Prof. Christof Holliger (Project coordinator)
Indo-Swiss Collaboration in Biotechnology (ISCB), SDC Berne
January 2005 – March 2008
Dr. Hans-Peter Kohler, EAWAG; Dr. Hansruedi Buser, FAW Wädenswil, Switzerland; Dr. R. Lal, University of Delhi, India; Dr. R. Jain, IMTECH, Chandigarh, India; Dr. B. Lal, TERI, New Delhi, India
The objectives of this follow-up project was to proof that the concepts developed during the previous project for bioremediation by bioaugmentation of HCH- and PNP-contaminated soil was indeed applicable. The intention was to test its feasibility in pot and small field-scale experiments. In addition, unsolved aspects of degradation of HCH isomers and PNP had to be investigated in order to be able to guarantee mineralization of all the compounds treated or at least transformation into harmless products
For the mass cultivation, a standard strategy was chosen, hence to start from a colony on agar plates, followed by cultivation in a culture tube, a culture flask, a 5-L bioreactor to finally carry out a large batch culture in a 100-L fermenter. Quality control of the biomass produced was done based on specific growth characteristic of the respective strain on agar plates that indicated whether a contaminant or the target strain did grow in the experiment. Whereas mass cultivation with Arthrobacter protophormiae strain RKJ100 was successful, the same was very problematic with Sphingobium indicum strain B90A. A possible explanation could be the slow growth of the latter strain. Despite medium composition improvements problems with mass cultivation in large fermenters remained. The only solution to this problem will be to accept contamination at this scale of cultivation since sufficient HCH-degrading bacterial cells are present in the biomass produced.
Two kinds of pilot-scale bioaugmentation field experiments were carried out. One consisted of plot experiments near the laboratory where a pit of one square meter and a depth of 20 cm was filled with contaminated soil from a contaminated agricultural field. The second experimental set-up consisted of a test in an agricultural field at a scale of 20-25 square meters. Controls with only addition of water and nutrients and, if appropriate, carrier material without immobilized pesticide-degrading bacteria were carried out.
To identify suitable field sites for pilot tests of the developed technology for HCH decontamination, different soils have been analyzed. These analyses showed that in agricultural fields the soil can be contaminated but at low levels. In soils taken from areas around production sites the pollution concentrations were on the other hand extremely high.
Pits for tests of the bioaugmentation strategy with Sphingobium indicum B90A were filled with HCH-contaminated soil taken from agricultural field situated near a lindane producing factory. HCH degradation of up to 95% was observed. S. indicum B90A was tracked by using modified conventional and molecular methods. The bacteria could survive up to 8 days, after that the number of cells decreased by 75-85%. To maintain a constant cell number, fresh inoculum was added every eighth day. Molecular fingerprint analysis of the microbial community indicated that the composition changed in all pits but independent whether corncob powder with or without S. indicum B90A was added to the soil or not. Hence, this indicated that the bioaugmentation bioremediation approach did not influence the diversity of the soil microbial community.
Pilot scale field experiments of S. indicum B90A bioaugmentation were carried out in agricultural fields in northern part of India. For this two HCH contaminated agricultural site, one at Chittora village, Muradnagar and second at Chinhat, Lucknow, were selected. The soils of the two sites were both polluted with HCH but with a quite different patterns with regard to the HCH isomers. Whereas the Chittora soil pollution was dominated by β-HCH followed by α-HCH, the Chinhat soil was mainly containing α- and γ-HCH. The bioremediation was successful for the latter soil, but no HCH degradation was observed for the soil dominated by β-HCH. Molecular fingerprint analysis of the soil microbial community did again not show any influence of the bioaugmentation with Sphingobium indicum. The changes in the microbial community composition observed were probably due to irrigation and tilling of soil. Survival tests showed that it was poor and that probably a regular application of the bacteria is needed for obtaining satisfying remediation results.
With Arthrobacter protophormiae strain RKJ100, two pilot-scale field trials for in-situ bioremediation of a naturally p-nitrophenol (PNP) contaminated soil were carried out at an agriculture field (Channjan Tea Estate, Jorhat, Assam). The field was found to be uniformly contaminated with PNP. Soil samples collected from other agriculture fields were also tested for PNP concentration, however, most of the other fields showed non-uniform distribution of the pollutant. Bioremediation studies were performed in plots of 2 m by 3 m and 8 m by 3 m as control and test plots, respectively. The degradation of PNP in plots bioaugmented with strain RKJ100 was enhanced many-fold as compared to the non-treated control plots. Degradation was more rapid in the upper 10 cm compared to the layer between 10 and 20 cm. The kinetics of PNP degradation at different soil depth was noticeably different. The apparent difference could be explained as a function of cell survival of bioaugmented strain that was better at 10 cm soil depth as compared to 20 cm. Molecular fingerprint analysis of the soil microbial community clearly indicated that the introduction of RKJ100 did not had an adverse effect on the microbial community structure. Minor changes in the community structure were detected at the beginning of the bioremediation process, but it was not a function of RKJ100 bioaugmentation. The microbial community at 10 cm soil depth and 20 cm soil depth exhibited strong tendency to stabilize towards the later time points of the bioremediation trials. The results of the second field trial, also performed at Channjan Tea Estate, were in strong agreement with those obtained with the first field trial as PNP degradation was found to be most efficient at top-soil followed by degradation at 10 cm, 20 cm and 30 cm soil depths.
To evaluate the applicability of bioaugmentation of strain RKJ100 as a bioremediation technology to different soils, microcosm studies were performed with soils collected from seven states of the country, namely Assam, Rajasthan, Gujarat, Maharastra, Karnataka, Andhra Pradesh and Tamil Nadu. The kinetics of PNP degradation in different soils were different with soil from Andhra and Maharastra having up to 90 % PNP degradation within 30 days and soil from Rajasthan having only 40% PNP degradation even after 45 days. The rate of survival of bioaugmented strain RKJ100 varied as well as the amount of quantifiable RKJ100 specific DNA indicating a possible cause of difference in kinetics of PNP degradation. Statistical analysis of the data confirmed that the difference in the kinetics of PNP degradation was highly correlated to the rate of RKJ100 cell survival, which itself was dependent on the indigenous microbial community structure of different soils.
A major issue of HCH-degradation was the observed incomplete dechlorination of b-HCH and d-HCH in pure culture tests. Additional degradation tests revealed the production of hydroxylated metabolites as initial products. The responsible enzyme for these transformations was LinB, the halidohydrolase that normally acts on the products of LinA. Two metabolites from b-HCH were identified as a pentachlorocyclohexanol (beta-1) and a tetrachlorocyclohexanediol (beta-2), the hydroxy metabolites observed during d-HCH incubations were identified as a pentachlorocyclohexanol (delta-1), a tetrachlorocyclohexanediol (delta-2), an unsaturated tetrachloro-2-cyclohexen-1-ol (delta-3) and a trichloro-2-cyclohexene-1, 4-diol (delta-4). This revealed that equatorial chlorines in b- and d-HCH were quite reactive toward hydroxylation with the appropriate enzyme of strain B90A. This is in contrast to the generally accepted dehydrochlorination reactions of HCHs where axial chlorines are far more reactive. b- and g-PCCH produced from a- and g-HCH by LinA were metabolized by LinB into several similar but structurally different cyclohexenols and cyclohexenediols. Interestingly, LinA was able to transform b- and d-PCCH to the unsaturated cyclohexenols, respectively, suggesting that it could catalyze hydroxylation reactions in addition to the widely known dehydrochlorinations. All these transformation studies show that the normally proposed degradation pathway for a- and g-HCH must perhaps be revised and that it has to be investigated whether the hydroxylated intermediates of the b- and d-HCH degradation are persistent in the environment and, if degradation is incomplete, whether they are toxic for living organisms.
In conclusion, it has been shown that the concept developed during the first project period has proven its feasibility in small and larger scale pilot studies. Field trials for remediation of PNP contaminated soils with bioaugmentation of degradative strain A. protophormiae strain RKJ100 have been successful and thus we have been able to develop a cheap bioremediation process for in situ bioremediation of soils contaminated with PNP. Unsolved questions to answer are mainly linked with HCH biodegradation since the mass cultivation was rather difficult and the fate and risk of metabolites formed from b-HCH and d-HCH are not yet known.