Title of Invention

A PROCESS FOR PRODUCTION OF CO-POLYMER PHB-CO-PHV BY BACILLUS CEREUS

Abstract The process for production of copolymer was developed by growing Bacillus cereus with inoculum size from 0.5 to 1.5 % in production medium of pH 6.0 to 9.0 containing complex nitrogen, simple sugars as the carbon source and minerals under shake culture condition at 30 to 37 °C for 24 to 60 h. The copolymer produced was having molecular weight 3.0X105, the ratio of hydroxybutyrate to hydroxyvalerate as 92:8, first melting temperature 128.9 and 167.3°C and was completely biodegradable. Agricultural wastes like orange peels could be used to enhance the copolymer production.
Full Text FORM 2
THE PATENT ACT - 197

COMPLETE SPECIFICATION
(See Section 10)
A process for production of copolymer, PHB-co-PHV
by Bacillus cereus
AGHARKAR RESEARCH INSTITUTE
G.G.Agarkar Road
Pune 411004, Maharashtra State. India
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed.

Title of Invention
A process for production of a copolymer, PHB-co-PHV by Bacillus cereus Names, Addresses and Nationality of the Applicants
MACS-Agharkar Research Institute, Gopal Ganesh Agarkar Road, Pune 411004, Maharashtra State, India.
Nationality - Indian
Field of invention
The present invention relates to the production of a co-polymer having potential application as biodegradable plastic. More particularly, the present invention relates to a co-polymer, PHB-co-PHV produced from a bacterial strain of Bacillus cereus MCM B- 1045
SUMMARY OF THE INVENTION:
Accordingly, the present invention provides a copolymer produced from a bacterial strain of Bacillus cereus ,MCM B-1045 having molecular weight in the range of 3 x105 and first melting temperature as 157 and 167°C, presence of butyric and valeric acid in 92:8 ratio.
The present invention further provides a process for the production of a copolymer which comprises in particular of 3-hydroxyalkanoate from a carbohydrate / simple sugar/mono or disaccharide, using a bacterium, Bacillus cereus, MCM B-1045, isolated from alkaline lake of Lonar, Dist. Buldhana, Maharashtra State.
Bacillus cereus was grown in nutrient rich medium for 18 h and the harvested cells were inoculated in the production medium containing a carbon source, nitrogen source and minerals, maintained at a pH in the range of 6.0 to 9.0, in submerged culture condition, at temperature ranging from 25 to 40°C, preferably under shaking condition. The cells were harvested after a period of 24 to 48 hrs followed by extraction of the polymer by solvent to obtain the copolymer.
It is identified as a copolymer of PHB and PHV based on analysis by GC, FTIR, NMR spectroscopy, Differential Scanning Calorimetry and Gel Permeation Chromatography.
The main advantage of the invention is the use of simple sugars for the production of copolymer. The copolymer has better thermoplastic characteristics as compared to the polyhydroxybutyric acid homopolymer.
Prior art and its defects:
Plastic is being used in day to day life in various forms but when discarded, it creates problem by persisting in the environment. It is reported that municipal solid waste is made up of 7% by weight or 18%) by volume of plastic, of which 50%o comes from packing material waste (Sasikala and Ramana, 1996). The disposal of plastic has become worldwide problem. Hence there is a need for the development of biodegradable polymer, which is ecofriendly.
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It is reported that the granules of polyhydroxybutyrate (PHB) which are synthesized and accumulated by some microorganisms, serve as an intracellular carbon and energy storage material (Brandl et al. 1990). These are formed under unbalanced growth conditions such as high C/N ratio, nutrient poor environment, osmotic pressure, UV radiation etc. Some of these bacteria are Pseudomonas, Bacillus, Cynanobacter, Alcaligenes eutrophus, etc. Alcaligenes eutrophus is used for industrial production.
The copolymers are mostly produced by Pseudomonas sp. (Anderson and Dawes, 1990) namely P. oleovorans, P. aeruginosa, P. jlourescens, P. putida, P. testosterone and P. extorguens. Similarly Bacillus species have been reported for the production of homopolymer namely polyhydroxybutyrate (Williamson, 1958). However the production of copolymer needs the presence of additional substrates e.g. Methylobacterium sp. requires valerate in addition to methanol (Yellore and Desai, 1998), Alcaligenes faecalis (NCIB 8156) requires acetate in addition to propionate, Pseudomonas putida (NCIB 9571) requires heptane and Pseudomonas aeruginosa (NCIB 8626) requires decanol (Haywood et al. 1989). Among the Bacillus species, Bacillus cereus is reported to produce copolymer in presence of valerate with glucose (Haywood et al. 1989). B. cereus (IC 143) (Labuzek, 2001) requires caprolactam for the production of tercopolymer, while Bacillus thuringiensis, B. mycoides (Borah, 2002), B. megaterium (Griebel 1968, McCool and Cannon, 1999) are reported to produce polymer of hydroxybutyrate.
Mixed culture of Azotobacter chrococcum and B. megaterium produced copolymer of PHB and PHV by utilization of 2% cane sugar (Dong et al. 2001).
In presence of n-amyl alcohol, many organisms are able to synthesize copolymer. (Yamane et al. 1993) from mixture of carbon sources.
It is reported that microorganisms produce granules as reserve food material when growing under stress. It was therefore thought worthwhile to explore alkaliphilic bacteria that were isolated from alkaline lake of Lonar for production of polymer, polyhydroxyalkanoate (PHA).
Objective of invention:
To develop a process for production of a copolymer by Bacillus cereus using simple carbohydrate.
Detailed description of the invention
The invention is described below in details
The organism Bacillus cereus is grown in nutrient broth for 18-24 h.
The production medium is prepared using essentially a sugar and an organic nitrogenous source. The medium is maintained in the pH range of 6.0 to 9.0. The organism Bacillus cereus MCM B-1045 is inoculated in the said production medium and incubated for 24 to 60 h in submerged culture condition. The cells are harvested and the copolymer is extracted by a solvent. The polymer produced is quantified by gravimetric method.
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In an embodiment of the present invention Bacillus cereus isolated from alkaline Lake of Lonar, Buldhana district, Maharashtra state, India was used for the production of a copolymer.
Bacillus cereus (MCM B-1045) is a gram positive rod, aerobic and alkaliphilic organism capable of growing optimally at pH 10.0. It grows in temperature range of 25-45°C and does not require any growth factor.
In another embodiment of the present invention Bacillus cereus was maintained on nutrient agar of pH 10.0. A 16 - 24 h old culture was inoculated in nutrient broth. After 18 h the cells were harvested and inoculated in production medium containing glucose as carbon source, casamino acid as nitrogen source and ferric chloride, ammonium chloride, potassium chloride, calcium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulphate as mineral salts. The flasks were incubated at 30°C under shake culture condition at 150 rpm for 48 h. The bacterial cells were harvested by centrifugation and lyophilized.
In yet another embodiment of the present invention the polymer was extracted from lyophilized cell mass using hot chloroform and precipitated by methanol. The polymer was characterized by studying Gas chromatography for the presence of hydroxy acids, 'H-NMR, FTIR, Differential Scanning Calorimetry for melting point and Gel permeation Chromatography for estimation of molecular weight of the polymer. The results are summarized in table
Characterization of the polymer produced by B.cereus
Differential Scanning Calorimetric analysis: melting point 157 and 167°C.
Molecular weight: 3.0 X 105
'H NMR analysis: hydroxy butyrate 92% and hydroxy valerate 8%.
The polymer was found to be biodegradable.
The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention.
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Example 1
Bacillus cereus (MCM B-1045) maintained on Nutrient Agar (NA) slant was transferred and grown in 100 ml Nutrient broth (NB) for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pallet was inoculated into 100 ml production medium (Table I) with 1% glucose as carbon source and 0.05% casamino acid as nitrogen source in 250 ml shaking flask at 30°C for 48h. The accumulation of intracellular PHA was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
Table 1: Composition of Production Medium, g/L
Na2HP04 8.7
NaH2P04 1.3
NH4CI 1.5
MgCI2 0.02
MnCl2 0.0006
KC1 0.2
FeCI3 0.002
CaCI2 0.001
Casamino acid 0.05
Glucose 10.0
Na2S04 0.1
pH 7.0
The polymer obtained was 20 mg 100mL-1
Example 2
The PHA production was studied using different carbon sources such as sucrose, trehalose, maltose and fructose which were incorporated individually in production medium.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% carbon source and 0.05% casamino acid as nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
The yield of cell mass and intracellular content of PHA is as given in Table 2.
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Table 2: Production of PHA using different carbon sources.

Carbohydrate source Dry Cell mass g 100-1 Polymer gl 00m L"1 % PHA
Glucose 0.142 0.020 14
Sugar from Market 0.115 J 0.034 29
Sucrose (Qualigens) 0.115 0.029 25
Trehalose 0.1043 0.022 21
Simple carbon source as sugar was found to yield maximum (29%) polymer.
Example 3
The PHA production was studied using different nitrogen sources such as yeast extract, peptone, beef extract and casamino acid which were incorporated in production medium.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% glucose as carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
Table 3: Production of PHA using different nitrogen sources

Nitrogen source Dry Cell mass g 100mL-1 Polymer g 100mL-1 % PHA
Yeast extract 0.131 0.026 20
Peptone 0.143 0.021 18
Beef extract 0.121 0.031 26
Casamino acid 0.142 0.022 16
The maximum (26%) PHA production was obtained using beef extract as nitrogen source in production medium.
Example 4
The production of PHA was carried out by supplementing dried and finely powdered peels of orange and banana separately at (i) 1% concentration in production medium as a sole carbon source and (ii) along with glucose.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% carbon source and or orange peels / banana peels powder and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile
6

blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
Table 4: Production of PHA by using of agricultural waste material by B. cereus

Substrate (1%) Dry Cell mass g 100ml/1 Polymer g 100mL-1 % PHA
Orange peels powder + glucose 0.133 0.0303 23
Banana peels powder + glucose 0.139 0.024 17
Glucose 0.148 0.020 13
PHA production was observed only in the production medium containing 1% orange peels & glucose and glucose & banana peels and glucose alone (Table 4) The production in presence of orange peel powder + glucose was higher as compared to glucose alone.
Agricultural waste and cheaper substrates like orange peel could be used to enhance PHA production.
Example 5
Effect of temperature on production of PHA
PHA production was carried out at different temperature viz. 30, 37 and 45°C.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21h at 30°C under shake culture condition. The broth was centrifuged and cell pallet was inoculated into 100 ml production medium with 1% carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C to 45°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
Maximum PHA production was at 37°C. However there was no production of the polymer at 45°C. Production of PHA at 30°C and 37°C is illustrated in Table 5
Table 5: Production of PHA at different temperature.

Temperature °C Dry Cell mass, g 100mL-1 Polymer, g 100mL-1 % PHA
30 0.141 0.021 15
37 0.068 0.017 25
45 0.007 - -
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Around 15 % polymer was formed at 30°C and 0.141 g dry cell mass while 25 % polymer was formed at 37°C and 0.068 g dry cell mass. Thus the polymer formation was better at
Example 6
Effect of pH on production of PHA
PHA production was carried out using production medium of different pH from 3 to 9. Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21h at 30°C under shake culture condition. The broth was centrifuged and cell pallet was inoculated into 100 ml production medium with 1% carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
There was no growth at pH 3, 4 and 5. The polymer was obtained at pH 6, 7, 8 and 9. The maximum polymer produced per g of dry cell mass was 29% at pH 6.0, while the actual polymer production in terms of yield in mg per 100 ml was higher at pH 7.0.
Table 6: Production of PHA at different pH.

pH Dry Cell mass g 100ml-1 Polymer g 100mL-1 % PHA
6 0.035 0.01 29
7 0.141 0.021 15
8 0.132 0.015 11
9 0.128 0.011 9
Although at pH 7.0 cell mass yield was higher than at pH 6.0, polymer content in individual cell was less as compared to cells grown at pH 6.0. The efficiency of organism to synthesis PHA is higher at pH 6.0.
Example 7
Effect of inoculum size on PHA production
PHA production was carried out by varying the inoculum size of B.cereus 2x106 to 6 x 106 cells/ml (0.5, 0.75, 1.00, 1.25, and 1.5%).
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted from the
8

lyophilized cell mass by hot chloroform and was precipitated with methanol. The polymer was estimated gravimetrically.
Table 7: Production of PHA using different inoculum densities.

Size of Inoculum Dry Cell mass g 100ml/1 Polymer g l00mL-1 % PHA
0.5 0.141 0.016 II
0.75 0.143 0.019 13
1.00 0.152 0.023 15-
1.25 0.154 0.023 15
1.50 1.156 0.023 15
The maximum polymer production was obtained using 1 % inoculum and further increase in inoculum density did not enhance PHA accumulation.
Example 8
Effect of incubation period on PHA production
PHA production was carried out at different time intervals i.e. 12, 24, 36, 48, 60 h. Bacillus cereus_ (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 12 to 60h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted from the lyophilized cell mass by hot chloroform and was precipitated with methanol. The polymer was estimated gravimetrically.
The dry cell mass and % polymer produced were recorded.
Table 8: Production of PHA at different incubation period.

Incubation time Dry Cell mass g 100mL-1 Polymer g 100mL-1 % PHA
12 0.022 - -
24 0.06 0.005 8
36 0.119 0.014 12
48 0.143 0.022 15
60 0.163 0.021 13
It is seen from Table 8 that the polymer production was highest (15%) at 48 h from 0.143 g dry cell mass.
9

Example 9
PHA production under the optimum conditions
PHA production was carried out under the optimum conditions, such as pH 6, temperature 37°C, incubation period 48 hrs, 1.0% inoculum, yeast extract as nitrogen source and sugar as carbon source.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated to have 1% inoculum density, into 100 ml production medium with 1% sugar as carbon source and 0.05% casamino acid as nitrogen source in 250 ml shaking flask at 37°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted by hot chloroform from the lyophilized cell mass and was precipitated with methanol. The polymer was estimated gravimetrically.
Table 9: Production of PHA under optimum conditions.

Dry Cell mass g 100mL-1 Polymer g 100mL –1 % PHA
Production under optimum conditions 0.076 0.03 39
Glucose+ casamino acid at 30 °C 0.142 0.02 14
The maximum (39 %) PHA production was obtained although the growth was less under optimal conditions when compared to that (14%) obtained without optimal conditions using glucose as carbon source and casamino acid as nitrogen source.
Example 10
Biodegradability of the polymer.
Bacillus cereus (MCM B-1045) from NA slant was transferred and grown in 100 ml NB for 21 h at 30°C under shake culture condition. The broth was centrifuged and cell pellet was inoculated into 100 ml production medium with 1% carbon source and 0.05% nitrogen source in 250 ml shaking flask at 30°C for 48h. The intracellular PHA accumulation was detected qualitatively by microscopic observation under ultraviolet light. PHA containing cells showed orange fluorescence after staining with Nile blue sulphate. The cells were harvested by cold centrifugation at 10,000 rpm. The intracellular PHA produced was extracted from the lyophilized cell mass by hot chloroform and was precipitated with methanol. The polymer was estimated gravimetrically and used for biodegradability testing.
Biodegradability of the polymer was assessed by ASTM method. The polymer was tested for its biodegradability by using different bacterial cultures.
In 50 ml capacity bottle, 30 ml of Davis Mingioli's medium with 1mg/ml polymer as carbon source was dispersed. The bottles were sealed and sterilized. The bottles were inoculated with
10

Bacillus subtilis MCM B-310, Pseudomonas putida MCM B-408, Micrococcus lyale MCM B-365 and Arthrobacter atrocyaneus MCM B-425 and incubated at 30°C under stationary conditions. The growth of the organisms and evolution of CO2 in the bottles was monitored at 24h interval. The cell mass increased and after 6-9 days of incubation evolution of CO2 was detected by GC.
The polymer was found to be mineralized by the cultures indicating its biodegradability.
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ADVANTAGES
The main advantages of the present invention are the following.
The production of copolymer by other species of Bacillus need presence of additional substrate along with glucose but production of copoloymer by Bacillus cereus is possible from glucose as sole carbon source.
Agricultural wastes like orange peels or banana peels could also be used for enhancing the production.

Distinguishing features
Present process
Bacillus cereus was able to synthesize Intracellular PHA when grown on glucose alone as carbon source

Earlier process
Copolymers are mostly produced by
Pseudomonas sp.
Bacillus sp., but there is requirement of
additional substrate like propionate or
glutamate along with glucose for the
production.

A process for microbial production of copolymer, PHB-co-PHV is developed.
Novelty of the Invention:
The production of an intracellular copolymer of PHB-co-PHV from simple sugars as a sole source of carbon by Bacillus cereus is reported.
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WE CLAIM
Claim 1: The process for production of copolymer was developed by growing Bacillus cereus with inoculum size from 0.5-1.5 % in nutrient medium of pH 6.0 to 9.0 containing complex nitrogen, carbohydrates as the carbon source and minerals under shake culture condition at 30 to 37°C for 24 to 60 h

Claim 2:

The organism claimed in claim 1, was isolated from the alkaline lake of Lonar, District Buldhana, Maharashtra state.



Claim 3:


The carbohydrate used in the production medium claimed in claim 1 included one of the sugars namely glucose, sucrose, trehalose and market sugar at 1% (w/v) concentration as the only carbon source.

Claim 4: The complex nitrogen sources used in the production medium claimed in
Claim 1 included peptone, beef extract, yeast extract and casaminoacid.
Claim 5: The PHA produced as claimed in claim 1 was identified as a copolymer of hydroxy butyric and hydroxyvaleric acid (3 PHB-co-3PHV) in the proportion of 92:8.


Name of natural person who has signed



13

References:
Anderson A.J and Dawes E.A. (1990), Occurrence, metabolism, metabolic role and industrial use of bacterial poloyhydroxyalkanoates. Microbial. Rev; 54(4), 450-472.
Borah B. (2002), The influence of nutritional and environmental conditions on the accumulation of poly-B-hydroxybutyrate in Bacillus mycoides RLJ B-017. J. Appl. Microbiol; 92, 776-783.
Brandl H, Gross R.A., Lenz R.W and Fuller R.C. (1990), Plastics from Bacteria and for Bacteria: Poly (be a-hydroxy-alkonates) as Natural, Biocompatible and biodegradable polyesters. Adv. Biochem. Eng; 41, 77-93.
Dong Z. (2001) Synthesis of poly (hydroxyalkalonic acid) using mixed Azotobacter chroococcum and Bacillus megaterium as catalyst. CN 1302823 (People's Republic of china).
Griebel Rod, Smith Z and Merrick J.M. (1968), Metabolism of poly-beta-hydroxy butyrate, purification, composition and properties of native poly-beta-hydroxybutyrate granules from Bacillus megatherium, Journal of Biochemistry; 7(10), 3676-3681.
Haywood G.N., Anderson A.J. and Dawes E.A. (1989), A survey of the accumulation of novel polyhyroxyalkonates by bacteria, Biotechnology letters; 11(7), 471-476.
Labuzek s and Radecka 1.(2001), Biosynthesis of PHB tercopolymer by Bacillus cereus UW 85, Journal of Applied Microbiology; 90,353-357.
Lee H.S.,Kim J.H., Park Y.S. and Kanz C.K. (2000), New microorganism Methylobacterim sp. and producing method for poly-hydroxy alkanoic acid, American Chemical Society.
McCool G.J. and Cannon M.C. (1999), Polyhroxyalkanoate inclusion body-associated proteins and coding regions in Bacillus megaterim, J.Bact. 181(2), 585-592.
Sasikala C.H. and Ramana CH.V. (1996), Biodegradable polyesters. Adv. Appl. Microbiol.:42, 97-218.
Williamson D.M. and Wilkinson J.F. (1958), The isolation and estimation of the poly-beta-hydroxy-butyrate inclusions of Bacillus species, Journal of General Microbiology: 19, 198-209.
Yellore V.S., Takur N.B and Desai A.J. (1999), Enhancement of growth and poly (3-hydroxybutyrate) production from Methylobacterim sp. ZP 24 by formate and organic acids. Lett. Appl. Microbiol; 29, 171-175.
Yamane T., Ueda S., magawa S., Tahara T., Torakazu T., Yoshiharu I, Hiroyuki U. (1993). Process for preparation of copolymer.
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ABSTRACT:
The process for production of copolymer was developed by growing Bacillus cereus with inoculum size from 0.5 to 1.5 % in production medium of pH 6.0 to 9.0 containing complex nitrogen, simple sugars as the carbon source and minerals under shake culture condition at 30 to 37 °C for 24 to 60 h. The copolymer produced was having molecular weight 3.0X105, the ratio of hydroxybutyrate to hydroxyvalerate as 92:8, first melting temperature 128.9 and 167.3°C and was completely biodegradable. Agricultural wastes like orange peels could be used to enhance the copolymer production.

Documents:

656-mum-2007-abstract(2-4-2007).pdf

656-mum-2007-abstract(granted)-(20-12-2010).doc

656-mum-2007-abstract(granted)-(20-12-2010).pdf

656-mum-2007-abstract.doc

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656-MUM-2007-CLAIMS(AMENDED)-(01-11-2010).pdf

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656-mum-2007-claims(granted)-(20-12-2010).doc

656-mum-2007-claims(granted)-(20-12-2010).pdf

656-mum-2007-claims.doc

656-mum-2007-claims.pdf

656-MUM-2007-CORRESPONDENCE(06-12-2010).pdf

656-mum-2007-correspondence(17-9-2007).pdf

656-MUM-2007-CORRESPONDENCE(29-11-2010).pdf

656-mum-2007-correspondence(ipo)-(20-12-2010).pdf

656-mum-2007-correspondence(ipo)-(27-11-2009).pdf

656-mum-2007-corresspondence-others.pdf

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656-mum-2007-form 1(2-4-2007).pdf

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656-mum-2007-form 2(complete)-(2-4-2007).pdf

656-mum-2007-form 2(granted)-(20-12-2010).doc

656-mum-2007-form 2(granted)-(20-12-2010).pdf

656-MUM-2007-FORM 2(TITLE PAGE)-(01-11-2010).pdf

656-mum-2007-form 2(title page)-(complete)-(1-11-2010).pdf

656-mum-2007-form 2(title page)-(complete)-(2-4-2007).pdf

656-mum-2007-form 2(title page)-(granted)-(20-12-2010).pdf

656-mum-2007-form 3(2-4-2007).pdf

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656-mum-2007-form-2.doc

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656-mum-2007-form-3.pdf

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656-MUM-2007-REPLY TO EXAMINATION REPORT(01-11-2010).pdf

656-MUM-2007-SPECIFICATION(AMENDED)-(01-11-2010).pdf

656-mum-2007-specification(amended)-(6-12-2010).pdf


Patent Number 244785
Indian Patent Application Number 656/MUM/2007
PG Journal Number 52/2010
Publication Date 24-Dec-2010
Grant Date 20-Dec-2010
Date of Filing 02-Apr-2007
Name of Patentee MACS-AGHARKAR RESEARCH INSTITUTE
Applicant Address G.G.AGARKAR ROAD, PUNE
Inventors:
# Inventor's Name Inventor's Address
1 SMITA SHRIKANT NILEGAONKAR MACS-AGAHRKAR RESEARCH INSTITUTE G.G.AGARKAR ROAD,PUNE 411004
2 M.PONRAJ MACS-AGAHRKAR RESEARCH INSTITUTE G.G.AGARKAR ROAD,PUNE 411004
3 PRADNYA PRALHAD KANEKAR MACS-AGAHRKAR RESEARCH INSTITUTE G.G.AGARKAR ROAD,PUNE 411004
4 SEEMA SHRIPAD SARNAIK MACS-AGAHRKAR RESEARCH INSTITUTE G.G.AGARKAR ROAD,PUNE 411004
5 JYOTI PRAKASH JOG NATIONAL CHEMICAL LABORATORY DR.HOMI BHABHA ROAD PUNE 411008
PCT International Classification Number C12Q1/04; C12Q1/04
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA