Effects of Culture Conditions on Poly(beta-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(beta-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH(2)PO(4), and 0.2% NH(4)Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.     

培养条件对毛栓菌漆酶分泌的影响

研究了碳源、氮源、愈创木酚、香兰素及培养条件对漆酶分泌的影响;结果表明,麦草粉作碳源、(NH_4)_2SO_4作氮源有利于漆酶的分泌,适宜浓度的愈创木酚和香兰素等对漆酶的产生有一定的作用;pH在3.0～8.0的范围内对漆酶的分泌影响差别不大,培养温度、接种量、通气量对漆酶的分泌有较大影响。     

Volatile Acid Production by Clostridium sporogenes under Controlled Culture Conditions

S ummary . Clostridium sporogenes , grown under controlled culture conditions in a complex medium, starts to produce volatile acids at the end of the logarithmic growth phase when sporulation begins. Since, however, the amount and type of acids depend on the nature of the medium used, it is doubtful whether the volatile acid pattern can be used for identification and classification of proteolytic Clostridia without controlling culture conditions.     

Production of l-Phenylalanine from trans-Cinnamic Acid with Rhodotorula glutinis Containing l-Phenylalanine Ammonia-Lyase Activity

An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid.     

Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.     

Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH₂PO₄, and 0.2% NH₄Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.     

Studies on Production of Nucleic Acid and its Related Compounds by Microorganisms

Adenosine was produced from adenine by means of fermentation using Bacillus subtilis Marburg 160-88 (st^r, try^-, pur^-). For the study on adenosine production, experiments concerning with pre-culture age, inoculum size, fermentation period, concentration of adenine, carbon source, nitrogen source and supplement of vitamins were carried out by test tube shaker. Furthermore the time course of fermentation was observed using jar fermentor. And it was proved that adenosine was produced about 1 mg/ml during the first 40 hrs of fermentation in the glucose mineral medium containing 1~2 mg/ml of adenine. This fermentation procedure seems to be one of economical methods for adenosine production.     

Culture Conditions of l-Isoleucine Fermentation from Acetic Acid

Culture conditions were studied for l-isoleucine production from acetic acid. Acetate and ammonium concentration in culture liquid exerted a great influence on the fermentation, and optimum concentration was 2–5 g/liter and 2–3 g/liter respectively. To maintain these conditions throughout the culture, it was necessary to supply intermittently a small amount of feeding solution which consisted of ammonium acetate and acetic acid. Molecular ratio of the former to the latter was 0.175, and total concentration of acetic acid was 700 g/liter.

Carbon dioxide showed an inhibitory influence on l-isoleucine production and adequate ventilation was necessary for satisfactory result. Maximum amount of l-isoleucine was 33.5 g/liter after 77-hr cultivation at 28°C and at pH 7.7. Production yield of l-isoleucine was 10% by weight from acetic acid.     

Reversal of Abscisic Acid-Induced Stomatal Closure by trans-Cinnamic and p-Coumaric Acid

Abscisic acid (ABA)-induced increase in stomatal diffusive resistance (SDR) in excised leaves of bean (Phaseolus vulgaris L. cv Pencil Pod) and maize (Zea mays L. cv Golden Bantam) is inhibited by low concentrations of trans-cinnamic acid (TCA) (1 micromolar) and p-coumaric acid (PCA) (10 micromolar) when given together with ABA (10 micromolar) in the transpiration stream through the cut end of the petiole or leaf blade. A concentration effect is observed both in the ABA action and its reversal by phenolic acids. Leaves having attained a high diffusive resistance in ABA solution recover rapidly when transferred to water. ABA (10 micromolar) induced closure of the stomata in onion, Allium cepa L. and Vicia faba epidermal peels. This is associated with loss of K⁺ from guard cells. In the presence of TCA (10 micromolar) and PCA (10 micromolar) K⁺ is retained in the guard cells with open stomata. The dark closure of stomata is also inhibited by TCA and PCA. It is suggested that these phenolic acids may inhibit the ABA effect by competing with or acting on some ABA-specific site, probably located on the plasma membrane, regulating flux of K⁺ ions. A weak association of ABA with the plasma membrane is envisaged because of the rapid recovery obtained upon transferral of the leaves to water.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

Using Phenotype MicroArrays to Determine Culture Conditions That Induce or Repress Toxin Production by Clostridium difficile and Other Microorganisms

Toxin production is a central issue in the pathogenesis of Clostridium difficile and many other pathogenic microorganisms. Toxin synthesis is influenced by a variety of known and unknown factors of genetics, physiology, and environment. To facilitate the study of toxin production by C. difficile, we have developed a new, reliable, quantitative, and robust cell-based cytotoxicity assay. Then we combined this new assay with Phenotype MicroArrays (PM) technology which provides high throughput testing of culture conditions. This allowed us to quantitatively measure toxin production by C. difficile type strain ATCC 9689 under 768 culture conditions. The culture conditions include different carbon, nitrogen, phosphorus, and sulfur sources. Among these, 89 conditions produced strong toxin induction and 31 produced strong toxin repression. Strong toxin inducers included adenine, guanosine, arginine dipeptides, γ-D-Glu-Gly, methylamine, and others. Some leucine dipeptides and the triple-leucine tripeptide were among the strongest toxin repressors. While some results are consistent with previous observations, others are new observations that provide insights into toxin regulation and pathogenesis of C. difficile. Additionally, we have demonstrated that this combined assay technology can be applied broadly to a wide range of toxin producing microorganisms. This study is the first demonstration of simultaneous assessment of a large number of culture conditions influencing bacterial toxin production. The new functional cytotoxin quantitation method developed provides a valuable tool for studying toxigenic microorganisms and may also find applications in clinical and epidemiological research.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Studies on Production of Biotin by Microorganisms

In a preceding paper we reported that Rhodotorula flava 194 effectively converted biotin to biotinamide. In a present paper the metabolism of desthiobiotin by R. flava 194 was studied under the same condition as in the conversion of biotin to biotinamide. Two desthiobiotin derivatives (Vitamer I and II) were isolated. Vitamer II (crystalline) was identified as bisnordesthiobiotin and Vitamer I was chromatographically determined as desthiobiotinamide.     

响应面法优化叶酸高产菌发酵培养基及发酵条件

采用响应面法对叶酸产生菌产朊假丝酵母Y2.12的发酵发酵培养基及发酵条件进行优化.首先,采用Plackett-Burman方法对影响发酵各因素的效应进行评价,筛选出有显著效应的3个因素：碳源乳糖、pH值和摇床转速;并通过Box-Benhnken设计和响应面分析法对这3个主要影响因素进行分析.结果表明,优化后这3个因素的最佳值为摇床转速196r/min、pH7和碳源乳糖含量为6.25%.采用优化后的发酵培养基及发酵条件进行摇瓶发酵,叶酸的含量可达23.14±0.13mg/L,取得了很好的优化效果.     

Studies on Production of Biotin by Microorganisms

Biotin derivatives with biotin activity for some biotin-requiring microorganisms have been isolated in crystalline form from the culture filtrate of strain 194, identified as Rhodotorula flava. The crystalline vitamer was identified as d-biotinamide.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzyme, comenic aldehyde dehydrogenase, which catalyzes the oxidation of comenic aldehyde to comenic acid was partially purified from cell extract of Arthrobacter ureafaciens K-1.

The enzyme was purified 31-fold at Sephadex G-100 filtration step, 112-fold at DEAE-Sephadex A-50 fractionation step, and recovery of the activity was 73.3% and 38.5% respectively.

NADP and magnesium ion were essential for the oxidation. The enzyme shows optimum activity at pH 7.8. Enzyme activity was extremely sensitive to sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. l-Cysteine or dithiothreitol protected the enzyme from p-chloromercuribenzoate inhibition. Carbonyl reagents, such as hydroxylamine and semicarbazide, inhibit the enzyme reaction by formation of addition compounds between carbonyl reagents and aldehyde group of the substrate. The enzyme was completely inactivated after heating for 5 min at 40°C The Km for 5-methoxy comenic aldehyde is 2.5×10^?6 m, and for NADP is 0.4×1O^?6 m. The reaction product, 5-methoxy comenic acid was identified by paperchromatography. The characterization of the enzyme has been carried out by using 5-methoxy comenic aldehyde as the substrate in stead of comenic aldehyde.     

Studies on Production of Biotin by Microorganisms

During the course of the study on biotin vitamers production by a hydrocarbon-utilizing bacterium, strain 5–2 (Pseudomonas sp.), it was found that crude RNA-alkali-hydrolyzate from yeast increased the accumulaion of biotin vitamers, most of which was determined as desthiobion, and that adenine in the crude RNA-alkali-hydrolyzate was a potent stimulator. Effect of adenine on biotin vitamers accumulation was observed in the medium with either hydrocarbon or glucose as a sole carbon source. The accumulation of total biotin vitamers by some other bacteria was also increased by adenine but that of true biotin was scarcely increased or inhibited by adenine.

The role of adenine on the accumulation of biotin vitamers was investigated with non-proliferating cells of strain 5–2, and it was supposed that adenine would not only inhibit the accumulation of true biotin but, as a result, cause the large accumulation of biotin vitamers which might be intermediates of biotin synthesis. When the medium was supplemented with excess biotin, complete repression occurred even in the presence of adenine.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzymatic oxidation of kojic acid to comenic aldehyde was found in the decomposition process of kojic acid by Arthrobacter ureafaciens strain (K-l), a kojic acid decomposing bacteria.

This enzyme was (probable a new type of non-heme iron protein) is assumed to catalyze the dehydrogenation of kojic acid, while the ferric ion contained in the enzyme is considered to serve as an acceptor of hydrogen released from kojic acid. The resulted ferrous ions are oxidized either by molecular oxygen under aerobic conditions or by NAD under anaerobic conditions, accompanying hydrogen peroxide in the former and reduced NAD in the latter. The enzyme was partially purified by using ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and column chromatography with DEAE-Sephadex A-50. The activity increased to 85 fold, compared with crude extracts and the recovery of the activity was 33.9%. The optimum pH of the reaction was 7.75. The enzyme was inactivated by PCMB, and unstable upon heat treatment. A loss of about 50% of the activity was caused by heating at 35%C for 5 min, but some reducing agents protected the enzyme from PCMB inhibition and the heat inactivation. Not only kojic acid, but also benzyl kojic acid or 5-methoxy kojic acid may be substrates. Km value for kojic acid was 1.43 × 10^?5m. The molecular weight of the enzyme was estimated to be about 55,000 and the enzyme contained about two atoms of iron in one molecule. The reaction mechanism for kojic acid oxidase is discussed.     

Stimulation of Bacteriocin Production by Dialyzed Culture Media from Different Lactic Acid Bacteria

The cross-effects of dialyzed postincubates (with a cut-off at 1000 Da) on the biomass and bacteriocin production of six strains of lactic acid bacteria were studied, and a predominance of stimulating responses was found, the characteristics of which suggested merely nutritional effects or the presence of precursor fragments of the bacteriocins. Additionally, cluster analysis of the detected responses provides an approach to define groups of highly compatible (potential consortia) or doubtfully compatible strains of lactic acid bacteria. Such a definition, which does not claim taxonomic value, has practical interest, however, in cases (e.g., silage production) in which it is convenient to use mixed inocula including strains able to establish positive interactions.