Title of Invention

"A PROCESS FOR THE PREPARATION OF POLYHYDROXYALKANOATES FROM UNHYDROLYSED STARCH USING A NOVEL STRAIN OF BACILLUS SP (MTCC 5150)"

Abstract A process for the production of polyhydroxyalkanoates from unhydrolysed starch using a novel strain of Bacillus Sp. The present invention relates to a process for the production of polyhydroxyalkanoates from unhydrolysed starch using a novel strain of Bacillus Sp. (MTCC5150). The invention relates to production of polyhydroxyalkanoate (PHA). biodegradable polyester by fermentation process. In the present invention starch hydrolysis and its utilization for polymer production is achieved by using a single bacterial culture, in a single step. Another novel feature is the production of copolymer of PHA by the bacterium in the presence of a single carbon source such as starch instead of a sugar and fatty acids as in the reported processes.
Full Text The present invention relates to a process for the production of polyhydroxyalkanoates from unhydrolysed starch using a novel strain of Bacillus Sp. (MTCC5150).
The invention relates to production of polyhydroxyalkanoate (PHA). biodegradable polyester by fermentation process. Synthetic plastics are known to be non-biodegradable and are accumulating in nature leading to environmental pollution. Several bacteria synthesize PHA and it has properties, which are similar to synthetic plastics, and hence has great potential to replace synthetic plastic in various packaging applications. The limitation to viable commercial process is mainly due to production costs This includes expensive carbon substrates, methods of isolation of PHA which is intracellular in nature, and use of co carbon substrates foi copolymer production.
Reference may be made to Polyhydroxyalkanoates: An overview: By C.S.K. Reddy, R. Ghai, Rashmi and V.C. Kalia, Bioresource technology, 87 (2003) 137-146, a most recent review wherein it is indicated that PHAs are produced through fermentation process followed by extraction and purification. It has been mentioned that PHA can be produced from a wide variety of substrates such as renewable resources: starch, cellulose; fossil resources: methane, mineral oil; byproducts: molasses, whey, glycerol; chemicals: propionic acid, and also carbon dioxide. The drawbacks in the usage of renewable resources are that the polymers such as starch and cellulose are to be hydrolysed prior to their use in the fermentation medium, which would increase the operational step and the cost of PHA production.
Reference may be made to Production of poly(3-hydroxybutyrate) from inexpensive substrates by B.S. Kim, Enzyme and microbial technology, 27 (2000), 774-777, wherein soluble starch has been used as carbon source

for the production of PHA using Azotobacter chroococcum. The starch used here is not native starch and is in the soluble form when used.
Reference may also be made to a Process for the production of
biopolymer by Lapointe R, Lambert, A., and Savard, L. (2002), U.S. Patent
application No. 200220031812, wherein, PHA is produced by Azotobactei
salinestris using enzymatically hydrolyzed potato tuber starch as
carbon source to produce polyhydroxybutyrate (PHB). Copolymer of Polyhydroxybutyrate-co-polyhydroxy valerate (65-90% of hydroxybutyrate and 10-35% hydroxyvalerate) was obtained by co feeding sodium valerate to the fermentation broth. The main draw back is that even though the raw material used is from renewable resource, enzymes such as amylases and amyloglucosidaes have been used for hydrolysis of starch. For copolymei production, co substrate such as sodium valerate is used in the fermentation otherwise only a homopolymer such as polyhydroxybutyrate is produced. This leads to extra processing steps and cost of PHA production.
The main objective of the present invention is to provide a process for the production of polyhydroxyalkanoates from unhydrolysed starch, which obviates the drawbacks as detailed above. Another objective of the present invention was to obtain a copolymer of PHA, which is better suited for packaging applications compared to a homopolymer.
Accordingly a process for the production of polyhydroxyalkanoates from unhydrolysed starch using a novel strain of Bacillus sp. (MTCC 5150) which comprises:
a) Growing a culture of Bacillus sp. (MTCC 5150), in a liquid medium comprising of a nitrogen source, a source of phosphate and

magnesium and a carbon source respectively such as herein described, and essentially containing a starch source preferably unhydrolysed starch selected from tapioca starch, potato starch, cornstarch, sorghum, rice, maize, at 100-250 revolutions per minute (rpm) in a shaker, for a period of 48-120 hours, at a temperature ranging from 25-35 degree C,
b) centrifuging the culture broth at 5000-6000 rpm for 15-20 minutes, to obtain the biomass,
c) extracting the biomass using sodium hypochlorite of 5 % to 10 % strength of available chlorine, at 30-40 degree C for 0.5-1 hours,
d) centrifuging the extract of step (c), at 5000-6000 rpm for 15-20 minutes, washing and dissolving the precipitate with acetone and chloroform,
e) precipitating the PHA from the chloroform by adding 1.5-2 volumes of hexane or isopropyl alcohol, getting PHA in solid form by conventional manner such as herein described and drying at 30-50 degree C to get desired polyhydroxyalkanoates (PHA).
In an embodiment of the present invention, the polyhydroxyalkanoates obtained is a copolymer of polyhydroxybutyrate and polyhydroxyvalerate in a ratio of 92:8.
In the process, Bacillus sp (MTCC 5150), a strain isolated from soil was deposited at the culture collection center (MTCC) of IMTCH, Chandigarh was used. The culture was inoculated into medium containing mineral salts of 1-2.2 g/l of Na2HP04, 1-1.5 g/l of KH2P04, 1-1.5 g/l of (NH4)2S04, 0.1-0.2 g/l of MgS04 7H20, 10-20 g/l of starch, and pH 6-7.5. The cultivation was carried out in a fixed or ambient temperature ranging from 25-35° C for a period varying from 48-12h. Cultivation was carried out

aerobically by placing the culture in a shaker at 150-250 rpm. After 120 h the cells are harvested by centrifugation at 5000-6000 rpm for 15-20 min and the sedimented cells were dried using acetone or by lyophilization. Dried cells were weighed and extracted by dispersion of sodium hypochlorite and chloroform in 1:1 ratio at 30-40° C for 1-4 h and the clear chloroform layerwas dried to get PHA,
Bacillus sp has been grown directly on starch to produce amylase which can breakdown starch into low molecular weight products which are utilized by the cells as carbon source for growth and metabolism. The cells under nutrient limitation conditions with excess carbon produce PHA. Hence providing starch io nutrient limiting medium would also result in its hydrolysis by the amylase enzyme and its uptake leading to the intracellular accumulation of the biopolymer-PHA.
The novelty of the present invention is that starch hydrolysis and its utilization for polymer production is achieved by using a single bacterial culture, in a single step. Another novelty features the production of copolymer of PHA by the bacterium in the presence of a single carbon source such as starch instead of a sugar and fatty acids as in the reported processes.
The following example is 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

Bacillus sp (MTCC 5150), a strain isolated from soil and deposited at the culture collection center (MTCC) of IMTCH, Chandigarh was used in the experiment.
Characteristics of the culture: The culture was: gram positive rods, motile, aerobic, hydrolyzed starch and casein, catalase positive, no growth observed at 65° C, growth seen in 10% NaCI, do not produce gas from sugars, VP test -ve, indole -ve, lecithinase +ve, citrate -ve, urease -ve, slow liquefaction of gelatin, arabinose and xylose were not fermented. The endospores were placed sub terminally, sporangia were swollen, the spore wall was thick and spores were oval.
The culture was transferred from the slant into nutrient broth and allowed to grow at 200 rpm for 18-24 h. The inoculum was transferred at 10% (v/v) into 500 ml flask that contained 100 ml of sterile medium. The composition of the medium used was: 2.2 g/l of Na2HP04, 1.5 g/l of KH2PO4, 1.5 g/l of (NH4)2S04, 0.2 g/l of MgS04 7H20, 20 g/l of cornstarch and pH 7.0. Sucrose and glucose at 20 g/l were used for comparison. The flasks were incubated at 250 rpm in a shaker, for a period up to 48-120 h at ambient temperature of 25-35° C. The bacterial cells were harvested by centrifugation at 6000 rpm for 20 min. The sediment was washed with water and acetone. PHA accumulated in the biomass was quantified by lysing the known amount of cells with sodium hypochlorite for 1hr at 37° C. This was followed by centrifugation at 6000 rpm for 20 min. The sediment was washed with ether, alcohol and acetone and later dissolved in chloroform. The clear chloroform layer was evaporated to dryness to obtain PHA. Quantity of PHA was calculated based on weight of dry biomass. For analysis of copolymer PHA was analyzed by gas chromatography. For this 10 mg lyophilized cells was subjected to methanolysis in sealed tubes with a solution consisting of 1 ml chloroform, 0.85 ml methanol and 0.15 ml cone. H2SO4at 100 °C for 140 min. Distilled

water was added to the cooled samples, the contents were homogenized and the bottom phase containing methylesters was used for GC analysis. The analytical parameters were: The detector used was flame ionization detector, 30 m DB-1 (fused silica gel - polymethyl siloxane) capillary column (internal diameter 0.25 mm and film thickness 0.25 microns), N2 (1 ml/min) was used as a carrier gas. The injector and the detector were at 170 °C and 220 °C, respectively. The program used was: 55 °C for 7 min; ramp of 4° C per min up to 100 °C; 10 °C per min rise up to 200 °C and hold at 200 °C for 10 min. Calibration was performed using standard P(HB-co-HV) containing 5 mol% of hydroxy valerate (Sigma) with benzoic acid as internal standard.
Data in table 1 indicate that yield of biomass and PHA was more in
medium containing cornstarch compared to glucose and sucrose
containing medium. The polymer contained copolymer of
polyhydroxybutyrate and polyhydroxyvalerate in 98:2 in sucrose medium and the ratio increased to 92:8 in the presence of starch.
Table 1: Production of PHA by Bacillus sp (MTCC 5150) in glucose, sucrose and starch.
(Table Removed)

The main advantages of the present invention are:
1. A renewable resource such as starch is used as a carbon substrate for PHA production.

2. Starch is used directly in the medium without prior liquefaction or
saccharification.
3. Hydrolysis of starch and its utilization by the bacterium is a single step process.
4. Instead of two carbon substrates used for PHA copolymer production, presently a single renewable substrates namely starch is used for copolymer production.






We claim:
1. A process for the production of polyhydroxyalkanoates from unhydrolysed starch
using a novel strain of Bacillus sp. which comprises:
a) Growing a culture of Bacillus sp. (MTCC 5150), in a liquid medium comprising of a nitrogen source, a source of phosphate and magnesium and a carbon source respectively such as herein described, and essentially containing a starch source preferably unhydrolysed starch selected from tapioca starch, potato starch, cornstarch, sorghum, rice, maize, at 100-250 revolutions per minute (rpm) in a shaker, for a period of 48-120 hours, at a temperature ranging from 25-35 degree C,
b) centrifuging the culture broth at 5000-6000 rpm for 15-20 minutes, to obtain the biomass,
c) extracting the biomass using sodium hypochlorite of 5 % to 10 % strength of available chlorine, at 30-40 degree C for 0.5-1 hours,
d) centrifuging the extract of step (c), at 5000-6000 rpm for 15-20 minutes, washing and dissolving the precipitate with acetone and chloroform,
e) precipitating the PHA from the chloroform by adding 1.5-2 volumes of hexane or isopropyl alcohol, getting PHA in solid form by conventional manner such as herein described and drying at 30-50 degree C to get desired polyhydroxyalkanoates (PHA).
2. A process for the production of polyhydroxyalkanoates from unhydrolysed starch
using a novel strain of Bacillus Sp. (MTCC 5150) substantially as herein described with
reference to the example.

Documents:

774-del-2005-Abstract-(27-04-2011).pdf

774-del-2005-abstract.pdf

774-del-2005-Claims-(27-04-2011).pdf

774-del-2005-claims.pdf

774-DEL-2005-Correspondence Others-(03-06-2011).pdf

774-del-2005-Correspondence-Others-(27-04-2011).pdf

774-del-2005-correspondence-others.pdf

774-del-2005-description (complete).pdf

774-DEL-2005-Form-1-(03-06-2011).pdf

774-del-2005-form-1.pdf

774-del-2005-form-18.pdf

774-del-2005-form-2.pdf

774-del-2005-Form-3-(27-04-2011).pdf

774-del-2005-form-3.pdf

774-DEL-2005-Petition-137-(03-06-2011).pdf


Patent Number 250788
Indian Patent Application Number 774/DEL/2005
PG Journal Number 05/2012
Publication Date 03-Feb-2012
Grant Date 27-Jan-2012
Date of Filing 31-Mar-2005
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 KOLAMEGEDDE SIDDAPPA LATHA KUMARI CENTRAL FOOD TECHNOLOGY RESEARCH INSTITUTE MYSORE, INDIA.
2 TUMKUR RAMACHANDRIAH SHAMALA CENTRAL FOOD TECHNOLOGY RESEARCH INSTITUTE MYSORE, INDIA.
PCT International Classification Number C07C
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA