Prof. Christof Holliger (project coordinator)
Indo-Swiss Collaboration in Biotechnology (ISCB), SDC Berne
September 2008 – December 2011
Dr. Hans-Peter Kohler, EAWAG; Dr. Thomas Poiger, Agroscope Wädenswil, Switzerland; Prof. R. Lal, University of Delhi, India; Dr. V. Raina, KIIT School of Biotechnology, Bhubaneswar, India.
Although the concept developed during the previous projects has been proven to be feasible even at pilot-scale with agricultural soil contaminated due to pesticide applications, there are different issues that have to be investigated further concerning HCH-contamination. Mass cultivation on cheap carbon and energy sources of Sphingobium indicum B90A has been very difficult and the fate or the toxicity of the metabolites produced from β-HCH and δ-HCH are still unsolved aspects of degradation of HCH isomers.
In the framework of another project carried out at the University of Delhi, several Sphingobium-like strains have been isolated that degrade all HCH-isomers very efficiently. An HCH-degrading consortium will be constituted from the different available pure cultures and tested for mass cultivation and HCH-degradation in soil microcosms.
We propose in addition to establish a survey of the presence of HCH-degradation metabolites at different HCH-contaminated site in soil and the aquifer underneath. In addition, the fate of these metabolites in HCH-degrading cultures will be studied in order to be able to fully understand the fate of all HCH-isomers. Furthermore, to at least guarantee transformation into harmless products, the toxicity of the degradation products will be elucidated with commercially available toxicity tests.
In order to establish a survey of the presence of such metabolites at different HCH-contaminated site in soil and the surrounding aquifers, soil and ground water samples were collected from agricultural fields (rice and vegetable) where HCH has been applied in recent years and from waste or dump sites of different pesticide manufacturing industries in and around Bhubaneswar and New Delhi. The hydroxylated metabolite B2 formed from β–HCH was detected in all soil samples but at much lower concentrations than remaining total HCH. The pentachlorocylohexenes (PCCHs) of α-, γ-, δ-HCH were not detected in any sample. The water samples also showed presence of hydroxylated β- and δ-HCH metabolites. The adapted method also revealed the presence of ε-HCH in all soil samples but was found to be absent in the analysed water samples. These results revealed that the metabolites were present; hence not all HCH has been mineralized at these contaminated sites.
No degradation of metabolites B2 and D2 was observed when they were incubated with different bacterial strains that are known to degrade HCH isomers and produce these metabolites. Any attempt to further convert these hydroxylated compounds by purified LinA and LinB enzymes failed. Thus, we can conclude that the hydroxylation of HCH and PCCH isomers produces very recalcitrant metabolites. Toxicity tests with synthetically produced metabolites in milligram amounts suggested that they seem not to be very toxic and therefore not of major concern. They were screened for their toxicity by the Microtox test and Yeast Estrogen Screen (YES) assay. Microtox, an assay indicating general toxicity against the bacterium Vibrio fischeri did not show general toxicity of either of the compounds. There was some estrogenic activity observed for metabolites B2 and D2 but at much higher concentrations than any concentration that has been measured in an environmental sample so far.
An experiment to biostimulate indigenous HCH degraders by treatment with different combinations of nutrient sources such as molasses, ammonium phosphate, molasses and ammonium phosphate, water and control was conducted. Residue analysis showed that a combination of carbon, nitrogen and phosphorus sources can lead to faster degradation of HCH residues within 12 months in comparison to the control pit. Hence based on the above results molasses and ammonium phosphate was used in field trials.
In order to improve the inoculum for bioaugmentation, application of a microbial consortium comprising of a group of new sphingomonad isolates, namely UM1, F2, IP26 and HDIPO4, along with Sphingobium indicum strain B90A was tested. The sphingomonads were isolated from high dose HCH-contaminated dumpsite soils and their survival in the field was expected to be better as a consortium. Large scale cultivation of isolates UMI, F2, IP26, HDIPO4 and of the consortium was standardized using mineral salt medium supplemented with 0.3% glucose. The survival of these strains immobilized on corncob powder as carrier material individually and as consortium was found to be feasible for a period of up to 60 days. Applying these corncob immobilized strains in pot experiments showed good degradation of HCH. Finally, field trials using a combined approach of biostimulation and bioaugmentation were carried out at the HCH contaminated dumpsite, Ummari village, Barabanki, Lucknow for a period of two months. Maximum degradation of HCH was observed with a combination of biostimulation and bioaugmentation (58%), followed by biostimulation (52%) and bioaugmentation (39%) alone.
In conclusion, we have shown in this final phase of our bioremediation project that the combined approach of biostimulation and bioaugmentation can be used for the decontamination of HCH-contaminated soil. Although the hydroxylated metabolites that are thus produced from β-HCH and δ-HCH are probably not further degraded, they can be considered as low risk for environmental and human health due to their low toxicity.