Title of Invention	"AN IMPROVED NON SOLVENT BASED PROCESS FOR EXTRACTION AND RECOVERY OF POLY-B-HYDROXYBUTYRATE (PHB)
Abstract	Present invention provides a simple non solvent based extraction and recovery process for PHB from microbial biomass . The process of the present invention involves following steps: the microbial cell biomass after PHB accumulation phase was harvested by centrifugation and thermally treated in the presence of aqueous ammonia. After ammonolysis, the suspension was centrifuged and the pellet was washed with sodium hypochlorite to obtain a white precipitate which, on drying, gave a white powder which was of 92 - 94% purity.

Title of Invention

"AN IMPROVED NON SOLVENT BASED PROCESS FOR EXTRACTION AND RECOVERY OF POLY-B-HYDROXYBUTYRATE (PHB)

Abstract

Present invention provides a simple non solvent based extraction and recovery process for PHB from microbial biomass . The process of the present invention involves following steps: the microbial cell biomass after PHB accumulation phase was harvested by centrifugation and thermally treated in the presence of aqueous ammonia. After ammonolysis, the suspension was centrifuged and the pellet was washed with sodium hypochlorite to obtain a white precipitate which, on drying, gave a white powder which was of 92 - 94% purity.

Full Text	AN IMPROVED NON SOLVENT BASED EXTRACTION AND RECOVERY PROCESS FOR PHB FROM MICROBIAL BIOMASS This invention relates to an improved non solvent based process for extraction and recovery of poly-B-hydroxybutyrate (PHB). Poly-B-hydroxybutyrate is a microbially produced polymer, which accumulates as amorphous granules inside the microbial cells. The physical properties of PHB resemble to that of polypropylene. Thus PHB can be used as a speciality plastic where biodegradabi1ity is an asset. PHB can be formed into films, fibres, sheets and moulded into various shapes by conventional plastic moulding techniques. PHB is quite a unique biopolymer which combines three exceptional properties : 1. Thermoplastic processabi1ity 2. 100% resistance to water 3. 100% biodegradabi1ity PHB could be used for applications similar to those of common plastics and would fit well into new waste management strategies. Recently much efforts have been put to commercial production of PHB. However, the use of microbially produced PHB is still restricted because of its high production cost. The expenditure involved in PHB recovery from microorganisms is the primary obstacle in its commercial exploitation. Downstream extraction of intracellular products like PHB requires separation of PHB from the rest of the biomass. A number of solvent extraction processes have been used to separate PHB from biomass, such as chloroform [Walker. J; Witton, J.R. and Alderson, B. (1982) Eur. Pat. Appl. 46,017], dichloroethane, [Barham, P.J. and Selwood, A. (1982). Eur. Pat. Appl., 58, 480], 1,1,2, trlchloroethane,[Vanlautera, N. and Gilaiw, J. (1982). U.S. Pat. 4,310,648], acetic anhydride, [Schmidt, J.; Biederman, B and Schmiechen, H. 1985), Ger (East) Pat. D.D., 223, 428] and propylene or ethylene carbonate, [Lafferty, R.M. and Helnzle, E. 1978, U.S. Pat., 4, 1381, 298]. The details of chloroform extraction is as under : The bactrial cells are harvested by centrifugation and freeze dried. Polyesters are extracted from the dried cells with hot chloroform in soxhlet apparatus. The period of chloroform extraction is usually 1 hr. In these solvent extraction technique several lipids are coextracted together with the polyesters from the cells. To remove lipids, the polyesters are then precipitated by slowly adding a suitable organic solvent such as diethylether, hexane, methanol or ethanol. Polyesters are then redissolved in chloroform and purified by reprecipitation with hexane. The process of solvent extraction is expensive and requires large inventories of solvents and heavy capital investments in solvent recovery plant. An alternative developed by researchers at Imperial Chemical Industry (ICI) involves thermal treatment of PHB containing biomass followed by enzymatic treatment and washing with anionic surfactant to dissolve unwanted biomass as illustrated by Homes & Lim [Homes, P.A. and Lim. G.B. (1985) Eur. Pat. Appl, 145, 233]. Williams, D. H. and Wilkinson, J.F. [(1958) J. Gen-mirobiol. 19, 198-203] has described a less complex procedure by using differential digestion method employing sodium hypochlorite. Although simple and effective, this method was avoided because it has been reported to cause severe degradation of PHB to lower molecular weight as outlined in following references [Alper, R.; Lunddgren, D.G.; Marchessault, R.H. and Gole, W.A. (1963). biopolymers, 1,545-556], [Berger, E.; Ramsay, B.A.; Ramsay, J.A. and chavarie, C. (1989) Biotechnology techniques, 3 (4), 227-232]. Another process involves the use of dispersion fluid composed of sodium hypochlorite and chloroform. [Sei Kwong Hahn.; yong keun chang; Beom Soo Kim and Ho nam Chang (1994), Biotechnology and Bioengineering, 44, 256-261]. Most of the hitherto known processes make use of solvent and thus are expensive and cumbersome. It was, therefore, imperative to develop a simple inexpensive method for extraction of PHB from microbial biomass using a non-solvent based process. Further the process should be gentle enough to preserve the integrity of polymer, and its molecular weight, so as to make it suitable for thermoplastic applications. The process must adequately remove the non-PHB residual mass and thereby rendering the final product 92-94% pure. This invention provides a cost-effective process for recovery of poly-B-hydroxybutyrate from microbial biomass without the use of solvents. PHB is of industrial interest because of its biodegradable, biocompatible and thermoplastic nature. PHB production on large scale warrants minimization of production cost. Hence the recovery process must dispense with the use of expensive solvents. The main objective of the present investigation was, therefore, to develop a simple nonsolvent based extraction and recovery process for PHB from microbial biomass avoiding the draw backs of hither to known processes. The process of investigation is based on our findings that when the microbial biomass is thermally treated in alkaline conditions, almost all the cells are lysed and the protein is released in the suspension in soluble form. When the suspension is centrifuged, a white pellet is obtained which, on analysis, confirmed the presence of mainly PHB and very little amount of residual mass. The pellet, when washed with sodium hypochlorite for a short duration, increased the purity of the isolated PHB further. The process of the present invention involves following steps: The microbial cell biomass after PHB accumulation phase was harvested by centrifugation and thermally treated in the presence of aqueous ammonia. After ammonolysis, the suspension was centrifuged and the pellet was washed with sodium hypochlorite to obtain a white precipitate which, on drying, gave a white powder which was of 92 - 94% purity. Accordingly, the present invention provides an improved non solvent based process for extraction and recovery of poly-B-Hydroxybutyrate (PHB), characterized in that thermal treatment of microbial biomass in presence of aqueous ammonia , which comprises culturing microbial biomass containing intracellular PHB , preferably culture of Alcaligenes eutrophus, by controlled conventional fermentation technique, in a nitrogen free mineral medium, harvesting biomass produced by centrifugation, then treating the said biomass with aqueous ammonia having concentration ranging 5N -10N, at a temperature in the range of 60° to 100°C for 60 to 120 min, separating the pellet so obtained by conventional manner followed by washing with sodium hypochlorite such as herein described for further purification, if desired , and drying to get Poly - B-Hydroxybutyrate as white powder. In a preferred embodiment of the invention, the final concentration of aqueous ammonia in the cell suspension may be 5N-10 N, the temperature of treatment may be in the range of 60☻C - 100°C and the treatment time may be in the range of 60-120 min. The recovery of PHB is expected to be around 92-94% . The process of this invention is illustrated by the following examples. However this should not constsued the limit of the scope of the invention. EXAMPLE: Alcaligenes eutrophus was grown in 1 L flasks with 250 ml Nutrient rich medium containing 10 g peptone, 10 g yeast extract, 5 g beef extract, 5 g ammonium sulfate, per litre of distilled water to obtain cell biomass (4g/L). This cell biomass was then transferred to nitrogen free mineral medium for intracellular accumulation of PHB. The composition of nitrogen free medium being Na2HP04 4.8 g/L, K2HP04 2.65 g/L, MgSO4 0.4g/L, Fructose 20 g/L, and Trace element solution 1 ml. Trace element solution contained H3BO3 0.3 g, CoCl2 6H20, 0.2 gf> ZnSO4 - 7H20 0.1g, MnClz-4H2O, 30 mg, Na2MoO4 . 2H20 30 mg, Nicl2 20 mg, CuSO4.5H20 10 mg. The cultivation was carried out on a rotary shaker at 150 rpm and 30oC. The pH of the medium was 7.0. PHB was accumulated to the extent of 2.5 g/L. The PHB containing biomass so obtained was then suspended in aqueous ammonia giving final concentration of 10 N. The temperature of the suspension was raised to 80*C and held for 60 min. After cooling the suspension was centrifuged to obtain whitish pellet which contained SOX PHB and other cell impurities. This PHB could be purified further to 92% by washing the pellet with sodium hypochlorite. Advantages of this invention are : That, even by eliminating solvent usage, it is possible to achieve higher recovery of PHB from the microbial biomass, the ammonia extraction fluid can be recycled in the fermentation process after adequate pH correction, thereby making the extraction process more economical. We claim: 1 . An improved non solvent based process for extraction and recovery of poly-B-Hydroxybutyrate (PHB), characterized in that thermal treatment of microbial biomass in presence of aqueous ammonia , which comprises culturing microbial biomass containing intracellular PHB preferably culture of Alcaligenes eutrophus, by controlled conventional fermentation technique, in a nitrogen free mineral medium, harvesting biomass produced by centrifugation, then treating the said biomass with aqueous ammonia having concentration ranging 5N - 10N, at a temperature in the range of 60° to 100°C for 60 to 120 min, separating the pellet so obtained by conventional manner followed by washing with sodium hypochlorite such as herein described for further purification, if desired , and drying to get Poly - B - Hydroxybutyrate as white powder. 2. An improved process as claimed in claim 1 wherein sodium hypochlorite used is containing 4 - 6% available chlorine. 3. An improved non solvent based process for extraction and recovery of poly-B-Hydroxybutyrate (PHB) substantially as herein described with reference to the examples.

Full Text

AN IMPROVED NON SOLVENT BASED EXTRACTION AND RECOVERY PROCESS
FOR PHB FROM MICROBIAL BIOMASS
This invention relates to an improved non solvent based process for extraction and recovery of poly-B-hydroxybutyrate (PHB). Poly-B-hydroxybutyrate is a microbially produced polymer, which accumulates as amorphous granules inside the microbial cells. The physical properties of PHB resemble to that of polypropylene. Thus PHB can be used as a speciality plastic where biodegradabi1ity is an asset. PHB can be formed into films, fibres, sheets and moulded into various shapes by conventional plastic moulding techniques. PHB is quite a unique biopolymer which combines three exceptional properties :
1. Thermoplastic processabi1ity
2. 100% resistance to water
3. 100% biodegradabi1ity
PHB could be used for applications similar to those of common plastics and would fit well into new waste management strategies.
Recently much efforts have been put to commercial production of PHB. However, the use of microbially produced PHB is still restricted because of its high production cost. The expenditure involved in PHB recovery from microorganisms is the primary obstacle in its commercial exploitation.
Downstream extraction of intracellular products like PHB requires separation of PHB from the rest of the biomass. A number of solvent extraction processes have been used to separate
PHB from biomass, such as chloroform [Walker. J; Witton, J.R. and Alderson, B. (1982) Eur. Pat. Appl. 46,017], dichloroethane, [Barham, P.J. and Selwood, A. (1982). Eur. Pat. Appl., 58, 480], 1,1,2, trlchloroethane,[Vanlautera, N. and Gilaiw, J. (1982). U.S. Pat. 4,310,648], acetic anhydride, [Schmidt, J.; Biederman, B and Schmiechen, H. 1985), Ger (East) Pat. D.D., 223, 428] and propylene or ethylene carbonate, [Lafferty, R.M. and Helnzle, E. 1978, U.S. Pat., 4, 1381, 298]. The details of chloroform extraction is as under :
The bactrial cells are harvested by centrifugation and freeze dried. Polyesters are extracted from the dried cells with hot chloroform in soxhlet apparatus. The period of chloroform extraction is usually 1 hr. In these solvent extraction technique several lipids are coextracted together with the polyesters from the cells. To remove lipids, the polyesters are then precipitated by slowly adding a suitable organic solvent such as diethylether, hexane, methanol or ethanol. Polyesters are then redissolved in chloroform and purified by reprecipitation with hexane.
The process of solvent extraction is expensive and requires large inventories of solvents and heavy capital investments in solvent recovery plant. An alternative developed by researchers at Imperial Chemical Industry (ICI) involves thermal treatment of PHB containing biomass followed by enzymatic treatment and washing with anionic surfactant to dissolve unwanted biomass as illustrated by Homes & Lim [Homes, P.A. and Lim. G.B. (1985) Eur. Pat. Appl, 145, 233]. Williams, D. H. and Wilkinson, J.F.
[(1958) J. Gen-mirobiol. 19, 198-203] has described a less complex procedure by using differential digestion method employing sodium hypochlorite. Although simple and effective, this method was avoided because it has been reported to cause severe degradation of PHB to lower molecular weight as outlined in following references [Alper, R.; Lunddgren, D.G.; Marchessault, R.H. and Gole, W.A. (1963). biopolymers, 1,545-556], [Berger, E.; Ramsay, B.A.; Ramsay, J.A. and chavarie, C. (1989) Biotechnology techniques, 3 (4), 227-232].
Another process involves the use of dispersion fluid composed of sodium hypochlorite and chloroform. [Sei Kwong Hahn.; yong keun chang; Beom Soo Kim and Ho nam Chang (1994), Biotechnology and Bioengineering, 44, 256-261].
Most of the hitherto known processes make use of solvent and thus are expensive and cumbersome. It was, therefore, imperative to develop a simple inexpensive method for extraction of PHB from microbial biomass using a non-solvent based process. Further the process should be gentle enough to preserve the integrity of polymer, and its molecular weight, so as to make it suitable for thermoplastic applications. The process must adequately remove the non-PHB residual mass and thereby rendering the final product 92-94% pure.
This invention provides a cost-effective process for recovery of poly-B-hydroxybutyrate from microbial biomass without the use of solvents. PHB is of industrial interest because of its biodegradable, biocompatible and thermoplastic nature. PHB production on large scale warrants minimization of production
cost. Hence the recovery process must dispense with the use of expensive solvents. The main objective of the present investigation was, therefore, to develop a simple nonsolvent based extraction and recovery process for PHB from microbial biomass avoiding the draw backs of hither to known processes. The process of investigation is based on our findings that when the microbial biomass is thermally treated in alkaline conditions, almost all the cells are lysed and the protein is released in the suspension in soluble form. When the suspension is centrifuged, a white pellet is obtained which, on analysis, confirmed the presence of mainly PHB and very little amount of residual mass. The pellet, when washed with sodium hypochlorite for a short duration, increased the purity of the isolated PHB further.
The process of the present invention involves following steps:
The microbial cell biomass after PHB accumulation phase was harvested by centrifugation and thermally treated in the presence of aqueous ammonia. After ammonolysis, the suspension was centrifuged and the pellet was washed with sodium hypochlorite to obtain a white precipitate which, on drying, gave a white powder which was of 92 - 94% purity.
Accordingly, the present invention provides an improved non solvent based process for extraction and recovery of poly-B-Hydroxybutyrate (PHB), characterized in that thermal treatment of microbial biomass in presence of aqueous ammonia , which comprises culturing microbial biomass containing intracellular PHB , preferably culture of Alcaligenes eutrophus, by controlled conventional fermentation technique, in a nitrogen free mineral medium, harvesting biomass produced by centrifugation, then treating the said biomass with aqueous ammonia having concentration ranging 5N -10N, at a temperature in the range of 60° to 100°C for 60 to 120 min, separating the pellet so obtained by conventional manner followed by washing with sodium hypochlorite such as herein described for further purification, if desired , and drying to get Poly - B-Hydroxybutyrate as white powder.
In a preferred embodiment of the invention, the final concentration of aqueous ammonia in the cell suspension may be 5N-10 N, the temperature of treatment may be in the range of 60☻C - 100°C and the treatment time may be in the range of 60-120 min. The recovery of PHB is expected to be around 92-94% .
The process of this invention is illustrated by the following

examples. However this should not constsued the limit of the scope of the invention.
EXAMPLE:
Alcaligenes eutrophus was grown in 1 L flasks with 250 ml Nutrient rich medium containing 10 g peptone, 10 g yeast extract, 5 g beef extract, 5 g ammonium sulfate, per litre of distilled water to obtain cell biomass (4g/L). This cell biomass was then transferred to nitrogen free mineral medium for intracellular accumulation of PHB. The composition of nitrogen free medium being Na2HP04 4.8 g/L, K2HP04 2.65 g/L, MgSO4 0.4g/L, Fructose 20 g/L, and Trace element solution 1 ml. Trace element solution contained H3BO3 0.3 g, CoCl2 6H20, 0.2 gf> ZnSO4 - 7H20 0.1g, MnClz-4H2O, 30 mg, Na2MoO4 . 2H20 30 mg, Nicl2 20 mg, CuSO4.5H20 10 mg.
The cultivation was carried out on a rotary shaker at 150 rpm and 30oC. The pH of the medium was 7.0. PHB was accumulated to the extent of 2.5 g/L.
The PHB containing biomass so obtained was then suspended in aqueous ammonia giving final concentration of 10 N. The temperature of the suspension was raised to 80*C and held for 60 min. After cooling the suspension was centrifuged to obtain whitish pellet which contained SOX PHB and other cell impurities. This PHB could be purified further to 92% by washing the pellet with sodium hypochlorite.
Advantages of this invention are : That, even by eliminating solvent usage, it is possible to achieve higher recovery of PHB from the microbial biomass, the ammonia extraction fluid can be recycled in the fermentation process after adequate pH correction, thereby making the extraction process more economical.

We claim:
1 . An improved non solvent based process for extraction and recovery of
poly-B-Hydroxybutyrate (PHB), characterized in that thermal treatment of microbial biomass in presence of aqueous ammonia , which comprises culturing microbial biomass containing intracellular PHB preferably culture of Alcaligenes eutrophus, by controlled conventional fermentation technique, in a nitrogen free mineral medium, harvesting biomass produced by centrifugation, then treating the said biomass with aqueous ammonia having concentration ranging 5N - 10N, at a temperature in the range of 60° to 100°C for 60 to 120 min, separating the pellet so obtained by conventional manner followed by washing with sodium hypochlorite such as herein described for further purification, if desired , and drying to get Poly - B - Hydroxybutyrate as white powder.
2. An improved process as claimed in claim 1 wherein sodium hypochlorite
used is containing 4 - 6% available chlorine.
3. An improved non solvent based process for extraction and recovery of
poly-B-Hydroxybutyrate (PHB) substantially as herein described with
reference to the examples.

Documents:

1254-del-1997-abstract.pdf

1254-del-1997-claims.pdf

1254-del-1997-correspondence-others.pdf

1254-del-1997-correspondence-po.pdf

1254-del-1997-description (complete).pdf

1254-del-1997-form-1.pdf

1254-del-1997-form-13.pdf

1254-del-1997-form-19.pdf

1254-del-1997-form-2.pdf

1254-DEL-1997-Form-3.pdf

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Patent Number

232481

Indian Patent Application Number

1254/DEL/1997

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

17-Mar-2009

Date of Filing

13-May-1997

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SUCHITRA GODBOLE	C-005 GANGA APARTMENT, MOHAN NAGAR, NAGPUR-440001, INDIA.
2	HATIM FATEHALI DAGINAWALA	C-005 GANGA APARTMENT, MOHAN NAGAR, NAGPUR-440001, INDIA.
3	TAPAN CHAKRABARTI	C-005 GANGA APARTMENT, MOHAN NAGAR, NAGPUR-440001, INDIA.
4	PURUSHOTTAM KHANNA	C-005 GANGA APARTMENT, MOHAN NAGAR, NAGPUR-440001, INDIA.

PCT International Classification Number

C08G 06306

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA