Process for the isolation of polyhydroxybutyrate from Bacillus mycoides RLJ B-017

EXAMPLE 2

[0053] Isolation

[0054] Activated sludge sample, from wastewater treatment plant was appropriately diluted and then spread on YPM-agar plates containing (g 1.sup.-1): yeast extract, 10; bactopeptone, 10; meat extract, 5; NaCl, 5; and agar, 20. The plates were incubated at 30.degree. C. for 72 h. Several colonies appeared on the plates and were screened for their PHB production. Isolate RLJ B-017, thus selected was used in this study.

[0055] Identification of the isolated strain

[0056] Morphological and taxonomic features of the selected isolate were examined by the established method (Sheath et. Al, 1986). The results are as shown Table 1 in Example 1.

[0057] Organism and Maintenance

[0058] Bacillus mycoides RLJ B-017, was grown at 30.+-.0.5.degree. C. and maintained at 4.degree. C. periodical transfer on YPM-agar slants.

[0059] Inoculum Preparation

[0060] The inoculum was grown on nutrient rich medium occupying 20% of the flask volume. The nutrient rich medium consists of (g 1.sup.-1): sucrose, 20; nutrient broths, 8; KH.sub.1PO.sub.4, 1.5; (NH.sub.4).sub.2SO.sub.4, 2.0, NaHPO.sub.4.12H.sub.2O, 2.239; MgSO.sub.4.7H.sub.2O, 0.2; CaCl.sub.2.2H.sub.2O, 0.02; FeSO.sub.47H.sub.2O, 0.01; and trace-element solution 1 ml 1.sup.-1. The trace element solution contained (g 1.sup.-1): ZnSO.sub.47H.sub.2O, 0.2; H.sub.3BO.sub.3, 0.6; MnCl.sub.2 4H.sub.2O, 0.06; CoCl.sub.2 6H.sub.2O, 0.4; CuSO.sub.44H.sub.2O, 0.02, NaMoO.sub.4.2H.sub.2O, 0.06. The pH was set at 7.2.

[0061] Cultivation conditions

[0062] The composition of basal culture media used was same as that of the inoculum media except nutrient broth was not added. Overnight grown inoculum was transferred directly in a 3L bioreactor containing the basal culture media of pH 7.2 with 2% molasses as carbon source. The temperature of the bioreactor was set between 30-35.degree. C. and its rpm was at 200. The cells were harvested by centrifugation after 24 hours corresponding to the end of exponential growth, and washed twice with distilled water.

[0063] Chemicals

[0064] Nutrient broth and all the other chemicals used were obtained from Difco Laboratories and Sigma Chemicals Co. and are of high purity analytical grade. However, Nutrient broth and all chemicals obtained from other commercial sources can also be used.

[0065] Processing of biopolymer

[0066] 10-25% (v/v) Sodium hypochlorite solution of pH 11 was prepared for processing. The cell pellet after centrifugation was treated with dispersions of sodium hypochlorite and chloroform (1:1 v/v). The treatment time was set between 2-3 hours at 30-35.degree. C. The suspension was then centrifuged at 10000 rpm for 15 minutes at 30.degree. C. Three separate phases were obtained. The lower phase containing PHB dissolved in chloroform was precipitated by adding ethanol. The precipitate was kept at cold (4.degree. C.) for 1 hr. and was recovered by centrifuging at 10000 rpm for 15 minutes at 4.degree. C. The pellet obtained was pressed to remove ethanol and dried at 60.degree. C. for 24 hrs. Molecular weight by viscosity method is 5,60,000 dalton, NMR data same as given in Example 1 and that of PHB obtained from M/s. Sigma chemicals in FIG. 2, Elemental analysis: C, 55.19%; H, 7.29% and N, 0.19% (PHB from Sigma Chemicals, USA: Mol weight 5,35,000 Dalton and elemental analysis: C, 55.64%; H, 72.5% and N, 0.59%).

EXAMPLE-3

[0067] Isolation

[0068] Activated sludge sample, from wastewater-treatment plant was appropriately diluted and then spread on YPM-agar plates containing (g 1-1): yeast extract, 10; bactopeptone, 10; meat extract, 5; and agar, 20. The plates were incubated at 30.degree. C. for 72 h. Several colonies appeared on the plates were screened for their PHB production. Isolate RLJ B-017, thus selected was used in this study.

[0069] Identification of the isolated strain

[0070] Morphological and taxonomic features of the selected isolate was examined by the established method (Sheath et. Al, 1986) The results are as shown Table 1 in example 1.

[0071] Organism and Maintenance

[0072] Bacillus mycoides RLJ B-017, was grown at 30.+-.0.5.degree. C. and maintained at 4.degree. C. by periodical transfer on YPM-agar slants.

[0073] Inoculum Preparation

[0074] The inoculum was grown on nutrient rich medium occupying 20% of the flask volume. The nutrient rich medium consists of (g 1.sup.-1): sucrose, 20; nutrient broth, 8; KH.sub.2PO.sub.4, 1.5; (NH.sub.4).sub.2SO.sub.4, 2.0; Na.sub.2HPO.sub.4.12H.sub.2O, 2.239; MgSO.sub.4.7H.sub.2O, 0.2; CaCl.sub.2.2H.sub.2O, 0.02; FeSO.sub.47H.sub.2O, 0.01. and trace-element solution 1 ml 1.sup.-1. The trace element solution contained (g 1.sup.-1): ZnSO.sub.47H.sub.2O, 0.2; H.sub.3BO.sub.3, 0.6; MnCl.sub.2 4H.sub.2O, 0.06; CoCl.sub.2 6H.sub.2O, 0.4; CuSO.sub.44H.sub.2O, 0.02; NaMoO.sub.4.2H.sub.2O, 0.06. The ph was set at 7.2.

[0075] Cultivation conditions

[0076] The composition of basal culture ivation conditions media used was same as that of the inoculum media except nutrient broth was not added. Overnight grown inoculum was transferred directly in a 3L bioreactor containing the basal culture media of pH 7.2 with 2% sucrose as carbon source. The temperature of the bioreactor was set between 30-35.degree. C. and its rpm was at 200. The cells were harvested by centrifugation after 24 hrs. corresponding to the end of exponential growth, and washed twice with distilled water.

[0077] Chemicals

[0078] Nutrient broth and all the other chemicals used were obtained from Difco Laboratories and Sigma Chemicals Co, and are of high purity analytical grade. However, Nutrient broth and all the other chemicals obtained from other commercial sources can also be used.

[0079] Processing of Bio-polymer

[0080] 10-25% (v/v) Sodium hypochlorite solution of PH 11 was prepared for processing. The cell pellet after centrifugation was treated with dispersions of sodium hypochlorite and chloroform (1.1 v/v). The treatment time was between 2-3 hours at 30-35.degree. C. The suspension was then centrifuged at 10000 rpm for 15 minutes at 30.degree. C. Three separate phases were obtained. The lower phase containing PHB dissolved in chloroform was precipitated by adding ethanol. The precipitate was kept at cold (4.degree. C.) for 1 hr. and was recovered by centrifuging at 10000 rpm for 15 minutes at 4.degree. C. The pellet obtained was pressed to remove ethanol and dried at 60.degree. C. for 24 hrs. Molecular weight by viscosity method was similar to mentioned above. NMR data were similar to shown in Example 1 and comparison oh .sup.1H NMR spectra with that of PHB obtained from M/s Sigma Chemicals in FIG. 2, Elemental analysis: similar to mentioned above Examples 1 and 2.

1TABLE 1 Morphological and taxonomical properties of Bacillus mycoides RLJ B-017 Sample Characterization Sample bacteria Characterization Bacteria Morphology Biochemical prop- erties Cell shape Rod Citrate utilization -Cell size (.mu.m) (1.0-1.2 .mu.m .times. Urease production + 3.0-5.0 .mu.m) Motility - Methyl red -Spore position Central Voges proskauer + Spore shape Ellipsoidal Nitrate reduction + Parasporal crystal - + Gram Staining + Acid production from Endospore + Glucose, Maltose, + Trehalose Cultural Galactose, Sucrose, + Characteristics Fructose Colony shape Rhizoid Arabinose, - Xylose Optimum 30.degree. C. Utilization of temperature Optimum pH 7.0 Rhamnose, Inositol, + Ribose Growth on nutrient + Galactose, Histidine, + agar Sucrose Growth on - Mannitol, Xylose, + MacConkey agar Arginine Growth at nutrient Raffinose + broth pH 5.0-7 0 + Salicin, Serine, - Methionine pH 8.0 - Glycerol Proline -Growth at NaCl Phenylalanine -2.5-7.0% .vertline. 8.5% -Growth at 5-20.degree. C., and -50.degree. C. 25-40.degree. C. +