# WILLOWS UNDER ARSENIC EXPOSURE: PHYSIOLOGICAL RESPONSES, ACCUMULATION AND DIFFERENTIAL GENE EXPRESSION IN ROOTS AND ABOVE GROUND PARTS

Aymeric Yanitch<fnref n="1" />, Emmanuel Gonzalez<fnref n="1" />, Frederic Pitre<fnref n="1" />,<fnref n="2" />, Simon Joly<fnref n="1" />,<fnref n="2" />, Michel Labrecque<fnref n="1" />,<fnref n="2" />

Corresponding author email: Biodiversity Center, 4101 Sherbrooke East, Montréal, Québec, Canada
aymeric.yanitch@umontreal.ca

## ABSTRACT

The toxicity of (arsenic) As in plants is mainly mediated by the competition between arsenate (AsV) and phosphate in metabolic processes. In addition, As could disrupt enzymatic activities by the binding of arsenite (AsIII) to thiol groups present in proteins. Regardless of the negative effect of As in plants metabolism, several species have shown the capacity to survive or avoid the stress associated to As. The objective of this study is to understand how some of them are even able to extract, degrade, or immobilize As contaminants.

Results from a four weeks hydroponic study with *Salix purpurea 'Fish Creek'* saplings showed that these shrubs are able to support up to 5 ppm As without showing any significant symptoms while taking up and accumulating As in its tissues. Physiological measurements, including photosynthesis, transpiration, and biomass production, were measured in plants exposed to 0, 5, 30 and 100 ppm of As. Preliminary differential gene expression analyses (RNA-seq) led to the identification of 164 differentially expressed genes in stem, 864 in leaves and 1348 genes in roots. These results will help us to develop a better understanding of the mechanism implied in As metabolism in willows and in plants in general.

<figure><img src="image_225.png" /></figure>

<footnote n="1">¹ Department of Biological Sciences, University of Montreal, Montréal, QC, Canada</footnote>

<footnote n="2">² Montreal Botanical Garden, Montréal, QC, Canada</footnote>
# EFFECTS OF HEAVY METALS ON PLANT RECOMBINANT GLUCOSE-6P-DEHYDROGENASE ISOFORMS

Alessia De Lillo<fnref n="1" />, Marco Lentini<fnref n="1" />, Daniela Castiglia<fnref n="2" />, Sergio Esposito<fnref n="1" />, Manuela Cardi<fnref n="1" />

Corresponding author email: alessia.dl87gmail.com

## ABSTRACT

Glucose-6-phosphate dehydrogenase (G6PDH - EC 1.1.1.49) is the key enzyme of the oxidative pentose phosphate pathway (OPPP); its reaction provides the reduction of NADP+ to NADPH, a co-factor essential in many metabolic processes in plant cells, and in response to biotic and abiotic stress, such as salinity, pathogen attack, ABA response, drought, nutrient starvation.

Previous studies suggested a possible correlation between metals and changes in total G6PDH activity. The plastidial P2-G6PDH isoform shows a redox regulation, localised at the level of two conserved cysteines; at least other 3 cys residues have been identified within its protein sequence, with structural and/or unknown functions.

We obtained mutagenized recombinant P2-G6PDH from *Populus trichocarpa*, changing the cysteine in serine, in order to investigate a possible direct effect of heavy metals in the regulation of G6PDH activity involving cys residues. The recombinant proteins were purified to apparent homogeneity, and the activities of the purified enzymes were measured in the presence of different elements (Ni, Cd, Pb, Cu, Zn).

Similar measurements were made on recombinant barley cytosolic G6PDH; this isoform presents several cysteines as well, but doesn't show any redox regulation.

Changes in the activity and kinetic properties of Cyt and P2-G6PDH were observed, suggesting peculiar role(s) in this regulation played by cys residues.

<figure><img src="image_226.png" /></figure>

<footnote n="1">¹Dipartimento di Biologia – Università di Napoli Federico II – Via Cinthia - 80126 Napoli, Italy</footnote>

<footnote n="2">²CNR-IBBR (Istituto di Bioscienze e BioRisorse) - Via Università, 133 - I-80055 Portici (NA), Italy</footnote>
PHYTOEXTRACTION POTENTIAL OF VETIVERIA ZIZANIOIDES FOR REMOVAL OF COPPER FROM CONTAMINATED SOIL IN PRESENCE OF VARIOUS ORGANIC AMENDMENTS

Sanjeev K. Singh<fnref n="1" />, Asha A. Juwarkar<fnref n="1" />, Jyotsna S. Meshram<fnref n="2" />

Corresponding author email: skr_singh@neeri.res.in

ABSTRACT

Copper (Cu) toxicity is a problem of both agricultural and environmental significance. Sources of Cu contamination include mining and smelting, urban, industrial and agricultural wastes, and the use of agrochemicals. The present study was undertaken to evaluate the growth response and phytoextraction capacity of *Vetiveria zizanioides* growing at different concentrations of Cu ranging from 500 to 2000 mg kg⁻<fnref n="1" /> without and with organic amendments viz. farm yard manure, mycorrhizae, *Azotobacter chroococcum*. The results indicate that the *Vetiveria zizanioides* was not survived in as such Cu contaminated soil (without amendment) at various concentrations. The test results also indicate that the *Vetiveria zizanioides* exhibited high tolerance to Cu in the soils and their normal growth attained even at Cu concentration of 2000 mg kg⁻<fnref n="1" /> after addition of organic amendments. Significant improvement in physico-chemical and microbiological properties in Cu contaminated soils was observed in presence of various organic amendment. The accumulation of Cu in roots was higher than the shoot of *Vetiveria zizanioides* and varied in the range of 50-70% depending upon the concentration of Cu in soils. The inoculation of plant with mycorrhizae and *Azotobacter chroococcum* increased the root length by 5-7 times and biomass by 8-10 times with respect to control (as such contaminated soil). Thus, *Vetiveria zizanioides* can be used as best potential accumulator of Cu from contaminated soil in presence of farmyard manure, *mycorrhizae* and *Azotobacter chroococcum*.

Keywords: phytoextraction, copper, farmyard manure, mycorrhizae, *Azotobacter chroococcum*, *Vetiveria zizanioides*

<figure><img src="image_227.png" /></figure>

<footnote n="1">¹ Scientists, Eco-System Division, CSIR-National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur-440020, India</footnote>

<footnote n="2">² Professor, Department of Chemistry, RTM Nagpur University, Nagpur-440001, India</footnote>
# PHYTOREMEDIATION POTENTIAL OF NATIVE FLORA OF CONTAMINATED SOIL OF VALE DAS GATAS MINE IN PORTUGAL

Paulo J.C. Favas<fnref n="1" />,<fnref n="3" />, João Pratas<fnref n="2" />,<fnref n="3" />

Corresponding author email: pjcf@utad.pt

## ABSTRACT

The objective of this study is to evaluate the phytoremediation potential of native flora of soils enriched with arsenic and heavy metals in the area of the old Vale das Gatas mine (Northern Portugal).

Very high maximum values for Pb (6,299 mg/kg), As (5,770 mg/kg) and W (636 mg/kg) were observed at soil and tailings of the Vale das Gatas mine area. The Cu-Mn-W-As-Pb-Zn association, which reflects the presence of mineralised veins in the area, is inversely correlated with pH.

In general, the content variations in plant materials were strongly related to the content variations in soils. It has also been verified that in contaminated locations or tailings, the concentration of metals in plant tissues is high due to the high metal concentrations in the soil. The leaves of *Agrostis castellana* and *Holcus lanatus* reflect the Cu, Pb and Ni pedogeochemical anomalies. The aerial parts of *Pteridium aquilinum* and *Juncus effusus* seem to be indicative of Zn anomalies in the soil. *Holcus lanatus* and *A. castellana* were the main accumulators of As, Cu, Fe and Pb and good accumulators of Zn. *Pteridium aquilinum* was a good accumulator of As, Pb and Zn. *Juncus effusus* appeared to be a Zn accumulator. The *P. pinaster* trees growing on the tailings and contaminated soils accumulated the studied elements in quantities greater than observed in plants of the areas representative of the local geochemical background. These values were also higher than those typically observed in this species. In the *P. pinaster* samples from tailings and contaminated soil locations, the older needles (2- and 3-years-old) show a tendency to accumulate higher concentrations of As, Fe, Zn, Pb and W while Ni and Cu were preferentially accumulated in young needles and stems (1-year-old).

The metal/metalloid concentrations higher than toxic level in some species indicate that internal detoxification metal tolerance mechanisms might also exist; therefore, their utility for phytoremediation is possible.

**Acknowledgements:** This study was partially supported by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE) and National Funds through the Portuguese Foundation for Science and Technology (PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).

<figure><img src="image_228.png" /></figure>

<footnote n="1">¹University of Trás-os-Montes e Alto Douro, UTAD, School of Life Sciences and the Environment, Quinta de Prados, 5000-801 Vila Real, Portugal, www.utad.pt</footnote>

<footnote n="2">²University of Coimbra, Department of Earth Sciences, 3001-401 Coimbra, Portugal</footnote>

<footnote n="3">³IMAR-CMA Marine and Environmental Research Centre, University of Coimbra, Portugal</footnote>
# PERFORMANCE OF *ATRIPLEX HALIMUS* L. IN ABSORPTION AND TRANSLOCATION OF Cr(III) AND Cr(VI), AND EVALUATION OF HIS TOLERANCE TO METALS

Belarbi Amaria<fnref n="1" />, Lotmani Brahim<fnref n="2" />

Corresponding author email: belarbi.biotech@gmail.com

## ABSTRACT

Metal pollution has become, due to industrial and technological advances in recent decades, a major threat to human health and environment. It is thus important to have effective tools to assess the bioavailability of metallic element and their environmental effects in contaminated soils. The purpose of the present work was to evaluate the tolerance and capacity to accumulate chromium in its two valence states, Cr(III) and Cr(VI) of *Atriplex halimus* L., grazing species, one of the most abundant perennial halophytes which is found as hedges in agricultural soils near a discharge of a bolts, cutlery and fittings factory (BCR, located in Oued Rhiou, Algeria).

The obtained results show a variation of the accumulation of chromium according to the plant organs, the metal form and the rate of chromium.

Chemical tests reveal an evolution of the chromium content in the aerial parts (leaves and stems) and the ground organs (roots) of *Atriplex halimus* L. This variation is related to the increase in dose and type of the used chromium.

It was also found that chromium absorption by plants treated with Cr(III) is more important than those treated with Cr(VI).

The obtained results also show that *Atriplex halimus* L. is more tolerant to trivalent chromium than to hexavalent chromium.

We finally found that this species present a large absorption capacity in roots and a low translocation to the above ground organs, which suggests the existence and evolution of the absorption, transport and detoxification mechanisms by storage or biochemical reactions in this species.

Keywords: *Atriplex halimus* L., heavy metals, chromium, accumulation, translocation, tolerance.

<figure><img src="image_229.png" /></figure>

<footnote n="1">¹ Faculté des Sciences Exactes et des Sciences de la Nature et de la Vie, Département Agronomie, Université Abdelhamid Ibn Badis de Mostaganem, Algeria</footnote>

<footnote n="2">² Faculté des Sciences Exactes et des Sciences de la Nature et de la Vie, Département Agronomie, Université Abdelhamid Ibn Badis de Mostaganem, Algeria</footnote>
# EFFICIENCY OF CADMIUM ACCUMULATION IN EUROPEAN AND HYBRID LARCH FOR USE IN PHYTOREMEDIATION

Bonet Amandine, Saladin Gaëlle, Gloaguen Vincent, Faugeron Céline

Laboratoire de Chimie des Substances Naturelles (EA 1069), Faculté de Sciences et Techniques de Limoges, France

Corresponding author email: celine.faugeron@unilim.fr

## ABSTRACT

The aim of this study was to compare the efficiency of cadmium (Cd) phytoextraction by two species of gymnosperms widely used in Limousin (France) for reforestation: a hybrid larch (*Larix x eurolepis Henry*) and one of its parents, European larch (*Larix decidua* Mill.). Larches were chosen because of their fast growth rate. The study focused on Cd, one of the most toxic metallic trace element (MTE) for plants and was conducted on two-years-old trees in greenhouse.

Larches were planted in November 2012 in pots containing a mixture of horticultural soil and sand (75/25, v/v). The temperature was maintained frost-free without artificial illumination and trees were watered with distilled water. After an acclimation of 3 months, larches were divided into 3 batches of 10 trees: a control group and two batches watered once with an aqueous solution of cadmium chloride, resulting in final concentrations of 50 and 100 µM Cd in soil, corresponding respectively to 12.5 and 25 mg.kg⁻¹ (the threshold for considering a polluted soil is 1-3 mg.kg⁻¹ according to the European Directive [1]). Plants were subsequently watered with distilled water and were harvested in November 2013 when needles just began to turn yellow. When trees were harvested, root system was briefly washed with water and then incubated for 2 hours in 20 mM EDTA in order to remove Cd adsorbed on root surface. Stem height and diameter (at the root collar) of each tree were measured just before potting in greenhouse and the day of harvest. Each tree was divided into 9 samples: taproot, roots, needles, buds, branches, bark and wood (for these organs, the last year was separated from the first two years). The obtained samples were weighed to determine their fresh weight and were then dried in a ventilated oven at 40°C to obtain their dry weight (DW).

Each dry sample was reduced in powder, mixed with 68% nitric acid and 30% H₂O₂ and mineralized by microwaves (Multiwave 3000, Anton Paar, Perkin Elmer). Cd concentration was then measured with a flame atomic absorbance spectrometer (HGA 900, Perkin Elmer) and the results were expressed in µg. g⁻¹ DW.

Results showed that the treatments with 50 or 100 µM Cd had no significant effect on European and hybrid larch growth. Indeed, stem height and collar root diameter, as well as fresh and dry biomass were not reduced. This result thus indicates that both larch species can tolerate a moderately polluted soil, what is required for further study. The measurements of Cd concentrations are in progress but the first results indicate that Cd would be mainly stored in root system, what is observed in non hyperaccumulating plant species and in most Cd hyperaccumulators [2]. Moreover, buds accumulated less Cd than other tested organs, what could be interpreted as a strategy of protection for the following year (in order to maintain development of young organs). Cd quantification in all organs will allow to determine which larch species is more suitable for Cd phytoextraction.

## References:

1. Council Directive of 12 June 1986 on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture (86/278/EEC), annexe IA, p 8.

2. Liu, Z., He, X., Chen, W., Yuan, F., Yan, K., Tao, D. (2009). Accumulation and tolerance characteristics of cadmium in a potential hyperaccumulator – *Lonicera japonica* Thunb. *Journal of Hazardous Materials*, 169, 170-175.

**Acknowledgements:** The financial support by a grant from the Regional Council of Limousin.

<figure><img src="image_230.png" /></figure>
# ACCUMULATION PATTERNS OF Cr(VI)/Cr(III) – QUALITATIVE AND QUANTITATIVE ANALYSIS OF XRF MAPS

Joanna Augustynowicz<fnref n="1" />, Zbigniew Gajewski<fnref n="1" />, Paweł Wróbel<fnref n="2" />, Anna Kołton<fnref n="1" />

Corresponding author email: augustyn@ogr.ur.krakow.pl

## ABSTRACT

The aims of this study were both the qualitative and quantitative analysis of Cr accumulation patterns in shoots of *Callitriche cophocarpa*. This globally-distributed, submersed macrophyte exhibits outstanding Cr phytoremediation capacity in an aquatic environment. Plants were immersed in 100 µM (5.2 mg/L) Cr solution for seven days, in environmentally-relevant conditions. Cr was applied separately at two stable forms as Cr(VI) and Cr(III), known from their diverse physicochemical properties as well as different influence on biota. The maps of Cr distribution in young leaves, mature leaves and stems, were obtained according to micro X-ray fluorescence spectroscopy (µXRF). This method is a non-destructive technique that allows studies of elements with a very narrow beam of X-rays, at concentration levels in µg/g range. The detailed analysis of XRF maps were done according to Image-Pro PLUS (Media Cybernetics) software.

Distinct patterns of Cr arrangements were found in respect to Cr speciation as well as the plant organ. Cr was found solely in spot-like structures when the shoots were exposed to Cr(III). However, Cr was visualized both in vascular bundles and spot-like structures after plant incubation in Cr(VI). The element was found exclusively in vascular bundles in the case of mature leaves exposed to Cr(VI). Preferentially, Cr deposition were observed in vascular bundles of Cr(VI)-treated stems, too. On the other hand the element was detected in high amount in spot-like structures in young leaves treated by hexavalent Cr. In this work we also demonstrated the statistically significant differences in the Cr concentrations dependent on the Cr valency and the type of plant organ. The concentration of Cr increased in the following order: Cr(VI) mature leaves < Cr(VI) young leaves < Cr(VI) stems < Cr(III) young leaves < Cr(III) mature leaves < Cr(III) stems. The relation between the tissue surface (%) that bounds Cr and Cr concentration (expressed in counts per second; cps) was described by the function: y=9.1lnx+8.4; where y – surface (%) occupied by Cr, and x – Cr content. According to this function we might conclude that the tissue structures of *Callitriche* responsible for Cr binding are already saturated in the conditions of our experiment; though, the maximal sorption capacity of the plant tissue toward Cr ions was not achieved.

This work has contribution into the knowledge concerning mechanisms of aquatic phytoremediators response to elevated concentration of Cr ions. It also provides information concerning application of particular organs of *Callitriche cophocarpa* in efficient Cr remediation.

**Acknowledgements:** The financial support by NCN project DEC-2011/03/B/NZ9/00952 is greatly appreciated.

<figure><img src="image_231.png" /></figure>

<footnote n="1">¹Institute of Plant Biology and Biotechnology, Faculty of Horticulture, University of Agriculture in Kraków, al. 29 Listopada 54, 31-425 Kraków, Poland</footnote>

<footnote n="2">²Department of Medical Physics and Biophysics, Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, al. Mickiewicza 30, 30-059 Kraków, Poland</footnote>
# HEAVY METALS AND METALLOIDS ACCUMULATION BY NATIVE FLORA OF MINING AREAS IN PORTUGAL: PHYTOREMEDIATION POTENTIAL

Paulo J.C. Favas<fnref n="1" />,<fnref n="4" />, João Pratas<fnref n="2" />,<fnref n="4" />, Mayank Varun<fnref n="3" />, Rohan D'Souza<fnref n="3" />, Manoj S. Paul<fnref n="3" />

Corresponding author email: pjcf@utad.pt

## ABSTRACT

The aim of this study is to evaluate the phytoremediation potential of native flora of soils enriched with metals and metalloids, in distinct abandoned mining areas of Portugal.

Analytical methods included colorimetry for W, atomic absorption spectrophotometry (AAS, Perkin-Elmer, 2380) for Ag, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn, hydride generation system (HGS) for As and Sb and fluorometry (Fluorat-02-2M analyzer, Lumex) for U.

Significant accumulation of heavy metals and metalloids in both soils and native wild flora suggests that metal contamination is a matter of great concern in the studied mining areas. The native flora displayed its ability to withstand high concentrations of heavy metals/metalloids in the soil. However, accumulation patterns of metals/metalloids in the plants tested differed. As metal concentrations in above ground parts were maintained at low levels, metal tolerance in most cases may mainly depend on their metal excluding ability.

However, metal/metalloid concentrations higher than toxic level in some species like Agrostis castellana (As, Fe), Cistus ladanifer (Cr, W), Cistus salvifolius (Ni, Pb), Digitalis purpurea (Sb, W, Zn), Helichrysum stoechas and Hypochaeris radicata (U), Holcus lanatus (As, Cu, Fe), Lonicera periclymenum, Mentha suaveolens and Phytolacca americana (Pb, Zn), Pinus pinaster (As, W, Zn), Polystichum setiferum and Solanum nigrum (Zn), Pteridium aquilinum (As), as well as the serpentine plant species Alyssum serpyllifolium subsp. lusitanicum, Lavandula stoechas subsp. sampaiana, Linaria spartea subsp. virgatula and Ulmus procera (Cr, Ni) and Bromus hordeaceus and Plantago radicata subsp. radicata (Ni) indicate that internal detoxification metal tolerance mechanisms might also exist; therefore, their utility for phytoremediation is possible.

Furthermore, the plants could grow and propagate in substrata with low nutrient conditions which would be a great advantage in the revegetation of mine tailings. It was also observed that despite lower accumulation, trees of the studied regions can be very effective due to their higher biomass.

*Acknowledgements:* This study was partially supported by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE) and National Funds through the Portuguese Foundation for Science and Technology (PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).

<figure><img src="image_232.png" /></figure>

<footnote n="1">¹ University of Trás-os-Montes e Alto Douro, UTAD, School of Life Sciences and the Environment, Quinta de Prados, 5000-801 Vila Real, Portugal, www.utad.pt</footnote>

<footnote n="2">² University of Coimbra, Department of Earth Sciences, 3001-401 Coimbra, Portugal</footnote>

<footnote n="3">³ St. John's College, Department of Botany, Agra 282 002, India</footnote>

<footnote n="4">⁴ IMAR-CMA Marine and Environmental Research Centre, University of Coimbra, Portugal</footnote>
# HEAVY METAL ACCUMULATION BY WILD PLANTS IN INDUSTRIALLY DISTURBED HABITATS OF THE MIDDLE URALS

Elena Zhujkova<fnref n="1" />, Tatiana Zhujkova<fnref n="2" />, Ludmila Leshukova<fnref n="1" />, Elvira Zinnatova<fnref n="1" />,<fnref n="2" />, Irina Kiselyova<fnref n="1" />

Corresponding author email: Irina.Kiselyova@mail.ru

## ABSTRACT

Heavy metals (HM), providing a cytotoxic, mutagenic and carcinogenic action are among the most dangerous pollutants. In plants HM inhibit photosynthesis and growth, change mineral nutrition and water balance. HM pollution demands the elaboration of the ways of their remediation and safe use. In connection with this the present study deals with the plant HM accumulation capacity both in roots and leaves to identify plant hyperaccumulators or excluders suitable respectively for phytoremediation, or cultivation in contaminated areas for economic purposes.

Accumulation capacity of *Tussilago farfara* L., *Plantago major* L., *Plantago media* L., *Taraxacum officinale* Wigg., *Melilotus albus* M., *Trifolium medium* L. were studied in 5 habitats near Nizhny Tagil (NT) and Kachkanar Mining and Processing Plant (KMPP). Soils, roots and shoots of plants were collected for HM determination – at least 10 soil samples and/or sludge, 10 flowering plants of each species separately underground and aboveground parts. The heavy metal content was determined by atomic absorption spectral analysis (PerkinElmer AAS 300). According to the level of pollution the habitats were defined as: background, buffer, impact.

The content of HM mobile ion forms in soil/sludge varied: 7 - 951 mg/g for Cu; 6-390 for Zn; 377-5700 for Fe; 0,2-124 for Co; 0,5-37 for Cr; 0.01-0.2 for Cd; 0.2-5 for Pb; 4-23 for Ni. The underground parts of plants accumulate 5-100 ug/g Cu; 8-150 ug/g Zn; 0,4-17 ug/g Co; 1-30 ug/g Cr and 110-3300 ug/g Fe. *P. major*, *P. media*, *T. officinale*, *T. farfara* revealed a high ability to accumulate copper in the roots, *P. major* – zinc, *M. albus*, *P. major*, *P. media*, *T. farfara*, and *T. officinale* - iron, *M. albus*, *P. major* - cobalt; *T. farfara*, *P. major*, *M. albus* - chromium. In most species the positive correlation between the content of HM in soils and in the roots was shown.

Accumulation coefficient (AC) less than 1 is usually observed at elevated HM levels in the soil due to the active work of the root barrier. AC ≥ 1 indicates unlimited diffusion or absorption of the metal ions into the plant. For plants from N.-Tagil sites (table) KH in most cases does not exceed 1, and with the growth of the toxic load decreased.

<table><caption>Table. AC for HM in wild plants from sites with different relative toxic load</caption><thead><tr><th rowspan="3">Species</th><th colspan="6">Relative toxic load (Nizhnij Tagil)</th></tr><tr><th>1,00</th><th>2,55</th><th>3,33</th><th>6,19</th><th>8,36</th><th>22,78</th></tr><tr><th colspan="6">AC<sub>Cu</sub>/AC<sub>Zn</sub></th></tr></thead><tbody><tr><td><i>P. major</i></td><td>0,7/0,9</td><td>0,8/0,4</td><td>0,4/0,4</td><td>0,3/0,4</td><td>0,3/0,2</td><td>0,1/0,2</td></tr><tr><td><i>T. farfara</i></td><td>2,0/0,4</td><td>1,3/0,1</td><td>0,3/0,2</td><td>0,2/0,1</td><td>0,20,1/</td><td>0,1/0,04</td></tr><tr><td><i>T. officinale</i></td><td>1,6/0,6</td><td>1,6/0,3</td><td>0,6/0,4</td><td>0,3/0,1</td><td>0,3/0,1</td><td>0,1/0,1</td></tr><tr><td><i>M. albus</i></td><td>0,5/0,9</td><td>0,4/0,2</td><td>0,1/0,2</td><td>0,8/0,6</td><td>0,1/0,1</td><td>0,02/0,1</td></tr><tr><td><i>T. medium</i></td><td>1,0/0,9</td><td>0,7/0,3</td><td>0,2/0,2</td><td>-/-</td><td>0,2/0,1</td><td>-/-</td></tr><tr><th colspan="7">Relative toxic load (KMPP)</th></tr><tr><td colspan="7">AC<sub>Cu</sub>/AC<sub>Zn</sub></td></tr><tr><td colspan="7">AC<sub>Cu</sub>/AC<sub>Zn</sub></td></tr><tr><td><i>T. officinale</i></td><td>23,6/8,32</td><td>14,2/4,1</td><td>30,8/14,4</td><td colspan="3">21,8/3,5</td></tr><tr><td><i>P. media</i></td><td>84,4/9,83</td><td>33,4/6,37</td><td>-/-</td><td colspan="3">52,7/10,4</td></tr></tbody></table>

Heavy metals concentration in soils, roots and shoots, accumulation and translocation coefficients allowed revealing different strategies in the accumulation of such essential elements as copper and zinc in plants. *P. major*, *P. media*, *T. officinale*, *T. farfara* have shown the high ability to accumulate Cu, *P. major*, *P. media* – Zn and *M. albus*, *T. officinale* – Fe. Species revealed difference in the effectiveness of soil/root and root/shoot barriers.

<figure><img src="image_233.png" /></figure>

<footnote n="1">¹Department of Plant Physiology and Biochemistry, Institute of Natural Sciences, Ural Federal University, Russia</footnote>

<footnote n="2">²Nizhniy Tagil State Social-Pedagogic Academy, Sverdlovsk Region, Nizhniy Tagil, Russia</footnote>
# PHYTOREMEDIATION OF URANIUM TAILINGS: ROLE OF CHELATORS IN TRIGGERING URANIUM ACCUMULATION IN WHEAT

**Bhagawatilal Jagetiya**

Laboratory of Plant Physiology and Biotechnology, Department of Botany, M.L.V. Government College, Bhilwara- 311 001, Rajasthan, India

Corresponding author email: bljagetiya@yahoo.com

## ABSTRACT

Radionuclides including toxic heavy metals mobilization through extraction from ores and processing for diversified uses has laid to the discharge of these harmful metals into the ecosystem. These pollutants pose a great risk to environment and human health. Conventional methods of remediation applied for contaminated environments have many limitations; alternatively, phytoremediation can serve as a prospective method for decontamination and rehabilitation of contaminated sites. Several approaches may be applied to further enhance the efficiency of metal phytoremediation. Chelator-assisted phytoextraction has been proposed as a potential tool for phytoremediation of U contaminated tailings. In the present study, a greenhouse experiment was set up to scrutinize the ability of citric acid (CA), oxalic acid (OA), nitrilotriacetic acid (NTA) and EDTA for phytoremediation of U tailings by wheat (*Triticum aestivum* L.). Uranium tailings were mixed with garden soil to yield 25:75 mixtures. Prepared pots were divided into four sets and treated with following different concentrations- 0.1, 0.5, 2.5 and 12.5 mmol kg⁻¹ soil amendments for each of the chelator whereas, control pots were remain untreated. Experiment was conducted in completely randomized block design with triplicates. The observations were recorded during the vegetative stage and effects of different concentrations of chelators were noted on growth parameters, which includes, shoot-root length and shoot-root fresh as well as dry mass. An optimum concentration for each of the chelator was recorded on the basis of biomass production, tolerance and accumulation potential. Uranium accumulation in different plant parts was recorded through pellet fluorometric method. Severe toxicity in the form of reduced growth and plant death was recorded at 12.5 mmol of each chelator. Lowest growth inhibition produced by chelators occurred in NTA which was followed by OA, moderate in CA and highest was recorded in EDTA treatments at their respective levels. Uranium uptake and accumulation were concentration dependent for each amendment applied. Maximum U uptake (3.4- fold) in the roots occurred at 2.5 mmol of CA while NTA proved to be the weakest for the same purpose. Chelator induced U uptake in the present study follows the order: CA> EDTA> OA> NTA.

**Keywords:** phytoremediation, uranium tailings, chelators, EDTA, NTA

<figure><img src="image_234.png" /></figure>
# IN IN VITRO CULTURE OF DITTRICHIA VISCOSA, MICROPROPAGATION AND ITS POTENTIAL USE IN PHYTOREMEDIATION

Carmine Guarino<fnref n="1" />, Angela Cicatelli<fnref n="2" />, Barbara Conte<fnref n="1" />, Serena Parrella<fnref n="2" />, Claudia Nittolo<fnref n="2" />, Valentina Spada<fnref n="1" />, Rosaria Sciarrillo<fnref n="1" />, Stefano Castiglione<fnref n="2" />

Corresponding author email: acicatelli@unisa.it; bbconte@yahoo.it

## ABSTRACT

Dittrichia viscosa (L.) Greuter is a ruderal plant species, widely distributed in the Mediterranean basin; it has a considerable biomass and is able to grow on heavy metal (HM) polluted soils, as reported by several authors [2,3,4,5]. However, only few reports indicate D. viscosa as a phyto-remediator plant, useful for reclamation of HM polluted soils. The principle aim of this work was to develop and set up a protocol for D. viscosa seed sterilization and for in vitro seedling culture. Furthermore, D. viscosa plants, derived from seed propagation, collected from three different natural populations located in central and southern Italy (Roma, Potenza, Crotone), were assessed for their genetic dissimilarity by RAPD analysis. On the basis of morphological characteristics, some clones were selected and tested to evaluate their capacity to grow on axenic medium in the presence of high Zn concentrations (up to 10, 20, 40, 60 and 100 fold of Zn present in the culture medium). Different tests carried out on Dittrichia seeds allowed us to define a simple and effective sterilization protocol, providing the combination of ethanol and sodium hypochlorite solutions, at 70 % and 1.0 % respectively. Seeds were sown on three different media: Murashige and Skoog (MS), Gamborg and Mohr (modified by Basile et al. [1]). Seedlings grew similarly on MS and Gamborg medium, while they showed an improved growth rate, in particular at root level, on modified Mohr medium. Plants were vegetatively reproduced transplanting nodal-internodal portions as microcuttings to fresh medium. Clonal propagation was extremely effective and allowed us to multiply vegetatively several seedlings of D. viscosa. When seedlings, grown in in vitro culture, reached 10-15 cm in length, were transferred to peat-based potting soil provided with a transparent plastic bag, necessary to limit the excess of seedling evapo-transpiration. Plants were regularly irrigated every 2-3 days with tap water and, after 15 days of greenhouse adaptation (23 °C; 14 h light and 10 h dark), plastic bags were removed from plants. The percentage of survival was larger than 80%. Some plants were vegetatively micropropagated on medium at increasing concentrations of Zn, as previously described. However microcuttings were able to produce new roots and to grow only at the lower Zn concentration (10 fold). The performed RAPD analysis on the three D. viscosa populations grown in vitro culture revealed a close genetic relationship between Roma and Potenza populations, whilst the Crotone population showed a clear genetic differentiation from the other two.

## References:

1. Basile A, Sorbo S., Lo' pez-Sa' ez J.A., Castaldo Cobianchi R, (2003) *Phytochemistry* 62, 1145-1151.

2. Fernández R., Bertrand A., Reis R., Mourato M.P., Martins L.L., González A., (2013) *Journal of Hazardous Materials* 244-245, 555-562.

3. Jiménez M.N., Bacchetta G., Casti M., Navarro F.B., Lallena A.M., Fernández-Ondoño E. (2011) *Ecological Engineering* 37, 392-398.

4. Marchiol L., Fellet G., Boscutti F., Montella C., Mozzi R., Guarino C. 2013. *Ecological Engineering* 53:343–353.

5. Nogales R., Benítez E. 2006. *Bulletin Environmental Contamination and Toxicology* 76, 538-544.

**Acknowledgements:** The work was supported by LANDE srl, Italy.

<figure><img src="image_235.png" /></figure>

<footnote n="1">¹ Department of Science and Technologies, University of Sannio, Benevento, Italy</footnote>

<footnote n="2">² Department of Chemistry and Biology; University of Salerno, Fisciano, Italy</footnote>
# EVALUATION OF ARSENIC REMOVAL POTENTIAL OF FIVE AQUATIC PLANTS GROWN SINGLY OR IN COMBINATIONS

Sudhakar Srivastava<fnref n="1" />#, Suvarna Sounderajan<fnref n="2" />, Ambuja Udas<fnref n="2" />, Penna Suprasanna<fnref n="1" />

## ABSTRACT

Arsenic (As) is a ubiquitously present highly toxic metalloid, which poses a serious threat to the environment and human health. Mitigating environmental As contamination is desirable. Exploiting the As accumulation potential of plants has been considered a potential solution; the technology is referred to as phytoremediation. In this backdrop, we opted for a combinatorial approach and concentrated on evaluating the potential of five aquatic plants, namely *Hydrilla verticillata*, *Ceratophyllum demersum*, *Lemna minor*, *Wolffia globosa* and *Eichhornia crassipes*, for growth and As

accumulation for one month in single and combined (in two plant combinations) culture. Plants (1 g L⁻<fnref n="1" />; total 4 g) were subjected to 4 L of 2500 µg L⁻<fnref n="1" /> As (as sodium arsenate; Na₂HAsO₄·7H₂O) in 10% Hoagland solution for a period of 30 d. In two plant combination treatments, 2 g of each plant was used. Total As in the plant material was estimated after digestion of oven-dried plants by using an

<figure><img src="image_236.png" /><figcaption>Figure 1. Arsenic concentration (µg g⁻<fnref n="1" /> DW) of plants grown singly or in combinations in control and arsenic treatment for 30 days. All values are means of triplicates ± SD.</figcaption></figure>

atomic absorption spectrophotometer. *Eichhornia* exhibited the maximum growth rate under control conditions. Under As stress, *Hydrilla* showed an increase in growth rate (19%) in comparison to control, while other plants demonstrated a decline. Among different combinations, *Ceratophyllum+Lemna*, *Hydrilla+Ceratophyllum* and *Hydrilla+Wolffia* showed higher growth in As (92%, 43%, and 33%, respectively) than under control conditions. As concentration (µg g⁻<fnref n="1" /> dw) was found to be maximum for *Hydrilla+Ceratophyllum* (2188) followed by *Ceratophyllum+Lemna* (1026) and *Ceratophyllum+Wolffia* (804) (Figure 1). Total As removal (µg) in 30 days was found to be the maximum for *Ceratophyllum+Lemna* (4365) followed by *Hydrilla+Ceratophyllum* (3326) and *Hydrilla+Wolffia* (1896). In all combinations of plants, *Hydrilla* always contributed more than 50% (52% to 78%), while *Eichhornia* less than 30% (17% to 28%). The study advocates the use of *Hydrilla* or *Ceratophyllum* as one plant of choice to be used in combination. It also proposes to screen for different plant combinations for better application of phytoremediation technique in field.

<figure><img src="image_237.png" /></figure>

<footnote n="1">¹ Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, Maharashtra, India</footnote>

<footnote n="2">² Analytical Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, Maharashtra, India</footnote>

<footnote>Present address: Institute of Environment & Sustainable Development, Banaras Hindu University, Varanasi-221005, U.P., India</footnote>
<figure><img src="image_238.png" /></figure>

PERSISTENT ORGANIC CONTAMINANTS
<figure><img src="image_239.png" /></figure>
PROPOSED METHODOLOGY FOR RESTORATION OF UNCONTROLLED LANDFILLS
IN INSULAR COMMUNITIES

Valentina Phiniketou, Antonis Zorpas

Cyprus Open University, Faculty of Pure and Applied Science, Environmental Conservation and Management,
P.O.Box 12794, 2252, Latsia, Nicosia, Cyprus

Corresponding author mail: antoniszorpas@yahoo.com ; antonis.zorpas@ouc.cy.cy

ABSTRACT

Despite the promotion of waste management being held throughout the world, unfortunately the disposal of untreated waste into final landfill sites is the most widely solution these days. Obviously, this cannot be part of an integrated waste management system where waste are recovered, reprocessed and reused as new or existing resources, since nothing is controlled.

Uncontrolled waste disposal on landfill sites causes pollution that can be hazardous on the environment and introduce several problems on socio-economic welfare of the planet, thus moving away from them is a major issue that requires immediate actions. Waste disposal pollution is caused by either normal operations, such as the emissions of landfill gas into the atmosphere and the leakage of leachate in subsoil (and then to the aquifer) or the accidence from subsidence and fires; either way they constitute serious threads to public health and the environment.

In this research we try to develop a methodology of restoration of landfill sites on large islands. If
landfill sites are problematic in mainland scenarios, in large islands the situation is even worse. The
limited surface of islands, the volume of waste which is steadily increased (due to fishing, tourism or
other sources) and the waste washed up from open seas, magnify the problem and makes them a
priority.

For the proposed methodology we used Cyprus as a case study. We evaluated different management plans for landfill restoration through multi-criteria analysis on the conditions found currently on the island. We examined the life cycle assessment (LCA) plan of uncontrolled landfill sites, their waste forms and the risk assessment of Cyprus landfills. Finally, we evaluated various recovery methods of soil and water contaminated with heavy metals (since these are very common in waste disposal sites) such as Permeable treatment walls, phytoremediation, biochemical process, and their suitability to Cyprus. Different scenarios were evaluated based on the effects of the pollution, the restoration and aftercare costs, the gas emissions, the simplicity of implementation, the perspective of land development, and, finally, for each open dump site the best option was chosen.

<figure><img src="image_240.png" /></figure>
# HYDROPONIC PHOTOBIOREACTORS FOR THE REMOVAL OF TOXIC ORGANIC POLLUTANTS

Elisha Tel-Or<fnref n="1" />, Itzhak Bilkis<fnref n="2" />, Yael Grunwald<fnref n="1" />, Eran Benyamini<fnref n="1" />

Corresponding author email: telor@agri.huji.ac.il

## ABSTRACT

Hydroponic bioreactors were recently developed for the removal of the toxic herbicide, atrazine, by Sorghum plants, and for the removal of bisphenol-A by *Cyperus corymbosus* and *Juncus acutus*.

Sorghum plants, were germinated and cultivated in a thin layer of solid nursery medium, developed their roots into a deeper solution containing high concentration of atrazine, up to 30mg/L.

The plants tolerated atrazine and gradually removed the atrazine from the medium, leading to its complete removal within 4 weeks. Aeration facilitated root activity. Root associated bacteria involved in atrazine removal were defined. ¹⁴C-atrazine breakdown products were identified in Sorghum shoots, including GSH-atrazine and atrazine metabolized products.

Bisphenol-A, recognized as an endocrine disruptor in mammals, fish and invertebrates, was tested for its remediation by the wetland plants, *Cyperus corymbosus* and *Juncus acutus*, set in a model hydroponic system of glass containers. Bisphenol-A, at concentrations between 50-200ppb, was remediated within 1-4 days. The mechanism of bisphenol-A remediation is currently investigated, aiming to identify mode of metabolism of the pollutant by the plants, involvement of root associated bacteria. We aim to determine the effectiveness of hydroponic bioreactors outdoors.

<figure><img src="image_241.png" /></figure>

<footnote n="1">¹ The R.H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel</footnote>

<footnote n="2">² Institute of Biochemistry and nutrition, The Hebrew University of Jerusalem, Rehovot 76100, Israel</footnote>
# FULL-SCALE REMEDIATION OF CONTAMINATED SLUDGE USING PLANTS - INVESTIGATIONS, RESULTS AND EXPERIENCES

André Gerth<fnref n="1" />, Diana Claus<fnref n="1" />, Helmut Dietze<fnref n="2" />, William Grosser<fnref n="2" />, Anja Hebner<fnref n="1" />

Corresponding author email: info@bioplanta-leipzig.de

## ABSTRACT

At a central waste water treatment plant of a city with about 530,000 inhabitants in Germany municipal as well as industrial waste water were treated. The sewage sludge was pumped to a site (56 ha) outside the city. On this site the water content of the sewage sludge was decreased and the sludge was used in agriculture afterwards. In 1990 the operation of the dewatering site was stopped and about 800,000 m³ of sewage sludge remained there in several lagoons.

An environmental impact assessment (EIA) was developed for the site. The EIA identified the need of remediation measures to prevent hazard risk. Different remediation techniques were compared using economical, social and environmental aspects. The biological *in situ* remediation using plants was chosen for remediation of the sludge lagoons. It is one of the biggest and best monitored phytoremediation projects of its kind in Europe.

On the site common reed (*P. australis*) is successfully used for the remediation of sewage sludge contaminated with metals (Cd, Cr, Cu, Ni, Pb, Zn). At the beginning of remediation (1996) the water content of the sludge was approximately 65%. These conditions were favourable for establishment of reed population as biological capping of the sludge lagoon. A special technology for the out planting, on coconut mats pre-cultivated reed plants, was developed and successfully used. A biomass yield of about 19 t dry matter × ha⁻<fnref n="1" /> by mowing the reed shoots in winter and a metal removal of 6.7 kg × ha × yr⁻<fnref n="1" /> could achieved.

Due to humification and mineralization of the sludge, linked to the remediation process, a decrease in plant density and biomass yield and a lower efficiency of metal removal were determined between 2005 and 2007. That's why BioPlanta has carried out extensively investigations based on crop management and agronomic practice to continue the phytoextraction in an optimal way and to improve the phytoremediation technology.

The selection of phytoremediating species has been shown to be the most important factor affecting the extent of metal removal regarding the metal content and yield. Using sunflower (*H. annuus*) an increase of total metal extraction by factor 8 was reached in comparison to reed. Sedge (*C. acutiformis*) shows a more efficient extraction for five out of six investigated metals (by factor 2 to 12) compared to reed. In field studies, BioPlanta proved that after application of N-P-K fertiliser reed showed a significant higher yield (2 to 3 fold), resulting in improved phytoextraction of metals by factor 3 to 14. Increase in yield was also achieved by mowing reed in winter, sunflower in autumn and rape in summer. Highest metal extraction (9.7 kg × ha⁻<fnref n="1" /> × yr⁻<fnref n="1" />) was reached combining agronomic practices with selected plant species, e.g. fertilization and mowing of reed in autumn. The application of plant supplements and herbicide revealed a plant and element specific increase of the phytoextraction efficiency.

As result of these investigations sunflower and also maize were used (instead of reed) for full-scale remediation on one of the four sludge lagoons. About 10 t dry matter × ha⁻<fnref n="1" /> of sunflower as well as 7 t dry matter × ha⁻<fnref n="1" /> of maize could be yielded. Due to the harvest of sunflower and maize about 2.3 kg × ha⁻<fnref n="1" /> × yr⁻<fnref n="1" /> and 3.5 kg × ha⁻<fnref n="1" /> × yr⁻<fnref n="1" /> of metals could be removed, respectively. Within 17 years remediation (1996 – 2013) in total 321 t dry matter were mowed and removed as well as 99.7 kg metals (92.6 kg Zn; 0.7 kg Ni; 4.4 kg Cu; 0.4 kg Cr; 0.5 kg Cd; and 1.1 kg Pb) were extracted using plants.

<figure><img src="image_242.png" /></figure>

<footnote n="1">¹ Vita 34 AG / Biotech Branch BioPlanta, Germany</footnote>

<footnote n="2">² Kommunale Wasserwerke Leipzig GmbH, Germany</footnote>
# ENVIRONMENTAL FATE OF PPCPs AT A 2,000 Ha MUNICIPAL WASTEWATER LAND-APPLICATION SITE

Andrew McEachran<fnref n="1" />, Damian Shea<fnref n="2" />, Elizabeth Guthrie Nichols<fnref n="1" />

Corresponding author email: egnichol@ncsu.edu

## ABSTRACT

The occurrence and fate of pharmaceutical and personal care products (PPCPs) in the environment are of increasing public importance due to their ubiquitous nature and documented effects on wildlife, ecosystems, and potentially humans. One potential, yet undefined, source of entry into the environment of PPCPs is via the land application of municipal wastewater onto permitted lands. The objective of this study is to determine the extent to which PPCPs are mitigated by or exported from managed tree plantations irrigated with municipal wastewater. A specific focus of the study is PPCP presence or removal in groundwater before surface water discharge at the catchment scale. The study site is a municipality that land-applies primary treated wastewater onto 2,000 ha of managed hardwood and pine plantations. Initial sampling and analysis includes grab sampling of groundwater throughout the study site and targeted analysis of a suite of PPCPs utilizing GC-MS and LC-MS/MS. Further study scope, methods, and preliminary results of targeted PPCPs in groundwater and surface waters will be presented.

**Acknowledgements:** We would like to thank NIEHS Superfund Research Program, the NC Department of Agriculture, NCSU Department of Forestry and Environmental Resources, and the NCSU Graduate Fellowship Program for financial support. Additionally, analytical assistance from Summer Xia (NCSU) and Wanda Bodnar (UNC) are greatly appreciated.

<figure><img src="image_243.png" /></figure>

<footnote n="1">¹North Carolina State University, Department of Forestry and Environmental Resources, College of Natural Resources, 3120 Jordan Hall, Raleigh, NC 27695-8008, USA</footnote>

<footnote n="2">²North Carolina State University, Department of Biological Sciences, College of Science, Toxicology Building 1220, Box 7633, Raleigh, NC 27695-8008, USA</footnote>
# MORPHOLOGICAL AND PHYSIOLOGICAL RESPONSES OF MAIZE (ZEA MAYS) EXPOSED TO FORMERLY MULTICONTAMINATED SOILS

Joan Dupuy<fnref n="1" />,<fnref n="2" />, Stéphanie Ouvrard<fnref n="1" />,<fnref n="2" />, Pierre Leglize<fnref n="1" />,<fnref n="2" />, Thibault Sterckeman<fnref n="1" />,<fnref n="2" />

Corresponding author email: pierre.leglize@univ-lorraine.fr

## ABSTRACT

Phytoremediation is promising, but depends on understanding contaminants' impact on plant functioning. We therefore focused on the impact of formerly contaminated soils on cultivated plants (Zea mays) especially on (i) the impact of polycyclic aromatic hydrocarbons (PAH) on plant functioning and (ii) the PAH transfer to plant. Cultivation was conducted under controlled conditions on former contaminated soils (coking plant, steel industry). Maize growth and organ development were assessed, by measuring both its morphological and nutrition parameters. PAH transfer was assessed by measuring soil total and available concentration using passive sampler method (polyoxymethylene membrane, POM) or resin extraction (Tenax®) and PAH concentration in roots and shoots. First results showed that, after five weeks, plants exposed to contaminated soils presented decreased biomasses and modified root architecture with fewer thin roots. PAH availability, using POM method, of all contaminated soils was low and was not well correlated to PAH concentration in roots. Other analyses are on going: nutrients up-taking, PAH concentration in shoots and available PAH using Tenax® extraction.

**Acknowledgements:** The financial support by IBRACS project funded by ADEME and Snowman network is greatly appreciated.

<figure><img src="image_244.png" /></figure>

<footnote n="1">¹ Université de Lorraine, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy, F-54518, France</footnote>

<footnote n="2">² INRA, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy, F-54518, France</footnote>
# IN SITU PHYTORECLAMATION OF SEWAGE SLUDGE AFTER ITS AMENDMENT WITH SOIL AND GRAVEL

Nataliia Suchkova<fnref n="1" />, Dimitrios Alifragkis<fnref n="2" />, Iakovos Ganoulis<fnref n="1" />, Efthymios Darakas<fnref n="1" />, Thomas Sawidis<fnref n="3" />, Ioannis Tsiripidis<fnref n="3" />, Felix Stolberg<fnref n="4" />

Corresponding author email: nsuchkov@civil.auth.gr

## ABSTRACT

Sewage sludge is often contaminated by heavy metals, organic pollutants, and pathogens. It is also characterised by a high water holding capacity and anaerobic conditions, and malodours associated with the presence of some volatile organic compounds (VOCs), even after digestion and alkaline stabilisation. These aspects present the main obstacles to applying sewage sludge to the land and explain why treated sewage sludge, if not landfilled or incinerated, often remains within the territory of Wastewater Treatment Plants (WWTPs) without further utilisation. Economic considerations and traditions may also be of relevance. In Greece, for example, owing to a lack of acceptance by farmers, most sludge is landfilled. According to Matthews (1999) [1] and IEEP (2009) [2] the country uses a very small quantity of sludge in agriculture (~10 %), and this mostly in the frame of research projects and pilot studies. In addition to agricultural use, trials have been performed in the timber industry [3], mine spoils reclamation [4], vermiculture [1] and cement industry [5]. However, this is still very much at the experimental stage in Greece. Meanwhile, the volume of sewage sludge is continuously growing, not only in landfills, but also within the area of Greek WWTPs, and this has an adverse effect on the environment and human health. In order to protect the environment, areas which already have an accumulation of sewage sludge should be reclaimed.

This paper reports on the possibility of using woody species, namely *Populus alba* L., *Robinia pseudoacacia* L., *Quercus pubescens* Willd. subsp. pubescens, and *Pinus pinea* L., for the reclamation of sewage sludge from the Thessaloniki WWTP (Greece) after its amending in different rations with soil of the neighbouring area and gravel. The establishment of the experimental site initiated in early January 2012. Seven ditches were dug using a tractor. Each ditch (13x1.5x0.8m) was filled with a mixture as follows: sludge/soil/gravel 33.33/33.33/33.33% applied as layers (T1), sludge/soil 50/50% (T2), sludge/soil 30/70% (T3), sludge non-amended (C4). In early March 2012 the treatments T1, T2, and T3 were planted with early mentioned plants. Four plants of each species separated by a distance of 0.5 m were used for each treatment. These plants were purchased from the Greek Forestry Service. The control units C1, C2, and C3 had corresponding mixture to T1-T3 respectively, but they were not planted. An automatic dripping irrigation system was installed in all experimental units and used for three hours early in the morning three times per week during the dry season. After six months since the plantation of the treatments, leaves of plants and samples of sludge mixture were collected for nutrients analyses. Macronutrients (Ca, Mg, K, P) trace elements (Mn, Cu, Zn, Fe, Cr, Ni, Pb) and Na were measured. The results showed that plants took up macronutrients at relatively high rates, in some cases reducing their excessive levels of concentration in the sludge by as much as 95 %. Trace elements were removed at a lower rate compared to hyperaccumulator plants, this was possibly affected by their reduced bioavailability in the substrate, but the bioconcentration factor, which was greater than one for most of the species and for most of the trace elements, indicated that the plants were capable of phytoextraction. It was concluded that the best effect on the rates of plants survival was produced by amending the sludge with greater amounts of soil (e.g., to 70% as for T3 and its own control C3). The fact that no native vegetation was established themselves in the unit C4, which was composed of 100 % sludge, means that it is

<figure><img src="image_245.png" /></figure>

<footnote n="1">¹School of Civil Engineering, Aristotle University of Thessaloniki, Greece</footnote>

<footnote n="2">²School of Forestry and Natural Environment, Aristotle University of Thessaloniki, Greece</footnote>

<footnote n="3">³School of Biology, Aristotle University of Thessaloniki, Greece</footnote>

<footnote n="4">⁴Urban Environmental Engineering, Management and Safety Department, A.N. Beketov National University of Municipal Economy in Kharkov, Ukraine</footnote>
impossible to introduce plants directly to fresh sludge with no amendments. The nutrients uptake did not differ significantly between the treatments, but did between the plant species. Thus, *P. alba* and *R. pseudoacacia* had better ability to concentrate macronutrients, while *Q. pubescens* – trace elements, reaching in some cases the threshold of hyperaccumulators for Cr (1,000 mg kg⁻¹).

References:

1. Matthews, P., 1999. Scientific and technical report No 4.

2. IEEP, 2009. Report on the implementation of the Sewage Sludge Directive 86/278/EEC.

3. Papapetrou P. D., 2002. Διδακτορική διατριβή. Α.Π.Θ. Θεσσαλονίκη (in Greek).

4. Brofas, G., Michopoulos, P., Alifragis, D., 2000. *J Environ Qual* 29, 811-816

5. Christoulas, G., Andreadakis, D., Kouzeli-Katsiri, A., Aftias, E., Mamais, D., 2000. *Wat Sci Tech* 42 (9), 29-36.

<figure><img src="image_246.png" /></figure>
# PHYTOREMEDIATION OF PHARMACEUTICALS IN WASTE WATERS – FROM LABORATORY TO REAL SCALE

Tomas Vanek<fnref n="1" />, Tereza Hudcova<fnref n="2" />, Jiri Syrovatka<fnref n="3" />, Petr Soudek<fnref n="1" />, Marie Pribylova<fnref n="1" />, Petr Marsik<fnref n="1" />

Corresponding author email: vanek@ueb.cas.cz

## ABSTRACT

In the recent time, the global production of the human and veterinary pharmaceuticals strongly increases together with their affordability not only in developed countries, but due their availability and decreasing prices also in Third World countries. Nevertheless, among the most polluted areas belong developed and industrial countries with high standard of living and population density.

The one of the most significant problems is persistence of the pharmaceutical residues and their metabolites in the environment. Many of them do not exhibit acute toxicity for water ecosystems, but have a cumulative effect for nontarget organisms.

Groundwater analysis showed, that the non-steroidal antiflogistics (e.g. diclofenac, ibuprofen and ketoprofen) together with paracetamol and antibiotics were the most abundant pharmaceuticals in the samples collected in Czech test localities.

The laboratory experiments with *in vitro* cultures of *Arabidopsis*, *Melilotus officinalis* proved presence of the several common metabolites of oxidation in cultivation medium contained common NSAID. Achieved result suggests the ability of the plant cells to transform human and veterinary pharmaceuticals to the products of the firsts detoxification step.

These results were verified in reed (Phragmites australis L.) cells, tissues and whole plants and finally tested in real scale in constructed wetland with combined vertical and horizontal flow designed for agriculture waste waters cleaning. Elimination of phosphorus, nitrogen and ammonia in this arrangement will be discussed too.

<figure><img src="image_247.png" /><figcaption>Fig. 1. CW in construction 2012.</figcaption></figure>

**Acknowledgements:** This study were supported from GACR 14-22593S and TACR TA01020573 grant

<figure><img src="image_248.png" /></figure>

<footnote n="1">¹ Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i. Rozvojová 263, 165 02 – Prague 6, Czech Republic</footnote>

<footnote n="2">² Dekonta a.s. Czech Republic</footnote>

<footnote n="3">³ Chramce Farm Czech Republic</footnote>
# PHYTOREMEDIATION OF PCBs CONTAMINATED AGRICULTURAL SOIL BY LEGUMINOSAE-GRAMINEAE INTERCROPPING

Chen Tu<fnref n="1" />, Ying Teng<fnref n="2" />, Yongming Luo<fnref n="1" />,<fnref n="2" />

Corresponding author email: ymluo@yic.ac.cn

## ABSTRACT

Phytoremediation of polychlorinated biphenyls (PCBs) contaminated soil by leguminosae (alfalfa) and gramineae (ryegrass and tall fescue) was studied in a field trial. All planted treatments had significantly higher PCBs removal efficiency compared with those of unplanted control after 270 days of in-situ phytoremediation. Alfalfa monoculture received the highest removal efficiency of soil PCBs by 59.6%. Analysis of PCBs congeners composition showed that the percentage of di-chlorinated biphenyl decreased in all planted treatments. Alfalfa produced the maximum biomass among the 3 plants. Total PCBs concentration in alfalfa roots reached 355.1 µg/kg, which was significantly higher than those in ryegrass and tall fescue. The phytoextraction efficiency of different treatment was in order of alfalfa > alfalfa-ryegrass-tall fescue > alfalfa-ryegrass > ryegrass > alfalfa-tall fescue > tall fescue. Furthermore, the comparison of the soil microbe taxonomic assignment at the level of phylum under different treatment showed that intercropping with alfalfa could increase the microbial richness and diversity, thus enhance the PCBs removal from the field soils. The results suggest that alfalfa may be an ideal candidate for the phytoremediation of PCBs contaminated soil.

<footnote n="1">¹ Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, China</footnote>

<footnote n="2">² Institute of Soil Science, Chinese Academy of Sciences, China</footnote>
# IMPACT OF EKKMI ADDITION TO SOIL ON WEATHERED *p*,*p'*-DDE ACCUMULATION IN XYLEM SAPS OF PLANTS

Mehmet Isleyen<fnref n="1" />, Jason C. White<fnref n="2" />, Kendra Ann Morrison<fnref n="3" />

Corresponding author email: [mehmet.isleyen@gmail.com](mailto:mehmet.isleyen@gmail.com)

## ABSTRACT

DDT (2,2-bis(chlorophenyl)-1,1,1-trichloroethane) was one of the most extensively used organochloride pesticides globally during the 1960s to 1970s and was widely applied in Turkey until being banned in 1985. In soil, *p*,*p'*-DDT can be biotically and abiotically converted to 2,2-bis(chlorophenyl)-1,1-dichloroethane (*p*,*p'*-DDD) and 2,2-bis(chlorophenyl)-1,1-dichloroethylene (*p*,*p'*-DDE) residues. Both *p*,*p'*-DDT and its metabolites are classified as persistent organic pollutants (POPs) and bind strongly to soil and sediment organic matter. Except phytoremediation, application of many in situ remediation technologies for POP contaminated soils are both ineffective and expensive. Previous studies have shown that *Cucurbita pepo* ssp. *pepo* (zucchini) has a significant and unique potential to accumulate weathered POPs such as *p*,*p'*-DDE, polychlorinated biphenyls (PCBs), and chlordane from contaminated soil, but that other plants, including closely related squash and melon species, do not have this ability. However, the mechanism of POP uptake by *Cucurbita pepo* ssp *pepo* remains unknown.

EKKMI is a new material produced from activated sludge of domestic wastewater treatment plants. This research focuses on DDE accumulations in xylem saps of *Cucurbita pepo* ssp. *pepo* (Zucchini) plants grown in both DDT contaminated soil and DDT contaminated soil amended with EKKMI. Pot experiments were conducted to assess the effect of EKKMI amendment on the phytoextraction of weathered *p*,*p'*-DDE from soil by plant systems. Soil contaminated with 526-691 ng/g *p*,*p'*-DDE was amended with EKKMI at 0 % (Control) and 1.0 % (w/w) levels and *Cucurbita pepo* (cv Raven and Zephyr) was then planted. The plants were harvested after 35-days of growth and *p*,*p'*-DDE concentrations were measured in xylem saps of Raven and Zephyr plants by SPME methods coupled with GC/μ-ECD.

Average plant biomass of Raven and Zephyr plants grown in EKKMI amended pots were 15 and 8 times higher than their control plants grown in unamended (0% of EKKMI) pots. For 1% EKKMI amended Raven plants, an average xylem *p*,*p'*-DDE concentration was measured as 0.59 µg/L which is almost 4 times more than that of xylem *p*,*p'*-DDE concentration obtained from Raven control plants. While average xylem *p*,*p'*-DDE concentration was 0.07 µg/L for control Zephyr plants, xylem contaminant concentration was increased approximately 8 times relative to control plants. Further investigation needs to be performed of the changes of DDE concentrations of plant compartments and metal contents in xylem sap of the cultivar and root to xylem interactions of the plants grown in unamended and amended pots.

<figure><img src="image_249.png" /></figure>

<footnote n="1">¹Environmental Engineering Department, Sakarya University, Sakarya, Turkey</footnote>

<footnote n="2">²Dept of Analytical Chemistry, Connecticut Agricultural Experiment Station, New Haven, USA</footnote>

<footnote n="3">³United States Environmental Protection Agency, Region 8, Denver, Colorado, USA</footnote>
USING PLANTS TO REMEDiate TNT AND RDX POLLUTION

Elizabeth L. Rylott<fnref n="1" />, Long Zhang<fnref n="2" />, Gengyun Zhang<fnref n="2" />, Quyen Nguyen<fnref n="2" />, Timothy J. Cary<fnref n="3" />, Antonio J. Palazzo<fnref n="3" />, Stuart E. Strand<fnref n="2" />, Neil C. Bruce<fnref n="1" />

Corresponding author email: liz.rylott@york.ac.uk

ABSTRACT

The containment and remediation of the explosive compounds 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military ranges are of high priority to the US Department of Defense. While RDX is highly mobile in the soil column, where containment and remediation strategies are urgently needed, highly phytotoxic TNT remains tightly bound to the organic material in the soil, hindering the use of plant-based approaches for remediation. RDX is now a significant threat to drinking sources such as those close to the US Massachusetts Military Reservation. Currently, there are no cost effective processes to contain RDX or remediate these vast areas of contaminated, vegetated land on military training ranges.

Uncontaminated
soil

RDX and TNT
(250 mg/kg)

<figure><img src="image_250.png" /></figure>

Unmodified
TNT detoxifying (nfsi)-line
RDX degrading (xplA)-line
Double nfst-xplA-line

<caption>**Figure 1.** Six-week-old Arabidopsis plants growing in soil contaminated with TNT and RDX [2].</caption>

While plants have an inherently low ability to degrade RDX, we have established that expression of a unique bacterial cytochrome P450, XplA and its partnering reductase, XplB [1,2] in the model plant species Arabidopsis (Arabidopsis thaliana) enables these plants to remove, and degrade, saturating concentrations of RDX from soil leachate. To enable plants to contain and metabolize RDX, they have to be able to withstand, and grow, in the presence of the co-contaminating, toxic TNT. To overcome this, we engineered Arabidopsis plants to express a bacterial nitroreductase, (NR) [3] and demonstrated that this conferred resistance to TNT. We then produced Arabidopsis plants expressing

all three genes. These NR-XplA-XplB-
expressing plants are both resistant to TNT
and able to metabolise RDX, using the
nitrogen obtained for growth (Figure 1) [4].
These genes have now been transferred into
switchgrass (Panicum virgatum), a species
native to temperate military training ranges
in the US. We are now in the process of
conducting field trials (Figure 2) to test the
ability of transgenic switchgrass plants to
remediate RDX.

<figure><img src="image_251.png" /><figcaption>Figure 2. Developing field trial plots in the US.</figcaption></figure>

References:

1. Jackson *et al.* (2007) *P.N.A.S.* 104, 16822-16827

2. Rylott *et al.* (2006) *Nat.Biotechnol.* 24, 216-9

3. Hannink *et al.* (2001) *Nat.Biotechnol.* 19, 1168-1172

4. Rylott *et al.* (2011) *et al. New Phytol.* 192, 405-413

Acknowledgements: This work is funded by the Strategic Environmental Research and Development Program (SERDP) and Environmental Security Technology Certification Program (ESTCP) of the US Department of Defence.

<figure><img src="image_252.png" /></figure>

<footnote n="1">¹Centre for Novel Agricultural Products, Department of Biology, University of York, York, YO10 5DD, UK</footnote>

<footnote n="2">²Department of Civil and Environmental Engineering, University of Washington, Seattle, WA, US</footnote>

<footnote n="3">³ERDC-CRREL, Hanover, NHL, US</footnote>
# ARABIDOPSIS GLUTATHIONE TRANSFERASES DETOXIFY TNT

Kyriakos Tzafestas<fnref n="1" />, Vanda Gunning<fnref n="1" />, Helen Sparrow<fnref n="1" />, Emily J. Johnston<fnref n="1" />, Andrew S. Brentnall<fnref n="2" />, Jennifer R. Potts<fnref n="2" />, Elizabeth L. Rylott<fnref n="1" />, Neil C. Bruce<fnref n="1" />

Corresponding author email: kt723@york.ac.uk

## ABSTRACT

The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant presents various health and environmental concerns. Due to its chemically resistant structure, TNT has proven to be highly recalcitrant to microbial degradation. In the present study we investigate the role of two glutathione transferases (GSTs), GSTU-24 and GSTU-25, from *Arabidopsis thaliana* (Arabidopsis), which are specifically upregulated in response to TNT exposure.

Microarray analysis on TNT-treated Arabidopsis plants and subsequent quantitative PCR analysis revealed eight upregulated GSTs, all in the Tau class (GST-U1, U3, U4, U7, U12, U22, U24 and U25), with GST-U24 and GST-U25 being the most up-regulated (252-fold and 177-fold respectively). Following affinity chromatography purification and characterisation of recombinant forms of both enzymes, we identified three distinct TNT-glutathionyl products. HPLC-based assays with subsequent mass spectrometry and NMR spectroscopy proved that GSTU-25 was able to produce 2-glutathionyl-4,6-dinitrotoluene, with the concurrent release of nitrite. This conjugate is chemically weaker than trinitrotoluene structures and is likely to be more susceptible to biodegradation.

In order to further investigate the detoxification abilities of these enzymes *in planta* the CaMV-35S promoter was used to drive the over-expression of GST-U24 and GST-U25 in Arabidopsis. These GST overexpressing lines exhibited significantly increased ability to withstand and detoxify TNT, displaying higher shoot and root biomasses than untransformed plants when grown in the presence of TNT. In addition, the overexpressing lines were able to remove more TNT from soil, with a corresponding reduction in glutathione levels, when compared to wild type plants.

In conclusion, GSTs contribute to the TNT detoxification pathway in Arabidopsis, while over-expression of plant GSTs confers enhanced resistance to TNT, along with an increased ability to remove and detoxify this environmental pollutant. Furthermore, the 2-glutathionyl-4,6-dinitrotoluene conjugate produced presents the opportunity for subsequent degradation and mineralization of TNT, rather than the indefinite storage of TNT-transformation products in the environment. Ultimately the GST-mediated detoxification pathway demonstrated here could be exploited in more robust plant species for the phytoremediation of TNT.

**Acknowledgements:** This work was funded by the Strategic Environmental Research and Development Program of the US Department of Defence. HS and EJ acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) and KT acknowledges funding from a Burgess studentship.

<figure><img src="image_253.png" /></figure>

<figure><img src="image_254.png" /></figure>

<footnote n="1">¹Centre for Novel Agricultural Products (CNAP), Department of Biology, University of York, York, YO10 5DD, UK</footnote>

<footnote n="2">²Department of Biology, University of York, York, YO10 5DD, UK</footnote>
# CONTRIBUTION OF ARBUSCULAR MYCORRHIZAL INOCULATION IN THE PHYTOREMEDIATION OF PAH POLLUTED SOILS: MOLECULAR CHARACTERIZATION OF THE PROTECTIVE EFFECT

Ingrid Lenoir, Joël Fontaine, Anissa Lounès-Hadj Sahraoui

Université Lille Nord de France, Lille, France. Université du Littoral Côte d'Opale (ULCO). Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV). 50, Rue Ferdinand Buisson. F-62228 Calais, France
*Corresponding author email: anissa.lounes@univ-littoral.fr*

## ABSTRACT

Soil contamination by polycyclic aromatic hydrocarbons (PAH) originating from intensive anthropic activities has now become a priority concern in industrialized countries. Indeed, some of these compounds exhibit mutagenicity and/or carcinogenicity leading to human toxicity and disrupting ecosystem functioning. Among remediation methods of polluted soils, phytoremediation has the advantage of being both ecological and economic. This phytotechnology uses the natural ability of plants to eliminate persistent organic pollutants directly and/or indirectly by stimulation of the rhizospheric degrading microflora activities via root exudation.

Nowadays, it is known that the arbuscular mycorrhization improves PAH dissipation [1,2,3] and allows plant protection against PAH toxicity [4]. Recent results have shown that PAH induced an increase of malondialdehyde (a biomarker of lipid alteration) but that mycorrhizal reduce this PAH-oxidative stress through inducing antioxidant enzyme system such as superoxide dismutase [4,5]. Moreover, the protective effect of mycorrhization against PAH is linked with some lipid modifications [6,7,8]. However, the molecular mechanisms involving in the tolerance against PAH toxicity in both symbiotic partners are still unknown. Thus, our work goes into this context and aims to study, both in the plant host and the mycorrhizal fungus, the expression of some genes involving in the resistance against reactive oxygen species (ROS) such as superoxide dismutase, peroxidase, glutaredoxin and pyridoxine; two DNA repair genes: transcription elongation factor II-S and Tyrosyl-DNA phosphodiesterase; one gene of metabolization of pollutants: glutathione S-transferase and two genes of lipid metabolism: sterol methyl oxidase and acetyl CoA but also verify the presence of oxidative stress by the dosage of H₂O₂ (ROS) in presence of benzo[a]pyrene (a high molecular weight PAH).

Our experiments have been carried out using a monoxenic culture system containing *Medicago truncatula* cv. *Jemalong* (A17) roots transformed with *Agrobacterium rhizogenes* and mycorrhized or not by the arbuscular mycorrhizal fungus (AMF) *Rhizophagus irregularis* in absence or presence of benzo[a]pyrene. Some plant antioxidant genes (superoxide dismutase and peroxidase) and the two DNA repair genes have been up-regulated in non mycorrhizal plants in presence of pollutant whereas no difference of these genes expressions were observed in mycorrhizal plants. Up-regulations of fungal genes involved in the protection against ROS (pyridoxine) and in the metabolization of pollutants (glutathione S-transferase) have also been observed. Taken together, these results suggested that arbuscular mycorrhization is able to give protection to *Medicago truncatula* against oxidative stress induced by benzo[a]pyrene.

**Keywords:** PAH, phytoremediation, arbuscular mycorrhizae, resistance gene expression, oxidative stress

## References:

1. Joner E.J., Johansen A., Loibner A.P., de la Cruz M.A., Szolar O.H., Portal J.M., Leyval C. (2001) *Environmental Science and Technology* 35 (13), 2773-2777.

2. Gao Y., Li Q., Ling W., Zhu X. (2011) Journal of Hazardous Materials 185 (2-3), 703-709.

3. Verdin A., Lounès-Hadj Sahraoui A., Fontaine J., Grandmougin-Ferjani A. et Durand R. (2006) *Mycorrhiza* 16, 397-405.

4. Debiane D., Garçon G., Fontaine J., Verdin A., Shirali P., Durand R., Grandmougin-Ferjani A. and Lounès-Hadj Sahraoui A. (2008) *Environmental and Experimental Botany* 64, 120-127.

5. Debiane D., Garçon G., Verdin A., Fontaine J., Durand R., Shirali P., Grandmougin-Ferjani A. et Lounès-Hadj Sahraoui A. (2009) *Phytochemistry* 70, 1421-1427.

6. Debiane D., Calonne M., Fontaine J., Laruelle F., Grandmougin-Ferjani A., Lounès-Hadj Sahraoui A. (2011) *Fungal Biology* 115, 782-792.

7. Debiane D., Calonne M., Fontaine J., Laruelle F., Grandmougin-Ferjani A., Lounès-Hadj Sahraoui A. (2012) *Journal of Hazardous Materials* 209-2010, 18-26.

8. Calonne M., Fontaine J., Debiane D., Laruelle F., Grandmougin-Ferjani A., Lounès-Hadj Sahraoui A. (2014) *Phytochemistry* 97, 30-37.

<figure><img src="image_255.png" /></figure>
# PCB REMOVAL IN LAGOON SEDIMENTS USING POPLAR AND WILLOW PHYTO PROCESSES

**Louis A. Licht**

President, Ecolotree Inc. North Liberty IA. USA

Corresponding author email: lou-licht@ecolotree.com

## ABSTRACT

This report details activities to reduce the mass of Polychlorinated Biphenyl (PCB) at the Altavista Virginia waste water Lagoon Site using phytoremediation (Phyto) techniques. PCB concentrations as high as 10,000 ppm have been measured in sediments deposited over 30 years ago. The Virginia Department of Environmental Quality has established a 50 ppm total PCB cleanup concentration goal.

Research at The University of Iowa has proven that a grass rhizosphere accelerates the PCB mineralization rate in planted soils. The ECap® poplar root system performs the same processes though deeper at full-scale. This project demonstrates how poplar and willow can grow a healthy plant rhizosphere in three feet of sediment within an operating lagoon. Roots support a more diverse microbe population. During the growing season, flooding and drying within the lagoon sediment pulse the water content, oscillating the redox potential.

Six ECap® plots were installed in 2012 – 2013 by placing a layer of soil blended using local materials over sediment. Unrooted poplar whips were then pushed through the soil and sediment layers to the top of the clay liner.

The planted poplar and willow trees grow well in the cover soil to increase water transpiration from sediment. The ECap® tree cover will help contain PCBs in the subsurface by keeping the surface soil layer intact while sustaining microflora intimate with roots.

Sediment samples taken before and after installing ECap plot #5 show PCB removal patterns. Native microbes in the rhizosphere appear to be achieving PCB 'disappearance' in sediment. Research is being proposed by The University of Iowa to define the microbial PCB transformation mechanisms and transport processes in a healthy poplar rhizosphere.

**Keywords:** PCB, sediment, rhizosphere, phytoremediation, poplar

<figure><img src="image_256.png" /></figure>

<figure><img src="image_257.png" /></figure>

<figure><img src="image_258.png" /></figure>
# PHYTOREMEDIATION OF CONTAMINATED SOIL WITH POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) BY SALICORNIA PERSICA

Mahleh Eghbalinejad<fnref n="1" />, Nayer-Azam Khosh Kholgh Sima<fnref n="2" />

*Corresponding author email: meghbalinejad@yahoo.com*

## ABSTRACT

Oil and salinity pollutions have been a serious problem of the modern age, due to loss of plant cover and soil organism diversity. In order to investigate the possibility of enhancing biodegradation of halophytes, this study was conducted to evaluate the mechanisms of natural biodegradation of crude oil under salinity conditions. For that, *Salicornia Persica* were collected from Nayband of Forest Park, Bushehr, Iran, and cultured in pots with soil texture of Sandy loamy. Six levels of crude oil, including 0, 0.4, 0.8, 1.6, 2.4 and 5.3% (w/w) and four levels of Salinity including 0, 200, 400, 600 mM NaCl were considered as treatments with 3 replications. PAHs concentrations of shoot and root were determined by (GC-MS) after 21 days of start of oil treatments. Results showed that the concentrations of crude oil and NaCl were significant on PAHs concentrations in shoot and root. Furthermore, by increase of NaCl concentration, uptake and concentration of PAHs compounds decreased considerably. Benzo (b) fluoranthene and Benzo ((ghi) perylene in shoot with 1874.461 and 1860.27, and Phenanthrene, Benzo((ghi)perylene in root with 2531.9988 and 2149.0945 µg/kg dw respectively were the most compounds. Absence of PAHs in control suggest that root was the main pathway for uptake of PAHs in this halophyte and it also seemed that phytoextraction and rhizodegradation have occurred as a major mechanism to remove contaminants. By this capacity of uptake PAHs from contaminated soil with crude oil, *Salicornia Persica* as potential candidate of native plant can be used as Bioaccumulator of marine pollution.

**Keywords:** contaminated soil, petroleum pollution, halophytic plant, accumulation, biodegradation, phytoextraction

<figure><img src="image_259.png" /></figure>

<footnote n="1">¹University of Tehran, Tehran, Iran</footnote>

<footnote n="2">²Agricultural Biotechnology Research Institute of Iran (ABRII), Tehran, Iran</footnote>
# DO CROSSTALKS WITH PRIMARY AND SECONDARY METABOLISM LIMIT OR STIMULATE THE DETOXIFICATION OF XENOBIOTICS?

## Jean-Paul Schwitzguébel

Laboratory for Environmental Biotechnology, EPFL, Lausanne, Switzerland

Corresponding author email: jean-paul.schwitzguebel@epfl.ch

## ABSTRACT

Because plants are static and live in a competitive and sometimes hostile environment, they have evolved efficient mechanisms that protect them from abiotic and biotic stresses. These mechanisms include detoxification and sequestration of xenobiotic compounds and of toxic trace elements, exploited in any phytoremediation process. However there must be a limit on the amount of pollutants that can be accumulated and detoxified without disrupting the normal plant biochemistry and physiology. This limit seems to depend not only on plant species, but also on the ecotype or cultivar. The presentation aims to highlight some biochemical mechanisms, suggested or supposed to of importance for the successful phytoremediation of organic contaminants.

Enzymes involved in xenobiotics detoxification are often linked to the redox chemistry of the cell. The activities of cytochrome P450 monooxygenase, peroxidase and glutathione transferase have implications on the regulation of cellular redox status, closely related to mitochondrial respiratory chains, also involved in maintaining the cellular and plant energy balance and carbon flow. For example, overloading a plant with high concentrations of xenobiotics requiring oxidation by P450 may compete with the normal functions of these enzymes. An increase in their activity may also impose a major demand on both intracellular O₂ and NAD(P)H pools, disturbing plant redox or energy status, and thus affecting both primary and energy metabolism.

Plant mitochondrial respiratory chains differ from the mammalian one. In addition to Complex I, they contain at least four other dehydrogenases that enable the controlled oxidation of matrix and cytoplasmic NAD(P)H. Furthermore, plant mitochondria are characterized by the presence of an alternative respiratory pathway, through which reducing equivalents can be transferred to oxygen. This pathway branches at the level of the ubiquinone pool and comprises a single enzyme, the alternative oxidase, not coupled to the synthesis of ATP and insensitive to cyanide. Increase in the expression and/or activity of the alternative oxidase has been observed during temporal events such as seed conditioning, leaf development, elevation of salicylic acid levels, thermogenesis, fruit ripening, oxidative stress, physical wounding and plant pathogenic attack. The alternative pathway is inhibited by benzhydroxamate compounds, antioxidants such as propylgallate and copper chelators such a disulfiram. The alternative oxidase is able to oxidise several added polyphenolic substrates and could also oxidise intracellular polyphenolic compounds. It is not yet known if it could directly participate in the metabolism of some xenobiotics.

Depending on the structure of the organic pollutant to be detoxified, the secondary metabolic processes in the plant could also be affected. Many plant specific metabolites, often involved in plant interactions with its environment, have a structure similar to xenobiotics, and detoxification of the latter does probably use, at least partially, the metabolic pathways of the former. For example, most of the natural anthraquinones are glycosylated, whereas glycosyl-transferases are known to be involved in the conjugation of many xenobiotics, probably including sulphonated anthraquinones. It is not yet known if the detoxification mechanisms of sulphonated aromatic compounds are unique and specific to anthraquinone producing plants.

Molecules involved in the conjugation of xenobiotics, like glutathione, also play a major role in normal plant metabolism. The presence of xenobiotic compounds can induce the biosynthesis of glutathione transferases and thus an increased use of glutathione. Plant glutathione level and redox status are thus affected under such conditions. On the other hand, phytochelatins are derived from glutathione and involved in the detoxification of trace elements with their -SH groups. In either case, implications on sulphur supply and metabolism are expected.

Experimental conditions do also affect plant physiology and biochemistry, and it is not obvious if results obtained with young and small plantlets under well-defined laboratory conditions can be extended to mature plants in the field. However, the large-scale implementation of phytoremediation will be successful only if the "right plant is used at the right place". Basic physiological and biochemical knowledge is thus required to select the most appropriate plant species, ecotype or cultivar, tolerant to the contaminants to be treated and able to accumulate and detoxify them without impacting its growth and survival.

<figure><img src="image_260.png" /></figure>
# EXOGENOUS OXYSTEROLS INCREASE THE NET PHOTOSYNTHETIC RATE, O₂ PRODUCTION AND CO₂ BINDING

Marek Kamlar<fnref n="1" />, Dana Hola<fnref n="2" />, Sarka Salajkova<fnref n="3" />, Marie Kocova<fnref n="2" />, Dana Tarkowska<fnref n="4" />, Pavel Drasar<fnref n="3" />, Olga Rothova<fnref n="2" />, Tomas Macek<fnref n="1" />

Corresponding author email: tomas.macek@vscht.cz

## ABSTRACT

An examination of the involvement of exogenously applied oxysterols, 24-epibrassinolide (24E) and the 20-hydroxyecdysone (20E) in the regulation of primary photosynthetic processes was performed together with the analysis of changes of the content of the above sterols in plant tissue. Phytoecdysteroids are steroid compounds present in many plant species (sometimes in rather large amounts), but their biological role is still far from being clear. We have found that the exogenous application of 20-hydroxyecdysone (20E) to leaves of New Zealand spinach causes stimulation of its net photosynthetic rate (PN) but does not positively affect the photosynthetic electron transport or the content of photosynthetic pigments. The increase in PN was observed shortly after 20E treatment and was statistically significant during the 4th and 6th hours after treatment but not later, which could be perhaps caused by a strictly short-term window of opportunity for ecdysteroids to significantly affect photosynthetic processes. To our knowledge, these results are the first to suggest a new potential biological function of phytoecdysteroids — regulation of photosynthesis. The effect of 20-hydroxyecdysone (20E) applied in low (10 nM) concentration on various photosynthetic parameters was made in spinach and maize. It also positively influenced the content of photosynthetic pigments in maize (not in spinach). The efficiency of the photosynthetic whole electron-transport chain responded negatively to the 24E or 20E treatment in both species, but there were interspecific differences regarding Photosystem (PS) II response: a positive effect on its oxygen-evolving complex and a slightly better energetical connectivity between PSII units were observed in maize whereas the opposite was true for spinach. The size of the pool of the PSI end electron acceptors was usually diminished due to 24E or 20E treatment. Plants treated with combination of both steroids mostly did not significantly differ from the control plants. We have demonstrated for the first time that 20E applied in low concentration can affect various parts of photosynthetic processes similarly to 24E and that brassinosteroids regulate not only PSII but also other parts of the photosynthetic electron transport chain – but not necessarily in the same way. The results confirm our previous results obtained with *in vitro* models, aimed at plant ecdysteroid-binding proteins identified using affinity chromatography (e.g. ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBisCO, oxygen evolving complex OEC and others). Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO₂ is fixed into organic matter, influence the water cleavage reaction evolving oxygen, and the chromatography analyses with MS detection show that application of exogenous 24-epibrassinolide can influence the ecdysteroid content in plant tissues.

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

<figure><img src="image_261.png" /></figure>

<footnote n="1">¹ Dept. of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague, Czech Republic</footnote>

<footnote n="2">² Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Czech Republic</footnote>

<footnote n="3">³ Dept. Natural Products, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague, Czech Republic</footnote>

<footnote n="4">⁴ Laboratory of Growth Regulators, Palacky Univ. and Institute of Experimental Botany ASCR, Olomouc, Czech Republic</footnote>
# BEFORE THE BLAST: THE INTERACTION BETWEEN PLANTS AND EXPLOSIVES

Paul V. Manley, II, Julie C. Zinnert, Donald R. Young

Department of Biology, Virginia Commonwealth University, USA

Corresponding author email: [manleypv@mymail.vcu.edu](mailto:manleypv@mymail.vcu.edu)

## ABSTRACT

Landmines are a global threat; at least 110 million landmines remain in place from conflicts in over 60 nations. Today, certain landmine detection methods place individuals in harm's way. Some risk can be mitigated using currently available remote sensing techniques when vegetation is present. As a landmine degrades and releases explosive compounds into the surrounding soil, nearby vegetation may absorb those compounds via bulk water movement into roots. Contaminant uptake causes plant stress which is detectable remotely using hyperspectral reflectance sensors. These sensors measure reflected light over a range of wavelengths which reveals information about the health status of a plant. Our study focused on using plants as bio-indicators to detect buried explosives. We exposed three species representing different functional types (*Ulmus alata* (tree), *Vitis rotundifolia* (vine), *Cyperus esculentus* (sedge)) to 500 mg kg⁻¹ of Composition B (Comp B; 60/40 mixture of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT)), a commonly used explosive, and took leaf fluorescence and reflectance measurements over a nine week period. These measurements were analyzed within and across species using standard reflectance indices to discern stress caused by exposure to Comp B in soil. Reflectance indices use specific wavelengths to reveal information about plant functional traits (i.e., pigment content, specific leaf area (SLA), and biomass). Our objective was to compare standard reflectance indices to specific wavelengths that are altered when vegetation responds to the presence of explosives in soil.

Plants in soil contaminated with Comp B exhibited changes in pigment content, SLA, and biomass as well as plant reflectance for *U. alata* and *V. rotundifolia*. *Cyperus esculentus* was not a good indicator for the presence of explosive compounds, only showing significance in one index at week five and another at week nine. Woody species *U. alata* and *V. rotundifolia* both showed significance in more indices representing changes in chlorophyll content, fluorescence, and SLA including the Photochemical Reflectance Index (PRI), $R_{750} - R_{710}$, $R_{740} - R_{850}$, and the Normalized Difference Vegetation Index (NDVI). By week five, leaves of the woody species exposed to Comp B were necrotic and senesced, which were then replaced by new growth. By week nine only two indices indicative of chlorophyll fluorescence revealed signs of stress, likely due to leaf drop and subsequent regrowth. Our next step is to use wavelengths that show differences due to the presence of Comp B to develop an explosives-specific index (ESI). Using plants from different parts of the globe and of various functional types will aid in the creation of an ESI for real-life application. Scaling up landmine detection efforts to the landscape level will decrease the inherent risk of current detection methods.

<figure><img src="image_262.png" /></figure>
# IN-SILICO STUDIES OF Cu-Zn SUPEROXIDE DISMUTASE ENZYME FROM HELIANTHUS ANNUUS WITH POLYCYCLIC AROMATIC HYDROCARBONS TO FIND OUT INSIGHT INTO ITS PHYTOREMEDIATION CAPABILITIES

Sudeep Roy<fnref n="1" />, Garima Soni<fnref n="2" />, Ashok Sharma<fnref n="2" />, Ivo Provaznik<fnref n="1" />

Corresponding author email: roy@feec.vutbr.cz Phone: +420774343910

## ABSTRACT

Cu-Zn superoxide dismutase is an enzyme that is reported to detoxify heavy metals and thus aid in phytoremediation. In this study an effort has been made to explore the wide potential of Cu-Zn superoxide dismutase enzyme found in *Helianthus annuus* for its capability to degrade pollutants of several industries which are mainly polycyclic aromatic hydrocarbons. It includes pollutants from iron, steel, rubber, stone and glass industries. Comparative genomics approach was employed in order to deduce the similarity between Aldehyde Keto Reductase [AKRs] related ESTs and gene corresponding to Cu-Zn superoxide dismutase [SOD1] in *Helianthus annuus*. This was followed by modeling of Cu-Zn superoxide dismutase protein. Motif and profile studies correlated the findings. Physico-chemical and functional characterization of the modeled protein was performed. The modeled protein is thermostable over wide range of temperatures and also has high aliphatic index. For molecular docking, a set of pollutants were selected from five different industries. The data were obtained from Environment Protection Agency [EPA] for iron, steel, rubber, stone and glass industries. Protein-ligand docking was carried out using Discovery Studio. Dibenzofuran, anthracene, Butyl benzyl phthalate, Tetrabromobisphenol, Mercaptobenzothiazole, Flumeturon and phenyl isocyanate dimer from different industries showed better binding capability with the Cu-Zn Superoxide Dismutase protein. These findings needs to be further tested in the field condition for its validation. These studies may open new avenues for utilizing the medicinal plants and their detoxifying properties for remediation of hazardous pollutants.

<footnote n="1">¹ Department of BioMedical Engineering, Faculty of Electronics and Communication, Brno University Of Technology, Antonínská 548/1, 601 90 Brno, Czech Republic</footnote>

<footnote n="2">² Biotechnology Division, Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research, Lucknow - 226015, India</footnote>
# ASSESSING THE USE OF *SALIX ALAXENSIS* FOR THE RHIZOREMEDIATION OF DIESEL-CONTAMINATED SOIL

Jessica Starsman<fnref n="1" />, Silke Schiewer<fnref n="1" />, Bill Schnable<fnref n="1" />, Mary Beth Leigh<fnref n="2" />

Corresponding author email: jkbay@alaska.edu

## ABSTRACT

Throughout Alaska there are many contaminated sites that cannot be reached by roads that require remediation. In the case of the remote community of Kaltag, Alaska tank farms were established in the early 1960's and since that time there have been multiple diesel spills. Rhizoremediation using native plants is a potentially cost-effective method for remediating soils contaminated with organic contaminants, but has not yet been extensively tested in Alaska. Native plants have the added benefit of being adapted to local conditions. The willow *Salix alaxensis* is native to Alaska and in previous studies has been shown to promote diesel biodegradation in soil and alter the diversity of active naphthalene degraders. These effects could potentially be related to the release of salicylic acid as a secondary root compound. Due to the presence of weathered diesel, the remote location and the costs of ex-situ remediation, the use of *Salix alaxensis* for rhizoremediation of the site was studied. Microcosm studies were performed with crushed *Salix alaxensis* root, mimicking root turnover, as well as salicylic acid and fertilizer on the biodegradation of fresh and weathered diesel and 1-chlorooctadecane in Kaltag soils. With cold weather dominating much of the year, half of the microcosms were incubated at 4°C as well as 20°C to simulate summer conditions. Diesel and 1-COD losses were quantified using GC-MS, respiration was monitored using respirometry, and populations of diesel-degraders were quantified using most probable number (MPN) methods. Community analyses using 16S rRNA sequencing are also planned. Preliminary results indicate that the soils possess substantial diesel-biodegradation abilities, and analyses of the effects of willows and other treatments are in progress. If found to be effective for biostimulation, the use of *Salix alaxensis* would be advantageous as cuttings could be taken directly from the local region. Future field-scale tests are planned in Kaltag to determine the effectiveness and costs of phytoremediation using native willows and other plants in relation to land farming methods.

**Acknowledgements:** The financial support of Alaska Department of Environmental Conservation and National Institutes for Water Resources.

<figure><img src="image_263.png" /></figure>

<footnote n="1">¹Institute of Northern Engineering, University of Alaska Fairbanks, Fairbanks, Alaska USA</footnote>

<footnote n="2">²Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska USA</footnote>
# A CONSISTENTLY ENHANCED PHOTOSYNTHETIC CAPACITY IMPROVES BRASSINOLIDE-MEDIATED PHENANTHRENE STRESS TOLERANCE

Golam Jalal Ahammed<fnref n="1" />, Jing Quan Yu<fnref n="2" />

Corresponding author email: abammed@zju.edu.cn

## ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants well-known for their mutagenic, carcinogenic and teratogenic activities. PAHs exist in the atmosphere in the form of solid and liquid aerosols. Because of lipophilic nature, PAHs are easily accumulated in the cuticle of leaf and thereby causing phytotoxicity. Plant growth regulators are used to improve plant tolerance in order to strengthen phytoremediation program as well. Brassinolide (a plant steroidal hormone) has been extensively documented for its role in abiotic stress tolerance. Previously, it has been reported that 24-epibrassinolide (EBR) alleviates root imposed phenanthrene (PHE) stress. However, role of EBR against PHE foliar spray-induced phytotoxicity is unknown. Here, we investigated the effects of EBR on photosynthetic apparatus at different time course challenged with PHE foliar spray in tomato.

Foliar parts of tomato plants were pretreated with 0.1 µM EBR and at the next day treated with 100 µM PHE inside specially constructed open-top chamber in green house. The foliar spray was repeated every 5 days for EBR and every 3 days for PHE and the experiment was terminated 25 days after first PHE application. At the end of treatment period, it was observed that PHE foliar spray significantly inhibited plant growth and biomass as indicated by reduced plant height, stem diameter, dry and fresh weight of shoot and root. Transmission electron microscopic observation showed obvious ultrastructural alterations especially in the chloroplast of leaf cell in PHE-treated plants. PHE-treated plants showed distorted or disorganized chloroplasts particularly in grana staking and development of plastoglobuli. Elevated levels of lipid peroxidation and accumulation of reactive oxygen species confirmed that PHE foliar spray caused an oxidative stress in the leaf. However, application of EBR remarkably improved plant growth and biomass, and lowered the oxidative damage to cell membrane and photosynthetic apparatus.

PHE foliar spray significantly reduced CO₂ assimilation rate (Pn), stomatal conductance and also transpiration rate (Tr) constantly throughout the experimental period. Supplementation of EBR not only improved Gs, but also Tr and consistently kept higher Pn in EBR+PHE-treated plant compared to PHE alone. Maximum photochemical efficiency and related chlorophyll fluorescence parameters studied at different time points during PHE treatment were all higher in EBR treated PHE-stressed plants. Activity of antioxidant and detoxification related enzymes such as superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, glutathione reductase, glutathione S-transferases, phenylalanine aminelyase and polyphenol oxidase etc. are all remarkably induced by EBR in PHE-stressed plants throughout the study period. Transcripts for different genes related to antioxidant enzymes are also in agreement with those observations.

<figure><img src="image_264.png" /></figure>

It could be concluded that EBR-mediated PHE foliar spray-induced phytotoxicity alleviation was associated with a consistent enhancement of photosynthetic capacity, antioxidant enzymes activity and redox homeostasis for a prolonged period of time in tomato. In addition, results of current study suggest that EBR may have a protective role against atmospheric PHE-induced phytotoxicity and EBR could be considered as a potential phytohormone for assisted phytoremediation to design a feasible and efficient clean up technology.

**Acknowledgements:** The financial support by National Basic Research Program of China (2009CB119000) and National Natural Science Foundation of China (31071790) is greatly appreciated.

<figure><img src="image_265.png" /></figure>

<footnote n="1">¹Dept of Horticulture, Zijingang Campus, Zhejiang University, Hangzhou 310058, P.R. China</footnote>

<footnote n="2">²Key Lab of Horticultural Plants Growth, Development and Quality Improvement, Ministry of Agriculture, P.R. China</footnote>
# EXPLOSIVES CONTAMINATED SOIL AS A PHYSIOLOGICAL FILTER AGAINST PLANT SUCCESSION AND ESTABLISHMENT

Stephen M. Via, Julie C. Zinnert, Donald R. Young

Department of Biology Virginia Commonwealth University, USA

Corresponding author email: viasm@vcu.edu

## ABSTRACT

Soils contaminated with explosive compounds pose a threat to individual organisms and whole ecosystems. While many studies have focused on individual species responses to explosive compounds there are relatively few community or ecosystem scale explosive contaminant studies. In an attempt to bridge this gap our goal was to investigate impacts on community structure and diversity in an area contaminated with three explosive compounds/mixtures (RDX, TNT, Composition B). Community data were collected from an experimental minefield which was cleared 17 years ago, contaminated with explosives, and allowed to revegetate naturally. Plots within contaminated and reference sites were established and presence/absence for both woody and herbaceous species was recorded. Indicator species analyses were used to identify species related to environmental variations. Ordination and cluster analyses were used to determine if contaminant presence caused shifts in community composition.

Diversity metrics (i.e. species richness, diversity, Shannon-Weiner indices) identified trends present in the communities which differentiated reference and contaminant plots. Species area curves showed that there were large separations between the plot richness with explosives compound contamination and reference plots in both the woody and herbaceous assemblages. Each contaminant type had a different set of woody and herbaceous indicator species. Ordination and cluster analyses both showed separation of reference and treatment areas based on community composition. Differences between reference and contaminant sites in species composition, diversity, and richness suggests that presence of explosive compounds in the soil is acting as a physiological filter which influences species establishment and success in the contaminated areas. In addition the various contaminants may act in different ways resulting in slightly modified communities. Explosive contaminated soils can have noticeable large scale impacts long after an initial contamination event.

<figure><img src="image_266.png" /></figure>
# APPLICATION OF PLANTS FOR BENZENE REMOVAL

Chairat Treesubsuntorn, Paitip Thiravetyan

School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10150, Thailand

Corresponding author email: paitip.thi@kmutt.ac.th

## ABSTRACT

An indoor gaseous benzene removal technology that has been widely researched is phytoremediation because plant can completely degrade benzene for use as a carbon source by P450 (mono-) or (di-) oxygenase. 8 ornamental plants that were screened for benzene removal showed that *D.sanderiana* can completely removal 20 ppm of benzene within 7 days, and the comparison between benzene removal by *D.sanderiana* under dark and light conditions found that benzene uptake under light conditions has higher benzene removal efficiency than under dark conditions. In addition, closing of stomata was found when *D.sanderiana* was grown under dark conditions. However, benzene removal by *D.sanderiana* still occurred under dark conditions. This result suggested that cuticle of plant could affect benzene uptake efficiency. To confirm the relation between benzene uptake and quantity of plant cuticle, 22 plant leaf grinding materials were screened for benzene adsorption and the quantity of crude wax in each plant species was studied. The results showed that *D. picta*, *A. aureum*, *F.religiosa*, *L. macrocarpa*, *A. scholaris* and *D.sanderiana* grinding leaf materials have high benzene removal efficiency, and the increase of crude wax quantity enhances benzene adsorption (r²=0.6512). Not only crude wax quantity but also wax composition was analyzed, and it was found that high quantity of α-linoleic acid might enhance high benzene adsorption efficiency (r²=0.4410). For industrial application, continuous benzene adsorption system and plant leaf material immobilized on glass beads were also designed. The result showed that removing 55 ppm of benzene continuously at retention times of 3-5 mins, *D. picta*, *A. aureum*, *A. scholaris* grinding leaf materials could remove around 70-90% of benzene, and the saturation of benzene on *A. aureum* and *A. scholaris* grinding leaf materials, which had the highest benzene adsorption capacity, was about 132 hours. The benzene adsorption mechanism was also studied by benzene desorption using solvent and it was found that more than 99% of benzene could be desorbed easily by hexane. This result confirmed that physical sorption is involved in benzene adsorption mechanism of plant leaf materials. In addition, functional groups on surface materials before and after benzene adsorption were analyzed by FTIR to confirm the physical sorption appearance. Although there are several technologies that have been developed to treat benzene in industries, these technologies usually generate secondary waste and require high cost for maintenance and waste disposal. The application of plant and plant materials is an attractive method that should be studied and developed.

**Acknowledgements:** The authors would like to thank the Thailand Research Fund and King Mongkut's University and Technology Thonburi (KMUTT) for their support for this research through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0319/2550).

<figure><img src="image_267.png" /></figure>
# COMPARISON OF STIMULATED PHYTOREMEDIATION OF PETROLEUM HYDROCARBONS IN AN AGED AND A FRESHLY SPIKED SOIL

Imran Hussain<fnref n="1" />,<fnref n="2" />, Bernhard Wimmer<fnref n="1" />, Gerhard Soja<fnref n="1" />, Angela Sessitsch<fnref n="1" />, Thomas G. Reichenauer<fnref n="1" />

Corresponding author email: thomas.reichenauer@ait.ac.at

## ABSTRACT

Remediation of organic contaminants in soils is a challenging problem in environmental science and engineering. Phytoremediation is a promising technology that uses plants and associated microorganisms for the remediation of petroleum hydrocarbons (PHC). *Lolium multiflorum* and *Lotus corniculatus* were used in a pot experiment in the greenhouse to compare phytoremediation in an aged contaminated soil (collected from a historically contaminated site, Austria) and a freshly spiked soil. The effect of simple plant growth in comparison to additional treatments with nutrients and biochar as well as inoculation of plants with PHC-degrading microorganisms was investigated. Plant performance was compared between the two contaminated and a non-contaminated soil.

Spiked and non-contaminated soils were made by mixing sand and loess in a proportion of 9:1 to obtain the same grain size distribution as the aged contaminated soil. 50 g of crushed coal was added per kg soil to simulate the soil from the contaminated site. The spiking concentration for diesel was 22.65 ml/kg soil to obtain the same petroleum hydrocarbon concentration that was measured in the aged contaminated soil (16292mg/kg soil). To stimulate phytoremediation nutrients, biochar and PHC-degrading microbial strains were added. Nutrients NH₄Cl (150 mg/100 g soil) and KH₂PO₄ (20 µg /100 g soil) were added, biochar was mixed with soil (5% w/w) and the microbial consortium (mixture of strains: Pantoea sp. strains, ITSI10 and Pseudomonas sp. strain, MixRI75) was added by seed inoculation before sowing.

Germination percentage (GP) was monitored weekly until three weeks after seed sowing. Biometric parameters (plant height, fresh and dry weight of shoots) and leaf chlorophyll content were recorded in periodic intervals. Soil samples were taken in regular intervals and PHC content was measured by GC-FID. The pot experiment started in spring 2014 and is planned until autumn 2015. The current results show that the germination rate of both plant species was reduced on aged contaminated soil, but not on spiked soil compared to the non-contaminated control. However 49 days after sowing biomass was significantly lower on spiked soil and on aged contaminated soil compared to the control soil. Amendment of nutrients and nutrients + microbial inoculation increased the biomass of *Lolium multiflorum*, but not in *Lotus corniculatus* in spiked soil. These amendments had no effect in aged contaminated soil. During the application of this abstract only one plant harvest was performed. In the presentation we will report about the development of plants and the degradation of PHCs in the first 6 months of the experiment.

<figure><img src="image_268.png" /></figure>

<footnote n="1">¹AIT Austrian Institute of Technology GmbH, Department of Health and Environment, Tulln, Austria</footnote>

<footnote n="2">²Department of Molecular Systems Biology, Faculty of Life sciences University of Vienna, Austria</footnote>
# RECLAMATION OF OIL SAND MINING SITES IN CANADA: OPPORTUNITIES AND CHALLENGES FOR PHYTOTECHNOLOGIES

**Jim Germida**<fnref n="1" />, **Natalie Blain**<fnref n="1" />, **Eduardo Mitter**<fnref n="2" />, **Sagar Chhabra**<fnref n="1" />, **Renato De Freitas**<fnref n="1" />, **Bobbi Helgason**<fnref n="3" />

*Corresponding author email: jim.germida@usask.ca*

## ABSTRACT

A vast majority of Canada's oil reserves are found in the Athabasca oil sands of Alberta as bitumen. Large-scale mining operations used to extract bitumen create disturbance footprints that require innovative reclamation strategies. Developing new phytotechnologies for reclamation of these mining sites might assist industry to meet regulatory and societal expectations. Nevertheless, despite opportunities for phytotechnologies, there are significant challenges in moving these technologies from theory to practice. Our research is focused on developing plant-microbe associations, especially those involving bacterial and fungal endophytes, as potential reclamation tools.

Our research sites were established in 2013-2014 at three field locations in Alberta. One is the Bitumount provincial historical site, which is considered the location of two of the world's first oil sand extraction plants. Despite no reclamation efforts by man, vegetation has colonized areas within this site, some of which contain hardened bitumen. The second is Wapisiw Lookout oil sands reclamation area at Suncor Energy Inc., which is planted to native grasses, shrubs. The third is the CNRL RA1 site where peat or LFH cover material is planted with various vegetation and fertilizer treatments. Plant and soil samples were collected in summer and fall 2013 and summer 2014 at all field sites. Soil samples were analysed for nutrients and residual hydrocarbons. Culture dependent and independent techniques were used to characterize microbial communities in soil and to assess the diversity of microbial endophytes in selected plant roots. An objective was to elucidate beneficial endophytes that might be used to assist plant growth at these sites.

As expected, unique and diverse microbial communities were detected in soils at all field sites and linked to variations in plant communities. Similarly, different plants harboured unique bacterial root endophytes. Many of these endophytes were only detected by culture independent techniques, and some isolated endophytes had hydrocarbon degrading genes. The relationship or benefit of specific bacterial endophytes to the growth of plants on these reclamation sites is unknown. This is being assessed in on-going studies to determine how these endophytes might contribute to remediation of hydrocarbons.

Recent and current studies on reclamation of oil sands mining sites in Canada demonstrate that phytotechnologies can be effective tools to establish plant cover. Nevertheless, reclamation of large areas of land can pose serious logistical challenges when using plants that benefit from specific microbial associations. Discovery of new, unique endophytes might lead to development of specific biological inoculants to assist plant establishment and growth at these sites. Development of inoculation strategies or deliver systems is required, as well as an understanding of the ecology of such associations.

**Acknowledgements:** The financial support of the Natural Science and Engineering Research Council of Canada and partners Suncor Energy Inc., and Canadian Natural Resources Limited, along with assistance from Alberta Culture is greatly appreciated.

<figure><img src="image_269.png" /></figure>

<footnote n="1">¹Department of Soil Science, College of Agriculture and Bioresources, University of Saskatchewan, Canada</footnote>

<footnote n="2">²Applied Microbiology Program, College of Agriculture and Bioresources, University of Saskatchewan, Canada</footnote>

<footnote n="3">³Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada</footnote>
# SUCCESSIONAL TRAJECTORIES OF STRUCTURAL AND CATABOLIC BACTERIAL COMMUNITIES IN OIL POLLUTED HYBRID *POPULUS RHIZOSPHERES*: A 2-YEAR FIELD STUDY

Shinjini Mukherjee<fnref n="1" />, Timo Sipilä<fnref n="1" />, Pertti Pulkkinen<fnref n="2" />, Kim Yrjälä<fnref n="1" />

Corresponding author email: shinjini.mukherjee@helsinki.fi

## ABSTRACT

Poplars have widely been used for rhizoremediation of a broad range of organic contaminants for the past two decades. Still, there is a knowledge gap regarding the *Populus* rhizosphere associated bacterial communities and their dynamics during the remediation process. It is envisaged that a detailed understanding of rhizospheric microbial population will greatly contribute to a better design and implementation of rhizoremediation. In a recent study, we demonstrated the successional patterns of bacterial communities in oil polluted aspen rhizosphere in a 10-week greenhouse experiment [1].

In order to further investigate the long-term succession of structural and catabolic bacterial communities associated with aspen rhizospheres in oil contamination, we carried out a field study. Hybrid aspen (*Populus tremula* x *tremuloides*) seedlings were planted in polluted soil excavated from an accidental oil-spill site. Rhizosphere and bulk/un-vegetated soil samples were collected for microbial community analyses at 7 different time-points during the course of 2 years and sampling time-points were chosen to cover the seasonal variation in the boreal climate zone. Structural bacterial communities were accessed by means of 16S rRNA amplicon pyrosequencing whereas catabolic communities were monitored by pyrosequencing of alkane hydroxylase genes and extradiol dioxygenase genes. The functional/catabolic genes in this study were aptly chosen to target the population of aromatic and aliphatic degraders as PAHs and alkanes are the most important constituents of crude oil.

We observed a clear succession of bacterial communities on both structural and functional levels during the study period. Two distinct phases- early and late, were observed in terms of the differences in the bacterial community structure during the successional process. Analysing the co-occurrence patterns 3 different marker genes during the course of succession provided important clues about the early and late phase community members along with the rhizosphere prevalent groups. Sphingomonas type extradiol dioxygenases and alkane hydroxylase homologs of *Rhodococcus* clearly dominated the early phase communities. These high dominance-low diversity functional gene communities underwent a transition to low-dominance-high diversity communities in the late phase. These results pointed towards the increased catabolic capacities and a change from specialists to generalist strategy of bacterial communities during the course of secondary succession. Additionally, a significant seasonal effect was visible on the bacterial community structure during the winter months.

## Reference:

1. Mukherjee S, Heinonen M, Dequvire M, Sipilä T, Pulkkinen P, Yrjälä K (2013) Secondary succession of bacterial communities and co-occurrence of phylotypes in oil-polluted Populus rhizosphere. Soil Biol Biochem 58, 188-197.

<figure><img src="image_270.png" /></figure>

<footnote n="1">¹ Molecular environmental microbiology group, University of Helsinki, Finland</footnote>

<footnote n="2">² Finnish forest research institute, Läyliäinen, Finland</footnote>
# CLEANSED PROJECT: SUSTAINABLE MANAGEMENT OF POLLUTED DREDGED RIVER SEDIMENTS

Serena Doni, Cristina Macci, Eleonora Peruzzi, Grazia Masciandaro

CNR, Institute of Ecosystem Study, Via Moruzzi 1, 56124 Pisa, Italy

Corresponding author email: serena.doni@ise.cnr.it

## ABSTRACT

The continuous stream of sediments, dredged from water bodies in order to maintain the navigation, is a common practice, but the fate of these sediments is an issue recognized worldwide.

The CLEANSED project (LIFE12 ENV/IT/000652) will demonstrate, evaluate and disseminate an innovative, integrated, multisector approach for the smart and sustainable management of polluted dredged river sediments.

The implementation of the project involved the dredging of sediments from Navicelli Canal in Pisa (Italy) and their characterization. These contaminated and nutrient poor sediments, after a phytoremediation process, developed in the framework of a previous EU project AGRIPORT (ECO/08/239065/S12.532262), were transformed into a valuable material with characteristics approaching those of an uncontaminated natural soil (techno-soil).

In the CLEANSED project, the AGRIPORT treated sediments, will be tested as growing substrate for the cultivation of ornamental plants with different saline stress tolerance, thus allowing to prevent the loss of soil due to plant nursing activities. On the other hand, fresh sediments will be tested, after a landfarming pre-treatment, as a filling material for the construction of a road under real working conditions. The whole cycle will take place within a real natural and productive district, i.e. the basin and alluvial plains of the Arno river, Italy.

**Acknowledgements:** Financial support by LIFE+ project CLEANSED ENV/IT/000652.

<figure><img src="image_271.png" /></figure>
# PHYTO-DEHYDRATION OF POLLUTED SEDIMENTS IN AN ARTIFICIAL POND

Dario Liberati<fnref n="1" />, Paolo De Angelis<fnref n="1" />, Paolo Sconocchia<fnref n="1" />,<fnref n="2" />, Andrea Sconocchia<fnref n="2" />

Corresponding author email: pda@unitus.it

## ABSTRACT

In a former plant using for the production of biogas the manure collected in the rural district, an artificial pond of about 2.6 hectares and 4 meters of depth was filled by the fine solid fraction residual after the digestion process.

The sediment is characterised by a water fraction of about 80% and the remaining solid fraction is made by sand (15%), lime (60%) and clay (25%), without any significant vertical stratification. The pH is around 8 and the electrical conductivity around 2.5 ms cm⁻<fnref n="1" />. The concentrations, on a dry weight basis, of organic carbon and nitrogen are around 25% and 5%, respectively. The only heavy metals present in high concentrations are the copper (around 1200 mg kg⁻<fnref n="1" /> d.w.) and the zinc (around 4000 mg kg⁻<fnref n="1" /> d.w.).

The first target of the remediation is the reduction of the water fraction (dehydration), to avoid that during heavy rains the spillage from the lagoon reaches the adjacent river. Secondary targets are related to the transformation of the quality of the sediments, increasing progressively the aerobic conditions of the whole mass and decreasing the methane emissions. As final target a naturalization of the area is under evaluation, with the creation of a new habitat of particular interest for the protection of the birds presents in the region.

The phytodehydration will be obtained by means of different plant species adapted to the wetland environment (and so to the anaerobic conditions of the sediment), “planted” on the surface, by floating frames realised in organic material. The frames will be fixed to the lagoon borders to avoid the movement of the plants under the pressure of the wind. Six species (*Tamarix gallica*, *Tamarix Africana*, *Phragmites australis*, *Juncus effusus*, *Iris pseudacorus* and *Carex gracilis*) and three planting technique (cutting and rooted cutting for *Tamarix* species, pre-sowed coconut material for the other species) will be tested. Each *Tamarix* species will be represented by four different genotypes. The space arrangements of the different species will guarantee a high resilience to the system and a continuous evapotranspiration activity.

In this paper we present the results obtained in the pilot phase, conducted in mesocosms, where we tested and evaluated the dehydration capacity of different species and genotypes, as well as the plant effect on the CO₂, CH₄ and N₂O emissions.

**Acknowledgements:** The financial support P.S.R. per l'Umbria 2007/2013 – Asse 1 – Misura 1.2.4: Progetto ZOOCOMPOST was greatly appreciated.

<figure><img src="image_272.png" /></figure>

<footnote n="1">¹Dept. for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Italy</footnote>

<footnote n="2">²Regional Agency for Environmental Protection of Umbria (ARPA Umbria), Italy</footnote>
# THE INFLUENCE OF ACTIVATED CARBON ON BIO/PHYTOREMEDIATION OF SOILS CONTAMINATED WITH CHLOROPHENOLS

**Elena R. Strijakova, Galina K. Vasilyeva**

Institute of physico-chemical and biological problems in soil science, RAS Pushchino Moscow region, Russia

*Corresponding author e-mail: ers_2410@yahoo.com*

## ABSTRACT

Chlorinated phenols is one of the most important groups of priority pollutants due to their high toxicity, mutagenicity and carcinogenicity. They are widely used as wood preservatives and in other fields of national economy. Although bioremediation and especially phytoremediation are the most economically effective and environmental friendly technologies they cannot be applied for remediation of highly contaminated soils because of their high microbial and phytotoxicity. Previously we developed the approach of adsorptive bioremediation for soils highly contaminated with such compounds like chloroanilines, petroleum hydrocarbons and others. This method is based on the use of activated carbon (separately or in combination with microbial preparations) for reduction of soil toxicity and mobility of contaminants during a soil bioremediation. The main subject of this investigation was to study the influence of activated carbon on the bio/phytoremediation rate of soils highly contaminated with chlorophenols. Vegetation experiments were carried out in a 2.7 L pots with 1.5 kg of soil samples (dry weight). Three types of soils (alluvial-meadow, grey forest and chernozem) were spiked with 4-chlorophenole (4CP, 5700 mg kg⁻¹) or mixture of 4-chloro phenol and 2,4,6-trychlorophenol (TCP) (totally 2400 mg kg⁻¹). After 1 day, granular activated carbon (GAC, Electrostal, Russia) was mixed with the contaminated soils at doses 0, 1, 2 or 4% (w/w), separately or in combination with chlorophenol-degrading microorganisms. For that purpose, biomass of bacterial strain *Rhodococcus opacus* GM-14 was grown in a minimal liquid medium with 4-chlorophenol and 2,4-dichlorophenol as sole carbon and energy sources. The soil samples were incubated under outdoor conditions on a soil surface covered with agrotextile to protect from rains. All samples (except untreated controls) were mixed weakly (when unplanted) and watered regularly when needed. In the end of the 1st and 2nd seasons (after 3 and 15 months) the pots were seeded with white clover (*Trifolium repens* L.) and in 2 months the grown plants were taken out and weighed. In addition, pure controls with uncontaminated soils and similar doses of activated carbon were also created. The soil samples were taken every 2 or 4 weeks for chemical and biological analyses. The 4CP and TCP were extracted from the soil with dichloromethane and their concentrations were determined by GC-MS. Besides, phytotoxicity of those soil samples was determined regularly by express-method on white clover germination.

It has been demonstrated that the soil amendment with activated carbon substantially reduced soil toxicity for chlorophenol-degrading microorganisms and phytoremediators, that remarkably accelerated degradation of the chlorophenols in all the soils. Removal efficiency of chlorophenols in soils amended with GAC in combination with *Rh. opacus* GM-14 was above 87-97% compared to 5-10% in control samples without any adsorbent independent on microbial preparation. Thus it was concluded that adsorptive bioremediation and phytoremediation are perspective approach for cleaning up chlorophenol contaminated soils.

<figure><img src="image_273.png" /></figure>
# BIODEGRADATION OF 2,4-DINITROTOLUENE BY SELECTED PLANT SPECIES

Radka Podlipná, Kateřina Moťková, Tomas Vanek

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 contamination of the environment by nitrocompounds is a worldwide environmental problem. 2,4-DNT is a toxic and recalcitrant by-product of 2,4,6-trinitrotoluene manufacturing, and is also used in dye processes and in the manufacture of polyurethane foams. 2,4-DNT is considered to be carcinogen to the humans. The adverse impact of this explosive on living organisms, its environmental persistence and low susceptibility to degradation demonstrates that the remediation of contaminated soil is essential. In our study we focused on the possibilities to use phytoremediation as a progressive cleaning method of the waste water and soil in the areas of present or former ammunition plant. In this contribution, we study the potential of plants to remove 2,4-DNT from contaminated soils and waters via phytoremediation. In the first step, the toxicity tests were performed. The germination of range of plants (hemp, flax, sunflower, mustard and maize) were not effected by the 2,4-DNT up to concentration 200mg/l except the maize, which was sensitive to concentration 50mg/l. Next, 2,4-DNT degradation in plant cells was studied too. First degradation products 2-aminonitrotoluene and 4-aminonitrotoluene were found both in the medium and in the plant cells. The mustard ecotoxicity test showed that the toxicity of these metabolites were in low concentrations (< 50mg/l) higher than the toxicity of parent compound, but the 2,4-diaminotoluene, the product of next reduction step, was less toxic in all concentration (0-200mg/l).

<figure><img src="image_274.png" /><figcaption>Degradation of 2,4-DNT by plant cell culture of reed (Phragmites australis).</figcaption></figure>

Acknowledgements: This study was supported from MYES of CR n. LH11048 grant

<figure><img src="image_275.png" /></figure>
# THE EFFECT OF STRESSFUL STIMULI (MECHANICAL AND CHEMICAL) ON THE EXPRESSION OF SECONDARY METABOLITES IN POPLAR BUDS

Libor Mrnka<fnref n="1" />, Tomáš Frantík<fnref n="1" />, Václav Čmolík<fnref n="2" />, Pavel Matějka<fnref n="2" />, Miroslav Vosátka<fnref n="1" />

Corresponding author email: libor.mrnka@ibot.cas.cz

## ABSTRACT

In response to biotic stress poplars synthesize a number of secondary metabolites. Some of them act as defense molecules against biotrophic and/or necrotrophic parasites. We tested the long-term effect of chemical and mechanical stresses on the expression of secondary metabolites in buds of two poplar clones from different poplar sections, *Populus nigra* clone Italica and *Populus trichocarpa* clone Fritzi Pauley. Both mechanical (perforation of leaves) and chemical (5mM salicylic acid) stimuli were applied two times in the end of the vegetation season. The buds were harvested after the trees had shed leaves.

A further aim of the study was to establish surface enhanced Raman spectroscopy (SERS) as a new technique for studying poplar secondary metabolites. This technique may complement the standard chromatographic techniques (HPLC, GC). For successful employment of SERS, the optimization of the extraction procedure with sequential application of carefully selected extractants and the proper choice of metal surface was crucial.

Preliminary SERS results confirm that the profile of secondary metabolites in the buds differed in the two examined clones. *P. trichocarpa* spectra were dominated by terpenoids while those of *P. nigra* were dominated by phenolic compounds. Further analyses should disclose whether stressful stimuli influenced the pattern of secondary metabolites in poplar buds.

**Acknowledgements:** The financial support by Technological Agency of the Czech Republic, project BIORAF – Biorefinery Research Centre of Competence contract No. TE01020080, is greatly appreciated.

<figure><img src="image_276.png" /></figure>

<footnote n="1">¹Department of Mycorrhizal Symbioses, Institute of Botany ASCR, Pruhonice, Czech Republic</footnote>

<footnote n="2">²Department of Analytical Chemistry, Institute of Chemical Technology Prague, Prague, Czech Republic</footnote>
# CONTENT OF TRACE ELEMENTS IN MAIZE ON SOIL CONTAMINATED WITH HEATING OIL AFTER APPLICATION OF NEUTRALIZING SUBSTANCES

Mirosław Wyszkowski, Veranika Sivitskaya

Department of Environmental Chemistry, University of Warmia and Mazury in Olsztyn, Poland

Corresponding author email: miroslaw.wyszkowski@uwm.edu.pl

## ABSTRACT

The purpose of this study has been to determine the effect of increasing rates of heating oil (0, 5, 10, 15 and 20 g kg⁻¹ of soil) and neutralizing substances (nitrogen, compost, bentonite, zeolite and calcium oxide) on the content of cadmium, lead, chromium and manganese in maize (Zea mays L.). The experiment with three replications was conducted in a plant house of the University of Warmia and Mazury in Olsztyn (north-eastern Poland). Soil in each pot was enriched with identical amounts of mineral fertilizers (100 mg N, 30 mg P, 100 mg K, 50 mg Mg, 0.33 B, 5 mg Mn and 5 mg Mo per kg of soil). In addition, each experimental treatment was enriched with mineral fertilizers in the following quantities: 100 mg N, 30 mg P, 100 mg K, 50 mg Mg, 0.33 B, 5 mg Mn and 5 mg Mo per kg of soil. Soil (9 kg) was first carefully mixed with heating oil and the aforementioned nutrients and substances and then placed in polyethylene pots. The detail specification of soil and substances used in experiment was given in our previous article [1]. A Polish cultivar of maize (Zea mays L.) called Reduta, grown at a density of 8 plants per pot, served as the experimental plant. Maize was harvested during the intensive stem elongation phase. At the same time, soil samples for analysis were collected. Having mineralized the plant material in 65% nitric acid, concentrations of the trace elements: cadmium, lead, chromium and manganese, were determined by the flame atomic absorption spectrophotometry (FAAS) on a SpectrAA240FS atomic absorption spectrophotometer. The results were processed statistically using two-factor analysis of variance ANOVA from a software package Statistica.

The increasing rates of heating oil and contamination alleviating substances produced different effects on the content of the analyzed elements in soil. In the non-amended series, the relationship between the increasing rates of heating oil and the cadmium content in plants followed a unidirectional trend, but the range of changes was relatively small. Under the higher rates of heating oil, the content of manganese increased by 64% up to the rate of 10 g kg⁻¹ of soil in maize compared to the control treatments (without heating oil). An increase in the concentration of lead in maize was observed under the influence of 15 g of oil per kg of soil. Higher rates of heating oil caused a decrease in the content of manganese and chromium in maize. In response to heating oil in soil, the content of chromium in maize rose by 89% (20 g oil kg⁻¹ of soil) relative to the treatment without this petroleum derivative.

Application of compost, bentonite, zeolite and CaO to soil contaminated with heating oil had a positive, limiting effect on the content of cadmium in maize. All the soil amending substances contributed to a considerable increase in the content of Pb⁺² in maize: compost by an average of 32%, nitrogen by 35%, bentonite by 63%, CaO by 105% and zeolite by 123% versus the series without any of these substances. As for chromium, all the substances except nitrogen slightly stimulated its higher concentrations in maize, and the highest quantities were determined in the series with zeolite (+18%) and CaO (+22%). The content of manganese in maize decreased significantly under the effect of neutralizing substances in comparison to the series without soil amending additives. The substance which most strongly inhibited the uptake of manganese by maize was bentonite, which reduced its content by 68%. Calcium oxide reduced the content of manganese in maize by 44%, compost by 22% and zeolite by 19%. Nitrogen was exceptional as it stimulated an increase in the content of this element in maize by an average 18% versus the series without neutralizing substances.

## References:

1. Wyszkowski M., Sivitskaya V. (2013) Effect of heating oil and neutralizing substances on the content of some trace elements in soil. *Fresenius Environmental Bulletin*, 22(4), 973-978.

**Acknowledgements:** The study has been performed as part of the research project No. N N305 305340 funded by the Polish National Science Centre.

<figure><img src="image_277.png" /></figure>
# THE INDUCED PHYTOREMEDIATION OF ORGANOCHLORIDE PESTICIDES-POLLUTED SOILS USING DERIVATIVE AND COMPOSITION OF OKSAN

A. Nurzhanova<fnref n="1" />, S. Kalugin<fnref n="2" />, R. Baizhumanova<fnref n="2" />, Ch. Seisenbai<fnref n="1" />, Y. Sailaukhanuly

Corresponding author email: gen_asil@mail.ru

## ABSTRACT

Phytoremediation is technology of restoration of polluted soil and water by using different plant species. Phytoremediation technology is applied directly in the area of contamination (in situ), helping to reduce costs and reduce exposure contaminated pollutant with the people and the environment. Most importantly, after phytoremediation soil does not lose its fertility. Consequently, this technology is ecologically safe and cost effective.

Exigency to develop phytoremediation technology of soils contaminated by pesticides arose from the fact that Kazakhstan as "environmental heritage" from the now defunct Soviet Union got many territories of the former storehouses of obsolete pesticides and surrounding land. In each region of the country, there are 5-20 the inactive former warehouses of pesticides. Soil around the warehouses has multicomponent pollution. Soil contains organochlorine pesticides and heavy metals.

Most important components of technology restoration of soil polluted with organochlorine pesticides with plants are phytoextraction and phytostabilization. Phytoextraction potential of the plant organism depends from the hydrophobicity of the pollutant. Degree of hydrophobicity (log Kow) largely determines the efficiency of absorption pollutants and their movements in the plants.

Low phytoextraction percentage promotes slow growth of plants and limited biomass production. The efficiency of phytoremediation of soil depends from the productivity of plants: t than larger biomass, consequently the higher accumulation of pesticides. In this regard, for increasing the phytomass of tolerable plants to pesticides and bioavailability of the pollutants from the contaminated soil to plant were used the new synthesized growth stimulator (derivatives of the oksan 2, 2-thiosemicarbazone dimetiltetragidropiran-4-one) and his compositions.

In the green and field was studied introducing derivatives of the oksane (SC in concentration 0,0001%) and their compositions (the wood charcoal 250 g + SC in concentration 0,0001%) in contaminated soil in period of germination on the physiological parameters of the plants (Xanthium sturmarium, Amaranthu retroflexus and Cucurbita pepo).

In green was found that the introduction of SC and their compositions in artificially polluted by pesticide (4.4' DDE metabolites in concentration 878±77µg kg⁻<fnref n="1" />) soil increased biomass to 2-4 times, rise the content of chlorophylls and carotenoids in the leaves, changing the ratio of chlorophylls a and b, and the chlorophylls and carotenoids, the coefficient of biological absorption, translocation coefficient and promote reduction of pesticides in soil as compared with control by 18%.

In field the introduction of SC and their compositions in soil polluted with pesticides (4.4' DDT and 4.4' DDE metabolites in concentration 1426±241µg kg⁻<fnref n="1" />) increased biomass plants and phytoextraction pesticides from soil to plant from 0.4% to 1.6% of pesticides from the square m<fnref n="2" />.

This work can be required for the development of pesticide remediation technologies for organchloride pesticides-contaminated sites.

<figure><img src="image_278.png" /></figure>

<footnote n="1">¹ Institute of Plant Biology and Biotechnology, 45Timiryezev st., 050040, Almaty, Kazakhstan</footnote>

<footnote n="2">² al-Farabi Kazakh National University, Almaty, Kazakhstan</footnote>
# IMPACT OF ORGANIC MATTER ADDITION TO SOIL ON WEATHERED p,p-DDE ACCUMULATION IN CUCURBITA PEPO CULTIVARS

## Pinar Sevim

Duzce University, Department of Environmental Engineering, Duzce, Turkey

Corresponding author email: pinar.sevim39@gmail.com

## ABSTRACT

Cucurbita pepo cultivars (cv:raven and zephyr) were grown in a 900 cm² mound at 2 m intervals containing soil with weathered DDE at 299-908 ng/g soil under field conditions. Soil removed from each mound (30 cm x 30cm x 25 cm) mixed with either 500 g or 1000 g organic matter as long as their unamended controls and each cultivar was planted into organic matter amended and unamended mounds. Cultivars were harvested at 42 days and DDE concentrations at plant compartments were analyzed by GC/μ-ECD.

A method previously published was used for extraction of plant tissues such as roots, shoots, leaves, fruits. For xylem sap collection, soil was oversaturated with tap water. After 3h, stem of each cultivar was cleaned of the soil particles and severed 5 cm above the soil surface and the severed stem was placed into a 100 mL amber glass vial. The sap volume was measured at the end of 8 h collection period and xylem sap DDE content measured by SPME method coupled with GC/μ-ECD.

The objective of this study was to assess the effect of organic matter amendments on the pyhtoextraction potential of cucurbita pepo cultivars under field conditions. Average shoot p,p'-DDE concentrations of raven grown in mounds with 500 g, 1000 g of organic matter amendments, and 0 g (unamended mound) were determined in as 2484 ng/g, 1621 ng/g, and 2068 ng/g (dry weight), respectively. For Zephyr, DDE contents for the same treatment types were 247 ng/g, 118 ng/g, 138 ng/g, respectively. Xylem sap p,p-DDE concentrations followed a similar pathway with shoot DDE concentrations of the cultivars grown in organic matter amended and unamended mounds under field condition.

**Acknowledgements:** The financial support by SAU-BAPK project 2013-50-02-016 contract No.

<figure><img src="image_279.png" /></figure>
# PHYTOREMEDIATION OF ORGANIC WASTE LEACHATE USING IPOMOEA AQUATICA IN TROPICAL COUNTRY

**Ramlah Mohd Tajuddin**

Faculty of Civil Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

Corresponding author email: ramlah2007@gmail.com, ramlah160@salam.uitm.edu.my

## ABSTRACT

This research was conducted to determine the performance of Ipomoea Aquatica as phytoremediator to treat leachate originated from vegetable waste from wet market in Malaysia which is considered as tropical country. Phytoremediation system was designed using Ipomoea Aquatica as phytoremediator which acted as a low cost and environmental friendly leachate treatment process. The performance of Ipomoea Aquatica to remove zinc, phosphorus and nitrate were tested and evaluated. It was also discovered that Ipomoea Aquatica able to reduce the concentration of Ammoniacal Nitrogen, Biochemical Oxygen Demand, Chemical Oxygen Demand and suspended solids by 1.88%, 1.71%, 2.49% and 4.44% respectively. The volume of vegetable leachate produced from a specified weight of solid vegetable waste was also determined. The optimum number of days for efficient removal performance capacity of Ipomoea Aquatica to treat leachate from vegetable waste was identified.

**Keywords:** phytoremediation, leachate treatment, vegetable waste, of Ipomoea Aquatica

<figure><img src="image_280.png" /></figure>
# PRELIMINARY EVIDENCES FOR THE ACCUMULATION OF DIOXINS AND FURANS IN PLANT TISSUES ON A PHYTOREMEDIATION SITE CONTAMINATED WITH ACC AND PCP

Frederic E. Pitre<fnref n="1" />,<fnref n="2" />, Hafssa Kadri<fnref n="1" />, Aymeric Yanitch<fnref n="1" />, Michel Labrecque<fnref n="1" />,<fnref n="2" />

Corresponding author email: frederic.pitre@umontreal.ca

## ABSTRACT

Wood preservatives like ACC and PCP are widely used by utility companies worldwide. Unfortunately, the use of wood treatments such as ACC and PCP produces leachates that degrade to As, Chrome and Copper, and dioxins and furans, respectively. These contaminants are highly toxic and new containment strategies are being explored. The objectives of this study was i) to determine which plant species can tolerate the mineralized, compacted and contaminated soil of a site used for storing wood-treated electric poles, and 2) evaluated the potential of selected plant species to extract or degrade contaminants originating from the leaching of the wood treatments.

A phytoremediation experiment was established in the summer of 2012 on the site of a facility used to handle electric poles treated with ACC and PCP. Four plant species (willow *Salix miyabeana*, tall fescue *Festuca arundinacea*, alfalfa *Medicago sativa*, indian mustard *Brassica juncea*) were established in a split-plot randomized block design, with fertilization as a treatment and repeated four times. After the second year of growth (i.e. 2013), the survival and the productivity of the plants were monitored and chlorophyll content evaluated. Leaf and root samples were also harvested and analysed for metal content, PCP, dioxins and furans.

The four species planted tolerated well the harsh conditions of the site and produced important quantities of biomass. Willows in particular had the highest biomass found. Fertilization had significant effects on the amount of above and belowground biomasses produced in all species. Similarly, stomatal conductance and chlorophyll content were significantly increased by fertilization. At the end of 2013, only willows were able to extract Cd, Cu and Zn from the soil. Surprisingly, preliminary analyses revealed that all the species planted accumulated dioxins and furans in their leaves and roots (willows > alfalfa > mustard > tall fescue).

The results obtained so far point toward the potential of willows to phytoextract specific metals. Moreover, the four plant species used in this study accumulated dioxins and furans in their roots and leaves. The next stages of the project is to evaluate to what extent the plant will accumulate large quantities of dioxins and furans and to investigate molecular processes potentially induced to degrade these compounds.

**Acknowledgements:** The financial support of this project was provided by grants from Hydro-Quebec, CRIBIQ and Mitacs.

<figure><img src="image_281.png" /></figure>

<footnote n="1">¹ IRBV, Montreal Botanical Garden, 4101 Sherbrooke East, Montreal, Quebec, H1X 2B2, Canada</footnote>

<footnote n="2">² IRBV, Biological sciences, University of Montreal, 4101 Sherbrooke East, Montreal, Quebec, H1X 2B2, Canada</footnote>
<figure><img src="image_282.png" /></figure>

PLANT-MICROBE INTERACTIONS
<figure><img src="image_283.png" /></figure>
# TRANSGENERATIONAL CHARACTERIZATION OF SEED ENDOPHYTIC BACTERIA OF A PLANT GROWING ON MINE RESIDUES

Ariadna Sánchez-López<fnref n="1" />, Jaco Vangronsveld<fnref n="2" />, Nele Weyens<fnref n="2" />, Ma. del Carmen A. González Chávez<fnref n="1" />, Rogelio Carrillo González<fnref n="1" />

Corresponding author email: jaco.vangronsveld@uhasselt.be

## ABSTRACT

Plant endophytic bacteria can be defined as bacteria that live within plant tissues without causing substantive harm to the host plant. Endophytes have been isolated from different plant organs: roots, leaves, stems, flowers, fruits and seeds. Much of the research on endophytic bacteria focused on roots, stems or leaves. More and more reports mention isolation of endophytic bacteria from seeds. In seeds cultivable bacteria are not as abundant as in other plant organs but most likely only a limited fraction of the endophytes are cultivable. It is mentioned that some endophytes that might be important during germination, or even seedling development, are transferred to next generations through seeds. Endophytic bacteria promote plant growth by similar mechanisms like soil plant growth promoting bacteria, helping host plant to overcome environmental stress. Additionally, it has been demonstrated that endophytic bacteria increase metal tolerance and affect metal translocation to the aerial plant parts.

In the mining district of Zimapan, Hidalgo state, central part of Mexico, the annual plant species *Crotalaria pumila* was found growing spontaneously on multi-metal contaminated mine residues. The plant accumulates, without toxicity symptoms, Zn, Cd, Pb and Cu in shoots at concentrations higher than those phytotoxic for other species. *Crotalaria pumila* has been observed for three consecutive years completing its life cycle and producing viable seeds. The persistence of the mentioned species could be attributed to the presence of beneficial endophytic bacteria transferred from one generation to another through the seeds. Therefore, the aim of this work is to study the composition and functions of seed endophytic bacteria of three generations of *C. pumila* growing naturally on mine tailings in a Mexican semiarid region (Zimapan). Seeds from three different generations (2011, 2012 and 2013) were collected from *C. pumila* plants growing on metal mine residues, as well as seeds from a non-contaminated site. Twelve different surface sterilization protocols including variations on concentration and exposition time to NaClO and ethanol were tested. Then, the seeds were crushed and dilutions were plated on solid growth medium. Cultivable strains were characterized genotypically and phenotypically. Phenotypical characterization of cultivable bacteria included tolerance to metals (Zn, Cd, Pb and Cu), siderophore, acetoin and indol-3-acetic acid production, 1-aminocyclopropane-1 carboxylate deaminase activity, ability to fixate N₂ and capacity to solubilize phosphate. Non-contaminated site seeds contained more colony forming units (cfu) per gram seed than those from multi-metal contaminated mine residues. More cfu were observed in seeds collected in 2013; however, in the three different seed generations similar bacteria were observed. The surface sterilization protocol was a key factor to obtain as many cultivable seed bacterial endophytes as possible. Besides cultivable strains, also the non-cultivable population were characterized using culture independent techniques.

<figure><img src="image_284.png" /></figure>

<footnote n="1">¹Edaphology, Colegio de Postgraduados Campus Montecillo, Texcoco, México</footnote>

<footnote n="2">²Environmental Biology, Hasselt University, Diepenbeek, Belgium</footnote>
AUTOCHTHONOUS BIOAUGMENTATION AS A STRATEGY TO ENHANCE Cu PHYTOREMEDIATION BY A SALT MARSH PLANT

Tânia Oliveira<fnref n="1" />,<fnref n="2" />, Ana Paula Mucha<fnref n="1" />, Izabela Reis<fnref n="1" />, Paula Rodrigues<fnref n="1" />, Carlos Rocha Gomes<fnref n="1" />,<fnref n="2" />, C. Marisa R. Almeida<fnref n="1" />

ABSTRACT

This work aimed to evaluate the potential of the salt marsh plant *Phragmites australis* for phytoremediation of Cu contaminated sediments and to evaluate if this potential can be enhanced by bioaugmentation with autochthonous microorganisms resistant to Cu.

Plants were collected in an estuarine area, placed in vessels and kept in greenhouses under tidal simulation. Sediments were contaminated with Cu (ca. 270 µg g⁻<fnref n="1" />) and autochthonous microbial consortia resistant to Cu (prepared in the laboratory) were added to half of the vessels. After two months, metal concentrations in plant structures and sediments were determined, as well as metal speciation on sediments. Plants physiological parameters (chlorophylls, carotenoids and thiolic compounds) and plant biomass weight were also determined to assess possible stress signs.

Plant accumulated significant amounts of Cu in all tissues although in higher amounts in its belowground structures. Cu induced some stress signs in the plants belowground structures, with lower biomass weight being observed, although thiolic compounds levels have increased. Belowground structures biomass weight was even lower when the microbial consortium with Cu resistant microorganisms was added probably due to the increase of the Cu bioavailable fraction in sediment observed when bioaugmentation was applied. However, in this case Cu translocation increased with higher amounts of Cu being observed in the stems of the plants. This increase did not caused any visual toxicity signs, being accompanied, for instance, by an increase in the production of thiolic compounds in the plant leaves, indicating that the plant had mechanisms to respond to the uptake of the metal.

Therefore, this work demonstrated that *P. australis* is capable of phytoremediate Cu contaminated sediments and that autochthonous bioaugmentation can change metal bioavailability in the rhizosphere, increasing the phytoextraction potential of this plant. This technique will contribute for the removal of this contaminant from contaminated sediments reducing its toxicity risk and contributing for an efficient risk management strategy of estuarine and coastal areas.

*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_285.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">²Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal
*Corresponding author email: calmeida@ciimar.up.pt*</footnote>
# METAGENOMIC ANALYSIS OF MICROBIAL COMMUNITIES FROM A Hg-CONTAMINATED RHIZOSPHERIC SOIL: DIVERSITY, STRUCTURE AND CO-OCCURRENCE NETWORK

Cyril Zappelini<fnref n="1" />, Battle Karimi<fnref n="1" />, **Julie Foulon**<fnref n="1" />, Damien Blaudez<fnref n="2" />, David Cazaux<fnref n="3" />, Etienne Yergeau<fnref n="4" />, Charles Greer<fnref n="4" />, Michel Chalot<fnref n="1" />

*Corresponding author email: michel.chalot@univ-fcomte.fr*

## ABSTRACT

The goal of phyto (dendro) remediation is to reinstall a green cover on highly anthropogenic/derelict soils, and when possible to valorize the woody biomass produced. A detailed understanding of the effect plants may have on the activities of rhizosphere microorganisms could help enhancing the productivity. We hypothesized that the composition of and interactions between bacterial and fungal communities under trees that have naturally recolonized the anthropogenic area are profoundly altered as compared to those from the undisturbed area. To test this hypothesis, we used a high-throughput DNA sequencing technology that generates large environmental datasets. We further applied modelling of co-occurrence network to investigate potential interactions between microbial taxa and to decipher complex interactions between different components of the microbial communities. In this study, rhizospheric soils were collected from a strongly anthropogenic and a control areas. Bacterial and fungal 16S rRNA and ITS genes were amplified and sequenced using next-generation sequencing technology (Ion Torrent). Soils were also analyzed for chemical and physical characteristics. Sequence data were processed using Mothur. Metagenomic semi-quantitative data on microbial communities were used to model the microbial networks of ecological co-occurrence interaction in control and tailing pond areas.

Detailed analyses of the structure of microbial communities revealed that 26.4 and 8.6% of bacterial and fungal genus, respectively, were over-represented in the tailing pond soil, corresponding to 40.0 and 19.6%, of bacterial and fungal DNA sequences, respectively. Conversely, 9.2 and 12.6% of bacterial and fungal genus, respectively, representing 24.8 and 17.5% of bacterial and fungal DNA sequences, were over-represented in the undisturbed soil. Interestingly, the two soils shared 64.4% and 78.8% of bacterial and fungal genus, respectively. Within the emergent bacteria genus in the tailing pond soil, all are only minor genus, representing individually less that 2% of total sequences. A noticeable exception concerned *Pseudomonas* species, from which 17.0% of sequences isolated from tailing pond soil derived, whereas only 0.9% of the sequences were derived from *Pseudomonas* species in undisturbed soil. Conversely, the bacteria genus MC18 is a major component of the undisturbed soil (2.3% of DNA sequences), whereas it did not significantly emerge in the tailing pond soil (0.007% of total DNA sequences). The global co-occurrence networks including bacterial and fungi genus consisted in largely connected communities with some variations between the undisturbed and the tailing pond soils. These results are further discussed at the whole community level, and the interactions between the different microbial compartments undiscovered.

**Acknowledgements:** The financial support by ANR project contract No ANR-10-INTB-1703-01-BIOFILTREE is greatly appreciated.

<figure><img src="image_286.png" /></figure>

<footnote n="1">¹ Laboratoire Chrono-environnement, UMR 6249, Université de Franche-Comté, Pôle Universitaire du Pays de Montbéliard, 4 place Tharradin, BP 71427, 25 211 Montbéliard</footnote>

<footnote n="2">² Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360 CNRS-Université de Lorraine, Faculté des Sciences et Technologies, Bld des Aiguillettes, BP 70239, 54506 Vandoeuvre-les-Nancy</footnote>

<footnote n="3">³ SOLVAY ELECTROLYSE France, Tavaux Cedex</footnote>

<footnote n="4">⁴ Biotechnology Research Institute, National Research Council of Canada, Montréal, QC, Canada</footnote>
# PROMISCUOUS DISPERSAL OF BIODEGRADATION GENES: A KEY TO IMPROVE ENDOPHYTE-ENHANCED PHYTOREMEDIATION?

Nele Weyens<fnref n="1" />, Esteban Martínez-García<fnref n="2" />, Víctor de Lorenzo<fnref n="2" />, Jaco Vangronsveld<fnref n="1" />

Corresponding author email: nele.weyens@uhasselt.be

## ABSTRACT

Because many bacteria possess a natural capacity to cope with contaminants, they can be exploited to improve the efficiency of phytoremediation. In case of phytoremediation of organic contaminants, plants can benefit from endophytes possessing appropriate degradation pathways and metabolic capabilities, leading to more efficient contaminant degradation and reduction of both phytotoxicity and evapotranspiration of volatile contaminants. We were able to obtain a 90% reduction in TCE evapotranspiration after inoculation of a TCE degrading poplar root endophyte in field grown poplar trees. To move this concept towards wide-ranging, large-scale applications, it is important that the positive effect that could be achieved after inoculation is **stable at the long term** and that the concept of endophyte-enhanced phytoremediation can easily be extrapolated to a broad variety of contaminants.

In order to achieve a stable endophyte-enhanced phytoremediation, it is essential that the degradation genes from the inoculated endophyte are transferred to members of the indigenous population. When these degradation genes are located on a plasmid, they can be transferred to other bacteria by means of horizontal gene transfer. The host range of the plasmid strongly determines the success of such a horizontal gene transfer while the frequency of the transfer is regulated by its dispersal capacity. Therefore, we designed a **broad host-range plasmid** for cloning and **promiscuous dispersal of biodegradative genes**.

Two plasmids with a broad host-range origin of replication from the SEVA (Standard European Vector Architecture) plasmid collection [1] were selected as a backbone in which different units can be introduced: the SEVA 221 and 251, equipped with the origin of replication of the RK2 and the RSF1010 plasmid respectively. Next to the broad host range and promiscuous dispersal, the stability of the plasmids is of high importance, the more, because the final introduction of the degradation genes will result in rather large plasmids which involve high energy needs for their host cell. To ensure the stability of the plasmids, the HOK/SOK toxin-antitoxin (TA) system was introduced into the SEVA plasmids 221 and 251 resulting in pSEVA221α and pSEVA251α. Next, the obtained plasmids were introduced into *Pseudomonas putida* W619, a plant growth promoting poplar endophyte, and were compared in a stability experiment. For pSEVA251α, 100% stability could be achieved, even after 100 generations.

In order to equip the SEVA251α plasmid with TCE degradation capacity, TCE degradation genes were isolated from the pTOM plasmid originating from *Pseudomonas putida* G4. These genes, together with a constitutive promoter, are cloned into pSEVA251α. In the final step, the complete plasmids (including the degradation genes) will be transferred to *Pseudomonas putida* W619, which can be used for inoculation experiments.

In future work, the pSEVA251α can be equipped with other desired degradation genes after which the entire plasmid can be transferred to the endophyte that will be used for inoculation. The broad host range and promiscuous dispersal of pSEVA251α together with the HOK/SOK TA system that optimizes the stability will ensure a successful transfer of the degradation genes to the indigenous population resulting in an improved phytoremediation efficiency that is stable at the long-term. Moreover, this rather easy way to extrapolate the concept (of using degrading endophytes to reduce phytotoxicity and evapotranspiration) to a broad variety of contaminants could move endophyte-enhanced phytoremediation towards a wide-ranging application potential.

## References:

1. R. Silva-Rocha, E. Martínez-García, B. Calles, M. Chavarría, A. Arce-Rodríguez, A. de las Heras, A. D. Páez-Espino, G. Durante-Rodríguez, J. Kim, P. I. Nikel, R. Platero, and V. de Lorenzo. (2013) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41 (D1): D666-D675.

**Acknowledgements:** This research was funded by the Fund for Scientific Research Flanders (FWO-Vlaanderen), post-doc grant for N.W., and has been financially supported by the UHasselt Methusalem project 08M03VGRJ.

<figure><img src="image_287.png" /></figure>

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

<footnote n="2">²Systems Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Cantoblanco-Madrid, Spain</footnote>
# WATER HYACINTH AND ITS ENDOPHYTIC BACTERIA AS A POTENTIAL TOOL FOR PHYTOREMEDIATION OF Ba

Arkadiusz Przybysz<fnref n="1" />, Jaco Vangronsveld<fnref n="2" />, Monika Małecka-Przybysz<fnref n="1" />, Sofie Thijs<fnref n="2" />, Sasha Truyens<fnref n="2" />, Magdalena Żmuda-Baranowska<fnref n="1" />, Stanisław Waldemar Gawroński<fnref n="1" />

Corresponding author email: arkadiusz_przybysz@sggw.pl

## ABSTRACT

**Background:** Energy is one of the biggest problems humanity is facing nowadays. Global energy demand is expected to double by the year 2050. The concern about the availability of energy has directed people towards shale-geologic formations containing imprisoned gas and/or oil within its pores. Technologies making shale gas extraction possible require huge amounts of water for drilling and fracking. In consequence, flowback waters contaminated by different pollutants, among many also heavy metals such as barium (Ba), are produced. Concentrations of Ba was found to reach on average 661 mg*dm⁻³ and maximum even up to 15500 mg*dm⁻³. One of the successful hyperaccumulators, efficiently absorbing heavy metals (Pb, Sr, Hg, Zn, Cd, Cu and Ni) and other contaminates (NO₃, NH₄ and phenols) from wastewater is water hyacinth (Eichhornia crassipes (Mart.) Solms). Fast growing and producing enormous amounts of biomass water hyacinth may be also considered as potential biofuel crop. In this study, usefulness of water hyacinth plants in Ba phytoextraction from contaminated water was evaluated and community of cultivable endophytic bacteria from its roots was characterized.

**Materials and Methods:** Water hyacinth plants were cultivated, under strictly controlled growth conditions, in plastic containers filled with nutrient solution (1/4 of modified Hoagland solution, pH 6.2) enriched with increasing concentrations of Ba (between 0 and 1500 mg*dm⁻³). Non-destructive measurements characterizing efficiency of photosynthetic apparatus (gas exchange, chlorophyll *a* fluorescence) were conducted in two week intervals. Biomass accumulation, reproductive capability and Ba content in particular plant organs were assayed at the end of the experiments. Cultivable endophytic bacteria strains were isolated from surface sterilized (active chloride and ethanol) roots of water hyacinth plants grown in 400 mg*dm⁻³ Ba and characterized (i) morphologically (size, colour, shape of the colony), (ii) phenotypically in plant growth promoting features (production of siderophores, organic acids and IAA, nitrogen fixation, phosphorus solubilization, activity of ACC deaminase and tolerance to selected heavy metals: Zn, Cu, Cd and Ni) and (iii) genotypically with amplified rDNA restriction analysis of 16S rRNA gene using HpyCH4 IV enzyme, sequencing and identification based on naïve Bayesian rRNA Classifier and Sequence Match at the Ribosome Database Project II.

**Results:** Obtained results showed that water hyacinth was able to grow exuberantly in concentrations not exceeding 500 mg*dm⁻³ Ba and accumulate extremely large quantities of this element, in concentration 1000 mg*dm⁻³ Ba even up to 35.4 and 83.50 g*kg⁻<fnref n="1" /> DW, for leaves and roots respectively. Lower Ba concentrations did not show any toxic effects on examined plants. However in higher concentrations (above 500 mg*dm⁻³ Ba) Ba had negative impacts on plant's reproductive capability and biomass production. Gas exchange (i.e. intensity of photosynthesis, stomatal conductance and transpiration rate) and values of the selected chlorophyll *a* fluorescence parameters were also lowered. Bacterial community associated with water hyacinth grown in the presence of Ba was dominated by Firmicutes, most of all with *Bacillaceae* family (the nearest strains from RDPII database was similar to *B. pumilus*, *B. cereus*, *B. aquimaris*, *B. subtilis*, *B. licheniformis*, *B. bombysepticus* and *B. amyloliquefaciens*). Much less represented were Proteobacteria (*Enterobacter asburiae*) and Actinobacteria (*Micrococcus* sp.). Bacteria communities isolated from water hyacinth roots hold important plant growth promotion features. Among isolates derived from plants exposed to Ba pollution 38% produced siderophores, 24% organic acids and 30% IAA. ACC deaminase activity, nitrogen fixation and phosphorus solubilisation was noted in 14, 28 and 53% of isolates, respectively. Vast majority of those bacteria were resistant to Zn, additionally *E. asburiae* could grow also in the presence of Cu and Cd.

**Conclusions:** These results support the conclusion that water hyacinth can be used in phytoextraction of Ba from polluted water and endophytic bacteria are most probably making this process more efficient.

**Acknowledgements:** This study was supported by the Warsaw Plant Health Initiative FP7-REGPOT-2011-1-286093.

<figure><img src="image_288.png" /></figure>

<footnote n="1">¹ Laboratory of Basic Research in Horticulture, Faculty of Horticulture, Biotechnology and Landscape Architecture, Poland</footnote>

<footnote n="2">² Centre for Environmental Sciences, Hasselt University, Belgium</footnote>
# INTERACTIONS PLANT – SOIL MICROORGANISMS FOR REMEDIATION OF METAL MINE RESIDUES

Ma. del Carmen A. González Chávez, Rogelio Carrillo González, Yazmin Stefani Perea Vélez, José Alfredo Nogues Iniesta, María Isabel Godínez Hernández

Edaphology, Colegio de Postgraduados Campus Montecillo, Texcoco, México

Corresponding author email: carmeng@colpos.mx

## ABSTRACT

Mining has been an important commercial activity in Mexico. As a result tons of mining waste is observed in many areas causing a potential risk not only for the environment, but also for the human population settled very near to mine tailings. In order to reduce the dispersion of potentially toxic elements (PTE) contained in tailings the establishment of a well-developed vegetation cover is required. However, mine tailings are drastically physico-chemical and biological altered environments and natural restoration may occur in many years. Microbe assisted phytoremediation and amendments are eco-friendly and cost-effective alternatives proposed to increase biological productivity; to improve fertility of mine tailings and induce sooner site remediation. The results obtained by using different plants such as *Jatropha curcas*, *Ricinus communis*, *Brassica napus*, *Medicago sativa*, *Acacia farnesiana*, *Senna multiglandulosa*, in combination with soil amendments such as perlite, vermicompost and biochar, as well as different beneficial soil microorganisms for instance arbuscular mycorrhizal fungi or plant growth promoting rhizobacteria (PGPR) will be presented. The tested plants were selected because they are found as naturally established plants in mine tailings (except *J. curcas*, *M. sativa* and *B. napus*), their tolerance to PTE and role to increase soil fertility, or their use as raw material for potential biodiesel production. Soil amendments were able to decrease PTE availability; perlite reduced Cd availability, biochar (5%) decreased availability of Cd (97%), Cu (32%), Pb (56%) and Zn (34%). Vermicompost at 10% or 15% also diminished availability of several PTE. Several biochemical properties were analyzed for the PGPR (indol acetic acid, ACC-deaminase, phosphate solubilization, tolerance to different PTE, siderophore production) under study and their effect on plant growth into mine tailings substrates amended with organic compounds. All these factors (amendments, tolerant plants, and selected microorganisms) represent a very useful tool to deal with the harsh environment such as mine tailings and propitiate their phytoremediation, but their efficiency varies depending of the combination.

<figure><img src="image_289.png" /></figure>
# THE COUPLING OF THE PLANT AND MICROBIAL METABOLISMS OF PAHs IN THE RHIZOSPHERE OF ALFALFA

**Anna Muratova, Ekaterina Dubrovskaya, Natalia Pozdnyakova, Olga Turkovskaya**

Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov, Russia

*Corresponding author email: amuratova@yahoo.com*

## ABSTRACT

Plant-microbial associations and symbioses, with a flexible metabolism and unique enzyme systems, have great advantages for survival in rugged environments, and their survival is due not only to an increase in tolerance to xenobiotics but also to an active removal of toxic substances from the habitat. Despite the increased use of plants and their associated microorganisms in the form of phytoremediation technology, biochemical interactions between partners in the rhizosphere that are aimed at the degradation of organic pollutants are not fully understood. We studied the catabolism of polycyclic aromatic hydrocarbon phenanthrene by several rhizosphere bacterial strains and the possibility of enzymatic oxidation of the PAH by the root exudates of alfalfa (Medicago sativa L.) in order to detect the possible coupling of the plant and microbial metabolisms under the rhizospheric degradation of the organic pollutant.

A comparative study of phenanthrene degradation pathways in the isolated PAH-degrading rhizobacteria *Pseudomonas stutzeri*, *P. viridiflava*, *Arthrobacter* sp., and *Ensifer meliloti* was conducted. Identification of the key metabolites formed during biotransformation of phenanthrene and its derivatives, including 9,10-phenanthrenequinone, 9-phenanthrol, 2-carboxybenzaldehyde, 1-hydroxy-2-naphthoic, salicylic and *o*-phthalic acids, was performed by using TLC, GC, HPLC, and mass spectrometry. It was found that the metabolic pathways of phenanthrene in these bacteria were different: *E. meliloti* catabolized phenanthrene through the salicylate pathway, while the remaining rhizobacteria converted this PAH through the *o*-phthalate pathway. 1-Hydroxy-2-naphthoic acid was the predominant metabolite in all strains.

Alfalfa plants were grown under sterile conditions in quartz sand in the presence and absence of phenanthrene for 3 weeks under controlled environmental conditions to obtain plant root exudates for study. The root exudates were concentrated by ultrafiltration, and the activity of peroxidases, tyrosinases and other oxidases was detected through an oxidation test with various substrates [2,20-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS), 2,7-diaminofluorene (2,7-DAF), *o*-dianisidine and 2,6-dimethoxyphenol], detectable by spectroscopy. It was found that the most marked activity was that of peroxidase, whereas the presence of oxidase and tyrosinase was detected on the verge of the test sensitivity. The optimal substrates for the detection of peroxidase activity of alfalfa root exudates were ABTS and DAF, and the optimum pH values were in the range 4.5-5.5. The presence of phenanthrene at a concentration of 0.03 g kg⁻¹ in the medium insignificantly reduced the peroxidase activity of the alfalfa root exudates.

Using alfalfa root exudates as a crude enzyme preparation, we found that the plant peroxidase in the presence of the synthetic mediator ABTS can oxidize phenanthrene (6-26%) and its microbial metabolites 9,10-phenanthrenequinone (22-27%), 1-hydroxy-2-naphthoic acid (28-100%), and 1-naphthol (92%). It was revealed that 9-phenanthrol significantly inhibited the peroxidase activity toward ABTS, which indicated a higher affinity of the enzyme to 9-phenanthrol and an indirect confirmation of PAH binding to the zymophore.

The results indicate the possibility of active participation of plants in the rhizospheric degradation of PAHs and their microbial metabolites, which makes it possible to speak about the coupling of the plant and microbial metabolisms of PAHs in the rhizosphere.

**Acknowledgements:** The financial support by RFBR project 13-04-02051 is greatly appreciated.

<figure><img src="image_290.png" /></figure>
# ENHANCED DEGRADATION OF POLYCHLORINATED BIPHENYLS USING MICROBE-ASSISTED PHYTOREMEDIATION

Jitendra K. Sharma, Rashmi R. Misra, 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

The presence of PCBs in the environment poses great risk to plants, animals and humans and their removal seems to be great ecological and economical problem due to their persistence in the environment. Thus, remediation of PCBs using microbes and plants represents one of the important mechanisms by which the concentration of this xenobiotic compound can be decreased and subsequently eliminated from the environment. However, it has been postulated that complete degradation of PCBs congeners can be achieved by use of microbial consortium along with plants i.e. microbe-assisted phytoremediation (MAP) rather than individual use of bacterial culture/consortium or plant. This study deals with MAP of PCBs (in Clophen oil) at microcosm level using bacterial consortium (T1) and combination of consortium and plant (T2) in the soil pots, contaminated with 2% Clophen oil along with a control (soil + 2% Clophen oil) over a period of 90 days. The plant used in this study was *Vetiveria zizanoides*. The samples were collected every 30th day and various physicochemical and microbial parameters were analysed to evaluate the degradation of PCBs in soil. The results showed decrease in total organic carbon (TOC), increase in chloride ion (Cl⁻), electrical conductivity (EC) in the treatment T2 with no significant changes in treatment T1 for the same as compared to control. The treatment showed increase in dehydrogenase activity in the following order; T2>T1>control. Even though, the results are yet to be confirmed by GC-MS analysis; the results of preliminary analysis performed in this study are encouraging. It can be inferred from the results that microbe-assisted phyto-remediation can be employed as an effective option for enhanced remediation of PCBs contaminated soil.

**Keywords:** PCBs, degradation, consortium, microbe-assisted phytoremediation, dehydrogenase activity

<figure><img src="image_291.png" /></figure>
# ISOLATION, CULTIVATION, AND CHARACTERIZATION OF ENDOPHYTES IN FUNCTION OF PHYTOREMEDIATION OF DDE-CONTAMINATED SOILS

Nele Eevers<fnref n="1" />, Bram Beckers<fnref n="1" />, Marijke Gielen<fnref n="1" />, Nele Weyens<fnref n="1" />, Jason C. White<fnref n="2" />, Jaco Vangronsveld<fnref n="1" />

Corresponding author email: nele.eevers@uhasselt.be

## ABSTRACT

2,2-bis(*p*-chlorophenyl)-1,1,1-trichloroethane (DDT) is a pesticide that was used worldwide from 1943, but has been forbidden in most western countries since the 1970s. In soil, it quickly degrades to 2,2-bis(*p*-chlorophenyl)-1,1-dichloroethylene (DDE). Both molecules are considered as persistent organic pollutants (POPs), which are highly recalcitrant in soil, resulting in a worldwide spread of DDE contaminated soils.

*Cucurbita pepo* has been shown to accumulate high concentrations of DDE from surrounding soil [1]. This research focuses on optimizing the phytoremediation process with the help of plant-associated endophytic bacteria. To enhance the extraction of DDE from the soil and improve its degradation in the plant, endophytic bacteria have to be isolated and cultivated. Previously, it has been shown that only about 1-5% of the total bacterial population can be isolated and cultivated successfully [2]. To increase the cultivable fraction of the total bacterial population, a first part of this research explores different isolation and cultivation techniques. Results reveal that the medium composition as well as the addition of plant extract can affect the extent and the diversity of the cultivable of the bacterial population.

To investigate the role of endophytes during phytoremediation of DDE, in the second part, a comparison was made between the endophytic population isolated from plants that were grown with 100 µg L⁻<fnref n="1" /> DDE and control plants. The isolated bacteria were identified genotypically using ARDRA and were characterized phenotypically using several tests concerning their plant growth promoting capacities such as the production of indol-3-acetic acid (IAA), siderophores, organic acids, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Bacteria that can be beneficial to plant growth were found in both control and exposed plants, however, a general trend was noticeable where endophytes isolated from DDE-exposed plants scored better in the tests for plant growth promoting capacities. Furthermore, a screening for possible DDE degradation by endophytic bacteria was performed. Endophytes isolated from DDE-exposed plants showed a DDE degrading capacity more often than endophytes isolated from plants grown without DDE.

## References:

1. White, J.C., Parrish, Z.D., Isleyen, M., Gent, M.P.N, Iannucci-Berger, W., Eitzer, B.D., and Incorvia Mattina, M.J., (2005). Uptake of weathered *p,p'*-DDE by plant species effective at accumulating soil elements. *Microchemical Journal* 81, 148-155.

2. Torsvik, V. and Øvreås, L., (2002). Microbial diversity and function in soil: from genes to ecosystems. *Current Opinion in Microbiology* 5, 2040-245.

<figure><img src="image_292.png" /></figure>

<footnote n="1">¹Hasselt University, Biology, Agoralaan building D, B3590 Diepenbeek, Belgium</footnote>

<footnote n="2">²Dept of Analytical Chemistry, Connecticut Agricultural Experiment Station, New Haven, USA</footnote>
# PLANT-MICROBIAL-SOIL SYSTEM OF Ni-HYPERACCUMULATING PLANTS: METAL AVAILABILITY ASSESSED VIA CONVENTIONAL SOIL ANALYSES AND DGT

Vanessa Álvarez-López<fnref n="1" />, Jakob Santner<fnref n="2" />, Petra Kidd<fnref n="1" />, Ángeles Prieto-Fernández<fnref n="1" />, Carmela Monterroso<fnref n="3" />, Andreas Kreuzeder<fnref n="2" />, Walter W. Wenzel<fnref n="2" />, Markus Puschenreiter<fnref n="2" />

## ABSTRACT

Hyperaccumulator plants have been described as good candidates for phytoextraction (or phytomining) techniques. The majority of these plant species have been identified as Ni hyperaccumulators, and their distribution is closely associated with serpentine soils. Phytomining of such Ni-rich soils provides an opportunity for the recovery of high-value metals with potential application in metallurgical processes. Success depends to a great extent on Ni availability to the roots and the ability of the plant to take up and accumulate this element in its shoots. Although controversial results can be found these plants have often been thought to access metal fractions not available to non-accumulating plants (thus increasing metal uptake), or that their root activity leads to a faster replenishment of soluble metal pools. Recent studies have also shown that hyperaccumulators harbor a higher number of metal-tolerant microorganisms compared to surrounding non-vegetated soil or non-accumulators, and that these plant-associated microorganisms can enhance metal availability for plant uptake. Here we characterize the plant-microbial-soil system of two Ni-hyperaccumulating subspecies of *Alyssum serpyllifolium*, both of which are endemic to the serpentine outcrops of the Iberian Peninsula. The objectives of this work were (1) to describe the culturable microbial community associated with these plants and to identify potential candidate microbial strains for application in phytomining; and (2) to further unravel the influence of this plant type on soil metal bioavailability and the potential implications in the efficiency of the phytomining process.

Plant and soil (non-vegetated and rhizosphere) samples were collected from *Alyssum serpyllifolium* subsp. *lusitanicum* growing in Samil and Morais (Tras-Os-Montes, N of Portugal) and Barazón (NW of Spain), and from *Alyssum serpyllifolium* ssp. *malacitanum* in Sierra Bermeja (S of Spain). For comparative purposes non-accumulating plants (*Dactylis glomerata*, *Holcus lanatus* and *Santolina semidentata*) growing at the same sites, and the non-hyperaccumulator *Alyssum serpyllifolium* ssp *serpyllifolium* growing in a calcareous soil in S. Nevada (S of Spain), were also sampled. Plant Ni accumulation was determined, and soil samples were used for physicochemical analysis and the isolation and characterization of plant-associated culturable bacteria (total bacterial density, metal tolerance, plant growth promoting traits and diversity indices). Metal availability and fractionation was assessed using simple extraction (Sr(NO₃)₂-, Ca(NO₃)₂-extractable and sequential extractions), while metal kinetics and soil replenishment capacity was assessed using the DGT (Diffusive Gradient in Thin Films) technique employing Chelex resin gels. DGT is a passive sampling technique and for diffusion-limited uptake the DGT-labile solute fraction (C_DGT) has been shown to predict plant uptake better than chemical extraction. Metal replenishment capacity in soils can be evaluated through the ratio C_DGT:[metal]_{soil solution}. DGT was also applied to plants grown in serpentine soil in rhizotrons for the chemical imaging of labile Ni distribution along the root axis and rhizosphere.

The *Alyssum serpyllifolium* subsp. *lusitanicum* population from Barazón showed the highest Ni accumulation in the shoot tissues. Total bacterial densities were higher in the calcareous soil, and the diversity of the culturable bacterial community associated with this non-hyperaccumulator was also

<figure><img src="image_293.png" /></figure>

<footnote n="1">¹ Instituto de Investigacións Agrobiolóxicas de Galicia (IIAG), Consejo Superior de Investigaciones Científicas (CSIC), Santiago de Compostela, Spain</footnote>

<footnote n="2">² University of Natural Resources and Life Sciences (BOKU), Department of Forest and Soil Sciences, Tulln, Austria</footnote>

<footnote n="3">³ Departamento Edafoloxía e Química Agrícola, Universidade de Santiago de Compostela (USC), Spain
*Corresponding author email: vanessa@iiag.csic.es*</footnote>
higher. However the densities of metal tolerant bacteria were higher in the serpentine soils and in the rhizosphere of the hyperaccumulator, and these were significantly correlated with soil Ni availability. Most bacterial strains belonged to the *Actinobacteria* phylum. Several strains were selected for PGP traits and Ni resistance, and promoted plant growth and Ni phytoextraction capacity in pot experiments. Simple extractants indicated a higher Ni availability in the rhizosphere of the hyperaccumulator, and this was corroborated by C~DGT~ values. Images showed Ni depletion in the rhizosphere of the hyperaccumulator and *H. lanatus*, but this was less pronounced in the grass species than the hyperaccumulator. On the other hand, a zone corresponding to an increase in Ni was seen in the rhizosphere of the non-hyperaccumulating *Alyssum* subspecies. Soil Ni availability and replenishment is discussed in relation to plant type and accumulation traits.

***Acknowledgements:*** The financial support of the FP-7 project GREENLAND (contract No. 266124) by the European Commission, and from the Spanish Ministerio de Economía y Competitividad and FEDER (project CTM2012-39904-C02-01), is greatly appreciated.

<figure><img src="image_294.png" /></figure>
# CHARACTERISATION OF ENDOPHYTIC BACTERIAL COMMUNITY IN THE HALOPHYTE *JUNCUS ACUTUS* L.

Evdokia Syranidou<fnref n="1" />, Nele Weyens<fnref n="2" />, Jaco Vangrosveld<fnref n="2" />, Nicolas Kalogerakis<fnref n="1" />

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

## ABSTRACT

Phytoremediation is the use of plants and their associated microorganisms to degrade toxic contaminants in soil, air or water. The idea of exploiting endophytic bacteria in phytoremediation strategies is becoming increasingly popular because endophytes can effect beneficially the plant growth and contribute to the plant higher tolerance towards biotic and abiotic stress. Halophyte species are widespread in the Mediterranean basin and are of special interest since these plants are adapted to cope with environmental stresses. *Juncus acutus* L. has a wide ecological range and was found to be tolerant to organic and inorganic contaminants.

The culturable endophytic community of this halophytic species (*Juncus acutus*) growing on Bisphenol-A contaminated soil and groundwater was assessed. The genotypic characterisation of the isolated strains using BOX-PCR was performed and the 16s rRNA gene of the isolates with distinct BOX-PCR profiles were amplified and sequenced. The ability of the isolates to grow in the presence of BPA as the sole carbon source was also investigated and the most promising isolates were tested for their capacity to degrade Bisphenol A. The strains were further phenotypically characterised for plant growth promoting characteristics (ACC deaminase, IAA production P solubilisation, siderophore and organic acid production) and trace metal tolerance (Zn, Ni, Cd, Pb). Bioaugmentation experiments were performed with selected endophytic bacteria in order to study the potential enhancement of BPA degradation rates.

Eighty endophytic strains were isolated from the roots and upper and lower part of the leaves of the halophyte. The majority of the strains (84%) exhibited an increased ability to tolerate BPA. Concerning plant growth promoting traits, many bacteria had at least one PGP characteristic. The potential benefits of bioaugmentation were investigated and the results indicate that the indigenous endophytic microbial populations of the halophyte may be exploited in a phytoremediation strategy.

**Acknowledgements:** This research was supported by European Union FP-7 project MINOTAURUS – Grant Agreement no. 265946.

<figure><img src="image_295.png" /></figure>

<footnote n="1">¹School of Environmental Engineering, Technical University of Crete, Greece</footnote>

<footnote n="2">²Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium</footnote>
# PLANT MICROBIOME POTENTIAL IN ENHANCING SOIL RHIZOREMEDIATION

Eleonora Rolli<fnref n="1" />, Gloria Agazzi<fnref n="2" />, Ramona Marasco<fnref n="1" />, Francesca Mapelli<fnref n="1" />, Elisabetta Franchi<fnref n="3" />, Daniele Daffonchio<fnref n="1" />, Sara Borin<fnref n="1" />

Corresponding author email: sara.borin@unimi.it

## ABSTRACT

Losses of contaminants during industrial and commercial operations, municipal and industrial waste treatment, oil extraction and production and inadequate storage often can result in soil contamination. More than 1,800,000 potentially contaminated sites have been identified in Europe. Among those, many are large scale sites affected by a mixed contamination e.g. by organic and inorganic pollutants, whose remediation implies technological and economic limitations on the choice of the remediation technique. The use of plants to extract and/or modify the pollutants (*phytoremediation*) together with root associated microbes to degrade organic pollutants or modify the heavy metals (*rhizoremediation*), has the advantages of being environmentally sustainable and based on a bioeconomy approach, applicable on a large scale, on extended sites and in long-term treatments, and suitable to mixed contamination. Microorganisms can be exploited for the magnification of the phyto- and rhizo- remediation efficiency by Plant Growth Promotion (PGP) activity. PGP bacteria are positively associated with the plant: they exert direct and indirect beneficial activities on the host plant by stimulating its growth and stress tolerance, receiving in turn a benefit from root exudates.

Aim of the work was to evaluate the potential of plant associated microbes for PGP enhanced rhizoremediation in a soil contaminated by heavy metals (Cu, Ni, As, Hg) and oil derived long chain (C>12) aliphatic hydrocarbons. A collection of 65 bacterial isolates was established from contaminated soil and from the rhizosphere of wild plants naturally selected in the most polluted area of the site, enriched on aliphatic hydrocarbons (C12, C13, C15, C16 and C17) as unique carbon source. The isolates, identified by 16S rRNA gene sequencing, belonged to different classes i.e. Actinomycetales, α- and β-Proteobacteria, Bacilli. The collection has been then functionally screened by i) resistance to heavy metals (Ni, Cu, Hg, As species), ii) PGP traits (modulation of phyto-hormone omeostasis; increase of nutrient availability; production of hydrolytic enzymes, EPS and organic volatile compounds), iii) presence of alkane degrading genes (alkane hydroxylase, *Alk*). Eighty percent of the strains showed to tolerate one or more metals in the growing medium and to hold at the same time multiple potential PGP traits.

The most promising strains, having potential degrading activity on alkanes and resistance to heavy metals and multiple PGP activities, have been selected and tested *in vivo* for the positive association with plants. Rhizocompetence of the strains was demonstrated as the ability of the bacterial cells to colonise the plant root of *Lupinus albus*, species with phytoextraction potential, and the model plant *Arabidopsis thaliana*. Direct PGP activity was tested on *Solanum lycopersicum* and *A. thaliana*. Five strains demonstrated to induce a statistically significant increase in length and fresh weight of stem of the bacterized plants, as compared with not inoculated ones. In *A. thaliana* plants growing in synthetic medium containing Cu and Ni, bacterial inoculation induced a change in the root architecture and an increase in the number of lateral roots and the overall root biomass.

The results of the work demonstrated that the microbiota of contaminated soil and in particular associated to the rhizosphere of naturally selected wild plants has the potential of sustaining soil rhizoremediation, particularly magnified by PGP activity. The most promising isolates belong to the genera *Gordonia*, *Sporosarcina* and *Cupriavidus*, and could be exploited on field as bioagumentation and PGP inocula for the bioremediation of soil polluted by hydrocarbons and heavy metals.

**Acknowledgements:** The financial support by Eni Syndial s.p.a. is greatly appreciated.

<figure><img src="image_296.png" /></figure>

<footnote n="1">¹Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Italy</footnote>

<footnote n="2">²Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Italy</footnote>

<footnote n="3">³Research Center for Non-Conventional Energy, Istituto Eni Donegani, San Donato Milanese (MI), Italy</footnote>
# ENDOPHYTIC BACTERIA CONTRIBUTE TO CARBAMAZEPINE REMOVAL IN PHRAGMITES AUSTRALIS

**Peter Schröder, Andrés Sauvêtre**

Helmholtz Zentrum München, German Research Center for Environmental Health, Department of Environmental Sciences, Research Unit Microbe-Plant Interactions, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

*Corresponding author email: peter.schroeder@helmholtz-muenchen.de*

## ABSTRACT

Carbamazepine is an anticonvulsant and mood-stabilizing drug used primarily in the treatment of epilepsy and bipolar disorder and prescribed widely in Europe and North America. Due to this fact, it is found ubiquitously as a persistent and recalcitrant contaminant, being one of the most prominent compounds in the list of hazardous PPCPs found in Waste Water Treatment Plants (WWTPs). Usual concentrations in WWTPs range between 1 µg/L in influents and 0.7 µg/L in effluents, indicating a remarkably low removal efficiency in activated sludge processes. Hence, the ultimate fate of this compound is the groundwater in many cases. Recent studies revealed concentrations up to 16 ng/L in confined and semi-confined groundwaters in France.

Up to now, current bioremediation approaches have focused on the role of plants in the uptake or absorption of carbamazepine from waters. Studies have been performed in edible plants (cucumber, soybean and *Brassica* sp.) and also in some grass and macrophyte species. Nevertheless, investigation about the degradation of carbamazepine by plants or their inhabiting microbes is scarce. However, since endophytes have been proposed to play an important role in phytoremediation, this study focuses on the identification and characterization of endophytic candidates for biodegradation of carbamazepine in wetland systems.

When *Phragmites australis* was exposed to carbamazepine (5 mg/L) for 9 days in semi-hydroponic conditions, different bacterial strains could be isolated from rhizomes and roots. Numerous of these strains were able to grow either in PDA or in R2A media supplemented with 10 µM carbamazepine. The species were identified by sequencing of the 16S rDNA. Furthermore, their growth in minimal medium with carbamazepine as sole carbon source was studied. Phylogenetic studies and identification of non-cultivable strains will shed more light on the behavior of the bacterial community in response to this important pharmaceutical.

<figure><img src="image_297.png" /></figure>
# PHYTOREMEDIATION OF SOIL CONTAMINATED WITH AROCLOR BY CHROMOLAENA ODORATA (L) KING & ROBINSON INOCULATED WITH RHIZOSPHERE ORGANISMS

## Harrison I. Atagana

Institute for Science and Technology Education, University of South Africa, Pretoria, South Africa

Corresponding author email: atagahi@unisa.ac.za

## ABSTRACT

*Chromolaena odorata* was planted on soil contaminated with 10, 30 and 50% Aroclor and inoculated with rhizosphere organisms to study the phytoextraction of PCB congeners present in the soil. Fertilizer (NPK 5%) was applied to the soil and watered regularly for ten weeks. Growth enzyme activities and changes in PCB concentrations in the contaminated soil and plant tissues were measured. Rhizosphere microorganisms were characterized by 16S rRNA base sequencing. Plant growth was not significantly impeded by the presence of Aroclor in the concentrations used in the experiments. Increases in enzyme activities in the soil were observed in all the experiments. Reduction of between 67 and 87% in PCB concentrations were observed at the end of the ten-week period. The concentrations of PCBs in the roots of all experimental plants were more than in the shoots. Rhizosphere organisms did not significantly enhance the growth of plants and PCB reduction in soil or PCB concentration in plant tissues. *Chromolaena odorata* has shown the potential to grow in Aroclor contaminated soil and extract PCB from such soil. However, it is not clear whether degradation of PCB occurred in plant tissues or in the soil. The difference in amount of PCB recovered from the soil and plant tissues may indicate that biodegradation has taken place. However, volatilization and translocation, or a combination of both may have occurred along with some degradation.

**Acknowledgements:** I want to acknowledge the financial support provided by the National Research Foundation of South Africa for this research project. I also want to acknowledge the University of KwaZulu-Natal for the use of their tunnels for the experiments.

<figure><img src="image_298.png" /></figure>
# CHARACTERIZATION OF ENDOPHYTIC BACTERIA FROM SELENIUM HYPERACCUMULATORS AND GM PLANTS

Martina Novakova<fnref n="1" />, Klara Richterova<fnref n="1" />, Lucie Musilova<fnref n="1" />, Lucian Staicu<fnref n="2" />, Jason Reynolds<fnref n="2" />, Iva Hrochova<fnref n="1" />, Martina Chovancova<fnref n="1" />, Katerina Demnerova<fnref n="1" />, Elizabeth Pilon-Smits<fnref n="2" />

Corresponding author email: suram@vscht.cz

## ABSTRACT

The goal of this work is to optimize the symbiotic synergism between plants and microbes resulting in more effective bio/phytoremediation processes. This approach is being performed for plants hyperaccumulating selenium (hyperaccumulators, HA) and genetically modified (GM) plants remediating organic or inorganic contaminants. We focus on the cultivable and also not cultivable (viable but not cultivable, VBNC) endophytic and rhizospheric bacterial communities responsible for contaminant accumulation and degradation.

An important aspect of this research is to compare the bacterial diversity of selenium hyperaccumulators and nonhyperaccumulators, to see how the bacterial community is shifted in HA and whether accumulation of selenium in plants can be facilitated by certain bacteria. Studied HA were *Stanleya pinnata* and *Astragalus bisulcatus* from Pine Ridge Natural Area, Fort Collins, Colorado, USA. Endophytic microorganisms were isolated from plant roots, stems and leaves. The most endophytes were isolated in the order roots>leaves>stems. Characterization of endophytes was performed by analysis of part of the 16S rRNA gene and MS MALDI-TOF Biotyper. Among others, the bacterial isolates were *Bacillus*, *Pseudomonas*, *Pantoea*, *Paenibacillus*, *Staphylococcus*, *Variovorax*. Isolates were tested for the ability to produce siderophores and to metabolize selenium and nitrogen compounds. The isolates were able to reduce selenite into elemental selenium and some were able to reduce nitrite into nitrogen. Further, the metagenomic DNA of endophytes was isolated in order to determine the bacterial community (VBNC bacteria). We used T-RFLP analysis that will be continued by pyrosequencing.

A similar approach has been performed to study the changes of bacterial diversity in GM plants, as compared with wild type plants. Studied plants were *Nicotiana tabacum* containing bacterial *bphC* gene. The results so far indicate that the rhizospheric bacterial community of GM plants has not been changed. Cultivatable endophytic bacteria have been isolated, and among these *Pseudomonas*, *Rhodococcus*, *Arthrobacter*, *Microbacterium* and *Leifsonia* have been identified. The metagenomic endophytic DNA from GM plants is under investigation.

**Acknowledgements:** This work was supported by grants MSMT KONTAKT LH12087 and GACR P501/12/P521. We acknowledge Jakub Ridl, Miluse Hroudova (IMG AS CR) and Ondrej Uhlik (ICT Prague) for pyrosequencing, and Tomas Frantik (Institute of Botany AS CR) for the factor analysis.

<figure><img src="image_299.png" /></figure>

<footnote n="1">¹Institute of Chemical Technology in Prague, Dpt of Biochemistry and Microbiology, Prague, Czech Republic</footnote>

<footnote n="2">²Colorado State University, Fort Collins, CO, USA</footnote>