Studies on the Low Temperature Fermentation

An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P 145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.

The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

With the Brevibacterium sp. P 145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate l-glutamic acid up to 5.88 mg/ml and l-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of l-glutamic acid and 2.54 mg/ml of l-alanine at 28°C.

The accumulation of l-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, l-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28° to 5°C or from 5° to 28°C, the amino acid accumulation was also changed to that of the final temperature. l-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.     

Fundamental Studies on the Aerobic Fermentation

The oxygen uptake rate of aggregated mycelia is decreased to an extent which, in the case of a typical spherical aggregate, could be estimated depending on its diameter, mycelial density, oxygen diffusivity, and so forth. Equations were presented in this paper to evaluate the oxygen uptake rate of an mold pellet. A favorable agreement was found between the calculation and the experiment.     

Studies on Oxidative Fermentation

It has been found that Gluconobacter liquefaciens metabolized 5-ketogluconic acid. In order to clarify metabolic pathways of this compound, the oxidation products by resting cells of this organism were investigated. Rubiginol, rubiginic, comenic, 2,5-diketogluconic, glycolic and tartronic acids were detected or identified in the reaction fluid. On the basis of these results and the data obtained by means of manometric experiments, the oxidation pathways of 5–ketogluconic acid were discussed.

Oxidation pathways of 5-ketogluconie acid by resting cells of Gluconobacter liquefaciens were further investigated. Arsenite inhibited the oxidation of this compound. The amount of carbonyl compounds in the oxidation products of 5–ketogluconic acid was increased by addition of 10^-3 m arsenite. Pyruvic and α-ketoglutaric acids were identified among these carbonyl compounds. Members of the tricarboxylic acid cycle were oxidized actively by resting cells or cell-free extracts of this organism. These results suggested the presence of the tricarboxylic acid cycle in the terminal oxidatjon of 5-ketogluconic acid by this organism.     

Studies on l-Threonine Fermentation

Fifteen strains of bacteria were treated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine to derive auxotrophic mutants, which were screened for their ability to produce l-threonine. A number of auxotrophs were derived from each strain. Among them, those which produced a large amount of l-threonine were found in Aerobacter aerogenes, Serratia marcescens and Escherichia coli, the members of the family Enterobacteriaceae. Nutritional requirements of these threonine producers were proved to be methionine, lysine, or α, ε-diaminopimelic acid (DAP).

In A. aerogenes and E. coli, double and triple auxotrophs were derived with futher mutational treatment. As a, rule, imposition of additional block led to the increase of l-threonine production. In E. coli, many triple auxotrophs (DAP^?, Met^?, He^?) and their isoleucine revertants were screened for their ability to produce l-threonine. Enhancement of l-threonine production was achieved with these mutants.

One of the isoleucine revertants, KY8280, was used to investigate some cultural conditions. As a result, l-threonine accumulation reached to a level of 13.8 mg/ml with the medium containing 7.5% fructose.     

Studies on Lysine Fermentation

In order to clarify the mechanism of l-lysine accumulation by Micrococcus glutamicus No. 901, a homoserine-auxotrophic mutant, the effects of various amino acids on the two enzymic reactions on the biosynthetic pathway of lysine, the phosphorylation of aspartate and the condensation of aspartic β-semialdehyde (ASA) with pyruvate, were studied using the cell-free extracts of the organism.

The aspartokinase received a multivalent inhibition by threonine plus lysine. Lysine exerted no feedback inhibition in its first step condensing reaction. From these results, the mechanism of the accumulation of lysine by the organism was discussed.     

Studies on the Low Temperature Fermentation

A marine yeast, strain MM313 was isolated from a marine sediment sample at depth of 1120 m. The organism was identified as a Candida sp. MM313. The yeast was able to utilize n-paraffin, n-C₁₀ to n-C₂₀. Regardless of its origin, the organism grew in a medium prepared with fresh water. However, the cell yield increased with increasing concentration of each salt in sea water in the medium and reached a maximum value at the concentration of 75%. The cultivation temperature for the maximum rate of growth and that for the maximum level of growth were 28° and 10°C, respectively. Several cultural conditions were investigated. The cell yields to n-paraffins were about 85% at 15°C after 4 days and 56% at 28°C after 3 days under optimal conditions.     

Studies on Histidine Fermentation

Mutants resistant to 1,2,4-triazolealanine (TRA) or 2-thiazolealanine (TA) were derived from Corynebacterium glutamicum ATCC-13761 by mutagenic treatment with N-methyl-N′-nitro-N-nitrosoguanidine. More than eighty percent of these mutants were found to accumulate a large amount of l-histidine in culture broth. Among these histidine producers, KY-10260 which was selected on TRA-containing agar, was used to investigate the cultural conditions for histidine production. The amount of histidine accumulation reached to a level of 6~8 mg/ml with a medium containing 15% molasses (as glucose) and 4.5% ammonium sulfate.

According to the similar procedure, some histidine producers were derived from other bacteria, Arthrobacter citreus, Brevibacterium flavum, Bacillus megaterium, Bacillus subtilis and Nocardia globerula.     

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.     

Studies on Lactic Acid Fermentation

Resting cells of l. fermentum convert glyceraldehyde to equimolar lactic acid and neither the evolution of carbon dioxide nor the uptake of oxygen was observed. Glyceraldehyde-3-phosphate and 3-phosphoglycerate were identified as intermediates which were equally labeled with inorganic P³² in reaction systems, and the presence of triokinase was suggested.     

Studies on l-Glutamic Acid Fermentation

In the previous paper, most of the enzymes of the Embden-Meyerhof-Parnas pathway and glucose-6-phosphate dehydrogenase have been demonstrated to be present in cell-free extracts of Brevibacterium divaricatum, No. 1627. In this paper, the presence of condensing enzyme, aconitase, TPN-linked isocitric dehydrogenase, succinic dehydrogenase, fumarase, DPN-linked malic dehydrogenase, TPN-linked malic enzyme, oxalacetic carboxylase, isocitritase and malate synthetase in cell-free extracts of this bacterium was also demonstrated. From these results it was concluded that a strain of Brevibacterium divaricatum which has been found to contain all of the enzymes of the tricarboxylic acid cycle, would be capable of forming the key enzymes of the glyoxylate bypass as well. It suggests that the accumulation of α-ketoglutarate involves the glyoxylate bypass besides the tricarboxylic acid cycle in this bacterium.     

Studies on l-Glutamic Acid Fermentation

Micrococcus glutamicus, a glutamate-produeing bacterium, is known to have strong activity of l-glutamic acid dehydrogenase which requires NADP as co-enzyme. In this paper, the NADP-speeifie l-glutamic acid dehydrogenase was purified from M. glutamicus by means of heat treatment with sodium sulfate, precipitation with acetic acid and diethyl-amino-ethyl (DEAE) cellulose column chromatography. The activity of the purified enzyme preparation reached 200-fold as high as that of the crude extract. Some properties of the purified enzyme were investigated. As a result, it was found that the highly purified enzyme preparation acted not only on l-glutamic acid (l-GA) but also on α, ε-diaminopimelic acid (α, ε-DAP) in the presence of NADP. Some of the probable consideration for the dehydrogenation of l-GA and α, ε-DAP are noted.     

L-缬氨酸发酵条件的研究

报道了L 缬氨酸高产菌XQ 6(Leu1AHVrα ABhr2 TAhr)摇瓶发酵条件的研究结果。试验结果表明 ,当发酵培养基中葡萄糖、(NH4 ) 2 SO4 、KH2 PO4 、MgSO4 ·H2 O、玉米浆和生物素的最适用量分别为 1 4 %、5 %、0 .1 %、0 .0 5 %、0 .5 %和2 5 μg/L时 ,经发酵培养 72h ,L 缬氨酸积累可达 5 8g/L ,最高为 62g/L。     

鸟苷补料分批发酵的研究

目的:以枯草芽孢杆菌TA208为出发菌株,研究了补料分批发酵方式下各种参数对鸟苷产量的影响。方法:采用补料分批发酵工艺,利用纸层析法测定发酵液中鸟苷的产量。结果:确定了葡萄糖、酵母粉和次黄嘌呤的最优补料方式,使鸟苷产量达到32.05g/L,较分批发酵方式提高了36.3%。结论:发酵工艺过程控制对发酵生产鸟苷具有重大影响。     

白灵菇液体发酵条件研究初报

通过对白灵菇液体摇瓶发酵的研究 ,确定了适宜白灵菇液体培养的条件为 :装液量为 15 0ml(5 0 0ml)、接种量为 10 %、pH值为 5 .0～ 6 .0、CMC浓度为 0 .4 % ;并通过发酵罐培养 ,研究其发酵过程中的pH值、溶氧值 (DO)的变化。     

Studies on the Amino Acids Fermentation of Pentose Materials

Fundamental studies on the cultural conditions for the amino acids production from pentoses and hexoses employing a strain of our new isolates named Brevibacterium pentoso-aminoacidicum nov. sp. were carried out.

As a result of these experiments, it became possible to obtain about 30% of alanine and 10% of L-glutamic acid based on xylose, and alanine from glucose with a yield of about 40% in the proper conditions.     

Studies on the Amino Acids Fermentation of Pentose Materials

This work was undertaken to determine whether the degradation of sugars by Brevibacterium pentoso-aminoacidicum nov. sp., a bacterium capable of producing amino acids from pentoses and hexoses, was due to constitutive or inducible enzymes. It was also intended to clarify the reason for the substrate specificity in the fermentation of sugars by this bacterium. After a series of experiments using washed resting cells grown on various kinds of sugars or their cell-free extracts, it was found that the enzymes involved in the degradation of pentoses were inducible, while those of hexose metabolism were constitutive. The activities of several enzymes related to the pathways of pentose metabolism were demonstrated and the substrate specificity of sugar degradation by this strain was explained satisfactorily by the inducer specificity of these enzymes.     

Studies on Erythorbic Acid Production by Fermentation

Screening was carried out for erythorbic acid (EA)-producing strains from about 5,000 newly isolated fungi and bacteria. Penicillium notatum FY 115 was screened out as most powerful EA producer. Only Penicillium, but no other genera, was obtained as EA producers from our screening program. Monospore selections and mutagenic treatments succeeded to elevate the yield of EA over 40% to glucose supplied. Various cultural conditions were studied, and pH change during fermentation process was proved to be most important for favorable EA production. Over 80% yield could be obtained when washed mycelium was used in dilute glucose solution.

Abundant accumulation of EA by the strain FY 115, Penicillium sp., in fermentation broth was studied, and EA, both free and Na-salt, was obtained as crystal in the yield of about 45% to glucose supplied, in the media of 8% glucose by jar fermentor, in considering the inhibitory effect of some metal ion.

Extraction processes were improved to elevate the yield and was developed the continuous multi-bed extraction system of anion-exchange resin, which resulted in the yield of 90.9% of EA from fermentation broth in sum total.