Indian Patents. 269325:"PROCESS FOR PRODUCTION AND QUANTITATION OF HIGH YIELD OF BIOBUTANOL "

Title of Invention	"PROCESS FOR PRODUCTION AND QUANTITATION OF HIGH YIELD OF BIOBUTANOL "
Abstract	The present invention relates to an improved process of production of high yield of butanol using Clostridium acetobutylicum ATCC 10132. The present invention in particular reports a strain with enhanced butanol tolerance under the optimized conditions.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES, 2003 PROVISIONAL SPECIFICATION (See Section 10; rule 13) "PROCESS FOR PRODUCTION OF HIGH YIELD OF BIOBUTANOL." RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India. The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:- FIELD OF THE INVENTION: The present invention relates to an improved process of production of high yield of butanol using Clostridium acetobutylicum ATCC 10132. The present invention in particular reports a strain with enhanced butanol tolerance under the optimized conditions. BACKGROUND OF THE INVENTION Butanol or butyl alcohol (sometimes also called biobutanol when produced biologically), is a primary alcohol with a 4 carbon structure and the molecular formula of C4H10O. It is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. Today, there is a paramount interest in producing fuels like butanol and ethanol using microorganisms by fermentation focusing on the environmental aspects and renewable nature of this mode of production. Butanol is a superior fuel and has more calorific value than ethanol (Qureshi and Blascher, 2000). Butanol has higher energy content (110,000 Btu's per gallon for butanol vs. 84,000 Btu per gallon for ethanol). It is six times less "evaporative" than ethanol and 13.5 times less evaporative than gasoline, can be shipped through existing fuel pipelines where ethanol must be transported via rail, barge or truck (Jones and Woods, 1986). Butanol is an important industrial solvent and potentially a better fuel extender than ethanol. Current butanol prices as a chemical are at $3.75 per gallon, with a worldwide market of 370 million gallons per year. The market demand is expected to increase dramatically if green butanol can be produced economically from low cost biomass. In addition to its usage as fuel, butanol can be used as a solvent for a wide variety of chemical and textile processes, in organic synthesis and as a chemical intermediate. It is also used as paint thinner and a solvent in other coating applications where it is used as a relatively slow evaporating latent solvent in lacquers and ambient-cured enamels. It finds other uses such as a component of hydraulic and brake fluids (Mutschlechner et al, 2000). It is also used as a base for perfumes, but on its own has a highly alcoholic aroma. Since the 1950s, most butanol in the United States is produced commercially from fossil fuels. The most common process starts with propene, which is run through an hydroformylation reaction to form butanal, which is then reduced with hydrogen to butanol. Butanol is produced by fermentation, from corn, grass, leaves, agricultural waste and other biomass. Production of industrial butanol and acetone via fermentation, using Clostridium acetobutylicum, started in 1916. Chime Wizemann, a student of Louis Pasteur, isolated the microbe that made acetone. Up until the 1920s, acetone was the product sought, but for every pound of acetone fermented, two pounds of butanol were formed. A growing automotive paint industry turned the market around, and by 1927 butanol was primary and acetone became the byproduct. The production of butanol by fermentation declined from the 1940s through the 1950s, mainly because the price of petrochemicals dropped below that of starch and sugar substrates such as corn and molasses. The labor intensive batch fermentation system's overhead combined with the low yields contributed to the situation. Fermentation-derived acetone and butanol production ceased in the late 1950s. Acetone butanol ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations. It was ranked second only to ethanol fermentation by yeast in its scale of production, and is one of the largest biotechnological processes ever known. The actual fermentation, however, has been quite complicated and difficult to control. ABE fermentation has declined continuously since the 1950s, and almost all butanol is now produced via petrochemical routes. In a typical ABE fermentation, butyric, propionic, lactic and acetic acids are first produced by C. acetobutylicum, the culture pH drops and undergoes a metabolic "butterfly" shift, and butanol, acetone, isopropanol and ethanol are formed. In conventional ABE fermentations, the butanol yield from glucose is low, typically around 15 percent and rarely exceeding 25 percent. The production of butanol was limited by severe product inhibition. Butanol at a concentration of 1 percent can significantly inhibit cell growth and the fermentation process. Consequently, butanol concentration in conventional ABE fermentations is usually lower than 1.3 percent. The key problem associated with butanol production is butanol toxicity /inhibition of the fermenting microorganism, resulting in low butanol titer in the fermentation broth. (Ezeji et al, 2007). Butanol is highly toxic to biological systems at quite low concentrations of 2% (Jones and Wood, 1986). This toxicity may be because butanol localizes in the plasma membrane and disrupts a number of physiological processes including membrane permeability, solute transport, maintenance of proton motive force, conformation and activity of intrinsic membrane proteins. Efforts are being made to improve the butanol tolerance level in different species of Clostridia with varying degree of success (Evan and Wang, 1988). Recent interest in the production of butanol has lead to re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture. In the past 20+ years, there have been numerous engineering attempts to improve butanol production in ABE fermentation, including cell recycling and cell immobilization to increase cell density and reactor productivity and using extractive fermentation to minimize product inhibition. Despite many efforts, the best results ever obtained for ABE fermentations to date are still less than 2 percent in butanol concentration, 4.46 g/L/h productivity, and a yield of less than 25 percent from glucose. Optimizing the ABE fermentation process has long been a goal of the industry. With that in mind, an alternative process was developed using continuous immobilized cultures of Clostridium tyrobutyricum and Clostridium acetobutylicum to produce an optimal butanol productivity of 4.64 g/L/h and yield of 42 percent. In simple terms, one microbe maximizes the production of hydrogen and butyric acid, while the other converts butyric acid to butanol. Compared to conventional ABE fermentation, this process eliminates acetic, lactic and propionic acids, acetone, isopropanol and ethanol production. The ABE fermentation process only produces hydrogen, butyric acid, butanol and carbon dioxide, and doubles the yield of butanol from a bushel of corn from 1.3 to 2.5 gallons per bushel. The drawbacks associated with such a process are two folds: having to maintain two sets of conditions for the two cultures, maintaining complete anaerobiosis in the immobilized system, dealing with the gases produced during the fermentation and their effect on the maintaining the integrity of the matrix used for immobilization. In conventional ABE fermentations, the butanol yield from glucose is low—between 15%-25%—and the butanol concentration in the fermentation is usually lower than 1.3%. (Butanol at a concentration of 1% can significantly inhibit cell growth and the fermentation process.) There have been numerous efforts over the years to improve butanol yield by using a variety of techniques to minimize product inhibition. In this respect, to develop a process for the maximum production and tolerance of this important fuel by process designing, standardization of media and fermentation conditions, strain improvement is of utmost importance (Agarwal et al, 2005). Physiological and nutritional factors such as initial sugar concentration, complex nitrogen sources, inoculum size, carbonate ion concentrations, pH and temperature of the growth medium are reported to be the most critical factors affecting both cell growth and butanol production (Samuelov etal, 1991;Nghiem^a/.,1997; Leeetal., 1999). US Patent 4,757,010 and European patent application EP 00111683 provides an improved strain of Clostridium for increased tolerance to butanol. JP03058782 provides Clostridium pasteurianum CA 101 stock (FERM P- 10817) as a mutant of genus Clostridium bacterium having analog resistance to fermented intermediate of butanol and butanol producibility. US patent 4,539,293 demonstrates the use of co-culture of microorganisms of the Clostridium genus, one favors the production of butyric acid and the other supports the formation of butanol. Japanese patent application JP 63157989 provides production of butanol by culturing a different strain Clostridium pasteurianum var. 1-53 (FERM P-9074) in a liquid medium containing a carbon source, a nitrogen source and other inorganic salts at 28-33 deg.C under slightly acidic pH condition in anaerobic state for 2-4 days. However the problems associated in these modified strains is that the use of genetically modified strains for fuel production cannot compete with the wild type as one needs to sterilize the feedstock to make sure that there is no competition for the genetically modified organisms. Further genetically modified organisms or various strains are expensive to develop and does not find relevance on high volume products. Various alternative in situ/online techniques of butanol removal including membrane-based systems such as pervaporation, liquid-liquid extraction, and gas stripping are used. US patent no. 4,777,135 describes a method of producing butanol by fermentation which comprises culturing under anaerobic conditions a butanol - producing microorganisms in a culture medium containing fluorocarbons. This process is not feasible on a commercial scale as the fluorocarbons are environmentally not safe US Patent 4,605,620 provides a process for butanol by using a medium containing carbohydrate and phosphate, wherein the experiments were performed with a total phosphate content of 1.0-0.4 mmoles. This process poses a restriction wherein the phosphate limiting medium is required. US patent 4,560, 658 provides a process for the production of butanol by fermentation of carbon containing compounds with Clostridium acetobutylicum wherein the fermentation is conducted in an aqueous medium containing a sufficient concentration of dissolved carbon monoxide. However the use of carbon monoxide make the process environmentally unsound. US patent 4,520,104 provides a process for the continuous production of butanol by fermentation of carbohydrates with C. acetobutylicum. This process combines continuous inoculum production at a high dilution rate and cycling the fermentation broth through material which adsorbs butanol whereby a vigorous cell population is maintained in the fermentation reactor for extended periods of time. The process is devised to remove the butanol produced in the broth so as to prevent its toxicity on the cells Japanese patent JP 62278989 provides a fermentation process for the production of acetone and butanol, by keeping a butanol-producing strain in resting state, adding a carbon source to the cell to effect the production of acetone and butanol in a short time, recovering and concentrating the butanol-producing strain, subjecting to the heat shock and adding to a fermentation tank Heat shock is required in the process.- to activate the spores of Clostridium and is pretty routine. Japanese patent application provides an anaerobic cellulolytic germ, e.g. Clostridium cellobioparum ATCC15832 or Ruminococcus albus ATCC27211, and Clostridium saccharoperbutylacetonicum are inoculated into a culture medium containing a material containing cellulose, e.g. wood, waste paper or pulp, as a main carbon source, and cultivated at 25-45°C and 4-9 pH under anaerobic conditions for about 2-20 days to collect the aimed compound, containing oxygen, and consisting essentially of butanol from the resultant culture. This process is time consuming and takes about 20 days for completion, hence not feasible on a large scale. Japanese patent 63269988 discloses butanol fermentation wherein yeast is subjected to autodigestion in a fermentation tank and proliferated prior to the inoculation of butanol-producing strain. The space in the fermentation tank becomes anaerobic and the temperature increases by the proliferation of yeast to perform butanol fermentation. An inefficient autodigestion would lead to contamination of the broth by the yeast US20050233031 provides a process for producing butanol which includes treating plant derived material to provide an aqueous liquor containing sugars in a fermentation process to produce a fermentation product. The process involves several steps and therefore cumbersome and tedious. Japanese Patent JP 200535328801 provides a method for producing butanol in which a culture solution is prepared by using a formulation of the food residue with the Japanese distilled spirit lees and water and butanol fermentation is carried out in the culture solution. The use of Japanese distilled spirit is limited to the production experiments performed in Japan. French patent FR2550222 provides a two stage process wherein a first stage of seeding with Clostridium acetobutylicum and a second stage of seeding with a yeast which produces ethanol, the second stage being commenced when the pH of the fermentation medium of the first stage has reached a minimum value. The invention applies in particular to the production of butanol, acetone and ethanol from sugarbeet and Jerusalem artichoke juices. The process requires pretreatment which makes it cumbersome. Although, there are reports where microbes have been exploited for the production of butanol by fermentation, yet an economically viable biosynthetic process for butanol production is yet to be developed (Jesse et ah, 2002). Thus looking into the need for a process which yields enhanced production of butanol, the present invention has focused on developing an ideal culture condition for the wild strain of Clostridium which will result in enhanced butanol tolerance and subsequently the in yields of butanol. OBJECT OF THE INVENTION It is the object of the present invention to provide an improved process for the production of high yield butanol using Clostridium acetobutylicum without any change in the strain of the microorganism. It is the object of the present invention to provide optimal fermentation conditions for enhanced production of butanol, using Clostridium acetobutylicum. It is the object of the present invention to provide a process with optimal fermentation conditions, which will result in increased butanol tolerance of the microorganism. It is the object of the present invention to provide a culture condition for high yields of butanol fermentation. It is the object of the present invention to provide a process for increased yields of butanol in a single batch fermentation conditions. It is the object of the present invention to provide a process for biobutanol using various biomass. It is the object of the present invention to provide a cost effective and industrially scalable process for butanol. SUMMARY OF THE INVENTION The present invention relates to an efficient process for the production of high yield of butanol using Clostridium acetobutylicum, without any change in the strain of the microorganism resulting in enhanced production of butanol. The present invention in particular aims at providing optimal culture conditions that would result in increased butanol tolerance of the microorganism. The present invention further aims at providing a cost effective and industrially scalable process for the production of butanol. The novelty of the present invention is production of high yields of butanol (upto 20 g/L) in a single batch process, without stripping the butanol produced. Thus process doesn't involve any fed-batch step which would involve extra step of addition of nutrients. Neither is any solvent-stripping required for reaching this high yield. Unlike many reported processes which employ continuous mode of fermentation thereby increasing the chances of contamination, the present process can be completed in a single batch mode. Careful optimization of the medium and acclimatisation has resulted in a strain that is capable of producing and tolerating such high yields of butanol in the broth. Thus, all these parameters make the process of the present invention more cost-effective. Further the inventors have also been able to successfully demonstrate the process at 5 L scale. In one aspect the present invention provides a process with increased butanol tolerance without the need of modifying the strain. In one preferred aspect the present invention provides tolerance to about 2.5% butanol concentration under optimized medium conditions directed to a process for providing the increased yield of butanol as provided in this invention. The most probable reason for its high tolerance to butanol may be that the process optimization has resulted in the final set of physio-chemical conditions under which the above mentioned limitations are alleviated. For example the redox opotential, osmolarity , electron flow may have been altered under the optimized conditions. Certain set of enzymes required for butanol tolerance and production may have been activated or induced under the optimized conditions. The culture may have adapted during the course of the optimization process to high butanol level. In one aspect the present invention provides a process with increased yields of butanol. In one preferred aspect the present invention reports a 9-fold increase in butanol production in 500 ml anaerobic bottles containing 300 ml of the optimized AnS medium as against the initial un-optimized AT medium (50 ml). In one aspect the present invention provides a process for evaluation of the biobutanol using various biomass. In one preferred aspect the seeds from jatropha and banana stems were used. The yield of biobutanol using the seeds and stems pretreated under different conditions such as alkaline, acidic and microwave digestion was also studied. In one aspect the present invention provides a process which can be scaled up on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. Fig.l: Shows the effect of different pH on butanol production by Clostridium acetobutylicum after 84 h of incubation. It was observed that though butanol was produced in the pH range 5.0 to 7.0 the optimal pH for production is 6.5, yielding 3.2 g /l of butanol in 84h. Fig. 2.Effect of different temperatures (°C) on butanol production by Clostridium acetobutylicum after 84 h of incubation Results on the effect of different temperatures (25, 33, 37, 39 & 45°C) showed that 3.0 g l\ of butanol was produced at 37 ± 2°C in 84 h. However at 25 and 55°C, no significant butanol production was observed Fig. 3.Effect of different carbon sources (2%) on butanol production. While studying the effect of nutritional factors, it was observed that none of the carbon sources tested supported as much butanol (3.2 gL-1) as was produced in the control viz. in glucose. This was followed by malt extract that supported 2.4 gL-1 of butanol in 84h Fig. 4. Effect of different concentrations of glucose on butanol production. It was found that 2.0% w/v glucose concentration supports maximum yield of 3.2 gL-'of butanol Fig.5: Effect of different concentrations of malt extract on butanol production. Concentration of malt extract, the second best sugar source, was varied in the medium (1.0- 10%). 4.82 gL-1 of butanol was produced when 5% malt extract was added along with 2% glucose in the AnS medium Fig.6 :Effect of different nitrogen sources (1.0%) on butanol production Beef extract was found to be the best nitrogen source among various nitrogen sources tested resulting in the production of 5.2gL_1 of butanol Fig. 7. Effect of different concentrations of beef extract on butanol production Optimization of concentration showed that 5.0 %w/v of beef extract is optimum for butanol production (7.8 gL-1) Fig. 8. Effect of different metal ions (0.5%) on butanol production. A significant increase in butanol production ( 11.1 gL-1) was achieved when the medium optimized so far was supplemented with Na2CO3 at 0.5%. This is followed by calcium ions that resulted in the production of 9.2gL-1 of butanol. Metal ion like Cu did not support any amount of butanol production Fig.9. Effect of different concentrations of sodium carbonate on butanol production. It was observed that 0.5% w/v of Na2CO3 is optimal for butanol production (1 l0gL-1). Fig.10. Effect of inoculum density on butanol production It was observed that, 14.5 gL-1 of butanol was produced at the inoculum density of 1%. However, with increase in the inoculum density beyond 2%, the production of butanol declined. Figure: 11 Fermentation profile of butanol at 5L scale The fermentation profile at 5 L scale indicates that the production of butanol is much faster at higher scale with the yield reaching 20 g/L in 48 h and reaching a plateau thereafter. DETAILED DESCRIPTION OF THE INVENTION Definitions: The term Butanol or biobutanol as used herein refers to n-butanol. The term butanol tolerance as used herein refers to the ability of the bacterium to survive and grow in the presence of ³ 1.3 % butanol in the medium The term Clostridium acetobutylicum refers to the bacteria that has the ability to produce butanol alongwith acetone and ethanol in a anaerobic fermentation The term yield as used herein refers to amount of butanol produced in the fermentation broth in g/L. As pH is one of the important factors that affect both growth and growth-associated production of molecules, butanol production was examined at different pH. The optimal pH for butanol production by Clostridium acetobutylicum in the present invention was 6.5. This is in accordance to the findings of Robson and Jones (1982), who reported that C acetobutylicum P262 showed good levels of solvent production within the pH range of 5.0-6.5. Similarly, Bielbl (1999) reported that Clostridium beijerinkii NCIMB 8052 showed much better growth and solvent production at pH 5.5 than at pH 5.0 or below. The present invention has found that temperatures of 37 ± 2°C is the optimal temperature for butanol production from Clostridium acetobutylicum ATCC 10132. This is in contrast to the earlier findings of McCutchan and Hickey (1954) who reported a decrease (upto 23%) in the solvent production by Clostridium sp. at 37°C as against the fairly constant yields of 31% at 30 and 33°C. The effect of carbon sources on butanol production by Clostridium acetobutylicum ATCC 10132, was studied and it was observed that glucose supported the highest butanol production. This was followed by malt extract, the second best carbon source. However, carbon sources like glycerol and sucrose supported a moderate amount of butanol. Sugars like rhamnose were not at all utilized by the strain. The most probable reason could be that the strain was unable to transport 2- deoxy glucose sugar. The study on the concentration of the sugars revealed that 2% glucose, supported 3.2 gL-1 of butanol. Similar has been reported by Biebl (1999) who observed maximum butanol production by C. acetobutylicum ATCC 824 in the medium containing 2.8% of glucose. However, in most of the studies, 6-7% has been found to be the optimum glucose concentration for butanol production. In this direction Parekh et a/.(1998) reported that 6.0% glucose in the medium yielded 10.0g/l of butanol from C.beijerinkii 8052 strain after 90h of incubation. Subsequently, when malt extract levels were varied in the medium in the range (1.0 - 10 % w/v) while keeping the concentration of glucose constant (at 2%), 4.8 gL-1 of butanol was produced at 5 % malt extract. Similarly, the effect of nutrition limitation on the onset and maintenance of solvent production has been investigated by a number of other workers. For example, Long et a/.(1984) reported that in the batch fermentation using Clostridium acetobutylicum P262, only acids were produced when the concentration of the carbon sources was limited. On supplementation of the medium with 5 % beef extract, a maximum of 5.3 gL-1 of n-butanol was produced as against the control (1% peptone) wherein 4.8 gL-1 of butanol production was observed. The most probable reason for beef extract being good source of nitrogen is because it not only provides nitrogen but also vitamins, and other nutrients which are essential for the growth of the microorganism Metal ions are known to play an important role in maintaining cellular metabolism and enzyme activities (Isar et al., 2006). A significant increase in butanol production was achieved when the medium optimized so far was supplemented with Na2CC>3. The reason could be that Na+ is a cofactor for most of the enzymes involved in the anaerobic pathway. Strobel et al. (1991) and Lee et al, (2000) reported that sodium ions are an important factor for the nutrient uptake. These ions are involved in the formation of transmembrane pH gradient, cell motility and intracellular pH regulation. Amongst different salts of sodium ion investigated, it was found that carbonate and bicarbonate were the most effective radicals for the production resulting in approximately 11.2 gL"1 of butanol. Change in the inoculum density from 1 - 2% did not significantly influence butanol production. However, an increase in inoculum density beyond 2% results in a decline in the production of the solvent. The most probable reason could be that as the inoculum size is increased beyond 2%, there is nutrition limitation. The present invention provides the effect of different physiological and nutritional parameters on butanol production by Clostridium acetobutylicum ATCC 10132. This strain initially produced 0.2 gL-1 of butanol in 84 h in Alternate Thioglycollate medium. However, when process optimization was employed, 20.0 gL-1 of butanol was produced in 300 ml of the optimized AnS medium consisting of Glucose (2%),Beef extract (5%), Malt extract (5%) Yeast extract (0.5%),K2HPO4 (0.3%), Na2CO3 (0.6%), (NH4)2SO4 (0.1%), CaCl2.2H2O (0.02%), MgCl2.7H2O (0.02%), Na2S(0.002%), at pH 6.5, 37° C, under static conditions (with gentle intermittent manual shaking) in 96h. Interestingly, it was also observed that the strain is tolerant to 2.5% butanol under optimized medium and conditions. The verification of the process in 300 ml medium clearly indicated that the process can be scaled up to higher size and about 20 gL-1 of butanol could be produced. The most probable reason for this increase in the yield could be the availability of more head space in bigger size bottles as against the smaller bottles. The Clostridium strain used in the present invention has shown tolerance to 2.5% butanol. The most probable reason for its high tolerance to butanol may be that the process optimization has resulted in the final set of physio-chemical conditions under which the above mentioned limitations are overcome. For example, the redox opotential, osmolarity, electron flow may have been altered under the optimized conditions. Certain set of enzymes required for butanol tolerance and production may have been activated or induced under the optimized conditions. The culture may have adapted during the course of study (optimization process). Since the actual tolerance level of this strain has never been reported earlier, it may also be the intrinsic un-exploited property of the strain. In several instances, a strain normally not reported to produce a biomolecule starts making it it in significant amounts after process optimization (Isar et al, 2006). Further studies on utilization of various biomass for butanol production using the conditions described above was performed. In particular , the biomass studied was jatropha seed cake and banana stem. Various pretreatments were given to the biomass prior to its use for the production of butanol. These pretreatments make the sugars from the biomass available for fermentation. The pretreatments include subjecting to fungal degradation, acid treatment, alkali treatment or microwave digestion. Jatropha seed cake was incubated with fungal culture Pluroteous osteratus at 23oC for a month and the biomass was extracted with a buffer. After extraction, the biomass was used as a supplement at different concentrations (1, 3, 5 and 10%) in Anaerobic sugar medium having 0.5% calcium carbonate. A yield of 8.4 g/1 of butanol was obtained after 48h using 3% of the fungal pretreated jatropha seed cake. In addition to this, when 1% soybean meal was added in AnS medium supplemented with 0.5 % calcium carbonate and 4 % of Beef extract a maximum yield of 10.5 g/L of butanol after 96h. Experiments were also done on Banana stem and jatropha seed pretreated with sodium hydroxide. The alkali treated biomass was supplemented at various concentrations in AnS medium having 0.5% calcium carbonate. At 1 % supplementation, a yield of 8.1 g/L butanol was obtained in AnS medium containing predigested banana stem as against 5.0 g/L butanol with predigested jatropha seed cake. With microwave digested biomass supplementation, a yield of 6.9 g/1 of butanol was obtained after 84h when 2% microwave treated banana stem was added to AnS medium having 0.5% calcium carbonate. With 1 % microwave digested jatropha seed cake supplementation, 7.0 g/1 of butanol was obtained after 84h. Supplementation with 0.1 N Sulphuric acid treated biomass in AnS medium having 0.5 % calcium carbonate resulted in a yield of 5.0 g/1 of butanol after 84h for 1% banana stem and a yield of 4.0 g/1 of butanol after 84h for 1% jatropha seed cake. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1: Organism and growth conditions Clostridium acetobutylicum ATCC 10132 was grown in 125 ml anaerobic bottles containing 50 ml of the Anaerobic Sugar (AnS) medium with the composition (gL-1 ): Glucose (20.0); Peptone (10.0); Yeast Extract (5.0); K2HPO4 (3.0); NaCl (1.0); (NH4)2SO4 (1.0); CaCl2.2H2O (0.2); MgCl2.6H2O (0.2); and Na2CO3 (1.0), pH 6.5. The medium was sterilized (15min, at 121°C) in glass bottles sealed with butyl rubber bungs. The headspace was filled with by N2, and Na2S.9H2O (0.02%) was added to remove traces of dissolved oxygen (Samuelov et al. 1991; Lee et al.,2000). The reduced medium was inoculated with 2 % seed inoculum and incubated at 37 ± 1°C for 96h with intermittent gentle shaking. EXAMPLE 2: Methods for estimation of butanol After the desired incubation period, the culture was withdrawn from the sealed vials using sterile disposable syringes and was centrifuged at 8000 x g in a Eppendorf centrifuge (model no 54151) for 10 min. Supernatant was filtered through 0.45(j. filter. The sample (20 ul) were analysed by HPLC, (Ehrlich et al, 1981) on a PRP 300X column (Hamilton) using acetonitrile and 0.5mM H2SO4 (9:1) as the mobile phase, at a flow rate of 1.5 ml/min at 37°C. Butanol was detected using a RI detector. EXAMPLE 3: Process in batch fermentation Various media tested for butanol production were Anaerobic sugar (AnS) medium, (Isar et al., 2006); Reinforced Clostridial (RC) medium, (Lin and Blaschek,1983); Soluble Starch Medium (SSM), (Moreira et al., 1981); Alternate Thioglycollate (AT) media, (Lin and Blaschek, 1983); Potato Starch media (PSM ), (Fouad et al.,1976). Amongst the media tested, AnS medium was the best yielding 3.2 g /l of butanol (Table 1). Butanol production was optimized in the AnS medium where the effects of different physiological and nutritional parameters were studied. Table:1 Butanol production (g/1) in different media Time (h) Media AnS RC AT SSM PSM 12 0.31 - - - - 24 0.43 - 0.02 - - 36 0.6 0.01 0.03 - - 48 1.2 0.02 0.09 0.01 0.09 60 2.1 0.05 0.1 0.04 0.09 72 2.2 0.10 0.2 0.07 0.10 84 3.2 0.13 0.2 0.09 0.11 96 3.0 0.11 0.1 0.08 0.09 Subsequently, the effect of pH (5- 7) and temperature (25 - 45 °C) was studied in the selected medium for butanol production. It was observed that the optimal pH for production is 6.5, yielding 3.2 g /l of butanol in 84h (Fig. 1). Results on the effect of different temperatures (25, 33, 37, 39 & 45°C) showed that 3.0 g /l of butanol was produced at 37 ± 2°C in 84 h(Fig. 2). Optimization of the nutritional parameters including carbon source, nitrogen source, metal ions required for maximum production of butanol was carried out. Various carbon sources employed include glucose, fructose, sucrose lactose, malt extract and glycerol at a concentration of 2.0 % w/v in the medium. None of the carbon sources supported as much butanol production (3.2 gL-1) as glucose in 84h (Fig. 3). Further, 2.0% w/v glucose gives the highest yield i.e. 3.2 gL-1 of butanol (Fig. 4). Approximately, 4.82 gL-1 of butanol was produced when 5% malt extract was added along with 2% glucose in the AnS medium (Fig 5). For nitrogen source optimisation, peptone (1% w/v) in the medium was replaced by different inorganic (ammonium hydrogen phosphate, ammonium chloride, sodium nitrate and urea) and organic nitrogen sources (yeast extract, beef extract, corn steep liquor, and tryptone) at the same concentration. Beef extract was found to be the best nitrogen source resulting in the production of 5.2 gL-1 of butanol (Fig.6). Optimization of concentration showed that 5.0 % w/v of beef extract is optimum for butanol production (7.8 gL-1) (Fig. 7). To assess the effect of metal ions, the carbonate / sulphate / chloride salts of different metal ions (Na+, Mg++, Ca++, Zn++, K+ Mn++) at 0.5% concentration were separately added in the medium. A significant increase in butanol production (11.1 gL-1) was achieved when the medium optimized so far was supplemented with Na2CO3 at 0.5%. Metal ion like Cu did not support any amount of butanol production (Fig.8). Among the various salts of sodium investigated including chloride, carbonate, sulphate and phosphate, carbonate was most effective for the production resulting in 11.2 gL-1 of butanol (Table 2). Further, upon optimizing the concentration of Na2CO3 , it was observed that 0.5% w/v is optimal for butanol production (11.0 gL-1) (Fig. 9). Table 2 : Effect of different salts of the selected metal ion on butanol production Mineral Salts Butanol, gL-1 Na2CO3 11.2 Na2SO4 9.1 NaCl 9.7 NaNO3 10.2 Na2HPO4 9.0 NaHCO3 11.1 Effect of inoculum size on butanol production was investigated. The optimized medium was inoculated with different inoculum size (1-10 %). Inoculum at 1 % was found to yield maximum butanol (14.5 gL-1) (Fig. 10). However, with increase in the inoculum density beyond 2%, the production of butanol declined EXAMPLE 4: Determination of butanol tolerance : The butanol tolerance level of the strain used in the present investigation was evaluated. The strain was inoculated in 50 ml of the optimized AnS medium containing different concentrations of butanol (0.5%, 1.0%, 1.3% 1.5%, 1.8%, 2.0%, 2.5%). Bottles were incubated for 96h at 37°C under static conditions with gentle intermittent manual shaking. The strain was found to tolerate upto 2.5 % butanol in the medium. EXAMPLE 5: Verification of the process in 300 ml medium : Butanol production in the optimized medium was validated in 500 ml anaerobic bottles containing 300 ml of the optimized medium. 1% of the inoculum was aseptically added with the help of syringe into the bottles and incubated at 37°C for 96h.A maximum of 20 gL-1 of butanol was produced. EXAMPLE 6: Scale up of the bio-butanol production at 5 L scale The butanol production in optimized medium using Clostridium acetobutylicum ATCC 10132 was scaled up to 5 L level in a 10L fermentor (Bioflow IV, NBS, USA). The optimized AnS medium was sterilized in situ at 110°C for 15 min. The medium was inoculated with 2 % of the seed inoculum (OD66onm = 0.6) and fermentation was carried out at 37±1°C for 84 h. The impeller speed was initially adjusted to 100 rpm and compressed sterile N2 was initially flushed for 30min to create anaerobic environment and was subsequently sparged intermittently into the fermentor at rate of 0.5 vvm. Samples were harvested periodically at an interval of 12h and analyzed for butanol production using HPLC and GC. The fermentation parameters such as temperature, N2 supply and agitation rate were continuously monitored and regulated. The butanol production started at 24 h in the fermentor and a maximum of 20.3 gL-1 of butanol was produced in 48h. Thereafter, there was no significant increase in the production of butanol (Figure 11 & Table 3) indicating a significant reduction in the production time of butanol at higher scale. Table 3 : Fermentation profile at 5 L scale Incubation period (h) Butanol Production (gL-1) 12 - 24 5.9 36 14.6 48 20.3 60 21.2 72 19.4 84 19.1 EXAMPLE 7: Study of the various biomass for biobutanol production The results obtained using various biomass is listed in Table 4. 1. Pretreatment of Jatropha seed with fungal culture Pluroteous osteratus Finely ground jatropha seeds (100 g, approx. mesh size 50 mm) was suspended in 50 ml of basal salt medium (0.5 % glucose, 0.1 % KH2PO4, 0.05 % MgSO4.7H2O, 0.05 % KCl, 0.05 % yeast extract). To this 50 ml each of stock solution I and stock solution II were added (Stock solution I: 0.02 % FeSO4.7H2O and Stock solution II: 0.016% Mn(CH3COO)2.4H20, 0.004% Zn(N03)2.4H20, 0.1 % Ca(N03)2.4H20, 0.006% CuSO4.5H20). The entire mixture was autoclaved at 121°C for 30 min and inoculated with 5-6 small malt extract (2%) agar blocks (lcmx 1cm) of two week old Pluroteous osteratus (grown at 25° C). The flask was incubated at 23°C for 30 days. After incubation, 500ml of 50mM citrate buffer (pH 5.0) was added and the contents were mixed thoroughly by shaking on the rotary shaker (200rpm) for 2h.The contents of the flask was squeezed using muslin cloth and the solid biomass was used as a supplement at different concentrations (1, 3, 5, and 10%) in 50 ml Anaerobic Sugar (AnS) Medium having 0.5% calcium carbonate contained in 125 ml of the sealed anaerobic bottles. A maximum yield of 8.4 g/1 of butanol was obtained after 48h using 3% of the fungal pretreated Jatropha seed cake. In addition to this, when 1% soybean meal was added in AnS medium supplemented with 0.5 % calcium carbonate and 4 % of Beef extract a maximum yield of 10.5 g/L of butanol after 96h. 2. Banana stem and jatropha seed pretreated with Sodium hydroxide digestion method (a) The banana stem was cut into small pieces and 100 g of these banana stem pieces were kept in 500 ml of 0.5M NaOH for 24 h with intermittent gentle shaking. After 24 h, these pieces were washed with running tap water to remove the alkali and were added to the AnS medium having 0.5% of CaCO3 at different concentrations for studying their effect on the production of butanol. The highest production of butanol 8.1 g/L was achieved in AnS medium containing 1% of the predigested banana stem. (b) The jatropha seeds were finely ground and were put into 500 ml of 0.5M NaOH for 24 h at room temperature. After 24 h, these pieces were washed with running tap water to remove sodium hydroxide. The different concentrations of pretreated jatropha seed cake were added to ANS media (containing 0.5% calcium carbonate).The highest production of butanol 5.0 g/L when 1% of the predigested jatropha seed was added to the AnS medium having 0.5% calcium carbonate. 3. Microwave Digestion for banana stem and Jatropha seeds (a) The banana stem was cut into the small pieces and partially digested in microwave oven for 10 min. The digested banana stem was ground in a mixer and finely ground banana was again digested in a microwave for 5 min. Sugar was analyzed by DNS method. A maximum yield of 6.9 g/1 of butanol was obtained after 84h when 2% microwave treated banana stem was added to AnS medium having 0.5% calcium carbonate. (b) The finely ground jatropha seed (20g) was added to 500 ml distilled water and cooked for 10 min in microwave oven. Microwave treated jatropha seeds were cooled down and again treated for 10 min in microwave oven. Sugar was analyzed by DNS method. The results showed that a maximum of 7.0 g/1 of butanol was obtained after 84h using 1% microwave treated jatropha seed cake in the AnS medium supplemented with 0.5% calcium carbonate and 0.5ml glycerol. 4. Banana stem and jatropha seed pretreated with 0. IN Sulphuric acid (a) The banana stem was cut into small pieces. Approximately, 100 g of these banana stem pieces were kept in 500 ml of 0.1N sulphuric acid for 24 h. These pieces were then washed with running tap water and were dried completely. The different concentration of this pretreated banana stem was then added to ANS media containing 0.5% calcium carbonate. The results showed that a maximum yield of 5.0 g/1 of butanol was obtained after 84h when 1% acid treated banana stem was added to AnS medium containing 0.5%) calcium carbonate. (b) The jatropha seed cake was ground. Approx. 50 g of the ground jatropha seeds were treated with 500 ml of 0.1N sulphuric acid for 24hours. After 24 h, these pieces were washed with running tap water to remove sulphuric acid. The predigested jatropha seed cake was dried completely. This pretreated jatropha seed cake was added to ANS media (having 0.5%) calcium carbonate) at different concentrations. A yield of 4.0 g/1 of butanol was obtained after 84h using 1%> acid pretreated jatropha seed cake. Table 4. Butanol production using biomass Biomass Pre-treatment Butanol, g/L Jatropha seed cake Fungal treatment with Pluroteous osteratus 8.4 0.5 M NaOH 5.0 0.1 N Sulphuric acid 4.0 Microwave digestion 7.0 Banana Stem 0.5 M NaOH 8.1 0.1 N Sulphuric acid 5.0 Microwave digestion 6.9 REFERENCES Agarwal, L., Isar, J., Saxena, R.K. (2005). Rapid screening procedures for identification of succinic acid producers. J Biochem Biophys Methods. 63 (1): 24- 32. Biebl, H.(1999). Comparative investigations of growth and solvent formation in 'Clostridium saccharoperbutylacetonicum' DSM 2152 and Clostridium acetobutylicum DSM 792. J. Ind. Microbiol. Biotechnol 22: 115- 120. Ehrlich, G.G., Georlitz, D.F., Bourell, J.H., Eisen, G.V. and Godsy, E.M. (1981). Liquid Chromatographic Procedures for fermentation products analysis in identification of anaerobic bacteria. Appl. Environ. Microbiol. 42(5): 878-885. Evan,P. J., and Wang,H.Y. (1988). Enhancement of butanol formation by Clostridium acetobutylicum in the presence of decanol- olely alcohol mixed extractants. Appl. Environ. Microbiol. 54: 1662-1667. Ezeji, T.C., Qureshi, N., Blsachek, H.P. (2007). Bioproduction of butanol from biomass: from genes to bioreactors. Curr.Opn Biotechnol. 18: 220- 227. Fouad M, Abou-Zeid AA, Yassein M. (1976). The fermentative production of acetone-butanol by Clostridium acetobutylicum. Acta Biol Acad Sci Hung. 27(2-3):107-17. Herrera, S. (2004). Industrial biotechnology-a chance at redemption. Nature Biotechnol 22 (6): 671-678. Isar, J., Agarwal, L., Saran, S., Gupta, P. and Saxena, R. K. (2006).Effect of process parameters on succinic acid production in Escherichia coli W3110 and enzymes involved in the reductive tricarboxylic acid cycle. Can. J. Microbiol. 52(9): 893-902. Jesse, T. W., Ezeji, T. C, Qureshi, N. and Blaschek, H. P. (2002). Production of butanol from starch based waste packing peanuts and agricultural waste. J. Ind. Microbial. Biotechnol. 29: 117- 123. Jones, D.T. and Woods, D.R.(1986). Acetone butanol fermentation Revisited. Microbiol. Rev.50 (4): 484-524. Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N. (1999). Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochem. 35:49-55. Lee, P.C., Lee, W.G., Kwon, S., Lee, S.Y., and Chang, H.N. (2000). Batch and continuous cultivation of Anaerobiospirillum succiniproducens for the production of succinic acid from whey. Appl. Microbiol. Biotechnol. 54 : 23-21. Lin, Y.L. and Blaschek, H.P. (1983). Butanol production by a butanol -tolerant train of Clostridium acetobutylicum in extruded corn broth. Appl. Environ Microbiol. 45: 966-973. Long, S., Long , D. T., and Woods , D. R.(1984). Initiation of solvent production , clostridial stage and endospore formation in Clostridium acetobutylicum P262. Appl. Microbiol. Biotechnol.,20: 256-261. McCutchan,W.N.,and Hickey, R.J. (1954). The butanol - acetone fermentation. Ind. Ferment. 1:347-388. Moon, S.H. and Parulekar, S.J. (1991). A parametric study of protease production in batch and fed-batch cultures ofBacillus fir-mus. Biotechnol. Bioeng.37, 467-483. Moreira , A. R., Ulmer, D.C., Linden, J.C. (1981). Butanol toxicity in the butylic fermentation. Biotechnol Bioeng. Symp. 11: 567-579. Mutschlechner O, Swoboda H, Gapes JR (2000). Continuous two-stage ABE-fermentation using Clostridium beijerinckii NRLL B592 operating with a growth rate in the first stage vessel close to its maximal value. J. Mol Microbiol. Biotechnol. 2(1): 101- 5. Nghiem, N.P., Davidson, B.H., Suttle, B.E., Richardson, G.R. (1997). Production of succinic acid by Anaerobiospirillum succiniciproducens. Appl Biochem Biotechnol 63-65: 565-576. Parekh, M., Formanek and Blaschek, H.P.(1998). Development of a cost effective glucose -corn steep medium for production of butanol by Clostridium beijerinckii. J. Ind. Microbiol Biotechnol. 21: 187-191. Qureshi, N. and Blaschek, H.P. (1999). Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijierinckii BAIOI and recovery by pervaporation. Biotechnol. Prog. 15: 594-602. Qureshi, N., Lolas, A., Blaschek, H.P.(2001). Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinkii BAIOI. J. Ind. Microbiol Biotechnol. 26: 290-295. Robson, P.M., and Jones, D.T. (1982). Production of acetone -butanol by industrial fermentation, In O. Chaude and Durand, G (ed.), Industrielle et Biotechnologie. p 169-214. Samuelov NS, Lamed R, Lowe S, Zeikus JG. (1991). Influence of CO2_HCO3 + levels and pH on growth, succinate production and enzyme activities of Anaerobiospirilium succiniciproducens. Appl. Environ. Microbiol. 57:3013-9. Strobel, H.J.,RiisseI, J.B. (1991). Role of sodium in the growth of a ruminal selenomonad. Appl Environ Microbiol. 57: 1663-1669. Zeikus, J.G. (1980). Chemical and fuel production by anaerobic bacteria. Ann Rev Microbiol 34: 423- 464. Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.

Full Text

FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"PROCESS FOR PRODUCTION OF HIGH YIELD OF BIOBUTANOL."
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre,
R-282, TTC Area of MIDC,
Thane Belapur Road, Rabale,
Navi Mumbai - 400 701
Maharashtra India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION:
The present invention relates to an improved process of production of high yield of butanol using Clostridium acetobutylicum ATCC 10132. The present invention in particular reports a strain with enhanced butanol tolerance under the optimized conditions.
BACKGROUND OF THE INVENTION
Butanol or butyl alcohol (sometimes also called biobutanol when produced biologically), is a primary alcohol with a 4 carbon structure and the molecular formula of C4H10O. It is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. Today, there is a paramount interest in producing fuels like butanol and ethanol using microorganisms by fermentation focusing on the environmental aspects and renewable nature of this mode of production. Butanol is a superior fuel and has more calorific value than ethanol (Qureshi and Blascher, 2000). Butanol has higher energy content (110,000 Btu's per gallon for butanol vs. 84,000 Btu per gallon for ethanol). It is six times less "evaporative" than ethanol and 13.5 times less evaporative than gasoline, can be shipped through existing fuel pipelines where ethanol must be transported via rail, barge or truck (Jones and Woods, 1986).
Butanol is an important industrial solvent and potentially a better fuel extender than ethanol. Current butanol prices as a chemical are at $3.75 per gallon, with a worldwide market of 370 million gallons per year. The market demand is expected to increase dramatically if green butanol can be produced economically from low cost biomass. In addition to its usage as fuel, butanol can be used as a solvent for a wide variety of chemical and textile processes, in organic synthesis and as a chemical intermediate. It is also used as paint thinner and a solvent in other coating applications where it is used as a relatively slow evaporating latent solvent in lacquers and ambient-cured enamels. It finds other uses such as a component of hydraulic and brake fluids (Mutschlechner et al, 2000). It is also used as a base for perfumes, but on its own has a highly alcoholic aroma.
Since the 1950s, most butanol in the United States is produced commercially from fossil fuels. The most common process starts with propene, which is run through an

hydroformylation reaction to form butanal, which is then reduced with hydrogen to butanol. Butanol is produced by fermentation, from corn, grass, leaves, agricultural waste and other biomass.
Production of industrial butanol and acetone via fermentation, using Clostridium acetobutylicum, started in 1916. Chime Wizemann, a student of Louis Pasteur, isolated the microbe that made acetone. Up until the 1920s, acetone was the product sought, but for every pound of acetone fermented, two pounds of butanol were formed. A growing automotive paint industry turned the market around, and by 1927 butanol was primary and acetone became the byproduct.
The production of butanol by fermentation declined from the 1940s through the 1950s, mainly because the price of petrochemicals dropped below that of starch and sugar substrates such as corn and molasses. The labor intensive batch fermentation system's overhead combined with the low yields contributed to the situation. Fermentation-derived acetone and butanol production ceased in the late 1950s.
Acetone butanol ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations. It was ranked second only to ethanol fermentation by yeast in its scale of production, and is one of the largest biotechnological processes ever known. The actual fermentation, however, has been quite complicated and difficult to control. ABE fermentation has declined continuously since the 1950s, and almost all butanol is now produced via petrochemical routes. In a typical ABE fermentation, butyric, propionic, lactic and acetic acids are first produced by C. acetobutylicum, the culture pH drops and undergoes a metabolic "butterfly" shift, and butanol, acetone, isopropanol and ethanol are formed. In conventional ABE fermentations, the butanol yield from glucose is low, typically around 15 percent and rarely exceeding 25 percent.
The production of butanol was limited by severe product inhibition. Butanol at a concentration of 1 percent can significantly inhibit cell growth and the fermentation process. Consequently, butanol concentration in conventional ABE fermentations is usually lower than 1.3 percent. The key problem associated with butanol production is

butanol toxicity /inhibition of the fermenting microorganism, resulting in low butanol titer in the fermentation broth. (Ezeji et al, 2007). Butanol is highly toxic to biological systems at quite low concentrations of 2% (Jones and Wood, 1986). This toxicity may be because butanol localizes in the plasma membrane and disrupts a number of physiological processes including membrane permeability, solute transport, maintenance of proton motive force, conformation and activity of intrinsic membrane proteins. Efforts are being made to improve the butanol tolerance level in different species of Clostridia with varying degree of success (Evan and Wang, 1988). Recent interest in the production of butanol has lead to re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture.
In the past 20+ years, there have been numerous engineering attempts to improve butanol production in ABE fermentation, including cell recycling and cell immobilization to increase cell density and reactor productivity and using extractive fermentation to minimize product inhibition. Despite many efforts, the best results ever obtained for ABE fermentations to date are still less than 2 percent in butanol concentration, 4.46 g/L/h productivity, and a yield of less than 25 percent from glucose. Optimizing the ABE fermentation process has long been a goal of the industry.
With that in mind, an alternative process was developed using continuous immobilized cultures of Clostridium tyrobutyricum and Clostridium acetobutylicum to produce an optimal butanol productivity of 4.64 g/L/h and yield of 42 percent. In simple terms, one microbe maximizes the production of hydrogen and butyric acid, while the other converts butyric acid to butanol. Compared to conventional ABE fermentation, this process eliminates acetic, lactic and propionic acids, acetone, isopropanol and ethanol production. The ABE fermentation process only produces hydrogen, butyric acid, butanol and carbon dioxide, and doubles the yield of butanol from a bushel of corn from 1.3 to 2.5 gallons per bushel. The drawbacks associated with such a process are two folds: having to maintain two sets of conditions for the two cultures, maintaining complete anaerobiosis in the immobilized system, dealing with the gases produced during the fermentation and their effect on the maintaining the integrity of the matrix used for immobilization.

In conventional ABE fermentations, the butanol yield from glucose is low—between 15%-25%—and the butanol concentration in the fermentation is usually lower than 1.3%. (Butanol at a concentration of 1% can significantly inhibit cell growth and the fermentation process.) There have been numerous efforts over the years to improve butanol yield by using a variety of techniques to minimize product inhibition.
In this respect, to develop a process for the maximum production and tolerance of this important fuel by process designing, standardization of media and fermentation conditions, strain improvement is of utmost importance (Agarwal et al, 2005). Physiological and nutritional factors such as initial sugar concentration, complex nitrogen sources, inoculum size, carbonate ion concentrations, pH and temperature of the growth medium are reported to be the most critical factors affecting both cell growth and butanol production (Samuelov etal, 1991;Nghiem^a/.,1997; Leeetal., 1999).
US Patent 4,757,010 and European patent application EP 00111683 provides an improved strain of Clostridium for increased tolerance to butanol. JP03058782 provides Clostridium pasteurianum CA 101 stock (FERM P- 10817) as a mutant of genus Clostridium bacterium having analog resistance to fermented intermediate of butanol and butanol producibility. US patent 4,539,293 demonstrates the use of co-culture of microorganisms of the Clostridium genus, one favors the production of butyric acid and the other supports the formation of butanol. Japanese patent application JP 63157989 provides production of butanol by culturing a different strain Clostridium pasteurianum var. 1-53 (FERM P-9074) in a liquid medium containing a carbon source, a nitrogen source and other inorganic salts at 28-33 deg.C under slightly acidic pH condition in anaerobic state for 2-4 days.
However the problems associated in these modified strains is that the use of genetically modified strains for fuel production cannot compete with the wild type as one needs to sterilize the feedstock to make sure that there is no competition for the genetically modified organisms. Further genetically modified organisms or various strains are expensive to develop and does not find relevance on high volume products.

Various alternative in situ/online techniques of butanol removal including membrane-based systems such as pervaporation, liquid-liquid extraction, and gas stripping are used.
US patent no. 4,777,135 describes a method of producing butanol by fermentation which comprises culturing under anaerobic conditions a butanol - producing microorganisms in a culture medium containing fluorocarbons. This process is not feasible on a commercial scale as the fluorocarbons are environmentally not safe
US Patent 4,605,620 provides a process for butanol by using a medium containing carbohydrate and phosphate, wherein the experiments were performed with a total phosphate content of 1.0-0.4 mmoles. This process poses a restriction wherein the phosphate limiting medium is required.
US patent 4,560, 658 provides a process for the production of butanol by fermentation of carbon containing compounds with Clostridium acetobutylicum wherein the fermentation is conducted in an aqueous medium containing a sufficient concentration of dissolved carbon monoxide. However the use of carbon monoxide make the process environmentally unsound.
US patent 4,520,104 provides a process for the continuous production of butanol by fermentation of carbohydrates with C. acetobutylicum. This process combines continuous inoculum production at a high dilution rate and cycling the fermentation broth through material which adsorbs butanol whereby a vigorous cell population is maintained in the fermentation reactor for extended periods of time. The process is devised to remove the butanol produced in the broth so as to prevent its toxicity on the cells
Japanese patent JP 62278989 provides a fermentation process for the production of acetone and butanol, by keeping a butanol-producing strain in resting state, adding a carbon source to the cell to effect the production of acetone and butanol in a short time, recovering and concentrating the butanol-producing strain, subjecting to the heat shock

and adding to a fermentation tank Heat shock is required in the process.- to activate the spores of Clostridium and is pretty routine.
Japanese patent application provides an anaerobic cellulolytic germ, e.g. Clostridium cellobioparum ATCC15832 or Ruminococcus albus ATCC27211, and Clostridium saccharoperbutylacetonicum are inoculated into a culture medium containing a material containing cellulose, e.g. wood, waste paper or pulp, as a main carbon source, and cultivated at 25-45°C and 4-9 pH under anaerobic conditions for about 2-20 days to collect the aimed compound, containing oxygen, and consisting essentially of butanol from the resultant culture. This process is time consuming and takes about 20 days for completion, hence not feasible on a large scale.
Japanese patent 63269988 discloses butanol fermentation wherein yeast is subjected to autodigestion in a fermentation tank and proliferated prior to the inoculation of butanol-producing strain. The space in the fermentation tank becomes anaerobic and the temperature increases by the proliferation of yeast to perform butanol fermentation. An inefficient autodigestion would lead to contamination of the broth by the yeast
US20050233031 provides a process for producing butanol which includes treating plant derived material to provide an aqueous liquor containing sugars in a fermentation process to produce a fermentation product. The process involves several steps and therefore cumbersome and tedious.
Japanese Patent JP 200535328801 provides a method for producing butanol in which a culture solution is prepared by using a formulation of the food residue with the Japanese distilled spirit lees and water and butanol fermentation is carried out in the culture solution. The use of Japanese distilled spirit is limited to the production experiments performed in Japan.
French patent FR2550222 provides a two stage process wherein a first stage of seeding
with Clostridium acetobutylicum and a second stage of seeding with a yeast which

produces ethanol, the second stage being commenced when the pH of the fermentation medium of the first stage has reached a minimum value. The invention applies in particular to the production of butanol, acetone and ethanol from sugarbeet and Jerusalem artichoke juices. The process requires pretreatment which makes it cumbersome.
Although, there are reports where microbes have been exploited for the production of butanol by fermentation, yet an economically viable biosynthetic process for butanol production is yet to be developed (Jesse et ah, 2002).
Thus looking into the need for a process which yields enhanced production of butanol, the present invention has focused on developing an ideal culture condition for the wild strain of Clostridium which will result in enhanced butanol tolerance and subsequently the in yields of butanol.
OBJECT OF THE INVENTION
It is the object of the present invention to provide an improved process for the production of high yield butanol using Clostridium acetobutylicum without any change in the strain of the microorganism.
It is the object of the present invention to provide optimal fermentation conditions for enhanced production of butanol, using Clostridium acetobutylicum.
It is the object of the present invention to provide a process with optimal fermentation conditions, which will result in increased butanol tolerance of the microorganism.
It is the object of the present invention to provide a culture condition for high yields of butanol fermentation.
It is the object of the present invention to provide a process for increased yields of butanol in a single batch fermentation conditions.

It is the object of the present invention to provide a process for biobutanol using various biomass.
It is the object of the present invention to provide a cost effective and industrially scalable process for butanol.
SUMMARY OF THE INVENTION
The present invention relates to an efficient process for the production of high yield of butanol using Clostridium acetobutylicum, without any change in the strain of the microorganism resulting in enhanced production of butanol. The present invention in particular aims at providing optimal culture conditions that would result in increased butanol tolerance of the microorganism. The present invention further aims at providing a cost effective and industrially scalable process for the production of butanol.
The novelty of the present invention is production of high yields of butanol (upto 20 g/L) in a single batch process, without stripping the butanol produced. Thus process doesn't involve any fed-batch step which would involve extra step of addition of nutrients. Neither is any solvent-stripping required for reaching this high yield. Unlike many reported processes which employ continuous mode of fermentation thereby increasing the chances of contamination, the present process can be completed in a single batch mode. Careful optimization of the medium and acclimatisation has resulted in a strain that is capable of producing and tolerating such high yields of butanol in the broth. Thus, all these parameters make the process of the present invention more cost-effective. Further the inventors have also been able to successfully demonstrate the process at 5 L scale.
In one aspect the present invention provides a process with increased butanol tolerance without the need of modifying the strain. In one preferred aspect the present invention provides tolerance to about 2.5% butanol concentration under optimized medium conditions directed to a process for providing the increased yield of butanol as provided

in this invention. The most probable reason for its high tolerance to butanol may be that the process optimization has resulted in the final set of physio-chemical conditions under which the above mentioned limitations are alleviated. For example the redox opotential, osmolarity , electron flow may have been altered under the optimized conditions. Certain set of enzymes required for butanol tolerance and production may have been activated or induced under the optimized conditions. The culture may have adapted during the course of the optimization process to high butanol level.
In one aspect the present invention provides a process with increased yields of butanol. In one preferred aspect the present invention reports a 9-fold increase in butanol production in 500 ml anaerobic bottles containing 300 ml of the optimized AnS medium as against the initial un-optimized AT medium (50 ml).
In one aspect the present invention provides a process for evaluation of the biobutanol using various biomass. In one preferred aspect the seeds from jatropha and banana stems were used. The yield of biobutanol using the seeds and stems pretreated under different conditions such as alkaline, acidic and microwave digestion was also studied.
In one aspect the present invention provides a process which can be scaled up on a large scale.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fig.l: Shows the effect of different pH on butanol production by Clostridium acetobutylicum after 84 h of incubation.

It was observed that though butanol was produced in the pH range 5.0 to 7.0 the optimal pH for production is 6.5, yielding 3.2 g /l of butanol in 84h.
Fig. 2.Effect of different temperatures (°C) on butanol production by Clostridium acetobutylicum after 84 h of incubation
Results on the effect of different temperatures (25, 33, 37, 39 & 45°C) showed that 3.0 g l\ of butanol was produced at 37 ± 2°C in 84 h. However at 25 and 55°C, no significant butanol production was observed
Fig. 3.Effect of different carbon sources (2%) on butanol production.
While studying the effect of nutritional factors, it was observed that none of the carbon sources tested supported as much butanol (3.2 gL-1) as was produced in the control viz. in glucose. This was followed by malt extract that supported 2.4 gL-1 of butanol in 84h
Fig. 4. Effect of different concentrations of glucose on butanol production.
It was found that 2.0% w/v glucose concentration supports maximum yield of 3.2 gL-'of butanol
Fig.5: Effect of different concentrations of malt extract on butanol production.
Concentration of malt extract, the second best sugar source, was varied in the medium (1.0- 10%). 4.82 gL-1 of butanol was produced when 5% malt extract was added along with 2% glucose in the AnS medium
Fig.6 :Effect of different nitrogen sources (1.0%) on butanol production
Beef extract was found to be the best nitrogen source among various nitrogen sources tested resulting in the production of 5.2gL_1 of butanol
Fig. 7. Effect of different concentrations of beef extract on butanol production

Optimization of concentration showed that 5.0 %w/v of beef extract is optimum for butanol production (7.8 gL-1)
Fig. 8. Effect of different metal ions (0.5%) on butanol production.
A significant increase in butanol production ( 11.1 gL-1) was achieved when the medium optimized so far was supplemented with Na2CO3 at 0.5%. This is followed by calcium ions that resulted in the production of 9.2gL-1 of butanol. Metal ion like Cu did not support any amount of butanol production
Fig.9. Effect of different concentrations of sodium carbonate on butanol
production.
It was observed that 0.5% w/v of Na2CO3 is optimal for butanol production (1 l0gL-1).
Fig.10. Effect of inoculum density on butanol production
It was observed that, 14.5 gL-1 of butanol was produced at the inoculum density of 1%.
However, with increase in the inoculum density beyond 2%, the production of butanol
declined.
Figure: 11 Fermentation profile of butanol at 5L scale
The fermentation profile at 5 L scale indicates that the production of butanol is much faster at higher scale with the yield reaching 20 g/L in 48 h and reaching a plateau thereafter.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term Butanol or biobutanol as used herein refers to n-butanol.
The term butanol tolerance as used herein refers to the ability of the bacterium to survive and grow in the presence of ³ 1.3 % butanol in the medium
The term Clostridium acetobutylicum refers to the bacteria that has the ability to produce butanol alongwith acetone and ethanol in a anaerobic fermentation

The term yield as used herein refers to amount of butanol produced in the fermentation broth in g/L.
As pH is one of the important factors that affect both growth and growth-associated production of molecules, butanol production was examined at different pH. The optimal pH for butanol production by Clostridium acetobutylicum in the present invention was 6.5. This is in accordance to the findings of Robson and Jones (1982), who reported that C acetobutylicum P262 showed good levels of solvent production within the pH range of 5.0-6.5. Similarly, Bielbl (1999) reported that Clostridium beijerinkii NCIMB 8052 showed much better growth and solvent production at pH 5.5 than at pH 5.0 or below.
The present invention has found that temperatures of 37 ± 2°C is the optimal temperature for butanol production from Clostridium acetobutylicum ATCC 10132. This is in contrast to the earlier findings of McCutchan and Hickey (1954) who reported a decrease (upto 23%) in the solvent production by Clostridium sp. at 37°C as against the fairly constant yields of 31% at 30 and 33°C.
The effect of carbon sources on butanol production by Clostridium acetobutylicum ATCC 10132, was studied and it was observed that glucose supported the highest butanol production. This was followed by malt extract, the second best carbon source. However, carbon sources like glycerol and sucrose supported a moderate amount of butanol. Sugars like rhamnose were not at all utilized by the strain. The most probable reason could be that the strain was unable to transport 2- deoxy glucose sugar.
The study on the concentration of the sugars revealed that 2% glucose, supported 3.2 gL-1 of butanol. Similar has been reported by Biebl (1999) who observed maximum butanol production by C. acetobutylicum ATCC 824 in the medium containing 2.8% of glucose. However, in most of the studies, 6-7% has been found to be the optimum glucose concentration for butanol production. In this direction Parekh et a/.(1998) reported that

6.0% glucose in the medium yielded 10.0g/l of butanol from C.beijerinkii 8052 strain after 90h of incubation.
Subsequently, when malt extract levels were varied in the medium in the range (1.0 - 10 % w/v) while keeping the concentration of glucose constant (at 2%), 4.8 gL-1 of butanol was produced at 5 % malt extract. Similarly, the effect of nutrition limitation on the onset and maintenance of solvent production has been investigated by a number of other workers. For example, Long et a/.(1984) reported that in the batch fermentation using Clostridium acetobutylicum P262, only acids were produced when the concentration of the carbon sources was limited.
On supplementation of the medium with 5 % beef extract, a maximum of 5.3 gL-1 of n-butanol was produced as against the control (1% peptone) wherein 4.8 gL-1 of butanol production was observed. The most probable reason for beef extract being good source of nitrogen is because it not only provides nitrogen but also vitamins, and other nutrients which are essential for the growth of the microorganism
Metal ions are known to play an important role in maintaining cellular metabolism and enzyme activities (Isar et al., 2006). A significant increase in butanol production was achieved when the medium optimized so far was supplemented with Na2CC>3. The reason could be that Na+ is a cofactor for most of the enzymes involved in the anaerobic pathway. Strobel et al. (1991) and Lee et al, (2000) reported that sodium ions are an important factor for the nutrient uptake. These ions are involved in the formation of transmembrane pH gradient, cell motility and intracellular pH regulation. Amongst different salts of sodium ion investigated, it was found that carbonate and bicarbonate were the most effective radicals for the production resulting in approximately 11.2 gL"1 of butanol.
Change in the inoculum density from 1 - 2% did not significantly influence butanol production. However, an increase in inoculum density beyond 2% results in a decline in

the production of the solvent. The most probable reason could be that as the inoculum size is increased beyond 2%, there is nutrition limitation.
The present invention provides the effect of different physiological and nutritional parameters on butanol production by Clostridium acetobutylicum ATCC 10132. This strain initially produced 0.2 gL-1 of butanol in 84 h in Alternate Thioglycollate medium. However, when process optimization was employed, 20.0 gL-1 of butanol was produced in 300 ml of the optimized AnS medium consisting of Glucose (2%),Beef extract (5%), Malt extract (5%) Yeast extract (0.5%),K2HPO4 (0.3%), Na2CO3 (0.6%), (NH4)2SO4 (0.1%), CaCl2.2H2O (0.02%), MgCl2.7H2O (0.02%), Na2S(0.002%), at pH 6.5, 37° C, under static conditions (with gentle intermittent manual shaking) in 96h. Interestingly, it was also observed that the strain is tolerant to 2.5% butanol under optimized medium and conditions.
The verification of the process in 300 ml medium clearly indicated that the process can be scaled up to higher size and about 20 gL-1 of butanol could be produced. The most probable reason for this increase in the yield could be the availability of more head space in bigger size bottles as against the smaller bottles.
The Clostridium strain used in the present invention has shown tolerance to 2.5% butanol. The most probable reason for its high tolerance to butanol may be that the process optimization has resulted in the final set of physio-chemical conditions under which the above mentioned limitations are overcome. For example, the redox opotential, osmolarity, electron flow may have been altered under the optimized conditions. Certain set of enzymes required for butanol tolerance and production may have been activated or induced under the optimized conditions. The culture may have adapted during the course of study (optimization process). Since the actual tolerance level of this strain has never been reported earlier, it may also be the intrinsic un-exploited property of the strain. In several instances, a strain normally not reported to produce a biomolecule starts making it it in significant amounts after process optimization (Isar et al, 2006).

Further studies on utilization of various biomass for butanol production using the conditions described above was performed. In particular , the biomass studied was jatropha seed cake and banana stem. Various pretreatments were given to the biomass prior to its use for the production of butanol. These pretreatments make the sugars from the biomass available for fermentation. The pretreatments include subjecting to fungal degradation, acid treatment, alkali treatment or microwave digestion.
Jatropha seed cake was incubated with fungal culture Pluroteous osteratus at 23oC for a month and the biomass was extracted with a buffer. After extraction, the biomass was used as a supplement at different concentrations (1, 3, 5 and 10%) in Anaerobic sugar medium having 0.5% calcium carbonate. A yield of 8.4 g/1 of butanol was obtained after 48h using 3% of the fungal pretreated jatropha seed cake.
In addition to this, when 1% soybean meal was added in AnS medium supplemented with 0.5 % calcium carbonate and 4 % of Beef extract a maximum yield of 10.5 g/L of butanol after 96h.
Experiments were also done on Banana stem and jatropha seed pretreated with sodium hydroxide. The alkali treated biomass was supplemented at various concentrations in AnS medium having 0.5% calcium carbonate. At 1 % supplementation, a yield of 8.1 g/L butanol was obtained in AnS medium containing predigested banana stem as against 5.0 g/L butanol with predigested jatropha seed cake.
With microwave digested biomass supplementation, a yield of 6.9 g/1 of butanol was obtained after 84h when 2% microwave treated banana stem was added to AnS medium having 0.5% calcium carbonate. With 1 % microwave digested jatropha seed cake supplementation, 7.0 g/1 of butanol was obtained after 84h.
Supplementation with 0.1 N Sulphuric acid treated biomass in AnS medium having 0.5 % calcium carbonate resulted in a yield of 5.0 g/1 of butanol after 84h for 1% banana stem and a yield of 4.0 g/1 of butanol after 84h for 1% jatropha seed cake.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: Organism and growth conditions
Clostridium acetobutylicum ATCC 10132 was grown in 125 ml anaerobic bottles containing 50 ml of the Anaerobic Sugar (AnS) medium with the composition (gL-1 ): Glucose (20.0); Peptone (10.0); Yeast Extract (5.0); K2HPO4 (3.0); NaCl (1.0); (NH4)2SO4 (1.0); CaCl2.2H2O (0.2); MgCl2.6H2O (0.2); and Na2CO3 (1.0), pH 6.5.
The medium was sterilized (15min, at 121°C) in glass bottles sealed with butyl rubber bungs. The headspace was filled with by N2, and Na2S.9H2O (0.02%) was added to remove traces of dissolved oxygen (Samuelov et al. 1991; Lee et al.,2000). The reduced medium was inoculated with 2 % seed inoculum and incubated at 37 ± 1°C for 96h with intermittent gentle shaking.
EXAMPLE 2: Methods for estimation of butanol
After the desired incubation period, the culture was withdrawn from the sealed vials using sterile disposable syringes and was centrifuged at 8000 x g in a Eppendorf centrifuge (model no 54151) for 10 min. Supernatant was filtered through 0.45(j. filter. The sample (20 ul) were analysed by HPLC, (Ehrlich et al, 1981) on a PRP 300X column (Hamilton) using acetonitrile and 0.5mM H2SO4 (9:1) as the mobile phase, at a flow rate of 1.5 ml/min at 37°C. Butanol was detected using a RI detector.

EXAMPLE 3: Process in batch fermentation
Various media tested for butanol production were Anaerobic sugar (AnS) medium, (Isar et al., 2006); Reinforced Clostridial (RC) medium, (Lin and Blaschek,1983); Soluble Starch Medium (SSM), (Moreira et al., 1981); Alternate Thioglycollate (AT) media, (Lin and Blaschek, 1983); Potato Starch media (PSM ), (Fouad et al.,1976). Amongst the media tested, AnS medium was the best yielding 3.2 g /l of butanol (Table 1). Butanol production was optimized in the AnS medium where the effects of different physiological and nutritional parameters were studied.
Table:1 Butanol production (g/1) in different media

Time (h) Media
AnS RC AT SSM PSM
12 0.31 - - - -
24 0.43 - 0.02 - -
36 0.6 0.01 0.03 - -
48 1.2 0.02 0.09 0.01 0.09
60 2.1 0.05 0.1 0.04 0.09
72 2.2 0.10 0.2 0.07 0.10
84 3.2 0.13 0.2 0.09 0.11
96 3.0 0.11 0.1 0.08 0.09
Subsequently, the effect of pH (5- 7) and temperature (25 - 45 °C) was studied in the selected medium for butanol production. It was observed that the optimal pH for production is 6.5, yielding 3.2 g /l of butanol in 84h (Fig. 1). Results on the effect of different temperatures (25, 33, 37, 39 & 45°C) showed that 3.0 g /l of butanol was produced at 37 ± 2°C in 84 h(Fig. 2).
Optimization of the nutritional parameters including carbon source, nitrogen source, metal ions required for maximum production of butanol was carried out. Various carbon sources employed include glucose, fructose, sucrose lactose, malt extract and glycerol at

a concentration of 2.0 % w/v in the medium. None of the carbon sources supported as much butanol production (3.2 gL-1) as glucose in 84h (Fig. 3). Further, 2.0% w/v glucose gives the highest yield i.e. 3.2 gL-1 of butanol (Fig. 4). Approximately, 4.82 gL-1 of butanol was produced when 5% malt extract was added along with 2% glucose in the AnS medium (Fig 5).
For nitrogen source optimisation, peptone (1% w/v) in the medium was replaced by different inorganic (ammonium hydrogen phosphate, ammonium chloride, sodium nitrate and urea) and organic nitrogen sources (yeast extract, beef extract, corn steep liquor, and tryptone) at the same concentration. Beef extract was found to be the best nitrogen source resulting in the production of 5.2 gL-1 of butanol (Fig.6). Optimization of concentration showed that 5.0 % w/v of beef extract is optimum for butanol production (7.8 gL-1) (Fig. 7).
To assess the effect of metal ions, the carbonate / sulphate / chloride salts of different metal ions (Na+, Mg++, Ca++, Zn++, K+ Mn++) at 0.5% concentration were separately added in the medium. A significant increase in butanol production (11.1 gL-1) was achieved when the medium optimized so far was supplemented with Na2CO3 at 0.5%. Metal ion like Cu did not support any amount of butanol production (Fig.8).
Among the various salts of sodium investigated including chloride, carbonate, sulphate and phosphate, carbonate was most effective for the production resulting in 11.2 gL-1 of butanol (Table 2). Further, upon optimizing the concentration of Na2CO3 , it was observed that 0.5% w/v is optimal for butanol production (11.0 gL-1) (Fig. 9).
Table 2 : Effect of different salts of the selected metal ion on butanol production

Mineral Salts Butanol, gL-1
Na2CO3 11.2
Na2SO4 9.1
NaCl 9.7
NaNO3 10.2

Na2HPO4 9.0
NaHCO3 11.1
Effect of inoculum size on butanol production was investigated. The optimized medium was inoculated with different inoculum size (1-10 %). Inoculum at 1 % was found to yield maximum butanol (14.5 gL-1) (Fig. 10). However, with increase in the inoculum density beyond 2%, the production of butanol declined
EXAMPLE 4: Determination of butanol tolerance :
The butanol tolerance level of the strain used in the present investigation was evaluated. The strain was inoculated in 50 ml of the optimized AnS medium containing different concentrations of butanol (0.5%, 1.0%, 1.3% 1.5%, 1.8%, 2.0%, 2.5%). Bottles were incubated for 96h at 37°C under static conditions with gentle intermittent manual shaking. The strain was found to tolerate upto 2.5 % butanol in the medium.
EXAMPLE 5: Verification of the process in 300 ml medium :
Butanol production in the optimized medium was validated in 500 ml anaerobic bottles containing 300 ml of the optimized medium. 1% of the inoculum was aseptically added
with the help of syringe into the bottles and incubated at 37°C for 96h.A maximum of 20 gL-1 of butanol was produced.
EXAMPLE 6: Scale up of the bio-butanol production at 5 L scale
The butanol production in optimized medium using Clostridium acetobutylicum ATCC 10132 was scaled up to 5 L level in a 10L fermentor (Bioflow IV, NBS, USA). The optimized AnS medium was sterilized in situ at 110°C for 15 min. The medium was inoculated with 2 % of the seed inoculum (OD66onm = 0.6) and fermentation was carried out at 37±1°C for 84 h. The impeller speed was initially adjusted to 100 rpm and compressed sterile N2 was initially flushed for 30min to create anaerobic environment and was subsequently sparged intermittently into the fermentor at rate of 0.5 vvm. Samples were harvested periodically at an interval of 12h and analyzed for butanol production

using HPLC and GC. The fermentation parameters such as temperature, N2 supply and agitation rate were continuously monitored and regulated.
The butanol production started at 24 h in the fermentor and a maximum of 20.3 gL-1 of butanol was produced in 48h. Thereafter, there was no significant increase in the production of butanol (Figure 11 & Table 3) indicating a significant reduction in the production time of butanol at higher scale.
Table 3 : Fermentation profile at 5 L scale

Incubation period (h) Butanol Production (gL-1)
12 -
24 5.9
36 14.6
48 20.3
60 21.2
72 19.4
84 19.1
EXAMPLE 7: Study of the various biomass for biobutanol production
The results obtained using various biomass is listed in Table 4.
1. Pretreatment of Jatropha seed with fungal culture Pluroteous osteratus
Finely ground jatropha seeds (100 g, approx. mesh size 50 mm) was suspended in 50 ml of basal salt medium (0.5 % glucose, 0.1 % KH2PO4, 0.05 % MgSO4.7H2O, 0.05 % KCl, 0.05 % yeast extract). To this 50 ml each of stock solution I and stock solution II were added (Stock solution I: 0.02 % FeSO4.7H2O and Stock solution II: 0.016% Mn(CH3COO)2.4H20, 0.004% Zn(N03)2.4H20, 0.1 % Ca(N03)2.4H20, 0.006% CuSO4.5H20). The entire mixture was autoclaved at 121°C for 30 min and inoculated with 5-6 small malt extract (2%) agar blocks (lcmx 1cm) of two week old Pluroteous osteratus (grown at 25° C). The flask was incubated at 23°C for 30 days. After incubation, 500ml of 50mM citrate buffer (pH 5.0) was added and the contents were mixed thoroughly by shaking on the rotary shaker (200rpm) for 2h.The contents of the

flask was squeezed using muslin cloth and the solid biomass was used as a supplement at different concentrations (1, 3, 5, and 10%) in 50 ml Anaerobic Sugar (AnS) Medium having 0.5% calcium carbonate contained in 125 ml of the sealed anaerobic bottles. A maximum yield of 8.4 g/1 of butanol was obtained after 48h using 3% of the fungal pretreated Jatropha seed cake.
In addition to this, when 1% soybean meal was added in AnS medium supplemented with 0.5 % calcium carbonate and 4 % of Beef extract a maximum yield of 10.5 g/L of butanol after 96h.
2. Banana stem and jatropha seed pretreated with Sodium hydroxide digestion
method
(a) The banana stem was cut into small pieces and 100 g of these banana stem pieces were kept in 500 ml of 0.5M NaOH for 24 h with intermittent gentle shaking. After 24 h, these pieces were washed with running tap water to remove the alkali and were added to the AnS medium having 0.5% of CaCO3 at different concentrations for studying their effect on the production of butanol. The highest production of butanol 8.1 g/L was achieved in AnS medium containing 1% of the predigested banana stem.
(b) The jatropha seeds were finely ground and were put into 500 ml of 0.5M NaOH for 24 h at room temperature. After 24 h, these pieces were washed with running tap water to remove sodium hydroxide. The different concentrations of pretreated jatropha seed cake were added to ANS media (containing 0.5% calcium carbonate).The highest production of butanol 5.0 g/L when 1% of the predigested jatropha seed was added to the AnS medium having 0.5% calcium carbonate.
3. Microwave Digestion for banana stem and Jatropha seeds
(a) The banana stem was cut into the small pieces and partially digested in microwave oven for 10 min. The digested banana stem was ground in a mixer and finely ground banana was again digested in a microwave for 5 min. Sugar was analyzed by DNS method. A maximum yield of 6.9 g/1 of butanol was obtained after 84h when 2%

microwave treated banana stem was added to AnS medium having 0.5% calcium carbonate.
(b) The finely ground jatropha seed (20g) was added to 500 ml distilled water and cooked for 10 min in microwave oven. Microwave treated jatropha seeds were cooled down and again treated for 10 min in microwave oven. Sugar was analyzed by DNS method. The results showed that a maximum of 7.0 g/1 of butanol was obtained after 84h using 1% microwave treated jatropha seed cake in the AnS medium supplemented with 0.5% calcium carbonate and 0.5ml glycerol.
4. Banana stem and jatropha seed pretreated with 0. IN Sulphuric acid
(a) The banana stem was cut into small pieces. Approximately, 100 g of these banana
stem pieces were kept in 500 ml of 0.1N sulphuric acid for 24 h. These pieces were then
washed with running tap water and were dried completely. The different concentration of
this pretreated banana stem was then added to ANS media containing 0.5% calcium
carbonate. The results showed that a maximum yield of 5.0 g/1 of butanol was obtained
after 84h when 1% acid treated banana stem was added to AnS medium containing 0.5%)
calcium carbonate.
(b) The jatropha seed cake was ground. Approx. 50 g of the ground jatropha seeds were
treated with 500 ml of 0.1N sulphuric acid for 24hours. After 24 h, these pieces were
washed with running tap water to remove sulphuric acid. The predigested jatropha seed
cake was dried completely. This pretreated jatropha seed cake was added to ANS media
(having 0.5%) calcium carbonate) at different concentrations. A yield of 4.0 g/1 of butanol
was obtained after 84h using 1%> acid pretreated jatropha seed cake.
Table 4. Butanol production using biomass

Biomass Pre-treatment Butanol, g/L
Jatropha seed cake Fungal treatment with Pluroteous osteratus 8.4
0.5 M NaOH 5.0
0.1 N Sulphuric acid 4.0
Microwave digestion 7.0
Banana Stem 0.5 M NaOH 8.1
0.1 N Sulphuric acid 5.0
Microwave digestion 6.9
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Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.

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Patent Number

269325

Indian Patent Application Number

2544/MUM/2007

PG Journal Number

42/2015

Publication Date

16-Oct-2015

Grant Date

15-Oct-2015

Date of Filing

24-Dec-2007

Name of Patentee

RELIANCE LIFE SCIENCES PRIVATE LIMITED

Applicant Address

DHIRUBHAI AMBANI LIFE SCIENCES CENTRE, R-282, TTC AREA OF MIDC, THANE- BELAPUR ROAD, RABALE, NAVI MUMBAI

Inventors:

#	Inventor's Name	Inventor's Address
1	JASMINE ISAR	DHIRUBHAI AMBANI LIFE SCIENCES CENTRE, R-282, TTC AREA OF MIDC, THANE- BELAPUR ROAD, RABALE, NAVI MUMBAI-400701
2	VIDHYA RANGASWAMY	DHIRUBHAI AMBANI LIFE SCIENCES CENTRE, R-282, TTC AREA OF MIDC, THANE- BELAPUR ROAD, RABALE, NAVI MUMBAI-400701
3	PRADEEP VERMA	DHIRUBHAI AMBANI LIFE SCIENCES CENTRE, R-282, TTC AREA OF MIDC, THANE- BELAPUR ROAD, RABALE, NAVI MUMBAI-400701

PCT International Classification Number

C12P7/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA