Bioremediation of Pesticide-Contaminated Soils and its Effect on Soil Functionality. Project ID: 1 April 2000 to 31 August 2004.

Transcription

1 Bioremediation of Pesticide-Contaminated Soils and its Effect on Soil Functionality ID: BR3 Duration: 1 April 2000 to 31 August 2004 Coordinator: Dr Christof Holliger Environmental Biotechnology Swiss Federal Institute of Technology Lausanne (EPFL) 1015 Lausanne, SWITZERLAND christof.holliger@epfl.ch Partners: Dr Jan Roelof van der Meer Université de Lausanne IMF Bâtiment de Biologie 1015 Lausanne, SWITZERLAND janroelof.vandermeer@imf.unil.ch Dr Rakesh Jain Institute of Microbial Technology Chandigarh , INDIA rkj@imtech.res.in Dr Rup Lal Department of Zoology University of Delhi Delhi , INDIA duzdel@del2.vsnl.net.in Dr Banwari Lal Microbial Biotechnology Tata Energy Research Institute (TERI) Darbari Seth Block New Delhi , INDIA banwaril@teri.res.in CO-WORKERS: Ms. Stéphanie Perez, EPFL, Switzerland stephanie.perez@epfl.ch Dr Lawlor Kirsten, EAWAG, Switzerland kirsten.lawlor@eawag.ch Dr. Om Vir Singh, IMTECH, India ovsingh@imtech.res.in Ms. Aparjita Basu, IMTECH, India Mr. Gunjan Pandey, IMTECH, India gp@imtech.res.in Ms. Shilpanjali Deshpande, TERI, India shilpanjalid@teri.res.in

2 RATIONALE Hexachlorocyclohexane (HCH), endosulfan, parathion, and methylparathion, which were banned or restricted in the US and in Europe years ago, are still applied in India to control insect pests affecting pulses and wheat. Unfortunately, the above-mentioned insecticides are not only very toxic but also poorly biodegradable. Their extensive application in Indian agriculture results in residual concentrations in almost any environment, drinking water, and in food. Consumption of selected insecticides in Indian agriculture (tonnes technical grade) Endosulfan HCH (BHC) Lindane Methyl Parathion A recent survey in Northern India revealed that the concentrations of insecticide residues in agricultural soil samples gives indeed rise to major concern. For HCH (all isomers), concentrations ranged between 14 and 158 ng/g in surface soil samples, and between ng/g in subsurface soil samples. Similar concentrations were measured for endosulfan. Even though the above-mentioned insecticides are mostly recalcitrant to biodegradation, a few bacteria have been reported to be able to utilise these pollutants as carbon and energy source. Others are capable to partially transform them. For example, only recently a Sphingomonas paucimobilis strain was isolated from the rhizosphere of sugar cane, which is able to degrade all isomers of HCH, even the very persistent b-isomer. Unfortunately, spontaneous natural biodegradation of these insecticides is very slow. Since contaminated sites represent severe health hazards for the population, faster and more efficient strategies for the decontamination of polluted soils are needed. The overall goal of this project is to develop and to test an efficient and cost effective biological treatment technique in order to remediate agricultural sites contaminated with HCH, endosulfan, parathion, and methylparathion, and to restore the functionality of the soil. This technique will be based on the introduction of specific microorganisms capable to completely degrade these insecticides. To achieve these goals, the following tasks will have to be tackled: Increasing the knowledge on the physiology, biochemistry, and genetics of the selected insecticide degrading bacteria Development of cultivation and storage techniques for the specific bacteria Determination of optimal field application methods and methods for monitoring the inoculants survival and efficacy for in-situ pesticide degradation Determination of the possible effects or improvements of introducing large quantities of microorganisms on soil functionality, by analysing microbial diversity and community structures All research activities shall be carried out under controlled laboratory conditions, in pot and tray experiments, and finally on the field scale. Genetically modified microorganisms will not be utilised.

3 Research Plan Dr. Rup Lal, University of Delhi, India Degradation of the individual HCH isomers by Sphingomonas paucimobilis: 1. Characterisation of the enzyme(s) responsible for the dehalogenation of the HCH isomers 2. Cloning, sequencing, and characterisation of the genes responsible for the degradation of HCH 3. Development of DNA probes to monitor HCH degrading bacteria Mass cultivation of HCH degrading bacteria Isolation of sulfan degrading bacteria Dr. Rakesh Jain, IMTECH, India Studies on the degradation of nitrophenolic compounds by Arthrobacter protophormiae, Pseudomonas cepacia and Rhodococcus rhodochrous. Isolation of new strains capable to degrade nitrophenolic compounds Studies on the genetics of the degradation of nitrophenolic compounds Development of DNA probes to monitor bacteria capable to degrade nitrophenolic compounds Dr. Christof Holliger, EPFL, Switzerland Bioaugmentation experiments in microcosms Bioaugmentation experiments in a closed soil system Studies on the cultivation, storage, and formulation of selected bacteria with regard to a subsequent field application Dr. Banwari Lal, TERI, India Bioaugmentation experiments in pots and trays Studies on bioremediation in the field Isolation and characterisation of endosulfan degrading bacteria

4 Mass cultivation of selected insecticide degrading bacteria Dr. Jan Roelof van der Meer, EAWAG, Switzerland Analysis of possible effects of pesticide application and of introduction of selected pesticide degraders on the soil microbial ecosystem by determining community characteristics: Analysis of microbial diversity Analysis of community structure (population abundance, spatial distribution) Determination of metabolic potential Studies on the expression of key genetic factor SUMMARY OF THE ACHIEVMENTS OF THE FIRST PROGRAM PHASE adapted from the summary provided by the project partners Pesticides form an integral component of agricultural practice in India in order to be able to produce and preserve the required amount of crops for such a densely populated country. Consequently, many cheap and effective insecticides such as hexachlor-cyclohexane (HCH), endosulfan, parathion, methyl-parathion, which were banned in many other countries are still being used or have only been banned recently in India. Their extensive use resulted in a widespread occurrence of residual insecticide concentrations in the Indian environment, and also in their accumulation in crops and food products. The overall goal of this project was to develop a bioremediation technology to treat pesticide contaminated soils by using specific inoculation of previously enriched pesticide degrading bacteria. Sphingomonas. paucimobilis B90A, a bacterium able to degrade four isomers of HCH, and Arthrobacter protophormiae RKJ100, a bacterium able to degrade para-nitrophenol (PNP), were chosen as model organisms. In addition, it was planned to enrich and isolate endosulfan degrading organisms. Research concentrated on the different steps typically necessary for a bioaugmentation process, e.g. mass cultivation of strains, storage of the produced biomass, formulation for application to the soil, and the conditions achieving optimal degradation of the target pesticide in soil. In addition, fundamental aspects of the pesticide degradation were investigated in order to assure that the pesticides were converted into harmless end products. For the pesticide endosulfan, only endosulfan-degrading strains could be enriched and initially characterized. Sphingomonas paucimobilis B90A degrades four HCH isomers, but only gammaand alpha HCH are completely dechlorinated. The strain mineralized 14 C-labeled gamma HCH completely to 14 C-CO 2. Strain B90A contains two copies of the lina gene. Both genes produced active enzymes in Escherichia coli, which can convert gamma-hch, but not beta-hch. The two LinA enzymes showed different activities for the two enantiomers of alpha-hch. Arthrobacter protophormiae RKJ100 was chosen as PNP degrading strain, PNP being a recalcitrant product formed from the organophosphorus pesticides parathion and methyl-parathion. Strain RKJ100 was capable of utilizing PNP as the sole source of carbon, nitrogen and energy. Degradation of PNP in this organism takes place through an oxidative route via the formation of p- benzoquinone and hydroquinone, which is further degraded via the betaketodipate pathway.

5 Endosulfan-degrading strains could be isolated during the project period, but it is not determined whether the degradation is complete. Therefore, at this stage they cannot be proposed for remediation of endosulfan-contaminated soils. The HCH- and PNP degrading strains grew well on molasses, indicating that it can be used as substrate for mass cultivation. Induction of the degradation pathways was not necessary to obtain pesticide degradation with cells cultivated on this substrate. Storage of cells was possible either as frozen cell pellet at - 20 C, or immobilized on corn cob powder at 4 C or ambient temperature. Microcosm experiments showed that pesticide degradation was optimal at 35% water holding capacity of the soil and with an incoculum size of 108 cells per g soil. In the case of S. paucimobilis, an additional carbon source had to be added to observe degradation, whereas A. protophormiae did not need a co-substrate. One square meter pilot tests showed that the concept developed during the first project period was suitable and led to the disappearance of the target pesticide.