# HALOPHYTE-ASSOCIATED PLANT GROWTH PROMOTING BACTERIA: KEY TOOL FOR LAND RECLAMATION AND RESTORATION

Francesca Mapelli<fnref n="1" />, Ramona Marasco<fnref n="1" />, Eleonora Rolli<fnref n="1" />, Marta Barbato<fnref n="1" />, Amel Guesmi<fnref n="2" />, Hanene Cherif<fnref n="2" />, Imene Ouzari<fnref n="2" />, Daniele Daffonchio<fnref n="1" />, Sara Borin<fnref n="1" />

Corresponding author email: francesca.mapelli@unimi.it

## ABSTRACT

Plant Growth Promoting (PGP) bacteria are known for their role in enhancing plant fitness both in conventional and extreme environments. PGP bacteria can directly and indirectly boost plant growth by providing nutrients and metabolites and by reducing the incidence of pathogenic attacks. Soil salinization and drought are among the most severe environmental stressors affecting crop production around the world. High salt content in the soil is a typical feature of arid and semiarid regions, hosting different halophyte species including those belonging to the genus *Salicornia*. Halophytes comprise several plant species of emerging interest as source of valuable products and services, including phyto-remediation and reclamation technologies. One of the most promising and sustainable approach to improve plant growth and production in salt and drought affected lands is represented by the selection of PGP bacteria able to cope with the extreme geochemical conditions of saline soils.

In this perspective we investigated by cultivation dependent methods and molecular fingerprinting the microbiome composition associated to *Salicornia rhizosphere* and bulk soils collected from Sebkhet and Chott hypersaline ecosystems in Central Tunisia. Denaturing Gradient Gel Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) fingerprinting were applied to depict the overall bacterial community structure of the hypersaline soils, displaying the occurrence of a rich and highly diverse microbiome associated to the *Salicornia* plants roots. A large collection of bacteria was established from bulk soils and *Salicornia* rhizospheres. Plate counts showed that culturable halophilic/halotolerant bacteria were abundant in the bulk soil as well as in the rhizosphere of *Salicornia* specimens. The PGP traits of the halophilic/halotolerant bacteria collection were assessed in vitro in presence of 5% of sodium chloride, to simulate those environmental condition that likely hamper the efficacy of PGP inocula selected from conventional ecosystems. Tolerance to abiotic stresses typical of desertic and semi-desertic areas, such as extreme temperature, salt concentration and low water availability, was widespread within the bacteria collection and represented a common trait in strains belonging to different phylogenetic groups. A high number of isolates showed the ability to influence the nutrients and hormonal balance in vitro at high salinity value. In addition, some strains expressed protease activity, potentially correlated to biocontrol capacity against phytopathogens. In particular, the screening allowed the identification of several strains resistant to the tested abiotic stresses and performing different PGP activities, including ammonia and indol-3-acetic acid production and phosphate solubilization. One of the best promising isolates, *Halomonas elongata* BDV11S17A, was successfully gfp-labelled and used to demonstrate its ability to colonize *Salicornia* roots under laboratory conditions. Furthermore, in vivo test were performed on a subset of bacteria, identifying strains able to sustain the growth of *Salicornia* plantlets.

The results confirmed that halophytes are valuable source of PGP bacteria. The halophilic/halotolerant strains isolated in the present study could be exploited to setup specifically designed biofertilizer to sustain plant growth in soils impacted by salinity and drought stresses in a sustainable perspective. According to the climate change scenario and the future extension of salt affected lands this powerful approach should be carefully considered for a successful land restoration through phytoremediation and to reclaim land traditionally considered unsuitable for agriculture.

**Acknowledgements:** The financial support by FP-7 EU project BIODESERT Grant agreement no. 245746 is greatly appreciated.

<figure><img src="image_300.png" /></figure>

<footnote n="1">¹ Università degli Studi di Milano, DeFENS, Via Celoria 2, 20133 Milano, Italy</footnote>

<footnote n="2">² Laboratory of Microorganisms and Active Biomolecules, University of Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia</footnote>
# ISOLATION AND CHARACTERIZATION OF PLANT GROWTH-PROMOTING RHIZOBACTERIA AND THEIR EFFECTS ON PHYTOREMEDIATION OF PETROLEUM-CONTAMINATED SALINE-ALKALI SOIL

Wuxing Liu<fnref n="1" />, Jinyu Hou<fnref n="1" />, Qingling Wang<fnref n="1" />, Linlin Ding<fnref n="1" />, Yongming Luo<fnref n="1" />,<fnref n="2" />, Caixian Tang<fnref n="3" />

Corresponding author email: [liuwuxin@issas.ac.cn](mailto:liuwuxin@issas.ac.cn)

## ABSTRACT

**Background:** Contamination of soil environment by petroleum hydrocarbons is becoming prevalent across the globe. Phytoremediation is a green technology that uses plants to remediate contaminated soils. Our previous study showed that growing tall fescue significantly increased microbial activity and the number of TPH-degraders of the soil. In additional, inoculating with Plant growth-promoting rhizobacteria (PGPR) may facilitate plant growth and development both directly and indirectly. Though earlier work has involved isolation of salt-tolerant rhizobacteria from halophytic environments, little is known about their tolerance to alkaline environments where the soils have high pH.

**Aims:** The objectives of the present study were: (1) to isolate and characterize efficient ACC deaminase producing PGPR from the rhizosphere of tall fescue grown in saline-alkali soils, (2) to evaluate other PGP activities of the most promising ACC deaminase producing isolates under various saline-alkaline stresses, and (3) to study the effect of the selected isolates on tall fescue growth and phytoremediation of a petroleum-contaminated saline-alkaline soil.

**Methods:** PGPR with ACC deaminase activity were isolated by a conventional method. The strain D5A isolated from rhizosphere soil of tall fescue was evaluated for their plant growth-promoting characters and effects of pH and salinity on the growth and IAA production of strain D5A. Different levels of Na₂CO₃ and NaHCO₃ stress on the growth of strain D5A was tested. A pot experiment was then conducted to study the PGPR assisted phytoremediation of petroleum contaminated soil.

**Results:** A total of 115 PGPR strains were isolated from the rhizosphere of tall fescue grown in petroleum-contaminated saline-alkaline soils. Of these, 5 strains shown ACC deaminase activity >1.0 M α-KB mg-1 h-1 were selected for further study and the isolate D5A presented the highest plant-growth-promoting activity and was identified as *Klebsiella* sp. It grew well on the LB medium containing 9% NaCl and at a pH range of 4-10. The inoculation of D5A significantly improved seed germination, biomass production and chlorophyll contents in leaves of tall fescue grown in the petroleum-contaminated saline-alkaline soil. Shoot dry weight was almost 3 times greater in the inoculated than non-inoculated treatment by the end of the experiment. The inoculation also increased root dry weight, root activity and chlorophyll contents by 73%, 101% and 170%, respectively. The TPH contents in the soil after 120 d decreased by 42%, 50% and 66% in the non-plant control, tall fescue and tall fescue + D5A treatments, respectively.

**Conclusions:** The isolate D5A showed the highest PGP activities and growth-promoting ability. It also had a good adaptability to extreme pH and high salinity and alkalinity level. The D5A was identified as Klebsiella sp. The inoculation of this isolate enhanced the phytoremediation efficiency by further 16% as well as promoted the growth of host plants in the petroleum-contaminated saline-alkaline soil. Thus, the D5A can be used as a potential inoculum to improve phytoremediation of the organic contaminants in petroleum-contaminated saline-alkaline soils. Further studies are needed to validate the results in multiple field sites.

**Acknowledgements:** We thank the National Natural Science Foundation of China (41001182), Jiangsu Provincial Natural Science Foundation of China (BK2012891) and National High Technology Research and Development Program of China (863 Program) (2012AA06A204) for financial support.

<figure><img src="image_301.png" /></figure>

<footnote n="1">¹Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China</footnote>

<footnote n="2">²Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China</footnote>

<footnote n="3">³AgriBio - Centre for AbriBioscience, La Trobe University, Melbourne Campus, Bundoora, Victoria 3086, Australia</footnote>
# PHYTOREMEDIATION OF SALT-AFFECTED SOILS USING HALOPHILIC BACTERIA

Muhammad Jamil<fnref n="1" />, Gulmeena Shah<fnref n="1" />, Tayybah Naz<fnref n="1" />, Salma perveen<fnref n="1" />, Ijaz Malook<fnref n="1" />, Eui Shik Rha<fnref n="2" />

Corresponding author email: dr.jamil@kust.edu.pk

## ABSTRACT

Salinity is a most important ecological constraint for cereal crop including rice nowadays. Phytoremediation have been used to remediate salt affected soil but the use of halophilic bacteria is limited. In this study, the salt resistant *Thalassobacillus denorans* (NCCP-58) and *Oceanobacillus kapialis* (NCCP-76), halophilic bacteria isolated from salt mines near Karak, Pakistan were used to evaluate its effect on rice variety (Basmati-385) in soil contaminated with different concentrations (control, 50, 100 and 150 mM) of NaCl. It has been observed that significant increase in germination percentage and germination rate occurred in seeds primed with bacterial strains as compared to non primed seeds. Root and shoot length was more in plants raised from primed seeds than non treated seeds. Plants raised from inoculated seeds showed a significant increase in fresh and dry weight of seedling after 15 days. Significant increase in photosynthetic pigment; chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid contents occurred in 28 days old plants inoculated with bacterial strains under different saline condition. Similarly significant increase occurred in total nitrogen and protein contents in inoculated plants. Ca⁺<fnref n="2" /> and K⁺ ion concentration significantly increased while Na⁺ ion concentration decreased in plants inoculated with bacterial strains as compared to non inoculated plants under different saline condition. Bacterial strains *Oceanobacillus Kapialis* (NCCP-76) was more responsive in term of physiological and biochemical parameters than *Thalassobacillus denounce Sp.* (NCCP-58). The corresponding strains have a positive effect in alleviating the salt stress in plants growing in saline condition.

<figure><img src="image_302.png" /></figure>

<footnote n="1">¹ Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology (KUST) Kohat 26000, Pakistan</footnote>

<footnote n="2">² Department of Well-being Resources, College of Agriculture and Life Science, Sunchon National University, Suncheon, Korea</footnote>
EFFICIENCY OF PSEUDOMONAS PUTIDA MTCC 4391 TO ENHANCE ARSENIC
REMEDIATION BY DIFFERENT PLANT SPECIES

Anshita Raj<fnref n="1" />, Shubhi Srivastava<fnref n="2" />, Nandita Singh<fnref n="1" />

Corresponding author email: anshitaraj_23@yahoo.co.in

ABSTRACT

A study was conducted to investigate the effect of arsenic tolerant bacteria *Psuedomonas putida*, having plant growth promoting characteristics, on growth and arsenic uptake by *P. vittata*, *A. capillus veneris*, *C. dentata* and *P. karka*. This arsenic tolerant bacterium mobilised arsenic from the treated soil and enhanced the arsenic uptake in the plant species. It was found, that soil inoculation of this bacterium significantly enhanced plant biomass and arsenic uptake from root to shoot. The present study also aimed to assess the role of arsenate reductase in detoxification of arsenic in the plant varieties. The results showed that due to increased accumulation of arsenic in plants by the inoculation of bacteria, arsenate reductase activity was also increased in the plant species. The results of the study denote that the arsenate tolerant bacteria have potential to enhance phytoremediation of arsenic-contaminated soils by plant species (*P. vittata*, *A. capillus veneris*, *C. dentata* and *P. karka*).

Acknowledgements: The authors are grateful to the Director, CSIR-NBRI for keen interest and continuous encouragement to conduct the experiments. AR thankfully acknowledges CSIR for awarding SRF grant.

<figure><img src="image_303.png" /></figure>

<footnote n="1">¹Eco-auditing Group, National Botanical Research Institute, Rana Pratap Marg, Lucknow- 226001, India</footnote>

<footnote n="2">²Microbiology Lab, Department of Botany, University of Calcutta, Kolkata 700019, India</footnote>
# EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI SPECIE ON MAIZE GROWTH AND HEAVY METALS UPTAKE

Ahmed Elgharably<fnref n="1" />, Nivien Allam<fnref n="2" />, Galal Elgharably<fnref n="1" />

Corresponding author email: age@au.edu.eg

## ABSTRACT

Consumption of plants accumulating heavy metals is a health risk for humans and animals. Several studies reported that arbuscular mycorrhizal fungi (AMF) have a significant effect on plant growth and can reduce plant uptake of heavy metals. However, promotion of heavy metals uptake in soils inoculated with AMF has been shown. Two different species of AMF (*Glomus geosporum*; Gg and *Glomus intraradices*; Gi) were isolated from heavy metals contaminated soils. Species of AMF might have different effects on plant growth and heavy metals uptake.

This study was conducted to elucidate the potential of AM fungi Ga and Gi on maize growth and heavy metals uptake.

Soil was collected from the area of El-Madabegh, Assiut, soils treated with wastewater over 50 years. Soil (3 kg), was placed into pots. Fifty gram of AMF inoculum of Gg, or Gi was placed below seeds of maize. A control soil (no AFM) was included. The soil was treated with 120 and 30 µg N-KNO₃ and P-KH₂PO₄ g⁻<fnref n="1" /> soil and irrigated to the field capacity throughout the experiment. After 45 days of growth, maize was harvested, and the shoot dry matter (DM) and content of Cd, Pb, Fe and Zn were determined.

Inoculation with AMF increased the dry weights of shoot and root with no significant difference between Gg and Gi treatments. Inoculation with Gi and Gg mycorrhizal fungi resulted in accumulation of higher concentrations of heavy metals in the maize roots than in the shoot.

With no AM inoculum, Pb, Cd, Fe and Zn concentrations in the shoot were approximately 67, 0.06, 370 and 197 µg and in the root were approximately 57, 0.37, 3197 and 232.9 µg g⁻<fnref n="1" /> DM, respectively. With Gg and Gi inoculation, Pb concentration in the shoot decreased by approximately 5 and 32 and that of Zn decreased by 9 and 28%, respectively. Inoculation with Gi resulted in lower Cd concentration in the shoot than in the root; the opposite is true for Gg inoculum. Shoot Fe concentration was optimal, but lower in the Gg compared to the Gi inoculated soil. In the root, compared to Gi, Gg inoculation resulted in higher Fe concentration.

AMF have a significant effect on maize growth in heavy metals contaminated soils, but maize uptake of heavy metals is dependent on AMF specie.

*Acknowledgements:* The financial support by Science and Technology Development Fund (STDF) project Heavy metals pollution and remediation in soils of Assiut, Egypt contract No. STDF 2153 is greatly appreciated.

<figure><img src="image_304.png" /></figure>

<footnote n="1">¹Department of Soils and Water, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt</footnote>

<footnote n="2">²Department of Botany and Microbiology, Faculty of Sciences, Assiut University, Assiut 71526, Egypt</footnote>
# MICROBE ASSISTED PHYTOREMEDIATION TECHNOLOGY FOR ENHANCED REMEDIATION OF OIL SLUDGE CONTAMINATED SOILS

**Sneha V. Nanekar**, Asha A. Juwarkar

Eco-System Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nehru Marg, Nagpur, 440020 India. +91 0712 2249764 (Tel), +91 0712 2249900 (Fax)

Corresponding author email: aa_juwarkar@neeri.res.in; juwarkar@gmail.com

## ABSTRACT

Oil sludge contamination due to increased use of petroleum products as an effect of increased urbanization and industrialization has emerged as a principal problem of concern. To improve the quality of contaminated soils/sites an eco-friendly option i.e. microbe assisted phytoremediation technology (MAPT) was adopted for remediation of the contaminated soil. An investigation was undertaken at CSIR-NEERI, Nagpur premises over a period of 180 days with an objective of remediating oil sludge contaminated soil using plant (Vetiveria zizaniodes), bulking agent (wheat husk), microbial consortium and nutrients. Oil sludge collected from oil refinery, was diluted to 1:1(wt/wt) ratio with soil and laid as top layer in eight plots of size 1m² over 15m² area. The overall microbial population was assessed by monitoring dehydrogenase activity and TPH degradation was estimated on gravimetric basis. Polyaromatic hydrocarbons were quantified on GC-MS. Plant analysis included the measurement of root and shoot length after 180 days. The results showed 85.21% degradation of TPH component in bulked treatment with consortium, plant and nutrients (T3[b]). TPH degradation in bioaugmented treatments with and without nutrients was 70.2% and 74.62% respectively as compared to control (36.38%). Furthermore, the dehydrogenase activity was found to increase initially till 4 months after which it remained more or less constant and finally was found to decrease slightly at the end of the study in all the treatments with treatment T3[b] showing highest activity. Also, root length and shoot length of vetiver grass increased by 88.04% and 90.74% respectively in T3[b] as compared to 73% and 85% respectively in treatment with plant and oil sludge only. It can be concluded that vetiver can serve as a promising plant for remediation of oil sludge contaminated soils owing to its tolerance to toxic contaminant. Also, the combination of amendments such as plant, bulking agent, microbial consortium and nutrients together can be taken as a solution which is both economic and eco-friendly to decontaminate/remediate oil sludge contaminated soils.

**Keywords:** MAPT, contamination, oil sludge, TPH, remediation, amendments

<figure><img src="image_305.png" /></figure>
# IMPROVEMENT OF GROWTH OF *HELIANTHUS TUBEROSUS* L. BY SOIL AND ROOT ENDOPHYTIC BACTERIA ON A Cd-Zn CONTAMINATED SOIL

**Blanca Montalbán**<fnref n="1" />, **Mª Carmen Lobo**<fnref n="1" />, **Sarah Croes**<fnref n="2" />, **Nele Weyens**<fnref n="2" />, **Jaco Vangronsveld**<fnref n="2" />, **Araceli Pérez-Sanz**<fnref n="1" />

Corresponding author email: [blanca.montalban@madrid.org](mailto:blanca.montalban@madrid.org)

## ABSTRACT

The utilization of high-biomass crops in phytotechnologies can help to stabilize or remove toxic metals in soils as well as produce a renewable energy resource on marginal lands that can no longer be used for food and feed production [1]. Some energy crops are effective in removing metals from soil, but show reduced biomass in the presence of toxic concentrations of metals. The interaction between plants and beneficial bacteria can enhance biomass production and metal tolerance of the plants. Some plant growth-promoting bacteria (PGPB) can improve biomass by different mechanisms such as nitrogen fixation, nutrient solubilization or production of phytohormones and siderophores. These PGPB can also reduce the metal-induced stress in plants, by decreasing the plant ethylene levels through 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity [2]. *Helianthus tuberosus* L. is an energy crop often used as bio-ethanol resource. Recent studies have shown the tolerance of this crop to toxic metals, particularly to Cd [3]. The aim of this work is to evaluate the effects of bacteria on the growth and metal uptake of *H. tuberosus* in the presence of Cd and Zn.

Cultivable bacteria were isolated from soil, rhizosphere, root and stem of *Brassica napus* growing on a Cd-Zn polluted soil in Belgium. These bacteria were tested for their metal resistance and potential plant growth-promoting characteristics (nitrogen fixation, phosphate solubilization, ACC deaminase activity, production of siderophores, organic acids, acetoin and indole acetic acid). Three Zn-resistant strains and four Cd-resistant strains were selected to inoculate roots of *H. tuberosus*, according to the best characteristics showed in plant growth-promoting tests. Tubers of two cultivars of *H. tuberosus* (VR and D19) were collected from an experimental field (IMIDRA, Madrid, Spain), transferred to plastic pots filled with quartz sand and watered with ½ diluted Hoagland's solution spiked with 0.1mM of Cd (CdSO₄·8H₂O) or 1mM of Zn (ZnSO₄·7H₂O). Control plants were fertilized with nutrient solution. Bacterial strains were grown in 869 liquid medium at 30°C. The bacterial suspension (10⁸ cfu mL⁻<fnref n="1" />) was sprayed into the pots after the appearance of the first roots. After three weeks of growing, plants were harvested. Fresh and dry weight, malondialdehyde (MDA) content and metal uptake were evaluated. In general, the effect of the bacterial strains on the growth of *H. tuberosus* was different depending on the metal and cultivar. In presence of Zn, the inoculation of *Pseudomonas* sp. 228 and 256 strains significantly increased the aerial biomass of both cultivars and decreased the MDA content in roots, with respect to non-inoculated plants. Regarding Cd, *Pseudomonas* sp. 262 and *Serratia* sp. 246 significantly increased the aerial biomass of VR cultivar and D19 cultivar, respectively. The inoculation of these bacterial strains could enhance biomass production of this crop in presence of Cd or Zn, improving the efficiency of the phytotechnology. Experiments on field soils are performed in order to evaluate the bacterial strains effects on the growth of *H. tuberosus* when metals and nutrients are less available and there is competence with endemic soil microorganisms.

## References:

1. Witters N., Mendelsohn R. O., Van Slycken S., Weyens N., Schreurs E., Meers E., Tack F., Carleer R., Vangronsveld J. (2012) Phytoremediation, a sustainable remediation technology? Conclusions from a case study. *I: Energy production and carbon dioxide abatement*. *Biomass and bioenergy* 39, 454-469.

2. Glick B. R. (2010) Using soil bacteria to facilitate phytoremediation. *Biotechnology Advances* 28, 367-374.

3. Long X., Ni N., Wang L., Wang X., Wang J., Zhang Z., Zed R., Liu Z., Shao H. (2013) Phytoremediation of Cadmium-Contaminated Soil by Two Jerusalem Artichoke (*Helianthus tuberosus* L.) Genotypes. *Clean – Soil, Air, Water* 41 (2), 202-209.

**Acknowledgements:** This work has been supported by EIADES PROGRAM S2009/AMB-1478, Centre for Environmental Sciences of Hasselt University and the predoctoral grant FPI-INIA2010 of Montalbán B.

<figure><img src="image_306.png" /></figure>

<footnote n="1">¹ Dept of Agro-environmental Research, IMIDRA, Spain</footnote>

<footnote n="2">² Centre for Environmental Sciences, Hasselt University, Belgium</footnote>
# POPLARS HOST DIFFERENT COMMUNITIES OF FUNGAL ENDOPHYTES IN HEAVY-METAL POLLUTED AND UNPOLLUTED SOILS

Christoph S. Schmidt, Libor Mrnka, Tomáš Frantík, Miroslav Vosátka

Department of Mycorrhizal Symbioses, Institute of Botany ASCR, Pruhonice, Czech Republic

Corresponding author email: christoph.schmidt@ibot.cas.cz

## ABSTRACT

The potential of endophytic fungi to enhance poplar growth and phytoremediation efficiency is a yet untapped resource. We investigated communities of fungal endophytes in *Populus maximowiczii* × *P. nigra* clone Max-4 at heavy metal polluted and unpolluted soils with a culture-dependent approach. Root and leaf endophytes were isolated from surface-sterilized poplar tissues using nutrient rich (2% MEA) and nutrient poor (SNA) media. The fungal isolates were grouped into morphotypes based on their colony morphology and growth characteristics.

The heavy metal-polluted site (Nové Podlesí, Czech Republic) displayed lower diversity and abundance of poplar endophytes than the unpolluted site (Průhonice, Czech Republic). Only 56 fungal endophytes belonging to 16 morphotypes could be isolated from the poplars grown at the polluted site whereas we were able to isolate 120 fungal endophytes of 33 different morphotypes from poplars growing at the unpolluted site. Isolated endophytes exhibit a typical long tail abundance with a higher diversity of rare morphotypes while the majority of isolates belong to few dominant morphotypes. The communities from the polluted and the unpolluted site appear to overlap only to a limited extent which is yet to be confirmed by molecular means. The obtained isolates will be molecularly identified by their ITS sequences and tested for their potential to improve growth of poplars and to enhance phytoextraction of heavy metals.

**Acknowledgements:** The financial support by Technological Agency of the Czech Republic, project Improvement of phytoremediation capacity and production potential of energy crops grown in contaminated and poor soils by means of endophytic and mycorrhizal symbionts contract No. TA03011184, is greatly appreciated.

<figure><img src="image_307.png" /></figure>
# REDUCTION OF Cr(VI) To Cr(III) BY THE ENDOPHYTIC BACTERIA OF THE HALOPHYTE *JUNCUS ACUTUS* L.

Helen Dimitroula, Evdokia Syranidou, Eleni Manousaki, Nicolas Kalogerakis

School of Environmental Engineering, Technical University of Crete, Greece

Corresponding author email: nicolas.kalogerakis@enveng.tuc.gr

## ABSTRACT

The idea of exploiting endophytic bacteria in phytoremediation strategies is becoming increasingly popular because endophytes can affect beneficial plant growth and contribute to a higher heavy metal tolerance. Many endophytes have been found to be equipped with various heavy metals tolerance mechanisms while they are able to take advantage of the specific niche provided by the plants. The microbial reduction of Cr(VI) to Cr(III) takes place intra-cellularly and has been demonstrated as a useful process. Therefore, the potential contribution of endophytic bacteria to plant detoxification from Cr(VI) deserves further investigation for phytoremediation applications to heavy metal contaminated environments.

This work is a part of EU's LIFE-ENVIRONMENT program where among other mitigation technologies, CWs with halophytic plants are being tested for Cr(VI) polluted groundwater remediation. More particular, the removal of Cr(VI) from polluted groundwater by constructed wetland planted with the halophytic plant *Juncus acutus* was investigated and the results reviled that the plant is a Cr(VI) tolerant and able to rhizofiltrate Cr(VI) with high efficiencies. As a second step the responsible mechanism for plant ability to accumulate and tolerate high concentrations of hexavalent Cr was examined through the investigation of the contribution role of plant's endophytic bacteria. For that purpose, Cr(VI) resistant endophytic bacteria were isolated from the halophyte and four isolated high Cr(VI) resistant strains were examined for their Cr(VI) reduction potential.

Resistance tests reviled that isolates from the leaf and the root were able to grow at high concentrations of hexavalent chromium. More specifically, the results of these experiments showed that six leaf isolates were able to tolerate up to 10 mg/L of Cr(VI) and two of them able to tolerate up to 100 mg/L of Cr(VI) (*Acidovorax* sp. strain U3 and *Ralstonia* sp. strain U36). Moreover, nine root isolates were found able to tolerate very high Cr(VI) concentrations (50 mg/L and 500 mg/L).

Furthermore, subsequent experiments were performed to test resistant strains ability to reduce Cr(VI) with two leaf (*Acidovorax* sp. strain U3 and *Ralstonia* sp. strain U36) and two root isolates (*Pseudomonas* sp. strain R16 and *Ochrobactrum* sp. strain R24). The data showed that all tested strains were found able to reduce Cr(VI) to Cr(III). For example, *Pseudomonas* sp. strain R16 was found able to completely reduce 100 mg/L of Cr(VI) to Cr(III) after 150 hours (figure).

<figure><img src="image_308.png" /><figcaption>Figure. Cr(VI) reduction and growth curves of the root endophytic bacterium *Pseudomonas* sp. strain R16 at 100 mg/L Cr(VI) initial concentration. Error bars correspond to standard deviation.</figcaption></figure>

**Acknowledgements:** This work was co-funded by the European Union in the LIFE10 ENV/GR/000601 project: CHARM - Chromium in Asopos Groundwater System: Remediation Technologies and Measures.

<figure><img src="image_309.png" /></figure>
# IMPROVING PHYTOREMEDIATION OF CHLORENDIC ACID BY EXPLOITING PLANT-ASSOCIATED BACTERIA

Inge Jambon, Nele Weyens, Robert Carleer, Jaco Vangronsveld

Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium

Corresponding author email: inge.jambon@uhasselt.be

## ABSTRACT

The possibility to remediate an industrial site contaminated with chlorendic acid, a fire retardant and expected carcinogen, by applying phytoremediation using poplar (Populus deltoides x (trichocarpa x deltoides) cv. Grimminge) is investigated. Phytoremediation of organic pollutants is however often inefficient because most plants are not able to completely metabolize xenobiotic compounds, potentially resulting in new environmental problems, and eventually evapotranspiration of unmetabolized, volatile, toxic compounds. Enrichment with degrading plant-associated bacteria can overcome this problem, since bacteria can metabolize organic components to a greater extent, thus leading to an improved contaminant-degradation. Moreover, plant-associated bacteria can promote plant growth, thereby enhancing the efficiency of phytoremediation even more.

To find suitable bacteria for enrichment during phytoremediation of chlorendic acid, the cultivable plant-associated bacterial community of the chlorendic acid-contaminated site was isolated and genotypically characterized by sequencing of the 16S rDNA. The greatest diversity was found in the shoots with 9 different genera isolated, followed by 6 genera in the soil, 4 in the rhizosphere and 3 in the roots. *Bacillus* sp. is the only genus that could be found in all 4 compartments. Subsequently, the tolerance of the isolated strains to chlorendic acid was tested which resulted in 34 of the 100 isolated strains being tolerant to 50 mg l⁻¹ chlorendic acid. 33 of these strains even showed a tolerance to 500 mg l⁻¹ chlorendic acid. The degradation capacity of the tolerant strains is explored.

Meanwhile the isolated strains are screened for different plant growth-promoting characteristics (e.g. production of the phytohormone indole-3-acetetic acid (IAA), nitrogen fixation, production of organic acids or siderophores which increase the uptake of iron). A consortium of bacteria will be selected, consisting of chlorendic acid-degrading and plant growth-promoting bacteria. The effect of inoculation of poplar with this consortium on the phytoremediation efficiency will be evaluated in a greenhouse experiment.

**Acknowledgements:** This project is funded by the Centre for Environmental Sciences, Hasselt University.

<figure><img src="image_310.png" /></figure>
# MODIFICATION OF MICROORGANISM COMMUNITY IN THE GRASS GROWING IN SOIL CONTAMINATED WITH PAHs

Juliusz Markowski, Marta Frymus, Stanisław W. Gawroński

Laboratory of Basic Research in Horticulture, Department of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Science

Corresponding author email: stanislaw_gawronski@sggw.pl

## ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are toxic to both humans and animals. Their significance increases with increasing number of aromatic rings. The most dangerous are the PAHs containing five and six aromatic rings, such as benzo[a]pyrene, showing a strong carcinogenic properties. They are also degraded very slowly, and therefore can remain in the soil for several years. Microorganism associated with roots of some plants can increase decomposition of PAHs in soil. Application microbial preparations containing the microorganisms capable to degrade PAHs or substance promote growth some microorganisms in soil can improve phytoremediation proces. High content of lignin-degrading enzymes in substrate used for the production oyster mushroom (*Pleurotus ostreatus*) fruit bodies causes that spent mushroom compost (SMC) can be used for bioremediation of soil. Formulation used for biological plant protection such as *Trichoderma harzianum* or effective microorganisms may have also positive impact to degradation PAHs.

The aim of this study was to estimate the effect of several microbiological formulation on composition of soil microorganism in rhizosphere of grasses recommended on the lawns along heavy traffic roads. In the experiment two dominant grass species on urban lawns was used: perennial ryegrass (*Lolium perenne* L. cultivar 'Solen') and red fescue (*Festuca rubra* L. cultivar 'Nimba') The plants were planted in 5 dm³ pots containing a mixture of horticultural substrate with 1% of top soil and 2,5% of sand (pH = 6.8, EC = 486 ms). Substrate was mixed a mixture of four aromatic hydrocarbons: phenanthrene, chrysene, benzo[k]fluoranthene and benzo(g,h,i)perylene (Sigma-Aldrich) in amount 2 mg kg⁻¹ soil dry matter. The experimental factors were [1] endogenous microflora, [2] fungus *Trichoderma harzianum* T22 [3] effective microorganisms (EM) [4] waste from oyster mushroom production (SMC). Soil without plants was used as reference. To improve microorganism growth in every month grass has been cut off, shredded and left in pot. With the nested-PCR reaction amplified 16S rDNA region which were then separated by electrophoresis in a denaturing gradient gel electrophoresis (DGGE, Dcode Universal Mutation Detection System, BIO-RAD). Cut from the gel bands were immersed in deionized water and then re-PCR reactions were performed. The resulting products were sent for sequencing. Obtained sequences were compared with BLAST database.

Most of sequences have been identified as uncultured bacterial clone. The impact of all substances and preparations used was more pronounced in samples from the rhizosphere of grasses than in pots without plants. Inoculation *Trichoderma* contributed to reduction of bacterial diversity in soil, only one band has been identified as uncultured delta proteobacteria clone. Results suggest that the addition of *Trichoderma* contributes to stimulate growth of a specific bacterial population shifting the microbial community. Addition of EM and SMS to soil led to increase microbiological diversity. In combination with the SMS identified species was *Bacillus subtilis*. DGGE is a good method that allows the assessment of changes in the microbial population but because of the large variation in soil identification of the individual species is difficult. Assessment of these changes on the efficiency bioremediation process requires further studies.

**Acknowledgements:** The work was financially supported by Warsaw Plant Health Initiative of REGPOT in the frame of 7FP EU # 286093.

<figure><img src="image_311.png" /></figure>
# RHIZODEGRADATION OF PHENANTHRENE BY A BACTERIAL STRAIN ISOLATED FROM AND REINOCULATED INTO THE RHIZOSPERE OF A HALOPHYTE SESUVIUM PORTULACASTRUM LINN.

Fang Yang, Yuanrong Luo, Lingfeng Huang

Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361005, China

Corresponding author email: huanglf@xmu.edu.cn

## ABSTRACT

*Sesuvium portulacastrum* Linn., a halophyte has been used in constructed eco-floating bed system (CEFBS) for phytoremediation of polluted water in Yundang Lagoon (a saline lagoon in Xiamen City, southeast China). A bacterium strain (R3) which was identified as *Novosphingobium resinovorum*, with the capability to degrade 82.94% phenanthrene (initial conc. 100 ppm) within 7 days, was isolated from the rhizosphere of *S. portulacastrum* planted on the CEFBS in Yundang Lagoon.

To investigate the rhizodegradation capacity of *S. portulacastrum* on PAHs with R3 reintroduced into the plant root system, a well designed microcosm approach was carried out, in which 4 treatments (i.e., indigenous rhizospore bacteria-removed plant, CK; indigenous rhizospore bacteria-removed plant with R3, S+R3; indigenous rhizospore bacteria-unremoved plant, NS; indigenous rhizospore bacteria-unremoved plant with R3, NS+R3; ) with 3 replicates were set. The result showed that the phenanthrene (initial conc. 20 ppm) reduction ratio in each treatment was 18.9%, 82.2%, 34.3% and 87.8% for CK, S+R3, NS and NS+R3, respectively. PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) and ETSA (Electron transport system activity) analysis indicated that R3 dominated the rhizosphere bacterial community and played the most important role in the process of phenanthrene rhizodegradation.

<figure><img src="image_312.png" /></figure>

Although the metabolism of PAHs rhizodegradation hasn't been uncovered, our study suggested that co-metabolism may be a plant-microbe interaction important to rhizoremediation.

**Acknowledgements:** The financial supports by Chinese Public Science & Technology Research Funds Project of Ocean (contract No. 201205009) and Science & Technology Research and Develop Funds Project of Shenzhen City (contract No. JCYJ20120830153030587) are greatly appreciated.

<figure><img src="image_313.png" /></figure>
# SELENIUM PHYTOTOXICITY IN CORN (ZEA MAYS L.): POTENTIAL ROLE OF MICROBES TO ALLEVIATE Se TOXICITY IN CEREAL CROP

## Muhammad Yasin, Muhammad Faisal

Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan

Corresponding author email: mohdfaysal@yahoo.com

## ABSTRACT

Selenium is an essential micronutrient element for bacteria, animals, humans and for some lower plants. Although, Se is not consider essential for growth of higher plant but it can be toxic for plants at higher concentrations. In this study, we tested phytotoxic effects of elevated levels of Se (3 mg kg⁻¹ of soil) on corn plant growth, biomass, soluble protein and enzymatic content. Microbial community in rhizosphere of plant can play an important role to tolerate the environmental stresses. Plants were inoculated with Se resistant bacterial strains YAK-1 (Genbankacc # JX203248, showed 99% homology to *Bacillus foraminis* strain) to observe the effects of inoculation on plant growth as wellas on Se toxicity. Such higher concentration of Se in soil caused significant reduction in plant shoot length (33%) and leaf soluble protein contents (22%), and significant increase in leaf acid phosphatase activity (30%) compared to control plants. Strain YAK-1 exhibited high resistance against Se (up to 20 mg sodium selenite mL⁻¹ of LB media) and showed following plant growth promoting characteristics; mixed organic acids production, ammonia production and hydrogen cyanide (HCN) production. In the absence of Se, bacterial inoculation resulted in increases in plant shoot length, plant dry weight, leaf acid phosphatase activity and soluble protein content.When Se-treated plants were co-cultivated in the presence of bacteria, plant shoot length increased significantly compared to only Se-treated plants, but it was not more than control plants. High concentration of Se in soil showed toxic effects on corn growth and inoculation with Se resistant rhizospheric bacteria can possibly alleviate Se toxicity in corn.

**Keywords:** bacteria, selenium, heavy metal resistance, plant-microbe interaction

<figure><img src="image_314.png" /></figure>
<figure><img src="image_315.png" /></figure>
<figure><img src="image_316.png" /></figure>

ECOSYSTEM SERVICES & ECORESTORATION
<figure><img src="image_317.png" /></figure>
# SOIL ECOSYSTEM SERVICES FOR THE EVALUATION OF A METAL PHYTOSTABILIZATION PROCESS ASSISTED WITH PGPBs

Aritz Burges<fnref n="1" />, Garazi Benito<fnref n="1" />, Fernando Blanco<fnref n="1" />, Unai Artetxe<fnref n="2" />, José Mª Becerril<fnref n="2" />, Lur Epelde<fnref n="1" />, Carlos Garbisu<fnref n="1" />

Corresponding author email: aburges@neiker.net

## ABSTRACT

Heavy metals are toxic to most plants at high concentrations, reducing plant development and, hence, compromising the efficiency of phytostabilization processes. One emergent way to overcome these limitations is the addition of endophytic plant growth-promoting bacteria (PGPB), which can positively affect plant growth and physiological status by, for instance, enhancing root development and increasing plant metal tolerance. Most importantly, the goal of any soil remediation technology must be not only to remove the contaminants from the soil or to render them harmless but to restore soil health. In this respect, microbial properties have been reported as most valuable bioindicators of soil health. Likewise, the human-centered concept of ecosystem services can be used to group these properties, facilitating the interpretation and providing long-term phytostabilization monitoring programs with the required stability through time.

In this study, we attempted to evaluate (i) the effect of applying endophyte PGPBs individually and in consortium (ii) the plant ecotype specificity of these endophytes and (iii) the combined effect of assisting the process with an organic amendment in a soil phytostabilization strategy through the measurement of soil ecosystem services. First, endophytic bacteria were isolated from a pseudometallophyte *Festuca rubra* native from an abandoned Pb/Zn mine tailing located in northern Spain, in order to examine their plant growth-promoting traits (ACC deaminase, IAA synthesis, siderophore production, phosphate solubilization, salinity tolerance and metal tolerance). Then, endophytic bacteria showing the best-performing traits were used (as individuals and in consortium) in a microcosm phytostabilization experiment with two different ecotypes of *Festuca rubra* (native and non-native of the mine tailing). Besides, half of the pots were amended with cow slurry. At the end of the growth period, plant biomass and metal contents in plant and soil were measured. Additionally, the effect of all these treatments on soil ecosystem services such as carbon storage, nutrient cycling and biodiversity preservation was determined through a variety of microbial properties with potential as bioindicators of soil health (soil microbial biomass, enzyme activities and community level genetic profiles, respectively).

The results obtained suggested that inoculated endophytic bacteria can have the potential to influence plant development and recovery of soil health in phytostabilization processes. Finally, soil microbial properties can be used to assess not only soil health but also the abovementioned ecosystem services.

**Acknowledgements:** The financial support by the Spanish Ministry of Economy and Competitiveness (FITOMINA project, AGL2012-39715-C03-02) is greatly appreciated.

<figure><img src="image_318.png" /></figure>

<footnote n="1">¹Soil Microbial Ecology Group, NEIKER-Tecnalia, E-48160 Derio, Spain</footnote>

<footnote n="2">²Department of Ecology and Plant Biology, University of the Basque Country, UPV/EHU, P. O. Box 644, E-48080, Bilbao, Spain</footnote>
# ECOLOGICAL ASPECTS OF PLANT TRACE ELEMENT (HYPER)-ACCUMULATION –
SELENIUM AS A CASE STUDY

Elizabeth A. H. Pilon-Smits, Ali F. El Mehdawi

Biology Department, Colorado State University, Fort Collins, CO, USA

## ABSTRACT

Plants that accumulate toxic trace elements are widely used in phytoremediation and biofortification. Such plants also occur in nature in areas that contain elevated levels of trace elements, either naturally or due to human causes. Some plant species even hyperaccumulate toxic elements to levels several orders of magnitude above those in surrounding vegetation. The ecological implications of these high levels of toxic elements in plants are important to study, particularly before large-scale phytotechnological applications. So far, there have been relatively few such studies. Here, an overview is presented of our studies on ecological aspects of selenium (Se) accumulation in plants.

Selenium (Se) is essential for mammals and a beneficial nutrient for plants. It is also toxic at higher levels, due to its similarity to sulfur and replacement of sulfur in proteins. Plants readily accumulate and assimilate Se via S transporters and enzymes. Some species native to seleniferous soils can even hyperaccumulate Se up to 1.5% of their dry weight. We have studied the effects of plant Se on interactions with herbivores, detrivores, pollinators, neighboring plants, and mutualistic and pathogenic microbes, using a combination of field surveys and laboratory experiments.

It was found that plant Se accumulation can profoundly affect ecological interactions (for a review see [1]). Plant Se accumulation (and likely volatilization) protects plants against a wide variety of pathogens and herbivores, owing to both deterrence and toxicity. In naturally seleniferous areas, however, specialist herbivores and pathogens are present that have evolved Se resistance and can exclusively utilize Se hyperaccumulators as food source. It was also found that hyperaccumulators may use Se for elemental allelopathy against Se-sensitive neighboring plants, since they enhance Se accumulation in neighbors. Se-tolerant plant neighbors, however, profit from their enhanced Se levels next to hyperaccumulators, owing to herbivore protection. Selenium readily accumulates in flowers and seeds, and does not deter floral visitors. Honey bees and bumble bees were found to collect Se-rich pollen and to accumulate Se in their tissues, warranting more investigation on health effects of floral Se on pollinators. Furthermore, Se hyperaccumulators and their surrounding seleniferous soil harbor a wide variety of Se-tolerant fungi and bacteria that may affect plant Se speciation, accumulation and tolerance.

These findings may be useful for phytoremediation and biofortification, not only of Se but also of other plant-accumulated toxic elements that may have similar ecological effects. The findings also have intrinsic scientific value, as they give insight into the importance of hyperaccumulators in their local ecosystems. In seleniferous ecosystems, Se hyperaccumulators may significantly affect local species composition, through their negative effects on Se-sensitive ecological partners and positive effects on Se-resistant partners. They may also facilitate Se movement in the food chain and Se cycling in the local ecosystem. Hyperaccumulators of other elements likely function similarly.

## References:

1. El-Mehdawi A.F., Pilon-Smits E.A.H. (2012) *Ecological Aspects of Plant Selenium Hyperaccumulation*. *Plant Biology* 14, 1-10.

**Acknowledgements:** Financial support for these studies was provided by US National Science Foundation to EAHPS.

<figure><img src="image_319.png" /></figure>
# PHYTOAVAILABILITY AND ECOLOGICAL RISK ASSOCIATED WITH CADMIUM IN AN ORGANIC MATTER RICH SOIL

Michele Parisien<fnref n="1" />, Allison Rutter<fnref n="2" />, Barbara Zeeb<fnref n="1" />

Corresponding author email: michele.parisien@rmc.ca

## ABSTRACT

Bioavailability is an important factor influencing the applicability of phytoextraction and the potential for risk at contaminated sites. Low contaminant bioavailability to plants leads to ineffective phytoextraction, while high bioavailability can lead to considerable plant uptake which in turn may lead to receptor exposure to that contaminant. When contaminant bioavailability is low, risk to receptors may be negligible due to decreased contaminant exposure from the plant-based food chain, and if exposure is decreased enough to eliminate risk, site remediation may not be necessary.

Cadmium (Cd) is a xenobiotic metal that can cause adverse health effects to humans, plants, and animals. Soil can become contaminated with Cd through the land application of sewage sludge products like compost, as at a site in Peterborough, Ontario, Canada. The concentration of Cd (≤ 21.4 µg·g⁻<fnref n="1" /> Cd) present in the soil at the site exceeds the standard set by the Ontario Ministry of the Environment (1.2 µg·g⁻<fnref n="1" /> Cd) and might therefore pose ecological risk. A phytoextraction feasibility study was conducted for this site using native and naturalized plant species (Brassica juncea, Chenopodium album, Helianthus annuus, Poa compressa) in a control treatment (<1.0 µg·g⁻<fnref n="1" /> Cd), a low Cd treatment (5.0±0.3 µg·g⁻<fnref n="1" /> Cd), and a high Cd treatment (16.5±1.2 µg·g⁻<fnref n="1" /> Cd) using soil collected from the site. None of the plants had a bioaccumulation factor ≥1 after 50 days of exposure and only B. juncea had a detectable concentration of Cd in its shoots (≤1.79 µg·g⁻<fnref n="1" /> Cd). Low Cd phytoavailability was determined to be due to the high soil organic matter content of the soil. If receptor exposure is low enough to eliminate risk, then remediation is not necessary. Therefore, an ecological risk assessment was conducted to determine the potential for risk to ecological receptors. Data for receptor exposure to Cd from plants was taken from the phytoextraction feasibility study, and a worm bioavailability study was conducted to determine exposure from soil invertebrates. Worms (Eisenia fetida) had mean bioaccumulation factors of 6 and 3 in the low and high Cd treatments, respectively. The hazard quotient (HQ) for all seven receptors (Scolopax minor, Turdus migratorius, Blarina brevicauda, Microtus pennsylvanicus, Sylvilagus floridanus, Procyon lotor, and Peromyscus maniculatus) was <1.0, indicating no potential risk to any of these groups.

The results of this study demonstrate the advantages of conducting a risk assessment when phytoextraction is found to be ineffective at a contaminated site due to low contaminant bioavailability, as receptor exposure might be sufficiently low to eliminate ecological risk, and therefore the necessity to remediate. The results of this risk assessment are contrasted to those at another site in Ontario, where soil contaminated with PCBs exhibited high bioavailability to *Cucurbita pepo* in a phytoextraction feasibility study, and the results of an ecological risk assessment indicate that phytoextraction would result in risk (HQ >1) to all receptor groups evaluated

Acknowledgements: NSERC CRD Grant to Drs. Barbara Zeeb and Allison Rutter

<figure><img src="image_320.png" /></figure>

<footnote n="1">¹Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, ON, Canada</footnote>

<footnote n="2">²School of Environmental Studies, Biosciences Complex, Queen's University, Kingston, ON, Canada</footnote>
# NATURALLY SELENIFEROUS SOILS: POTENTIAL SOURCE OF SELENIUM FOR BIOFORTIFIED FOOD CROPS

**Muhammad Yasin**<fnref n="1" />,<fnref n="3" />, **Ali Farag El Mehdiwi**<fnref n="3" />, **Muhammad Faisal**<fnref n="1" />, **Courtney E. Jahn**<fnref n="2" />, **Marie F.S. Turner**<fnref n="2" />, **Elizabeth A. H. Pilon-Smits**<fnref n="3" />

*Corresponding author email: yasin_mmg@yahoo.com*

## ABSTRACT

Selenium (Se) is an essential micronutrient for human beings and considered an important preventative agent against different cancers, AIDS, and cardiac- and immune system-related disorders. Worldwide, Se deficiency is much more common than Se toxicity. Selenium-accumulating crop plants may be used to extract Se from seleniferous soils and to provide dietary adequate Se levels (55µg/day) in low-Se areas.

In this study Se accumulator Indian mustard (*Brassica juncea*) was tested for its ability to extract Se from naturally seleniferous shale rock-derived soil collected from Fort Collins, CO, USA. Two bacterial consortia, C1 and C2 (four highly Se-tolerant *Bacillus* strains in each, isolated from soil) were tested for their capacity to promote plant growth, Se accumulation, non-protein thiols in plants, photosynthesis rate, water use efficiency and stomatal conductance.

The Se concentration in the *B. juncea* plants was very high (711 mg kg⁻<fnref n="1" /> DW in leaves and 358 mg Se kg⁻<fnref n="1" /> DW in seeds). Plants inoculated with Se-resistant PGPR consortia C1 and C2 overall stimulated plant growth in this seleniferous soil. Plants inoculated with the C1 consortium showed significantly increased dry biomass, number of leaves, main inflorescence node length and seed weight in comparison to control plants. The C1-inoculated plants showed increased levels of non-protein thiols (antioxidants), enhanced photosynthesis and reduced stomatal conductance. Plants inoculated with C2 exhibited significantly enhanced non protein thiols, significantly reduced photosynthesis and reduced stomatal conductance compared to control plants. Selenium levels in the pod, leaf and seed tissue were enhanced in C1-inoculated plants and reduced in C2-inoculated plants, compared to control plants.

In conclusion, *B. juncea* may be used to extract Se from seleniferous soil for the production of Se-fortified plant material, and inoculation with bacterial consortium C1 further enhances the efficiency of this process.

*Keywords: *Bacillus* spp., selenium, ICP, non protein thiols, photosynthesis*

<figure><img src="image_321.png" /></figure>

<footnote n="1">¹ Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore-54590, Pakistan</footnote>

<footnote n="2">² Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523-1177, USA</footnote>

<footnote n="3">³ Department of Biology, Colorado State University, Fort Collins, Colorado 80523-1878, USA</footnote>
# PHYTOTECHNOLOGIES AND ECORESTORATION IN AGRICULTURAL MATRICES OF NEW ZEALAND

**Nicholas Dickinson**

Department of Ecology, Lincoln University, Lincoln 7647, Christchurch, New Zealand

Corresponding author email: nicholas.dickinson@lincoln.ac.nz Ph. +64 3423 0728

## ABSTRACT

This paper evaluates the integration of phytotechnologies and ecological restoration to mediate the environmental footprint of agriculture in three contrasting landscapes in South Island, New Zealand. The agricultural matrix that accounts for 50% of the country's land area has occurred through its conversion from native vegetation cover in little more than the past 200 years. Earlier vegetation clearance and burning by Polynesians was followed by European introduction of ungulate grazing, cultivation, new plant species, fertilisation and irrigation. Within a relatively short time period, the original soil template that supported native species has been and continues to be profoundly modified. Restoration of native species on these soils presents new and different challenges both for plants and ecologists.

The aim of the research described in this paper is to understand the role played by biodiversity in soil biogeochemistry, rather than vice versa. Firstly, it is described how soil physico-chemistry has been modified through agricultural practices in both lowland and montane environments, with a focus on major nutrients, key trace element deficiencies and soil biota. Experimental results are then shown of how plant litters and rhizospheres of native plants substantially differ from forage crops in their modification of soil hydrology, pH, soluble and gaseous nitrogen and trace element dynamics. A significant interplay between soil biota and native plant rhizospheres also modifies soil biogeochemistry. Beneficial effects are demonstrated of native plants on marginal land, including riparian zones, hedgerows and paddock fence lines. Case studies are described that include a conversion of plantation forestry to intensive irrigated dairy, restoration of a coastal sand plain forest impacted by mining and agriculture, and an upland sheep farm.

The findings shows that selection of plant traits from New Zealand's unique biodiversity palette provides an opportunity to use phytotechnology to resolve some of the environmental constraints currently impacting agriculture, thereby future-proofing and adding value to agricultural production systems. This is introducing a new paradigm of incorporation of nature conservation into mainstream farm planning, in a country where more than 80% of the native flora and fauna of New Zealand is endemic and found nowhere else. Restoration of biodiversity into intensive agricultural systems may contribute advantageously to a primary industry with a branding based on its clean and green credentials.

<figure><img src="image_322.png" /></figure>
# DECREASE IN SOIL ORGANIC MATTER FROM PALAEO TO PRESENT DAY SOILS IN SOUTH AFRICA – A CHALLENGE FOR PHYTOREMEDIATION

Bea Hurter¹, Pieter W. van Deventer²

¹,² School of Geo- and Spatial Sciences / Soil Science, North-West University, Potchefstroom, South Africa

Corresponding author email: 22160396@nwu.ac.za

## ABSTRACT

The recent geology of the last 65 Ma is known as the Cenozoic era which is subdivided into the quaternary and tertiary periods. The soils of the Cenozoic era contain the nutrients necessary to sustain flora, fauna and human life. Carbon, known as an indicator element in ecological and soil qualities, is present in the A-horizons of both present day and palaeo soils. The soil organic matter (SOM) content benchmarks the environmental status of that point in time when the specific horizon was exposed.

The objectives of this study were a) to compare contemporary and palaeo climatic conditions with geological deposits dated and characterized by means of C¹⁴, pollen and other methods, b) to determine the ecosystem health and status of different geological epochs, c) to use this data to predict future scenarios, and d) to discuss the use of long-term eco-management systems such as phytoremediation and precision farming in order to secure soil carbon.

Data for this study were obtained from the SOM content of different palaeo soil horizons exposed in the Florisbad Holocene deposit in South Africa. Sedimentary layers associated with varying climatic conditions of the last 400,000 years are represented at this site. These different layers were sampled and analysed for SOM by means of the Loss on Ignition (LOI) method. Data from soil organic matter analyses, together with pollen indicators and other dating information on the Holocene deposits were correlated with data from SOM analyses of the A-horizon of the current soils found in the same climatic zone and geological province. Results indicated that the SOM percentage in the present day A-horizon is lower than that found in the majority of the palaeo soils.

The palaeo deposits have a depth of 4.5 m and consist of five palaeo sedimentary layers, with the first as the top layer. Relative to the present day A-horizon in the area (not palaeo deposits) was used as a bench mark, a higher ecosystem functionality of 9.77% SOM was obtained for layer 1, 18.83% SOM for layer 2, 66.96% SOM for layer 3, and 16.87% SOM for layer 4. The bottom (fifth) layer had the lowest ecosystem functionality with a decreased value of 43.69% SOM relative to the present day A-horizon. The carbon sink in the present day soil surface is therefore lower than that found in the past. These decreased levels of carbon in the A-horizon may be due to agricultural activity, deforestation, overgrazing and/or adverse climatic changes.

To conclude: Based on these findings, phytoremediation could play an important role in ensuring soil carbon sequestration in disturbed areas (such as barren crop fields) or in anticipated global warming events. The negative impact of climate change in South Africa could be delayed by means of eco-restoration, re-forestation and habitat creation; this would facilitate an increase in SOM in present day A-horizons as well as improve the ecosystem functionality and quality of soils.

**Keywords:** cenozoic deposits, soil organic matter, palaeo soil horizons, carbon sequestration, phytoremediation programs

**Acknowledgements:** Financial support from the Technology and Human Resources for Industry Programme (THRIP) of the National Research Foundation (NRF) [South Africa] as well as from AGES (Potchefstroom) is greatly appreciated.

<figure><img src="image_323.png" /></figure>
# PLANT ESTABLISHMENT, COMMUNITY STABILIZATION, AND ECOSYSTEM DEVELOPMENT ON OILS SANDS SOFT TAILINGS

Stephen Ebbs, Lilyan Glaeser, Melissa House, Dale Vitt

Department of Plant Biology and Center for Ecology, Southern Illinois University, Carbondale, IL USA

## ABSTRACT

The excavation of bitumen from the oil sands regions of western Canada has a disruptive influence on the natural ecosystems of the region and creates a significant challenge for reclamation efforts. In particular, the removal of the naturally occurring peatlands represents a loss of one of the world's largest carbon sinks. Working cooperatively with a major oil sands company, a project is underway to evaluate strategies for re-establishing peatlands on soft tailings from the oil sands mining. Sandhill Watershed is a 50 ha artificial wetland/upland complex north of Fort McMurray, AB. Within this watershed, a 17 ha area represents a constructed wetland where a layer of stockpiled peat has been established over the high saline oil sand soft tailings. The restoration trajectory of this fen complex is being monitored to evaluate the establishment and early performance of select native plant species established in the fen in comparison to plants growing naturally at reference sites.

Arrays of 20 native species were planted in plots in the fen complex in 2012 and 2013 while select sedge and grass species were planted throughout the remainder of the wetland subsection of the fen. Physiological performance of a subset of four plant species in the plots was evaluated in the summer of 2013. The four species evaluated were water sedge (Carex aquatilus), black-girdle bulrush (Scirpus atrocinctus), seaside arrowgrass (Triglochin maritima), and bog birch (Betula glandulifera). Physiological performance was assessed using a CI-340 portable photosynthesis system to quantify parameters such as net photosynthesis, evapotranspiration, and water use efficiency. Pore water was also sampled within each plot at depth intervals through the peat layer to the tailing interface to examine the vertical migration of base cations (particularly Na) in the wetland. The hydrological characteristics of the wetland plots were also determined and the plots grouped in categories based on the common soil moisture regimes. Comparable measurements were taken on plants of the same species growing at nearby reference sites.

<figure><img src="image_324.png" /></figure>

<figure><img src="image_325.png" /><figcaption>Fig. 1. Net photosynthesis rate and water use efficiency for water sedge (C. aquatilis) plants in the Sandhill fen wetland complex in response to the four moisture profiles across the plots.</figcaption></figure>

Significant differences in physiological performance parameters were observed within each species in response to the moisture profiles across the plots. Parameters such as net photosynthesis were generally significantly different between the plots with consistent moisture and those that were drier (e.g., Fig. 1). Surprisingly, the range of data values obtained for the four species on the wetland plots were similar to the range observed for the comparable plants growing at the natural sites in the area. These results suggest that even under the worst moisture regime, the wetland fen plants were still physiologically active and that activity was comparable to plants at the benchmark sites.

This effort is one of only a few peatland reclamation efforts and one of the first to attempt to create a peatland directly on soft tailings. Additional monitoring is planned for the summer of 2014. This monitoring will examine plants established in 2012 and assessed in 2013 as well as the new plants established in 2013. In addition, data will be collected on net productivity and organic matter production to assess how the physiological performance results relate to biomass production and organic matter accumulation. The results are expected to provide the oil sands industry with information on the trajectory of this reclamation and to provide information useful in planning and implementing future wetland fen reclamation efforts.

**Acknowledgments:** The financial support for this research provided through a grant from Syncrude Canada is greatly appreciated. We are also very thankful for logistic support from Jessica Clarke and Carla Wytrykush (Syncrude Canada).

<figure><img src="image_326.png" /></figure>
# REVEGETATION OF MUCK DUMPING SITES IN INDIAN HIMALAYAN REGION

RD Singh<fnref n="1" />, Gopichand<fnref n="1" />, SK Vats<fnref n="1" />, RK Ogra<fnref n="1" />, Arvind Gulati<fnref n="1" />, Anil Sood<fnref n="1" />, RR Pawar<fnref n="2" />, Jaspreet Singh<fnref n="2" />, Sharad Bhatnagar<fnref n="2" />, Vikrant Jaryan<fnref n="1" />, RL Meena<fnref n="1" />, Ranjeet Singh<fnref n="1" />, Om Parkash<fnref n="1" />, Rakesh Goel<fnref n="2" />, AK Singh<fnref n="2" />, PS Ahuja<fnref n="1" />

Corresponding author email: rdsingh@ihbt.res.in

## ABSTRACT

Eco-restoration of the muck dumping sites in command area of a hydro-electric power generation project is a matter of serious concern from ecological and social points of view. Realizing the importance of the technological interventions under such situation, CSIR-IHBT took up revegetation of 10 closed dumping sites (DS) of National Hydro-electric Power Corporation's (NHPC) Parbati Hydroelectric Project Phase-II in Kullu district, Himachal Pradesh, India, in 2010. These dumping sites are located at 1400 to 2220 m amsl covering about 9 ha. Owing to the steep slope of the dumping sites, a network of retaining walls made of stacked stone-filled gabions tied together with mild steel wire-net was made throughout the dumping sites for their stabilization. Considering the agro-climatic conditions of the dumping sites 11 tree species were selected for plantation. The saplings of the tree species were planted in gunny bags placed in pits at the sites containing garden soil, organic manure and peat moss. The consortium of selected plant growth promoting rhizo-bacteria (PGPR) formulated by the Institute was applied at collar level of the tree saplings. After plantation, pelleted seeds of potential herbs and shrubs were also spread on the surface of the dumping sites. About 1 year after planting, observations were recorded on rate of establishment of the saplings at each dumping site. The rate of survival ranged from 69.2 % (at DS-01) to 96.6 % (at DS-13b). The average survival rate was 85.4 %. Higher establishment and height was attained by *Robinia pseudoacacia* L.

<figure><img src="image_327.png" /></figure>

<footnote n="1">¹ CSIR-Institute of Himalayan Bioresource Technology, Palampur, HP-176061, India</footnote>

<footnote n="2">² National Hydroelectric Power Corporation Ltd., Faridabad, Haryana-121003, India</footnote>
<figure><img src="image_328.png" /></figure>

PLANT-NANOPARTICLE INTERACTIONS
<figure><img src="image_329.png" /></figure>
# INVESTIGATING POSITIVE AND NEGATIVE IMPACTS OF ENGINEERED NANOPARTICLES ON PLANT GROWTH AND DEVELOPMENT

**Stephen Ebbs**<fnref n="1" />, **Scott Bradfield**<fnref n="1" />, **Pawan Kumar**<fnref n="1" />, **Xingmao Ma**<fnref n="2" />

## ABSTRACT

There are an increasing number of engineered nanomaterials emerging in the international market and a wider range of potential applications for those nanomaterials. Nanomaterials are being included in a variety of consumer products as well as industrial and agricultural chemicals in order to improve the stability and properties of the formulations and to improve their efficacy of those products and chemicals. Multiple benefits for such nano-enhanced products have been proposed. There are also concerns that broader use of nanomaterials, coupled with accidental spills and discharges into industrial and municipal waste streams, may facilitate the uncontrolled release of such materials into the environment. Such concerns have been magnified by studies showing adverse effects of some nanomaterials on plants and animals. In light of the expected growth of the nanotechnology industry, there is a pressing need to evaluate both positive and negative impacts of nanomaterials on living organisms. Collaborative research currently underway is examining the direct impacts of select nanomaterials on agricultural and food plants, as well as indirect impacts that may be caused by such materials.

Research on the potential positive effects of nanomaterials on plants have focused on potential benefits to TiO₂ and SiO₂ to the growth and yield of agronomic crops such as maize and soy as well as horticultural applications to promote plant establishment and reduce failure to pathogen infection during propagation. In the former case, studies have been conducted that examined the impact of foliar TiO₂ application to examine the physiological effect on photosynthesis and the repercussions for seed yield and seed quality. Studies to date have demonstrated a transient increase in photosynthesis in corn and soy in response to some foliar treatment strategies. Yield analysis is currently underway to assess the impact on seed production and the commercial characteristics of those seeds (e.g., oil and protein percentage). Similarly, plant propagation studies using wandering jew have shown that some nanomaterials reduce rooting of plant cuttings while other nanomaterials have no adverse effect. The effect of nanomaterial pretreatment on rooting, plant establishment, and pathogen resistance are being evaluated.

Research is also examining the adverse effects of nanomaterials, either directly to plants or indirectly to animals that might consume plants that have come in contact with nanomaterials. Experiments underway with an array of food plants are examining the phytotoxicity of nanomaterials. Recent results, for example, from root vegetables such as carrot and parsnip have shown that exposure to increasing concentrations of CuO or ZnO interfere with plant water relations (Fig. 1). Additional studies are examining the impact of nanomaterial exposure on root plasma membrane integrity and leakage (e.g., Fig. 2). Tissues from these and additional studies are being used in a physiologically-based extraction test to assess the release of the nanomaterials during human digestion to estimate the potential exposures that might results from consumption of such plant foods. An additional line of research is examining the interaction between specific nanomaterials and other soil contaminants (e.g., heavy metals). In particular, efforts are focusing on possible synergistic phytotoxicity of nanomaterials and heavy metals to plants. Additional efforts are focused on determining the extent to which nanomaterials increase the accumulation of metals and vice versa and the mechanism of interaction. The implications of the interaction between nanomaterials and heavy metals for dietary exposure to these compounds will also be assessed.

<figure><img src="image_330.png" /><figcaption>Cumulative Transpiration</figcaption></figure>

<figure><img src="image_331.png" /><figcaption>Fig. 1: Inhibition of evapotranspiration by carrot and parsnip in response to CuO.</figcaption></figure>

<figure><img src="image_332.png" /><figcaption>Treatment</figcaption></figure>

<caption>Fig. 2: Electrolyte leakage from lettuce roots after 60 days of watering with AgNPs or Ag⁺.</caption>

**Acknowledgments:** Financial support for this research was provided through USDA Grant 2011-06426.

<figure><img src="image_333.png" /></figure>

<footnote n="1">¹Department of Plant Biology and Center for Ecology, Southern Illinois University, Carbondale, IL USA</footnote>

<footnote n="2">²Department of Civil and Environmental Engineering, Southern Illinois University Carbondale, IL USA</footnote>
# PHYTOTOXICITY OF MULTIWALL CARBON NANOTUBES AND IMPACT ON THE UPTAKE OF COEXISTENT PESTICIDES BY LETTUCE (LACTUCA SATIVA L.)

Helmi Hamdi<fnref n="1" />, Roberto De La Torre-Roche<fnref n="2" />, Joseph Hawthorne<fnref n="2" />, Jason C. White<fnref n="2" />

Corresponding author email: helmi.hamdi@certe.rnrt.tn

## ABSTRACT

The field of nanotechnology is projected to achieve a global market value of $3 trillion by 2020, with engineered nanomaterials (NM) being currently used in a wide variety of commercially available products; including medical devices, plastics, textiles, cosmetics, electronics and agrichemicals. Carbon NM such as C₆₀/C₇₀ fullerenes, single/multiwall carbon nanotubes (S/MWCNT), have been the focus of much interest with regard to agricultural applications, although the scientific literature reporting their phytotoxic effects is rather thin and conflicted. Besides, given CNT chemical and physical properties, interactions with coexisting organic chemicals can be anticipated. In this research study, the effect of non-functionalized and amino-functionalized multiwall carbon nanotube (CNT) (NF-MWCNT, NH₂-MWCNT) exposure, as well as the impact of CNT presence on coexistent pesticide accumulation, was investigated in lettuce (Lactuca sativa L. cv. Green Grand Rapids). Lettuce seeds were sown directly into CNT-amended vermiculite (1000 mg L⁻<fnref n="1" />) to monitor phytotoxicity during germination and growth. During growth, lettuce seedlings were subsequently exposed to chlordane (cis-chlordane, trans-chlordane, trans-nonachlor) and *p*,*p*'-DDE (all at 100 ng/L) in the irrigation solution for a 19-d growth period. CNT exposure did not significantly influence seed germination (82-96%) or plant growth. Similarly, pesticide exposure had no impact on plant growth, total pigment production or tissue lipid peroxidation. After 19 d, the root content of total chlordane and *p*,*p*'-DDE was 390 and 73.8 µg g⁻<fnref n="1" />, respectively, in plants not exposed to CNTs; the shoot levels were 1.58 and 0.40 µg g⁻<fnref n="1" />, respectively. The presence and type of CNT significantly influenced pesticide availability to lettuce seedlings. Non-functionalized CNT decreased the root and shoot pesticide content by 88% and 78%, respectively, but amino-functionalized CNT effects were significantly more modest, with decreases of 57% in the roots and 23% in the shoots, respectively. The presence of humic acid completely reversed the reduced accumulation of pesticides induced by amino-functionalized CNT, likely due to strong competition over adsorption sites on the NM. These findings have implications for food safety and for the use of engineered NM in agriculture, especially with leafy vegetables.

**Keywords:** NF-MWCNT, NH₂-MWCNT, pesticides, uptake, lettuce

**Acknowledgement:** Helmi Hamdi would like to deeply thank the Council for the International Exchange of Scholars (CIES) for supporting his travel and stay at the CAES, New Haven, CT, USA, under the Fulbright Visiting Scholar Program (Grant ID 68120784). The authors gratefully acknowledge T. Arsenault and K. Prapayotin-Riveros for technical assistance. We also acknowledge USDA AFRI grant 2011-67006-30181.

<figure><img src="image_334.png" /></figure>

<footnote n="1">¹Water Research and Technology Center, University of Carthage, P.O.Box 273, Soliman 8020, Tunisia</footnote>

<footnote n="2">²Department of Analytical Chemistry, the Connecticut Agricultural Experiment Station (CAES), 123 Huntington Street, New Haven, CT 06504, USA</footnote>
TOXICOGENOMICS OF CdS QDs INTERACTIONS WITH *ARABIDOPSIS THALIANA*

Luca Pagano, Marta Marmiroli, Davide Imperiale, Nelson Marmiroli

Department of Life Sciences, University of Parma, Italy

Corresponding author email: marta.marmiroli@unipr.it

ABSTRACT

Nanotechnology is a rapidly growing industry and engineered nanomaterials (NMs) are applied in
several areas such as electronics, biomedicine, pharmaceuticals, cosmetics, food production. The
scarce knowledge about uptake, interaction with cells, toxicity, is hindering the full application of
NMs, as shown by recent decisions of the European Commission and European Food Safety
Authority. The aim of this work was to develop a toxicogenomics approach for risk assessment of
NMs, focusing on quantum dots cadmium sulfide nanoparticles (CdS QDs) using *Arabidopsis thaliana*
(L.) Heynh as model system. Two mutant lines of Arabidopsis have been selected as resistant to lethal
concentrations of CdS QDs, and the phenotypes and genotypes have been characterized. The global
gene expression profile in the two mutants have been analyzed using Affymetrix GeneChip
Arabidopsis ATH1Genome Array, showing differences in the panel of induced and repressed genes
not only among the mutants and the wild type, but also between the two mutants. Genes of
particular interest in both mutants identified with the transcriptomic approach were validated by
Real Time PCR. The results obtained from the two approaches (mutant-based and whole-genome) in
plant could offer a possible hypothesis concerning the tolerance/resistance mechanism in which the
CdS QDs are involved. Furthermore results found suggested that CdS QDs and Cd²⁺ could exploit
different pathways of tolerance/resistance. It was also observed a possible epistatic activity by which
the up- or down-regulation of one or few gene could lead to a cascade of other genes influenced by
the CdS treatments. The use of model organisms might offer new strategies for the risk assessment
of NMs within the frame of environment and health. Therefore, plant-based model systems are able
to provide information about genetic and physiologic targets of broad interest also in other
organisms related to the risks posed by the exposition and contact with NMs. Our approach, which
merges the classical top-down (from mutant phenotype to genotype) and bottom-up (from gene to
function) approaches, allows to shed light on function and functionality of our targets: from
observing phenotype to knowing what lays genetically behind that phenotype.

<figure><img src="image_335.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_336.png" /></figure>

A. experimental set-up and result; B. RT qPCR heatmap resulted from validation of the genes isolated in microarray experiments.

<figure><img src="image_337.png" /></figure>
# PLANT UPTAKE AND TRANSLOCATION OF RADIOACTIVELY LABELLED TiO₂ AND CeO₂ NANOPARTICLES

**Stanislav Smrček**<fnref n="1" />, **Ján Kozempel**<fnref n="2" />, **Uwe Holzwarth**<fnref n="3" />, **Elena Bellido Vera**<fnref n="3" />, **Matteo Dalmiglio**<fnref n="3" />, **Neil Gibson**<fnref n="3" />

Corresponding author email: stanislav.smrcek@natur.cuni.cz

## ABSTRACT

Nanotechnology and nanomaterials have been widely applied in the technological and consumer products during the last years. The addition of nanoparticles significantly improves quality of the many consumer products. On the other hand the fate of these nanoparticles in the environment after use is not well documented and their health risks are not clear. The fundamental problem of nanoparticles in the environment is the possible entry into a food chains of humans and animals preferably by the contamination of plant food and water sources. The investigation of nanoparticles plant uptake and translocation is complicated by the presence of naturally occurring nanoparticles which cannot be reliably distinguished from naturally occurring ones. For these purpose it seems to be unique the use of radioactively labelled nanoparticles for plant uptake studies. In these experiments the determined radioactivity originating from the tested nanoparticles can be measured unambiguously and with high sensitivity. The method is especially useful for the rapid testing of uptake and translocation of nanoparticles by agriculturally interesting plants.

The TiO₂ and CeO₂ nanoparticles labelled with ⁴⁸V and ¹³⁹Ce were prepared by direct cyclotron activation of corresponding nanomaterials using a Scanditronix MC-40 cyclotron as described previously. Phytoextraction of these labelled nanoparticles was tested using *in vitro* cultures of common cultivated plants (Zea mays, Helianthus annuus, Cucumis sativus). The nanoparticles were added to the 2-3 weeks old sterile seedlings cultures cultivated on half-strength Murashige-Skoog medium. After next 7 days the resulting plants were removed, root system gently washed with distilled water and the radioactivity measured using electronic autoradiography.

The results show that both used nanoparticles are adsorbed and/or absorbed by the root system and some translocation was observed using electronic autoradiography. Although about 99 % of radioactivity was typically found in the root area, the remaining activity was distributed between stem and leaf parts. This fact uniquely determined possibility of contamination of plants with nanoparticle material.

It can be concluded, that the nanoparticles can contaminate food chains and the health risk of these contamination must be evaluated. In these simple methodics we cannot yet distinguish if the contamination is caused with true nanoparticles or if the measured radioactivity is from dissolved ionic forms which could be produced by the interaction of the nanoparticles with some acidic compounds in plant tissues. Experiments with electron microscopy cannot distinguish between naturally occurring and artificial nanoparticles. For these purposes the advanced double labelled nanoparticles will be prepared and used in uptake experiments.

**Acknowledgements:** This project was funded by the QualityNano under contract No.: JRC-TAF-79, and its support is greatly appreciated. Ján Kozempel acknowledges partial support from the Ministry of education, youth and sports of the Czech Republic, under grant No.: LK21310 and Stanislav Smrček acknowledges partial support from the Ministry of education, youth and sports of the Czech Republic, under project MSM0021620857.

<figure><img src="image_338.png" /></figure>

<footnote n="1">¹ Charles University in Prague, Faculty of Science, Albertov 6, 12000 Prague, Czech Republic</footnote>

<footnote n="2">² Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Břehová 7, 11519 Prague, Czech Republic</footnote>

<footnote n="3">³ Nanobiosciences Unit, European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Via E. Fermi 2749, I-21027 Ispra, Italy</footnote>
# INTERACTION BETWEEN METALLIC NPs AND SALT MARSH PLANTS: IMPLICATIONS FOR PHYTOREMEDIATION

Federico Andreotti<fnref n="1" />,<fnref n="2" />, Cátia Caetano<fnref n="1" />,<fnref n="3" />, Ana Paula Mucha<fnref n="1" />, Paula Rodrigues<fnref n="1" />, Carlos Rocha Gomes<fnref n="1" />,<fnref n="3" />,
**C. Marisa R. Almeida**<fnref n="1" />

Corresponding author email: calmeida@ciimar.up.pt

## ABSTRACT

Increased use of metallic nanoparticles (NPs) raises the probability of these NPs to reach the environment. Health and environmental effects of common metals are well-known, however, when metals take the form of NPs, consequential hazards based on shape and size are yet to be explored. Although a lot of information exists regarding plants interaction with metals there is a lack of information among metallic NPs interaction with plants, including with salt marsh plants.

This work aims to study the interactions among a Cu metallic NPs (CuO) and the salt marsh plants *Halimione portulacoides* and *Phragmites australis* comparing with the interactions observed when the selected metal is in its ionic form. In addition, it aims to evaluate the potential of these plants for the phytoremediation of these NPs. The chosen plants have shown to uptake considerable amount of Cu when in its ionic form.

Experiments were conducted with sediment elutriate, a simplified natural medium, being the plant expose for 6 days to medium contaminated either with CuO or with Cu (II). To evaluate metal uptake, total metal concentrations were determined by atomic absorption spectroscopy in plant tissues at the beginning and at the end of the experiments.

Results indicate that *H. portulacoides* and *P. australis* had the ability to accumulate Cu in its roots, being the accumulation, respectively, 10 and 4 times lower when the metal was in the nanoparticle form. Regarding *H. portulacoides* no metal translocation was observed when NPs were added to the medium. But for *P. australis* metal translocation occur when the metal was added either in ionic or in NP form, indicating that interactions between plants and NP differ with the plant species.

These facts need to be taken in consideration when applying these plants for the phytoremediation of contaminated sediments.

**Acknowledgements:** This work was partially funded by the Project ECORISK (reference NORTE-07-0124-FEDER-000054), co-financed by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

<figure><img src="image_339.png" /></figure>

<footnote n="1">¹CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal</footnote>

<footnote n="2">²Department of Agriculture and Environment Sciences, Faculty of Agriculture University of Milan, Italy</footnote>

<footnote n="3">³Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal</footnote>
# CO-EXPOSURE TO ENGINEERED NANOPARTICLES ALTERS THE TOXICITY AND ACCUMULATION OF PERSISTENT PESTICIDES IN AGRICULTURAL CROPS

Jason C. White<fnref n="1" />, Roberto De la Torre-Roche<fnref n="1" />, Joseph Hawthorne<fnref n="1" />, Helmi Hamdi<fnref n="2" />, Baoshan Xing<fnref n="3" />, Lee A. Newman<fnref n="4" />, Xingmao Ma<fnref n="5" />

## ABSTRACT

Although the use of engineered nanomaterials (NM) has increased, the fate and effects of these substances in the environment is poorly understood. As an emerging class of contaminants, there is significant potential for NM interactions with co-existing chemicals. Direct molecular interactions between NM and co-contaminants may change the availability and activity of either or both molecules in soils and water. In addition, NM may alter the ability of biota such as plants to tolerate or bioaccumulate co-existing chemicals. This uncharacterized level of co-contaminant interactions may be of particular concern in agricultural systems, where food chain contamination and subsequent human exposure may occur.

A number of studies have been initiated in which the impact of NM co-exposure on the toxicity and accumulation of persistent pesticides by plants has been evaluated. Exposure systems include both model media (vermiculite) with freshly added contaminants and soils with field-weathered pesticide residues. In one set of experiments, the accumulation of DDE by zucchini, soybean and tomato grown in fullerene ($C_{60}$)-amended vermiculite increased by 30-65% compared to controls. In a second vermiculite-based trial, nonfunctionalized multi-wall carbon nanotubes (CNT) decreased the chlordane and DDE accumulation in lettuce by 83%, but amino-functionalized CNT effects were significantly more modest, with decreases of only 40%. Interestingly, humic acid completely reversed the reduced accumulation of pesticides induced by amino-functionalized CNT, likely due to strong competition over adsorption sites on the nanomaterial. However, in a third study Ag nanoparticles (NP) suppressed DDE uptake by zucchini and soybean grown in vermiculite. Interestingly, NP Ag resulted in greater suppression of DDE accumulation that did equivalent bulk or ion exposures and data suggests that Ag-mediated closure of aquaporins may be responsible for decreased uptake.

In soil with 0.20 mg/kg weathered DDE, $C_{60}$ co-exposure at 1000 mg/Kg on had little impact on residue accumulation by pumpkin. A second more comprehensive study involved corn, soybean, zucchini, and tomato being grown in soil containing weathered chlordane (2 mg/kg) and DDE (0.2 mg/kg) that was amended with 0, 500, 1000, or 5000 mg/Kg $C_{60}$ or CNT. CNT co-exposure consistently reduced chlordane and DDE accumulation by up to 80% in a concentration dependent manner for all species, depending on plant type and CNT concentration. However, $C_{60}$ had species- and pesticide-specific effects on residue accumulation, ranging from complete suppression of DDE uptake (corn/tomato) to 35% increases in chlordane accumulation (tomato/soybean).

<figure><img src="image_340.png" /><figcaption>Figure 1. Effect of CNT or $C_{60}$ co-exposure (0-5000 mg/Kg) on the uptake of chlordane components (ng) by tomato.</figcaption></figure>

The finding that engineered nanomaterials can significantly alter the accumulation of pesticides in food crops may have significant implications for food safety, as well as for the movement of pesticides and other organic contaminants through the environment.

*Acknowledgements:* The financial support of USDA AFRI grant 2011-67006-30181 and the Fulbright Visiting Scholar Program (Grant ID 68120784) is greatly appreciated.

<figure><img src="image_341.png" /></figure>

<footnote n="1">¹Department of Analytical Chemistry, CT Agricultural Experiment Station, New Haven, USA</footnote>

<footnote n="2">²Water Research & Technology Center, University of Carthage, Soliman, Tunisia</footnote>

<footnote n="3">³Stockbridge School of Agriculture, University of Massachusetts, Amherst, USA</footnote>

<footnote n="4">⁴Department of Environmental & Forest Biology, SUNY College of Environmental Science & Forestry, Syracuse, USA</footnote>

<footnote n="5">⁵Department of Civil and Environmental Engineering, Southern Illinois University Carbondale, Carbondale, USA
*Corresponding author email: Jason.White@ct.gov*</footnote>
NANOPARTICLE EFFECT ON PHYSIOLOGY AND METABOLISMUS OF PLANTS

Tomas Vanek, Petr Soudek, Radka Podlipná, Martin Vagner, Přemysl Landa, Radomíra Vaňková

Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i. Rozvojová 263, 165 02 –
Prague 6, Czech Republic

Corresponding author email: vanek@ueb.cas.cz

ABSTRACT

The use of nanoparticles (NPs) in commercial products and industrial applications has increased greatly in recent years although understanding of the interaction mechanisms at the molecular level between NPs and biological systems, is largely lacking.

Our work is focused to the study of effect of nanoparticles to the higher plant metabolisms, both in laboratory and real conditions to elucidate potential of phytoremediation methodology for removing NPs from environment. In laboratory conditions nanoparticles of TiO2, ZnO2, AlO2, Fullerenes and Graphite fibers were tested using *Arabidopsis thaliana* as a model systems.

Generally, all nanoparticles decreases plant cells viability – the most toxic effect was found for ZnO2, where only 67% of starting viability was detected. All nanoparticles (with exception of graphite fibers) decreased the production of ethylene. Part of their unfavorable effects might be disturbance of defense pathways in tobacco cells, probably *via* disturbance of ion homeostasis.

All nanoparticles exhibit negative effect on cell division and stimulate various stress responses, e.g. antioxidant system and ethylene formation, while microarray data (1) confirm stimulation of antioxidant system as well as general stress response and down-regulation of genes related to cell division.

Proteomic study using DIGE methodology confirms overexpression of proteins, related to achieved microarray data.

Effect of selected nanoparticles to plant photosynthesis were studied too, preliminary data were achieved and will be discussed during presentation.

<figure><img src="image_342.png" /></figure>

<table><caption>Effect of ZnO on Arabidopsis thaliana proteome</caption><thead><tr><th>SSP</th><th>Mr (kD)</th><th>pl</th><th>ZnN</th><th>ZnO</th></tr></thead><tbody><tr><td>504</td><td>51.50</td><td>6.10</td><td>262676</td><td>852887</td></tr><tr><td>605</td><td>56.00</td><td>6.00</td><td>1634991</td><td>327465</td></tr><tr><td>1508</td><td>52.40</td><td>5.80</td><td>734266</td><td>260054</td></tr><tr><td>1803</td><td>70.80</td><td>5.76</td><td>1369409</td><td>569686</td></tr><tr><td>3206</td><td>42.20</td><td>5.49</td><td>202688</td><td>640728</td></tr><tr><td>4702</td><td>69.40</td><td>5.45</td><td>743103</td><td>332577</td></tr><tr><td>5102</td><td>34.60</td><td>5.40</td><td>3696451</td><td>248656</td></tr><tr><td>5104</td><td>37.80</td><td>5.39</td><td>14154</td><td>155830</td></tr><tr><td>5201</td><td>40.87</td><td>5.39</td><td>1568642</td><td>617504</td></tr><tr><td>5301</td><td>45.80</td><td>5.38</td><td>883489</td><td>364221</td></tr><tr><td>5401</td><td>47.20</td><td>5.39</td><td>558103</td><td>246864</td></tr><tr><td>5502</td><td>54.67</td><td>5.39</td><td>878202</td><td>144947</td></tr><tr><td>5706</td><td>67.00</td><td>5.28</td><td>676446</td><td>137559</td></tr><tr><td>7604</td><td>62.20</td><td>5.00</td><td>511651</td><td>208465</td></tr><tr><td>7606</td><td>60.10</td><td>4.97</td><td>2179761</td><td>920518</td></tr><tr><td>7701</td><td>65.70</td><td>5.11</td><td>562316</td><td>230274</td></tr><tr><td>7803</td><td>72.80</td><td>5.10</td><td>994702</td><td>2265996</td></tr><tr><td>8706</td><td>64.40</td><td>4.00</td><td>183923</td><td>51886</td></tr></tbody></table>

Reference:

1. Landa, P., Vaňková, R., Andrlová, J., Hodek, J. Maršík, P., Štorchová, H., White, J. C. Vaněk, T. (2012) *Journal of Hazardous Materials* 241, 55-62

Acknowledgements: This work was supported by LH11047Myes and FR/TI3/778 project

<figure><img src="image_343.png" /></figure>
# TROPHIC TRANSFER POTENTIAL OF RARE EARTH ELEMENT (REE) OXIDE NANOPARTICLES THROUGH TERRESTRIAL FOOD CHAINS

**Jason C. White**<fnref n="1" />, **Roberto De la Torre-Roche**<fnref n="1" />, **Joseph Hawthorne**<fnref n="1" />, **Sanghamitra Majumdar**<fnref n="2" />, **Jorge Gardea-Torresdey**<fnref n="2" />, **Baoshan Xing**<fnref n="3" />, **Lee A. Newman**<fnref n="4" />, **Xingmao Ma**<fnref n="5" />

## ABSTRACT

Although the use of engineered nanomaterials (NM) has increased dramatically, the fate and effects of these substances in the environment is poorly understood. Given that NM may represent an emerging class of contaminants, there is significant potential for NM interactions with terrestrial biota such as plants, as well as for bioaccumulation and biomagnification of these materials through food chains that may or may not include humans. In spite of this concern, very few studies have addressed the potential trophic transfer and biomagnification of NM in terrestrial environments. This lack of understanding on NM bioaccumulation may be of particular concern in agricultural systems, where food chain contamination and subsequent human exposure may occur.

Studies have been initiated in which 0 or 1000 mg/kg bulk or nanoparticle (NP) cerium oxide (CeO₂) or lanthanum oxide (La₂O₃) was added to an agricultural loam. Zucchini was then grown in the cerium-amended or control soils for approximately 28 days; lettuce was grown in the lanthanum oxide-amended soils for 40 d. At harvest, the element content of the root and shoot tissue was determined by ICP-MS. Fresh shoot tissue that had been exposed to 0 or 1000 mg/kg bulk or NP CeO₂ or La₂O₃ was used to feed herbivorous cricket populations for 14-21 days. Select cricket tissues were digested and the element content was determined by ICP-MS. Live crickets that had consumed zucchini or lettuce shoots exposed to 0 or 1000 mg/kg bulk or NP REE metal oxides were then used to feed carnivorous mantid or wolf spider populations. The Ce content of the carnivores was also determined by ICP-MS. Scanning/Transmission electron microscopy with energy dispersive X-ray spectroscopy will be conducted on select plant and animal tissues.

Zucchini plants exposed to NP CeO₂ had significantly greater Ce content than plants exposed to the bulk metal oxide (figure). Root Ce concentrations were 3.9 times greater in the NP-exposure; notably, all root tissues were rinsed with dilute HNO₃ to remove surface retained ceria prior to actual digestion and analysis for absorbed element. This data represents one of the few reports showing enhanced NP accumulation by plants under soil-grown conditions as compared to an equivalent bulk material. Crickets consuming NP-contaminated leaf tissue had approximately 20.0 ng/g Ce whereas those consuming bulk-contaminated tissue only had 10.3 ng/g. Although these levels are 10-100 times below the leaf tissue levels, the data suggest that trophic transfer does occur and that it may be particle size specific. Interestingly, cricket feces collected from insects consuming bulk and NP-contaminated leaves approached 240 and 650 ng/g, respectively, and increased over time. This suggests that significant Ce may enter the insect but that elimination may be effective. Although only limited mantid data has been obtained, individuals consuming bulk and NP-exposed crickets contained 3.2 and 7.6 ng/g, respectively. Again, these levels suggest trophic transfer, although not biomagnification. Interestingly, limited sampling of mantid feces showed Ce at levels up to 99 ng/g.

<figure><img src="image_344.png" /></figure>

<figure><img src="image_345.png" /></figure>

The finding that engineered NM are accumulated at significantly greater levels from soil that corresponding bulk materials and that particle-size specific trophic transfer may occur has significant implications for food safety, as well as for assessing the risk of NM released to the environment.

**Acknowledgements:** The financial support of USDA AFRI grant 2011-67006-30181 is greatly appreciated.

<figure><img src="image_346.png" /></figure>

<footnote n="1">¹Department of Analytical Chemistry, CT Agricultural Experiment Station, New Haven, USA</footnote>

<footnote n="2">²Department of Chemistry, University of Texas-El Paso, El Paso, USA</footnote>

<footnote n="3">³Stockbridge School of Agriculture, University of Massachusetts, Amherst, USA</footnote>

<footnote n="4">⁴Department of Environmental & Forest Biology, SUNY College of Environmental Science & Forestry, Syracuse, USA</footnote>

<footnote n="5">⁵Department of Civil and Environmental Engineering, Southern Illinois University Carbondale, Carbondale, USA
*Corresponding author email: Jason.White@ct.gov*</footnote>
IMPACT OF CERIUM OXIDE NANOPARTICLES ON THE PHYSIOLOGICAL AND BIOCHEMICAL PROCESSES OF *BRASSICA RAPA* IN THREE GENERATIONS

Xingmao Ma<fnref n="1" />, Stephen D. Ebbs<fnref n="2" />, Qiang Wang<fnref n="1" />

Corresponding author email: ma@engr.siu.edu or ebbs@plant.siu.edu (1230 Lincoln Drive, MC 6603, Carbondale, IL)

ABSTRACT

The intrinsic ability of cerium oxide nanoparticles (CeO₂-NPs) to form oxygen vacancy makes this material a popular component in a plethora of commercial products. Its application as a fuel additive in particular entails possible substantial release of this nanoparticle to the environment and warrants an intensive study on its environmental health and safety impact. Even though the short term impact of CeO₂-NPs on the environmental health, including plant health has been evaluated, the long term, multi-generational impact of CeO₂-NPs to plants is still unclear. The main aim of this study was to evaluate the potential impact of CeO₂-NPs on a suite of physiological and biochemical processes of *Brassica rapa* after continuous irrigation of the plants with different concentrations of CeO₂-NPs (0, 10, 100 and 1,000 mg/L) over three generations. The results showed that plants in the second and third generation displayed slower plant growth, smaller biomass, and fewer and smaller silique and seeds. Plants in the later generations also contained higher evidence of reactive oxygen species, specifically H₂O₂. An examination of the activities of three enzymatic antioxidants (SOD, CAT and GPX) involved in the generation and detoxification of H₂O₂ indicated that the activities of these enzymes showed a generational pattern. Overall, the results implied that the second and third generations of plants experienced higher oxidative stress and the photosynthetic processes in later generations are more susceptible to the impact of CeO₂-NPs than the first generation plants. This study provided the first evidence that that plant response to CeO₂-NPs varied between different generations of plants and long term evaluation on the phytotoxicity of engineered nanomaterials to plants is critical.

Keywords: cerium oxide nanoparticle, oxidative stress, ROS, multigenerational impact

<figure><img src="image_347.png" /></figure>

<footnote n="1">¹ Civil and Environmental Engineering, Southern Illinois University Carbondale, USA</footnote>

<footnote n="2">² Department of Plant Biology, Southern Illinois University Carbondale, USA</footnote>
# GENE REGULATIONS AND ANTIOXIDANT ENZYME RESPONSES IN *ARABIDOPSIS THALIANA* TO NANOPARTICLE CERIUM AND INDIUM OXIDE EXPOSURE

Chuanxin Ma<fnref n="1" />, Hong Liu<fnref n="1" />, Baoshan Xing<fnref n="1" />, Craig Musante<fnref n="2" />, Jason C. White<fnref n="2" />, Om Parkash Dhankher<fnref n="1" />

Corresponding author email: parkash@umass.edu

## ABSTRACT

Rare earth elements (REEs) nanoparticles (NPs) have been widely applied in various products such as polishing agents, cosmetics and catalyst. As the demands and use of nanoproducnts increased annually, the released NPs could inevitably cause a series of environmental problems. However, the risk assessment of nanotoxicity in plants is still at the initial stage. In this study, the effects of cerium oxide (CeO₂) and indium oxide (In₂O₃) NPs exposure on *Arabidopsis thaliana* were investigated. Our previous study has already demonstrated that CeO₂ NPs could induce several folds of MDA, an important indicator for plant's response to stresses. In order to fully understand defense system of A. *thaliana* to both NPs exposures, altered antioxidant enzyme activities, which are able to scavenge reactive oxygen species (ROS) induced by both NPs were measured at 250 and 1000 ppm treatments. Except superoxide dismutase (SOD), several antioxidant enzymes were highly induced to defend nanotoxicity. These antioxidant enzymes include catalase (CAT) and ascorbate peroxidase (APX), both of which mainly contribute to destructing ROS; phenylalanine ammonialyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD), which are involved to secondary metabolism in plants; glutathione S-transferase (GST) and glutathione reductase (GR), involved in sulfur assimilation and metabolism, were significantly induced at CeO₂ NPs exposure. At the molecular level, expression of genes central to the stress response such as the sulfur assimilation and glutathione (GSH) metabolic pathway was determined by qPCR. Meanwhile, expression of altered genes related to antioxidant enzymes, which have abilities of scavenging ROS, was also analyzed. Further experiments are currently underway to measure the ROS productions in response to CeO₂ NPs. DNA damages in A. *thaliana* caused by nanotoxicity is determined by analyzing the levels of 7,8-dihydro-8-oxoguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde). Last, but not the least, nutrient elements, especially cations, will be measured in order to indicate whether NPs could disrupt elements transportation at different exposure concentrations. These studies will be highly useful in understanding the fate, transport, and toxicity of manufactured NPs in the agricultural crops and to further develop strategies for mitigating the toxicity of these NPs in food crops.

<figure><img src="image_348.png" /></figure>

<footnote n="1">¹Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA</footnote>

<footnote n="2">²Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA</footnote>
# EVIDENCES OF GENOTOXICITY AND PHYTOTOXICITY IN ZEA MAYS AND HORDEUM VULGARE EXPOSED TO CeO₂ AND TiO₂ NANOPARTICLES

Alessandro Mattiello, Filip Pošćić, Rita Musetti, Massimo Vischi, Luca Marchiol

Dept of Agriculture and Environmental Sciences, University of Udine, Italy

Corresponding author email: alessandro.mattiello@uniud.it

## ABSTRACT

In recent years, remarkable progress has been made in developing nanotechnologies. This has been leading to a fast-growth of a commercial application which involves the use of a great variety of manufactured nanomaterials (NMs). So far, it is estimated that the research on the environmental health and safety of nanomaterials receives less than 5% of the funding spent to develop new nanomaterials [1]. There is a small but growing body of scientific studies showing that some NMs are toxic to commonly used environmental indicators such as algae, invertebrate and fish species. It was hypothesized that the plant uptake of NMs varies largely depending on the type of plant and the size and chemical composition of the NMs [2]. However, very few studies on NMs-plant interactions have been published, so far. For this reason there is great concern regarding potential NMs impact to agriculture and the food supply.

Potential early phytotoxic and genotoxic effects of CeO₂ and TiO₂ nanoparticles compared to bulk counterparts and control are currently investigated in barley (Hordeum vulgare 'Pilastro') and maize (Zea mays 'Synthesis') seeds/seedlings.

The early phytotoxic effects are measured by treatment of triplicate samples of 10 sterile seeds of barley and maize in Petri dishes with suspensions of CeO₂ (<50 nm) and TiO₂ (<25 nm) nanoparticles and the respective bulk counterparts at 0 (control), 500, 1000 and 2000 ppm for 7 days. At the end of treatment the relative seed germination inhibition, relative root growth inhibition and metal uptake by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) in the roots and seeds with coleoptile are measured. Genotoxicity of nanoparticles are assessed by Randomly Amplified Polymorphism DNA (RAPDs) on seedlings previously germinated in sterile milli-Q water. Furthermore, TEM observations of root, seed and coleoptile tissues are performed. Mitotic index (MI) are measured in the same condition except for the exposure period which will be 24 hours.

The expected results will provide information about the potential early genotoxicity and phytotoxicity on CeO₂ and TiO₂ on two important crops.

## References:

1. Environ Toxicol Chem 2012 31, 3-14.

2. J Agric Food Chem 2011 59, 3485-3498

<figure><img src="image_349.png" /><figcaption>Figure 1: Germination percentage rate of barley seeds treated with Ce³⁺/Ce NP.</figcaption></figure>

<figure><img src="image_350.png" /><figcaption>Figure 2: Meristematic cells of barley root tip treated with Ce³⁺/Ce NP 2000 ppm. In this picture there are 12 cells in mitotic phase.</figcaption></figure>

<figure><img src="image_351.png" /><figcaption>Figure 3: Meristematic cells of barley root tip treated with Ce³⁺ 2000 ppm. In this picture there are no cells in mitotic phase.</figcaption></figure>

<figure><img src="image_352.png" /></figure>
BIOTISATION AND NANOTECHNOLOGY FOR PHYTOREMEDIATION

Turnau K.<fnref n="1" />,<fnref n="2" />, Ogar A.<fnref n="2" />, Wężowicz K.<fnref n="2" />, Plazuk E.<fnref n="2" />, Stojakowska A.<fnref n="3" />, Malarz J.<fnref n="3" />, Rozpadek P.<fnref n="2" />,<fnref n="4" />

*Corresponding author email: katarzyna.turnau@uj.edu.pl*

ABSTRACT

The selection of the most efficient microbial consortia for the particular plant species grown in specific environmental conditions plays a key role in developing phytoremediation technologies. During recent years (within the project MAESTRO 2011/02/NZ9/137, financed by the National Science Centre, Poland) a wide variety of microbes were isolated from Polish flotation wastes containing high concentration of Zn and Pb. The collection of isolated microorganisms included bacteria and mycorrhizal fungi. They were tested using: *Verbascum* spp. *Helianthus annuus*, *Hieracium pilosella* and *Cichorium intybus* under greenhouse conditions. Differences in plant responses depended on plant species and fungal strains. For *Cichorium intybus* the most significant differences were found for quercetine and kaempferol derivatives. AM fungi have repeatedly been demonstrated to alleviate metal toxicity and reduce stress in plants. In the presence of arbuscular mycorrhiza a new antioxidant homeostasis is established. In chicory a decrease in SOD activity and an increase in the activity of catalase and peroxidase was observed, indicating the reduction in the production of superoxide. However, differences in the concentration of medically important compounds in M+ and M- chicory grown on polluted soil was not as evident. Fe nanoparticles and benthonite was added/supplemented into the substratum to verify their ability to improve *H. annuus* vitality.

<figure><img src="image_353.png" /></figure>

<footnote n="1">¹Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland</footnote>

<footnote n="2">²Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland</footnote>

<footnote n="3">³Institute of Pharmacology PAN, Smętna 12, 31-343 Kraków, Poland</footnote>

<footnote n="4">⁴Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland</footnote>
# DIRECT TOXIC EFFECTS OF SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES ONTO MICROALGAL CHLORELLA SP.

Derek Juinn Chieh Chan<fnref n="1" />, Joanna Wan Yii Tai<fnref n="1" />, Pey Yi Toh<fnref n="1" />, Bee Wah Ng<fnref n="1" />,<fnref n="2" />

Corresponding author email: ch_derekchan@eng.usm.my

## ABSTRACT

Superparamagnetic iron oxide nanoparticles (SPION) have an increasing number of applications, such as in biomedical (Singh et al., 2010; Buyukhatipoglu and Clyne, 2010; Hoskins et al., 2012), wastewater treatment (Kong et al., 2012; Kakihara et al., 2004; Yavuz et al., 2006) and also in microalgae harvesting through magnetophoresis (Xu et al., 2011; Lim et al., 2012; Toh et al., 2012; Liu et al., 2009; Prochazkova et al., 2012; Cerff et al., 2012; Hu et al., 2013). One of the attractive property of the SPION is that it exhibits magnetic responsive only after the introduction of magnetic field. However the largely utilization of SPION for environmental remediation tend to induce SPION losses to the surrounding, especially into the aquatic system, due to inefficient recovery system. In this study, *Chlorella* sp. microalga is used as the model system to evaluate the toxicity of SPION as microalgae are recognized as the indispensable base of food chain in aquatic system (Lavens and Sorgeloos, 1996). The study has showed the growth of the *Chlorella* sp. cells is inhibited by the dispersion of SPION throughout the culture medium. A 20 mg/L dosage of SPION showed a cell growth inhibition of 19.2 ± 4.1 % with respect to the control group. The primary inhibition effect is due to the shading effect of SPION by blocking out the light illumination and inhibits the photosynthesis. The secondary toxicity comes from the internalization of SPION which induces cellular oxidative stress. It promotes lipid peroxidation and the disruption of cytoskeleton protein and carbohydrate in cells (Long et al., 2012; Singh et al., 2010).

The yield of total lipid, protein and carbohydrate at day 7th of exposure exhibit a reduction of 19.7 %, 65.7 % and 29.9 % respectively compared to the control group with a dosage of 20 mg/L SPION.

**Acknowledgements:** This work is supported by Fundamental Research Grant Scheme (FRGS) (Grant No. 203/PJKIMIA/6071271) and Research University Postgraduate Research Grant Scheme (USM-RU-PRGS) (Grant No. 1001/PJKIMIA/8045037) from Universiti Sains Malaysia and the Membrane Science and Technology cluster USM. Toh, P.Y. was supported by the My PhD scholarship from Ministry of Higher Education of Malaysia.

<figure><img src="image_354.png" /></figure>

<figure><img src="image_355.png" /></figure>

<figure><img src="image_356.png" /></figure>

<figure><img src="image_357.png" /></figure>

<figure><img src="image_358.png" /></figure>

<footnote n="1">¹School of Chemical Engineering, Universiti Sains Malaysia, Nibong Tebal, Penang 14300, Malaysia</footnote>

<footnote n="2">²School of Biological Sciences, Universiti Sains Malaysia, Minden, Penang 11800, Malaysia</footnote>
# RESPONSE SURFACE METHODOLOGY APPROACH FOR TRACE ELEMENTS AND NUTRIENTS ADSORPTION ONTO NANO-MAGHEMITE

Domingo Martínez-Fernández<fnref n="1" />, Deniz Bingöl<fnref n="2" />, Michael Komárek<fnref n="1" />

Corresponding author email: domingo.marfer@gmail.com

## ABSTRACT

Iron nano-oxides are important scavengers of contaminants, being a good choice for contaminated soils because of their sorption properties [1]. Nano-maghemite (NM) has been investigated recently due to its abundance and effectiveness at removing As, Cu<fnref n="2" />⁺, Zn<fnref n="2" />⁺, and Pb<fnref n="2" />⁺ from contaminated water, and as an in-situ remediation material.

An experiment was carried out to study the competition for adsorption between trace elements (TEs) and the nutrients K and N following the application of NM (iron nano-oxide; Fe₂O₃) to a soil solution (the 0.01 mol L⁻<fnref n="1" /> CaCl₂ extract of a TEs-contaminated soil). The soil was collected from the former mining area of Mokrsko (Czech Republic) affected by high TEs concentrations [2]. Using MINITAB software version 16.1.1. (MINITAB Inc., USA), response surface methodology (RSM) with a central composite design (CCD) of 13 runs (in triplicate) was designed for KNO₃ and NH₄NO₃ (from 0 to 6 mmol L⁻<fnref n="1" />). After the nutrients were mixed with the soil solution in the different treatments, NM was added to obtain 1% w/v NM in the soil-nutrients-solution. After agitation (24 h in darkness), the pH was determined, samples were centrifuged and filtered using 0.45-µm nylon filters (efficient due to aggregation). Samples were analysed using ICP-OES (Varian, VistaPro, Australia). The CCD allowed to obtain statistically-significant quadratic models to predict the concentrations of TEs remaining in solution in the presence of NM and nutrients, simultaneously. Adsorption response surface plots were obtained to graphically illustrate their concentrations in the equilibrium within the experimental region (Figure).

According to the models, K⁺ and NH₄⁺ were important factors for Ca, Fe, Mg, Mn, Na, and Zn adsorption by NM

(R²adj> 95%, except for Zn with R²adj= 87%), but KNO₃ had an dominant effect. Ca, Mg, and Mn concentrations showed a tendency to be lower in the presence of NM and nutrients. For Na adsorption, KNO₃ was not a statistically-significant factor at the 95% confidence level (p-value > 0.05), since it was unchanged in the presence or absence of KNO₃. The available Zn depended inversely on the K⁺ concentration and proportionally on the NH₄⁺ concentration (Figure F), with a quadratic interaction (higher adsorption capacity of NM at 6 mmol L⁻<fnref n="1" /> KNO₃ and 2.5 mmol L⁻<fnref n="1" /> NH₄NO₃). So, the presence of K⁺ could have facilitated the adsorption of Zn on the NM until reaching a limit, from which both NH₄⁺ and K⁺ equally decreased Zn adsorption. No significant differences were detected for pH, Al, As, Cd, or Cu (R²adj<0.8), and their quadratic models were not created. The interaction between nutrients and TEs in the soil solution may be crucial for the effectiveness of NM during the phytoremediation of contaminated soils.

<figure><img src="image_359.png" /></figure>

## References:

1. Komárek, M., Vaněk, A., Ettler, V. 2013. Chemical stabilization of metals and arsenic in contaminated soils using oxides - A review. *Environmental Pollution* 172, 9-22.

2. Drahota, P., Rohovec, J., Filippi, M., Mihaljevič, M., Rychlovský, P., Červený, V., Pertold, Z. 2009. Mineralogical and geochemical controls of arsenic speciation and mobility under different redox conditions in soil, sediment and water at the Mokrsko-West gold deposit, Czech Republic. *Science of the Total Environment* 407, 3372-3384.

Acknowledgements: To Zuzana Michálková and Hana Šillerová for their help with analyses. Supported by Postdok ČZU (ESF/MŠMT CZ.1.07/2.3.00/30.0040), and by the Czech Science Foundation (GAČR 503/11/0840).

<figure><img src="image_360.png" /></figure>

<footnote n="1">¹Czech University of Life Sciences Prague, Faculty of Environmental Sciences, Department of Environmental Geosciences, Prague, Czech Republic</footnote>

<footnote n="2">²Kocaeli University, Faculty of Science and Arts, Department of Chemistry, Kocaeli, Turkey</footnote>
# PROTEOMICS OF *ARABIDOPSIS THALIANA* MUTANTS RESISTANT TO CdS QUANTUM DOTS (CdS QDs)

Davide Imperiale, Marta Marmiroli, Luca Pagano, Nelson Marmiroli

Department of Life Sciences, University of Parma, Italy

Corresponding author email: marta.marmiroli@unipr.it

## ABSTRACT

The industry of nanotechnology is a steeply growing and nanomaterials (NMs) have an application in many industrial fields, from electronics, to biomedicine, pharmaceuticals, cosmetics, and food production. The scant knowledge regarding uptake, interaction with cells, toxicity, is hampering the complete application of NMs, as shown by recent decisions of the European Commission and European Food Safety Authority. The aim of our work was to develop a toxicogenomics approach to assess the risk posed by NMs, focusing on quantum dots cadmium sulfide nanoparticles (CdS QDs) using *Arabidopsis thaliana* (L.) Heynh as model system.

In a previous study, two mutant lines of Arabidopsis, have been selected as resistant to lethal concentrations of CdS QDs (80 mg/L), and the phenotypes and genotypes have been extensively characterized both on molecular and physiological side (Marmiroli et al. 2014).

Proteomic analysis has been performed on crude protein extracts, obtained from whole 15-days seedlings of one of the foregoing mutants, *atnp02*, and on the wild type, grown on agarized MS, both treated with 80 mg/L CdS QDs and non-treated. Proteins with different isoelectric point (pI) and hydrophobicity have been separated by a 2D liquid chromatography technique (ProteomeLab PF2D, Beckman), and an analysis using MultiVue Software (Eprogen) has been carried out on qualitative/quantitative differentially abundant protein peaks between wild-type and mutant plants in both conditions. Proteins whose abundance was statistically different in response to the experimental conditions were identified by MALDI-TOF/MS to infer their possible role in the plant response to CdS QDs, in particular resistance.

One integrated study using a global proteomic approach to study stress response due to nanoparticles in Arabidopsis plants, together with physiological, biochemical and transcriptomic analyses, leads to a better understanding of some of the genetic, molecular and physiological mechanisms at the basis of nanoparticle stress response. The nature of the mutant resistance to CdS QDs can be clarified by comparing the transcriptomic analysis, which indicate the RNA abundance, with the actual quantity of proteins synthetized in the plant as effectors of the resistance phenotype.

<figure><img src="image_361.png" /></figure>
# INVESTIGATION OF UPTAKE (ENDOCYTOSIS), AND TRANSLOCATION OF GOLD NANOPARTICLES IN PLANTS

Meaghan Kern, Guangshu Zhai, Katherine Walters, Jerald Schnoor

## ABSTRACT

The exact mechanism by which nanoparticles enter and translocate in plant cells is still unclear. One potential mechanism is endocytosis. Previous research has shown the nanoparticles enter suspended plant cells through endocytosis and endocytosis can be inhibited by temperature (4°C) and the chemical inhibitor Wortmannin. In this study, we investigate endocytosis as the main nanoparticle uptake mechanism into whole plants of hydroponically grown *Helianthus annuus* and *Populus* spp. The inhibition of endocytosis is expected to inhibit the uptake of nanoparticles into *H. annuus* and a decrease or absence in gold nanoparticles is expected.

Preliminary research of TEM imaging on hybrid poplar has shown that gold nanorods enter and translocate from the roots to leaves of woody hybrid poplars (Figures 1 and 2). Gold nanorods were taken up and transported from roots to leaves (Figures 1 and 2). However, gold nanorods were found to maintain their shapes in the roots (Figure 1A-C) and to change their shapes during transport to leaves (Figure 2A-D). However, further objectives are to investigate the specific mechanism by which nanoparticles enter plant cells through attempting to inhibit endocytosis in woody and herbaceous dicots.

<figure><img src="image_362.png" /><figcaption>Figure 1: TEM images of root after 6 day exposure of gold nanorods (10×34nm). A: Gold Nanorod in the roots; B: magnified panel A; C: magnified panel B; D: TEM of commercial gold nanorod (10×34nm).</figcaption></figure>

<caption>Figure 2: TEM images of leaf after 6 day exposure of gold nanorods (10×34nm). A: Gold Nanorod (10×34nm) in the leaves; B: magnified panel A; C: magnified panel B; D: magnified panel A.</caption>

*H. annuus* seeds were germinated in soaked paper towels and transplanted to an Erlenmeyer flask and grown in the growth chamber (16 hours daylight and 8 hours night). 0.10 Hoagland solution was used as the main hydroponic solution. Three different sets of experiments were set up for each of the three nanoparticle sizes (5, 25, 50 nm), with 2 replicates per treatment. Four different treatments were set-up: control with no nanoparticles, 4°C, 4°C with 120 µmol of Wortmannin, and control with nanoparticles. When seedlings of similar appearance have 4 true leaves, they were either dosed or not dosed with nanoparticles. Sunflowers with 4 true leaves were dosed with 4 mL of suspended gold nanoparticles. After 4 days, roots, stems and leaf samples were used for TEM imaging to look for the presence and absence of nanoparticles. In addition, ICP-MS analysis examines the metal concentration of the plant tissue of the woody and herbaceous plant material.

<figure><img src="image_363.png" /></figure>

<footnote n="1">¹ Civil and Environmental Engineering, University of Iowa, Iowa City, Iowa, USA</footnote>

<footnote n="2">² W.M. Keck Phytotechnologies Laboratory, University of Iowa, Iowa City, Iowa, USA</footnote>
# UPTAKE AND ACCUMULATION OF ZnO AND CuO ENGINEERED NANOPARTICLES IN CARROT AND PARSNIP

Pawan Kumar<fnref n="1" />, Xingmao Ma<fnref n="2" />, Stephen Ebbs<fnref n="1" />

Corresponding author email: pawank@siu.edu

## ABSTRACT

With the advancement in the field of nanotechnology, the production and the usage of engineered nanoparticles (ENPs) has increased exponentially over the recent years. This has attracted global attention both in the industrial and scientific world and there are increasing efforts to investigate the release, toxicity and environmental fate of ENPs. The interaction of ENPs with plants is a subject of continued study and there is a growing body of research in this area. The present study investigated the phytotoxic effects of ZnO and CuO ENPs on carrot and parsnip and accumulation of metals in the edible portion of these plants.

Six month old hydroponically grown plants of each species were treated for 10 days with increasing concentrations of one of the two ENPs at concentrations (of 0, 10, 100, 500, or 1000 ppm) in DI water. An ionic treatment, whose concentration was determined through prior efforts by quantifying the maximum dissolution of these ENPs in DI water at the aforementioned concentrations, of the plants with the corresponding ion ($Zn^{2+}$ or $Cu^{2+}$) was also included to distinguish between the nanoparticle and the ionic effects. Nutrients were supplied through foliar application during the treatment period and an array of physiological measurements was taken.

The plants accumulated the corresponding metals from ENPs in their storage organs. The metal concentration changed in a dose dependent manner and showed some saturation at higher concentration treatments. The peels for both the plant species accumulated significantly higher concentrations of metal than the flesh. There was no effect on the relative chlorophyll content of leaves or the biomass of the storage organ, but there was a significant difference in the total amount of water transpired during the treatment period suggesting an imbalance in water relations. Visually, the plants treated with some of the highest ENP concentrations showed a loss of turgor and leaves appeared were unable to support their own weight when compared to controls.

<figure><img src="image_364.png" /></figure>

Overall, this study has provided information describing the impact of ENPs on these two plants and the accumulation of Zn and Cu. The results have potential implications for agriculture, remediation and human health.

Currently, efforts are underway to determine the spatial distribution and chemical form of these ENPs in the storage organs of these plants. Nutritional bioaccessibility will also be assessed using a simulated gastric extraction to assess the release of metals from the edible portion of these plants in the digestive tract of humans. The results will contribute to the ongoing assessment of the possible risk of these ENPs to human health from consumption of these plants.

**Acknowledgements:** The financial support by USDA Grant 2011-06426 is greatly appreciated.

<figure><img src="image_365.png" /></figure>

<footnote n="1">¹Department of Plant Biology, Southern Illinois University, Carbondale, IL USA</footnote>

<footnote n="2">²Department of Civil and Environmental Engineering, Southern Illinois University, Carbondale, IL USA</footnote>
# IRON AND ZINC NANOPARTICLES IN MICROBIALLY ASSISTED BIOREMEDIATION OF Zn-Pb POST-FLOTATION WASTES

Rafał Ważny<fnref n="1" />, Ewa Plażuk<fnref n="2" />, Inna Zhebrak<fnref n="2" />,<fnref n="3" />, Anna Ogar<fnref n="2" />, Teresa Anielska<fnref n="2" />, Szczepan Zapotoczny<fnref n="4" />, Piotr Rozpądek<fnref n="2" />,<fnref n="5" />, Katarzyna Turnau<fnref n="1" />,<fnref n="2" />

Corresponding author email: rafal.wazny@uj.edu.pl

## ABSTRACT

Almost 250 000 sites in Europe require urgent remediation. Nanoparticles have great potential to reduce the toxicity of heavy metals released to ground waters from post-flotation and industrial wastes. Although, remediation technologies are still in experimental or pilot stage, our recent studies, within the UMBRELLA EU project, provided several microorganisms that can support growth of plants on metal enriched substrata. Iron colloids can remove dissolved metals by immobilization/adsorption and transformation of elements on their surface. Nanoparticles can be produced either in chemical processes or naturally, by living organisms including plants, bacteria and fungi. In the experiment we used *Helianthus annus*, *Iris pseudoacorus* and their symbiotic *Cadophora* sp. which is an endophyte isolated from plants inhabiting metal polluted sites and the water plant specific, mycorrhizal *Glomus spp*. In addition we tested cyanobacteria *Leptolyngbya frigida* isolated from a Zn waste in Sardinia for its ability to form Zn-based nanoparticles. Nanoparticle size determines its remediation potential. Visible/UV absorption spectra were recorded using a Hewlett-Packard HP 8452A diode-array spectrophotometer. The average size of nanoparticles reached about 100 nm.

The obtained data can be used for optimizing remediation technologies in artificial wetlands allowing efficient and sustainable metal removal and immobilization. This environmentally-friendly technology, based on plant-microbe consortia may be a useful alternative systems commonly used in environmental engineering.

**Acknowledgements:** The financial support by NSC project MAESTRO contract No. 2011/02/A/NZ9/00137 and by Foundation for Polish Science, International PhD Projects Program co-financed by the EU European Regional Development Fund (MPD/2009-3/5) is greatly appreciated.

<figure><img src="image_366.png" /></figure>

<footnote n="1">¹Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland</footnote>

<footnote n="2">²Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland</footnote>

<footnote n="3">³Department of Botany, Yanka Kupala State University, Grodno, Belarus</footnote>

<footnote n="4">⁴Physical Chemistry Department, Jagiellonian University, Kraków, Poland</footnote>

<footnote n="5">⁵Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland</footnote>
# NANO-HEAVY METAL INTAKE, TRANSPORT, ACCUMULATION AND TOXICOLOGY OF FRAGARIA X ANANASSA

Zeynep Zaimoglu<fnref n="1" />, Reyhan Erdogan<fnref n="2" />, Hatice Gulen<fnref n="3" />, Fuat Budak<fnref n="1" />, Dilek Budak<fnref n="4" />

## ABSTRACT

Phytoremediation can be defined as the combined use of plants, soil amendments like solid waste disposal sites and agronomic practices to remove pollutants. This technique has many advantages compared with, other remediation procedures: low economic costs, the generation of recyclable residues, and the possibility of being applied to soils and waters, causing minimum environmental impact. Techniques involve the elimination of the pollutants from the soil, the toxic elements accumulating in the harvestable parts of the plants.

Nanotechnology is a fast emerging discipline not only in physics and chemistry but also in the field of biology. In view of the tremendous applications of nanotechnology, there is a motivation among scientists to carry out research in this most vital discipline. Chemists are highly interested in synthesizing nanoparticles of different dimensions employing many of the precious metals. Already scientists have started exploiting the bio-based synthesis of nano-metals using leaf extracts and microorganisms (bacteria and fungi).

This paper reports on the accumulation of nano-silver and its morpho-physiological effects in Fragaria vesca leaves shoots and roots. This research examines the effects of four nano-silver concentrations on the growth and chlorophyll contents of Fragaria vesca leaves shoots and roots, to evaluate the ability of young and mature laminae to take up nano-silver, to detect and partially characterize the nano-silver-binding biomolecules in the laminae, as a biochemical tolerance mechanism to nano-silver exposure.

<figure><img src="image_367.png" /></figure>

<footnote n="1">¹ Environmental Engineering Department, Faculty of Engineering and Architecture, Cukurova University, Adana, Turkey</footnote>

<footnote n="2">² Department of Landscape Architecture, Faculty of Agricultural Engineering, Akdeniz University, Antalya, Turkey</footnote>

<footnote n="3">³ Horticulture Department, Faculty of Agricultural Engineering, Uludag University, Bursa, Turkey</footnote>

<footnote n="4">⁴ Agricultural Economy Department, Faculty of Agricultural Engineering, Cukurova University, Adana, Turkey
*Corresponding author email: zeynepzaimoglu6@gmail.com*</footnote>
<figure><img src="image_368.png" /></figure>
<figure><img src="image_369.png" /></figure>

HUMAN EXPOSURE & RISK
<figure><img src="image_370.png" /></figure>
# ECOLOGICAL BENCHMARK VALUES FOR Cd IN BRAZILIAN SOILS: A PHYSIOLOGICAL AND ECOTOXICOLOGICAL APPROACH

Ingrid Fernanda Santana Alvarenga<fnref n="1" />, Gabriel Caixeta Martins<fnref n="2" />, Luiz Roberto Guimarães Guilherme<fnref n="2" />

Corresponding author email: guilherm@dcs.ufla.br

## ABSTRACT

Trace elements, such as Cd, are naturally present in soils at different concentrations, with higher contents normally found in soils affected by anthropogenic activities. The increasing consciousness regarding soil pollution in Brazil has led the National Environment Council to develop a national legislation (Directive Conama 420/2009) proposing reference values for assessing soil quality. These values – ranked based on increasing risks – were set as “background”, “target” (ecotoxicological benchmarks), and “investigation”. Background values were supposed to be set individually for each State, while investigation values were defined nationwide based on human health risk assessments. Provisional target values, which are indicative of a level at which there is a sustainable soil quality, have also been set countrywide. Yet, each State was supposed to develop its own research in order to validate the proposed nationwide target value (e.g., 1.3 mg Cd kg⁻<fnref n="1" /> soil). Based on this, we have established an experiment with plants in order to propose target value for Cd in Brazilian soils using a combined physiological and ecotoxicological approach for risk assessment. With that we intend to find out how high the concentration of this metal could go without, however, compromising soil quality and function, i.e., with negligible risks to the ecosystem.

The experiment was conducted in a greenhouse, using two representative soils from the State of Minas Gerais, as follows: Oxisol (clay 24%; sand 64%; pH 4.8; P 1.13 mg dm⁻³; K 32 mg dm⁻³; Ca 0.3 cmolc dm⁻³; Mg 0.1 cmolc dm⁻³; Al 0.6 cmolc dm⁻³; effective CEC 1.1 cmolc dm⁻³; V 9.6%; OM 1.6%), and Inceptsol (clay 31%; sand 47%; pH 5.3; P 2.6 mg dm⁻³; K 34 mg dm⁻³; Ca 1.6 cmolc dm⁻³; Mg 0.4 cmolc dm⁻³; Al 0.5 cmolc dm⁻³; effective CEC 2.6 cmolc dm⁻³; V 34%; OM 2.9%). Tests followed the OECD-208 normative, using four metal-sensitive plant species (Zea mays, Oryza sativa, Phaseolus vulgaris, Raphanus sativus). Plants were exposed to increasing doses of Cd (0.4; 0.72; 1.29; 2.3; 4.1; 13.6; 24.4 mg kg⁻<fnref n="1" />) during 21 days. In addition to the variables commonly evaluated in these kind of studies (e.g., fresh and dry shoot weight, or shoot height), we have also measured the following endpoints for the ecological assessment: germination, emergence, plant growth, enzymatic activity (antioxidant system), shoot Cd concentration, chlorophyll index, photosynthesis, stomatal conductance, transpiration, photosystem II efficiency, and relative rate of electron transport. Following these measurements, we calculated the effective concentration causing 50% inhibition of each evaluated endpoint (EC50). All accepted EC50 values (those following a normal distribution) were then plotted to derive a 5th percentile value (HC5), which is defined as that concentration expected to protect 95% of the assessed endpoints.

Toxicity symptoms were more severe for plants growing in the Oxisol, compared with the Inceptsol. Raphanus sativus did not germinate with 24.4 mg kg⁻<fnref n="1" /> of Cd in the Oxisol and all other measured endpoints were also affected, yet with different intensities. While the plants cultivated in the Inceptsol showed mild toxicity symptoms, we have noticed significant variations in the enzymatic activity of the antioxidant system for plants exposed to Cd in this last soil. Using the accepted EC50 values, we could get HC5 values (mg Cd kg⁻<fnref n="1" /> dry soil) of 2.15 (Oxisol), 10.35 (Inceptsol), and 2.65 (both soils together). The different values observed for HC5 are mainly associated with soil properties affecting the available fraction of Cd in the soil (e.g., pH, OM, and clay content). Finally, derivation of the new proposed target value was possible by adding the Cd background reference value for Minas Gerais State soils (0.4 mg kg⁻<fnref n="1" />) to the HC5, obtaining, as a result, a new proposed ecotoxicological screening benchmark soil value (target value) of Cd for soils of Minas Gerais State (mg Cd kg⁻<fnref n="1" /> dry soil): 2.55 (Oxisol), 10.75 (Inceptsol), and 3.05 (both soils together). The last value is in accordance with a previous target value set for Cd in Brazilian soils (3.0 mg Cd kg⁻<fnref n="1" />, for the State of São Paulo).

We conclude suggesting that adding new physiological endpoints as additional variables to assess ecological risks of contaminated soils might be relevant for improving the sensitivity of assays with plants, thus helping us to define a more robust target value (ecotoxicological screening benchmark) for Cd in soils. Also, taking into consideration the high soil heterogeneity found in Brazil, it is recommended to have a range of target values based on different soil properties.

Acknowledgements: The financial support provided by FAPEMIG, CAPES, and CNPq.

<figure><img src="image_371.png" /></figure>

<footnote n="1">¹Biology Department /Plant Physiology Sector, Federal University of Lavras, Brazil</footnote>

<footnote n="2">²Soil Science Department, Federal University of Lavras, Brazil</footnote>
# MOLECULAR AND BIOCHEMICAL CHARACTERIZATION AND MODE OF ACTION OF SAP13 IN PROVIDING TOLERANCE TO MULTIPLE ABIOTIC STRESSES

Parul Tomar, Anirudha Dixit, Om Parkash Dhankher

Stockbridge School of Agriculture, University of Massachusetts, 230 Stockbridge Rd, Amherst, MA 01003, USA

Corresponding author email: parkash@umass.edu

## ABSTRACT

Global climate change and environmental stresses are the main reasons for the decline of crop production worldwide. In the past years, a major focus has been on improving plant species and their tolerance towards these stresses but not much has been achieved because of the limited knowledge of the gene/network of genes that might be involved in providing tolerance to such multiple abiotic stresses. Recently, members of Stress Associated Protein (SAP) family in plants have been shown to impart tolerance to multiple abiotic stresses. There are 14 SAP genes in Arabidopsis thaliana and these proteins contain A20, AN1 and C2H2 zinc finger domains. AtSAP13, a member of the SAP family carries two AN1 zinc finger domains and an extra Cys2-His2 domain. When overexpressed in Arabidopsis and Brassica, it has been shown to provide tolerance to multiple abiotic stresses such as toxic metals (As, Cd, Zn), drought, and salt. However, the mode of action of this SAP member in providing tolerance to multiple abiotic stresses is largely unknown. In-silico analysis of the promoter sequences upstream of ATG start codon of AtSAP13 using PLACE database predicted the presence of various abiotic stress related cis regulatory elements. We hypothesized that the expression of AtSAP13 gene might be regulated via the interaction of cis-elements present in the AtSAP13 promoter with abiotic stress related trans factors via protein-DNA interactions under different abiotic stresses. Through yeast one hybrid assay, we have proved this hypothesis and identified several transcription factors such as DREB, ERE, ZIP, HSE etc that are interacting with the AtSAP13 promoter. These interactions are being analyzed through Electrophoretic mobility shift assay (EMSA) to understand the molecular and biochemical functioning of AtSAP13. Candidate transcription factors interacting with AtSAP13 will be characterized further using both forward and reverse genetic approaches. Further, Camelina sativa, a member of Brassicaceae family and closely related with Arabidopsis, has been proposed as an ideal biofuel crop. In order to improve its adaptability to wider geographical ranges and marginal land, we aim to characterize and overexpress endogenous SAP13 in C. sativa in providing tolerance to various stresses. We have identified and cloned CsSAP13 in C. sativa. Resulting transgenic plants will be analyzed for enhanced biomass, seed and oil yield under multiple abiotic stresses. The knowledge and information gained will not only be applied on agricultural crops to engineer crops that will be better able to withstand such abiotic stresses and still produce sustainable yield but will also help to grow crops for food and biomass production on barren lands, thus making them more cultivable over time. Therefore, the proposed research could have a significant impact on global food security, biofuel production, and human and environment health enhancement.

<figure><img src="image_372.png" /></figure>
# TOXIC ARSENIC COMPOUNDS REMAIN IN NATIVE PLANT SPECIES FROM ARSENIC POLLUTED SOILS

Sara García-Salgado, M. Ángeles Quijano

School of Civil Engineering, Technical University of Madrid, Spain

Corresponding author email: sara.garcia@upm.es

## ABSTRACT

Arsenic is considered a toxic element for plants. However, the discovery of arsenic resistant and hyperaccumulating plant species has increased the interest in understanding the distribution of arsenic species in these environmental matrices. Plants are able to take up both arsenate (As(V)) and arsenite (As(III)) from soils, via phosphate transporters and some aquaporin channels, respectively, but both forms of arsenic are toxic to cellular metabolism of plants. Methylated arsenic species can be taken up by plants from soils as well, but generally less efficiently than inorganic species. Some authors have suggested that the synthesis of phytochelatins (PCs) is involved in As(III) detoxification in terrestrial plants. The chelating properties of these peptides lead to arsenic complexes which are less reactive and therefore less toxic for plants than the free arsenic ions [1]. Arsenic can also be present in plants as non-extractable arsenic species, strongly bound to lipids or to cell wall constituents. Nevertheless, the arsenic remained in plants as free inorganic or methylated ions has a greater interest due to its higher mobility and toxicity, and therefore, its ecological impact and risk to health.

The aim of this work consisted on the determination of the fraction of arsenic present as toxic forms (inorganic and methylated species) present in native terrestrial plants from polluted soils by former mining activities (Mónica mine, NW Madrid, Spain), with high total arsenic concentration levels (up to 3,500 µg g⁻¹) [2], due to their higher mobility and the risk associated to their reintegration into other environmental compartments. For this purpose, roots and aboveground parts were analysed separately, to assess possible transformations from translocation processes. Extractions were carried out with deionized water by microwave-assisted extraction, at a temperature of 90 °C and three extraction steps of 7.5 min each. Total extracted arsenic concentrations were determined by ICP-AES, showing extraction percentages from 9 to 39%. Speciation studies were performed by HPLC-(UV)-HG-AFS [3], and they showed the main presence of As(V) (up to 350 µg g⁻¹), followed by As(III), in both plant parts. Monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO) were also found only in some plants. On the other hand, the use of 0.5 mol L⁻¹ acetic acid as extractant led to higher extraction percentages (33-87%), but lower column recoveries, probably due to the extraction of arsenic compounds different to toxic free ions studied, which may come from biotransformation mechanisms carried out by plants to reduce arsenic toxicity. However, As(V) concentrations increased up to 800 µg g⁻¹ in acid medium, indicating the probable release of As(V) from organoarsenic compounds and therefore a higher potential risk for the environment [4].

From the easily soluble, water-extractable arsenic species concentration levels, it can be drawn that between 70 and 89% of the total arsenic in plants must have been biotransformed, so it is present under the form of different arsenic compounds. Some of these compounds are likely to be extracted with acetic acid, which seems to induce the release of As(V) from organoarsenic compounds, showing a higher potential risk for the environment. This high level of biotransformation of most part of arsenic could explain the survival capacity of the plant species studied, despite their high total arsenic concentrations. Still, elevated arsenic concentration levels remain as toxic forms, predominantly as arsenate, reaching up to 190 µg g⁻¹ considering roots and aboveground parts separately, and 350 µg g⁻¹ considering the sum of both plant parts (more than double in acid medium), which may constitute an environmental risk due to its possible reintegration to the environment. Therefore, the study and control of native plants growing in arsenic polluted soils is a relevant factor for environmental safe.

## References:

1. F.J. Zhao, J.F. Ma, A.A. Meharg and S.P. McGrath, *New Phytol.*, 2009, 181, 777-794.

2. Sara García-Salgado, D. García-Casillas, M.A. Quijano-Nieto and M.M. Bonilla-Simón, *Water Air Soil Pollut.*, 2012, 223, 559-572.

3. Sara García-Salgado, M.A. Quijano and M.M. Bonilla, *Anal. Chim. Acta*, 2012, 714, 38-46.

4. Sara García-Salgado, M. Ángeles Quijano, *Environ. Sci.: Processes Impacts*, 2014, Advance Article (DOI: 10.1039/C3EM00624G; first published online 10 Feb 2014).

**Acknowledgements:** This work was financially supported by Ministerio de Educación y Ciencia (project CTM2007-66432) and Technical University of Madrid (project 188/Q105815-102).

<figure><img src="image_373.png" /></figure>
# PHYTOREMEDIATION AND ENVIRONMENTAL RISK ASSESSMENT: A NEW APPROACH

Nelson Marmiroli<fnref n="1" />, Elena Maestri<fnref n="1" />, Valeria Giovannelli<fnref n="2" />

Corresponding author email: elena.maestri@unipr.it

## ABSTRACT

Phytotechnologies applied to environmental remediation are considered green technologies. Nevertheless, despite the wide range of their potential applications and clear advantages, few research activities have been focused on their potential impacts. EPA has listed some of the possible adverse effects and highlighted the need of a Risk assessment applied to phytoremediation processes. Taking into account that phytoremediation is a continuum of different processes, the Environmental Risk Assessment (ERA) procedure applied to phytoremediation should be carried out on a case by case basis, meaning that its results would depend on the plants, the potential receiving environment(s) and the contaminant. At the same time the whole assessment has to be science based; it requires a complex procedure that needs to be transformed into an operational and standardized tool. In this paper we propose a methodology that aims to answer to these requirements. It has been developed starting from an Operating Model for the Environmental Risk Assessment (OMERA) applied to the environmental release of Genetically Modified Plants (M. Lener, V. Giovannelli, et al. 2013. Applying an operating model for the environmental risk assessment in Italian Sites of Community Importance (SCI) of the European Commission Habitats Directive (92/43/EEC), *Bulletin of Insectology* 66:257-267). It is mainly addressed to scientists, companies, regulators, environmentalists, also aiming to provide the stakeholders with a scientifically correct source of information. The ERA structure moves from the assumption that the occurrence of a risk is strictly related to the presence of four components: a) source b) diffusion factors c) migration routes d) receptors. Source is the site where the organism is released and/or enabled to express its harmful characteristics; diffusion factors are linked to biological characteristics of the plant; migration routes are linked to chemical, physical, biological characteristics of the receiving environment; receptors include humans, animals and ecosystems. The ERA structure comprises two main components: a conceptual model represented as a flowchart and an electronic Questionnaire (eQ) driven by a relational database (Microsoft Office Access). The eQ includes two kinds of question sets: the first is descriptive and allows the characterization of the topic to be analysed in each step; the second type is a so called on/off question, asking for a yes/no/don't know answer that can open or close specific paths of risk definition. According to a precautionary approach, when a "don't know" answer is given, highlighting a lack of knowledge, the system automatically selects the path that represents the worst-case scenarios. The on/off questions allow the user to follow the flow chart as a decision tree starting from the source, through all the components of the conceptual model, and leading to the identification of potentially affected receptors (if any) and related risks impacts (if any).

Principles end structure of OMERA have been modified to be adapted to phytoremediation. For example, the source characterization includes the contaminant, the phytotechnology applied and the potential use of amendments. In case of phytoremediation, the use of alien species is considered as a secondary source of risks and specifically analyzed. The resulting eQ is composed by almost 300 questions, which are not all activated on a case by case basis. Once the eQ is compiled the system will produce a report containing all the information and data inserted and a list of the identified potential effects. The OMERA methodology will be applied to different risk scenarios to be validated. It is useful to stress that the proposed model can be applied both *ex ante* and *ex post* release: in the *ex ante* phase to identify potential effects, in the *ex post* release phase to select Risk Management (RM) procedures, to set up monitoring activities, and to verify the success of the RM procedures applied.

Phytoremediation is a technology that meets requirements for a sustainable development, but as every human activity is not risk free; furthermore as for other issues it would be really useful and constructive to have the possibility to analyze and to collect research data and results in a standard way. The application of the OMERA method can be considered a tool for collecting and elaborating data in a standardized way, and would help the harmonization of the Environmental Risk Assessments performed by different actors.

<figure><img src="image_374.png" /></figure>

<footnote n="1">¹Dept. of Life Sciences, University of Parma, Italy</footnote>

<footnote n="2">²Dept. of Nature Protection, Italian National Institute for the Environmental Protection and Research, Italy</footnote>
# EUROPEAN AND NATIONAL LEGISLATION WITH REGARDS TO THE DIFFERENT STEPS IN A PHYTOREMEDIATION APPROACH FOR METAL-CONTAMINATED LAND

Hoppenbrouwers Marianne<fnref n="1" />, Witters Nele<fnref n="1" />, Vangronsveld Jaco<fnref n="1" />, Mench Michel<fnref n="2" />, Bert Valerie<fnref n="3" />, Gaucher Rodolphe<fnref n="3" />, Vanheusden Bernard<fnref n="1" />

Corresponding author email: nele.witters@uhasselt.be

## ABSTRACT

When applying phytotechnologies for management of metal-contaminated land, many questions are raised with regard to legislative issues. Within the REJUVENATE and GREENLAND projects we try to answer them using a flow approach in which each step when considering and applying phytotechnologies is examined: (1) describing the land status and use, (2) planting/sowing, (3) growing, (4) harvesting and (5) processing the crops, and (6) using the remainders. The focus is on European legislation as a basis for national rules (Directives) or as directly enforceable (Regulations), followed by legislation of the Member States and then, if relevant, regional legislation. Strategic communications of the Commission and proposals for new legislation are considered only when it is probable that these will become formal obligations in the near future. National legislation has been assessed in detail for all countries represented within the GREENLAND project.

In the first step, the status and use of the land, and the implications with regards to soil threshold values and the use of crops are defined. In the second step (planting/sowing), international regulation on crop selection, and more specifically on invasive and exotic species, and genetically modified organisms, as well as on soil management, e.g. soil amendments for stabilisation versus the enhancement of 'bioavailability' are considered. With regards to step three (growing), the principles of good agricultural practice are checked, e.g. concerning the use of pesticides. The classification of the harvested crop (step four) based on European and national legislation is complemented with an expert view. For the fifth step (processing), we focus on energy conversion of the biomass (e.g. pyrolysis) and on requirements with regards to input and output threshold values. In a final step we concentrate on the use or disposal of the remainders which contain metals in increased concentrations, such as the biochar.

The result of a complete analysis of legislation applicable throughout the entire phytoremediation cycle allows us to provide an objective overview to policy makers and regulators, who are now often overwhelmed and maybe even misled by the whole range of potentially related legislation.

**Acknowledgements:** The authors are grateful for financial support from the OVAM within the European Snowman Network (REJUVENATE), and the European Commission under the Seventh Framework Programme for Research (FP7-KBBE-266124, GREENLAND)

<figure><img src="image_375.png" /></figure>

<footnote n="1">¹Centre for Environmental Sciences (CMK)/Hasselt University, Belgium</footnote>

<footnote n="2">²UMR BIOGECO, Institut National de la Recherche Agronomique (INRA)/University of Bordeaux, France</footnote>

<footnote n="3">³Institut National de l'Environnement industriel et des Risques (INERIS), France</footnote>
# DOUBLE WIN: PHYTOREMEDIATION SAFEGUARDS INCOME WHILE MINIMIZING HEALTH RISKS IN AN AGRICULTURAL REGION

Witters Nele<fnref n="1" />, Van Passel Steven<fnref n="1" />, Vanheusden Bernard<fnref n="1" />, Ruttens Ann<fnref n="2" />, Mendelsohn Robert O.<fnref n="3" />, Meers Erik<fnref n="1" />,<fnref n="4" />, Tack Filip<fnref n="4" />, Vangronsveld Jaco<fnref n="1" />

Corresponding author email: nele.witters@uhasselt.be

## ABSTRACT

In the Campine region (North-East of Belgium) a large agricultural area is contaminated with mainly cadmium, lead and zinc, and legal food threshold values and soil standards for cadmium are often exceeded. Uptake by crop plants is a significant route of cadmium transfer into the human food chain. Besides human health risks, there are economic concerns, i.e. for the farmers facing contaminated land who can no longer grow high income vegetables for the risk of their harvest being confiscated. Phytoremediation has often been suggested as an alternative remediation technology because it is a solar driven, low impact option that can be carried out at much lower cost than conventional methods. Its actual implementation lags behind because until now there does not exist a decision tool for the (gradual) integration of phytoremediation crops in an agricultural system. In this paper, food safety values are used to decide on the optimal introduction in time of different phytoremediation crops and vegetables based on phytoextraction capacities and economic benefits of these crops. The tool is then applied on the Campine region where about 280 km<fnref n="2" /> is contaminated with mainly cadmium, while the region largely depends on agriculture. Results show that in the given region, (1) based on food threshold values, phytoremediation is chosen 15% over growing the allowed crop at the current cadmium concentration, and that (2) using the decision model based on food threshold values, safe use of the land including phytoremediation when economically optimal, results in costs that are twice as low as conventional remediation. Given the fact that in the Flemish region there are 85,000 potentially contaminated lands, we show that considering and applying phytoremediation when appropriate results in considerable cost savings, while minimizing human health risks.

<figure><img src="image_376.png" /></figure>

<footnote n="1">¹Centre for Environmental Sciences (CMK), Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium</footnote>

<footnote n="2">²CODA-CERVA (Veterinary and Agrochemical Research Centre), Leuvensesteenweg 17, 3080 Tervuren, Belgium</footnote>

<footnote n="3">³School of Forestry & Environmental Studies, Yale University, 195 Prospect Street, New Haven, CT 06511, USA</footnote>

<footnote n="4">⁴Department of Applied Analytical and Physical Chemistry, Ghent University, Coupure 653, 9000 Ghent, Belgium</footnote>
# THE EFFECT OF SILICON ON THE UPTAKE AND TRANSLOCATION OF ARSENIC IN TOMATO (SOLANUM LYCOPERSICUM L.)

M. Marmiroli<fnref n="1" />, V. Pigoni<fnref n="1" />, M.L. Savo-Sardaro<fnref n="1" />, N. Marmiroli<fnref n="1" />

Corresponding author email: marta.marmiroli@unipr.it

## ABSTRACT

The metalloid element Arsenic (As) is toxic to most life forms; in humans, its ingestion has been associated with a whole series of pathologies leading to its classification as a carcinogenic. Si, the second most abundant element in the earth's crust occurs within soils mainly in the form of inert quartz or crystalline silicate; however, monosilicic acids are soluble and thus available to plants and microbes. Absorbed Si is beneficial for plant growth, largely because of its role in combating biotic and abiotic stress. Tomato is a major horticultural crop in both Europe and the US, but its fruit can be compromised by As contamination. Feeding of Si to tomato crops has been used to alleviate drought and salinity stress, but to date no attempt has been made to correlate Si supplementation with As uptake and its translocation to the aerial part of the plant. Here, we show that Si treatment can indeed influence As uptake in tomato. In addition, we demonstrate that tomato cultivars differ from one another with respect to their capacity to take up and translocate As, whether or not the plants are supplied with soluble Si. During previous experiments on eight different tomato cultivars we were able to discriminate that four of them translocate As to the berries, with and without Silicon treatments, in different concentrations according to the cultivar.

Transcription profiling related to three genes associated with the abiotic stress response was carried out for cvs. Aragon and Gladis, which were able to concentrate As inside the fruit, even when Si was administered. We choose the genes: Gluthatione Reductase gene (LeGR), Heat Shock Protein gene (Hsp90-1) and Phytochelatin Synthase gene (Phyt1200), because in previous studies they were demonstrated to be involved in As stress response, for RT-qPCR experiments. The relative abundance of each transcript was calculated from the average Ct of each amplification according to the 2^(-ΔΔCt) method. Exposure to NaAsO2 up-regulated Glutathione Reductase (LeGR) in the leaf to a greater extent in cv. Aragon than in cv. Gladis. The up-regulation of phytochelatins synthase (Phyt1200) in response to exposure to NaAsO2 was less intense than for LeGR, and the two cultivars did not respond differentially. For LeHsp90-1, the presence of NaAsO2 in the growth medium did not induce any change in transcript abundance in either cultivars.

<figure><img src="image_377.png" /></figure>

Transcript abundance of selected stress-associated genes as assessed by qPCR. The data have been normalized to the abundance of the reference expressed sequence (exp (SGN-U346908) endogenous) utilized as internal control

<figure><img src="image_378.png" /></figure>

<footnote n="1">¹Department of Life Sciences, University of Parma, 43124 Parma, Italy</footnote>
<figure><img src="image_379.png" /></figure>
<figure><img src="image_380.png" /></figure>

MOLECULAR ENGINEERING
<figure><img src="image_381.png" /></figure>
# USE OF POPLAR FOR REHABILITATION OF METAL POLLUTED SOILS

Van Anh Le Thi<fnref n="1" />, Mathieu Pottier<fnref n="1" />, Annabelle Dejardin<fnref n="2" />, Gilles Pilate<fnref n="2" />, Sébastien Thomine<fnref n="1" />

Corresponding author email: lethi@isv.cnrs-gif.fr

## ABSTRACT

Phytoextraction is the use of plants to remove heavy metals from soils contaminated by industrial activities, such as smelting or disposal of high metal wastes. Poplar is well adapted for phytoextraction because it tolerates growth on contaminated soils and produces high biomass. In addition, poplar is also suitable for molecular genetic studies because its genome was sequenced in 2006 and it is amenable to transgenic technologies. However, previous reports indicated that metal accumulation was the highest in leaves causing metal return to the soil after leaf abscission. In order to circumvent this problem, genetic engineering can be used to limit metal accumulation in leaves or direct metal accumulation in poplar trunks using relevant metal transporter genes under the control of tissue-specific promoters.

We first focused our study on the functional characterization of suitable metal transporter genes. We selected candidate transporters potentially controlling metal storage in the vacuole, the main compartment where heavy metals are accumulated in plant cells. The NRAMP (Natural Resistance Associated Macrophage Protein) and the IREG (Iron Regulated Gene) metal transporter families, which are still poorly characterized in poplar, have been investigated. Based on the knowledge gained from their homologues in *Arabidopsis thaliana*, PtNRAMP3.1 and PtNRAMP3.2 are putatively involved in metal release from the vacuole while PtIREG1 is putatively involved in metal sequestration in the vacuole. We showed that both PtNRAMP3.1 and 3.2 are functional metal transporters but only PtNRAMP3.2 is targeted to the vacuolar membrane. In contrast, PtNRAMP3.1 is targeted to intracellular compartments. In addition, the function of PtIREG1 putative metal transporter gene was investigated by heterologous expression in yeast: PtIREG1 was shown to be able to complement the hypersensitivity of mutant strains to Ni but not to other metals, including Co, Cd, Fe, Mn and Zn.

Transgenic poplars overexpressing PtNRAMP3.1 or PtNRAMP3.2 are being assessed for metal tolerance and accumulation. When grown on non-contaminated soil, wild-type and transgenic poplar lines exhibit clear differences in leaf metal concentrations (Cu, Fe, Mg, Mn and Zn). Metal accumulation will also be measured in transgenic poplar lines grown on metal-contaminated soil. Arabidopsis and poplar plants overexpressing PtIREG1 as a fusion with the fluorescent protein GFP (PtIREG1-GFP) are being generated. This should allow identification of the subcellular compartment where PtIREG1 resides and analysis of the effect of PtIREG1 expression on metal tolerance and accumulation. Besides, PtIREG1-GFP under control of the CAD promoter, which targets expression in poplar xylem cells, was introduced in poplar to specifically drive metal accumulation in wood.

**Acknowledgements:** This work was supported by the Agence Nationale de la Recherche (PHYTOPOP Project ANR-06-ECOT-O15-01, the CNRS and a fellowship to Van Anh Le Thi from the Vietnamese Government to the PhD training program in France for future lecturers-researchers of University of Science and Technology of Hanoi (USTH).

<figure><img src="image_382.png" /></figure>

<footnote n="1">¹Institut des Sciences du Végétal, CNRS UPR 2355, 91198 Gif-sur-Yvette Cedex, France</footnote>

<footnote n="2">²Unité Amélioration Génétique et Physiologie Forestières, UR0588, INRA-Val de Loire, 45075 Orléans Cedex 2, France</footnote>
# PREPARATION OF TRANSGENIC PLANTS STIMULATING BACTERIAL DEGRADATION OF PCBs

Veronika Kurzawova, Martina Chovancova, Jitka Viktorova, Tomas Macek, Martina Novakova

Institute of Chemical Technology in Prague, Dpt of Biochemistry and Microbiology, Prague, Czech Republic

Corresponding author email: suram@vscht.cz

## ABSTRACT

Polychlorinated biphenyls (PCBs) pose a serious problem as a widely spreaded contaminants. A possible choise for remediation can be the use of microorganisms, plants and their consortia. Some bacteria aerobically degrade PCBs using the pathway determinated in biphenyl operon. Expression of biphenyl operon genes is inducible and it was found that several plant secondary metabolites including flavonoids and terpenes can serve as efficient inducers. In this work we investigated that plant secondary metabolite quercetin can serve also as an inducer of *bphA* gene, a gene of bacterial biphenyl operon. Concerning these results we decided to prepare transgenic plants of *Nicotiana tabacum* overexpressing gene for flavonoid-3'-hydroxylase (*AtF3'H*) from *Arabidopsis thaliana*, the enzyme converting plant indigenous kaempferol to quercetin.

Gene for flavonoid-3'-hydroxylase (*AtF3'H*) was fused with sequence for hexahistidine tail and the tagged gene was cloned under the root specific promoter in the pGreen0029 vector. Prepared construct was introduced into the *Agrobacterium tumefaciens* for plant transformation. Because of the root specific promoter the expression of *AtF3'H* gene would be targeted to plant roots with final overproduction of the quercetin in the root and its release to the rhizosphere. This should lead to stimulation of PCB degradation by rhizospheric bacteria. Prepared transgenic plants are nowadays under the investigation.

**Acknowledgements:** The work was supported by GACR P501/12/P521.

<figure><img src="image_383.png" /></figure>
# PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF N₂-FIXING BACTERIA NODULATING LEGUMINOUS SPECIES ON SOILS FROM MINING AREAS

Wesley de Melo Rangel<fnref n="1" />⁻<fnref n="4" />, Paulo Ademar Avelar Ferreira<fnref n="2" />, Silvia Maria de Oliveira Longatti<fnref n="3" />, Daiane Silva Bonaldi<fnref n="3" />, Fatima Maria de Souza Moreira<fnref n="3" />, Jaco Vangronsveld<fnref n="4" />

Corresponding author email: wesleyrangeu@gmail.com

## ABSTRACT

Mining soils are often devoid of natural recolonization, necessitating human intervention to restore a vegetation cover. A promising technique for rehabilitation of these areas is the phytoremediation. The success of phytoremediation is associated with microbial and their essential biological processes taking place in the rhizosphere or inside the plants, which benefits their growth and development. Among the microbial processes biological N₂ fixation (BNF) is highlighted, it is only performed by a group of prokaryotes able to convert the atmospheric N₂ into NH₄. Among these prokaryotes, bacteria that establish mutual symbiosis with leguminous plants (N₂ fixing bacteria nodulating leguminous - NFBNL) are extremely important. The nodulated plants incorporate C and N to the soil, and increase their ability to absorb nutrients such as N, fixed by bacteria, which makes them more tolerant to environmental stresses.

Therefore, the aims of this study were (i) isolate, (ii) characterize phenotypically and (iii) identify NFBNL species from nodules of leguminous plants growing on gold mine soils, contaminated with arsenic, and zinc mine soils, contaminated with Zn, Cd and Pb.

The strains were isolated from nodules of *Crotalaria spectabilis* and *Stizolobium aterrimum* growing on gold mine soils, and nodules of *Leucaena leucocephala* growing on zinc mine soils, in Brazil. Then the strains were reinoculated in their host plants to check their nodule induction. Further, they were characterized phenotypically for their production of organic acids, indol-acetic acid, and siderophores, and 1-aminocyclopropane-1-carboxylate (ACC-deaminase), and also for their capacity to solubilize phosphate, and perform nitrogen fixation.

**Acknowledgements:** The financial support by Edital 20/2008 PRONEX - Programa de Apoio a Núcleos de Excelência, Processo FAPEMIG CAG - APQ-00118-09, Edital MCT/CNPq/CT-Agro/CT-Hidro/MAPA-SDC-SPAE nº 44/2008 - Recuperação de Áreas Degradadas, Processo CNPq 577513/2008-7, and Edital 30/2013 MCTI/CNPq/CT-Biotec - Pesquisa, Desenvolvimento e Inovação em Biotecnologia para solução de problemas ambientais, Processo CNPq 402858/2013-1, the research productivity fellowship granted to F. M. S. Moreira by the National Council for the Scientific and Technological Development (CNPq), and the student's fellowships granted by the CNPq and the Commission for Improvement of Higher Education Staff (CAPES) are greatly appreciated.

<figure><img src="image_384.png" /></figure>

<footnote n="1">¹Biology Department, Agricultural Microbiology Post Graduate Program, Federal University of Lavras (UFLA), PO Box 3037, Lavras, Minas Gerais, 37.200-000, Brazil</footnote>

<footnote n="2">²Soils Department, Federal University of Santa Maria, CEP: 97.105-900, Santa Maria, Rio Grande do Sul, Brazil</footnote>

<footnote n="3">³Soil Science Department, Soil Science Post Graduate Program, Federal University of Lavras (UFLA)</footnote>

<footnote n="4">⁴Centre for Environmental Sciences, Hasselt University, B-3590 Diepenbeek, Belgium</footnote>
# PREPARATION OF TRANSGENIC PLANTS WITH IMPROVED RESISTANCE TO STRESS

Jitka Viktorova, Lucia Kazimirova, Petra Lovecka, Tomas Macek

Dept of Microbiology and Biochemistry, Institute of Chemical Technology in Prague, CZ

Corresponding author email: prokesoj@vscht.cz

## ABSTRACT

Osmotin was discovered in 1987 in cells of *Nicotiana tabacum* adapted to high level of salt. Later, it was found in other plant species, both monocots and dicots. This protein is involved in plant responses to biotic and abiotic stress. The antimicrobial activity against some fungi was already published as well as its cryoprotective and osmolyte role.

For the purpose of subsequent genetic manipulation, osmotin (*OSM*) gene was amplified using PCR with the genome DNA of Virginia tobacco (*Nicotiana tabacum*) as the template. The *OSM* fused with the oligonucleotide coding for hexahistidine tag (*His-OSM*) was expressed in *Escherichia coli* BL21 cells. After verification of the successful expression and localization, the recombinant osmotin was purified and renaturated. The native conformation of the isolated protein was confirmed using MALDI TOF MS, infrared and Raman spectroscopy.

The recombinant osmotin was subsequently used for testing its antimicrobial activity against food and human pathogens. Significant antimicrobial activity was detected against the causers of candidiasis and otitis (*Candida parapsilosis* and *Candida tropicalis*), and against food pathogens *Debaryomyces hansenii*, *Torulaspora globosa* and *Saccharomycodes ludwigii*, which cause for example souring of wines and musts. Simultaneously, the hemolytic activity and toxicity of osmotin against human embryonic kidney cell was examined. The results demonstrated harmlessness of osmotin to human cells at high concentrations.

The model plant species tobacco served as the second expression system. Coding sequence of *His-OSM* was inserted into the vector pGreen0029 under the control of cauliflower mosaic virus constitutive promoter. The obtained plasmid was transferred into *Agrobacterium tumefaciens* C58-C1 cells. The successful transient expression of *His-OSM* gene was verified by agrobacterial infiltration in both dicotyledonous and monocot plants. Transgenic plants bearing *His-OSM* gene were prepared by the agrobacterial cocultivation method. The presence of the transgene was detected on the DNA level and the transgene copy number was determined by quantitative PCR. The mRNA quantification demonstrated strong expression of the transgene.

The observation of seeds germination and chlorophyll content in the presence of NaCl showed higher tolerance of the transgenic tobacco plants to osmotic stress. Tobacco plants with increased production of osmotin can serve as a useful model for the preparation of agricultural crops with higher tolerance to biotic and abiotic stress. The first attempts to transformation of flax (*Linum usitatissimum* L.) and potato (*Solanum tuberosum* L.) plants by osmotin transgene have been performed.

**Acknowledgements:** The financial support by GACR project No. 501/11/1650 is greatly appreciated.

<figure><img src="image_385.png" /></figure>
<figure><img src="image_386.png" /></figure>

BIOCHAR
<figure><img src="image_387.png" /></figure>
# INVESTIGATIONS WITH BIOCHAR AND HALOPHYTES IN THE PHYTOREMEDIATION OF A HIGH SALINITY LANDFILL

Kaitlin McSorley<fnref n="1" />, Barbara A. Zeeb<fnref n="1" />, Allison Rutter<fnref n="2" />

Corresponding author email: kmcsorle@rmc.ca

## ABSTRACT

Soil salinity is an issue that affects the structure and productivity of nearly 1 billion ha of soil worldwide. The landfilling of cement kiln dust (CKD) represents an anthropogenic cause of soil salinity, and contributes to the creation of marginal salinized lands. Preliminary investigations of a CKD impacted landfill in Bath, Canada, indicate chloride concentrations nearly 1000 times that of average Canadian soils. The objectives of this research are to determine the ability of halophytic grasses to grow at a CKD impacted landfill as well as their ability to extract soil chloride. Greenhouse trials with CKD soil and a 10% (w/w) biochar amendment were conducted to evaluate the effects on the germination and growth of 13 warm and cool season halophytic perennial grass species. In addition, this research investigates the effects of biochar on high salinity soil and water through sorption experiments to determine its ability to reduce electrical conductivity (EC) and sodium adsorption ratio (SAR). Results from greenhouse germination and transplant studies along with two seasons of field work will be presented and discussed. Phytoremediation represents a low cost, low maintenance, *in situ* remediation option that could reduce the impact of industry induced soil salinity, while at the same time making use of marginal salinized soils to grow halophytic perennial grasses.

*Acknowledgements:* The financial support by The Natural Sciences and Engineering Research Council of Canada for their grant to Dr. Barbara Zeeb and Dr. Allison Rutter is greatly appreciated.

<figure><img src="image_388.png" /></figure>

<footnote n="1">¹Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Canada</footnote>

<footnote n="2">²School of Environmental Studies, Biosciences Complex, Queen's University, Kingston, Canada</footnote>
# BIOCHAR IMMOBILIZES RISK ELEMENTS AND IMPROVES WILLOWS BIOMASS PRODUCTION ON HEAVILY CONTAMINATED SOIL

Kateřina Břendová, Jiřina Száková, Pavel Tlustoš

Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague – Suchdol, Czech Republic

Corresponding author email: brendova@af.czu.cz

## ABSTRACT

Currently, soil contamination is one of the most serious problems of soil protection, while the so-called old loads are the most significant threats to human health. Willows were described as suitable plants for phytoremediation, however extremely high content of risk element restrict biomass production of these trees [1]. The availability of risk elements can be possibly reduced by soil additives. Recently, the attention has been focused on the research of unconventional soil amendment additives - biochar. In the last decade, biochar ability to bind undesired contaminants, organic [2] and inorganic materials, heavy metals [3,4] has been widely described

The aim of this study was to observe the effect of elevated rates of biochar application on risk elements transport through the soil profile, then the effect of biochar amendment on plant growth as well contaminant accumulation in willow tissues was evaluated.

The experiment was established at greenhouse controlled conditions. To observe risk element content in leachate, the lysimeter (40 cm) pots were used. *Salix Smithiana* was chosen as an experimental crop. The leachate was analysed (ICP-OES)) each 3 weeks during vegetation. The trees were harvested twice, firstly in July, secondly in early October to test maximum accumulation potential. The twigs and leaves were analysed separately. The experiment consists of 4 treatments: control (no applied biochar), and rates of 5%, 10%, and 15% of biochar from total mass of soil. At each treatment two willows cuttings were planted.

The results showed that 5% of biochar significantly increased biomass production; the increasing biochar rates didn't influence biomass production. Willows leaves at control treatment showed extreme phytotoxic signs. Biochar application significantly decreased lead uptake into plant tissues by 53% both in leaves and twigs when 5% of biochar was applied at first harvest, at second harvest by 77% in leaves. However zinc and cadmium uptake wasn't restricted significantly at 5 % treatment. Cd uptake significantly decreased by 32%, and 34% in leaf tissues at treatment of dose of 15% biochar amendment at first harvest. In leachate, both cadmium and zinc content decreased to values below detection limits at all biochar treatments and lead wasn't detected at these treatments.

Thanks to this study, we can summarize that biochar appears to be a very effective regulator of availability of observed risk elements and improver agent for biomass production of remediation plants.

## References:

1. Vysloužilová M., Tlustoš P., Száková J. (2003) Plant Soil Environment 49, 542-547

2. Jones D. L., Edwards-Jones G., Murphy D. V. (2011) Soil Biol Biochem 43, 804-813

3. Uchimyia M., Klasson K. T., Wartelle L. H., Lima I. M. Chemosphere 82, 2011, 1438-1447

4. Zhang, Z.; Solaiman, Z. M.; Meney, K.; Murphy, D. V.; Rengel, Z. J Soil Sediment 2012 DOI: 10.1007/s11368-012-0571-4

**Acknowledgements:** The financial support by CIGA project contract No. 20132007 is greatly appreciated.

<figure><img src="image_389.png" /></figure>
# EFFECTIVENESS OF BIOCHAR FOR INCREASING SOIL QUALITY AND REDUCING METAL BIOAVAILABILITY OF THREE DIFFERENT SOILS

**Markus Puschenreiter**<fnref n="1" />, **Wolfgang Friesl-Hanl**<fnref n="2" />, **Gerald Dunst**<fnref n="3" />, **Mario Wagner**<fnref n="4" />, **Franz Zehetner**<fnref n="1" />, **Jasmin Karer**<fnref n="2" />, **Anna Wawra**<fnref n="1" />,<fnref n="2" />, **Jakob Fessl**<fnref n="1" />, **Christoph Hoefer**<fnref n="1" />, **Gerhard Soja**<fnref n="2" />

*Corresponding author email: markus.puschenreiter@boku.ac.at*

## ABSTRACT

Anthropogenic activities, including intensive agriculture and industrial activities, have led to a general decline of soil quality in the last decades. In this context, the loss of organic matter in soils and the contamination with heavy metals are two major problems. Since large areas are affected, low-cost and environmentally friendly approaches are needed for the remediation of degraded or polluted soils. The application of amendments may improve the soil quality, increase productivity and reduce the bioavailability of pollutants. Biochar has often been considered as an efficient and effective soil additive. In an on-going Austrian research project, several types of biochar are tested on two agricultural soils low in organic matter and one agricultural soil contaminated with Pb, Zn and Cd. For all three soils, the suitability of the biochar-amended soils for renewable biomass production using maize or Miscanthus is tested.

In a preliminary experiment, the following types of biochar were tested in different application rates: (i) cellulosic fiber sludge + cereal husks; (ii) Poplar wood chips biochar; (iii) Miscanthus biochar. All three types were produced by the company Sonnenerde and enriched with compost, and nitrogen ((NH₄)₂SO₄). On the contaminated soil, also lime and a 50:50 mixture of gravel sludge and siderite bearing material was used for comparison. The effectiveness was assessed by measuring plant biomass of *Lolium multiflorum* and nitrogen availability and uptake, and for the contaminated soil by additionally determining extractable metal concentrations in soil and metal uptake in plants (Cd, Cu, Ni, Pb, Zn). The three most effective treatments were subsequently also realised in two-year field experiments and in a parallel greenhouse pot experiment. Either maize or Miscanthus was planted in spring of 2013. Maize plants were harvested in October and Miscanthus plants were harvested in November. In parallel, soil samples were obtained for analysing C, N, and metals for the contaminated soil.

Preliminary results show that in case of the two soils initially low in organic matter, the nitrogen-enriched biochar were most effective in terms of increasing nitrogen supply, increasing organic matter content in soil and increasing plant biomass production. On one soil, the addition of nitrogen-enriched biochars led to a doubling of shoot biomass production compared to the untreated and unfertilized control.

On the contaminated soil, both the poplar biochar and the gravel sludge + siderite bearing material mixture were most effective in terms of reducing metal extractability and uptake in the plant biomass (*Lolium multiflorum*). In the case of Zn a significant decrease after lime, poplar BC and gravel sludge with siderite bearing material was detected. However, Zn remained above the phytotoxicity level of 200 mg kg⁻<fnref n="1" />; lime treatment reduced the Zn concentration in *Lolium multiflorum* to 513 mg kg⁻<fnref n="1" />, gravel sludge to 531 mg kg⁻<fnref n="1" /> and poplar BC to 560 mg kg⁻<fnref n="1" /> while in the control plot 713 mg kg⁻<fnref n="1" /> Zn were measured.

The results of the field experiment and the parallel greenhouse test will provide further information on the effectiveness over a longer period (i.e. 2 years). So far, it has been found that a mixture of biochar and inorganic additives were most effective in increasing biomass productivity of Miscanthus on the metal-contaminated soil in comparison to an equivalent nutrient supply with mineral fertilizer (+80 %).

**Acknowledgements:** The financial support by Austrian Research Promotion Agency (FFG), contract No. 836538, is highly appreciated.

<figure><img src="image_390.png" /></figure>

<footnote n="1">¹ University of Natural Resources and Life Sciences Vienna, Department of Forest and Soil Sciences, Peter Jordan Straße 82, A-1190 Wien; and Konrad Lorenz Straße 24, A-3430 Tulln, Austria</footnote>

<footnote n="2">² AIT Austrian Institute of Technology GmbH, Health & Environment Department, Konrad Lorenz Straße 24, A-3430 Tulln, Austria</footnote>

<footnote n="3">³ Sonnenerde Gerald Dunst - Kulturerden GmbH, Oberwarterstraße 100, A-7422 Riedlingsdorf, Austria</footnote>

<footnote n="4">⁴ Mario Wagner, Kaiser Franz-Josef-Straße 6, A-1230 WIEN</footnote>
# NANOSCALE INTERACTIONS BETWEEN ENGINEERED NANOMATERIALS AND BLACK CARBON (BIOCHAR) IN SOIL

Jason C. White, Joseph Hawthorne, Roberto De la Torre-Roche, **Alia D. Servin**

Department of Analytical Chemistry, CT Agricultural Experiment Station, New Haven, USA

*Corresponding author email: Alia.Servin@ct.gov*

## ABSTRACT

The increasing manufacturing and use of engineered nanomaterials (NMs) has resulted in potentially excessive yet poorly characterized environmental exposure. In agricultural systems, exposure may occur directly through the application of nano-enabled agrichemicals or indirectly through the wide scale amendment of soils with NM-contaminated biosolids. NMs that have been released into these environments will interact with soil constituents, including black carbon (BC), that may subsequently affect particle fate and toxicity to soil-based biota such as plant and earthworm species. Biochar is a form of black carbon that is being proposed as an amendment to soils so as to improve soil fertility and enhance crop production. Due to the highly porous micro- and nano-structure of biochar, one can anticipate potentially significant molecular interactions with nanomaterials. However, the interactions of biochar with NMs and the subsequent effect on the bioavailability and toxicity of these materials to representative crop species (zucchini, soybean, corn, lettuce) and soil invertebrates (earthworms) is unknown.

Studies have recently been initiated in which biochar from several different feedstocks and synthesis conditions were added separately to an agricultural and residential soil at three amendment rates of 0, 0.5 and 5% (v/v). Dispersed cerium oxide (CeO₂) or Ag nanoparticle solutions were added to containers to yield final concentrations of 0, 500, 1000 or 2000 mg/kg. Plant or earthworm species were subsequently added to the soils and were harvested after 28 days of exposure. To date, results from a single experiment have been analysed, although numerous additional experiments are currently underway. For soybean, there were minimal effects of pecan shell-derived biochar presence on Ce uptake or plant biomass; in several instances, the trend was for decreased NP uptake with char amendment, but there were no effects of statistical significance. Perhaps the most interesting finding was a significant decrease in pigment content/production in soybean upon nanoceria exposure, regardless of char presence. This is particularly interesting given the lack of biomass effects on soybean. Additional results focused on different types of biochar, plant species, and nanoparticles will be presented, as well as experiments focused on exposed earthworm species.

The results of these investigations will serve agriculture by providing a mechanistic foundation for the fate and bioavailability of NMs in biochar amended soil that will aid in the development of accurate nanotoxicity risk assessments.

*Acknowledgements:* The financial support of USDA NIFA grant 2011-03518 is greatly appreciated.

<figure><img src="image_391.png" /></figure>
# OPPORTUNITIES AND DRIVERS FOR BIOFUELS AND BIOCHAR PRODUCTION BY VALORISING AGRI-FOOD SOLID PROCESSING RESIDUES AND WASTES

D. Rovas, A. Zabaniotou

Biomass Group, Department of Chemical Engineering, Aristotle University of Thessaloniki, Univ. Box 455, 54124 Thessaloniki, Greece

Corresponding author email: azampani@auth.gr

## ABSTRACT

Food processing industry faces increasing regulatory pressure to reduce environmental impacts and provide sustainable management and use of wastes. The topic is gaining increased attention worldwide by the new advances in the integrated management of food processing waste of the global food & drink Industry, satisfying energetic needs of the production. However, most bioenergy systems are characterized by low efficiencies resulting in an increased cost of operation. Significant improvement in efficiencies could be achieved by developing systems that generate both power and heat. While, residues could be a useful energy source, they can also play a fundamental role in preserving soil fertility and contributing to carbon sequestration into the soils. For material and energy recovery efficiencies, a trade-off between the two targets should be accomplished. Small distributed gasification systems or pyrolysis systems could be a win-win solution. Gasification-based CHP integrated in a symbiotic way with the processing plant, can fulfil the needs on energy by using the residues while pyrolysis process can be optimized for the recovery of biochar and biofuels production. The application of biochar to the soil stabilizes a carbon fraction thus reducing the release of CO₂ in the atmosphere. This work presents a case study applied to an olive mill being a rural activity in the Mediterranean areas

**Acknowledgements:** This research has been co-financed by the European Union LIFE SMART, by European Union- European Regional Development Fund and Greek Ministry of Education/EYDE-ETAK through program ESPA 2007-2013 / EPAN II / Action "SYNERGASIA" (Project 1165) and European Social Fund - ESF) and General Secretariat for research and Technology through the Operational Program ". Acronym: Fertenergy.
# USE OF BIOCHAR AS A POST ANAEROBIC DIGESTION TREATMENT FOR AMMONIUM-NITROGEN REMOVAL FROM PIGGERY SLURRY

Kizito Simon<fnref n="1" />,<fnref n="2" />, Wu Shubiao<fnref n="2" />, Lei Ming<fnref n="2" />, Dong Renjie<fnref n="2" />

Corresponding author email: saviokizito@gmail.com or wushubiao@cau.edu.cn

## ABSTRACT

In China, anaerobic digestion (AD) with biogas supply is the major livestock manure treatment procedure and the estimated amount of biogas residues from livestock biogas projects is more than 200 million tons every year. Farm spreading for land fertilization is the most common disposal method for the residual slurry, however, due to the high loads of nitrogen in the slurry; farm land carrying capacity is often exceeded especially in off peak cropping seasons. Unless novel research solutions are sought to recover nutrients and recycle water, there is a higher risk of adverse pollution from these wastes. The purpose of this study therefore was to investigate NH₄-N sorption capacity of biochar from piggery liquid manure after anaerobic digestion.

The biochar was produced from mixed wood cuttings (WDB) at temperatures of 500°C and risk husks (RHB) at 300°C. Proximate Analysis was used to determine the Moisture content (MC), Ash, Fixed carbon (FC) and Volatile content (VC) of the Biochar in accordance to the ASTM D1762-84 standard method for chemical of wood charcoal analysis (ASTM, 1990). The untapped bulk density was determined as the mass of uncompressed biochar that filled a volume of 10ml tubes whereas the pH and electro conductivity (EC) were determined after 2hours shaking of mixture of 1g biochar in 20ml of double distilled at a constant water bath temperature of 90°C using pH and EC reader (FE20 METTLER TOLEDO, Switzerland and Orion Model 115A, Thermal Fisher Scientific) respectively. The surface area and total pore volume of the biochar were determined using BET method with N₂ absorption at 77K based on multipoint calculation technique. In addition, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR) techniques were used to further characterize the biochar morphology and chemistry. We also characterized the slurry on some important parameters that could affect our study. The studied parameters were determined using the APHA (1998) standard for wastewater analysis and they included; pH, TSS, BOD₅, NH₄N, PO₄-P and COD. The bulky density, % MC, % ash, % VC and % FC of the WDB were; 0.4994g/ml, 4.3%, 5.87%, 8.92% and 85.21% for WDB whilst for RHB it were 0.298g/ml, 4.3%, 45.96%, 44.67% and 17.34% respectively. The BET surface area, total pore volume, pH and EC for WDB were 273.63m²g⁻<fnref n="1" />, 0.176mlg⁻<fnref n="1" />, 9.8 and 411µscm⁻<fnref n="1" /> respectively whilst for RHB it were 10.995m²g⁻<fnref n="1" />, 0.0308mlg⁻<fnref n="1" />, 7.8 and 300 µscm⁻<fnref n="1" />. The slurry TSS was 150-175mg/L, pH ranged from 8.0-8.5, BOD₅ was 850-920mg/L, and NH₄-N was 1380-1450 mg/L, whereas PO₄-P and COD ranged from 15-25mg/L and 4000-4200 mg/L respectively. Ammonium sorption was studied using synthetic NH₄Cl [analytical grade] and the Anaerobic Digestion Slurry (ADS). From batch equilibrium and kinetics experiments the amount of NH₄-N absorbed as a function of biochar dosage, biochar particle size (0.25-1.25mm), initial effluent concentration, contact time, initial pH, and temperature was quantified after equilibration for 24 hours in an end-to end shaker with a constant water bath temperature of 318 K using 50ml of effluent solution (adjusted pH of 7.0) loaded onto 1g of biochar.

From the results, absorption was higher with pure NH₄Cl solution than with the slurry. The lower absorption with the slurry could be attributed to presence of other competing ions such metals present in the slurry. Furthermore, there was positive increase in absorption with pH, temperature, initial concentration, and contact time for two types of biochar. Wood biochar performed better than RHB with pure NH₄Cl but the difference with the slurry was not big as it would be expected considering the difference in BET surface area. The maximum adsorption of NH₄-N at 1400mg/L in the slurry was 44.64 ± 0.602 mg·g⁻<fnref n="1" /> and 39.8 mg·g⁻<fnref n="1" /> ± 0.54 for WDB and RHB respectively. The Langmuir model fitted the sorption data better with R<fnref n="2" /> = 0.973(NH₄Cl) and 0.897(slurry) for RHB and 0.986 (NH₄Cl) and 0.8902 (slurry) for WDB. From the results, we conclude that biochar has potential to reduce nitrogen loading in piggery farm waste water and we suggest that biochar filters can be developed and used as the first line effluent recipients prior to release into lower N concentration degradation systems.

<figure><img src="image_392.png" /></figure>

<footnote n="1">¹PhD Candidate, School of Engineering, China Agricultural University</footnote>

<footnote n="2">²Key Laboratory for Bioenergy Engineering and Low Carbon Technology, School of Engineering, China Agricultural University, 100083 Beijing China</footnote>
# BIOCHAR INFLUENCE ON CeNPs LEACHING AND PLANT UPTAKE IN LEPIDIUM SATIVUM FROM PACKED-LYSIMETER EXPERIMENT

Guido Fellet, Alessandro Mattiello, Rita Musetti, Luca Marchiol

Department of Agriculture and Environmental Science, University of Udine, Italy

Corresponding author email: [guido.fellet@uniud.it](mailto:guido.fellet@uniud.it)

## ABSTRACT

The use of metal-nanoparticles (MeNPs) has been increasing over the last few years. Their presence in soil may be detrimental to the entire ecosystem; it has been shown that they may have negative effects on organisms' reproduction, mobility and survival. The magnitude of such impact depends also on the MeNPs mobility, other than the MeNPs bioavailability. Nanoparticles interact with the soil aggregates and both the physical and chemical properties of the soil influence the MeNPs mobility.

Porous media can interfere with the mobility of contaminants in soil, hence with MeNPs. Being the biochar a porous material with charged surface groups, it is expected to have an influence on the balance of the MeNPs transport in natural soils. Biochar notoriously changes the soil properties such as pH, EC and others that may have an impact on the MeNPs aggregation and mobility.

Biochar may immobilize the soil MeNP by increasing the cation exchange capacity but also by straining due to the high porosity. The difference in MeNPs mobility will be considered in soil amended with two biochars which differ only for the quenching process, that is, water or dry quenching. In the first case, we hypothesize that water quenching, by removing the ash from the pyrolyzed biomass and increasing the BET and porosity of the biochar, affects the interaction with the MeNPs and reduces the leaching.

Soil collected from a residential area in NE of Italy was mixed with Ce-NPs at 1,000 mg kg⁻¹ and amended with biochar from wood-pellets quenched with either water or without water at the dose of 5% (dw/dw). Twelve column-lysimeters packed with the soil mixtures will be seeded with watercress (*Lepidium sativum*). The leachates will be collected and analysed to evaluate the amount of NPs leached from the soil treated with the two biochars. At the end of the experiment, the plants will be harvested and analysed to measure the Ce uptake and the presence of CeNPs at cellular level as well as in the leachate will be verified by means of transmission and scanning electron microscopy.

<figure><img src="image_393.png" /></figure>
# A STUDY ON THE CHARACTERISTICS OF BIO-CHAR WITH THE SLUDGE OF SEWAGE BY LOW TEMPERATURE HYDROTHERMAL CARBONIZATION

Minah Oh<fnref n="1" />, Woori Jo<fnref n="1" />, Seungjin Oh<fnref n="1" />, Seong-Kyu Park<fnref n="2" />, Jai-Young Lee<fnref n="1" />

Corresponding author email: leejy@uos.ac.kr

## ABSTRACT

This study proposed a low temperature hydrothermal carbonization to treat and recycle sewage sludge. Such a reaction was used to identify the optimal conditions to produce bio-char. A chemical activation process was also introduced to develop the micro pores of the produced bio-char. To produce bio-char, the original material, sewage sludge, of 50 g was put in a reactor and experimented at 220, 230 and 240°C for 1, 2, 3, 5, 8, and 10 hours. In Batch 1., the optimal reaction temperature for the chemical activation of bio-char was determined by varying the reaction temperature at 500, 600, and 700°C for 60 minutes each. In Batch 2., based on the identified optimal activation temperature, the optimal reaction time for the chemical activation of bio-char was determined by doing an experiment for 30, 60, 90, and 120 minutes. As a result, the optimal production conditions of bio-char and activated bio-char were correlated with the iodine adsorption capacity. The iodine absorption capacity was assessed according to the experimental method prescribed in KS M 1802 of Korean Industrial Standard. The optimal conditions to produce bio-char and activated bio-char were 230°C and 8 hours and 600°C and 60 minutes, respectively. Those conditions were used to conduct an isothermal adsorption experiment for removal efficiency. Heavy metals such as arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), zinc (Zn) and nickel (Ni) were analyzed under the standard method for waste in Korea. Lead recorded the highest heavy metal adsorption efficiency of bio-char, being followed by copper, cadmium, zinc, and nickel. Arsenic was hardly adsorbed overall. The heavy metal absorption efficiency of activated bio-char was shown more than 80% in all the heavy metals except for arsenic, which was not adsorbed relatively and recorded adsorption efficiency about 20~40%.

**Acknowledgements:** This project is supported by Korea Ministry of Environment (MOE) as "The Eco-Innovation 21 Project (2013000150004)" is greatly appreciated.

<figure><img src="image_394.png" /></figure>

<footnote n="1">¹Department of Environmental Engineering, The University of Seoul, Korea</footnote>

<footnote n="2">²Kofirst R&D Center, KF E&E Co. Ltd., Korea</footnote>
<figure><img src="image_395.png" /></figure>

GREEN ROOFS / COVERS / SUSTAINABILITY
<figure><img src="image_396.png" /></figure>