The glucose is broken down into C3 and C2 fragments by glutamic acid producing microorganisms through the Embden Meyerhof-Parnas (EMP) pathway and the pentose-phosphate pathway and the fragments are channeled into the tricarboxylic acid (TCA) cycle.
The reactions of EMP pathway are more common under conditions of glutamic acid production. The key precursor of glutamic acid is α – ketoglutarate, which is formed in the TCA cycle via citrate, isocitrate and α-ketoglutaric acid, which is then converted into L-glutamic acid through reductive amination with free NH4+ ions. The last step is catalysed by the NADP dependent glutamate dehydrogenase.
The NADPH2 required at this stage of the reaction is furnished through the proceeding oxidative decarboxylation of isocitrate dehydrogenase. The NADPH2 is then regenerated by the reductive amination of α-ketoglutarate.
(i) Effect of Permeability on Glutamic Acid Production:
Production and excretion of glutamic acid is dependent on cell permeability.
Increased permeability in glutamic acid producing bacteria can be accomplished by one of the following ways:
(a) Through biotin deficiency.
(b) Through the addition of penicillin.
(c) Through the addition of saturated fatty acids or fatty acid derivatives.
(d) Through the oleic acid deficiency in oleic acid auxotrophs.
(e) Through the glycerol deficiency in glycerol auxotrophs.
(ii) Conditions of Production:
The following factors affect the glutamic acid fermentation:
(a) Carbon source,
(b) Nitrogen source,
(c) Growth factors,
(d) Oxygen supply, and
(e) pH of the medium
(a) Carbon Source:
A wide variety of carbohydrates are used as carbon source in the fermentation process. Glucose and sucrose are frequently used. However, starch hydrolysates, fructose, maltose, ribose and xylose are also used less frequently. Moreover sucrose, sugarcane molasses and sugar beet molasses can also be used. Both the molasses contain high biotin content (0.4-1.2 mg kg-1 in cane molasses and 0.02-0.08 mg kg-1 in beet molasses). Penicillin or fatty acid derivatives (e.g. Tween-66) must be added to the fermentation medium.
When these molasses are used in the medium preparation because they increase the cell permeability of L-glutamic acid. For industrial production, generally cane molasses or starch hydrolysate are used.
(b) Nitrogen Source:
Ammonium sulphate, ammonium chloride, ammonium phosphate, aqueous ammonia, ammonia gas and urea have been used as nitrogen source. Although large amount of ammonium ions are necessary, a high concentration of it inhibits the growth of the microorganism as well as the yield of L-glutamic acid. Therefore, suitable amount of ammonia is added, as the fermentation progresses. These salts also help in the pH control.
(c) Growth Factors:
The important growth factor is biotin. Its optimal concentration depends upon the carbon source used. In media with 10% glucose, its requirement is 5 mg liter-1. In media with lower glucose concentration, it is considerably lower. Some strains require L-cystine as an additional growth factor.
(d) Oxygen Supply:
The oxygen concentration should neither be too low nor too high. Optimal L-glutamic acid yields are obtained at kb value of 3.5 x 10-6 mole 02 atm-1 min-1 ml-1. Excretion of lactate and succinate occurs under oxygen deficiency, whereas excess oxygen under ammonium ions deficiency causes growth inhibition and production of α-ketoglutarate. In both the cases, glutamic acid yields are low.
Optimum pH for growth and glutamic acid production is 7.0-8.0 and it is controlled by the addition of ammonium salts.
(iii) Fermentation Production:
Important features of L-glutamic acid production are precised in Fig. 5.7.
L-glutamic acid can be produced commercially by the following ways:
1. By a two stage fermentation process where α-ketoglutaric acid is produced by one microorganism, is then converted into L-glutamic acid by another microorganism.
2. By one stage fermentation process employing only one microorganism.
The fermentation process of L-glutamic acid is described below:
(i) Inoculum production,
(ii) Preparation of medium,
(iii) Fermentation process,
(iv) Harvest and recovery
(i) Inoculum Production:
A medium with the following composition is prepared for inoculum production.
A suitable strain of C. glutamicum from a stock culture is selected and is inoculated into the above sterilized medium. The culture is incubated upto 16 hours at 35°C. After sufficient growth occurs, approximately 6% by volume of inoculum is added to the production fermenter.
(ii) Preparation of Medium:
A production medium is prepared with the following composition:
The medium with the above composition is sterilized and employed for the production of L-glutamic acid.
(iii) Fermentation Process:
The fermentation is carried out, approximately, for 40-48 hours at 30°C temperature. The pH is adjusted to 7.0-8.0. The urea is added intermittently during the fermentation. Approximately 50% of the supplied carbohydrate is converted into L-glutamic acid. The general flow diagram for the industrial production of L-glutamic acid is illustrated in Fig. 5.8.
(iv) Harvest and Recovery:
The same process of recovery that is employed for L-lysine is also employed for the harvest and recovery of L-glutamic acid.
Glutamic acid can also be produced through biotransformation of racemic mixture of D-and L-hydantoin-5-propionic acid with the help of hydantoinase. Simultaneously D-hydantoin is converted to L-hydantoin-5-propionic acid in the presence of hydantoin racemase (Fig. 5.9).