Abstracts workshop 2.pdf

A Solid Waste Contaminated Site in the Negev Desert – Case Study
U. Manandhar, R. Vulkan, O. Barazani, P. Sathiyamoorthy and A. Golan-Goldhirsh Ben-Gurion University of the Negev The Jacob Blaustein Institute for Desert research, Department of Dryland Biotechnologies Desert Plants Biotechnology Laboratory, Sede Boker Campus 84990, Israel A solid-waste-contaminated site in the Negev Desert is dominated by big shrubs of Nicotiana glauca. The concentration of certain heavy metals (Cu, Zn and Pb) was more than an order of magnitude higher than in non-contaminated desert soil. The effect of the contaminated soil on selected heavy metal-tolerant desert plants (Bassia indica, Mesembryanthemum nodiflorum, Nicotiana gluaca and Atriplex halimus) was conducted in a pot experiment. Plants were grown for four weeks in contaminated and uncontaminated soils, and in each of the soils with the addition of EDTA, compost or EDTA+compost. In each of the two soil types, the fresh and dry weights of B. indica and M. nodiflorum was not significantly different among the treatments. Whereas, fresh and dry weights of N. glauca grown in uncontaminated soil were higher than those grown in contaminated soil. Fresh and dry weights of A. halimus grown in soil amended with compost was significantly higher than that grown in uncontaminated soil. Metal analysis showed that in EDTA and EDTA+compost treatments, Zn and Cu concentrations in shoot of B. indica, N. glauca and A. halimus were higher than in control and compost treatments. Among all plants tested, N. glauca accumulated the highest concentration of copper and zinc (approx. 700 mg/Kg DW). It appears a species of choice for further studies. Suitability of Birch and Hybrid Poplar
for Bioremediation of Polyaromatics and Heavy Metals
K. Yrjäläl, C. Fortelius2, M.-L. Äkerman2, A. Tervahauta3, S. Kärenlampi3, N. Stenval4, P. Pulkkinen4 and K. Haahtelal1 lDepartment of Biosciences, University of Helsinki, Finland ([email protected]) 2Espoo-Vantaa Institute of Technology, Finland 3Department of Biochemistry, University of Kuopio, Finland 4Finnish Forest Research Institute, Vantaa Research Center, Vantaa, Finland About 1-15 t waste oil per gas station is annually produced in Finland and oil wastes have frequently been reported to escape to Nature. The cold climate in Finland creates exceptional challenges for remediation of polluted sites. Any type of biological pollutant degradation is slower than in warmer climates. The spread of the pollutant is also relatively slow, which provides more time for planning and executing cleaning activities. Micro propagated birch clones, Betula pendula, were used in microcosm experiment where synthetic mixtures of polyaromatics (PAH) were used to test the suitability of a Wales (Great Britain) and a Harjavalta (Finland) birch clone for remediation. A PAH mixture containing anthrascene, phenanthrene, fluoranthene, and pyrene was added to soil either 200 or 1200 ppm. The micro propagated birch clones were grown in pots containing either 0.25 or 0.5 kg sand and compost mixture. The temperature, humidity and CO2concentration were continually registered in the growth chamber. The growth of 10 hybrid poplar clones, Populus tremulalpopulus tremuloides, was tested in a soil microcosm containing waste ash. The clones were propagated from root cuttings, allowed to form suits and beginning of roots, and replanted to waste ash contaminated soil. The effect of the ash on shoot and root growth was monitored. Enhanced Metal-uptake of Clover by Coinoculated, Ni-adapted Beneficial
Mycorrhiza and Helper Brevibacillus strains
A. Vivas1, B. Biró2, I. Vörös2, R. Azcon1 and J.-M. Barea1 1Dept. Soilmicrobiol. and Symbiotic Systems, Estación Experimental del Zaidin,CSIC, Granada, Spain 2Lab. of Rhizobiology, Research Institute for Soil Science and Agricultural Chemistry of the Hungarian Academy of Sciences, Budapest, Hungary
The synergistic and stress-tolerant ability of some beneficial microbes, such as the arbuscular
mycorrhizal fungi (AMF) and nitrogen- fixing Azospirillum or Rhizobium bacteria from the
rhizosphere of higher plants are well-documented (Biró et al, 2000). The potential
enhancement of the phytoremediation processes by some metal-adapted Brevibacillus helper
strains of the mycorhizosphere is being reported in this study.
A Ni-adapted arbuscular mycorrhizal fungal strain was isolated from a long-term field experiment of the RISSAC, Hungary. Various rates (0, 30-, 90- and 270 mg kg-1 dry soil) of the Ni(sulphate) was added into the calcareous chernozem soil by 7 years prior the isolations. Abundance of the countable Ni-tolerant bacterial flora was also assessed, the most typical colonies was isolated and characterised by mRNS molecular techniques. Metal tolerance ability of the isolates was checked in vitro with orbital shaker and spectrophotometer in nutrient solution contaminated with 20-, 40-, 60- and 80 µgml-1 Ni-sulphate. Single- or tripartite inoculation of the Ni-tolerant microbes (mycorrhiza and bacteria) was performed in a pot experiment. Red clover (Trifolium pratense L) was used as host for testing the metal- and dry matter accumulation-potential and the rate of AMF colonisations. Heat-sterilised, control or inoculated metal-spiked (270 mg kg-1 Ni) soil was used for 45 days growth of the clover hosts. Data were analysed statistically and significant differences are being presented. Nickel-adapted endomycorrhiza inoculum was found to be tolerant to the 270 mgkg-1 soil-applied Ni rates. The same beha viour could be detected for the identified, Ni- isolated Brevibacillus sp. strain in vitro. As a consequence of the Ni-tolerant ability an increased growth was detected at the highest Ni-doses, in comparison with the non-adapted and autentic control strain. Nodulation, biomass-production and macroelement contents were increased by the single inoculation of both the adapted bacterium and the AM fungi. In case of the tripartite inoculation a synergistic, further intensified beneficial effect was found with a five- fold increase of the Ni- uptake ability. In contrast, only a slight stress-buffer effect could be realised by the non-adapted authentic mycorrhiza strain on the clover growth. The use of rhizo-biological tools and the adaptation ability of beneficial microbes are being great potentials for the enhanced phytoremediations. Supported by the Hungarian Scientific Research Fund (OTKA), the EU-Kp5 and by the bilateral agreement between the CSIC and the HAS. References
Biró, B., Köves-Péchy, K., Vörös I., Takács T., Eggenberg, P., Strasser, R.J. (2000): Interrelation between Azospirillum and Rhizobium nitrogen-fixers and arbuscular mycorrhizal fungi in the rhizosphere of alfalfa at
sterile, AMF-free or normal soil conditions. J. Appl. Soil Ecol. 15: 159-168.
Interactions between Crop Growth, Contaminants and Soil Ecosystem
Alterra, Green World Research, P.O. Box 47, 6700 AA Wageningen, The Netherlands Interactions between crop- growth, contaminating heavy metals, orga nic contaminants, and soil ecosystems were investigated in field and pot experiments (Figure 1). Organic contaminants
Does crop-growth affect the rate of degradation of organic contaminants such as mineral oil and polycyclic aromatic Hydrocarbons (PAH’s)? If yes: by means of what mechanisms and under which conditions. Supposed mechanisms: a) Root growth enhances transport of water, oxygen, nutrients and substrates towards microbes degrading the organic contaminants and thus reduces microbial growth limitations in a physical way. b) Growing roots exudate easily decomposable organic substrates and enhance microbial activity by reducing substrate limitations. With respect to mechanism b the concepts of priming and co- metabolism will be discussed. Priming is the stimulation of degradation of resistant organic material by addition of easily decomposable substrates (Dalenberg and Jager, 1981 and 1989; De Nobili et al., 2001). Co-metabolism is the process by which microbes in the obligate presence of a growth substrate transform a non- growth substrate. In a pot-experiment with PAH/oil contaminated sediment species from the later stages of landfarmed plots, different species interacted in a different way with crops in their effects on soil ecosystems (Table 2a and 2b). This indicates that probably growing crops depend on the type of soil (sediment) in their effect on the soil ecosystem and thus in their potential to enhance contaminant degradation (Harmsen and Bouwman, 2002) Heavy metals
Heavy metals affect crop-growth and soil ecosystems because some of them are essential for living organisms (Cu, Zn etc.), and, under conditions of increased concentrations, they all are more or less toxic to organisms and crops. Slight increases in heavy metal concentrations affect the lag period preceding the start of mineralization of certain substrates, the ratio of respired to biomass-incorporated substrate-C, the numbers of heavy metal resistant bacteria. However sensitive bacteria may be for increases in heavy metal concentrations, the soil ecosystem as a whole still functions when vegetation collapses for toxicity reasons; the subsequent degradation of the soil ecosystem is then caused mainly by lack of substrate. Effects of crop growth on heavy metal speciations and on the soil ecosystem were investigated in a pot experiment (a) and in the field (b). a) In moderately Cu-contaminated sandy arable soil with reduced crop production. Growth of a Cu-tolerant grass variety on this soil remediated various injured biological parameters – bacterial growth and bacterivorous nematodes – and decreased the copper activity in the soil solution (Figure 1 and Table II) b) In heavily Zn (Cd, Pb, Cu) – contaminated sandy nature soil where vegetation had disappeared. Growth of Zn-tolerant grass varieties, in combination with soil additives largely rehabilitated the soil ecosystem and reduced the (bio) availability of the heavy metals (Table 3, Table 4) (Bouwman et al., 2001) References
Bouwman, L.A., J. Bloem, P.F.A.M. Römkens, G.T. Boon, and J. Vangronsveld, 2001: Beneficial effects of the growth of metal tolerant grass on biological and chemical parameters in copper – and zinc contaminated
sandy soils. Minerva Biotecnologica 13, 19-26.
Dalenberg, J.W., and G. Jager, 1981: Priming effects of small glucose additions to 14C -labelled soils. Soil Biol. Biochem. 13, 219-223.
Dalenberg, J.W., and G. Jager, 1989: Priming effects of some organic additions to 14C-labelled soil. Soil Biol. Biochem. 21, 443-448.
De Nobili, M., M. Contin, C. Mondini, and P.C. Brookes, 2001: Soil microbial biomass is triggered into activity by trace amounts of substrate. Soil Biol. Bichem., 33, 1163-1170.
Harmsen, J. and L.A.Bouwman, 2002: Bioremediation of polluted sediment: a matter of time or effort? (II). In : R.E. Hinchee, A. Porta and M. Pellei (Editors) Remediation and Beneficial Reuse of Contaminated Sediments. Battelle Press, Columbus (in press). Potential for using Microbial Inoculants to Enhance
the Phytoremediation of Polluted Soils
School of Biomedical and Life sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
Phytoremediation is a relatively new technology that uses plants and their associated
microflora to decontaminate polluted soil and groundwater. To be effective, plants need to be
able to take up large quantities of the pollutants or have microbial communities associated
with them that are capable to detoxify polluting substances by degradation or by rendering
them non-soluble. In either case, phytoremediation is positively correlated to root growth and
root proliferation. Besides selection of suitable plant species, the key to effective
phytoremedfiation technologies lies therefore in the development of methods that enhance
root growth in contaminated environments.
In cases where the concentrations of polluting substances are phytotoxic to the plant, the rhizosphere micro-flora can be of benefit to the plant by detoxification of the rhizosphere itself, allowing plant roots to proliferate. For example, some rhizosphere fungi, such as Fusarium and Trichoderma, are capable of catabolising free cyanide and by doing so increase plant growth. Inoculation of plants with such strains will therefore stimulate plant growth, thus enhancing the remediation of polluted soils. Similarly metal bioavailability to the plant could potentially be reduced by bacteria, such as Ralstonia species, that cause the formation of insoluble metal carbonates, making the metal unavailable to the plant. Besides providing a detoxification mechanism, a variety of microorganisms associated with the rhizosphere are known to stimulate root growth directly by: • Providing the plant with extra nutrients (mycorrhizae, N2-fixing organisms), • Production of ACT-deaminase which reduces the levels of ethylene in the rhizosphere • Production of hormones that stimulate plant growth. We have investigated the effect of the rhizosphere bacterium Pseudomonas fluorescens F113, which produces the antibiotic 2,4 diacetylphloroglucinol (DAPG). Besides being an effective biological control agent against a variety of root pathogens, inoculation of pea plants with the DAPG producing strain resulted in increased root growth and increased release of organic acids by the roots into the rhizosphere. As a result of inoculation the DAPG producing P. fluorescens, nodulation of the roots with introduced Rhizobium strains was increased four fold, while there was a strong indication that VA mycorrhizal development was enhanced too. No such effects were observed using a non-DAPG producing mutant strain. Inoculation with plant growth promoting organisms might therefore have potential in enhancing the phytoremediation of polluted soils. Why it may be Critical to rely on Cattail for Phytoremediation
GSF-National Research Center for Environment and Health, Inst. for Soil Ecology, Dept. of Rhizosphere Biology, Ingolstädter Landstraße 1; D-85764 Neuherberg Cattail (Typha latifolia and T. angustifolia) are two freshwater monocots with Europe wide distribution. They are found abundantly in creeks, along swampy riverbanks and in drainages of agricultural areas. In the latter case, Typha is found very effective in removing nitrogen and phosphorus from the drainage water. Due to the strengthening of the water directive of the European Community the need to improve and maintain high quality standards for sewage treatment effluents during the next years is of importance. Plant-based treatment systems may offer an adequate supplement to existing technologies. In order to test for Typha´s suitability for the removal and metabolism of organic xenobiotics especially from sewage water we performed a set of in vitro experiments. Thypha latifolia (L.) and Typha angustifolia (L.) were investigated for a) the general detoxification capacity of organic xenobiotics; and b) the fate and specific breakdown of two persistent substances: bis (2-ethylhexyl)-phthalate (DEHP) (a plasticizer) and Lamotrigine (an antiepileptical medicament). These are substances of high environmental concern. Preliminary results indicate that Typha plants possess peroxidase activity and glutathione S-transferase (GST) activity for the conjugation of several xenobiotic model substrates (i.e. CDNB, DCNB etc.) in leaves, rhizomes and roots. Total GST activity seems to be shared by several GST-isoforms. Whereas the activity of some Glutathione S-transferase remains unaffected following the application of xenobiotics in induction experiments, other are induces by both chemicals and medicaments. In studies with Typha roots and rhizomes, the removal DEHP and Lamotrigine was observed. This disappearance seems to be connected to the activity of GSTs or Glucosyltransferases. The significance of this effect for the utilization of Typha in sewage treatment is discussed. Further studies using contaminated wastewater are planned. Sunflower Growth and Tolerance to Arsenic
is increased by the Rhizospheric Bacteria Pseudomonas fluorescens
S. Shilev1, A. Fernández1, M. Benlloch2 and E.D. Sancho1 EMIR-UCO, 1Departamento de Microbiología, 2Departamento de Agronomía Escuela Técnica Superior de Ingenieros Agrónomos y Montes, Universidad de Córdoba Apdo. 3048, 14080 – Córdoba, Spain Sunflower (Helianthus annuus) is one of the plant species which can be used in phytoremediation (Salt et al., 1995) due to its middle tolerance to heavy metals and to its rapid growth and high biomass production. In order to exploit the ability of some rhizospheric bacteria to increase plant tolerance to biotic and abiotic stresses, we have isolated, selected and identified several bacterial species tolerant to heavy metals (Shilev et al., 2001). The effect of these bacteria populations on plant growth, arsenic accumulation and tolerance in sunflower have been evaluated and results presented here. Pseudomonas fluorescens partially inhibited plant growth reduction caused by arsenic and promoted its accumulation in roots and shoots. References
Salt, D. E., M. Blaylock, N. Kumar, V. Dushenkov, B. D. Ensley, I. Chet and I. Raskin, 1995: Phytoremediation: A novel strategy for the removal of toxic metals from the environment using plants.
Biotechnology 13, 468-474
Shilev, S., J. Ruso, M. Puig, M. Benlloch and E. D. Sancho, 2001: Rhizospheric bacteria promote sunflower (Helianthus annuus L.) plant growth and tolerance to heavy metals. Minerva Biotecnologica 13:1, 37-39
Tolerance and Sensitivity to Arsenic is determined
by Arsenic Fluxes in Rhizospheric Bacteria
S. Shilev1, M. Benlloch2 and E.D. Sancho1 EMIR-UCO, 1Departamento de Microbiología, 2Departamento de Agronomía Escuela Técnica Superior de Ingenieros Agrónomos y Montes, Universidad de Córdoba Apdo. 3048, 14080 – Córdoba, Spain Rhizospheric bacteria have a beneficia l effects on the growth of plants in contaminated soils. In this sense, we have isolated rhizospheric bacteria that promote sunflower growth and tolerance to arsenic (Shilev et al., 2001). We have studied arsenic fluxes in tolerant (Pseudomonas fluorescens) and sensitive (Bacillus sp.) rhizobacteria. The results showed that the initial uptake was similar in both microorganisms, but, after several minutes in the presence of the metalloid, the tolerant strain was able to extrude the toxic element. After 60 minutes, suspended in arsenic (100-3000 ppm), the tolerant bacteria accumulated less than 1 µg/mL, while the sensitive strain accumulated more than 3 times this concentration. In addition, preliminary results indicate important differences in the efflux processes. References
Shilev, S., J. Ruso, M. Puig, M. Benlloch and E. D. Sancho, 2001: Rhizospheric bacteria promote sunflower (Helianthus annuus L.) plant growth and tolerance to heavy metals. Minerva Biotecnologica 13:1, 37-39

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