Title of Invention	"A PROCESS FOR PREPARATION OF LINDANE (BHC OR BENZENE HEXACHLORIDE) FREE INDUSTRIAL EFFLUENTS/WATER, USING A NOVEL FORMULATION OF MICROBIAL CONSORTIUM"
Abstract	A process for preparation of lindane (BHC or benzene hexachloride) free industrial effluents/water, characterized in using a novel formulation of microbial consortium in powder form comprising equal proportions for organisms consisting of novel mutated strains of pseudomonas and rusarium having characteristics as herein described, the said process comprises treating lindane industry effluents water bodies and other contaminated material having lindane residue with powder of said formulation of microbial consortium in a treatment plant, under agitation at 150 rpm for a time period ranging 3-24 h to get lindane free effluents or water.

Title of Invention

"A PROCESS FOR PREPARATION OF LINDANE (BHC OR BENZENE HEXACHLORIDE) FREE INDUSTRIAL EFFLUENTS/WATER, USING A NOVEL FORMULATION OF MICROBIAL CONSORTIUM"

Abstract

A process for preparation of lindane (BHC or benzene hexachloride) free industrial effluents/water, characterized in using a novel formulation of microbial consortium in powder form comprising equal proportions for organisms consisting of novel mutated strains of pseudomonas and rusarium having characteristics as herein described, the said process comprises treating lindane industry effluents water bodies and other contaminated material having lindane residue with powder of said formulation of microbial consortium in a treatment plant, under agitation at 150 rpm for a time period ranging 3-24 h to get lindane free effluents or water.

Full Text	This invention relates to a process for preparation of lindane (BHC or benzene hexachloride ) free industrial effluents /water using a novel formulation of microbial consortium. This invention particularly relates to the development of a formulation for elimination of lindane residues from industry effluents and water bodies through biodegradation. Lindane is the insecticidal component of the most extensively used organochlorine pesticide which is commonly known as benzene hexachloride (BHC) or gammaxane. This is the gamma-isomer of hexachlorocyclohexane (gamma-HCH). Due to extensive and indiscriminate use of this powerful insecticide this has emerged as one of the major environmental pollutants in India and other third world countries. This compound is recalcitrant and persists in the environment for several years. Partial degradation of lindane to various intermediary metabolites in soil, particularly in flooded fields, has been reported. Refernce may be made to Deo, P.G., Karanth,N.G and Karanth,N.G.K., 1994, Crit. Rev. Microbiol. 20: 57-78 and Kumar,S., Mukerji,K.G. and Lal.R., 1996, Crit. Rev. Microbiol. 22: 1-26. The drawbacks are that! toxic intermediates are generated which accumulate, thus not resulting in complete detoxification. However, reports on complete mineralization of gamma-hexachlorocyclohexane by microorganisms under aerobic conditions are very few. Reference may be made to Kunhi, A.A.M. 1995. In R.Sankaran and K.S. Manja (eds) Microbes for Better Living. MICON-94 and 34th Ann.Conf.AMI (Nov. 1994), DFRL, Mysore for a review of this. Pseudomorzas pauciflioJbilis SS88 (Senoo,K. and Wada, H. 1989, Soil Sci. Plant Nutr. 35: 79-87), Pseudomonas sp. (Sahu,S.K., Patnaik, K.K., Sharmila, M and Sethunathan, N. 1990, Appl .Environ.Microbiol. 56: 3620-3622) and Sphingomonas paucimobilis (Bhuyan, S. Sreedharan, 3., Adhya, T.K. and Sethunathan, N. 1993, Pestic.Sci. 38: 49-55) have been,shown to degrade lindane. The drawbacks are that all these organisms degrade lindane at very low concentrations (8-11 ug/ml). Hence, it is imperative to develop a process by which high concentrations of lindane can be degraded. Mixed microbial cultures can be acclimated to higher and higher concentrations of lindane in the laboratory and a consortium that can degrade lindane residues from industry effluents and contaminated water bodies can be developed. The main object of the present invention is to provide a process for the preparation of a formulation useful for the degradation of benzenehexachloride (BHC). Another object of the present invention is to prepare a formulation for the treatment of HCH-industry effluents for elimination of lindane and other pesticide residues. In the drawings accompanying this specification Figure-1 represents Degradation of 25 ug/ml lindane by the defined mixed microbial culture CFR2002 -0- growth (total biomass protein) , -0-residual lindane and -X- inorganic chloride released. Figure 2 represents Mineralization of 50 ug/ml of lindane by the defined mixed microbial culture CFR2002. All symbols are as in Fig. 1. Figure 3 represents Degradation of 100 ug/ml of lindane by the defined mixed microbial culture CFR2002. All symbols are as in Fig.l. Figure 4 represents Effect of pH of the medium on degradation of 10 ug/ml of lindane by the defined mixed microbial culture CFR2002. And Figure 5 represents Effect of incubation temperature on the degradation of 10 ug/ml of lindane by the defined mixed microbial culture CFR2002. The cultures were incubated at different temperature for 20 h without shakinq. Accordingly the present invention provides a process for preparation of lindane (BHC or benzene hexachloride ) free industrial effluents /water , characterized in using a novel formulation of microbial consortium in powder form comprising equal proportions for organisms consisting of novel mutated strains of pseudomonas and fusarium having characteristics as herein described , the said process comprises treating lindane industry effluents water bodies and other contaminated material having lindane residue with powder of said formulation of microbial consortium in treatment plant, under agitation at 150 rpm for a time period ranging 3 - 24 h to get lindane free effluents or water. The enzymatic formulation obtained is intracellular in nature . In an embodiment of the present invention benzenehexachloride (BHC) used may be alpha-, beta-, gamma-, delta- and epsilon-isomers or mixture thereof and employed in the range of 10-100 ppm. In another embodiment of the present invention the cultivation is effected at room temperature under shaking conditions for at least 48 h. In yet another enboditnent of the present invention, the cultivation is effected in standard -culture media at a wide pH range of 5.0-8.0. A mixed microbial culture (consortium CFR2002) was prepared by incorporating nine bacterial strains (different species of Pseudojnonas) and a fungal strain (Fusarium sp.) (Table 1) and was mutated to degrade increasing concentrations of gamma-HCH in the medium. This defined mixed culture could completely mineralize 100 ug/ml lindane within 48 h in shake flasks. The mixed culture could degrade lindane at a fairly wide range of pH (4.0-8.5) and temperature (5-60°C). This microbial mixed culture can be effectively deployed for treatment cf lindane industry effluents, water bodies, soil and other materials contaminated with lindane residues. A novel process was developed for elimination of lindane residues from industrial effluents and other contaminated materials using a mixed microbial culture. A microbial consortium was developed by enrichment of a mixture of BHC-contaminated soil (collected from sugarcane fields) and sewage (collected from Mysore Municipal Corporation Sewage Treatment Plant) in shake flasks containing gamma-HCH as the sole source of carbon and energy. The microbial consortium that got established in the medium containing 10 ug/ml of gamma-HCH was resolved into several bacterial and fungal strains. Individual strains were tested for their ability to degrade lindane. All the bacterial strains and a fungal strain were able to degrade 10 ug/ml of gamma-HCH and failed to degrade higher concentrations. Nine bacterial strains identified as different species of Pseudomonas and the fungal strain belonging to Fusarium were mixed and the mixed culture could degrade higher concentrations of gamma-HCH. The culture characteristics of the different isolates when grown on nutrient agar plates are as follows: 1. Pseudomonas sp. CFR 1010 - Gram -ve motile rods, colonies buff coloured, round (4-7 mm dia.) with irregular margin, umbonate, transluscent, aerobic with irregular, fluorescent margin, oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, does not reduce nitrate, has ortho-mode of ring cleavage, grows at 4\|C and 30\|C, produces fluorescent pigment, degrades lindane. 2. Pseudo/nonas sp. CFR 1011 - Gram -ve short motile rods, colonies limon yellow coloured, round (1-2 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 pigments, has ortho-mode of ring cleavage, does not grow at 4\|C, but grows at 30\|C, degrades lindane. 3. Pseudomonas sp. CFR 1012 - Gram -ve motile rods, colonies lemon yellow coloured, round (1.25-2.0 mm dia.) convex, transluscent, aerobic, smooth, fluorescent, oxidase +ve, catalase +ve, does not. hydrolyse starch and gelatin, ferments dextrose, but not mannitol, produces H^S, indole reaction ve, does not produce fluorescent pigments, has meta-mode of ring cleavage, grows at 4\|C and 30\|C, degrades lindane. 4. Pseudomonas sp. CFR 1013 - Gram -ve motile large rods, colonies buff coloured, round (7-10 mm dia.) umbonate, transluscent, aerobic, smooth, shining, L-form, oxidase +ve, catalase +ve, doesnot hydrolyse starch and gelatin, ferments dextrose, but not mannitol, does not produce H2S, indole reaction -ve, does not produce fluorescent pigments, has meta-mode of ring cleavage, grows at 4\|C and 30\|C, degrades lindane. 5. Pseudomonas sp. CFR 1014 - Gram -ve, motile, coccobacilli, colonies buff coloured, smooth (0.75-1.0 mm dia.) convex, transluscent, aerobic, fluorescent, oxidase +ve, catalase +ve, does not hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, does not produce fluorescent pigment, has ortho-mode of ring cleavage, does not grow at 4\|C, but grows at 30\|c, degrades lindane. 6. Pseudomonas sp. CFR 1015 - Gram -ve, motile, coccobacilli, colonies whitish yellow coloured, smooth (0.5-1.0 mm dia.) convex, transluscent, aerobic, fluorescent, oxidase +ve, catalase +ve, does not: hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, does not produce fluorescent pigments, has ortho-mode of ring cleavage, does not grow at 4\|C, but grows at 30 \| C, degrades lindane. 7. Pseudomonas sp. CFR 1016 - Gram -ve, motile rods, colonies whitish, round (1.0-1.25 mm dia.) convex, transluscent, aerobic, smooth, shining, fluorescent, 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 pigments, has ortho-mode of ring cleavage, does not grow at 4\|C, but grows at 30\|C, degrades lindane. 8. Paeudomonas sp. CFR 1017 - Gram -ve, motile, coccobacilli, colonies yellowish coloured, round (1.0-1.5 mm dia.) 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 pigments, has meta-mode of ring cleavage, does not grow at 4\|C, but grows at 30\|C, also grows sparingly at 41\|C, degrades lindane. 9. Pseudomonaa sp. CFR 1018 - Gram -ve, non-motile rods, colonies 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, grows at 30\|C, degrades lindane. 10. Fusarium sp. CFR 217 - White cottony mycelial growth (on Potato Dextrose Agar), pinkish orange on the under surface of the colony, mycelium septate, branching, growth ridges abserved, spores two to three types including sickle-shaped ones, multicellular and septate. The mutants of the above cultures have been developed by subjecting them to increasing concentrations of lindane used as a sole source of carbon and energy, by conventional enrichment techniques. Gamma-HCH used was 99% pure and was obtained from Sigma Chemical Company, U.S.A., 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 enrichment and for growth of the individual cultures as well as the mixed culture (Consortium CFR2002) contained (per litre of distilled water) 0.675 g KH2P04; 5.455 g.Na2HPO4; 0.25 g NH4NO3; 0.2 g MgSO4. 7H2O; 0.1 g Ca (NO3)2 and 1 ml of trace mineral solution containing (mg/ml) FeSO4.7H20, 1.0; MnS04.H2O, 1.0; CuCl2.2H2O, 0.25; Na2MoO4.2H2O, 0.25; and H3BO3 0.10. The pH of the medium was 7.5. Required amount of gamma-HCH was added to the medium as described later. For studying the effect of pH on gamma-HCH degradation, the required pH was adjusted by altering the buffer salt concentrations or by the additions of acid (HNO3) or alkali (NaOH) solutions. Nutrient agar plates were used for resolving the different bacterial strains from the consortium as well as for the study of their taxonomy. Nutrient agar medium contained 3.0 g beef extract, 5 g peptone, 5 g NaCl and 20 g agar agar per litre of distilled water (pH 7.0). Fungal strains were isolated and grown on potato-dextrose agar (infusion of 200 g potato, 20 g dextrose and 20 g agar-per litre, pH 5.5) . The cultivation of individual strains as well as the consortium in shake flasks was carried out in 50 ml of mineral medium containing required amount of growth substrate (gamma- HCH), taken in 250 ml Erlenmeyer flasks and incubating at 30\|C on a rotary shaker (150 rpm), For maintaining the individual strains as well as the mixed culture, mineral agar plates/slopes containing gamma-HCH (25 ug/rnl) were used. The culture was incubated for 4-5 days at 30\|C and then preserved at 4\|C. The mixed culture was maintained in liquid culture also, after growing it in mineral salts medium with 25 ug/ml gamma-HCH for 24 h and storing it at 4JC. The growth of the mixed culture was determined by estimating the total protein in the biomass by a modified method of Lowry (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. J.Biol.Chem 193: 265-285) as follows. The cells were harvested by centrifugation, washed with distilled water and suspended in 3.4 ml of distilled water 0.6 ml of 20% NaOH was added, mixed and digested in a boiling water bath for 5 min. Total protein in cooled sample was determined by Lowry's method using Folin-Ciocalteau reagent. Quantitative estimation of chloride was done by mercury (II) thiocyanate method (Bergmann, J.G. and Sanik Jr.J. 1957. Anal.Chem. 29: 241-243). To 2 ml of aliquot of the culture supernatant 0.2 ml of 0.25M ammonium iron (III) sulphate [NH4Fe(SO4)2.12H2O] in 9 M nitric acid was added followed by the addition of 0.2 ml of a saturated solution of mercury (II) thiocyanate in ethanol. Absorbance of the sample solution was measured at 460 nm by a spectrophotometer (Shimadzu UV-160 A, Japan). The amount of chloride was computed from a standard a curve prepared for NaCl. Quantitative determination of the substrate residue (lindane) was done either by thin layer chromatography (TLC) or by gas chromatography with 63Ni electron capture detector (BCD). The culture broth was extracted thrice with equal volume of a mixture of hexane: acetone (8:1) by mixing thoroughly each time. The solvent layer was pooled, evaporated and redissolved in convenient volume of hexane. Known volume was loaded on to silica gel G coated plate (0.3 mm) and developed in cyclohexane. The plate was air dried and gamma-HCH was detected by spraying the plate with 2% solution of o-tolidin in acetone. 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 vs. square root of the area, prepared for pure gamma-HCH. For gas chromatographic determination of the gamma-HCH residues, the hexane extract was passed through a florisil column to remove impurities. Shimadzu ISA (Japan) gas chromatograph with 63Ni detector was used. The conditions used were as follows: Column; 2 mm l.D. and 200 mm length packed with 1.5% OV-17+1.95 QF1 on 80-100 mesh chromosorb W,; carrier gas, nitrogen; flow at 40 ml/min,, column temperature 210\|C, injector temperature 230\|C and detector temperature 300\|C. The enrichment of a mixture of BHC-contaminated sugar cane field soil (collected from the outskirts of Mysore City) and sewage (collected from the Mysore City Municipal Corporation sewage treatment plant) was done in shake flasks using a mineral salts medium containing (per litre) 0.675 g KH2P04; 5.455 g Na2HPO4; 0.25 g NH4N03; 0.2 g MgS04.7H20; 0.1 g Ca(N03)2 and 1 ml of trace minerals solution containing (in mg/ml) FeSO4. 7H2O, 1.0; MnS04.H20, 1.0; NaMoO4, 0.25; H3BO3, 0.1 and CuCl2.2H20, 0.25). Gamma-HCH (lindane) was used as the sole source of carbon and energy at 10 ug/ml concentration. A cloth-filtered aqueous suspension of soil and sewage was added as inoculum (5 ml) to 50 ml medium taken in 250 ml conical flasks. The culture was incubated at 30\|C on a rotary shaker (150 rpm). After one week 5 ml of the culture broth was inoculated to fresh 50 ml medium having the same composition and 10 ug/ml of gamma-HCH. After 5 such transfers the broth was analysed for chloride release. Stoichiometric amount of Cl~ was detected in the medium indicating complete mineralization of the added gamma-HCH. Appropriately diluted culture broth was plated on nutrient agar and potato-dextrose agar. Nine different bacterial strains and one fungal strain was isolated. These individual strains were screened for their ability to degrade gamma-HCH. All the bacterial strains and the fungal strain degraded upto 10 ug/ml of gamma-HCH when inoculated individually. However, they failed to degrade higher concentrations of the substrate. When all the bacterial strains and the fungal strain were inoculated degradation of 25 ug/ml of gamma-HCH was observed. The individual bacterial strains were identified according to Bergey's Manual of Determinative Bacteriology and all of them were found to belong to the genus Pseudomonas (Table 1). All these strains were distinctly different in their morphological and biochemical characteristics. The fungus was identified as a species of Fusarium (Table 1). Table 1: The list of microorganisms present in the gamma-HCH degrading mixed culture CFR2002 (Table Removed) The cultures are deposited in CFTRI Culture Collection Centre. The mixed microbial culture, consortium CFR2002 was acclimatized to higher and higher concentration of gamma-HCH (from 25 ug/ml to 100 ug/ml). With acclimation the rate of degradation of gamma-HCH improved significantly. The time taken for complete mineralization of 100 ug/ml of gamma-HCH by the unacclimated mixed culture was 14 days. The acclimated culture took only 48 h for complete degradation of 100 ug/ml of gamma-HCH with concomitant release of stoichiometric amount of chloride. 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 To test the efficiency of the defined mixed culture to completely degrade gamma-HCH (lindane) the following experiments were done: 25 ml mineral medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HPO4, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g.Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a rotary shaker at 150 rpm at 30\|C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.l (Sheet No.l) of the drawing accompanying this specification. Complete disappearenc'e of the substrate and release of 100% Cl was observed after 24 h incubation. Biomass almost doubled during this period. These observations clearly indicate that gamma-HCH (25 ug/ml) was completely mineralized. The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying. EXAMPLE 2 Degradation of 50 ug/ml gamma-HCH by the mixed culture, consortium CFR2002 was studied in shake flasks as follows: 25 ml mineral 'medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HPO4, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a. rotary shaker at 150 rpm at 30 \|C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. Inoculum used was the cells collected from the previous experiment (Example I). The cells were pelleted,washed thoroughly amd added as inoculum. About 98% of the substrate (50 ug/ml gamma-HCH) disappeared and more than 80% Cl" was released withing 24 h of incubation. Complete disapperence of the substrate and 100% Cl~ release occured after 48 h (Fig 2., Sheet No.2) of the drawing accompanying this specification. The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying. EXAMPLE 3 The degradative ability of the mixed culture CFR2002 to mineralize 100 ug/ml of gamma-HCH was tested. 25 ml mineral medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HP04, 0.25 g NH4NO3, 0.2 g MgS04.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum was added and the flasks were incubated on a rotary shaker at 150 rpm at 30\|C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. Substrate concentrations were 100 ug/ml and the inoculum size was 25 ug protein/ml equivalent of cells. The cells collected from the previous experiment were washed and used as inoculum. Almost complete disappearence of the substrate (100 ug/ml) and release of stoichiometric amounts of Cl~ was observed within an incubation period of 48 h. The biomass increased from 25 ug to about 150 ug protein per ml during this period. The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying. EXAMPLE 4 The effect of pH on the degradation of lindane by the mixed culture CFR-2002 was studied by varying the pH of the medium from 3.0 to 11.0. The pH was altered by changing the concentrations of the buffering salts KH2PO4 and Na2HPO4 or by adjusting with HN03 or NaOH solution at the two extremities, 2B ml mineral medium wens taken in 250 ml flasks. The mineral medium contained required amounts of KH2PO4, and Na2HP04 to obtain the required pH, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added arid inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a rotary shaker at 150 rpm at 30\|c. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. 10 ug/ml of gamma-HCH was added as substrate. The cultures were incubated for 20 h on a rotory shaker at 30\|C. The mixed culture wat) able t,o degrade gamma-HCH at a wide pil range from 3.0-9.0 (Fig 4. sheet No.4). However, the best activity was observed at pH ranging from 6.5 to 7.5. The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying. EXAMPLE 5 The effect of temperature on degradation of gamma-HCH by the microbial mixed culture CFR2002 was studied by growing the cultures at 5,8,20,25,30,35,40,50 and 60°C under stationary conditions. 10 ml mineral medium containing 10 ug/ml substrate was taken in 100 ml conical flasks and were inoculated with cells grown previously with 100 ug/ml substrate, after a thorough wash. They were incubated at different temperatures without shaking, for 20 h. Degradation of the added substrate, as indicated by Cl~ release, was observed at a wide range of temperature (from 5° to 60°C), the optimum degradation being afe 35°C (Fig.5; Sheet No.5). The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying. The main advantages of the present invention are: 1. The formulation can be in the powder form 2. The formulation can be applied to effluent treatment plants for elimination of BHC residues. 3. The formulation works at room temperature. 4. The formulation works at a wide range of pH. 5. BHC to the tune of 100 ug/ml gets completely mineralised. We claim: 1. A process for preparation of lindane (BHC or benzene hexachloride) free industrial effluents /water , characterized in using a novel formulation of microbial consortium in powder form comprising * equal proportions for organisms consisting of novel mutated strains of pseudomonas and fusarium having characteristics as herein described , the said process comprises treating lindane industry effluents water bodies and other contaminated material having lindane residue with powder of said formulation of microbial consortium in a treatment plant, under agitation at 150 rpm for a time period ranging 3 - 24 h to get lindane free effluents or water. 2. A process as claimed hi claiml wherein BHC used may be alpha-, beta-, gamma-, delta- and epsilon-isomers or mixture thereof and employed in the range of 10 — 100 ppm. 3. The process preparation of lindane (BHC) free industrial effluents/water , characterized in using a novel formulation of microbial consortium substantially as herein described with reference to the examples.

Full Text

This invention relates to a process for preparation of lindane (BHC or benzene hexachloride ) free industrial effluents /water using a novel formulation of microbial consortium.
This invention particularly relates to the development of a formulation for elimination of lindane residues from industry effluents and water bodies through biodegradation.
Lindane is the insecticidal component of the most extensively used organochlorine pesticide which is commonly known as benzene hexachloride (BHC) or gammaxane. This is the gamma-isomer of hexachlorocyclohexane (gamma-HCH). Due to extensive and indiscriminate use of this powerful insecticide this has emerged as one of the major environmental pollutants in India and other third world countries. This compound is recalcitrant and persists in the environment for several years. Partial degradation of lindane to various intermediary metabolites in soil, particularly in flooded fields, has been reported. Refernce may
be made to Deo, P.G., Karanth,N.G and Karanth,N.G.K., 1994, Crit. Rev. Microbiol. 20: 57-78 and Kumar,S., Mukerji,K.G. and Lal.R., 1996, Crit. Rev. Microbiol. 22: 1-26. The drawbacks are that! toxic intermediates are generated which accumulate, thus not resulting in complete detoxification. However, reports on complete mineralization of gamma-hexachlorocyclohexane by microorganisms under aerobic conditions are very few. Reference may be made to Kunhi, A.A.M. 1995. In R.Sankaran and K.S. Manja (eds) Microbes for Better Living. MICON-94 and 34th Ann.Conf.AMI (Nov. 1994), DFRL, Mysore for a review of this. Pseudomorzas pauciflioJbilis SS88 (Senoo,K. and Wada, H. 1989, Soil Sci. Plant Nutr. 35: 79-87), Pseudomonas sp. (Sahu,S.K., Patnaik, K.K., Sharmila, M and Sethunathan, N. 1990, Appl .Environ.Microbiol. 56: 3620-3622) and Sphingomonas paucimobilis (Bhuyan, S. Sreedharan,
3., Adhya, T.K. and Sethunathan, N. 1993, Pestic.Sci. 38: 49-55)
have been,shown to degrade lindane. The drawbacks are that all
these organisms degrade lindane at very low concentrations (8-11 ug/ml). Hence, it is imperative to develop a process by which high concentrations of lindane can be degraded. Mixed microbial cultures can be acclimated to higher and higher concentrations of lindane in the laboratory and a consortium that can degrade lindane residues from industry effluents and contaminated water bodies can be developed.
The main object of the present invention is to provide a process for the preparation of a formulation useful for the degradation of benzenehexachloride (BHC).
Another object of the present invention is to prepare a formulation for the treatment of HCH-industry effluents for elimination of lindane and other pesticide residues.
In the drawings accompanying this specification Figure-1 represents Degradation of 25 ug/ml lindane by the defined mixed microbial culture CFR2002 -0- growth (total biomass protein) , -0-residual lindane and -X- inorganic chloride released. Figure 2 represents Mineralization of 50 ug/ml of lindane by the defined mixed microbial culture CFR2002. All symbols are as in Fig. 1. Figure 3 represents Degradation of 100 ug/ml of lindane by the defined mixed microbial culture CFR2002. All symbols are as in Fig.l. Figure 4 represents Effect of pH of the medium on degradation of 10 ug/ml of lindane by the defined mixed microbial culture CFR2002. And Figure 5 represents Effect of incubation temperature on the degradation of 10 ug/ml of lindane by the defined mixed microbial culture CFR2002. The cultures were incubated at different temperature for 20 h without shakinq.
Accordingly the present invention provides a process for preparation of lindane (BHC or benzene hexachloride ) free industrial effluents /water , characterized in using a novel formulation of microbial consortium in powder form comprising equal proportions for organisms consisting of novel mutated strains of pseudomonas and fusarium
having characteristics as herein described , the said process comprises treating lindane industry effluents water bodies and other contaminated material having lindane residue with powder of said formulation of microbial consortium in treatment plant, under agitation at 150 rpm for a time period ranging 3 - 24 h to get lindane free effluents or water.
The enzymatic formulation obtained is intracellular in nature .
In an embodiment of the present invention benzenehexachloride (BHC) used may be alpha-, beta-, gamma-, delta- and epsilon-isomers or mixture thereof and employed in the range of 10-100 ppm.
In another embodiment of the present invention the cultivation is effected at room temperature under shaking conditions for at least 48 h.
In yet another enboditnent of the present invention, the cultivation is effected in standard -culture media at a wide pH range of 5.0-8.0.
A mixed microbial culture (consortium CFR2002) was prepared by incorporating nine bacterial strains (different species of Pseudojnonas) and a fungal strain (Fusarium sp.) (Table 1) and was mutated to degrade increasing concentrations of gamma-HCH in the medium. This defined mixed culture could completely mineralize 100 ug/ml lindane within 48 h in shake flasks. The mixed culture could degrade lindane at a fairly wide range of pH (4.0-8.5) and temperature (5-60°C). This microbial mixed culture can be effectively deployed for treatment cf lindane industry effluents, water bodies, soil and other materials contaminated with lindane residues.
A novel process was developed for elimination of lindane
residues from industrial effluents and other contaminated
materials using a mixed microbial culture. A microbial consortium
was developed by enrichment of a mixture of BHC-contaminated soil
(collected from sugarcane fields) and sewage (collected from
Mysore Municipal Corporation Sewage Treatment Plant) in shake
flasks containing gamma-HCH as the sole source of carbon and
energy. The microbial consortium that got established in the
medium containing 10 ug/ml of gamma-HCH was resolved into
several bacterial and fungal strains. Individual strains were
tested for their ability to degrade lindane. All the bacterial
strains and a fungal strain were able to degrade 10 ug/ml of
gamma-HCH and failed to degrade higher concentrations. Nine
bacterial strains identified as different species of Pseudomonas
and the fungal strain belonging to Fusarium were mixed and the
mixed culture could degrade higher concentrations of gamma-HCH.
The culture characteristics of the different isolates when grown on nutrient agar plates are as follows:
1. Pseudomonas sp. CFR 1010 - Gram -ve motile rods, colonies
buff coloured, round (4-7 mm dia.) with irregular margin,
umbonate, transluscent, aerobic with irregular, fluorescent
margin, oxidase +ve, catalase +ve, does not hydrolyse starch
and gelatin, does not ferment dextrose and mannitol, does not
produce H2S, indole reaction -ve, does not reduce nitrate,
has ortho-mode of ring cleavage, grows at 4|C and 30|C,
produces fluorescent pigment, degrades lindane.
2. Pseudo/nonas sp. CFR 1011 - Gram -ve short motile rods,
colonies limon yellow coloured, round (1-2 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 pigments, has ortho-mode of ring cleavage, does not grow at 4|C, but grows at 30|C, degrades lindane.
3. Pseudomonas sp. CFR 1012 - Gram -ve motile rods, colonies
lemon yellow coloured, round (1.25-2.0 mm dia.) convex,
transluscent, aerobic, smooth, fluorescent, oxidase +ve,
catalase +ve, does not. hydrolyse starch and gelatin, ferments
dextrose, but not mannitol, produces H^S, indole reaction
ve, does not produce fluorescent pigments, has meta-mode of
ring cleavage, grows at 4|C and 30|C, degrades lindane.
4. Pseudomonas sp. CFR 1013 - Gram -ve motile large rods,
colonies buff coloured, round (7-10 mm dia.) umbonate,
transluscent, aerobic, smooth, shining, L-form, oxidase +ve,
catalase +ve, doesnot hydrolyse starch and gelatin, ferments
dextrose, but not mannitol, does not produce H2S, indole
reaction -ve, does not produce fluorescent pigments, has
meta-mode of ring cleavage, grows at 4|C and 30|C, degrades
lindane.

5. Pseudomonas sp. CFR 1014 - Gram -ve, motile, coccobacilli,
colonies buff coloured, smooth (0.75-1.0 mm dia.) convex,
transluscent, aerobic, fluorescent, oxidase +ve, catalase
+ve, does not hydrolyse starch and gelatin, does not ferment
dextrose and mannitol, does not produce H2S, indole reaction
-ve, does not produce fluorescent pigment, has ortho-mode of
ring cleavage, does not grow at 4|C, but grows at 30|c,
degrades lindane.
6. Pseudomonas sp. CFR 1015 - Gram -ve, motile, coccobacilli,
colonies whitish yellow coloured, smooth (0.5-1.0 mm dia.)
convex, transluscent, aerobic, fluorescent, oxidase +ve,
catalase +ve, does not: hydrolyse starch and gelatin, does not ferment dextrose and mannitol, does not produce H2S, indole reaction -ve, does not produce fluorescent pigments, has ortho-mode of ring cleavage, does not grow at 4|C, but grows at 30 | C, degrades lindane.
7. Pseudomonas sp. CFR 1016 - Gram -ve, motile rods, colonies
whitish, round (1.0-1.25 mm dia.) convex, transluscent,
aerobic, smooth, shining, fluorescent, 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 pigments, has ortho-mode of ring
cleavage, does not grow at 4|C, but grows at 30|C, degrades
lindane.
8. Paeudomonas sp. CFR 1017 - Gram -ve, motile, coccobacilli,
colonies yellowish coloured, round (1.0-1.5 mm dia.)
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 pigments, has meta-mode of ring
cleavage, does not grow at 4|C, but grows at 30|C, also
grows sparingly at 41|C, degrades lindane.

9. Pseudomonaa sp. CFR 1018 - Gram -ve, non-motile rods,
colonies 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, grows at 30|C, degrades lindane.
10. Fusarium sp. CFR 217 - White cottony mycelial growth (on
Potato Dextrose Agar), pinkish orange on the under surface of
the colony, mycelium septate, branching, growth ridges
abserved, spores two to three types including sickle-shaped
ones, multicellular and septate.
The mutants of the above cultures have been developed by subjecting them to increasing concentrations of lindane used as a sole source of carbon and energy, by conventional enrichment techniques.
Gamma-HCH used was 99% pure and was obtained from Sigma Chemical Company, U.S.A., 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 enrichment and for growth of the individual cultures as well as the mixed culture (Consortium CFR2002) contained (per litre of distilled water) 0.675 g KH2P04; 5.455 g.Na2HPO4; 0.25 g NH4NO3; 0.2 g MgSO4. 7H2O; 0.1 g Ca (NO3)2 and 1 ml of trace mineral solution containing (mg/ml) FeSO4.7H20, 1.0; MnS04.H2O, 1.0; CuCl2.2H2O, 0.25; Na2MoO4.2H2O, 0.25; and H3BO3 0.10. The pH of the medium was 7.5. Required amount of gamma-HCH was added to the medium as described later. For studying the effect of pH on gamma-HCH degradation, the required pH was adjusted by altering the buffer salt concentrations or by the additions of acid (HNO3) or alkali (NaOH) solutions.
Nutrient agar plates were used for resolving the different bacterial strains from the consortium as well as for the study of their taxonomy. Nutrient agar medium contained 3.0 g beef extract, 5 g peptone, 5 g NaCl and 20 g agar agar per litre of distilled water (pH 7.0). Fungal strains were isolated and grown on potato-dextrose agar (infusion of 200 g potato, 20 g dextrose and 20 g agar-per litre, pH 5.5) .
The cultivation of individual strains as well as the consortium in shake flasks was carried out in 50 ml of mineral medium containing required amount of growth substrate (gamma-
HCH), taken in 250 ml Erlenmeyer flasks and incubating at 30|C on a rotary shaker (150 rpm), For maintaining the individual strains as well as the mixed culture, mineral agar plates/slopes containing gamma-HCH (25 ug/rnl) were used. The culture was incubated for 4-5 days at 30|C and then preserved at 4|C. The mixed culture was maintained in liquid culture also, after growing it in mineral salts medium with 25 ug/ml gamma-HCH for 24 h and storing it at 4JC.
The growth of the mixed culture was determined by estimating the total protein in the biomass by a modified method of Lowry (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. J.Biol.Chem 193: 265-285) as follows. The cells were harvested by centrifugation, washed with distilled water and suspended in 3.4 ml of distilled water 0.6 ml of 20% NaOH was added, mixed and digested in a boiling water bath for 5 min. Total protein in cooled sample was determined by Lowry's method using Folin-Ciocalteau reagent.
Quantitative estimation of chloride was done by mercury (II) thiocyanate method (Bergmann, J.G. and Sanik Jr.J. 1957. Anal.Chem. 29: 241-243). To 2 ml of aliquot of the culture supernatant 0.2 ml of 0.25M ammonium iron (III) sulphate [NH4Fe(SO4)2.12H2O] in 9 M nitric acid was added followed by the addition of 0.2 ml of a saturated solution of mercury (II) thiocyanate in ethanol. Absorbance of the sample solution was measured at 460 nm by a spectrophotometer (Shimadzu UV-160 A, Japan). The amount of chloride was computed from a standard a curve prepared for NaCl.
Quantitative determination of the substrate residue (lindane) was done either by thin layer chromatography (TLC) or by gas chromatography with 63Ni electron capture detector (BCD). The culture broth was extracted thrice with equal volume of a mixture
of hexane: acetone (8:1) by mixing thoroughly each time. The solvent layer was pooled, evaporated and redissolved in convenient volume of hexane. Known volume was loaded on to silica gel G coated plate (0.3 mm) and developed in cyclohexane. The plate was air dried and gamma-HCH was detected by spraying the plate with 2% solution of o-tolidin in acetone. 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 vs. square root of the area, prepared for pure gamma-HCH.
For gas chromatographic determination of the gamma-HCH residues, the hexane extract was passed through a florisil column to remove impurities. Shimadzu ISA (Japan) gas chromatograph with 63Ni detector was used. The conditions used were as follows: Column; 2 mm l.D. and 200 mm length packed with 1.5% OV-17+1.95 QF1 on 80-100 mesh chromosorb W,; carrier gas, nitrogen; flow at 40 ml/min,, column temperature 210|C, injector temperature 230|C and detector temperature 300|C.
The enrichment of a mixture of BHC-contaminated sugar cane field soil (collected from the outskirts of Mysore City) and sewage (collected from the Mysore City Municipal Corporation sewage treatment plant) was done in shake flasks using a mineral salts medium containing (per litre) 0.675 g KH2P04; 5.455 g Na2HPO4; 0.25 g NH4N03; 0.2 g MgS04.7H20; 0.1 g Ca(N03)2 and 1 ml of trace minerals solution containing (in mg/ml) FeSO4. 7H2O, 1.0; MnS04.H20, 1.0; NaMoO4, 0.25; H3BO3, 0.1 and CuCl2.2H20, 0.25). Gamma-HCH (lindane) was used as the sole source of carbon and energy at 10 ug/ml concentration. A cloth-filtered aqueous suspension of soil and sewage was added as inoculum (5 ml) to 50 ml medium taken in 250 ml conical flasks. The culture was incubated at 30|C on a rotary shaker (150 rpm). After one week 5 ml of the culture broth was inoculated to fresh 50 ml medium
having the same composition and 10 ug/ml of gamma-HCH. After 5 such transfers the broth was analysed for chloride release. Stoichiometric amount of Cl~ was detected in the medium indicating complete mineralization of the added gamma-HCH. Appropriately diluted culture broth was plated on nutrient agar and potato-dextrose agar. Nine different bacterial strains and one fungal strain was isolated. These individual strains were screened for their ability to degrade gamma-HCH. All the bacterial strains and the fungal strain degraded upto 10 ug/ml of gamma-HCH when inoculated individually. However, they failed to degrade higher concentrations of the substrate. When all the bacterial strains and the fungal strain were inoculated degradation of 25 ug/ml of gamma-HCH was observed.
The individual bacterial strains were identified according to Bergey's Manual of Determinative Bacteriology and all of them were found to belong to the genus Pseudomonas (Table 1). All these strains were distinctly different in their morphological and biochemical characteristics. The fungus was identified as a species of Fusarium (Table 1).
Table 1: The list of microorganisms present in the gamma-HCH degrading mixed culture CFR2002
(Table Removed)
The cultures are deposited in CFTRI Culture Collection Centre.
The mixed microbial culture, consortium CFR2002 was acclimatized to higher and higher concentration of gamma-HCH (from 25 ug/ml to 100 ug/ml). With acclimation the rate of degradation of gamma-HCH improved significantly. The time taken for complete mineralization of 100 ug/ml of gamma-HCH by the unacclimated mixed culture was 14 days. The acclimated culture took only 48 h for complete degradation of 100 ug/ml of gamma-HCH with concomitant release of stoichiometric amount of chloride.
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
To test the efficiency of the defined mixed culture to completely degrade gamma-HCH (lindane) the following experiments were done: 25 ml mineral medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HPO4, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g.Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a rotary shaker at 150 rpm at 30|C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.l (Sheet No.l) of the drawing accompanying this specification. Complete disappearenc'e of the substrate and
release of 100% Cl was observed after 24 h incubation. Biomass almost doubled during this period. These observations clearly indicate that gamma-HCH (25 ug/ml) was completely mineralized.
The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying.
EXAMPLE 2
Degradation of 50 ug/ml gamma-HCH by the mixed culture, consortium CFR2002 was studied in shake flasks as follows: 25 ml mineral 'medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HPO4, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a. rotary shaker at 150 rpm at 30 |C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. Inoculum used was the cells collected from the previous experiment (Example I). The cells were pelleted,washed thoroughly amd added as inoculum.
About 98% of the substrate (50 ug/ml gamma-HCH) disappeared and more than 80% Cl" was released withing 24 h of incubation. Complete disapperence of the substrate and 100% Cl~ release occured after 48 h (Fig 2., Sheet No.2) of the drawing accompanying this specification.
The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying.
EXAMPLE 3
The degradative ability of the mixed culture CFR2002 to mineralize 100 ug/ml of gamma-HCH was tested. 25 ml mineral medium was taken in 250 ml flasks. The mineral medium contained 0.675 g KH2PO4, 5.455 g Na2HP04, 0.25 g NH4NO3, 0.2 g MgS04.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added and inoculated with the cells of the mixed culture. The inoculum was added and the flasks were incubated on a rotary shaker at 150 rpm at 30|C. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. Substrate concentrations were 100 ug/ml and the inoculum size was 25 ug protein/ml equivalent of cells. The cells collected from the previous experiment were washed and used as inoculum.
Almost complete disappearence of the substrate (100 ug/ml) and release of stoichiometric amounts of Cl~ was observed within an incubation period of 48 h. The biomass increased from 25 ug to about 150 ug protein per ml during this period.
The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying.
EXAMPLE 4
The effect of pH on the degradation of lindane by the mixed culture CFR-2002 was studied by varying the pH of the medium from 3.0 to 11.0. The pH was altered by changing the concentrations of the buffering salts KH2PO4 and Na2HPO4 or by adjusting with HN03 or NaOH solution at the two extremities, 2B ml mineral medium wens taken in 250 ml flasks. The mineral medium contained required amounts of KH2PO4, and Na2HP04 to obtain the required pH, 0.25 g NH4NO3, 0.2 g MgSO4.7H2O, 0.1 g Ca(NO3)2 and 1 ml of trace mineral solution per litre of distilled water. The substrate, gamma-HCH was added at 25 ppm (25 ug/ml). The required amount of the substrate was dissolved in 50 ul of acetone and was added at the bottom of dry, sterile flask inside a laminar hood and kept open till the acetone was completely evaporated. Then 25 ml mineral medium was added arid inoculated with the cells of the mixed culture. The inoculum added was cells equivalent to 64 ug protein per ml. The flasks were incubated on a rotary shaker at 150 rpm at 30|c. Samples were drawn at regular intervals of 3 h and analysed for growth (total biomass protein), inorganic chloride (Cl~) and residual substrate. 10 ug/ml of gamma-HCH was added as substrate. The cultures were incubated for 20 h on a rotory shaker at 30|C.
The mixed culture wat) able t,o degrade gamma-HCH at a wide pil range from 3.0-9.0 (Fig 4. sheet No.4). However, the best activity was observed at pH ranging from 6.5 to 7.5.
The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying.
EXAMPLE 5
The effect of temperature on degradation of gamma-HCH by the microbial mixed culture CFR2002 was studied by growing the cultures at 5,8,20,25,30,35,40,50 and 60°C under stationary conditions. 10 ml mineral medium containing 10 ug/ml substrate was taken in 100 ml conical flasks and were inoculated with cells grown previously with 100 ug/ml substrate, after a thorough wash. They were incubated at different temperatures without shaking, for 20 h.
Degradation of the added substrate, as indicated by Cl~ release, was observed at a wide range of temperature (from 5° to 60°C), the optimum degradation being afe 35°C (Fig.5; Sheet No.5).
The microbial mass was separated by centrifugation at 8000 rpm for 10 min and lyophilised by freeze drying.
The main advantages of the present invention are:
1. The formulation can be in the powder form
2. The formulation can be applied to effluent treatment plants
for elimination of BHC residues.
3. The formulation works at room temperature.
4. The formulation works at a wide range of pH.
5. BHC to the tune of 100 ug/ml gets completely mineralised.

We claim:
1. A process for preparation of lindane (BHC or benzene hexachloride) free
industrial effluents /water , characterized in using a novel formulation of
microbial consortium in powder form comprising * equal proportions for
organisms consisting of novel mutated strains of pseudomonas and fusarium
having characteristics as herein described , the said process comprises treating
lindane industry effluents water bodies and other contaminated material having
lindane residue with powder of said formulation of microbial consortium in a
treatment plant, under agitation at 150 rpm for a time period ranging 3 - 24 h to
get lindane free effluents or water.
2. A process as claimed hi claiml wherein BHC used may be alpha-, beta-, gamma-,
delta- and epsilon-isomers or mixture thereof and employed in the range of 10 —
100 ppm.
3. The process preparation of lindane (BHC) free industrial effluents/water ,
characterized in using a novel formulation of microbial consortium substantially
as herein described with reference to the examples.

Documents:

2147-del-1998-abstract.pdf

2147-del-1998-claims-(cancelled).pdf

2147-del-1998-claims.pdf

2147-del-1998-complete specification (granted).pdf

2147-del-1998-correspondence-others.pdf

2147-del-1998-correspondence-po.pdf

2147-del-1998-description (complete).pdf

2147-DEL-1998-Drawings.pdf

2147-del-1998-form-1.pdf

2147-del-1998-form-13.pdf

2147-del-1998-form-2.pdf

2147-del-1998-form-4.pdf

2147-del-1998-form-9.pdf

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Patent Number

189721

Indian Patent Application Number

2147/DEL/1998

PG Journal Number

16/2003

Publication Date

19-Apr-2003

Grant Date

27-Jan-2004

Date of Filing

24-Jul-1998

Name of Patentee

COUNCIL OF SCEINTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	ANEBAGILU ABDULLA MOHAMMAD KUNHI	CENTRAL FOOD TECHNOLOGICAL. RESEARCH INSTITUTE, MYSORE 670 013. INDIA
2	CHARIVUVILAYIL DANIEL ELCEY	CENTRAL FOOD TECHNOLOGICAL. RESEARCH INSTITUTE, MYSORE 670 013. INDIA
3	HARAVEY KRISHNAN MONONMANI	CENTRAL FOOD TECHNOLOGICAL. RESEARCH INSTITUTE, MYSORE 670 013. INDIA

PCT International Classification Number

C12N 9/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA