Biosynthesis of Vitamin B 12 Osnaider Gmez Meja Maitreyi Sadanand Joshi Vitamin B12/ Cobalamin Why is Vitamin B 12 so Necessary? No innate production by human metabolism, has to be consumed from outside source Major role in DNA synthesis and regulation Deficiency leads to: - pernicious anemia - fatigue - depression - poor memory, etc. Microbial synthesis possibilities Possible Microorganisms to be utilized: 1. Propionibacterium freudenreichii 2. Propionibacterium shermanii 3. Streptomyces griseus 4. Pseudomonas denitrificans Organisms (1),(2) & (3) are anaerobic while (4) follows aerobic pathway Process Requisites Bacteria: Pseudomonas denitrificans Substrate: Glucose, Corn steep liquor, Beet molasses, Maltose syrup Temperature: 30 o C pH: Duration of fermentation: 2-3 days in pre culture and 7 days in actual production fermenter Aeration Reactor type: Batch(shake flask) on laboratory scale & Fedbatch on commercial scale Synthesis Pathway How Does it Work Commercially ? Commercial Synthesis First phase culture in 150 L seed fermenter Secondary fermentation in 9000 L seed culture L fermenter for actual vitamin B12 production Production Process Fermentation Separation Purification 1. Fermentation Fermentor L 28 C 0,5 v/v.min 180 RPM 40 hrs. Fermentor L 28 C 0,4 v/v.min 130 RPM 30 hrs. Fermentor L 32 C 0,3 v/v.min 130 RPM 180 hrs. 80 90 L 5000 L L Substrate input 2. Separation Heater C pH 6,5 8,5 30 min FIltration Microorganism retention Precipitation Stabilization of the vitamin molecule Cyanide/ thiocyanate Cyanide/ thiocyanate Tannic acid Or cresol 80% purity 3. Purification Crystallization Addition of organic solvent. Purification Solvent extraction (Cresol, CCl4, water/Butanol) Ion exchange Activated Carbon Final product: Food and pharmaceutical Application 198,27 mg/L Pure Vitamin B 12 Pure Vitamin B 12 Alternative Technologies for Separation Column chromatography Aqueous two phase extraction for avoiding an organic solvent in purification Reagent Choices to boost production Economics point of view Cost effective fermentation Mostly used carbon source Undesirable components & unstable fermentation Refined sugar Gives high yield High price Beet molasses Sucrose Maltose syrup and Corn Steep Liquor The medium cost generally occupy almost 50% of most fermentation products. Avoid unstable fermentation Half price of the Sucrose Substrate feeding Yields obtained with sucrose are the highest but an expensive source Core steep liquor and maltose syrup give better yield Beet molasses: moderate yield but the cheapest source pH control strategy With glucose as a feed pH could be well maintained at 7- 8, but with beet molasses it shoots up to 9.1 Stable pH could be achieved by using glucose and high production levels are obtained Effect of Betaine feed Feeding Betaine (N,N,N-trimethylglycine) in the range 57 g/l during 50140 h of fermentation stimulates synthesis Beet molasses contain high concentration of Betaine Glucose as a substrate with betaine addition improves production SUMMARY Process overview : 2.Separation 3. Purification 1. Fermentation 1. Works in 3 stages of fermentation at about o C & at the pH of for 250 hrs 3. Solvent extraction, Ion exchange, Crystallization 2. Heating, filtration and precipitation Pure Vitamin B 12 Pure Vitamin B 12 Substrate input Overall optimized operation Glucose as a substrate, good for pH control while higher production rates with maltose syrup Small addition of betaine can bring a big change in production rate Step wise reduction of Dissolved Oxygen improves production rate, but too low concentrations inhibit the cell growth Under the well controlled conditions, maximum production rate of 4.60 mg/L could be obtained Advantages and Disadvantages PROS Easier synthesis with high quality product as compared to chemical synthesis High productivity compared to other microorganisms when performed with bacterium Pseudomonas denitrificans Well optimized process with intelligent choice of reagents can make the synthesis affordable CONS The use of cyanide and solvents Use of food products for industrial manufacturing is still debated in terms of food security Slow process (10 days) References Xia W., Chen W., Peng WF., Li KT. (2014). Industrial vitamin B 12 production by Pseudomonas denitrificans using maltose syrup and corn steep liquor as the cost- effective fermentation substrate. Bioprocess Biosyst Eng 38: Martens J., Barg H., Warren MJ., Jahn D. (2001). Microbial production of vitamin B 12. Appl Microbiol Biotechnol (2002) 58: Li KT., Liu DG., Li YL., Chu J., Wang YH., Zhuang YP., Zhang SL., (2008). Improved large-scale production of vitamin B 12 by presudomonas denitrificans with betaine feeding. Bioresource Technology 99 (2008) LI KT., Liu DG., Chu J., Wang YH., Zhuang YP., Zhang SL., (2008). An effective and simplified pH-stat control strategy for industrial fermentation of vitamin B 12 by pseudomonas denitrificans. Bioprocess Biosyst Eng (2008) 31: Thank You