Title of Invention	"A PROCESS FOR THE DEGRATION OF TECHNICAL HEXACHLOROCYCLOHEXANE"
Abstract	The present invention relates to a process for the degradation of technical hexachlorocyclohexane in soil using a formulated microbial consortium. Complete degradation of technical hexachlorocyclohexane is achieved using the developed process.

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The present invention relates to a process for the degradation of technical hexachlorocyclohexane. This invention particularly relates to the development of bacterial inoculants for the elimination of tech-HCH from contaminated soil through biodegradation. Hexachlorocyclohexane (HCH, has been one of the most extensively used broad spectrum organochlorine insecticides, which amounts to nearly 47% of total pesticides used in India (David, D.V. 1992, Pest Management and Pesticides: Indian Scenario, Namrata Publications, Madras, India). Commercial formulations of HCH contain a mixture of α-,ß-,γ- and δ- isomers at 65-70, 5-20, 10-15 and 3-4 % respectively, among which the y- isomer is the only active insecticide (Bachmann.A, Walet.P, de Bruin. W, Huntgens. J.L, Roelofsen. W and Zender. A,J,B, Appl.Environ. Microbiol. 54:143-149). All the isomers persist for several years and cause environmental concern. The residues of technical grade HCH have been detected in soil, water and air (Deo.P.G, Karanth. N.G and Karanth. N.G.K, Biodegradation of hexachlorocyclohexane isomers in soil and food environments. Crit. Rev. Microbiol, 20:57-58). The residues of HCH have been found in adipose tissue and also in breast milk (tanabe.S, Gondaira. F, Subramanian. A, Ramesh. A. Mohan. D, Kumaran, P, Venugopalan. V.K and Tatsukawa. R., 1990, J.Agric. Food Chem. 38:899-903). Almost all food stuffs including processed foods in India have been shown to contain high levels of HCH (Kannan. K, Tanabe. R., Ramesh. A,, Subramanian. A and Tatsukawa, R. 1992, J. Agric. Food Chem. 40: 518-524). These residues enter the human body through food chain from contaminated soil, animal feed, poultry feed, etc. Reference may be made to Deo.P.G., Karanth. N.G. and Karanth. N.G.K 1994, Grit. Rev. Microbiol. 20: 57-78; Haider. K 1979, Z. Naturforsch. 34: 1066-1069; Jagnow.G., Haider. K. and Ellwardle. P.C. 1977. Arch. Microbiol. 115:285-292; Kunhi. A.A.M., 1995, MICON lntemational.;MacRae. I.C., 1989, Rev. Environ. Contam. Toxicol. 109:1-18;Ohisa.N and Yamaguchi. M 1979. Soil Biol. Biochem. 11: 645-649; Raghu.K. and MacRae. I.C 1966, Science, 154:263; Sethunathan. N. , Baustida. E. and yoshida. J. 1969, Can. J. Microbiol., 15: 1349- 1354, wherein degradation of HCH under anaerobic conditions in air- logged soils and lake sediments have been reported. In all these cases, accumulation of intermediary metabolites have been reported, implying incomplete degradation of HCH. Reference may be made to Bachmann. A., de Bruin. D., Jumelet J.C., Rijnaarts. H.H.N and Zender. A.J.B., 1988a, Appl. Environ. Microbiol., 54: 548-554;Bachmann. A., walet. P., wijnen. P., de Bruin. W., Huntjers. J. L. M., Roelofsen. W. and Zender. A.J.B., 1988b. Appl. Environ.Microbiol. 54: 143- 149; Bhuyan. S., Sahu. S. K., Adhya. T. K. and Sethunathan. N. 1992. Biol.Pert. Soils. 12: 279- 284; Bhuyan. S., Sreedharan. B., Adhya. T. K. and Sethunathan. N. 1993. Pest. Sci. 38: 450- 463; Huntenjens. J. L. M., Brouwer. W., Grobben. K., Jasma. O., Scheffer. F. and Zender. A. J. W. 1988; Holf et a/. (Ed.), Kluwer Academic Publications; Imai. R., Nagata., Senoo. K., Wada. H., Fukuda. M., Takagi. M. and Yano. K. 1989. Agric. Biol. Chem. 53: 2015- 2017; Sahu. S. K., Patnaik. K. K., Bhuyan. S., Bindu. S., Kurihara. N., Adhya. T. K. and Sethunathan. N. 1995. J. Agric. Food Chem. 43: 833- 837; Senoo. K. and Wada. H. 1989. 35: 79- 87; Thomas. J. C., Berger. F., Jacquir. M., Bernillon. D., Baud- Grasset. F., Truffaut. N., Normand. P., Vogei. T. M. and Simonet. P. 1996. J. Bacteriol. 178: 6049- 6055, wherein, few bacterial strains such as Sphingomonas paucimobillis, Pseudomonas versicularis and Pseudomonas spp. could degrade different isomers of HCH under aerobic conditions. Reference may be made to Kunhi A. A. M. 1995. MICON Internationai-1994, wherein, mixed microbial culture/ microbial consortium isolated from HCH-contaminated soil was able to degrade HCH isomers in shake flasks. Reference may be made to an Indian patent 2448/DEL/95, wherein, a microbial consortium isolated from soil by enrichment technique could degrade HCH upto 100ppm after acclimation. Reference may be made to an Indian patent 2147/ DEL/ 98, wherein a microbial consortium was isolated from HCH- contaminated soil by long- term enrichment technique. This consortium when acclimated with y- HCH was able to degrade 100ppm of y- HCH in shake flasks. Reference may be made to an Indian patent (Submitted), wherein a microbial consortium, obtained by pooling a-, p-,y- and 5- HCH - degrading consortia was acclimated with technical grade HCH. The resultant consortium was able to degrade 25 ppm of tech.- HCH in soil. Drawback in all the above works is that the HCH- degraded by axenic cultures is very low and degradation time is long. Only single isomers have been used in many cases. In all these cases, the microorganisms are added as such and no inoculant formulations are available. Hence, it is imperative to develop an inoculant formulation capable of degrading tech- HCH in soil. The main objective of the present invention is to provide a process for the preparation of inoculants formulation useful for the degradation of tech-hexachlorocyclohexane in soil Another object of the present invention is to prepare inoculant formulations for the treatment of soil for the elimination of tech- HCH residues. Yet another object of the invention is to provide a formulation by lyophilization of microbial mass. Still another object of the present invention is to provide a formulation which degrades tech-HCH completely. Accordingly the present invention provides a process for the preparation of inoculants formulation useful for the degradation of tech-hexachlorocyclohexane in soil which comprises, (i) growing the individual isolates of the consortium may be selected from Pseudomonas fluorescens CFR- 1023, Ps. Putida CFR- 1024, Ps. fluorescens CFR- 1025, Burkholderia pseudomalleii CFR- 1026, Ps. putida CFR-1027, Flavobacterium sp. CFR- 1028, Vibrio alginolyticus CFR- 1029, Ps. aeruginosa CFR- 1030, Ps. stutzeri CFR- 1031, Ps. fluorescens CFR- 1032, in wheat bran hydrolysate/ glucose- yeast extract medium containing tech- HCH, (ii) separating the microbial mass by conventional methods and mixing them at equal ODeoo, (ill) aseptically lyophilizing the mixture to get the mixture in powder form, (iv) inducing the mixture with tech- HCH to get pesticide degrading properties and (v) inoculating them to powdered sterile Sphagnum or sterile wheat bran and palletizing them followed by drying them, to get the said pesticide degrading preparation. In an embodiment of the present invention, the growing of individual isolate may be effected at a temperature ranging from 22-28°C for a period of 44-48 h. In another embodiment of the present invention, the medium used may be selected from 0.06-0.07% KH2PO4, 0.5-0.6% Na2HPO4> 0.02-0.03% NH4NO3, 0.02-0.03% MgSO4 7H2O, 0.01-0.02% Ca(NO3)2, 0.45-055% cone. H2SO4 supplemented with 1 ml of mineral solution containing 0.1% FeSO4, 0.1% MnSO4 H20, 0.025% CuCI2.2H20, 0.025% Na2Mo04 2H20 and 0.01% H3BO3, with a final pH in the range of 7-8. Yet in another embodiment of the present invention, the inoculant formulation may be prepared from Sphagnum mass or wheat bran at a temperature in the range of 26~28°C for a period ranging from 21-30 days. Still in another embodiment of the present invention, inoculant preparation may be stored at a temperature ranging from 7-9° C. In another embodiment of the present invention, the tech-HCH may be employed upto 25 ng/g soil. Yet in another embodiment of the present invention, the tech-HCH may be selected from a, p, y, and 6- isomers. Still in another embodiment of the present invention, the pellets may be air-dried. In another embodiment of the present invention, the mixture of isolates may be induced with tech-HCH for a period ranging from 22-26 h. Yet in another embodiment of the present invention, the formulations used for complete degradation of tech-HCH may be employed upto 500 ^ig protein/g soil. Still in another embodiment of the present invention, the complete degradation of tech-HCH may be effected in a period ranging from 118-122 h using the lyophilized formulation or sphagnum pellets. In another embodiment of the present invention, the complete degradation of tech-HCH may be effected in a period ranging from 142-146 h using wheat bran pellets. Yet in another embodiment of the present invention, the inoculant preparation may be stable. In the drawings accompanying this specification, Figure 1. represents the TLC pattern of technical grade HCH. 1= Origin, 2= 5-HCH, 3= B- HCH, 4= y-HCH and 5= a- HCH. Figure 2 represents the gas chromatogram of technical grade HCH. A = solvent, b= a- HCH, c= y- HCH, d= B- HCH, e= 6-HCH. Figure 3 represents the degradation of technical grade HCH by the inoculant formulation. All the symbols are same as in Figure 2. Figure 4 represents the inoculant formulation prepared using Sphagnum mass and wheat bran. Figure 5 represents the inoculant formulations prepared using Sphagnum mass and wheat bran that are pelletted and air- dried. The mixed microbial consortium (CFR 2003) was prepared by incorporating 10 bacterial strains (Table 1) and was mutated to degrade increasing concentrations of tech- HCH. This defined mixed culture could degrade 25 ng tech- HCH by 120 h in soil. The inoculant formulations were obtained by inoculating the mixed culture to sterile Sphagnum mass or wheat bran separately at 10 cells/ml based on plate count and incubating them at ambient temperature (25° to 28°C) for 30 days. The pellets of inoculant formulations were obtained by inoculating the mixed microbial culture to sterile Sphagnum mass or wheat bran (separately) and making them in to pellets of approximately 0.5g (dry weight) and air-drying them. These inoculant formulations could be effectively deployed for the treatment of tech-HCH in soil. A novel process was deployed for the elimination of tech-HCH from soil and contaminated material using inoculant formulations of mixed microbial culture. The microbial consortium was deployed by enrichment of mixture of HCH-contaminated soil (collected from sugarcane field) and sewage (collected from Mysore Municipal Corporation sewage plant in shake flasks containing individual isomers of HCH, the four different consortia were pooled and then inoculated to tech-HCH(5 ng/ml). Then the consortium that got established was resolved into individual members and identified as 7 Pseudomonas spp. one each of Burkholderia sp., Flavobacterium sp., and Vibrio sp. Bacterial isolates have been deposited at Central Food Technological Research Institute Culture Collection Centre and are having characteristics as herein described, The culture characteristics of the different isolated when grown on nutrient agar plates are as follows: 1. Pseudomonas fluorescens CFR 1023 Gram -ve rods, colony buff coloured, round (4 -7 mm dia.), umbonate, transluscent, aerobic with irregular fluorescent margin , oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, produces H2S, indole reaction -ve, does not reduce nitrate, produced fluorescent pigment and degrades HCH. 2. Pseudomonas putida CFR 1024 Gram -ve short motile rods, light lemon yellow coloured, round (1to2 mm dia.) with smooth fluorescent margin , umbonate, transluscent, aerobic, oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, produces H2S, indole reaction -ve, does not produce fluorescent pigment, and degrades HCH. 3. Ps. fluorescens CFR-1025, Gram -ve, motile rods, colonies light lemon yellow coloured, round (1.5- 3.0mm dia.), convex, transluscent, aerobic, fluorescent, oxdase +ve, catalase +ve, does not hydrolyse starch and gelatin, ferments dextrose, does not ferment mannitol, produces H2S, indole reaction -ve, does not produce fluorescent pigment and deggrades HCH. 4. Burkholderia pseudomalleii CFR- 1026, Gram -ve, motile large rods, colonies buff coloured, round (1.2mm dia.), umbonate, transluscent, smooth, shining, oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, ferments dextrose but not mannitol, indole reaction -ve, does not produce fluorescent pigment, degrades HCH. 5. Ps putida CFR- 1027 Gram -ve motile coccobacilli, colonies buff coloured, smooth (1.0-3.0 mm dia.), convex, transluscent, aerobic, fluorescent, oxidase +ve, does not hydrolyse starch and gelatin, does not ferment glucose and mannitol, does not produce H2S, indole reaction -ve, does not produce fluorescent pigments and degrades HCH. 6. Flavobacterium CFR 1028, Gram -ve, non-motile rods, yellow coloured, round (1.0 -1.2 mm dia.), raised, opaque, aerobic, smooth, oxidase +ve, catalase +ve, convex, does not produce H2S, indole reaction -ve, does not produce fluorescent pigments, degrades HCH. 7. Vibrio alginolyticus CFR- 1029, Gram -ve, motile small rods, whitish yellow coloured, smooth, convex, transluscent, aerobic (1.0- 3.0mm dia.), oxdase +ve, catalase +ve, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, degrades HCH. 8. Ps. aeruginosa CFR- 1030, Gram -ve, coccobacilli, spreading colonies, aerobic, produces green pigment into nutrient agar medium, oxidase +ve, catalase +ve, motile, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, degrades HCH. 9. Ps. stutzeri CFR- 1031, Gram -ve, motile rods, whitish, round colonies (1.0- 1.25mm dia.), convex, transluscent, aerobic, shining, fluorescent, oxidase +ve, catalase +ve, does not hydrolyse starch, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, degrades HCH. 10. Ps. fluorescens CFR 1032, Gram -ve, motile coccobacilli, buff coloured, round (1.0-1.5 mm doa.), umbonate, transluscent, aerobic, oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, ferments dextrose and mannitol, indole reaction -ve, produces H2S, produces fluorescent pigment, degrades HCH. The mutants of the above cultures have been developed by subjecting them to increasing concentrations of tech-HCH used as a sole source of carbon and energy, by conventional technique. Technical grade HCH (tech- HCH) was obtained from Hindustan Insecticides, Mumbai, India. Other chemicals used in the media and reagents were of analytical grade and were obtained from standard Indian companies The basal mineral medium used for the enrichment and for growth of the individual cultures as well as the mixed cultures (consortium, CFR- 2003) contained (per litre of distilled water), 0.675g KH2PO4, 5,455 g Na2HPO4, 0.25 g NH4N03, 0.2 g MgS04. 7H2O, 0.1 g Ca(N03)2 and 1 ml of mineral solution (containing mg/ml) FeS04, 1.0, MnSO4. H2O 1.0, CuCI2.2H20, 0.25,Na2Mo042H2O, 0.25,, H3B03, 0.10 and cone. H2S04 5 ml. The pH of the medium was 7.5. Required quantity of HCH was added to the medium as described later. The inoculant formulation was prepared in Sphagnum mass and wheat bran. Around 200 gm quantity of powdered Sphagnum mass was dispensed into plastic bags after neutralizing the Sphagnum to pH 7.0 with CaC03. The neutralized Sphagnum was sterilized by autoclaving for 15 mins at 121°C, 15 Ibs pressure for three successive days. The neutralized Sphagnum was inoculated with 80ml of cell suspension containing 109" cells ml"1 based on plate count. Bags were incubated aseptically for 21- 30 days at ambient temperature (26- 28 °C). Packaged inocula were stored at 8 °C until further use. Same protocol was used to prepare inoculant formulations from wheat bran. To prepare inoculant pellets, thrice autoclaved Sphagnum mass/ wheat bran was mixed with bacterial suspension, palletized and air- dried. Air-dried pellets were packaged and stored at 8 °C until further use. Nutrient agar plates were used for resolving different bacterial strains from the consortium as well as to study their taxonomy. Nutrient agar contained 3g beef extract, 5g peptone, 5g NaCI and 20g agar-agar per liter of distilled water (pH 7.0). Individual isolates of the consortium were grown in 50 ml of wheat bran hydrolysate or glucose-yeast extract medium in 250 ml Erlenmeyer flasks containing 0.5 % reducing sugars and incubating at 30 °C on a rotary shaker (150 rpm). After 72 h of growth, the cells were harvested, pooled at equal ODeoo, induced with tech- HCH for 24 h and used as inoculum after washing the harvested cells with 0.1% tween 80 and minimal medium. This induced consortium was used in the preparation of inoculant formulations. For the degradation of tech-HCH in soil, 80 g sterile soil was taken in plastic cup and spiked with known concentration of tech-HCH in acetone. Then the above developed consortium / inoculant formulation was inoculated at known colony forming units (cfus). The growth of the culture in shake flasks was determined by estimating protein of alkali -digested sample. In the inoculant formulation and in the soil sample growth was estimated by determining the colony forming units (cfus) on nutrient agar. Quantitative determination of the substrate residues (tech-HCH) was done by thin layer chromatography or gas chromatography, with 63Ni electron capture detector. The soil sample was extracted thrice with equal volumes of acetone : ethylacetate (1 : 1), acetone : ethyl acetate (5: 95), and ethyl acetate. The solvent layers were pooled, evaporated and the residue was redissolved in a convenient volume of acetone. Known volume of the acetone containing the residue was loaded on to a silica gel -G coated plate (0.3 mm) and developed in cyclohexane. The plates were air dried and HCH isomers were detected by spraying the plate with 2% solution of o-tolidine in acetone and exposing to sunlight. The spots were delineated by marking with a needle and the area was measured. The concentration was computed from a standard plot of log concentrations versus square root of the area, prepared for reference standard of tech-HCH. For gas chromatography, 63Ni electron capture detector was used. The conditions used were as follows: column 2mm ID and 200 cm length, packed with OV-17 and 1.95 QF-1 on 80 - 100 mesh chromosorb W, carrier gas nitrogen, flow at 30 ml / min, column temperature 210° C, injector temperature 230° C, detector 320° C.The solvent fraction was passed through florisil column to remove impurities. The enrichment of HCH-contaminated soil and sewage water with different isomers of HCH was done in 250 ml conical flasks in a shaker at 150 rpm. The microbial consortium that got established in shake flasks with individual isomers of HCH was mixed together and acclimated with 5 µg/ ml tech- HCH. Acclimation to higher concentrations was carried out after ensuring that the previously added concentration has been degraded completely. At 25 ng/ ml tech- HCH concentration in shake flasks, all the isomers of tech- HCH were found to be degraded completely. The individual bacterial strains of the consortium were identified by Microbact Gram Negative Bacterial identification System. Table 1 gives the composition of the tech- HCH acclimated bacterial consortium. Table 1: The list of the microorganisms present in the tech-HCH degrading consortium (Table Removed) The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 The mineral medium contained 0.675g KH2PO4, 5.455g Na2HP04, 0.250 g NH4N03 in one litre distilled water. 80g of soil was taken in plastic cups and 25 |ug tech- HCH/ g soil was added and mixed well. The inoculant formulation prepared in Sphagnum mass was added at levels containing 109 cells/ g soil. The final moisture level was maintained at 15%. Only the consortium was used in one set of experiments to compare the degradation with inoculant formulation. The cups were incubated at 25- 30 °C in a wooden chamber. Sampling was done at regular intervals and analysed for growth (CPU) and residual substrate. The results are presented in Table 2. Complete disappearance of all the four isomers of tech-HCH was observed by 120 h of incubation. The observations were same in both the types of inoculant formulations prepared using Sphagnum . Table 2: Degradation of tech- HCH using microbial inoculant formulations with Sphagnum mass. (Table Removed) Example 2 The mineral medium contained 0.675g KH2PO4, 5.455g Na2HP04, 0.250g NH4N03 in one litre distilled water. 80g of soil was taken in plastic cups and 25 ^g tech- HCH/ g soil was added and mixed well. The inoculant formulation prepared in wheat bran was mixed to the spiked soil at the levels containing 109 cells/ g soil. The final moisture level was maintained at 15%. The cups were incubated at 25-30 0(19HCa wooden chamber. Sampling was done at regular intervals and analysed for growth (CPU) and residual substrate. The results are presented in Table 3. Complete disappearance of all the four isomers of tech- HCH was observed by 144 h of ubation. Table 3.: Degradation of tech- HCH using inoculant formulations with wheat bran. (Table Removed) Example 3 The stability of the inoculant formulations was studied by determining the number of organisms surviving after known period of storage. It was observed that the inoculant formulations prepared in Sphagnum mass was stable compared to that of wheat bran. The individual members did not survive after one month of storage in wheat bran pellet. The results are presented in Table 4. Table 4.: The stability of inoculant formulations during different periods of storage (Table Removed) Example 4 The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HP04, 0.250 g NH4N03. 80 g soil was taken in plastic cups and 25 ^ig tech-HCH /g soil was added and mixed well. The consortium was added at 2 |ig protein /g soil through 2000 ng protein /g soil. The final moisture level in the soil was maintained at 15%. The cups were incubated at 25 - 30 ° C in a wooden chamber. Sampling was done at regular intervals and analysed for growth (cfus) and residual substrate. The results are presented in table 5. Complete disappearance of the substrate was observed when 500 fj,g protein /g soil was used. All four isomers of HCH disappeared completely by 120 h of incubation. Table 5 :Effect of inoculum level on the degradation of tech-HCH in soil. (Table Removed) Example 5 The mineral medium contained 0.675 g KH2P04, 5.455 g Na2HPO4, 0.250 g NH4NO3. 80 g soil was taken in plastic cups and 25 |ig tech-HCH /g soil was added and mixed well. The consortium was added at 500 jxg protein /g soil. The final moisture level in the soil was maintained at 15%. The cups were incubated at 25 - 30 ° C in a wooden chamber. Sampling was done at regular intervals and analysed for growth (cfus) and residual substrate. The results are presented in table 6. It was observed that degradation was observed even at a low tech-HCH concentration of 2 jag /g soil. The time taken for degradation of tech-HCH increased with increase in tech-HCH concentration. 25^g tech-HCH /g soil was completely degraded by 120 h. But higher concentration were not completely degraded. Table 6: Degradation of different concentrations of tech-HCH by the microbial consortium (Table Removed) The main advantages of the present invention are: 1. The inoculant preparation is stable for longer time. 2. The inoculant is prepared using cheap agricultural wastes. 3. The inoculant preparation can be applied to soil for elimination of tech- HCH. 4. The inoculant preparation can eliminate tech- HCH completely from contaminated soil samples. We claim: 1. A process for the degradation of technical hexachlorocyclohexane, wherein the steps comprising: [a] growing the individual isolates of the consortium consisting of Pseudomonas fluorescens CFR-1023, Pseudomonas putida CFR-1024, Pseudomonas fluorescens CFR-1025, Burkholderia pseudomalleii CFR-1026, Pseudomonas putida CFR-1027, Flavobacterium species CFR-1028, Vibrio alginolyticus CFR-1029, Pseudomonas aeruginosa CFR-1030, Pseudomonas stutzeri CFR-1031 and Pseudomonas fluorescens CFR-1032 in wheat bran hydrolysate glucose yeast extract medium such as herein described containing 5 to 25 microgram per ml of technical hexachlorocyclohexane at a temperature ranging from 22 to 28 degree C for a period of 4 to 48 hours; [b] separating the microbial biomass from the growth medium as obtained in step [a] by known methods; [c] aseptically lyophilizing the biomass as obtained in step [b] to get the microbial mixture in a powder form; [d] inducing the powder as obtained in step [c] with 5 to 25 microgram per ml of technical hexachlorocyclohexane for a period of 22 to 26 hours; [e] inoculating 1 to 10 wt% of the induced powder as obtained in step [d] to powdered sterile Sphagnum or sterile wheat bran and pelletizing them at a temperature of 26 to 28 degree C for a period of 21 to 30 days followed by drying; [f] adding 2 to 1000 microgram protein per gram of the soil of the pellets as obtained in step [e] to soil contaminated with technical hexachlorocyclohexane and incubating for a period of 118 to 146 hours to obtain completely degraded technical hexachlorocyclohexane. 2. A process as claimed in claim 1, wherein the growth medium contains 0.06 to 0.07 % KH2PO4, 0.5 to 0.6% Na2HPO4, 0.02 to 0.03 % NH4NO3, 0.02 to 0.03 % MgSO4 7H2O, 0.01 to 0.02 % Ca[NO3]2, 0.45 to 0.55 % conc H2SO4 supplemented with 1 ml of mineral solution containing 0.1% FeS04, 0.1% MnSO4 H2O, 0.025% CuCI2.2H2O, 0.025% Na2MoO4 2H2O and 0.01% H3BO3 with a final pH in the range of 7.0 to 8.0. 3. A process as claimed in claim 1, wherein the inoculant preparation is stored at a temperature of 7 to 9 degree C. 4. A process as claimed in claim 1, wherein the concentration of technical hexachlorocyclohexane in soil is 25 micro gram per ml. 5. A process as claimed in claim 1, wherein the technical hexachlorocyclohexane comprises alpha, beta, gamma and delta isomers. 6. A process for the degradation of technical hexachlorocyclohexane substantially as herein described with reference to the foregoing examples.

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278-DEL-2002-Abstract-(13-08-2008).pdf

278-del-2002-abstract.pdf

278-DEL-2002-Claims-(13-08-2008).pdf

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278-del-2002-description (complete)-13-08-2008.pdf

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