Glucose-ammonium Sulfate Medium Research Articles

Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH-stress strategy

To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. When C. utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1.5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5.5 to 1.2 at 24 h and maintained at 1.2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02-08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1.2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197.3 mg l-1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2.11 to 2.67% (w/w), and the total GSH concentration could reach 737.1 mg l-1 and increased by 24.9%. This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources.

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C6), 3-hydroxyoctanoate (HO or C8), and/or 3-hydroxydecanoate (HD or C10) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97-99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.

Possible physiological roles of aspartase, NAD- and NADP-requiring glutamate dehydrogenases of Pseudomonas fluorescens.

The levels of aspartase, NADP- and NAD-requiring glutamate dehydrogenases (GDHs) in Pseudomonas fluorescens grown under various nutritional conditions were determined. NADP-GDH showed the highest value on glucose-ammonium sulfate medium and markedly lower values on amino-acid and casamino-acids media, while the reverse was found for the NAD-GDH, as in the case of other microorganisms with two GDHs. Aspartase did not show a marked variation between the media examined. Glucose nutritionally induced NADP-GDH but suppressed NAD-GDH; and it had no effect on aspartase, which was slightly induced by casamino acids. Transfer of the cells grown on glucose-ammonium sulfate medium to casamino-acids medium clearly increased the levels of NAD-GDH and aspartase, while addition of chloramphenicol to the media abolished the increases, suggesting that the increases were due to de novo synthesis of the enzyme proteins. These results indicate that the aspartase of this microorganism has a different function from those in others, including Escherichia coli.

Lipid accumulation inTrichodermaspecies

Two filamentous fungi, Trichoderma harzianum and Trichoderma viride, were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride. In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.

Lipid accumulation in Trichoderma species

Two filamentous fungi, Trichoderma harzianum and Trichoderma viride, were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient ( C/N = 60 ) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride. In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

More than 300 mg/liter of orotic acid was found to accumulate in the supernatants of the cultures of wild type strains of E. coli K12. The pyrimidine precursor was accumulated in a synthetic medium such as glucose-ammonium sulfate medium. The substance was isolated from the culture, crystallized, and identified as orotic acid. Orotic acid was excreted mainly during logarithmic phase of the bacterial growth. Yeast extract or nutrient broth stimulated bacterial growth, but suppressed orotic acid accumulation. E. coli strains other than K12 failed to accumulate orotic acid.The results suggest that the accumulation of orotic acid is specific to E. coli K12.

Inhibitory effects of ethidium bromide on the growth kinetics of Klebsiella aerogenes, adaptation to ethidium and cross-resistance to proflavine.

The aerobic growth ofKlebsiella aerogenes adapted to a chemically defined glucose ammonium sulphate medium was studied in this medium containing ethidium bromide (EB). The viability of sensitive bacteria on solid medium decreased markedly with EB above 150 mg/liter, and only a fraction of the viable inhabitants of a colony grown with EB at 500 mg/liter survived after a second transfer on to this concentration. In liquid medium containing EB above 30 mg/liter, the logarithmic phase was composite, consisting of a slower first mode followed by a faster second mode, and with EB above 50 mg/ liter composite growth was preceded by a lag. This lag was preceded by a mass doubling and one cell division with EB below 100 mg/liter; at higher concentrations only a mass doubling occurred, and the bacteria became elongated.

&lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt;と&lt;i&gt;Micrococcus&lt;/i&gt; sp. の混合培養に関する研究(第2報)

(1) Saccharomyces cerevisiaeとMicrococcus sp.の混合培養において,高濃度のイノシトールを培養の途中に添加すると混合培養酵母の生育が促進された. (2) Micrococcus sp.の単菌培養では生育が進んでも培地中のイノシトール濃度に変化がみられなかった.両菌の混合培養においては,酵母の単菌培養の場合よりも,培地からのイノシト一ル消費がゆるやかであった. (3) 高濃度のイノシトールを含む培地では,イノシトールのない培地に比べて,酵母の生育は促進されたが.Micrococcus SP.の生育は遅延された. (4) 酵母とMicrococcus sp.の接種菌濃度をかえて混合培養した場合,酵母に対してMicrococcus sp.の接種菌量が多いと酵母の生育が抑えられ,逆に酵母の接種菌量を多くするとMicrococcus sp.の生育が抑制された. (5) 以上のことより,Micrococcus sp.と酵母の生育における拮抗作用の要因について論じた.

&lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt;と&lt;i&gt;Micrococcus&lt;/i&gt; sp. の混合培養に関する研究(第1報)

When Saccharomyces cerevisiae was cultured with shaking in a glucose-ammonium sulfate medium free from myoinositol, cells excreted a considerable amount of myoinositol to the medium at the late stationary phase of growth. Under optimal conditions, the yield of myo-inositol, that was excreted, exceeded 30μg/ml of the culture medium. When the cells were grown in the medium containing a small amount of added myoinositol (1μg/ml), growth was slightly stimulated but the excretion of myo-inositol decreased considerably as compared to the culture without myo-inositol. Time course study of myo-inositol formation by the yeast cells revealed that myo-inositol was synthesized concomitant with growth and existed predominantly as bound forms in the early stationary phase cells and that it was released thereafter from the cells as free and bound forms upon a further incubation, since no significant change in the amount of total myoinositol was detectable throughout this period. Present address: Laboratory of Food and Nutrition, Fukushima Women's Junior College, Fukushima

The phosphatase activity of Bacterium lactis aerogenes

In the early stages of a growth cycle of Bact. lactis aerogenes in a glucose-ammonium sulphate medium the phosphatase activity of the cells rises to high levels from which it later declines, as though by progressive dilution with less active material. The decline is not prevented by maintenance of a constant pH, nor by periodic dilution with fresh medium so as to keep the cell count in unit volume near a steady mean. The pattern of variation depends on the medium: for example, amino acid additions lower the maximum, replacement of glucose by acetate prevents the decline, absence of molecular oxygen leads to a low activity throughout.

The phosphatase activity ofBacterium lactis aerogenes

In the early stages of a grow cycle ofBact. lactis aerogenesin a glucose-ammonium sulphate medium the phosphatase activity of the cells rises to high levels from which it later declines, as though by progressive dilution with less active material. The decline is not prevented by maintenance of a constant pH, nor by periodic dilution with fresh medium so as to keep the cell count in unit volume near a steady mean. The pattern of variation depends on the medium : for example, amino acid additions lower the maximum, replacement of glucose by acetate prevents the decline, absence of molecular oxygen leads to a low activity throughout.

Variations in dehydrogenase and deaminase activity during bacterial growth.

Apart from a gradual decay in the stationary phase, the glucose and succinic dehydrogenase per unit mass are roughly constant (at high and low values, respectively) during the growth cycle of Bact. lactis aerogenes in a glucose-ammonium sulphate medium. Asparagine deaminase per unit mass rises steeply towards the end of the growth cycle as the medium grows acid, an increased amount tending to compensate the lower specific activity at the adverse pH. During the early stages of a growth cycle started with an inoculum of this high deaminase content the enzyme activity per unit mass rapidly falls by dilution with new material of lower content. During the long lag shown by the cells on an initial transfer to a medium containing succinate in place of glucose the asparagine deaminase content rises and, after growth begins, falls again. The glucose dehydrogenase remains unchanged, while the succinic dehydro­genase, once growth begins, rapidly rises to a value very much higher than the original—requiring, however, several subcultures in succinate to reach its optimum. Succinate-adapted cells do not again show the abnormal initial increase in deaminase. On retransfer to a glucose medium succinate-adapted cells rapidly lose their high succinic dehydrogenase activity, but only very slowly develop a lag in succinate. Adaptation to succinate appears to involve more than one enzyme system.

Variations in catalase activity during a bacterial growth cycle

The variation in the relative rate of catalase synthesis byBact. lactis aerogeneshas been studied by measuring the enzyme activity of samples of cells taken at intervals from growing cultures, under different conditions. In the normal growth cycle in a glucose-ammonium sulphate medium the rate of catalase synthesis is small during the early part of the logarithmic phase, but the content per cell increases rapidly as the pH of the medium falls. The increased activity is not a direct result of the acidity itself, but may be attributed to modification of the cell metabolism under acid conditions so that more catalase is required. Continued growth under anaerobic conditions tends to reduce catalase activity, although the characteristic variation of the relative rate of synthesis through the growth cycle is maintained. The variation of the rate of formation of the enzyme in cells growing in a succinate medium is similar to that in glucose, although synthesis is now appreciable even in the early stages of growth.