ASHIKA RAVEENDRAN 2ND MSC BIOTECHNOLOGY

FERMENTATIVE PRODUCTION OF

VITAMINS AND

AMINO ACIDS

VITAMINSVitamins are essential micronutrients

required in trace quantities that cannot be synthesized by mammals

They are essential for metabolism for all living organisms

Apart from their nutritional –physiological roles as growth factor ,vitamins are increasingly being introduced as food/feed additives ,as medical therapeutic agents .

Today many processed foods, feeds pharmaceuticals, cosmetics and chemical contain extraneously added vitamins or vitamin related compounds.

Presently few of the vitamins are chemically synthesized or via extraction processes

With growing consumer consciousness led to substituting with biotechnological processes.

FAT SOLUBLE VITAMINSVitamin A Vitamin D Vitamin E Vitamin K

Vitamin E Most abundant among fat soluble vitamins and has

the highest antioxidant activity in vivo.

In nature, only photosynthetic organisms are capable of producing α-tocopherol.

In humans, ∞-tocopherol is believed to play a major role in prevention of light induced pathologies of the skin, eyes and degenerative disorders such as atherosclerosis, cardiovascular diseases and cancer.

Industrial application of ∞-tocopherol includes its use in preservation of food, in cosmetics and sunscreens

Currently ∞-tocopherol is obtained by chemical synthesis and by extraction from vegetable oils

Extraction from oil is not efficient ,as these typically contains low levels of ∞-tocopherol.

Several strains of freshwater microalgae Euglena gracilis Z and marine microalgae Dunaliella tertiolecta produce

∞-tocopherol in concentrations higher than conventional foods.

production of high amounts of vitamin E has been successfully demonstrated by E. gracilis Z. using two-step culture.

In the first step of the batch culture, E. gracilis Z. was photo- heterotrophically cultivated in modified Oda and modified Hunter media at high light intensity.

When the cells reached late exponential phase, they were separated, washed and resuspended in the same volume of Cramer and Mayers (CM) medium for the second step of cultivation.

The two-step cultures using high cell densities gave high productivity of antioxidant vitamin

Vitamin K.

Vitamin KVitamin K₂ Vitamin K₂Vitamin K₁

Vitamin K is an essential cofactor for the post-translational conversion of glutamic acid residues of specific proteins in blood and bone to γ-carboxyglutamic acid.

Fermentative Production of Vitamin K₂ Tani and Taguchi have reported that as

much as 182 mg/L MK was produced using detergent supplement culture and a mutant of Flavabacterium.

Lactic acid bacteria are reported to produce MK with the yield of 29–123 g/L MK-7, MK-8, MK-9 and MK-10.

In fermented soybeans, Bacillus subtilis produces menaquinones, the major component being MK-7 and the minor one being MK-6.

Sumi studied production of MKs by the fermentation of okara with seven different natto bacilli.

The highest production rate of 36.6 mg/g was seen in the Chinese natto strain followed by (in mg/g of okara-natto wet mass): 14.2 in Naruse, 11.9 in Asahi, 6.8 in Takahashi, 1.9 in Miyagino (natto bacilli for food production), and 5.2 in Nitto and 1.9 in Meguro (natto bacilli for medicine) after incubation for 4 days at 37 °C.

The water-soluble vitamin K was isolated as a dark yellow powder by DEAE Sepharose chromatography and membrane filter fractionations.

WATER SOLUBLE VITAMINSBiotinRiboflavin cyanocobalamin

VITAMIN B 12 Cyanocobalamin, by definition vitamin B12, is

the industrially produced stable cobalamin form which is not found in nature.

Vitamin B12 is obtained exclusively by fermentation process.

Important dietary component, requirement 0.001 mg/day.

Cyanocobalamin consist of a cobinamide linked to a nucleotide.

Cobinamide – cobalt linked to cyanide grp, surrounded by 4 reduced pyrrole ring.

Nucleotide – 5 , 6 – dimethyl benziminazole

VITAMIN B 12 BIOSYNTHESIS

MICROORGANISMS IN INDUSTRIAL PRODUCTION OF VIT. B12Streptomyces griseus , S. olivaceus ,

Bacillus megaterium , B. coagulans , Pseudomonas

denitrificans , Propionibacterium freudenreichii , P.

shermanii and a mixed fermentation of a Proteus spp

and a Pseudomonas sp.

Manufactured by submerged fermentation

Aeration and agitation of medium essential

Fermentation process completed in 3 to 5 days

VIT.B12 PRODUCTION USING Streptomyces olivaceus NRRL B-1125

PREPARATION OF INOCULUMPure slant culture of Streptomyces olivaceus

NRRL B-1125 is inoculated and grown in 100 to 250 ml of inoculum medium.

Seeded flask are kept on shaker for incubation .

Flask cultures are used to inoculate large amount of inoculum media arranged in series of tank .

2 or 3 successive transfers are made to obtain required amount of inoculum cultures.

Inoculum of production tank must be 5% of the volume of production medium

PRODUCTION MEDIUMConsist of carbohydrate ,proteinaceous material ,

and source of cobalt and other salts .

Sterilization of medium batch wise or continuously . Batch – medium heated at 250°F for 1 hrContinuous – 330°F for 13 min by mixing with live

steam.

COMPONENTS AMOUNT (%)

Distillers solubles 4.0Dextrose 0.5 to 1CaCO3 0.5COCl2.6H2O 1.5 to 10 p.p.m.

TEMPERATURE , PH , AERATION AND AGITATIONTemperature : 80°F pH: At starting of process pH falls due to

rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium

pH 5 is maintained with H2SO4 and reducing agent Na2SO4 .

Aeration and agitation : Optimum rate of aeration is

0.5 vol air/vol medium/min. Excess aeration cause foaming.

ANTIFOAM AGENT , PREVENTION OF CONTAMINATIONAntifoam agent : soya bean oil , corn oil, lard oil and silicones (sterilized before

adding) .Prevention of contamination : essential

to maintain sterility , contamination results in reduced yields ,

equipments must be sterile and all transfers are carried out under aseptic conditions

Yield : yield of cobalamin are usually in the range of 1 to 2 mg. per litre in the fermented broth

DOWNSTREAM PROCESS OF VITAMIN B12

DISSOLVED VITAMIN B 12

CONCENTRATION OF CELL MASS

CREAM CONCENTRATION OF CELL MASS

EX(TRACTION Eg :alcohol such as methanol

Dissolved vitamin B12

Chromatography

Pure vitamin B12

Crystallisation from organic solvents

RIBOFLAVINRiboflavin, or vitamin B2, is used for human nutri-

tion and therapy and as an animal feed additive. Its deficiency in humans is correlated with loss of

hair, inflammation of skin, vision deterioration, and growth failure.

This vitamin has also been found to be successful in treatment of migraine and malaria .

Riboflavin has been produced commercially by chemical synthesis, by fermentation and by a combination of fermentation and chemical synthesis.

MICRO –ORGANISMS IN INDUSTRIAL PRODUCTION OF VIT. B2Although bacteria (Clostridium sp.) and

yeasts (Candida sp.) are good producers, two closely related ascomycete fungi, Eremothecium ashbyii and Ashbya gossypii, are considered the best riboflavin producers.

Ashbya gossypii produces 40 000 times more vitamin than it needs for its own growth.

OTHER MICRO -ORGANISMS PRODUCING VIT. B 2Gene amplification and substitution of wild type

promoters and the regulatory regions with strong constitutive promoter from Bacillus subtilis have resulted in increased riboflavin production .

Lactococcus lactis MG 1363 strain using both direct mutagenesis and metabolic engineering for simultaneous overproduction of both folate and riboflavin

Improved strains for the production of riboflavin were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes

PREPARATION OF INOCULUMStarts from slants or spores dried on

sand.After 1 or 2 stages, further propagation is

carried on 1 or 2 tank inoculum stages

PRODUCTION MEDIUMFermentor 10,000 to 1,00,000 gals range.

Production medium designed according to type of micro- organism.

Ashbya gossypii : sources - palm oil ,corn steep liquor, glucose,

molasses , whey, collagen , soya oil , glycine.

Stahmann et al. reported riboflavin yields in excess of 15 g/L of culture broth in a sterile aerobic submerged fermentation of Ashbya gossypii with a nutrient medium containing molasses or plant oil as

major carbon source.

Ertrk et al. studied fermentative production of riboflavin by Ashbya gossypii in a medium containing whey.

The quantities of riboflavin produced by Ashbya gossypii in whey with different supplements.

Supplement Quantity of riboflavin (mg./L)

Bran 389.5Glycine + peptone 120Sucrose 87.5Glycine 78.3Yeast extract 68.4Peptone 23.2Soyabean oil 17.5

For Eremothecium ashbyii - still slops from alcohol industry with skim milk , soya bean meal or casein(protein source),maltose/ sucrose/glucose (carbohydrate source).

low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin

Organic wastes like beef extract, hog casings, blood meal or fish meal supported the production of riboflavin from Eremothecium ashbyii NRRL 1363.

Recent studies with wild type of E. ashbyii have yielded 3.3 g/L of riboflavin using molasses and peanut seed cake as carbon and nitrogen source, respectively

CONDITIONSpH : 6 to 7.5Temperature : 26 to 28 °C Fermentation : submerged

aerated fermentation Fermentation time : 96 to 120 hrsAeration & agitation required.Yield : 3 to 6 g or more / litre

DOWNSTREAM PROSSESSING OF RIBOFLAVIN Riboflavin is recovered from the broth by

centrifu- gation after inactivation of the microorganisms by heat.

Pasteurization of the broth ensures that no viable cells of the production organism are present in the final product.

After heating, the cell mass is separated from fermentation broth by centrifugation.

Differential centrifugation leads to separation of cells and riboflavin crystals because of differences in size and sedimentation behaviour.

Riboflavin is then recovered from cell-free broth by using evaporation and vacuum drying.

VITAMIN C L-ascorbic acid finds its use mainly in food

industry, being a vitamin as well as an antioxidant.

Majority of commercially manufactured L-ascorbic acid is synthesized via Reichstein process using D-glucose as a starting material

Approximately 50 % of synthetic ascorbic acid is used in vitamins supplements and pharmaceutical preparations.

Because of its antioxidant properties and its potential to stimulate collagen production, it is also widely used as an additive to cosmetics.

REICHESTEIN PROCESS

.

Reichestein Process

Diacetone-L-Sorbose

L-Sorbose

D-Sorbitol

D-Glu

2-Keto-L-Gluconic

acid methyl ester

L-Ascorbic

acid

Sorbitol pathway

2-keto-D-gluconic acid pathway

Yeast Based Fermentative ProcessesSaccharomyces cerevisiae and

Zygosaccharomyces sp. produce L-ascorbic acid intracellularly when incubated with L-galactose.

Over-expression of the D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase in Saccharomyces cerevisiae enhances this ability significantly.

FERMENTATION BY ALGAE Skatrud and Huss described a method that involved

initial growth of Chlorella pyrenoidosa ATCC53170 in a fermentor with a carbon source that is sufficient for the cells to grow to an intermediate density. At the depleted stage, additional carbon source was added sequentially or continuously to maintain the carbon source concentration below a predetermined level until the addition is terminated. This resulted in the production of 1.45 g/L of L-ascorbic acid.

Euglena gracilis Z. is one of the few microorganisms which simultaneously produce antioxidant vitamins such as carotene (71 mg/L), vitamin C (86.5 mg/L) and vitamin E (30.1 mg/L).

BIOTIN (VITAMIN H) Biotin (vitamin H) is one of the most fascinating

cofactors involved in central pathways in pro- and eukaryotic cell metabolism.

While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves.

Biotin is added to many food, feed and cosmetic products.

Majority of the biotin sold is synthesized chemically.

The chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes

Biosynthesis of Biotin

The conversion of dethiobiotin to biotin has not been resolved.

bioF gene

bioD gene

bioB gene

bioA gene

Ogata et al. screened microorganisms and demonstrated that the bacterium B. sphaericus can excrete significant quantities of biotin synthetic pathway intermediates from precursor, Pimelic acid.

Microbial fermentation of amino acids

Glutamic acid Microbial production of l-glutamic acid has

been extensively studied by a large number of research investigators.

The most popular Coryneform species include

C.glutamicum, Corynebacterium, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divarticum, Brevibacterium ammoniagenes, Brevibacterium thio- genetalis, Brevibacterium saccharoliticum, and Brevibacterium roseum .

Other glutamic acid-producing organisms include Escherichia coli, Bacillus megaterium, Bacillus circulans, Bacillus cereus, and Sarcina lutea.

Industrially, glutamic acid is usually manufactured by batch/fed-batch submerged fermentation processes using genetically modified strains of Corynebacterium or Brevibacterium.

MEDIA COMPOSITION The seed medium composition can be

used: glucose (8%), NH4Cl (0.5%), corn steep liquor (0.3%), K2HPO4 (0.5%), KH2PO4 (0.5%), MgSO4·7H2O (0.03%), CaCO3 (1.0%), and deionized water to make 100%.

The pH of the medium -7.2 The inoculated flasks are grown in an

orbital shaker incubator maintained at 30°C and 230 rpm for 15 h.

The entire contents of the one flask is then transferred to a 2.0 L capacity fermenter with 500 mL of sterile nutrient medium containing molasses (20%), KH2PO4 (0.5%), KH2PO4 (0.5%), MgSO4·7H2O (0.3%), urea (0.8%), CaCO3 (1.0%), and deionized water to make 100%.

In most cases, the optimum pH of the medium was recorded as 7.0.

The fermentation is usually initiated with continuous agitation and aeration for 48 h at 30°C

INDUSTRIAL APPLICATIONS AND THERAPEUTIC ROLEThe greatest application of glutamic acid and its

salt is in the food industry as a flavor enhancer. To aid in peptic ulcer healingOne of the leading roles of glutamic acid in

pharmaceuticals is that of a neurotransmitter. The blockage of NMDA receptors can greatly

affect the memory and overall mental performance of an individual.

Glutamic acid and aspartic acid have the capability to combine with NMDA receptors thus increasing cation conductance, depolarizing the cell membrane, and deblocking the NMDA receptors.

LYSINEl-Lysine is one of the leading and

most exploited amino acid among the essential amino acids list.

l-lysine can be synthesized from α-aminoadipic acid by yeast and Neurospora mold, or from diaminopimelic acid (DAP) by E. coli

FERMENTATIVE PRODUCTION OF LYSINE

Organism: mutant strain of C.glutamicum In commercial-scale starches, molasses and

glucose are mostly used as the carbon source. Care must be taken to create a balance

between carbon and nitrogen sources such as corn steep liquor, soybean cake acid hydrolysate, yeast extract, peptone, and the like

Inorganic salts such as KH2PO4, K2HPO4, MgSO4·7H2O,FeSO4·7H2O, ZnSO4·7H2O, MnSO4·7H2O, (NH4)2 SO4.

Other nutrients are biotin and vitamin B1.

In most cases, the optimum pH of the medium has been 7.2 and temperature at 30°C.

The seed stage cultivation requires around 24 h, whereas the fermentation stage is complete by approximately 96 h.

After this, harvesting is done and the product l-lysine is recovered using some suitable and economical method

INDUSTRIAL APPLICATIONS AND THERAPEUTIC ROLE It is an important additive to animal feed for

optimizing the growth of pigs and chickens.In the food industry, l-lysine is used in a

number of dietary or nutritional supplements that are popularly used by athletes, weight lifters, bodybuilders, and even some individuals to boost their energy level and protect their muscles from deterioration.

l-lysine is also recommended for the treatment of some viral infections, for example, herpes simplex, cold sores, shingles, and human papillomavirus infections such as genital warts and genital herpes

TRYPTOPHAN No scientific reports were available

relating to the microbial direct production of tryptophan. During this period, more attention was given by researchers looking into the possibility of tryptophan production

With the introduction of efficient strains of Corynebacterium and E. coli, now tryptophan is largely produced by fermentation

FERMENTATIVE PRODUCTION Genetically modified strain of C. glutamicum

that is capable of producing tryptophan.Fermentation medium may be prepared from

molasses (30%), corn-steep liquor (0.7%), KH2PO4 (0.05%), K2HPO4 (0.15%), MgSO4·7H2O (0.025%), (NH4)2SO4 (1.5%), and calcium carbonate (1%).

In addition vitamin B1, biotin, l-phenylalanine, and l-tyrosine.

pH adjusted to 6.81.0 mL of 20% silicon RD in deionized water

is added as antifoam.

The fermenters can be harvested after 72 h.

Product recovery is usually done using ultracentrifugation at around 10,000 rpm, followed by treatment with cation exchange resin and decolorization with activated carbon.

After further centrifugation, the mixture can be subjected to drying under a vacuum dryer.

INDUSTRIAL APPLICATIONS AND THERAPEUTIC ROLETryptophan has a wide range of

applications in the feed and pharmaceutical industries.

As an essential amino acid with a unique indole side chain, which indicates its use as a precursor for a number of neurotransmitters in the brain.

Its application in the chemical synthesis of some antidepressant drugs

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