l-Glutamic Acid Fermentation with Molasses

An l-glutamic acid (l-GA)-forming bacterium. Microbacterium ammoniaphium was cultured in the molasses medium with or without poiyoxyethylene fatty acid esters to obtain l-GA-accumulating cells or non-accumulating cells, respectively.

Then protoplast-like bodies (PLB) were prepared from each group of cells by reacting them with egg white lysozyme.

l-GA-accumulating reaction by the PLB was carried out under high and low osmotic pressures.

From the results of the experiment, it was shown that the difference in the ability of l-GA accumulation between l-GA-accumulating cells and non-accumulating cells was attributed mainly to the difference in the nature of the cell membrane.

Further, the relationship between the molar ratio of saturated fatty acids/unsaturated fatty acids which was reported previously and the nature of the membrane was discussed.

The lipid composition of the cell membrane from Microbacterium ammoniaphilum was determined by thin-layer and column chromatographies to make clear the relation between the extracellular accumulation of l-glutamic acid and the lipid in the cell membrane. When polyoxyethylene fatty acid ester was added to the beet medium and a large amount of l-glutamic acid was accumulated, the increase of the saturated fatty acid (C₁₆, C₁₈) in the neutural lipid fraction and the decreases of the phospholipid fraction and the unsaturated fatty acid $({\text{C}}_{18}^{1=})$ in the neutral lipid fraction were recognized.     

l-Glutamic Acid Fermentation

Structure of a sugar lipid produced by an oleic acid-requiring mutant of Brevibacterium thiogenitalis was studied and established as (I).

Relation between biotin and oleic acid was studied using a biotin-requiring organism accumulating l-glutamic acid and its blocked mutants lacking the biosynthetic system of biotin or/and oleic acid. The results support the following considerations. Biotin is not formed from oleic acid and does not substantially affect the growth of l-glutamic acid-accumulating bacteria and their productivity of l-glutamic acid.

Consequently, biotin serves only for the synthesis of fatty acids in the present organisms. The essential factor for their growth and metabolism is an unsaturated fatty acid like oleic acid and not biotin. And also, saturated fatty acids have substantially no relation with their growth and metabolism like accumulation of l-glutamic acid.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Tween 60 (polyoxyethylene sorbitan monostearate) has been found to be the most effective derivative of fatty acid in accumulating l-glutamic acid in biotin-sufficient medium. The effect was exceedingly subject to the influence of the addition time of the ester, and this was observed also on the growth curve of Brev. lactofermentum. Changes of the growth curve caused by the varied addition time of the ester corresponded to those by the concentration of biotin in the medium that did not contain Tween 60. The patterns of fermentation course in the two corresponding conditions, such as biotin 3 μg/l and biotin 20 μg/l-Tween 60 mg/ml, agreed closely with each other. It seemed that identical cells were grown on the conditions. The only difference between the cells was observed as to the contents of intracellular biotin. Although l-glutamic acid was not accumulated by biotin-sufficient cells, cells with sufficient biotin and capable of accumulating l-glutamic acid were obtained in the presence of Tween 60, in which case the ester neither prevented the cells from taking up biotin nor controlled the level of intracellular biotin.     

l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

Brev. lactofermentum rapidly took up biotin from culture medium and stored it in the cells. The saturation level of the stored biotin (3.8 × 10⁴ molecules/cell) exceeded the level required for the maximum growth by ten times, and the minimum level (1.3 × 10³ molecules/cell) was the most adequate to the accumulation of l-glutamic acid. The stored cellular biotin over the minimum level was metabolically available in the subsequent culture lacking in supplemented biotin. The cellular biotin was gradually reduced to the minimum level with the multiplication of the cells, and them the accumulation of l-glutamic acid was observed. This relation between the level of cellular biotin and the accumulation of l-glutamic acid was impaired by the addition of Tween 60 or some saturated fatty acid. In the presence of biotin and Tween 60 the biotin-saturated cells turned into cells capable of accumulating l-glutamic acid keeping the maximum level; and in the same medium the cells having the minimum amount of biotin took up biotin and then were saturated with it, and yet the cells preserved the acid-accumulating property. It was confirmed with the use of bioautographic technique and avidin test that the biotin released from the cells by acid hydrolysis was identical with authentic d-biotin.     

l-Glutamic Acid Fermentation with Molasses

When an l-Glutamic acid (l-GA)-forming bacterium, Microbacterium ammoniaphilum, was cultured in the molasses medium with the addition of penicillin to accumulate large quantity of l-GA extracellularly, no significant differences were observed in the phospholipid quantity and the fatty acid composition which were found between the l-GA-accumulating cells grown either in the molasses medium with addition of polyoxyethylene fatty acid ester (POEFE) or in the glucose medium with the addition of biotin.

Moreover, it was shown that, in the molasses-POEFE system, the amount of l-GA accumulated was nearly constant, independent of the extracellular osmotic pressure caused by the presence of NaNO₃ or β-alanine, while, in the molasses-penicillin system, the amount varied inversely to the osmotic pressure.

From these results, it is assumed that either chemical or mechanical process can eliminate the permeability barrier in the cell membrane, thus allowing the extracellular accumulation by l-GA-forming bacteria.     

Culture Conditions for the Production of l-Glutamic Acid from n-Paraffins by Glycerol Auxotroph GL-21

A novel process for the microbial production of l-glutamic acid on an industrial scale was successfully established by using a glycerol auxotroph.

The most suitable carbon source for producing L-glutamic acid was n-paraffins (C₁₃–C₁₅). The production of L-glutamic acid was not affected by a large amount of biotin or oleic acid in the absence of penicillin, and occurred maximally at the glycerol concentration of 0.02% at pH 6.6. The most effective temperature was 28°C.

Under optimal conditions in a 200 liter fermentor, the mutant produced 72 g/liter of L-glutamic acid. On the other hand, the parent produced 53 g/liter of L-glutamic acid in the presence of penicillin.

It is believed that the low productivity of L-glutamic acid by the parent strain was mainly due to the occurrence of the marked decrease in the viable cell counts at the later phase of the fermentation caused by the action of penicillin added.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

Change in Permeability to l-Glutamic Acid in the Glycerol Auxotroph GL-21

In this study, the mechanism of the extracellular accumulation of l-glutamic acid by the glycerol auxotroph was partially clarified. Whenever Corynebacterium alkanolyticum GL–21 (glycerol auxotroph) accumulated a large amount of l-glutamic acid in the fermentation broth, the content of its cellular phospholipids was not more than 50% of that of C. alkanolyticum No. 314 (prototroph).

Moreover, biotin, oleic acid or thiamine had no influence on the cellular phospholipid content of the auxotroph.

Under limited supply of glycerol, the efflux of l-glutamic acid in the auxotroph was extremely enhanced, but its enzyme activities participating in l-glutamic acid biosynthesis remained at the same level as those of the prototroph.

From the results, it is considered that the regulation of phospholipid content gave rise to the destruction of the permeability barrier to l-glutamic acid in the cell membrane.     

Induction of Antibiotic Formation in Streptomyces sp. No. 362 by the Change of Cellular Fatty Acid Spectrum

Microorganisms which require oleic acid for the formation of antibiotics were screened. Streptomyces sp. No. 362, one of the selected organisms, produced antimicrobial substances only when oleic acid, palmitic acid or the high concentration of l-glutamic acid (or l-glutamine) was supplemented to the medium. The cellular fatty acid composition was changed by the supplement of these fatty acids, but not by l-glutamic acid (or l-glutamine). Antibiotic-producing cells had about 4 to 10 times larger amino acid pools, especially l-glutamic acid pool, and hexosamine pools. The ability for l-glutamate uptake of cells grown in the oleic or palmitic acid supplemented medium was markedly enhanced and the efflux of the accumulated l-glutamate was reduced. The antibiotic produced by this strain was identified as one of the streptothricin-group antibiotics and the role of these additives in the antibiotic formation is discussed.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Relation between Fatty Acid Composition of Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum

Relation between fatty acid composition of cellular phospholipids and the excretion of L-glutamic acid was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When grown on n-hexadecane, the proportion of unsaturated fatty acids was higher in L-glutamic acid-accumulating cells than in L-glutamic acid-nonaccumulating cells. When grown on fructose or acetic acid, the reverse relation was observed. Moreover, cells containing no oleic acid produced L-glutamic acid from n-pentadecane.

These results suggest that the membrane permeability to L-glutamic acid is not always controlled by the cellular content of unsaturated fatty acids.     

l-Glutamic Acid Fermentation with Molasses

The relation between oleate and biotin to the extracellular accumulation of l-glutamate in Microbacterium ammoniaphilum was studied. And it was suggested that oleate was the essential constituent for the bacterial cell structure, and, at the same time, it participated in the cellular permeability of l-glutamate. On the other hand, biotin was recognized to play a role on the synthesis of cellular fatty acid, mainly oleate and palmitate. Through the discussion above mentioned, the reason was made clear that biotin was not necessary for the bacterial growth or the extracellular accumulation of l-glutamate, if oleate had been added.     

Studies on the Behaviors of Impurities on the Crystallization of l-Glutamic Acid

The behaviors of impurities such as amino acids and inorganic salts at the time of crystallization of l-glutamic acid were investigated; and it was concluded that amino acids which co-existed in the solution of l-glutamic acid followed the crystals of l-glutamic acid persistently, and the contamination mechanism would not be clarified by the adherence of mother liquor or the formation of liquid foams in the crystals, or by the mixed crystal formation, but by a physical adsorption on the crystal surfaces.     

Studies on the Metabolism of d-Amino Acid in Microorganisms

It is confirmed by a new method for the determination of d-glutamic acid, that Aerobacter strain A rapidly metabolizes d-glutamic acid, while it only shows feeble metabolic activity towards l-glutamic acid when it is grown on a dl-glutamate-K₂HPO₄ medium. A specific d-glutamic oxidase is demonstrated in the cell-free extracts of Aerobacter strain A. This enzyme seems to be different from d-glutamic-aspartic oxidase obtained from Aspergillus ustus by the authors, since the former has no activity towards d-aspartic acid.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

l-Glutamic Acid Fermentation

A study was made on the differences between Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant D-248 in some physiological characteristics.

The most important difference of the characteristics was found in their intracellular fatty acid contents. Namely, the cellular oleic acid content of D-248 was scarcely affected by biotin but limited by the oleic acid which was added to the medium.

On the other hand, various enzyme activities and rates of oxygen uptake for several organic acids were found to be slightly different between the two strains.

These observations suggest that oleic acid has an important role for the production of l-glutamic acid.

The effect of biotin and oleic acid on the cellular fatty acid contents, and the relation between the cellular fatty acid contents and the productivity of l-glutamic acid were investigated using Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant, D-248.

While the synthesis of palmitic acid in D-248 was stimulated by biotin and competitively reversed by oleic acid added to the culture medium, the level of cellular oleic acid was scarcely affected by biotin but regulated by oleic acid in the medium.

For the productivity of L-glutamic acid, the most important factor was the level of cellular oleic acid, and the effect of cellular palmitic acid was considerably weak. This relation was subjected to a figuration and able to be expressed on the whole as one exponential-like curve. An amount of over 70 per cent of cellular fatty acids was distributed in the phospholipid fraction and its fatty acid composition was almost the same as that of whole cells.     

Chemical Studies on the Antibiotic Esperin

Esperin is an acidic antibiotic with a molecular formula of C₃₉H₆₇N₅O₁₁ and, on hydrolysis with acid, it afforded l-aspartic acid, l-glutamic acid, l-valine, l-leucine, d-leucine and 2-tridecenoic acid. By treatment with alkali, esperin was transformed to esperinic acid, C₃₉H₆₉N₅O₁₂, which was shown to be β-hydroxytridecanoyl-glutamyl-aspartyl-valyl-leucyl-leucine. From chemical and physical studies, esperin was proved to be the lactone of esperinic acid, represented by the formula III.     

Studies on l-Glutamic Acid Fermentation

The authors have carried out a series of studies on l-glutamic acid fermentation with a strain of Brevibacterium divaricatum nov. sp. in the previous papers.

In this paper, some metabolism of l-glutamic acid and oxidative decomposition of several organic acids concerning the tricarboxylic acid cycle by the resting cells have been studied. The results suggest that l-glutamic acid is one of the final fermentative products of this bacterium, and the tricarboxylic acid cycle is working as a glutamic acid forming cycle.

The presence of glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, DPN-linked glyceraldehyde-3-phosphate dehydrogenase, and TPN-linked glucose-6-phosphate dehydrogenase in cell-free extracts of this bacterium was also demonstrated.