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Glutamic acid fermentation

Glutamic acid fermentation produces glutamic acid through microbial fermentation. Corynebacterium glutamicum is commonly used to produce glutamic acid through fermentation. Glutamic acid has various applications including as a flavor enhancer in foods as monosodium glutamate, in treating neurological diseases, and for producing polyglutamic acid which has industrial uses. Downstream processing after fermentation includes filtration, crystallization or ion exchange chromatography to purify the glutamic acid.

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GLUTAMIC ACID FERMENTATION
INTRODUCTION • Glutamic acid was discovered by Ritthausen in 1866. • In 1908 professor Ikeda found that the material enhancing the taste of sea weed (tangle) soup came from a sodium salt of glutamic acid (monosodium glutamate). • Since then monosodium glutamate has been widely used in both the food industry and by the general public as a flavor-enhancing agent. • The demand of glutamic acid has increased rapidly. Before 1956 glutamic acid was mainly obtained by the hydrolysis of plant proteins . gluten
• In 1956 Japanese investigators reported the possibility ofproducing glutamic acid by rnicrobiological Means, and began on an industrial scale the production of this amino acid
333.8 °CBoiling point
APPLICATIONS • Gluatamic acid is Important in brain metabolism hence various analogues of glutamic acid are used in treating various neuropathic diseases. • Since 1908, monosodium glutamate has been used as a flavor enhancing agent both by the food industry and the general public. • Poly glutamic acid (PGA) is a naturally occurring anionic polymer that is biodegradable, edible, and non-toxic towards human and environment . • It is a good candidate for various industrial applications including thickener, bitterness reliving agent , Cryoprotectant. • Curable biological adhesive • heavy metals absorbers .
• Cosmetics - L-Glutamic Acid is widely used as Hair restorer in Cosmetics. - As Hair restorer: in treatment of Hair Loss. - As Wrinkle: in preventing aging
Microorganisms • Corynebacteriumspp. (C. glutamicum; C. lilum) Brevibacterium spp. (B. divericartum: B. alanicum) Microbacterium spp. (M. flavumvar. glutamicum) Arthrobacter spp. (A. globiformis; A. aminofaciens)
PRODUCTION OF GLUTAMIC ACID BY WILD TYPE BACTERIA • (i) Organisms: Wild type strains of the organisms of the four genera mentioned above are now used for the production of glutamic acid. • The preferred organism is however Corynebacterium glutamicum. The properties common to the glutamic acid bacteria are: • (a) they are all Gram-positive and non-motile; •(b) they require biotin to grow;
• (c) they lack or have very low amounts of the enzyme alpha-ketoglutarate, which is formed by removal of CO2 from isocitrate formed in TCA cycle (citric acid cylce). • Since alpha -ketoglutarate is not dehydrogenated it is available to form glutarate by reacting with ammonia. • (ii) Conditions of the fermentation: The composition of a medium which has been used for the production of glutamic acid is as follows (%): glucose, 10; corn steep liquor 0.25; enzymatic casein hydrolysate 0.25; K2HPO(Dipotassium phosphate )4 0.1, Mg. SO4, 7 H2 O, 0.25; urea, 0.5. • It should be noted that besides glucose, hydrocarbons have served as carbon sources for glutamic acid production. • The optimal temperature is 30° to 35° and a high degree of aeration is necessary.
• (iii)Biochemical basis for glutamic acid production: Studies by several workers have clarified the basis for glutamic acid production as summarized below. • (a) Glutamic acid production is greatest when biotin is limiting; that is, when it is sub optimal. When biotin is optimal, growth is luxuriant and lactic acid, not glutamic acid, is excreted. • The optimal level of biotin is 0.5 mg per gm of dry cells; with higher amounts glutamic acid production falls. • (b) The isocitrate-succinate part of the TCA cycle is needed for growth. It is only after the growth phase that glutamic acid production becomes optimal. • (c) An increase in the permeability of the cell is necessary so as to permit the outward diffusion of glutamic acid, essential for high glutamic acid
• This increased permeability to the acid can be achieved in the following ways: • (i) ensuring biotin deficiency in the medium • (ii) treatment with fatty acid derivatives, • (iii) ensuring oleic acid deficiency in mutants requiring oleic acid (C16 - C18). • (iv)addition of penicillin during growth of glutamic acid bacteria, Cells treated in one of the first three ways above have cell membranes in which the saturated to unsaturated fatty acid ratio is abnormal, therefore the permeability barrier is destroyed and glutamic acid accumulates in the medium. • The major factor in glutamic acid production by wild type organism is thus altered permeability. • Treatment with penicillin prevents cell-wall formation. Cell wall inhibiting antibiotics such as penicillin and cephalosporin have enabled the use of molasses .which are rich in biotin for glutamic acid production.
FERMENTOR PRODUCTION OF AMINO ACID • 21.6.1 Fermentor Procedure Starting from shake flasks the inoculum culture is grown in shake flasks and transferred to the first seed tank (1,000–2,000 liters) in size. • After suitable growth the inoculum is tansferred to the second seed tank (10,000– 20,000 liters), • which serves as inoculum for the production tank (50,000–500,000 liters). • The fermentation is usually batch or fed-batch . • In batch cultivation all the nutrients are added at once at the beginning of the fermentation, except for ammonia. • which is added intermittently to help adjust the pH, and fermentation continues until sugar is exhausted.
• In a fed-batch process, the fermentor is only partially filled with medium and additional nutrients added either intermittently or continuously until an optimum yield is obtained • . The fed-batch appears preferable for the following reasons: (a) Most amino acid production requires high sugar concentrations of up to 10%. If all were added immediately, acid would be quickly produced which will inhibit the growth of the microorganisms and hence reduce yield • (b) Where auxotrophic mutants are used, excess supply of nutrients leads to reduced production due to overgrowth of cells or feed back regulation by the nutrient. (c) During the lag phase of growth, the oxgen demand of the organism may exceed that of the organism leading to reduced growth
21.6.2 Raw Materials • The main raw materials used are cane or beet molasses and starch hydrolysates from corn or cassava as glucose. • In the US, the preferred carbon source is corn syrup from corn, whereas in Europe and South America it is beet molasses. • As nitrogen source, inorganic sources such as ammonia or ammonium sulfate is generally used. • Phosphates, vitamins and other necessary supplements are usually provided with corn steep liquour.
FERMENTATION The usual culture medium for glutamic acid fermentationcontains a carbon source such as glucose, the acid hydrolysate ofstarch, molasses, or a mixture of these substances. A nitrogen source such as urea, and other chemicals is present. The prepared culture medium is sterilized in a fermentor by steam. When the temperature of the medium cools down to 30°C, the micro-organism is added to the fermentor in a proper inoculum size. The micro-organism is incubated for thirty-six to forty-eight hours during which time the Ph(7-7.8), temperature(30-35) , and aeration rate are carefully controlled.
21.6.4 Down Stream Processing • After fermentation, the cells may be filtered using a rotary vacuum filter. Sometimes filtration can be improved by using filter aids. • These filter aids, usually, kiesselghur which are based on diatomaceous earth, improve the porosity of a resulting filter cake leading to a faster flow rate. • Before filtration a thin layer is used as a precoat of the filter (normally standard filters). The extraction method of the amino acid from the filtrate, depends on the level of purity desired in the product. • However two methods are generally used: the chromatographic (ion exchange) method or the concentration-crystallization method.
Crystallization • Crystallization is often used as a method to recover the amino acid. Due to the amphoteric character (contains both acidic and basic groups) of amino acids, their solubility is greatly influenced by the pH of the solution and usually show minima at the isoelectric point (zero net charge). • Since temperature also influences the solubility of amino acids and their salts, lowering the temperature can be used in advance as a means of obtaining the required product. • Precipitation of amino acids with salts, like ammonium and calcium salts, and with metals like zinc are also commonly used.
Ion exchange • resins have been widely used for the extraction and purification of amino acids from the fermentation broth. • The adsorption of amino acids by ion exchange resinsis strongly affected by the pH of the solution and by the presence of contaminant ions. • There are two types of ion exchange resins; cation exchange resins and anion exchange resins. • Cation exchange resins bind positively charged amino acids (this is in the situation where the pH of the solution is lower then the isoelectric point (IEP) of the amino acid),
• whereas anion exchange resins bind negatively charged amino acids (pH of the solution is higher than IEP). • Extraction of the bound amino acid(s) is done by introducing a solution containing the counterion of the resin. • • Anion exchange resins are generally lower in their exchange capacity and durability than cation exchange resins and are seldom used for industrial separation. • In general, ion exchange as a tool for separation is only used when other steps fail, because of its tedious operation, small capacity and high costs
According to the findings of the scientific panel of the PFA, Chinese salt contains Monosodium glutamate (MSG) which can cause headaches, fatigue, palpitations, nausea and vomiting, sweating, flushing and numbness of the face, more so among people who are sensitive to it. It can also cause hypertension and is extremely hazardous for pregnant women, the panel found. According to some reports, regular use of this ingredient can cause long- term issues like high blood pressure, autism, hormonal imbalance, epilepsy, food allergies, asthma, reduction in bile formation, cancer and possible sterility in females.
Glutamic acid fermentation