FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPEC1FICA TION
(See section 10, rule 13)
1. Title of the invention
A PROCESS FOR PRODUCTION OF ORGANIC ACID
2. Applicant(s)
Name Nationality Address
TATA CHEMICALS LTD INDIA BOMBAY HOUSE, 24 HOMIMODI STREET,
MUMBAI-400001
3. Preamble to the description
COMPLETE SPECIFICA TION
The following specification particularly describes the invention and the manner in which it is
to be performed.

The document generally discloses an isolated microorganism capable of producing organic acid. In particular the document discloses an isolated facultative anaerobe microorganism capable of producing succinic acid and lactic acid.
BACKGROUND
Organic acids such as lactic acid and succinic acid have wide applications in food, pharmaceutical, agriculture and chemical industries as biodegradable polymers, food additives, and intermediate products of other compounds.
Succinic acid a C4-dicarboxylic acid (butanedioic acid) is currently used in the preparation of bulk chemicals such as surfactants, ion chelators and food additives as well as in the preparation of fine chemicals including supplements used in pharmaceuticals, antibiotics and vitamins. Succinic acid is predicted to be one of the future platform chemicals because huge variety of bulk chemicals such as 1,4-butanediol, tetrahydrofuran, y-butyrolactone, biodegradable polymers etc. can be produced from it by chemical conversion.
Lactic acid a C3 monocaboxylic acid is used as a food additive, in pharmaceuticals and for the production of other organic chemicals, including ethyl lactate, acrylic acid, propylene glycol, and polyactide.
Acetic acid is a C2 monocarboxylic acid, also known as ethanoic acid, is widely used in food industry as it gives vinegar its sour taste and pungent smell. The largest single use of acetic acid in chemical and polymer industry is in the production of vinyl acetate monomer, closely followed by acetic anhydride and ester production.
Propionic acid is also a C3 monocaboxylic acid, widely used as a preservative for both animal feed and food for human consumption. In more specialized applications, it is also used to make pesticides and pharmaceuticals. The esters of propionic acid have fruit-like odors and are sometimes used as solvents or artificial flavorings.
Currently, succinic acid and lactic acid are produced through petrochemical process, which raises concerns about environmental pollution and sustainable development. The production of these organic acids by microbial fermentation provides a simple and environmentally friendly process for industrial production of these organic acids. Microorganisms have been isolated and studied for succinic acid and lactic acid production from different sources. Among them three naturally occurring bacteria Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens and Mannheimia

succiniciproducens are known to produce succinic acid as a major fermentation product along with acetate, ethanol, formate and lactate as co-products at various levels under anaerobic conditions.
However, most microorganisms currently available for production of succinic acid and iactic acid are anaerobic microorganisms that require zero oxygen concentration to survive. The need of strictly anaerobic conditions requires additional expensive equipments for the production of these organic acids adding to the cost of their production.
Therefore, there is a need for a microorganism that can tolerate oxygen and is capable of producing succinic acid and lactic acid at a high yield. There is also a need for a process that would allow for microbial production of succinic acid and lactic acid in a simple and efficient manner.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:
The accompanying drawing illustrates the preferred embodiments of the invention and together with the following details description serve to explain the principles of the invention.
Figure 1 is a microscopic observation of the isolated bacterium having the access number MTCC 5488.
SUMMARY
An isolated bacterial strain having accession number MTCC 5488 is disclosed.
In accordance with an aspect, a process for producing organic acids by biotransformation is also disclosed. The process includes inoculating a fermentation medium with a 2-6% inoculum of microbial strain having accession number MTCC 5488 in an anaerobic environment. The process further includes purging the fermentation medium with one or more of air, carbon dioxide or nitrogen followed by incubating the same at a temperature in the range of 32-40°C, and obtaining organic acids from the fermentation medium.
In accordance with an aspect, a method for isolating a bacterial strain is also disclosed. The method includes inoculating bovine rumen fluid in a nutrient broth having 1% glucose and incubating the same at temperature in the range of 32-40°C. The process further includes sub-culturing the culture from the preceding step in a fresh nutrient broth having 1% glucose and incubating the same at a temperature in the range of 32-40°C. The

process further includes inoculating the culture from the preceding step on a nutrient agar plate and incubating the same in an anaerobic environment at a temperature in the range of 32-40°C. The process further includes inoculating a colony from the nutrient agar plate in a tryptic soy broth and incubating the same in an anaerobic environment at a temperature in the range of 32-40°C and isolating a bacteria) strain producing 1gm/lt of succinic acid and lactic acid.
DETAILED DISCRIPTION
To promote an understanding of the principles of the invention, reference will be made to the embodiment illustrated in the drawing and specific language will be used.to describe the same, it will nevertheless be understood that no limitation of scope of the invention is thereby intended, such alterations and further modifications in the described method and such further applications of the principles of the inventions as illustrated therein being contemplated as would normally occur to one skilled in art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
An isolated facultative anaerobe capable of producing organic acid is disclosed. More particularly an isolated microbial strain capable of producing one or more of succinic acid, lactic; acid, acetic acid and propionic acid is disclosed. The microbial strain has been isolated from bovine rumen fluid of buffalo.
To isolate the microbial strain, bovine rumen fluid was collected from buffalo. The 10% (volume/volume) bovine rumen fluid was used as an inoculum to inoculate nutrient broth containing 1 percent glucose. The nutrient broth containing 1 percent glucose inoculated with the bovine rumen fluid is incubated at a temperature in the range of 32 to 40°C. By way of specific example, the incubation is preferably done at 37°C temperature. The incubation is carried out for a period of 48 to 72 hours. By way of specific example, the incubation is carried out for 72 hours. After 72 hours this culture was sub-cultured in fresh sterilized nutrient broth with 1 percent glucose and incubated at a temperature in the range of 32 to 40 °C and for a period of 48 to 72 hours. Specifically, the incubation is carried out for 48 hours at 37°C to obtain a final grown culture.

By way of specific example, the nutrient broth used for culturing of microbial strain comprises of 5gm/lt peptic digest of animal tissues, 5gm/lt sodium chloride, 1.5gm/lt of beef extract and 1.5gm/lt of yeast extract.
The final grown culture is transferred to a nutrient agar plate and incubated in an anaerobic environment at a temperature in the range of 32 to 40 °C. The culture may be diluted with a phosphate buffer before inoculation on the nutrient agar plate. By way of specific example, the final grown culture was diluted to 10-4 in phosphate buffer and 0.1 ml of this diluted sample was spread on nutrient agar plates. The plates were incubated in an anaerobic jar with carbon dioxide atmosphere at a temperature of 37°C. A colony from the nutrient agar plate is inoculated in a nutrient broth and incubated in an anaerobic environment at a temperature in the range of 32 to 40 °C. By way of specific example single, well isolated colonies were picked up and transferred to test tubes containing 5 ml sterilized tryptic soy broth and incubated in an anaerobic jar with carbon dioxide atmosphere for 48-72 hours at 37°C. After 48 hours of incubation, the succinic acid concentration was analyzed by high performance liquid chromatography (HPLC, Aminex, HPX - 87H column). A strain producing more than 1 grams/litre of succinic acid and lactic acid was selected. The isolated strain was found to be gram-negative rod or coccobacillus, non-spore forming and facultative anaerobe.
The isolated microbial strains were further characterized genetically using 16S rRNA (ribosomal RNA) gene sequencing. By way of a specific example, genomic DNA was isolated from the screened strains using Zymo Research® kit and used as template in the Polymerase Chain Reaction (PCR) to isolate the 16S rDNA fragment. This Polymerase Chain Reaction (PCR) was carried out using 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'- TACGGYTACCTTGTTACGACTT - 3') primers, the primer numbers corresponding to that of the E. coli gene. The PCR fragments so obtained were then cloned into pTZ57R vector (Fermentas®) and sequenced using M13 forward and reverse primers. The sequence similarity of the 16S rDNA to other known strains was analyzed using the database at http://greengenes.lbl.gov/. The strains showed closest similarity to Corynebacterium vitaeruminis strain with 99.13 % similarity score.
The isolated microbial strain was capable of producing both succinic acid and. lactic acid by fermentation of various carbohydrates. Furthermore, the isolated microbial strain may also additionally produce acetic acid and propionic acid by controlling the environment, as illustrated in examples 5 and 6.

In accordance with an aspect, the growth of isolated microbial strain was observed at temperature of 32°C to 40°C. By way of specific example the optimal growth temperature was found to be 37°C. In accordance with an aspect, growth of the isolated microbial strain was observed at pH of 5.5 to 9.5. Optimal growth was observed at pH of 7.5.
The isolated microbial strain has been deposited for patent purposes in the The Microbial Type Culture Collection and Gene Bank at IMTECH, Chandigarh, India and has been given the accession number MTCC 5488.
A process for production of organic acid is disclosed. In particular, a process for production of succinic acid and lactic acid is disclosed. The process may also be used to produce acetic acid and propionic acid in addition to the succinic acid and lactic acid.
The process comprises of culturing the isolated microbial strain having accession number MTCC 5488 or a mutant or variant thereof capable of producing organic acids including succinic acid and lactic acid in a suitable fermentation medium containing carbohydrate in the presence of carbon dioxide for a predefined period of time till the carbohydrate present in the medium is converted into succinic acid and lactic acid.
A 2 to 6% inoculum of the microbial strain having accession number MTCC 5488 is inoculated on the fermentation medium in an anaerobic environment.
In accordance with an aspect, the 2-6% inoculum comprises of isolated microbial strain, in tryptic soy broth.
In accordance with an aspect, the fermentation medium may include any carbohydrate that can be fermented by the isolated bacterial or its mutant or variant. Such carbohydrate may include but is not limited to glucose, sucrose, dextrose, fructose, lactose, soluble starches, corn syrup, cane juice, sweet sorghum juice, molasses, sugar syrup or biomass hydrolyzates of cane bagasse.
In accordance with an aspect, the fermentation medium in addition to carbohydrates may contain an appropriate carbon source, suitable minerals, salts, cofactors, buffers and other components suitable for the growth of the bacterial culture and promotion of the enzymatic pathway necessary for production of succinic acid and lactic acid.
By way of specific example, the fermentation medium comprises of 20 grams/litre of glucose, 10 grams/litre of peptone, 5 grams/litre of yeast extract, 3 grams/litre of

K2HP04, 1 grams/litre of NaCl, 0.02 grams/litre of CaCl2.2H20, 0.02 grams/litre of MgCl2.6H20 and 1 grams/litre of Na2C03.
The pH of the fermentation medium may be between 5.5 to 9.5 and preferably at a pH of 7.
In accordance with an aspect, the process for production of organic acids can be performed with fed batch fermentation process with intermittent or continuous supply of carbon source.
The incubation of the microbial culture may be carried out for a predefined period of time in either batch, fed-batch or continuous culture conditions in the presence of carbon dioxide.
The incubation may be carried out at a temperature in the range of 32°C to 40°C and preferably the incubation is carried out at a temperature of 37°C.
The organic acids, such as succinic acid and lactic acid produced may be separated from the fermentation medium by any known method.
By way of specific example:
A loopful of culture of the isolated microbial strain bacterial strain growing on tryptic soy agar plates was inoculated into test tubes containing 5 ml of sterilized tryptic soy broth and incubated in an anaerobic jar with a carbon dioxide atmosphere for 24 hours at 37°C temperature. After 24 hours, 2 to 6 % (volume/volume) inoculum was used to inoculate in 150 ml anaerobic bottles containing 50 ml media consisting of 20 grams/litre of glucose, 10 grams/litre of peptone, 5 grams/litre of yeast extract, 3 grams/litre of K2HP04, 1 grams/litre of NaCl, 0.02 grams/litre of CaCl2.2H20, 0.02 grams/litre of MgCl2.6H20 and 1 grams/litre of Na2C03. The pH of the production fermentation media was adjusted to 7.0 before autoclaving. After inoculation, carbon dioxide gas was purged into media for 5 minutes and then inoculated bottles were incubated at 37°C temperature and 160 rpm on a rotary shaker. After 24 hr hours of incubation, lactic acid and succinic acid concentrations were determined by HPLC, which were found to be of 4.13gm/lt and 1.98gm/lt respectively.
The following examples are provided to explain and illustrate certain preferred embodiments of the process of the invention.

Example 1:
Isolation and selection of organic acids producing bacteria:
To isolate microbial strain, bovine rumen fluid was inoculated at 10% concentration into 500ml nutrient broth containing 1% glucose. This culture was incubated under anaerobic conditions with carbon dioxide atmosphere at 37° C for 72hrs. After 72 hours this culture was sub-cultured into fresh sterilized nutrient broth with 1 % glucose and incubated for 48 hrs at 37°C to obtain the final grown culture. The final grown culture was diluted to 10-4 in phosphate buffer and 0.1 ml of this diluted sample was spread on nutrient agar plates. The plates were incubated in an anaerobic jar with Carbon dioxide atmosphere at a temperature of 37°C. Single, well isolated colonies were picked up and transferred to test tube containing 5 ml sterilized tryptic soy broth and incubated in an anaerobic jar with carbon dioxide atmosphere for 48 hours at 37°C. After 48 hours of incubation, the succinic acid concentration was analyzed by high performance liquid chromatography (HPLC, Aminex, HPX - 87H column). A strain producing more than 1 g/1 of succinic acid & lactic acid was selected.
Example 2:
Characteristics of Corynebacterium vitaeruminis MTCC 5488: The isolated strain Corynebacterium vitaeruminis MTCC 5488 had coccobacillus cell morphology with non-spore forming and facultative anaerobic growth characteristics (Figure 1). This strain MTCC 5488 was capable of producing both succinic acid and lactic acid by fermentation of various carbohydrates. In accordance with an aspect, the growth of isolated strains was observed at temperature of 32°C to 40°C. By way of specific example the optimal growth temperature was found to be 37°C. In accordance with an aspect, growth of the isolated strain was observed at pH of 5.5 to 9.5. Optimal growth was observed at pH of 7.5. The biochemical profile of isolated bacteria was studied using rapid carbohydrate kit (HiMedia Laboratories Pvt. Ltd.). To accomplish that, initially single, well isolated colonies were picked up and transferred to test tube containing 5 ml sterilized tryptic soy broth and incubated in an anaerobic jar with carbon dioxide atmosphere for 24 hours at 37°C under static condition. After 24 hrs of growth the 50 micro liters of cell suspensions was transferred to carbohydrate test strips. These inoculated strips were incubated at 37°C under static condition for 24 hrs. During incubation, carbohydrates are fermented to acid which reduces the pH detected by the

color change of an indicator present in the strips. The results of this biochemical profile test as shown in table 1 can be used for the strain identification. Results show that isolated strain utilized and produced acids from dextrose, fructose, galactose, sucrose, mannose and cellobiose. Isolated strain also utilized salicin and ONPG.
Table 1: Biochemical profile of Corynebacterium vitaeruminis MTCC 5488

Substrate Biochemical profile of Corynebacterium MTCC 5488 vitaeruminis
Lactose -
Xylose -
Maltose -
Fructose +
Dextrose +
Galactose +
Raffinose -
Trehalose -
Melibiose -
Sucrose +
L-Arabinose -
Mannose +
Inulin -
Sodium Gluconate -
Glycerol -
Salicin +
Glucosamine -
Dulcitol -
Inositol -
Sorbitol -
Mannitol -
Adanitol -
α-M ethyl-D-glucoside -
Ribose -

Rhamnose -
Cellobiose +
Melezitose -
α-Methyl-D-mannoside -
Xylitol -
ONPG +
Esculin -
D-Arabinose -
Citrate -
Malonate -
Sorbose -
Example 3:
Identification of the selected bacteria producing organic acid: The isolated microbial strain producing organic acids was identified using 16S rDNA (ribosomal DNA) gene sequencing. By way of a specific example, genomic DNA was isolated from the selected strain using Zymo Research kit and used as template in the Polymerase Chain Reaction (PCR) to isolate the 16S rDNA fragment. This Polymerase Chain Reaction (PCR) was carried out using 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'- TACGGYTACCTTGTTACGACTT - 3') primers, the primer numbers corresponding to that of the E. coli 16S rDNA gene. The PCR fragments so obtained were then cloned into pTZ57R vector (Fermentas®) and sequenced using Ml3 forward and reverse primers. The sequence similarity of the 16S rDNA to other known strains was analyzed using the database at http://greengenes.lbl.gov/. The strains showed closest similarity to Corynebacterium vitaeruminis strain with 99.13 % similarity score. This strain was deposited with accession number 5488 at the Microbial Type Culture Collection (MTCC) in IMTECH, Chandigarh, India.
Example 4:
Fermentation of Corynebacterium vitaeruminis under Anaerobic Conditions with carbon dioxide gas
To study the effect of anaerobic conditions with carbon dioxide gas on organic acid production using isolated microbial strain, the batch fermentation was carried out in 5 L

reactor. Inoculum was grown in Tryptic Soy broth (BD) media for 15 - 20 hrs at 37°C under static conditions. Following media composition was used to carry out fermentation in 5 L. Glucose 20 g/1, Yeast Extract 5 g/1, Peptone 10 g/1, K2HP04 3 g/l, NaCl lg/1, CaCl2.2H20 - 0.02 g/1; MgCl2.7H20 - 0.02 g/1 . 15-20 hr old inoculum (10 % v/v) was transferred to the reactor and fermentation was carried out for 24 hrs under following conditions: Temperature - 37°C±0.5°C, carbon dioxide flow rate - 0.1 - 0.25wm, Agitation speed - 300 rpm. The pH of the fermentation medium was maintained at 6.5 -7.0 using 5 N NaOH. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations. The Cell growth was measured by taking absorbance at 600nm wavelength (OD60o) using a photometer. The concentrations of organic acid (succinic acid, acetic acid, propionic acid and lactic acid) and residual sugars were determined by high performance liquid chromatography (HPLC, Aminex, HPX - 87H column) under the following conditions: mobile phase - 0.008 N H2S04, Flow rate - 0.6 ml/min, Temperature - 35°C, Detector - refractive index. The measured concentrations of residual glucose and organic acids are correlated to initial volume. Final results are shown in table 2. Under anaerobic conditions (in the presence of carbon dioxide) culture produced lactic acid and succinic acid in the ratio of 8:1. Yield of lactic acid is much higher (98 %) than succinic acid (12 %). Acetic and propionic acid concentrations were not found at the end of fermentation.
Table 2:

Parameters Results at the end of fermentation (19 hr)
Biomass (OD600) 7.3
Residual Glucose (g/1) 0.0
Amount of succinic acid produced (g/1) 2.46
Amount of lactic acid produced (g/1) 19.6
Ratio of Lactic acid & succinic acid 8:1
Succinic acid yield (%) 12
Lactic acid yield (%) 98
Succinic acid Productivity (g/l.h) 0.13
Lactic acid Productivity (g/l.h) 1.03

Example 5:
Fermentation of Corynebacterium vitaeruminis under Anaerobic Conditions with nitrogen gas
To study the effect of anaerobic conditions with nitrogen gas on organic acid production using isolated microbial strain, the batch fermentation was carried out in the same manner as in example 4 except that nitrogen gas supplied at a flow rate of 0.1 - 0.25 vvm and the initial glucose concentration i.e 30 g/1. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 3. Under this condition the culture has produced mixture of organic acids such as lactic acid, succinic acid, acetic acid and propionic acid with concentrations of 6.04 g/1, 2.98 g/1, 2.86 g/1 and 11.89 g/1 respectively. Culture of isolated microbial strain grew very fast under this condition with the OD of 11.72 which is higher than the cell growth obtained in the presence of carbon dioxide gas as in example 4. Results show that anaerobic condition with nitrogen gas favors the propionic acid pathway along with succinic, acetic and lactic acid.
Table 3:

Parameters Results at the end of fermentation (21 hr)
Biomass (OD600) 11.72
Residual Glucose (g/1) 0.0
Amount of succinic acid produced (g/1) 2.98
Amount of lactic acid produced (g/1) 6.04
Amount of Acetic acid produced (g/1) 2.86
Amount of Propionic acid produced (g/1) 11.89
Example 6:
Fermentation of Corynebacterium vitaeruminis under aerobic and anaerobic conditions with carbon dioxide gas
To study the effect of aerobic and anaerobic conditions with carbondioxide gas on organic acid production using isolated microbial strain, the batch fermentation was carried out in the same manner as in example 4 except that air was supplied at a flow rate of 0.1 -0.25 wm for initial 10 hrs of fermentation to maintain aerobic condition and after that

carbon dioxide gas was purged at the rate of 0.1 — 0.25 vvm to maintain anaerobic conditions. In this example the initial glucose concentration was 48 g/1. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 4. Under this condition the culture has produced mixture of organic acids such as lactic acid, succinic acid, acetic acid and propionic acid with concentrations of 12.14, 5.82 g/1, 2.88 g/1 and 15.42 g/1 respectively. Culture of microbial strain grew very fast under this condition with the OD of 11.6 which is higher than the cell growth obtained in the presence of carbon dioxide gas as in example 4. Results show that combination of aerobic and anaerobic condition with carbon dioxide favors the propionic acid and lactic acid pathway along with succinic and acetic acid.
Table 4:

Parameters Results at the end of fermentation (21 hr)
Biomass (OD600) 11.6
Residual Glucose (g/1) 0.0
Amount of succinic acid produced (g/1) 5.82
Amount of lactic acid produced (g/1) 12.14
Amount of Acetic acid produced (g/l) 2.88
Amount of Propionic acid produced (g/1) 15.42
Example 7:
Substrate Inhibition
To study the tolerance to high concentration of glucose (substrate) the fermentation of Corynebacterium vitaeruminis was studied at different concentrations of glucose (20 g/1, 40 g/1 & 50 g/1) in 5 L reactor under the same conditions as described in example 4. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 5. Results clearly demonstrate that fermentation media containing glucose concentration more than 40 g/1 affect the cell growth and overall yield of main product i.e. lactic acid.

Concentrations of byproducts were also increased as the glucose concentration increased from 20 to 50 g/1.
Table 5:

Parameters Glucose 20 g/1 Glucose 40 g/1 Glucose 50 g/1
Biomass (OD600) 7.3 9.82 7.05
Residual Glucose (g/1) 0.0 3.95 5.73
Amount of succinic acid produced (g/1) 2.46 2.95 8.34
Amount of lactic acid produced (g/1) 19.6 24.3 28.13
Amount of Acetic acid produced (g/1) 0.0 0.32 1.4
Amount of Propionic acid produced (g/1) 0.0 0.86 3.72
Ratio of Lactic acid & succinic acid 8:1 8.2:1 3.4:1
Succinic acid yield (%) 12 8.7 17.5
Lactic acid yield (%) 98 72 59.0
Succinic acid Productivity (g/l.h) 0.13 0.13 0.12
Lactic acid Productivity (g/l.h) 1.03 1.05 0.4
Example 8:
Fermentation of Corynebacterium vitaeruminis with biomass hydrolyzates (from Cane bagasse) as an alternative carbon source
One of the key objectives of this example is the development of cost effective fermentation medium to obtain maximum product yield using biomass hydrolyzates of cane bagasse as an alternative carbon source. The batch fermentation was carried out in the same manner as in example 4 except that biomass hydrolyzate was used as a base medium with glucose concentration of 22 g/\. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 6. Lactic acid was the only product at the end of fermentation with the concentration of 18 g/1. However the productivity of lactic acid (0.64 g/l.h) was relatively low compared with that obtained from glucose fermentation due to long fermentation time. These results suggest that lactic acid can be produced

economically and efficiently by the fermentation of Corynebacterium vitaeruminis from inexpensive biomass hydrolyzates. Table 6:

Parameters Results at the end of fermentation (28 hr)
Biomass (OD600) 6.8
Residual Glucose (g/1) 0.0
Amount of succinic acid produced (g/1) 0.0
Amount of lactic acid produced (g/1) 18.03
Lactic acid yield (%) 84.0
Lactic acid Productivity (g/l.h) 0.64
Example 9:
Fermentation of Corynebacterium vitaeruminis with Corn steep liquor as a low cost nitrogen source
Corn steep liquor (CSL), a byproduct of the corn wet milling industry, is one of the cheapest complex nitrogen sources available. For the economical production of organic acid from glucose, the feasibility of CSL as a nitrogen source for the growth of Corynebacterium vitaeruminis was examined. The batch fermentation was carried out in the same manner as in example 4 except that CSL (10 g/1) was used as a main nitrogen source in the fermentation medium with small amount of yeast extract and peptone to stimulate rapid growth and fermentation. Initial glucose concentration in this example was 26 g/1. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 7. These results suggest that CSL can be used as good alternative nitrogen source for Corynebacterium vitaeruminis when glucose is used as a carbon source.
Table 7:

Parameters Results at the end of fermentation (24 hr)
Biomass (OD600) 5.1
Residual Glucose (g/1) 1.04
Amount of succinic acid produced (g/1) 6.7

Amount of lactic acid produced (g/1)
15.6
Ratio of Lactic acid & succinic acid 2.3:1
Succinic acid yield (%) 27
Lactic acid yield (%) 62
Succinic acid Productivity (g/l.h) 0.28
Lactic acid Productivity (g/l.h) 0.65
Example 10:
Fermentation of Corynebacterium vitaeruminis with sodium carbonate as a Neutralizing agent.
The main objective of this study is to replace sodium hydroxide with low cost sodium carbonate so that the entire process for making organic acids will be more economical. The experiment was carried out in the same manner as in example 4 except the sodium carbonate was used to maintain the pH of the fermentation medium to 7.0. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in example 4. Final results are shown in table 8. Lactic acid was the main product at the end of fermentation with the concentration of 16 g/1. However the productivity of lactic acid (0.7 g/l.h) was relatively low compared with that obtained with sodium hydroxide as a neutralizing agent (Example 4).
Table 8:

Parameters Results at the end of fermentation (24 hr)
Biomass (OD6oo) 6.8
Residual Glucose (g/1) 0.11
Amount of succinic acid produced (g/1) 2.1
Amount of lactic acid produced (g/1) 16.04
Ratio of Lactic acid & succinic acid 7.6:1
Succinic acid yield (%) 9.8
Lactic acid yield (%) 73.0
Succinic acid Productivity (g/l.h) 0.09
Lactic acid Productivity (g/l.h) 0.7

Fed-batch Fermentation:
The results of example 7 have shown that substrate inhibition was a clear limitation to the organic acid production by Corynebacterium vitaeruminis in batch fermentation. Fed batch fermentation was designed to maintained sugar concentration at a lower level (less than inhibitory concentration) during the fermentation process so that cells can grow faster and organic acid production phase can be prolong due to elimination of the substrate inhibition. Two types of feeding were used in present invention which are explained in the following two examples.
Examples 11:
Fed-batch fermentation of Corynebacterium vitaeruminis with intermittent feeding of glucose
The Fed-batch fermentation was carried out in 5 L reactor. Inoculum was grown in Tryptic Soy broth (BD) media for 15 - 20 hrs at 37°C under static conditions. Following media composition was used to carry out fermentation in 5 L. Glucose 20 g/1, Yeast Extract 5 g/1, Peptone 10 g/1, K2HP04 3 g/1, NaCl lg/1, CaCl2.2H20 - 0.02 g/l; MgCl2.7H20 - 0.02 g/1 . 15-20 hr old inoculum (10 % v/v) was transferred to the reactor and fermentation was carried out for 24 hrs under following conditions: Temperature -37°C+0.5°C, carbon dioxide flow rate - 0.1 -0.25wm, Agitation speed - 300 rpm. The pH of the fermentation medium was maintained at 6.5 - 7.0 using 5 N NaOH. Till 17 hr the fermentation was carried out in a batch mode after that glucose was intermittently added to reactor. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations. The Cell growth was measured by taking absorbance at 600nm wavelength (OD6oo) using a photometer. The concentrations of organic acid (succinic acid, acetic acid, propionic acid and lactic acid) and residual sugars were determined by high performance liquid chromatography (HPLC, Aminex, HPX - 87H column) under the following conditions: mobile phase - 0.008 N H2S04, Flow rate - 0.6 ml/min, Temperature - 35°C, Detector - refractive index. The measured concentrations of residual glucose and organic acids are correlated to initial volume. Maximum concentrations of cell growth and organic acids are shown in table 9. The intermittent addition of glucose did not improve the cell growth but improved the acid concentration at the end of fermentation.

Table 9:

Parameters Results at the end of fermentation (70 hr)
Biomass (OD600) 8.3
Residual Glucose (g/1) 1.52
Amount of succinic acid produced (g/1) 7.54
Amount of lactic acid produced (g/1) 30.16
Ratio of Lactic acid & succinic acid 4:1
Succinic acid yield (%) 17.25
Lactic acid yield (%) 69
Succinic acid Productivity (g/l.h) 0.11
Lactic acid Productivity (g/l.h) 0.43
Example 12:
Fed-batch fermentation of Corynebacterium vitaeruminis with continuous feeding of glucose
The fed-batch fermentation was carried out in the same manner as in example 11 except that glucose (37 %) was fed at the rate of 0.5 ml/min to the reactor continuously from 10 hours to 35 hours to maintain the limiting concentration of glucose (5 g/1). In this fermentation the initial glucose concentration was 15 g/1. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid, acetic acid, propionic acid and lactic acid concentrations by methods described in method section. Results are shown in table 10. The continuous feeding of glucose improved the cell concentration and organic acid concentrations at the end of fermentation.
Table 10:

Parameters Results at the end of fermentation (70 hr)
Biomass (OD6oo) 12.1
Residual Glucose (g/1) 5.1
Amount of succinic acid produced (g/1) 8.4
Amount of lactic acid produced (g/1) 38.3
Ratio of Lactic acid & succinic acid 4.5:1

Succinic acid yield (%) 17.0
Lactic acid yield (%) 79.0
Succinic acid Productivity (g/l.h) 0.24
Lactic acid Productivity (g/l.h) 1.1
Example 13:
Effect of inoculum age on the production of organic acids (Lactic acid & Succinic acid) in fed-batch fermentation of Corynebacterium vitaeruminis
The experiment was carried out in the same manner as in example 12 except the age of inoculum that was 24 hours old at the time of transfer from flask to reactor. The glucose was fed to the reactor continuously from 20 hours to 94 hours to maintain the limiting concentration of glucose (5 g/1). In this fermentation the initial glucose concentration was 10 g/1. Samples were withdrawn at regular time interval during fermentation and analyzed for cell concentration, residual glucose, succinic acid and lactic acid concentrations by methods described in method section. Final results are shown in table 11.
Table 11:

Parameters Results at the end of fermentation
Biomass (OD6oo) 1.14
Amount of succinic acid produced (g/1) 10.33
Amount of lactic acid produced (g/1) 21.71
Ratio of Lactic acid & succinic acid 2.1:1
Succinic acid yield (%) 33%
Lactic acid yield (%) 67%
Succinic acid Productivity (g/l.h) 0.11
Lactic acid Productivity (g/1.h) 0.23

SPECIFIC EMBODIMENTS ARE DESCRIBED BELOW
An isolated bacterial strain having accession number MTCC 5488 for production of organic acids.
A process for producing organic acids by biotransformation comprising inoculating a fermentation medium with a 2-6% inoculum of microbial strain having accession number MTCC 5488 in an anaerobic environment, purging the fermentation medium with one or more of air, carbon dioxide or nitrogen followed by incubating the same at a temperature in the range of 32-40°C, and obtaining organic acids from the fermentation medium.
Such process(s), wherein the fermentation medium comprising glucose 20gm/lt, peptone l0gm/lt, yeast extract 5gm/lt, dipotassium phosphate 3gm/lt, sodium chloride lgm/lt, calcium chloride dehydrate 0.02gm/lt? magnesium chloride hexahydrate 0.02gm/lt and sodium carbonate 1 gm/lt.
Such process(s), wherein the process is a fed-batch fermentation process with intermittent or continuous supply of the glucose in the fermentation medium.
Such process(s), wherein the innoculum of the microbial strain having accession number MTCC 5488 is in a tryptic soy broth.
Such process(s), wherein incubation is carried out for a period of 20-28 hours.
Such process(s), wherein pH of the fermentation medium is preferably maintained
at 7.
Such process(s), wherein the purging of carbon dioxide or nitrogen is done to create an anaerobic environment.
Such process(s), wherein the purging of carbon dioxide produces succinic acid and lactic acid.
Such process(s), wherein the purging of nitrogen produces succinic acid, lactic acid, acetic acid and propionic acid.
Such process(s), wherein air is purged followed by carbon dioxide to create both an aerobic and anaerobic environment.
Such process(s), wherein the organic acids produced are succinic acid, lactic acid, acetic acid and propionic acid.

FURTHER SPECIFIC EMBODIMENTS ARE DESCRIBED BELOW
A method for isolating a bacterial strain comprising inoculating bovine rumen fluid in a nutrient broth having 1 % glucose and incubating the same at temperature in the range of 32-40°C, sub-culturing the culture from the preceding step in a fresh nutrient broth having 1% glucose and incubating the same at a temperature in the range of 32-40°C, inoculating the culture from the preceding step on a nutrient agar plate and incubating the same in an anaerobic environment at a temperature in the range of 32-40°C, inoculating a colony from the nutrient agar plate in a tryptic soy broth and incubating the same in an anaerobic environment at a temperature in the range of 32-40°C and isolating a bacterial strain producing lgm/lt of succinic acid and lactic acid.
Such method(s), wherein the isolated bacterial strain has accession number MTCC
5488.
INDUSTRIAL APPLICABILITY
The isolated microbial strain as described produces succinic acid and lactic acid as a major fermentative product. Moreover, as the isolated microbial strain is a facultative anaerobe tolerant to oxygen, it allows for the production of succinic acid and lactic acid under aerobic / anaerobic conditions in the presence of carbon dioxide. This eliminates the problem of process instability which usually occurs due the presence of oxygen in the conventional fermentation process of producing succinic acid and lactic acid using obligate anaerobic microorganism.
The isolated microbial strain as described is capable of utilizing various renewable agricultural feedstocks including cane juice, sweet sorghum juice, molasses and sugar syrup for the production of succinic acid and lactic acid.
Moreover, in order to make the process more cost effective and economical, without compromising the yield of products other low cost carbon and nitrogen sources can be used such as biomass hydrolyzates of cane bagasse and corn steep liquor for the production of organic acids.
Moreover, purging of nitrogen gas in order to provide anaerobic conditions promotes growth of microbial strain which in turn results in higher synthesis of proteins and enzymes which favours propionic acid and acetic acid production along with lactic acid and succinic acid.

Furthermore, the process for production of organic acids also involves carbon dioxide fixation since microorganisms need carbon dioxide as one of the substrate for producing the organic acids. Thus in addition to being based on renewable resources, bio-based succinic acid and lactic acid production has the environmental benefit of using green house gas (carbon dioxide) as a substrate.

We claim:
1. An isolated bacterial strain having accession number MTCC 5488 for production of organic acids.
2. A process for producing organic acids by biotransformation, comprising:
a. inoculating a fermentation medium with a 2-6% inoculum of microbial
strain having accession number MTCC 5488 in an anaerobic environment;
b. purging the fermentation medium with one or more of air, carbon dioxide
or nitrogen followed by incubating the same at a temperature in the range
of 32-40°C;and
c. obtaining said organic acids from the fermentation medium.
3. A process for producing organic acids as claimed in claim 2, wherein the fermentation medium comprises of: glucose 20gm/lt, peptone l0gm/lt, yeast extract 5gm/lt, dipotassium phosphate 3gm/lt, sodium chloride lgm/lt, calcium chloride dehydrate 0.02gm/lt, magnesium chloride hexahydrate 0.02gm/lt and sodium carbonate lgm/lt.
4. A process for producing organic acids as claimed in claim 2, wherein the process is a fed-batch fermentation process with intermittent or continuous supply of the glucose in the fermentation medium.
5. A process for producing organic acids as claimed in claim 2 wherein the innoculum of the microbial strain having accession number MTCC 5488 is in a tryptic soy broth.
6. A process for producing organic acids as claimed in claim 2, wherein incubation is carried out for a period of 20-28 hours.
7. A process for producing organic acids as claimed in claim 2, wherein pH of the fermentation medium is preferably maintained at 7.

8. A process for producing organic acids as claimed in claim 2 wherein the purging of carbon dioxide or nitrogen is done to create an anaerobic environment.
9. A process for producing organic acids as claimed in claim 8, wherein the purging of carbon dioxide produces succinic acid and lactic acid.
10. A process for producing organic acids as claimed in claim 8 wherein the purging of nitrogen produces succinic acid, lactic acid, acetic acid and propionic acid.
11. A process for producing organic acids as claimed in claim 2 wherein air is purged followed by carbon dioxide to create both an aerobic and anaerobic environment.
12. A process for producing organic acids as claimed in claim 11, wherein the organic acids produced are succinic acid, lactic acid, acetic acid and propionic acid.
13. A method for isolating a bacterial strain comprising:
inoculating bovine rumen fluid in a nutrient broth having 1% glucose and
incubating the same at temperature in the range of 32-40°C;
sub-culturing the culture from the preceding step in a fresh nutrient broth having
1% glucose and incubating the same at a temperature in the range of 32-40°C;
inoculating the culture from the preceding step on a nutrient agar plate and
incubating the same in an anaerobic environment at a temperature in the range of
32-40°C;
inoculating a colony from the nutrient agar plate in a tryptic soy broth and
incubating the same in an anaerobic environment at a temperature in the range of
32-40°C; and
isolating a bacterial strain producing lgm/lt of succinic acid and lactic acid.
14. A method for isolating a bacterial strain as claimed in claim 13, wherein the isolated bacterial strain has accession number MTCC 5488.
15. An isolated bacteria] strain substantially as herein described with reference to and as illustrated by the accompanying figure.

16. A process for producing organic acids by biotransformation substantially as herein described with reference to and as illustrated by the accompanying figures.
17. A method for isolating a bacterial strain substantially as herein described with reference to and as illustrated by the accompanying figures.