UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTIMPACTS OF HYDROPHILIC POLYSACCHARIDES ON BACTERIALATTACHMENT AND MEMBRANE BIOFOULING
Yosuke Tashiro1, Keitaro Yoshida2, Thithiwat May2 and Satoshi Okabe2
Affiliation 1Shizuoka University, Hamamatsu, Shizuoka 432-8561 Japan,
2
Hokkaido University, Sapporo, Hokkaido 060-8628 Japan
Email: tytasir@ipc.shizuoka.ac.jp (Y. Tashiro)
Membrane bioreactors (MBRs) become important options for wastewater treatment andreclamation. However, membrane fouling hampers the widespread application of MBRs due tosevere flux decline and high-energy consumption, leading to operational and maintenance cost
increase and frequent chemical washes. Membrane fouling is complex and occurs as a result of
accumulation of bacterial cells, extracellular polymeric substances (EPS) and other soluble
microbial products on membrane surfaces and membrane pore clogging. The impacts ofspecific EPS components required for biofilm formation on initial bacterial attachment to MF
membranes and subsequent membrane fouling are not well understood. Therefore, further
studies are obviously required to better understand the interactions between EPS andmembranes.
In this study, we focused on a hydrophilic polysaccharide, which is one of EPS produced byseveral γ-proteobacteria and has unique characteristics of repressing bacterial attachment at the
beginning stage of biofilm formation and developing voluminous three-dimensional biofilms at
the later stage. Here we investigated the effects of the hydrophilic polysaccharide on bioulingon several properties of membrane materials and obtained new insights into the association
between bacterial EPS production and biofouling. We established a novel screening system todetect the bacterial attachment on several kinds of membrane used for the wastewater treatment
and found that the hydrophilic polysaccharide inhibits the attachment on hydrophobic
membranes. Moreover, we built a bench-scale cross-flow filtration system and investigated theeffect of the hydrophilic polysaccheride on the transmembrane pressure, the accumulated
biomass on membrane and the filtration resistance. The results showed that the hydrophilicpolysaccharide causes severe membrane clogging regardless of low biomass on the membrane.
Thus, roles of polysaccharides in biofouling process are complex, and biomass on membraneand biofouling are not coincidental.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTPOPULATION DYNAMICS AND CELL-TO-CELL INTERACTION IN A MODELMICROBIAL ECOSYSTEM
Kenshi Suzuki1, Fatma Azwani2, Yuma Inuzuka3, Yosuke Tashiro1, and Hiroyuki Futamata1
1,3
Shizuoka University, Hamamatsu, 432-8561 Japan,
2
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
f0330226@ipc.shizuoka.ac.jp, f0012019@ipc.shizuoka.ac.jp, tytasir@ipc.shizuoka.ac.jp,
achkawa@ipc.shizuoka.ac.jp, thfutam@ipc.shizuoka.ac.jp

Abstract. Cell-to-cell interactions have a crucial role in microbial ecosystems. Since naturalenvironments have a huge diversity of microorganisms interacting each other, it is too complex
to analyze all things in microbial ecosystems. In this study, mixed chemostat cultures wereconstructed to analyze the dynamics and succession of microbial ecosystem by using threedifferent strains, Pseudomonas sp. C8, Ralstonia sp. P10, and Comamonas testosteroni R2.
Real-time PCR was used to monitor the population densities of three strains. In a mixed culture
containing three strains, they showed equilibrium population densities at early stage. However,C8 and R2 became dominants and P10 was about two orders of magnitude lower than those of
C8 and R2 in the transition to carbon starved condition. The other hand, R2 only dominated in
coculture with C8. Furthermore, P10 and R2 showed equilibrium population densities in theirs
coculture. These results suggested that these strains shared the role in this model ecosystemand some interactions play important roles. To understand bacterial interaction, wecharacterized the substance to affect the growth. A supernatant from a strain used in this study
was filtrated by 0.2 µm membrane and was added (10% [v/v]) in the other culture. These
analyses revealed that the supernatant from C8 (C8-supernatant) repressed significantly thegrowth of other strains. Interestingly, the growth repressed with C8-supernatant was almost
completely recovered by addition of FeCl3. Although the growth of R2 was strongly repressedby C8, R2 became dominant in the model ecosystem without addition of FeCl3. This result
suggested that R2 has another recovering mechanism from the growth repression with C8supernantant. To obtain a clue for revealing this interesting subject metabolomic analysis iscurrently under investigation.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTANALYSIS OF THE GROWTH REPRESSING COMPOUNDS SECRETED FROMPSEUDOMONAS SP.C8
Yuma Inuzuka1, Kenshi Suzuki2, Fatma Azwani3,Yosuke Tashiro2, Hajime Kobori4, Wei Tao
Qui4, Hirokazu Kawagisi4, and,Hiroyuki Futamata2
Affiliation: 1, 2Shizuoka University, Hamamatsu 432-8561 Japan, 3Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, 4Shizuoka University, Shizuoka, 422-8529, Japan
Email: f0012019@ipc.shizuoka.ac.jp, f0330226@ipc.shizuoka.ac.jp
tytasir@ipc.shizuoka.ac.jp, thfutam@ipc.shizuoka.ac.jp
Microbes have been contributed to wide-ranging industries. Although most microbial
industrials are based on mixed microbial communities, it remains poorly understood how
microbes interact there. To maintain efficient microbial activities in microbial industries, it isimportant to understand cell-cell interactions in complex microbial ecosystems. In theprevious study, the interaction of phenol-degrading bacteria including Pseudomonas sp. C8,
Ralstonia sp. P-10 and Comamonas testosteroni R2 was analyzed and we found that thesupernatant of C8 (C8SP) strongly represses the growth of P-10 and R2. The aim of this study
is to understand how they affect complex microbial ecosystems. To analyze the effect of C8SP
on environmental microorganisms, microbes derived from the paddy soil was inoculated to an
inorganic medium with 0.2 mM phenol in the presence and absence of 10%(v/v) C8SP. Whenphenol was completely consumed in the culture, 10%(v/v) culture was transferred to a fresh
medium with or without C8SP respectively, and this step was repeated 15 times. In theanalysis of microbial community dynamics, bacterial community structures were finally stable
at different equilibrium, suggesting that C8SP is useful for controlling microorganism
community structures. Of C8SP fractionated with different molecular weight, the fraction lessthan 5000 Da exhibited the growth repression. C8SP was partitioned with n-hexane-, ethyl
acetate-, n-butanol-, and H2O-soluble components. A growth test using R2 showed that thegrowth repressing compound(s) was in the H2O-soluble components. Interestingly, of the
H2O-soluble components fractionated with different molecular weight, some fractions
exhibited the growth repression of strain R2 but not strain P-10. Identifying growthcontrolling compounds and resolving the mechanism how C8SP affects the growth of
microorganisms would establish a new strategy to control complex microbial communities.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTELECTRONIC FLOW FOR EFFICIENT REDUCTIVE DECHLORINATION OF
TRICHLOROETHENE IN A ENRICHMENT CULTURE
Kenji Suzuki1, Hiroyuki Morioka Naruaki Sato, Teruaki Hanji, Yosuke Tashiro, andHiroyuki FUTAMATA2
1&2
Shizuoka University, Hamamatsu 432-8561 Japan
Affiliation
Email: f0912072@ipc.shizuoka.ac.jp, tytasir@ipc.shizuoka.ac.jp, thfutam@ipc.shizuoka.ac.jp

Abstract. Chlorinated ethenes, such as trichloroethene (TCE), are persistent groundwaterpollutants. Bioremediation strategies for these contaminanted sites are based on reductive
dechlorination. To develop efficient reductive dechlorination, it requires an understanding of
the electron flow in microbial ecosystem. A dechlorinating microbial enrichment culture (LS)was constructed with citrate as electron donor, which converted TCE to ethene via cis-1,2dichloroethene and vinyl chloride (VC). The 5th transfer culture LS completely dechlorinated
TCE to ethene in about 3 weeks. Methane production rate increased after completion ofdechlorination. It took over 3 months to dechlorinate TCE completely in the 6th LS, mainly
because of slow VC-dechlorinating rate. Methane was produced more in the dechlorination
process. Accumulation and decrease of acetate corresponded to methane production in the 5th
and 6th LS. Archaeal communities in 5th LS and 6th LS were compared to clear how methane
was produced. Clone library analyses targeting archaeal 16S rRNA gene revealed thathydrogentrophic methanogen Methanocorpusculm spp. were dominant archaea in the 5th LS,
and aceticlastic methanogen Methanosaeta spp. were dominant in the 6th LS. Real-time PCR
analysis showed similar population densities of Dehalococcoides in both LSs. These results
indicated that the interspecies hydrogen transfer between anaerobic acetate oxidizers andDehalococcoides might be important for efficient dechlorination. Furthermore it wassuggested that there is need to obviate the competition between anaerobic acetate oxidizers andaceticlastic methanogens. The effect of lactate on dechlorination property of 9th LS wasinvestigated, and the result showed that the addition of lactate promotes dechlorination of vinyl
chloride (VC), suggesting that lactate was powerful tools for controlling electronic flow,
resulted in efficient TCE-dechlorination.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTEFFECT OF EXTERNAL RESISTANCE ON THE ELECTRICITY GENERATION
AND MICROBIAL COMMUNITY STRUCTURE IN MICROBIAL FUEL CELLS
Hiroko Kubota, Yutaka Kato, Kei Suzuki, and Hiroyuki Futamata

Shizuoka University, Hamamatsu, Japan, 432-8561
f0012046@ipc.shizuoka.ac.jp, f5345018@ipc.shizuoka.ac.jp, and thfutam@ipc.shizuoka.ac.jp

Abstract Format. Effects of external resistance on microbial ecosystem and electricityproducing properties in microbial fuel cells (MFCs) were investigated. Two kinds of MFCs
were constructed with lake sediment as inoculum, lactate as electron donor and different
external resistances, 10 Ω (LR-MFC) and 1000 Ω (HR-MFC). Polarization curve analyses
revealed the electricity producing potential of these MFCs. By comparing with the potential of
LR-MFC, HR-MFC enabled to decrease and maintain the internal resistance lower, resulted in
the production of higher power density. Cyclic voltammetry analyses revealed that theelectricity producing property of LR-MFC changed from indirect extracellular electron transfer(EET) to direct EET, while direct EET had occurred in HR-MFC. These results indicated thatexoelectrogens-biofilm had developed on the surface of anodes in both MFCs.
Multidimensional scaling (MDS) analyses based on PCR-DGGE profiles revealed thatbacterial community structures of LR- and HR-MFCs were almost similar until day 38, after
which these bacterial community structures were significantly different from each other andwere stable at different dynamic equilibrium with fluctuation. The dynamic equilibrium of
HR-MFC fluctuated less than that of LR-MFC, suggested that selective bias for bacterial
enrichment on the anode of HR-MFC was stronger than that of LR-MFC. Clonal analyses
targeting 16S rRNA gene showed that Geobacter spp. dominated approximate 20% and 32%
of biofilm communities on the anode of LR- and HR-MFCs at day 197, respectively. However,the proportion of Geobacter spp to analyzed clone number decreased 5% and 0% on the anodeof LR- and HR-MFCs at day 427, respectively. On the other hand, Firmicutes dominated 40 to
60% of biofilm communities on the anode of LR- and HR-MFCs at day 427. These results
suggested that higher external resistance is useful for controlling the exoelectrogen ecosystem,
in which Firmicutes instead of Geobacter was selectively enriched on the surface of anode.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTPRODUCING MECHANISM OF RECHARGEABLE BIOMINERAL
Yusuke Chiba, Kei Suzuki, Hiroki Mochihara, Yusuke Tashiro and Hiroyuki Futamata
Shizuoka University, Hamamatsu, Japan, 432-8561
yusuke070809@yahoo.co.jp, f5345018@ipc.shizuoka.ac.jp, h.mochihara@yahoo.co.jp,
tytasir@ipc.shizuoka.ac.jp and thfutam@ipc.shizuoka.ac.jp
Abstract. Microorganisms that were obtained from the biofilm on the surface of anode in an
MFC produced precipitate under anaerobic conditions with lactate as electron donor and ferric
citrate as electron acceptor. Interestingly, the current density was increased approximate 80folds by addition of the precipitate into anode chamber in MFC. Furthermore, electrochemical
analyses demonstrated that the precipitate was one of rechargeable biomineral. Scanningelectron microscope (SEM) and energy dispersive X-ray spectrometry (EDX) analyses
revealed that the shape was flat and polygon and that the biomineral consisted of phosphorus,
iron, calcium, titanium, and oxygen derived from a medium. When the biomineral was
produced, the sulfate was consumed completely. In parallel, the concentration of ferric ion in asupernatant decreased, and the concentration of ferrous ion in the biomineral increased
concomitantly. The biomineral was not produced under the condition of pH 4.7 and the sulfur
in the supernatant was not consumed. The biomineral was produced under the absence ofsulfate but the iron in the biomineral was almost ferric. These results indicated that the
reduced sulfate (sulfide) was required for reduction of ferric. Denaturing gradient gelelectrophoreses analysis was performed to investigate what kinds of microorganisms playmajor role in biomineralization. The results showed that Pelosinus sp. and Desulfovibrio sp.
dominated in the compound-producing culture. These results suggested that sulfate-reducers
and iron-reducers played major role in the biomineralization including chemical reaction.
However, it is unknown that how bacteria produce the biomineral, how the biomineral ischarged and what kinds of chemical reaction occur. These interesting subjects are currently
under investigation.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTDEVELOPMENT OF THE EFFECTIVE WASTEWATER TREATMENT PROCESS BY
CONTROLLING ELECTRONIC FLOW
Takuya Hosokawa1, Yosuke Tashiro2, Hiroyuki Futamata2
1,2
Shizuoka University, Hamamatsu, 432-8561 Japan
f0012116@ipc.shizuoka.ac.jp, tytasir@ipc.shizuoka.ac.jp, thfutam@ipc.shizuoka.ac.jp
Activated sludge process under aerobic conditions is used in wastewater treatment. However,there are some problems, including cost and greenhouse gas generation. Anaerobic wastewater
treatment would be a way to solve these problems by inhibiting excess generation of activatedsludge. But anaerobic wastewater treatment needs higher treatment efficiency under low
concentration of chemical oxygen demand (COD) towards the practical use. To exploit theefficient anaerobic wastewater treatment, microbial fuel cells (MFCs) were constructed to control
electronic flow under anaerobic condition. We prepared 6 reactors containing microbes derived
from paddy soil, and investigated the effect of biominerals (BM), which is formed by microbes
under anaerobic conditions, on electricity generating capacity. COD removal efficiency indicatesthe performance of wastewater treatment, and is important factor for continuously treatment. In
the MFC without BM (Reactor IV), COD remained approximate 1500 mg/L and propionic acidwas accumulated. On the other hands, the MFC with BM (Reactor V) showed that 76% of COD
was removed for 10 days. The amount of coulomb obtained from Reactor V was about 800-folds
higher than Reactor IV. In theory, Reactor V was capable of repressing 36% of the generation ofexcess sludge compared with aerobic condition. These results suggested that the electronic flowin the system changed by adding BM, suggested the potential for effective wastewater treatment
under anaerobic condition.

UPM-SHIZUOKA UNIVERSITY
INTERNATIONAL COLLOQUIUM 2014
March 3rd 2014
Agriculture Hall, Faculty of Agriculture,
Universiti Putra Malaysia

ABSTRACTINTERSPECIFIC CELL-CELL COMMUNICATION WITH MEMBRANE VESICLESSECRETED BY BACTERIA
Yusuke Hasegawa1, Hiroyuki Futamata2 and Yosuke Tashiro2
Affiliation 1Department of Physical Engineering, Faculty of Engineering, Shizuoka University,
Department of Applied Chemistry and Biochemical Engineering, Graduate School ofEngineering, Shizuoka University
Email: f0012105@ipc.shizuoka.ac.jp, thfutam@ipc.shizuoka.ac.jp andtytasir@ipc.shizuoka.ac.jp
Abstract: Many bacteria secrete membrane vesicles (MVs), which are bacterial extracellularparticles in the size of 50-250 nm-diameter and have liposome structures. It has been known thatMVs have a role in the transfer of several signals for the cell-cell communication. However, itremains unknown how they behave among interspecies bacteria. This study aims to understandwhat type of the behavior MVs show in heterogeneous communities and to find out a mechanismof MV fusion to the cell. Here we screened high MV-producing bacterial strains with a highthroughput MV quantitative method and characterized their MVs with the transmitted electron
microscopic (TEM) imaging analysis. Buttiauxella agrestis JCM 1090T, which was the highest
MV-producing bacterium tested in our experiments, was used as a model MV donor and theattachment of MVs on bacterial cells was investigated by flow cytometry and TEM imaging
analysis. The result showed that MVs derived from B. agrestis JCM 1090T easily attach to own
cells as compared to other bacterial cells, suggesting that B. agrestis JCM 1090T MVs are used as
an intraspecies communication tool rather than interspecies. Here we also established a method ofanalyzing the interaction between MVs and cells. It will hold the promise of building a system, inwhich MVs convey useful materials to target cell. The final goal of this study is to control
specific cells in heterogeneous communities with bacterial MVs.