Studies on Oxidative Fermentation. Part XII

Two substances besides rubiginol, of positive ferric chloride reaction were isolated from a shaking culture of Gluconoacetobacter liquefaciens, using glucose as the substrate. One of them was found to be a new γ-pyrone derivative and the composition was concluded to be 3,5-dihydroxy-l,4-pyrone-2-carboxylic acid. The other substance was found to be comenic acid. Assumptions were made with regard to the mechanism of the formation of these γ-pyrone derivatives from glucose.     

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 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 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 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.     

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 the the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

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 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.     

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。     

鸡腿菇液体深层发酵工艺条件的研究

目的：研究碳源、氮源、接种量、初始pH值、温度等对鸡腿菇深层发酵的生物量和多糖产量的影响。方法：通过摇瓶培养确定了该菌株的发酵优化条件,在此条件下,获得了较高的多糖产量。结论：结果表明葡萄糖、酵母浸粉有利于鸡腿菇菌体生长和胞外多糖的形成,在初始pH值6、接种量10％、温度26℃、250ml摇瓶装液量l00ml的条件下,鸡腿菇深层发酵结果最佳,在此基础上进行摇瓶发酵曲线测定,确定了鸡腿菇适宜发酵周期为144h,胞外多糖最高可达1．98g/L。     

微生物混合发酵的研究及应用

由于不同微生物之间的正相互作用,人们发现应用两种或两种以上微生物混合发酵能更好地解决实践中的许多问题。在过去几年中,对微生物的混合发酵的应用以及其中微生物之间的相互作用机理的研究取得了明显进展,主要有以下4个方面:(1)对生物质的降解利用;(2)对环境污染物的降解;(3)生产特定的代谢产物;(4)混合发酵的工艺。综述了微生物混合发酵的应用及相关机理、涉及的微生物和影响因素。     

茁霉多糖发酵及提取工艺条件研究

目的：茁霉多糖是一种新型多功能生物材料,该文拟对出芽短梗霉发酵茁霉多糖及其提取条件进行初步探索。方法：通过摇瓶培养确定了该菌株的发酵优化条件,在此条件下,获得了较高的多糖产量,同时通过醇提的方法对茁霉多糖的提取进也行了研究。结果：初步确定了茁霉多糖最佳发酵与提取条件。结论：摇瓶转速和发酵初始pH值是多糖发酵的重要影响因素,它们与多糖的合成密切相关。     

黑木耳深层发酵工艺条件的研究

研究了碳源、氮源、接种量、初始pH、温度等对深层发酵黑术耳的生物量和多糖产量的影响,结果表明葡萄糖、酵母膏、豆饼粉有利于黑木耳菌体生长和胞外多糖的形成。在初始pH5．3、接种量10％、种龄6d,温度28℃、250ml摇瓶装液量80ml的条件下,黑木耳深层发酵结果最佳,在此基础上进行摇瓶发酵曲线测定。确定了黑木耳适宜发酵周期为96h,胞外多糖最高可达5.01g/L。     

红谷霉素生料发酵工艺的研究

红谷霉素是链霉菌702发酵所产的一种生物活性物质,具有较强抗细菌活性。在摇瓶条件下,对链霉菌702生料发酵生产红谷霉素进行研究。采用单因素实验探索生料发酵工艺,筛选出摇瓶生料发酵条件为7.5%的种子接种量和添加抗菌剂1.5ml。在此条件下进行生料发酵,使红谷霉素摇瓶发酵产量达到1.46g/L,比常规灭菌发酵产量提高25.7%,实验结果表明红谷霉素生料发酵是一种潜在可行的发酵方法,不但可以很好的节约发酵成本,还可以提高发酵产量。     

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.