Bioremediation of pesticide-contaminated soils and its effect on soil functionality


Prof. Christof Holliger, Roberta Pagliarini, Stéphanie Perez

Funding agency

Indo-Swiss Collaboration in Biotechnology (ISCB), SDC Berne

Project period

March 2000 – August 2004


Dr. J.R. van der Meer, EAWAG ; Dr. R. Lal, University of Delhi, India ; Dr. R. Jain, IMTECH, Chandigarh, India ; Dr. B. Lal, TERI, New Delhi, India


Pesticides form an integral component of agricultural practise 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 hexachloro-cyclohexane (HCH), endosulfan, parathion, and methyl-parathion, which were banned in many other countries, are still being used or have been banned only recently in India. Their extensive use resulted in a widespread occurrence of residual insecticide concen-trations in the Indian environment, and also in their accumulation in crops and food products. The overall goal of this project is to develop a bioremediation technology to treat pesticide contami-nated Indian soils by using specific inoculation of previously en-riched pesticide degrading bacteria (bioaugmentation). Sphingomonas paucimobilis B90A, a bacterium able to degrade four isomers of HCH, and Arthrobacter protophormiae, a bacterium able to degrade para-nitrophenol, the hydrolysis product of parathion and methyl-parathion, were chosen as model organisms. In addition, it was planned to enrich and isolate endosulfan-degrading organisms.


Research has concentrated on the different steps typically necessary for a bioaugmentation process. e.g. mass cultivation of the pesticide-degrading 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 to harmless endproducts. For the pesticide endosulfan, only endosulfan-degrading strains could be enriched and initially characterized.

Sphingomonas paucimobilis B90A degrades four HCH isomers, but only gamma- and alpha- HCH are completely dechlorinated. The strain mineralised 14C-labeled gamma-HCH completely to 14C-CO2. Strain B90A contains two copies of the linA gene. Both genes pro-duced active enzymes in Escherichia coli, which could 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 p-nitrophenol (PNP)-degrading strain, PNP being a recalcitrant product formed from organophosphorous pesticides parathion and methylparathion. 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 β-ketoadipate pathway. Endosulfan-degrading strains could be isolated during the project period, but it is not yet whether the pesticide is used as carbon and en-ergy source or as sulfur source, and whether degradation is complete. Therefore, they cannot now 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 molasses. Storage of cells was possible either as frozen cell pellet at -20°C, or immobilized on corncob powder at 4°C or ambient temperature. Microcosm experiments showed that pesticide degrada-tion was optimal at 35% water holding capacity of the soil and with an inoculum size of 10E8 cells per g of 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.