation of cellulase and xylanase gene expression in T. reesei.
Further experiments are needed to clarify the role of ACEII and its target genes. We are currently
analyzing the target genes of ACEII using microarray analysis to reveal genome-wide responses tooverexpression of ACEII. The ACEII overexpressing strains constructed in this study would begood candidates to dissect the true function of ACEII.
4. Conclusion
We have successfully developed novel T. reesei strains expressing tagged-ACEII under thecontrol of the cbh1 and the xyn3 promoters. The strains expressed markedly higher amounts of ace2
5

Transactions on GIGAKU 1(2012)01031/1-6transcripts in cellulase-inducing conditions, and the maximum level of the expression was estimatedas 570-fold higher for the cbh1 construct and as 20-fold higher for the xyn3 construct. Analysis ofcellulase production profiles and the gene expression profiles revealed the possible involvement of
ACEII in the regulation of cellulase and xylanase gene expression in T. reesei. Our study provides
the first insights into ACEII overexpresion in T. reesei.

Acknowledgments
This work was supported in part by a grant to W. Ogasawara from the NEDO (The New Energy
and Industrial Technology Development Organization) project of the Ministry of Industrial andTechnology of Japan, and JST-JICA.

References[1] Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J.,
Chertkov, O., Coutinho, P.M., Cullen, D., Danchin, E.G., Grigoriev, I.V., Harris, P., Jackson, M.,
Kubicek, C.P., Han, C.S., Ho, I., Larrondo, L.F., de Leon, A.L., Magnuson, J.K., Merino, S.,
Misra, M., Nelson, B., Putnam, N., Robbertse, B., Salamov, A.A., Schmoll, M., Terry, A.,
Thayer, N., Westerholm-Parvinen, A., Schoch, C.L., Yao, J., Barbote, R., Nelson, M.A., Detter,
C., Bruce, D., Kuske, C.R., Xie, G., Richardson, P., Rokhsar, D.S., Lucas, S.M., Rubin, E.M.,
Dunn-Coleman, N., Ward, M., Brettin, T.S., Nat. Biotechnol. 26 (2008) 553-560.
[2] Schmoll, M., Kubicek, C.P., Acta. Microbiol. Immunol. Hung. 50 (2003) 125-145.
[3] Xu, J., Nogawa, M., Okada, H., Morikawa, Y., Appl. Microbiol. Biotechnol. 54 (2000) 370-375.
[4] Stricker, A.R., Mach, R.L., de Graaff, L.H. Appl. Microbiol. Biotechnol. 78 (2008) 211-220.
[5] Furukawa, T., Shida, Y., Kitagami, N., Ota, Y., Adachi, M., Nakagawa, S., Shimada, R., Kato,
M., Kobayashi, T., Okada, H., Ogasawara, W., Morikawa, Y., 2008. Identification of thecis-acting elements involved in regulation of xylanase III gene expression in Trichoderma reesei
PC-3-7. Fungal Genet. Biol. 45, 1094-1102.
[6] Aro, N., Salheimo, A., Ilmen, M., Penttilä, M., J. Biol. Chem. 276 (2001) 24309-24314.
[7] Kawamori, M., Morikawa, Y., Takasawa, S., Appl. Microbiol. Biotechnol. 24 (1986) 449-453.
[8] Hynes, M.J., Corrick, C.M., King, J.A., Mol. Cell. Biol. 3 (1983) 1430-1439.
[9] Rahman, Z., Shida, Y., Furukawa, T., Suzuki, Y., Okada, H., Ogasawara, W., Morikawa, Y.,
Appl. Microbiol. Biotechnol. 82 (2008) 899-908.
[10] Furukawa, T., Shida, Y., Kitagami, N., Ota, Y., Adachi, M., Nakagawa, S., Shimada, R., Kato,
M., Kobayashi, T., Okada, H., Ogasawara, W., Morikawa, Y., Fungal Genet. Biol. 45 (2008)
1094-1102.
[11] de Vries, R.P., van de Vondervoort, P.J.I., Hendriks, L., van de Belt, M., Visser, J., Mol. Genet.
Genomics 268, (2002) 96-102.
[12] Marui, J., Kitamoti, N., Kato, M., Kobayashi, T., Tsukagoshi, N., FEBS Letters 528 (2002)
279-282.
[13] Stricker, A.R., Trefflinger, P., Aro, N., Penttilä, M., Mach, R.L., Fungal Genet. Biol. 45 (2008),
436-445[14] Rauscher, R., Würleitner, E., Wacenovsky, C., Aro, N., Stricker, A.R., Zeilinger, S., Kubicek,
C.P., Penttilä, M., Mach, R.L., Eukaryot. Cell 5 (2006), 447-456.
[15] Strauss, J., Mach, R.L., Zeilinger, S., Hartler, G., Stöffler, G., Wolschek, M., Kubicek, C.P.,
FEBS Lett. 376 (1995), 103-107.
[16] Zeilinger, S., Ebner, A., Marosits, T., Mach, R., Kubicek, C.P., Mol. Genet. Genomics. 266(2001), 56-63.
[17] Saloheimo, A., Aro, N., Ilmen, M., Penttilä, M., J. Biol. Chem. 275 (2001), 5817-5825.
[18] Nitta, M., Furukawa, T., Shida, Y., Mori, K., Kuhara, S., Morikawa, Y., Ogasawara, Y., Fungal
Genet. Biol. 49 (2012), 388-397.

(Received: 30 May, 2012, Accepted: 18 June, 2012)6

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Thermal Gradient Creep of Ni-based Superalloy Coatings
Subramanian Rajiv Gandhi, Ryohei Nomura, Masakazu Okazaki*,
Satoshi Yamagishi
Department of Mechanical Engineering, Nagaoka University of Technology,1603-1 Kamitomioka-machi, Nagaoka 940-2188, Japan*E-mail: okazaki@mech.nagaokaut.ac.jp
Thermo-mechanical fatigue tests were carried out for thermal barrier
coatings on three different substrate materials SUS316 stainless steel alloy,
Mar-M247 and Inconel 738 Ni-based alloys, under a combined condition
where the external load was superimposed on thermal cycling in acontrolled manner. Special attention was paid to specific failure modes
under this kind of complicated TMF loadings. Finally, the failure
mechanism and crack density were investigated for all these materials.

1. Introduction
Gas turbines are extensively used for aircraft propulsion and in power generation. The most
severely loaded parts in a turbine are the rotor blades, which are made up of nickel-based superalloys[1]. These alloys are being used extensively because of their excellent creep rupture strength, rupture
ductility, and thermal fatigue [2]. However, when these alloys are exposed to elevated temperatures,
their oxidation resistance becomes a more pivotal factor to dominate turbine blade longevity.
Thermal barrier coatings are more essential to increase the lifetime and efficiency of highly loaded
turbine blades and vanes at elevated temperature by reducing the average metal temperature andmitigating the detrimental effects of hot spots. They are regarded as one of the most successful
innovations and applications of coatings in aircraft industry. Thermal barrier coatings (TBCs) consistof three layers; the prevalent outer layer is yttria-stabilized zirconia (YSZ), acting as the thermal
barrier coating, an oxidation resistant bond coat, consisting of a metallic alloy like MCrAlY and anickel-based superalloy substrate [3-4]. When superalloys along with thermal barrier coatings are
exposed to high temperature, the turbine blade system is subjected to high thermo-mechanicalfatigue (TMF) loading. Understanding of resistance to this kind of damage has gained more
importance in recent years. The basic principles of failure seem to be understood under idealizedlaboratory conditions, however, a detailed analysis of the mechanisms and the possibilities of
avoiding damages are still lacking. Especially, under a significant thermal gradient condition in
actual combustion gas environment, unexpected modes of failures may be experienced. There issuch an aspect that few adequate test beds impede us to get these understandings.
In this study, a novel test system has been originally developed which enables us to apply thecomplicated TMF loadings to superalloy–TBC specimen under a significant thermal gradient in an
actual combustion gas environment. By employing this system, failure mechanism was investigatedfor TBCs on three different substrate materials Mar-M247 and Inconel 738 Ni-based alloys, SUS316
stainless steel alloy. The TBC had nominally the same composition for all those substrate materials.
Finally, failure mechanism and crack density were investigated for all these materials.
2. Experiment
Test System
A new test facility illustrated in Fig. 1 has been developed, where the system consists of a
combustion part and a test section. In the former part, a simulated combustion atmosphere is
produced employing a type of combustor, so that the test section inlet temperature (TSIT) reaches up
to 1500C maximum. Associating this with a servo-electro hydraulic testing machine, the static or
cyclic mechanical loading is superimposed in a synchronized manner to the thermal cycling. The
system temperature is controlled by TSIT; the temperatures at some representative positions in the1Transactions on GIGAKU 1(2012)01032/1-6

specimen, e.g., upstream and downstream gas temperatures just above the specimen surface (as wellas the inside temperatures in some cases), are continuously monitored. The temperatures measured
for the present TBC specimen are summarized in Table 1 where the TSIT was cyclically changed
between 1100/750C with a frequency of 300/300 s in heating/cooling duration time, respectively.
Thus, the new test system successfully gives external load associated with a significant thermal
gradient inside the specimen.

Fig. 1 Illustration of newly developed test system

Table 1 Summary of thermal gradient at the representative parts inside the TBC specimen whenthermal cycle was applied between 1100/750C (in TSIT) by means of a new test facility developed
Side stream

Up stream

Down stream

Test

sectionGas temp.

Interface**

Gas temp.

Interface**

Gas temp.

Interface**

inlet

gas

above TBC

temp. inabove TBC

temp. inabove TBC

temp. intemperature*

specimen

TBC

specimen

TBC

specimen

TBC

( C)

surfacespecimensurfacespecimensurfacespecimen

( C)

( C)

( C)

( C)

( C)

( C)

1100

990

950

960

930

900

880

750

675

650

665

640

630

620

* Changed cyclically with a frequency of 300/300 s in heating/ cooling, respectively.
** Bond coat/ substrate interface (mounted R type thermocouples)
Outline of Test
Solid cylindrical substrate specimens were prepared from Mar-M247 polycrystalline and Inconel
738 Ni-based alloys, SUS316 stainless steel alloy. The TBC specimens used, whose gauge section
dimension is explained in Fig. 2. The TBC specimen consists of three layers; a Ni-base superalloy,
bond coat (of CoNiCrAlY alloy, or AMDRY9951, 100 m thick produced by low pressure plasma
spraying), and a top coat (made with 8 wt% YSZ, or METCO 204NS, 500 m in thickness byatmospheric plasma spraying). Employing the above new testing system, the TMF tests were carriedout, where a static, or constant axial stress was superimposed on the thermal cycling, which issummarized in Table 2, between 1280 and 700C in TSITs with a frequency of 1/360 s.

2Transactions on GIGAKU 1(2012)01032/1-6

Fig. 2

TBC specimen

Table 2 Summary of thermal cycles and mechanical load
Substrate

MarM 247

Inconel 738

SUS316

Thermal700–1280C

700–1280C

700–1280C

750–1113C

750–1113C

170 MPa

170 MPa

100 MPa

60 MPa0 MPacycling
Mechanicalloading

3. Results and Discussion
3.1. Thermal Cycles with Mechanical Load
The crack initiation and growth of TBC coating were observed after a significant number ofthermal cycles.The average number of thermal cycles to failure for TBCs on Mar-M247, Inconel 738,
and SUS316 substrates was 99 cycles, 50 cycles at 170 MPa, and 130 cycles at 60 MPa, respectively,
as illustrated in Figs. 3, 4, and 5. TBCs on Mar-M247 substrates averaged about twice the thermal
cycle life as those on Inconel 738 substrates. The micrograph observations clearly illustrate thatcrack growth occurred perpendicular to the external load direction (Fig. 6). In addition, notablechanges in shape of the test piece were observed. The length of the specimen during the test isextended with the number of cycles. At the end of 130 cycles, a permanent strain of 1.4% was
measured in SUS316, 0.35% strain in Mar-M247 at 99 cycles, and 0.82% strain in Inconel 738 at 50
cycles as illustrated in Fig. 7. The phenomenon of the TBC failures is deeply related with
deformation of the substrate. When the coating material is subjected to external load at elevatedtemperature, both coating and substrate start to deform. Due to variation in the thermal expansion
coefficient between the substrate and the TBC, the thermal stresses increase at the interface. Finally,
crack initiate in the coating and growth occurs uniformly throughout the specimen. Hence,
deformation of the substrate is also one of the main factors for the failure of TBC coatings.

Fig. 3 Micrographs of the TBC surface after 99
thermal cycles with 170 MPa tensile loadingat 700/1280C (Mar-M247-1 TBC
specimen)

Fig. 4 Micrographs of the TBC surface after 50
thermal cycles with 170 MPa tensile loadingat 700/1280C (Inconel 738 TBC specimen)

3

Transactions on GIGAKU 1(2012)01032/1-6

Fig. 5 Micrographs of the TBC surface after 130
thermal cycles with 60 MPa tensile loadingat 700/1280C (SUS316 TBC specimen)

Fig. 6 Micrographs of the TBC surface after 130
thermal cycles with 60 MPa tensile loadingat 700/1280C (SUS316 TBC specimen)

Fig. 7 Schematic illustrations on damage evolution in the respective TBC specimens

3.2. Microstructural Analysis
Microstructural features of TBC specimens were examined before the thermal cycle (Fig. 8) andafter failure. Evolution of the TBC microstructure during the thermal cycle for TBCs on SUS316
substrates is shown in Fig. 9 after 130 thermal cycles. Three notable changes occurred at the
TBC–substrate interface during thermal exposure [5]. First, there was a continuous increase in the
thickness of the TGO layer between the bond coat and the YSZ top coat. Second, the interface
roughness progressively increased. Third, interface delamination defects formed between the TGO
and the top coat [6-8].
TBCs on inconel 738 and Mar-M247 substrates exhibited similar to those observed in Fig. 9 for
TBCs on SUS316 substrates. The interface at which spalling occurred during the thermal cycle isshown in Figs. 9, 10, and 11. And, also shows regions from which the TBC spalled from the bondcoat and regions where the TBC remained intact. There was evidence that the interface crack
originated at the YSZ–TGO interface (above the alumina) and, only during the course of thecatastrophic crack propagation that led to spallation for the SUS316, Inconel 738, and Mar-M247
substrates occasionally ran below the alumina at the TGO–bond coat interface. The delamination
defects tended to become deeper and longer with increased number of cycles.

4Transactions on GIGAKU 1(2012)01032/1-6

Top coat

Top coat

TGO
Bond coat

TGO

Fig. 9 TBC surface after 130 thermal cycled with
60 Mpa tensile loading at 700/1280C(SUS316)

Substra

Top

TG

TGO

Bond

Fig. 8 Micrographs of SUS316 TBC specimen
before thermal cycle

Bond coatTop coatBond
Fig. 11 Micrographs of the TBC surface after 99
thermal cycles with 170 MPa tensile loadingat 700/1280C (Mar-M247 specimen)

Fig. 10 Micrographs of the TBC surface after 50
thermal cycles with 170 MPa tensile loadingat 700/1280C (Inconel 738 TBC specimen)

3.3. Crack Density Measurement
Two types of cracks were observed within the coating after the thermal cycles, vertical oriented(parallel to the load axis) and horizontally oriented (perpendicular to the load axis). These cracks
were found to occur specially at the interface between TGO and top coat [9], [10]. Horizontallyoriented cracks density is greater than vertical oriented cracks. Figure 12 shows very strong channel
cracking in the coating. Normally, this kind of cracking occurs perpendicular to the loading direction.
The crack density was very large about 0.81 at downstream side rather than upstream side about 0.50
(Figs. 12 and 13). This difference is may be due to the large compressive stress on the downstreamside when there is some thermal gradient. However, this phenomenon is not understood completely.
The horizontally oriented crack density measured by the following equation.

Z crack density 

No. of cracks in the Z direction (n)Gage length (mm)5SubstraTransactions on GIGAKU 1(2012)01032/1-6

Fig. 12 Micrograph of horizontal
cracks at upstream sideTGO

To
p coatFig. 13 Crack density measurement withthermal gradient at 1280/700C

4. Conclusion
A new test system has been originally developed that enables us to apply the complicated TMF
loadings to superalloy and TBC specimen under a significant thermal gradient in an actual
combustion gas environment. By employing this system, the following results have been found. The
failure mechanism was investigated for TBCs on three different substrate materials Mar-M247 andInconel 738 Ni-based alloys, SUS316 stainless steel alloy. From these results, it was clearlyobserved that Mar-M247 can withstand more thermal cycles and stresses than other materials tested.
The phenomenon of the TBC failures was deeply related with deformation of the substrate, which
was due to variation in temperature between upstream and downstream. From the SEM observation,
three notable changes occurred at the TBC–substrate interface during thermal exposure. First, there
was a continuous increase in the thickness of the TGO layer between the bond coat and the YSZ top
coat. Second, the interface roughness progressively increased. Third, interface delamination defects
were formed between the TGO and the top coat. Two types of cracks were observed within thecoating after the TMF and creep test, those were vertical oriented and horizontally oriented. Fromthat result, the crack density was higher under the tests with thermal gradient than those without
thermal gradient.
References[1] N.P. Padture, M. Gell, and E.H. Jordan, Science, 296 (2002) 280.
[2] B. Sarhan and M. Bartsch, Key Eng. Mater., 333 (2007)137.
[3] G.W. Goward, Surf. Coat. Technol., 108–109 (1998) 73.
[4] Y.H. Sohn, J. Am. Ceram. Soc., 85 [8] (2002) 2065.
[5] A.A. Ruud, J. Am. Ceram. Soc., 84(7) (2001)1545.
[6] J.P. Singh, J. Am. Ceram. Soc., 84 [10] (2001) 2385.
[7] C.G. Levi, E. Sommer, and S.G. Terry, J. Am. Ceram. Soc., 86 [4] (2003) 676.
[8] P. Ke, Q. Wang, W. Hua, and J. Gong, J. Am. Ceram. Soc., 90 [3] (2007) 936.
[9] H. Chen, Y. Liu, and Y. Gao, J. Am. Ceram. Soc., 93 [6] (2010) 1732.
[10] A.G.Evans and J.W.Hutchinson, Surf. Coat. Technol., 201 (2007) 7905.
(Received: 23 March, 2012, Accepted: 27 April, 2012)6

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Characteristics and Directed Evolution of Trichoderma reesei
Endoglucanase III (Cel12A)– A Review
Hirofumi Okada*, Hikaru Nakazawa, Wataru Ogasawara, Yasushi Morikawa
Department of Bioengineering, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: hirofumi@vos.nagaokaut.ac.jp

This paper reviews shortly characteristics of the Trichoderma reesei
endoglucanase III (Cel12A) and improvement of the enzymatic properties bydirected evolution. EG III gene contains a 702-bp open reading frame that
encodes a 234-amino-acid peptide with the 16-amino-acid signal sequence.
The deduced protein sequence has significant homologies with glycosidehydrolase family 12. The protein has been expressed as a mature form in
Escherichia coli strains JM109 or Rosetta-gami B (DE3) pLacI. The
site-directed mutagenesis experiment of the enzyme demonstrated that
Glu116 and Glu200 are important for catalytic function as the mucleophilic
and acid-base residues, respectively. It is also described that directed
evolution allowed Cel12A to enhance the stability and specific activity.

1. Introduction
Cellulose, which is a major constituent of plant cell walls, is the most abundant form of biomasson earth and can be converted into bioethanol and other chemical products via fermentation
following hydrolysis to fermentable sugars. Cellulose is composed of linear homopolymers of
D-glucose linked by b-1,4 bonds and includes crystalline structures stabilized by intermolecular andintramolecular hydrogen bonds.
Glycoside hydrolases, including plant cell-wall hydrolases, such as cellulases and hemicellulases,
are conveniently classified into a number of families based on their amino-acid sequence similarities[1]. With the accumulation of three-dimensional structural data, the related families have beengrouped into clans, since their members share a common fold and the position of their catalytic
residues is conserved despite the dissimilarities of their sequences. In addition, it has been found thatmost glycoside hydrolases are modular enzymes consisting of a discrete catalytic domain and acarbohydrate binding module such as a cellulose binding domain [1]. Glycoside hydrolases cleave
their target substrates by either a single or double displacement mechanism, resulting in the
inversion of the product, respectively [2].
Cellulase, including cellobiohydrolase (CBH), endoglucanase (EG), and b-glucosidase,hydrolyzes the b-1,4-D-glycoside bonds of cellulose. CBH releases cellobiose from either the
reducing or non-reducing ends of the cellulose chain, whereas EG randomly degrades the internalbonds of the amorphous region of cellulose. Cellulolytic enzymes are widely distributed among
plants, animals, bacteria, and fungi. The filamentous fungus Trichoderma reesei is known as a potent
cellulase producer because of its remarkable ability to secrete cellulolytic enzymes. It has beenreported that this fungus possesses two CBH genes, cbh1 and cbh2, and eight EG genes, egl1 to egl8[3], and that its CBH I and II and EG I to VII are secreted proteins. In this review, we reportmainly the enzymatic characteristics and the improvement of EG III (Cel12A).

Transactions on GIGAKU 1(2012)01033/1-6

2. Molecular Characterization of EG III
EG III purified from the culture of T. reesei PC-3-7 has a molecular mass of 25 kDa and a
specific activity of 11.2 U/mg for carboxy methyl cellulose (CMC) as a substrate and a slight activity
toward Avicel [4]. The enzyme is inactive toward xylan, p-nitrophenyl b-D-glucopyranoside(PNPG), and chitosan. Analysis of hydrolysis products of cellooligosaccharide, phosphoric
acid-swollen cellulose (PASC) and Avicel revealed that EG III finally gave a small amount of
glucose, cellobiose and cellotriose. Cellotriose could not be completely hydrolyzed after prolonged
incubation with the enzyme. EG III exhibited synergism with CBH I in degradation of Avicel.
EG III gene contains a 702-bp open reading frame that encodes a 234-amino-acid peptide as shown in
Fig. 1 [5]. The mature protein presumably starts at amino acid 17, glutamine, which seemed to bepyroglutamylated [4]. The N-terminal 16-amino-acid peptide of the immature protein showed thetypical structure of a signal peptide that contains the region with a high hydrophobic index following
a positively charged amino acid. The 234 amino acid protein deduced from the nucleotide sequence
has a molecular mass of 25,158 Da for the unprocessed form, and if the initial 16 amino acids are
excluded, the calculated molecular mass of the mature protein is 23,480 Da, which is in good
agreement with the biochemical data. When the EG III sequence was compared with those available
from the databases, FI-CMCase from Aspergillus aculeatus, CMCase-I from A. kawachii, and CelS
from Erwinia cartovora subsp. cartovola showed 56, 47, and 26 % homology with EG III at theamino acid level, respectively (Fig. 1). These are all EGs which belong to glycoside hydrolase (GH)family 12. GH12 family is further classified into four subgroups; archaea, bacteria, fungal EG, andfungal xyloglucanase subgroups (Fig.2). EG III is categorized as fungal EG subgroup.

Fig. 1 Alignment of the EG III sequence with those of GH12 EGs.
Asterisks represents the conserved Glu residues among family-12 cellulases

Transactions on GIGAKU 1(2012)01033/1-6

Fig. 2 Pylogenetic tree of GH12 enzymes.

3. Identification of catalytic residues in EG III
To identify the catalytic residues in EG III, the mature part of egl3 cDNA was expressed in E.
coli JM109 using the tac promoter. The E. coli transformant produced EG III protein largely as
insoluble inclusion bodies in the cytoplasm of the cells. The insoluble fraction was solubilized withurea and EG III was purified by cation chromatography to electrophoretic homogeneity. The purified
enzyme was immunologically and enzymologically identical to EG III purified from T. reesei (Table
1)[6].
Table 1 Properties of the EG IIIs from T. reesei and E. coli
T. reesei
Molecular mass (kDa)
25
Specific activity (U/mg)
12.7
Optimum pH
5.5
Optimum temperature (ºC)
55
pH stability
5.0-5.5
Temperature stability
up to 45ºC

E. coli
25
13.1
5.5
55
5.0-5.5
up to 45ºC

Two glutamic acid residues, Glu-116 and Glu-200 of T. reesei EG III are conserved amongfamily-12 cellulases (Fig. 1) and presumed to act as the nucleophile and the acid/base in the double
displacement mechanism. To confirm this, Glu-116 and Glu-200 were separately replaced with Asp
or Gln by site-directed mutagenesis. A double mutant, E116D:E200D, having two aspartates at theseputative catalytic residues was also prepared. All the mutants were expressed in E. coli as inclusion

Transactions on GIGAKU 1(2012)01033/1-6

bodies, which were solubilized and purified according to the same procedure as that for thewild-type (wt) EG III. Based on the results of SDS-PAGE analysis, the mutant enzymes wereobtained with similar yields to that of the wt enzyme, and the molecular mass of each mutant wasestimated to be the same as that of the wt EG III (data not shown). Comparison of CD spectra of themutants with that of the wt enzyme in the far-UV regions did not reveal any significant difference,indicating that the mutations did not affect the fold of the enzyme (data not shown).
Specific activity and kinetic parameters, Km and kcat, of the wt EG III and the mutants were
determined using CMC as a substrate (Table 2). The specific activities of the Asp mutants, E116D
and E200D, were 1.7% and 0.4% of that of the wt EG III, respectively, while the double mutant
E116D:E200D displayed little activity. The kcat values of the mutant enzymes E116D and E200D
were 50- and 200-fold lower than that of the wt enzyme, respectively, which corresponded to thedecreases in specific activity. On the other hand, both of the Gln mutants, E116Q and E200Q, hadlittle activity, namely 0.01-0.02% of that of the wt enzyme, strongly suggesting that these glutamic
acids play an important role in the catalysis of hydrolysis The Km value of the E116D mutant (4.2
g/L) was twofold higher than that of the wt EG III (2.0 g/L), while that of the E200D mutant (0.5g/L) was reduced to one fourth of that of the wt EG III. Furthermore, the catalytic efficiency(kcat/Km) of the mutant enzyme E116D was more than two orders of magnitude lower than that of the
wt enzyme. Reduction of catalytic efficiency was less pronounced when E200 was replaced by D.
The results observed in other double-replacement-mechanism glycosidases [7] indicate that the
acid/base residues usually less contribute to catalysis than the nucleophile residues. From thesefindings, it is plausible that Glu-116 and Glu-200 act as the nucleophile and the general acid/base,respectively, in the catalysis of hydrolysis.
Table 2 Specific activities and kinetic parameters of wild-type and mutant enzymes with CMC as a
substrate
EnzymeSpecific activity
Km (g/L)
kcat (s-1)
kcat/Km (L/g・s)(U/mg)(%)
Wild type
13.1
(100)
2.00
5.50
2.8
E116D
0.214
(1.7)
4.17
0.106
0.025
E200D
0.0483(0.40)
0.500
0.02650.053
E116:E200D
1.82×10-3（0.015）−−−E116Q
1.61×10-3（0.013）−−−E200Q
2.39×10-3（0.019）−−−4. Directed evolution of EG III
Recently, improvement of individual cellulase enzymes has been attempted by protein
engineering (rational protein engineering or directed evolution) in order to enhance cellulose
degradation ability in T. reesei [8, 9]. It is difficult to obtain the desired enzyme by rational protein
engineering because enzyme structure-function relationships are poorly understood. Directed
evolution following random mutagenesis, which is not dependent on an understanding ofstructure-function relationships appears to be a better approach. The use of E. coli as a host for
directed evolution of cellulase has obvious advantages. E. coli is amenable to molecular genetic
analysis, grows rapidly, and is transformed at high frequency. EG III was slightly expressed as anactive enzyme in the soluble fraction in E. coli JM109, but most of the recombinant protein was
found in an inclusion body as described in section 3 [6]. Improvement of E. coli expression system
to increase expression of EG III as an active form was performed with the pET expression vector
system [10]. The egl3 gene was subcloned from the E. coli expression vector into pET22b.
Rosetta-gami B (DE3) pLacI exhibited the highest amount of CMCase activity among eight E. coli
strains. The optimal temperature for maximal productivity of EG III was 37ºC (0.76 U/ml broth).
Furthermore, in order to increase yields of functional EG III from E. coli, a simple purification
method was developed. The method was based on the differential precipitation of E. coli proteins at
pH4.0 that does not precipitate functional EG III. Thus, recombinant EG III from E. coli was easily

Transactions on GIGAKU 1(2012)01033/1-6

separated from native E. coli proteins and inactive form EG III, yielding single band in SDS-PAGE.
Enzymatic characteristics of the purified recombinant enzyme were the same as those of the nativeand renatured recombinant enzymes in Table 1. Employment of Rosetta-gami B (DE3) pLacI as arecombinant host and differential precipitation at acidic pH greatly facilitate development of
improved EG III, through high-throughput screening of candidate enzymes obtained by directed
evolution without refolding an inclusion body.
To improve the specific activity of EG III, directed evolution in E. coli was performed using
error-prone PCR and activity screening in E. coli RosettaBlue (DE3) pLacI. This E. coli strain was avery suitable host for directed evolution because a very low basal activity of wild-type EG III wasdesirable for the identification of the mutant enzymes with enhanced enzymatic activity byhigh-throughput screening on plates. The clone harboring the highest stability and the highest
specific activity produced the mutant enzyme, 2R4 with multiple mutations, G41E, T110P, K173M,
Y195F, P201S, and N218I, which was selected via the second-round mutagenesis after the optimal
first-round mutagenesis and screening. It produced 130-fold higher amount of the 2R4 enzyme than
the transformant producing the wild-type EG III [11]. 2R4 showed broad pH stability ranging from
pH4.4 to pH8.8 and complete thermotolerance at 55ºC for 30 min compared with those of thewild-type EG III with pH stability ranging from pH4.4 to pH5.2 and absolute thermosensitivity at
55ºC for 30 min. kcat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of
the wild type, though the Km was twice of that of the wild type (Table 3).
Table 3 Kinetic parameters of 2R4 mutant
EnzymeSpecific activity(U/mg)
Wild type
22
2R4
26

kcat
(min-1)630±10860±30Km(g/L)
1.5±0.2
3.0±0.4

kcat/Km(L/g・min)440±50290±20

Enzyme stabilization extends the duration of enzyme activity and the range of the reaction
temperature, which is important for industrial application of enzymes, for example, laundry
detergents, biomass saccharification, or the stonewashing of denim.

Acknowledgments
This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports, Culture and Technology, Japan, and by the grant from the ResearchInstitute of Innovative Technology for the Earth (RITE) project.
References[1] B. Henrissat, G. J. Davies, Plant Physiol. 124 (2000) 1515.
[2] C. S. Rye, S. G. Withers, Curr. Op. Chem. Biol. 4 (2000) 573.
[3] P. K. Foreman, D. Brown, L. Dankmeyer, R. Dean, S. Diener, N. S. DunnColeman, F.
Goedegebuur, T. D. Houfek, G. J. England, A. S. Kelly, H. J. Meerman, T. Mitchell, C.
Mitchinson, H. A. Olivares, P. J. M. Teunissen, J. Yao, M. Ward, J. Biol. Chem. 278 (2003)
31988.
[4] Y. Morikawa, A. Takahashi, K. Yano, M. Yamasaki, H. Okada, Genetics, Biochemistry andEcology of Lignocellulose Degradation (eds. K. Shimada et al.) UNI Publishers, (1994) pp 458.
[5] H. Okada, K. Tada, T. Sekiya, K. Yokoyama, A. Takahashi, H. Tohda, H. Kumagai, Y.
Morikawa, Appl. Environ. Microbiol. 64 (1998) 555.
[6] H. Okada, K. Mori, K. Tada, M. Nogawa, Y. Morikawa, J. Mol. Cat. B: Enzymatic 10 (2000)
249.
[7] S. L. Lawson, W. W. Wakarchuk, S. G. Withers, Biochemistry 36 (1997) 2257.
[8] A. G. Day (Genencor International IU) Patent (2003) 2,004,016,760.
[9] S. Teter, J. Cherry, Ward, C., A. Jones, P. Harris, J. Yi (Novozymes Biotech. IU) Patent (2004)
20050048619.

Transactions on GIGAKU 1(2012)01033/1-6[10] H. Nakazawa, K. Okada, R. Kobayashi, T. Kubota, T. Onodera, N. Ochiai, N. Omata, W.
Ogasawara, H. Okada, Y. Morikawa, Appl. Microbiol. Biotechnol. 81 (2008) 681.
[11] H. Nakazawa, K. Okada, T. Onodera, W. Ogasawara, H. Okada, Y. Morikawa, Appl. Microbiol.
Biotechnol. 83 (2009) 649..
[12] T. Honma and T. Komatsu, Opt. Express 18 (2010) 8019.
[13] F. Suzuki, T. Honma, and T. Komatsu, Mater. Chem. Phys., 125 (2011) 377.
[14] F. Suzuki, K. Ogawa, T. Honma, and T. Komatsu, J. Solid State Chem., 185 (2011) 130.
[15] H. Tanaka, T. Honma, Y. Benino, T. Fujiwara, and T. Komatsu, J. Phys. Chem. Solids, 64(2003) 1179.
[16] T. Honma, Y. Benino, T. Fujiwara, R. Sato, and T. Komatsu, J. Phys. Chem. Solids, 65 (2004)
1705.

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Electrostatic Layer-by-Layer Polymer Membranes for Heavy Metal
Ion Desalination
Wei Min Zhou, Huitan Fu, Motohiro Tagaya, Takaomi Kobayashi *Department of Materials Science and Technology, Nagaoka University of Technology,1603-1 Kamitomioka-cho, Nagaoka 940-2188, Japan*E-mail: takaomi@nagaokaut.ac.jp
In the present review, we introduced the electrostatic self-assembly (ESA)
modified membrances by forming ionic alternate layers of cetyl trimethyl
ammonium chloride (CTAC) and poly(acrylic acid) (PAA) onto charged
copolyacrylonitrile membranes. Along with surface characterization of the
ESA multilayer, the desalination properties of the membranes with ESA
multilayer were evaluated by investigating the metalion removal capability.
Based on the experiment results, positively charged membrane terminatedwith CTAC on the ESA layer showed considerable high rejection for Fe3+.
Alternately deposited cationic chitosan microspheres showed effective ESA
layer of PAA onto the desalination by charged polyacrylonitrile (PAN)
membrane. It was confirmed that the alternative multilayer of chitosan micro
sphere and PAA deposited on the base membrane surface presented removal
capability of Cu2+. Especially, membranes with chitosan microspheres / PAA
ESA layers on the surface showed relatively higher adsorption capability ascompared with membranes with chitosan / PAA ESA layers.

1. Introduction
It has been known that layer-by-layer (LbL) self-assembly is a spontaneous and reversibleorganization process of interacting organic and polymeric components by their aggregation into
ordered structures on substrate. This technique has been widely applied for the fabrication ofmultilayer films of organic and polymeric compounds, organic-inorganic hybrid structures, larger
objects such as latex particles, and even purely inorganic thin films. By combining various
functional materials for the formation of self-assembly multilayer, the LbL technique has led to awide range of novel materials for various applications [1,2]. It is noticed that the LbL fabrication isusually guided by a driving force of hydrophobic interaction [3], hydrogen-bond [4], covalentbonding [5], and electrostatic interaction [6] between assembled compounds. Among those driving
forces, electrostatic interaction between oppositely charged molecules becomes a very fascinating
and attractive approach because of its simplicity and efficiency. The technique of alternate
layer-by-layer assembly of cationic and anionic polyelectrolytes, generally referred to electrostatic
self-assembly (ESA) in 1991 [7]. They took advantage of the charge-charge interaction between
oppositely charged layers to create electrolytes multilayer. The polyelectrolyte conformation andlayer interpenetration were due to an idealization of the surface charge reversal with each adsorption
step which was based on the electrostatically driven multilayer build-up depicted [8,9]. Because thesubstrate surface contained negatively or positively charged due to the alternate deposition ofpolycation or polyanion on the transmission of charged particles like ions through self-assembly
polyelectrolyte multilayer membrane might be affected by the charged surface. Selectively ion
transport behavior through ESA composite multilayer films was studied by Take [8] and Schelenoff[9]. The results suggested that highly specific ion separation was achieved and was affected by the
charge and size of ion that pass through the composite multilayer. These researches made thepolyelectrolytes multilayers interesting for the application of water desalination like heavy metal ion
removal.
In this review, we introduce the ESA modified membranes by forming electrostatic alternate
layers of cetyl trimethyl ammonium chloride (CTAC) and poly(acrylic acid) (PAA) onto charged
copolyacrylonitrile membranes (Fig. 1). Along with surface characterization of the ESA multilayer,1Transactions on GIGAKU 1(2012)01034/1-6

infrared analysis was carried out. Finally, we evaluated the adsorptive properties of ESA multilayer
for Fe3+ and Fe2+ and ESA membranes with chitosan microspheres / PAA for removing of Cu2+.
OH
CH2

-

CH
CN

CH2
m

H2O

O

CH
m

C
NH2

OH
CH2
O

C
NH2

Fig. 1 Preparation procedure of ESA
multi-layer on polymer membrane having
negative charge.

-

CH

CH2
m

H2O

O

CH

O

+

NH3

m

C
-

Fig. 2 Chemical reaction of PAN treatedby alkali solution.

2. Preparation of multilayer composite surfaces prepared by ESA technique anddesalination behaviour
As presented in Fig. 3, quaternized N,N-dimethylethyl ammonium bromide (EDAMA) was
polymerized with AN for preparation of cationic base membrane. AA and the quaternized EDAMA
were used for their functional groups in order to provide charged sites for polyacrylonitrile basemembranes. Also, poly(acrylonitrile-co-acrylic acid) [P(AN-co-AA)] membrane was used as anegatively charged base, while poly(acrylonitrile-co-N,N-dimethyethyl ammonium bromideP(AN-co-EDAMA) was used as a positively charged substrate [10,11]. Their base membranes ofP(AN-co-AA) and P(AN-co-EDAMA) were prepared by phase-inversion of their respective
dimethyl sulfoxide solutions in water [12]. The base membranes of P(AN-co-AA) orP(AN-co-EDAMA) were orderly dipped in positive CTAC and negative PAA aqueous solutions.
After each deposition step, the resulting membranes were rinsed thoroughly with water. Suchdeposition process was repeated to obtain desired number of self-assembled layers.
As presented in Fig. 3, the base membrane surface was modified alternately by layer-by-layer
formation of CTAC and PAA via ESA treatment. In Fig. 4, the FT-IR spectrum indicated that for the
positively charged substrate of P(AN-co-EDAMA), the ESA treatment was performed in thedeposition cycle order of PAA/CTAC/PAA/CTAC. As a result of the first deposition of the PAA layer,
the band strength of the carboxylic group at about 1720 cm-1 became stronger. This confirmed theassembly of the PAA layer onto the positively charged base membrane. However, after thedeposition of CTAC layer, the carboxylic groups bound on the CTAC layer resulted that the
carboxylic acid absorption peak became weaker relative to that of P(AN-co-EDAMA)/PAA. A
similar tendency repeated itself in accordance with the self-assembly of the first cycle of PAA layer
and CTAC layer.
Since the alternate ESA layers on the membrane surface contained charged groups, it was
expected that the permeate behavior of metal ions through the modified membrane could be affectedby the ESA layers formed on the membrane surface [13,14]. Thus, the desalination of the ESA
modified membranes was evaluated by testing the permeability for metal ion solution. Fig. 5 shows
rejection for metal ions of Fe3+ and Fe2+ of resultant membranes of the P(AN-co-AA)/CTAC/PAA
system and with solid lines for Fe3+ and dotted lines for Fe2+. It could be seen that the membranemodified with ESA layers showed considerable high rejection for metal ion of Fe3+ and the rejection
for Fe3+ was higher than for Fe2+. This means that Fe2+ has a bigger ion radius of about 0.78 nm thanthe 0.64 nm of Fe3+. Additionally, the change tendency of the rejection curves of the two systems forboth metal ions behaved oppositely to each other as revealed in Fig. 3. These indicate that
P(AN-co-AA) base membrane having COO- on the surface was negatively charged, whileP(AN-co-EDAMA) base membrane having quatemary ammonium groups on the surface was2Transactions on GIGAKU 1(2012)01034/1-6

positively charged. Their different charge properties of base membrane surface resulted in different
cycle order of self-assembled multilayer deposited on the base membrane surface, as performed incycle of CTAC/PAA/CTAC/PAA for the former and PAA/CTAC/PAA/CTAC for the latter. The
opposite change tendency of the permeability of resultant membranes further confirmed theinfluence of the ESA terminal layer on its permeability.

CTAC

(a)

P(AN-co-AA)/CTAC/PAA system

Reflection (%)PAA(0) P(AN-co-EDAMA)(1) P(AN-co-EDAMA)/PAA(2) P(AN-co-EDAMA)/PAA/CTAC(3) P(AN-co-EDAMA)/PAA/CTAC/PAA(4) P(AN-co-EDAMA)/PAA/CTAC/PAA/CTAC

3500 3000 2500 2000 1500 1000
(b)

500

Wavenumber (cm-1)

P(AN-co-EDAMA)/PAA/CTAC system

Fig. 3 Illustration images of layer-by-layer
assembling of CTAC and PAA on negatively
charged P(AN-co-AA) membrane surface (a)and on positively charged P(AN-co-EDAMA)
membrane surface (b).

Fig. 4 FT-IR spectra of base membrane andmembrane with self-assembled layers.

b)

Rejection for Fe ions (%)

Rejection for Fe ions (%)a)

Number of layers on membrane surface

Number of layers on membrane surface

Fig. 5 a) The effect of AA fraction contained in base membrane on the rejection of
Fe3+ (—) and Fe2+ (…) for P(AN-co-AA)/CTAC/PAA system (cast solution
concentration for base membrane: 15wt%; pH of metal ions solution: 7). b) The effectof P(AN-co-EDAMA) base membrane on the rejection of Fe3+ (— ) and Fe2+ (… ) forP(AN-co-EDAMA)/PAA/CTAC system. (EDAMA contained in base membrane: 2mol%; pH of metal ions solution: 7).
In different amounts of AA segments, three kinds of P(AN-co-AA) copolymers prepared with a
AA fraction of 10 mol%, 15 mol% and 26 mol% [15], were used as base membranes for ESA
treatment. Then, the surfaces were modified with ESA layers of CTAC and PAA to study the effectof AA fractions contained in the base membrane on the removal properties of modified membranes.
It was found that, when the base membrane used for self-assembly treatment contained higher AA
mol%, the corresponding resultant membranes showed higher removal properties for both Fe3+ andFe2+. This means that base membrane which contained higher AA mol% has more ionized carboxyl
charged sites on the surface.
3

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3. Self-assembly functionalized membranes with chitosan microsphere / polyacrylic acidlayers.
In order to use negatively charged PAN, we converted the CN groups to negatively charged
COO- group on the PAN membrane surface, the chemical reaction (Fig. 2) was performed by using
KOH solution. The FT-IR measurement was carried out to study the chemical composition of the
PAN membrane surface before and after the KOH treatment. In the spectrum of the PAN membrane
treated by KOH for 24 h, the characteristic absorption peak for the CN group at about 2300 cm-1
became weaker while the characteristic peak assigned to carboxyl group in the range from 2800 cm-1
to 3700 cm-1 appeared stronger. Especially, the band at 1700 cm-1 assigned to the carbonyl groupfrom COO- existed in the FT-IR spectrum. So, it was concluded that the PAN membrane surface wasnegatively charged due to the formation of carboxyl groups resulting from the KOH treatment.
Negatively charged
membrane surface
Chitosa n

Chitosan solution

Reflection (%)

PAAPolyanion (PAA) solution

Polycation
chitosan or chitosan
microspherelayers(0) PAN treated by KOH(1) PAN/Chito san(2 ) PAN/Chito sa n/PAA(3) PAN/Chito san/PAA/Chitosa n(4 ) PAN/Chito san/PAA/Chito san/PAA

Repeat steps for desired
number of layers

4000

Polyanion

3000

2000

1000

Wavenumber (cm-1)(PAA) layers

PAN/Chitosan microsphere/PAA system

Fig. 6 Illustration images of layer-by-layer
assembling of chitosan/PAA and chitosan
microsphere/PAA layers onto charged basemembrane.

Fig. 7 FT-IR spectra of membranes with
self-assembled layers.

Fig. 6 shows the surface of treated PAN membrane which was negatively charged due to the
KOH treatment. Then, the positively charged chitosan and negatively charged PAA werealternatively coated to form self-assembly layers onto the charged PAN membrane. Also, chitosan
polycation microspheres cross-liked by sulfate groups were used instead of chitosan to form
electrostatical self-assembled layers onto the negatively charged base membrane. The deposition
process of the alternate formation of chitosan or chitosan microspheres and PAA layers was repeated
4 times.
The surface of membranes having alternate ESA multilayer was investigated by using reflection
FT-IR spectroscopy (Fig. 7) for PAN/Chitosan/PAA system and PAN/chitosan microspheres/PAA
system, respectively. It was found that the characteristic band of CN group from the charged PANbase membrane at about 2300 cm-1 became weaker due to the deposition of ESA layers of chitosan
microgel and PAA. In addition, the characteristic band at about 1170 cm-1 corresponding to theprimary alcoholic group of chitosan. The peak at about 1400 cm-1 was assigned to the stretchingvibration of amide band from chitosan appeared in the spectra, especially for cycled 1 and 3, forwhich the surface was terminated with chitosan layer or chitosan microspheres layer. In the cases ofcycle 2 and 4 for surfaces terminated with PAA layer, similar spectra with PAA were observed and it
was believed to be resulted by the coverage of PAA on membrane surface. It was also noted for the
PAN/Chitosan microsphers/PAA system, that the disappearance of the peak of the CN group fromthe charged PAN base membrane was drastic due to the coverage of the ESA layers. Surface
roughness of the chitosan microspheres/PAA membranes were represented in Fig. 8. The surface
roughness of -1 and 0 which referred to membranes before and after NaOH treatment, indicated thatthe membrane surface became rougher due to the existence of hydrophilic carboxyl groups resulting
from NaOH treatment. Here, the -1 symbol means that the PAN membranes treated. It was also4Transactions on GIGAKU 1(2012)01034/1-6

Surface roughness (nm)Adsorption capacity for Cu2+ (10-6g/g)

apparent that the surface morphology was dramatically altered by the formation of the chitosan
microspheres/PAA ESA multilayer. While the corresponding surface roughness values was 8.0, 23.1,
15.0, 39.9, and 36.4 nm for the chitosan microspheres/PAA membranes with cycle layers of 0, 1, 2, 3
and 4, respectively. Therefore, the introduction of the chitosan microspheres for the ESA multilayer
could be effectively for roughing the base of membrane surfaces.

Number of self-assembed layers on membrane surface
Number of ESA layers on membrane surface

Fig. 8 Influence of numbers of self-assembled
layers onto the membrane surface on the surface.

H 2O

Fig. 9 Effect of the copper solution pH on
the adsorption capacity of the resultant
membranes of PAN/chitosan microsphere/PAA
system.

Cu 2+
HO
HOH 2C

HO

O

NH 3

H2 O

H 2O

H 2O

Cu2+
HO
HOH2 C

NH2

O

NH2

O
O


O
CH 2 OH

NH3

COO

O
CH2 OH

COO

Fig. 10 Illustration images of the chelation for Cu2+ by the assembled chitosan on negatively
charged membrane surface.
It is known that chitosan could be applied for adsorption of metal ions since the amino group iscapable of binding metal ions as served as coordination sites. For example, Juang et al. [16] reported
high selectivity for Cu2+ ion was observed by chitosan adsorbents. Chang et al. [17] found that thechitosan-modified microsphere showed the largest equilibrium adsorption capacity at pH 5.5.
Therefore, in the present work, we studied the binding capability for Cu2+ ion of the resultant
membrane at various pHs. Fig. 9 shows the effect of the pH of Cu2+ ion solution on the Cu2+
adsorption of resultant membranes with Chitosan microspheres/PAA multiple-layers. The adsorption
amounts of the Cu2+ ion were plotted at the cycle number for the ESA multilayer formation. The odd
layers were for the chitosan or chitosan microspheres layers exposed on the membrane surface
outmost and evens were for PAA layers on the outmost. It is seen that when the adsorption was
carried out at pH 3, the membranes terminated with PAA layers showed higher adsorption capacity
rather than those with terminal chitosan or chitosan microspheres layers. But, when the pH increased,
the change regulation could not be seen at pH 7 in both (a) and (b) systems. In the case of thechitosan microspheres/PAA system, the values of the adsorption capacity were significantly
decreased to be in the range of about 50 ppm / g-membrane. However, at pH 5 and 6, the values of
the adsorption capacity increased with increasing the cycled number of the ESA operation and then5Transactions on GIGAKU 1(2012)01034/1-6

ranged in about 200-240 ppm/g-membrane. It was thought that the adsorption capacity of theresultant membrane depended on the isoelectric point of chitosan, which was at about 6.3. At below
the pH, the protonation of amine groups of chitosan could occur, while at higher pH the group
behaved as non-protonated group. However, the binding ability of chitosan for copper ions was
mainly due to the amine groups which were served as coordination sites for the sequestration ofcopper ion [18]. At lower pH, most of the amine groups in the chitosan segments was protonated andnot available for copper uptaking by chelation. Thus, the values of the adsorption capacity decreased
with decreasing pH. However, as the protonated chitosan at low pH was able to bind anions byelectrostatic attraction [19], according with Fig. 10.

4. Conclusion
In the present reviews, layer-by-layer electrostatic self-assembly technique was used for the
preparation of functional polymeric membranes with metal ion removal capability. The fabrication
process and properties of the resultant membranes were mentioned by using data of FT-IR spectra indetail. The negatively charged P (AN-co-AA) membrane and positively charged P (AN-co-EDAMA)
were used as the substrates for the ESA treatment. The desalination behavior of the ESA multilayer
modified resultant membranes was studied by evaluating the metal-ion removal properties for Fe3+
and Fe2+. Also, membranes with chelation capability were prepared by the alternate self-assembledof chitosan layer and PAA layer on PAN membrane surface. Taking advantage of the adequate
surface area and a large amount of loading sites for metal ions, chitosan microspheres wereintroduced to use as cationic species for complex formation with Cu2+ ion by the ESA multilayer.
The result of adsorption experimental for Cu(II) showed that the layer-by-layer deposition of
chitosan/PAA or chitosan microspheres/PAA on charged base membrane surface was functionallyequipped it with chelating ability for Cu2+.
Acknowledgment
We thank the supporting of Program for Project supported by forming a Hub for Human
Resources Development and New Industry Creation Building a Sustainable Society Through HighlyInteractive, Cooperative Educational Research with Pacific Rim Countries.
References[1] S. Zapotoczny, M. Golonka, and M. Nowakowska, Macromol. Rapid Commun., 26 (2005) 1049.
[2] D. K. Kweon, S. B. Song, and Y. Y. Park, Biomaterials, 24 (2003)1595.
[3] E. Lojou, and P. Bianco, Langmuir. 20 (2004) 748.
[4] V. Kozlovskaya, S. Ok, S. Sousa, M. Libera, S. A. Sukhishvili, Macromolecules 36 (2003) 8590.
[5] Y. S. Kim, J. Y. Koo, and H. M. Kim, Phys. Rev. Lett., 100 (2008) 256105/1-4.
[6] S. P. Jiang, Z. Liu, and Z. Q. Tian, Adv. Mater., 18 (2006) 1068.
[7] G. Decher, Science, 277 (1997) 1232.
[8] L. Krasemann and B. Tieke, Langmuir, 16 (1999) 287.
[9] T. R. Farhat, and J. B. Schlenoff, J. Am. Chem. Soc., 125 (2003) 4627.
[10] Y. Ding, B. Bikson, and J. K. Nelson, Macromolecules, 35 (2002) 912.
[11] T. Kobayashi, H. T. Fu, Q. Cui, and H. Y. Wang, J. Appl. Polym. Sci., 110 (2008) 3234.
[12] T. Kobayashi, K. Kumagai, Y. Nosaka, H. Miyama, N. Fujii, and H. Tanzawa, J. Appl. Polym.
Sci., 43 (1991)1037.
[13] M. Y. Arica, M. Yilmaz, and G. Bayramoglu, J. Appl. Polym. Sci., 103 (2007) 3084.
[14] D. X. Wang, M. Su, Z. Y. Yu, X. L. Wang, M. Ando, and T. Shintani, Desalination., 175 (2005)
219.
[15] X. L. Wang, T. Tsuru, S. Nakao, and S. Kimura, J. Membr. Sci., 135 (1997) 19.
[16] R. S. Jang, and H. J. Shao, Adsorption, 8 (2002) 71.
[17] Y. C. Chang, S. W. Chang, and D. H. Chen, Reac. Func. Polym., 66 (2006) 335.
[18] L. Dambies, C. Guimon, S. Yiacoumi, and E. Guibai. Colloids Surf. A., 77 (2000) 203.
[19] E. Guibal, Sep. Purif. Technol., 38 (2004) 43.
(Received: 23 March, 2012, Accepted: 27 April, 2012)6