[001] Field of invention relates to continuous fermentative production of vitamin B 12 in single reactor system. It also relates to co-production of vitamin B 12 and one or more organic acid/s by continuous cell recycle in single reactor system. Background of the invention

[002] Vitamin B 12 (cobalamin) is an important cofactor in the metabolism of carbohydrates, lipids, amino acids and nucleic acids. Vitamin B 12 has various applications in the food and medicine industries. Vitamin B 12 is, moreover, a therapeutic agent used in chemotherapy. The advanced knowledge about cobalamin to participate in maintaining nervous system (Tanaka, 2013) and involvement in many physiological processes has made it a demanding molecule. Its role has been particularly established in treatment of diabetic retinopathy, diabetic peripheral neuropathy, peripheral nerve neuropathy, schizophrenia, Alzheimer's (Smolek et al., 2013; Thaipisuttikul and Galvin, 2012), trigeminal neuralgia, megaloplastic anemia and facial paralysis in Bell plasy's syndrome (Ganesan et al., 2012). These credentials and rising demands of the compound urges for its direct, easy and bulk production.

[003] Generally vitamin B 12 is prepared by fermentation. The two main corresponding genera of microorganisms employed for its preparation at industrial level are Propionibacterium and Pseudomonas. Several Propionibacterium species are capable of producing vitamin B 12 in large scale fermentation processes.

[004] Various aerobic and anaerobic fermentation processes are known for the production of vitamin B 12. Certain reports suggest the presence of air for production of precursor (Rehm, 1980), or for its attachment as the lower ligand to the cobalamin molecule (Berry and Bullerman, 1966). Providing air has improved yield and productivity in Vitamin B 12 fermentation (Santana-Castillo et. al., 1985). Other references suggest that presence of air inhibits key enzymes in the vitamin B 12 pathway (Schwartz, 1973) and that anaerobiosis results in high vitamin B 12 production (Quesada-Chanto et al., 1998). In either fermentation processes for production of vitamin B 12, aerobic or anaerobic, other organic acids are formed such as propionic acid, acetic acid and succinic acid. These acids inhibit cell growth in fermentation broth and thereby reduce the yield of vitamin B 12. To overcome formation of inhibitory organic acids during fermentation, cyclic aerobic-anaerobic operation are suggested so that acids formed during anaerobic phase are consumed in aerobic phase; however final yield of vitamin B 12 is low (Ye et al., 1996).

[005] US6492141 describes process for production of vitamin B 12. Process describes following steps (a) culturing strain in one fermentor anaerobically; (b) transferring the said culture to second fermentor and subjecting to oxygen; (c) adding fresh culture medium to first fermentor; and (d) repeating (a), (b) and (c) steps for producing vitamin B 12. Patent describes non-continuous process in two fermentor system, which makes operating system complicated and increases operating cost.

[006] US2753289A describes use of mixed culture for the production of vitamin B 12. Mixed culture of an organism of the genus Propionibacterium and an organism of the genus Lactobacillus is used for production of vitamin B 12. Process described herein requires longer fermentation time (more than 72 hours), for production of vitamin B 12. Miyano et al. (2000) has also suggested co- culturing by use of acid assimilating strain in vitamin bl2 fermentation, along with a vitamin B 12 producer, Propionibacterium.

[007] Currently existing systems for production of vitamin B 12 and organic acids from Propionibacterium uses two or more series of fermentors. The processes for production of each of the said products operate best at different pH (5.5-6.5 for acids and 6.5-7) for vitamin bl2. Also, reports suggest raising temperature of the reaction by at least 2-12°C for vitamin B12 production (US 6492141). Most of the reported work has vitamin B 12 production in batch fermentation having total reaction time of 168 hours or more and with longer time of aeration (72-84hours). To overcome above mentioned drawbacks, there is need to develop a system of continuous process for production of vitamin B 12, which is robust, with lesser retention time, low operating cost and economically viable. Object of the invention

[008] An object of the present invention is to provide continuous fermentative process for the production of vitamin B 12 in single reactor system.

[009] Another object of the invention is to provide high cell density culture in single reactor system for continuous fermentative production of vitamin B 12 by continuous cell recycling.

[010] Yet another object of the invention is to provide continuous process for co- production of vitamin B 12 and one or more organic acid/s.

[011] Further object of the invention is to provide system and process for production of vitamin B 12 and other value added products.

Summary of invention

[012] One aspect of present invention is to provide continuous process for production of vitamin B 12 in single reactor system by using high density cell culture. The process is carried out by growing Propionibacterium freudenreichii shermanii under anaerobic condition. Fresh nutrient media with precursor is continuously fed in fermentation broth to produce vitamin B 12. Vitamin B 12 in fermentation broth is separated and isolated by known conventional methods. During the process intermittent aeration provided for a limited time period, which is otherwise maintained under anaerobic conditions. Further invention also provides process for production of vitamin B 12, one or more organic acid and other value added products.

Description of the drawings

[013] Figure 1 shows single reactor system for continuous production of vitamin B 12, one or more organic acid/s and other value added products, wherein a reactor (1) is connected to a feed reservoir (2). Cell separator system (3) is connected to reactor (1); wherein fermentation broth from the reactor (1) is continuously removed and passed through the cell separator system (3) to obtain light phase and heavy phase; wherein the heavy phase is partially or completely recycled to the reactor (1). Light phase is collected in light phase reservoir (4). Aeration system (5) such as sparger is used to supply nitrogen gas so as to maintain anaerobic condition and air to maintain aerobic condition as per requirement. Impeller (6) is used to maintain cells in suspension and for uniform mixing of feed in reactor vessel. Further the system is provided with temperature and pH monitoring and controlling unit (7).

Detailed description

[014] The terms used in the specifications are defined as follows- [015] The term "Vitamin B 12" or "cobalamin" used herein refers to broad meaning so as to include cyanocobalamin, hydroxocobalamin, methylcobalamin and 5'desoxyadenosylcobalamin, characterised by a cyano, hydroxyl, methyl or δ'- deoxyadenosyl radical respectively. The terms "Vitamin B12" or "cobalamin" can be used interchangeably. [016] The term "Feed" used herein refers to medium comprising required nutrients or combination of nutrients and precursor, devoid of culture. Terms "feed", "medium" and "nutrient medium" can be used interchangeably.

The term "cell density" used herein refers to cell concentration. Cell density is calculated on the basis of cell weight, both wet cell weight and dry cell weight. [017] The term "High cell density" used herein refers to high cell concentration, wherein concentration of cells is calculated on wet cell weight and/or dry cell weight basis. The concentration of cells on wet cell weight basis is more than 50g/L, further more than lOOg/L, preferably more than 200g/L and even up to 400g/L. The concentration of cell on dry cell weight basis is more than 20g/L, further more than 50g/L, preferably more than lOOg/L, more preferably 200g/L. Terms "High cell density", "High density culture", "High cell density culture", "High cell biomass", "High density cell biomass", "High cell cultivation" can be used interchangeably.

[018] The term "Single reactor system" used herein refers to system in which one or more reactors connected and/or operated in series or in parallel or any order. Further it can be batch or continuous or fed-batch process. Term "single reactor system" and "single reactor" are same and can be used interchangeably.

[019] The term "Heavy phase" used herein refers to solid fractions obtained after passing fermentation broth through cell separation system; wherein heavy phase comprises cell mass, solid matters. [020] The term "Light phase" used herein refers to liquid fractions obtained after passing fermentation broth through cell separation system; wherein light phase comprises vitamin B 12, organic acid/s, value added products and other metabolites.

[021] Present invention provides process for fermentative production of intracellular and extracellular vitamin B 12 using high density cell biomass of microorganism by continuous cell recycle in single reactor system. It also provides a process for co-production of vitamin B 12 and one or more organic acid/s. The process is simple and advantageous as it is carried in single reactor system, uses constant temperature and pH for controlling acid concentrations that allows production of vitamin B 12. Present invention also provides continuous and simple process for production of vitamin B 12 with higher productivity and yield in short time.

[022] In an embodiment of the present invention provides continuous process for the production of Vitamin B 12, wherein microorganism is selected from Propionibacterium freudenreichii, Rhodopseudomonas protamicus, Propionibacterium shermanii-, Propionibacterium freudenreichii shermanii, Pseudomonas denitrificans, Nocardia rugosa, Rhizobium cobalaminogenum, Micromonospora sp., Streptomyces olivaceus, Nocardia gardneri, Butyribacterium methylotrophicum, Pseudomonas sp., Arthrobacter hyalinus. More preferably microorganism selected is Propionibacterium freudenreichii shermanii.

[023] A continuous process for co-production of vitamin B 12 and organic acids in cell recycle high cell density single reactor system with high productivity, wherein said process comprises: i. growing Propionibacterium freudenreichii shermanii cells in fermentation media in a reactor under anaerobic condition; ii. concentrating cells of step (i) by known conventional methods to obtain concentrated cells in the range of 20 g/1 to 200g/l (on dry cell weight basis); iii. continuously feeding the concentrated cells obtained from step (ii) with fresh nutrient medium with or without precursor in fermentation media, and continuously passing the fermentation broth through a cell separator system while maintaining constant fermentation broth volume in the reactor to obtain a light phase comprising vitamin B 12 and/or organic acids, and a heavy phase comprising cells; wherein constant fermentation broth volume is maintained by partially or completely recycling heavy phase to the reactor and/or by supplying fresh nutrient feed in the reactor system at the same rate as to the fermentation broth passed through the cell separator system with hydraulic retention time (HRT) in the range of 4 to 24 hrs; iv. providing controlled intermittent aeration to the fermentation broth in the reactor during step (iii) for a specified time period; and v. separating light phase from heavy phase by known conventional methods; and processing of light phase to obtain vitamin B 12 with concentration in the range of 9 μg/ml to 76 μg/ml and productivity in the range of 1.1 μg/ml.h to 9.1 μg/ml.h., and organic acids with concentration in the range of 7 g/1 to 50 g/1 and productivity in the range of 0.2 g/l.hr to 2.66 g/l.hr.

[024] An embodiment of the present invention relates to a continuous process for production of vitamin B 12 with high productivity in single reactor system, wherein process comprises following steps:

(i) growing Propionibacterium freudenreichii shermanii cells in a reactor under complete anaerobic condition;

(ii) concentrating cells of step (i) by known conventional methods;

(iii) continuously feeding the cells in the fermentation broth of step (ii) with fresh nutrient medium also comprising precursor for production of vitamin

B 12 and continuously passing the fermentation broth through a cell separator system to obtain a light phase comprising vitamin B 12 and other metabolites and a heavy phase comprising cells; wherein the heavy phase is partially or completely recycled back to the reactor so as to maintain constant fermentation broth and cell volume in the reactor; (iv) optionally bleeding cell biomass for maintaining constant cell viability in reactor;

(v) providing intermittent aeration to the fermentation broth in the reactor during step (iii) for a designed time period which is otherwise maintained under anaerobic conditions; and

(vi) processing of light phase for separation and isolation of vitamin B 12 and other metabolites.

[025] An embodiment of the invention provides Propionibacterium freudenreichii shermanii, wherein is strain procured from American type culture collection having accession number ATCC 13673.

[026] In an embodiment of the present invention there is provided process for production of vitamin B 12, wherein process is carried under anaerobic condition. Anaerobic conditions are maintained by sparging nitrogen gas in a reactor system.

[027] Other embodiment of the invention provides growing Propionibacterium freudenreichii shermanii in anaerobic condition till it reaches late exponential phase and/or early stationary phase. Further, cells in late exponential phase and/or early stationary phase are concentrated by known conventional methods such as but not limited to centrifugation, filtration etc.

[028] Another embodiment of the invention provides feeding nutrient medium in the same reactor for further growth of the cells. Nutrient medium is selected from but not limited to carbon source, nitrogen source, minerals, salts, buffers, macroelements and micro-elements known to those skilled persons in the art.

[029] In an embodiment of the invention there is provided nutrient medium, wherein nutrient medium comprises carbon source selected from but does not limit to carbohydrates such as glucose, fructose, lactose, sucrose, starch, biomass hydrolysates, cellulose, lignocellulose, dairy waste such as whey permeate, organic acid, organic salts, molasses, glycerol, and/or combination thereof and other suitable carbon source know to the person skilled in the art.

[030] In an embodiment of the invention there is provided nutrient medium, wherein nutrient medium comprises nitrogen source selected from but not limited to peptones, yeast extract, casein, casein hydrolysates, corn steep liquor, animal waste like poultry droppings as well as products derived from soyabean, cotton seed, rape seed such as flour, hydrolysates, flakes, meal, and inorganic salts of ammonium ion, nitrates, nitrites, urea, protein, casein, amino acids, organic nitrogen sources, other conventional nitrogen sources, and combination thereof.

[031] Other embodiment of the invention provides concentrating cell mass, wherein after reaching late exponential phase and/or early stationary phase of the microbe, cells are harvested and concentrated by known conventional method such as but not limited to centrifugation and filtration etc.

[032] Another embodiment of the invention provides feeding nutrient to concentrated cell mass in the same reactor for suitable number of cycles till desired cell concentration is reached in fermentation broth to obtain high density cell biomass. High density cell mass is obtained by cell concentration, wherein cell concentration step is repeated for 2-10 cycles.

[033] Yet another embodiment of the invention provides high density cell culture, wherein concentration of cells on wet cell weight basis is more than 50g/L, further more than lOOg/L, preferably more than 200g/L and even up to 400g/L. The concentration of cell on dry cell weight basis is more than 20g/L, further more than 50g/L, preferably more than lOOg/L, more preferably up to 200g/L.

[034] An embodiment of the invention provides continuous process for production of vitamin B 12, wherein fermentation is carried in anaerobic condition. Further intermittent aeration is provided during cell recycling, otherwise system is maintained in anaerobic condition.

[035] Other embodiment of the invention provides feeding fresh medium in the fermentation broth, wherein fresh medium comprises precursor. Precursor promotes the production of the vitamin B 12. Precursor is selected from δ-amino levulinic acid (ALA), 5,6-dimethyl benzimidazole (DMBI), ortho-phenylene diamine, l,2-dimethyl-4,5-diaminobenzene. Most preferably, precursor used is 5,6-dimethyl benzimidazole (DMBI).

[036] Another embodiment of the invention provides continuously feeding the cells in the fermentation broth with fresh nutrient medium comprising precursor for production of vitamin B 12 and continuously passing the fermentation broth through cell separator system to obtain a light phase comprising vitamin B 12 and other metabolites and a heavy phase comprising cells; wherein the heavy phase is partially or completely recycled back to the reactor so as to maintain constant fermentation broth volume in the reactor. [037] Yet another embodiment of the present invention provides cell separator system, wherein fermentation broth removed from reactor is subjected to cell separator system to obtain heavy phase comprising cell mass and light phase comprising vitamin B 12 and other metabolites. Further heavy phase comprising high density cell mass is partially or completely recycled back to the reactor, and light phase comprising vitamin B 12 and other metabolite is processed further. The cell separator system is selected from known conventional methods and equipments such as but not limited to centrifugal separator, membrane separator. Time required for a reactor volume to get replaced i.e. hydraulic retention time (HRT) with fresh feed is 4 to 24 hours.

[038] Further embodiment of the invention provides maintaining constant fermentation broth volume in a reactor, wherein nutrient feed is supplied in the reactor system at the same rate as to the fermentation broth passed through the cell separator system.

[039] Other embodiment of the invention provides method for recovery of the nutrient from light phase and/or heavy phase and further can be recycled to the reactor.

[040] An embodiment of the invention provides continuous process for intracellular and/or extracellular production of vitamin B 12. Extracellular vitamin B 12 is obtained in light phase, wherein the light phase is processed further to separate vitamin B 12. Intracellular vitamin B 12 is obtained by lysing high density cell mass and processing it further.

[041] Another embodiment of the invention provides lysing of cell mass to obtain intracellular vitamin B 12, wherein cells are disrupted to release vitamin B 12 inside cell. Cell lyses/disruption can be achieved by but not limited to chemical cell lyses (Triton X-100™, Tween 20™, Tween 80™, sodium dodecyl sulfate, acid treatment) or physical cell lyses (manual grinding, sonication, microwave assisted lysis, bead beating, high pressure cell lysis, liquid nitrogen homogenization, freeze thaw) or enzymatic cell lysis (lysozymes) or combination thereof. Cell disruption is attained preferably with physical lysis treatment, more preferably acid treatment, yet more preferably using concentrated sulfuric acid. Vitamin B 12 released from the cell is processed further for isolation. [042] Yet another embodiment of the invention provides continuous process, wherein high density cell mass is recycled back to reactor. During the course of cell recycle, intermittent aeration is provided to the system, which is otherwise maintained in anaerobic condition. Intermittent aeration is provided for at least 24 hr, preferably at least 18 hr, more preferably 2 hr.

[043] In some embodiment of the invention there is provided immobilization of microbial cell for production of vitamin B 12 and other value added products.

[044] An embodiment of the present invention provides processing of vitamin B 12 in light phase and/or released from the disrupted cell mass, wherein vitamin B 12 is isolated by conventional methods known to the person skilled in the art but not limited to filtration, crystallization, centrifugation, precipitation, evaporation, decantation, simple distillation, fractional distillation, chromatography and or combination thereof. The isolated vitamin B 12 is devoid of cell mass and other metabolites.

[045] An embodiment of the present invention provides process for production of vitamin B 12, wherein concentration of vitamin B 12 ranges from 9 μg/ml to 170 μg/ml, depending on alterations in conditions provided.

[046] Other embodiment of the invention provides fermentation process, wherein fermentation is carried at pH in the rage of 6 to 7, more preferably 6.5 and temperature in the range of 25 to 40°C, more preferably 30°C.

[047] An embodiment of the present invention relates to a continuous process for co-production of vitamin B 12 and one or more organic acid/s in single reactor system, wherein process comprises following steps:

(i) growing Propionibacterium freudenreichii shermanii cells in a single reactor system under complete anaerobic condition, wherein growing cells produce one or more organic acid/s;

(ii) concentrating cells of step (i) by known conventional methods;

(iii) continuously feeding the cells in the fermentation broth of step (ii) with fresh nutrient medium also comprising precursor for production of vitamin B 12, and continuously passing the fermentation broth through a cell separator system to obtain a light phase comprising vitamin B 12, one or more organic acid/s and other metabolites, and a heavy phase comprising cells; wherein the heavy phase is partially or completely recycled back to the reactor so as to maintain constant fermentation broth and cell volume in the reactor;

(iv) optionally bleeding cell biomass for maintaining constant cell viability in reactor

(v) providing intermittent aeration to the fermentation broth in the reactor during steps (iii) for a designed time period which is otherwise maintained under anaerobic conditions; and

(vi) processing the light phase to separate vitamin B 12, one or more organic acid/s and other metabolites.

[048] Other embodiment of the invention provides process for co-production of vitamin B 12 and one or more organic acid/s, wherein organic acids are selected from but not limited to propionic acid, succinic acid, acetic acid, carbonic acid, lactic acid, formic acid. Organic acid/s are extracellularly produced throughout the fermentation process. Further organic acid/s are processed by known conventional methods.

[049] In another embodiment of the invention there is provided process for production of value added products, wherein said value added products comprises but not limited to organic acid/s, sugar/s such as oligosaccharides, disaccharides, trehalose etc.

[050] Another embodiment provides co-production of vitamin B 12 and organic acid/s in fermentation broth, wherein fermentation broth is subjected to a cell separator system to obtain heavy phase comprising cells and light phase comprising vitamin B 12 and organic acid/s and other metabolites. Further, cells in heavy phase can be recycled to the reactor.

[051] Yet another embodiment provides further processing of the light phase to separate vitamin B 12 and organic acid/s, wherein further separation of organic acid/s can be carried by conventional methods known to a person skilled in the art such as but not limited to filtration, crystallization, centrifugation, precipitation, evaporation, decantation, simple distillation, fractional distillation, chromatography and/or combination thereof.

[052] Another embodiment of the invention relates to single reactor system for continuous production of vitamin B 12 and other value added products, wherein system comprises: i) a reactor- for production of vitamin B 12 and other value added products, wherein reactor is connected to feed reservoir;

ii) feed reservoir- from which nutrient medium is fed to the said reactor; iii) cell separator system- subjecting fermentation broth to obtaining light phase comprising vitamin B 12 and other value added products and heavy phase comprising cell mass; further heavy phase is partially or completely recycled to the said reactor;

iv) Light phase reservoir- to collect the light phase obtained from the cell separator system;

v) An aeration system- such as sparger is used to supply nitrogen gas so as to maintain anaerobic condition and air to maintain aerobic condition as per requirement.

vi) Impeller- to maintain cells in suspension and for uniform mixing of feed in reactor vessel.

[053] Yet another embodiment of the present invention as provided in figure 1 relates to single reactor system for continuous production of vitamin B 12 and other value added products, wherein system comprises:

i) a reactor (1) connected to a feed reservoir (2), wherein nutrient medium from fed reservoir is fed to reactor;

ii) cell separator system (3) connected to reactor (1); fermentation broth from the reactor (1) is continuously passed it through the cell separator system (3) for obtaining a light phase and heavy phase; wherein the heavy phase is partially or completely recycled to the reactor (1);

iii) light phase reservoir (4) for collecting the light phase obtained from the cell separator system (3)

iv) an aeration system/air sparger for providing anaerobic condition and intermittent aeration in reactor (1)

[054] In an embodiment of the system there is provided pH and temperature monitoring and controlling unit.

[055] Another embodiment of the invention provides impeller system used to maintain cells in suspension and for uniform mixing of feed in reactor vessel. Examples

[056] Examples described herein are for illustration of the invention and should not restrict scope of the invention.

[057] Given below are the illustrative examples of method for co production of Vitamin B 12 and organic acids from P.freudenreichii shermanii ATCC 13673, based on the current invention. It is to be understood, that these examples ae given by way of illustration and not by way of limitation.

Example 1:

Continuous high cell density cell recycle cultivation of Propionibacterium freudenreichii shermaniiATCC 13673

[058] 300mL of Propionibacterium freudenreichii shermaniiATCC 13673 grown in static flask condition for 48hrs was centrifuged and the cell pellet inoculated in 1.5L of fermentation medium. The inoculum accounted to an average dry cell weight (DCW) of lg/L. The fermentation medium consisted of 2% glucose, 1% yeast extract, 2g/L KH ₂ P0 ₄ , 4g/L (NH ₄ ) ₂ HP0 ₄ , lOmg/L NaCl, lOmg/L CaCl ₂ .6H ₂ 0, lOmg/L CoCl ₂ , lOmg/L MgS0 ₄ .6H ₂ 0, 2.5mg/LMnS0 ₄ .H20 and5mg/L FeS0 ₄ .7H ₂ 0. A process for biomass build up was initiated by stepwise increment of glucose concentration in the same reactor with same inoculum. As the cells reached near stationary phase (DCW of 6+0.5g/L), the reactor content was concentrated to 1L reactor volume to achieve DCW of lOg/L. Feed medium containing 1% glucose was fed to the cells and recycle mediated by cell separator. Feed glucose was increased when the DCW reached 25g/L, and the cell biomass increased further to >75g/L. Further feeding the cells with 3% glucose increased DCW to >100g/L. Beyond lOOg/L DCW where high cell density (HCD) was achieved and was advantageous for vitamin B 12 formation, the feed was also supplemented with cobalamin precursor 5,6-dimethylbenimidazole (DMBI). Increasing substrate concentration to 4% aided in biomass build up to about 130g/L. DCW increased with increase in glucose concentration to 5%. Feeding 6.5% glucose resulted in >170g/L dry biomass. Feed containing 7.5% glucose fed to the cells generated at 200g/L DCW. Each feed was fed for three constant hydraulic retention times (HRTs) of metabolite formation.

Example 2: Co- production of Vitamin B12 and organic acids at different glucose concentrations at high cell densities

[059] In the process described in example 1, metabolite profiling was done at each glucose concentration. The titres of organic acids with different glucose concentrations are mentioned in Table 1 :

Table 1: Organic acid production with different glucose concentrations

[060] For vitamin B 12 production a glucose feeding of 5% was selected to keep the organic acids concentrations less than 40g/l. With 5% glucose and DMBI in the feed, hydroxocobalamin (OH-B 12) was produced in the light phase (64.23 g/mL).

Example 3:

Process optimization for vitamin B12 production

[061] Cell growth viz a viz vitamin B 12 concentrations are inhibited by higher acid concentrations. To maintain low acid concentration for vitamin B 12 production as explained in example 2, an experiment similar to example 1 was carried out. 300mL of Propionibacterium freudenreichii shermanii ATCC 13673 grown in static flask condition for 48hrs was centrifuged and the cell pellet inoculated in 1.5L of fermentation medium. The inoculum accounted to an average dry cell weight (DCW) of lg/L. The fermentation medium consisted of 2% glucose, 0.6% yeast extract, 0.5g/L KH ₂ P0 ₄ , lg/L (NH ₄ ) ₂ HP0 ₄ , lOmg/L NaCl, lOmg/L CaCl ₂ .6H ₂ 0, lOmg/L CoCl ₂ , lOmg/L MgS0 ₄ .6H ₂ 0, 2.5mg/L MnS0 ₄ .H20 and 5mg/L FeS0 ₄ .7H ₂ 0. Biomass build up was carried out by cell concentration and broth dilution using cell separator as explained in example 1. Feed supplemented to the cells during the process of recycle contained 3% glucose and lOOmg/L DMBI. The complete process of attaining >200g/L DCW was achieved in 10 days. The biomass so obtained was further used for vitamin B 12 production with precursor in the feed medium. In this process, the organic acids was maintained <30g/L with PA concentration of 13+lg/L.

[062] The same method of cell biomass build up was applied to a species of a related dairy microbe, Lactobacillus. It was seen that the cells could not attain as high biomass as in case of P.shermanii.

Example 4:

Physical parameters for vitamin B12 and organic acid co- production

[063] 300mL of Propionibacterium freudenreichii shermanii ATCC 13673 grown in static flask condition for 48hrs was centrifuged and the cell pellet inoculated in 1.5L of fermentation medium. The inoculum accounted to an average dry cell weight (DCW) of lg/L. The batch fermentation medium consisted of 2% glucose, 0.6% yeast extract, 0.5g/L KH ₂ P0 ₄ , lg/L (NH ₄ ) ₂ HP0 ₄ , lOmg/L NaCl, lOmg/L CaCl ₂ .6H ₂ 0, lOmg/L CoCl ₂ , lOmg/L MgS0 ₄ .6H ₂ 0, 2.5mg/L MnS0 ₄ .H20 and 5mg/L FeS0 ₄ .7H ₂ 0. It was established from similar to experiment 1 that organic acid production is best at 30°C, while maintaining a pH of 5.5. Biomass Whereas, vitamin B 12 production requires an increment in temperature from 28-30°C to 32- 34°C, while maintaining a pH range of 5.5-6.5, where 6.5 is the best suited for vitamin B 12 synthesis.

Example 5:

Determination of precursor DMBI concentration

[064] 150-200g/L DCW of P.shermanii ATCC 13673 was recycled in a fermentation broth with TOA maintained at <30g/L. Medium devoid of DMBI was fed to the cells for 4 days. Vitamin B 12 was not produced or was synthesized below HPLC detectable quantity, in the absence of DMBI. To the same cells, when feed containing 200 mg/L DMBI was supplied and light phase collected over the next 4 days, 44^g/ml of OH-B 12 was produced, as quantified using HPLC. In a similar experiment, with 3% glucose and lOOmg/L DMBI as explained in example 4, -70 mg/L OH-B 12 was produced. Nearly 59% of the total DMBI provided is consumed for cobalamin production to give a stable vitamin B 12 production.

Example 6:

Controlled aeration for vitamin B12 and organic acid production

[065] In the process described in example 3, different aeration pattern was investigated for vitamin B 12 production. Different aeration conditions were provided to the HCD P.shermanii ATCC 13673 culture. It was observed that increased aeration affected vitamin B 12 production by P.shermanii and resulted in no formation of cobalamin. On the other hand, little or complete anaerobic condition provided by nitrogen sparging aided in vitamin B 12 formation by the culture. The details of the aeration pattern and the amount of vitamin B 12 formed are as given in the table 2 below:

Table 2: Controlled aeration for cobalamin production

[066] The study indicated that 2hr aeration/day is best suited for vitamin B 12 production, where the 2 hrs should be at a fixed interval, such that the cycle is divided evenly as 2 hrs aeration + 22 hrs nitrogen + 2 hrs aeration + 24 hrs nitrogen and so on. For organic acids production no aeration was required and high productivities were obtained as described in table 1.

Example 7:

Use of soy hydrolysate as nitrogen source for organic acid production from

P '.shermanii

[067] Potential of soya in the form of soy hydrolysate has been determined by substituting it as the sole nitrogen source for growth of P. shermanii. The amount of soy hydrolysate that was required to meet the nitrogen content equivalent to yeast extract was estimated by Kjeldahl method. The culture could use soy hydrolysate in place of yeast extract and produced organic acids with 0.53g/g yields.

Example 8:

Process scale up at laboratory scale for organic acids production

[068] Fermentation was carried in a 5L continuous stirred tank reactor, under the optimized conditions of media, pH, temperature, acid concentration, air sparging duration and precursor addition. Cell pellet of 300mL of Propionibacterium freudenreichii shermanii ATCC 13673 grown in static flask condition for 48hrs was inoculated in 1.5L of fermentation medium. The inoculum accounted to an average dry cell weight (DCW) of lg/L. The fermentation medium consisted of 2% glucose, 0.6% yeast extract, 0.5g/L KH ₂ P0 ₄ , lg/L (NH ₄ ) ₂ HP0 ₄ , lOmg/L NaCl, lOmg/L CaCl ₂ .6H ₂ 0, lOmg/L CoCl ₂ , lOmg/L MgS0 ₄ .6H ₂ 0, 2.5mg/L MnS0 ₄ .H20 and 5mg/L FeS0 ₄ .7H ₂ 0. Biomass build up was carried out by cell concentration and broth dilution using cell separator as explained in example 1. The total working volume was raised to a final of 5L sequentially, by the mentioned method. Further recycle was maintained till termination of the fermentation run using the cell separator. Organic acids concentration was estimated in light as well as well as in heavy phase. The experiment demonstrated the culture's ability to produce organic acids in high yields (0.74g/g) and productivities (2.66g/L.h).