Effects of culture conditions on yield of Shiga-like toxin-IIv from Escherichia coli

The effects of selected culture conditions on production of Shiga-like toxin-II variant by an edema disease strain of Escherichia coli (412) and E. coli TB1 (pCG6) containing the cloned genes for Shiga-like toxin-II variant were examined. Incubation time, culture media, incubation temperature, starting pH of the culture medium, aeration, static culture, anaerobiosis, carbon sources, amino acids, antibiotics, and mitomycin C were investigated. The study showed that Shiga-like toxin-II variant was primarily cell associated and that strain TB1 (pCG6) produced as much as 100 times more toxin than did strain 412. Culture conditions that resulted in the greatest yield of Shiga-like toxin-II variant were incubation at 37 degrees C for 24 h with shaking in syncase broth initially adjusted to pH 8.5. Aerobic culture with shaking resulted in higher yields of Shiga-like toxin-II variant than did static aerobic or anaerobic culture. Addition of various carbon sources or amino acids, or tetracycline, lincomycin, or trimethoprim:sulfadoxine did not increase yields of toxin. The amount of Shiga-like toxin-II variant in supernatant preparations from strain TB1 (pCG6) was significantly increased by addition of mitomycin C to the culture medium.     

Production of Tumor-Inhibitory L-Asparaginase by Submerged Growth of Serratia marcescens

Production of a tumor-inhibitory asparaginase by submerged fermentation with Serratia marcescens ATCC 60 was studied to ascertain optimal nutritional conditions for large-scale production leading to enzyme purification studies. Five strains of S. marcescens were screened in shake-flask studies and were found to produce 0.8 to 3.7 IU/ml 48 hr after inoculation. The requirements for asparaginase production with S. marcescens ATCC 60, the high producing strain, included the following: 4% autolyzed yeast extract medium (initial pH 5.0), an incubation temperature of 26 C, and limited aeration for a zero level of dissolved oxygen during the fermentation. Addition of various carbohydrates to the fermentation medium did not enhance yields. The peak cell population in the fermentation medium and the maximal asparaginase yields occurred simultaneously. Highest enzyme yields were found when the pH of the fermentation cycle rose to approximately 8.5. Yields of 4 IU of asparaginase/ml of cell suspension have been obtained consistently in 40 to 42 hr from 10-liter volumes (500 ml/4-liter bottle) produced on a reciprocating shaker. Scale-up to a 60-liter fermentor yielded 3.1 IU/ml in 35 hr.     

The role of pH in the 'glucose effect' on prodigiosin production by non-proliferating cells of Serratia marcescens

The production of prodigiosin by non-proliferating cells of Serratia marcescens is inhibited by addition of glucose or different carbon sources to the induction medium. The induction in acidic external pH, mimicking the effects produced by the carbon sources, reduced prodigiosin synthesis, and the prodigiosin production seems to be related to the length of the low pH period. Buffering at pH 7·5 increased pigment production in media with repressing carbon sources. This study reveals that the inhibitory effect of carbon sources on prodigiosin production may be due to a lowering of the pH of the medium.     

The effect of pH on prodigiosin production by non-proliferating cells of Serratia marcescens

The synthesis of prodigiosin by non-proliferating cells of Serratia marcescens was examined at various pH values between 5.5 and 9.5. During incubation in unbuffered medium, pH changed and prodigiosin production was similar regardless of the initial pH. Variations in pigment production were noted when buffers were employed in cultures of non-proliferating cells. The optimum pH for prodigiosin production was 8.0–8.5. Proline oxidase was also measured. The results suggest that the effect of pH may be related to the amount of proline which can be incorporated into prodigiosin.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

Production of γ-linolenic acid by Mortierella isabellina grown on hexadecanol

AIMS: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol. METHODS AND RESULTS: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied. The production of gamma-linolenic acid reached 2.44 mg ml-1 (271 mg g-1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23 degrees C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5 degrees C. CONCLUSION: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.     

粘质沙雷氏菌发酵生产D-乳酸的研究

本文对粘质沙雷氏菌发酵生产D-乳酸进行了研究。以粘质沙雷氏菌G1(Serratia marcescens G1)为出发菌种,摇瓶试验确定了发酵培养方式:前12 h为菌体生长阶段,有氧培养,温度28℃,pH值7.0;后36 h为D-乳酸合成积累阶段,无氧培养,温度44℃,pH值6.0。且发现使用葡萄糖为碳源时更有利于D-乳酸的合成积累。采用缺失2,3-丁二醇合成能力的基因工程菌株R1为出发株,经筛选后得到耐受较高浓度乳酸盐的菌株R150,以R150为发酵菌种,在3.7 L发酵罐上采用两阶段发酵法,并通过增加起始菌体浓度的方法,发酵生成的D-乳酸浓度达到83.5 g/L,光学纯度达到98.9%。本研究成果为使用粘质沙雷氏菌发酵生产D-乳酸的深入研究打下了基础。     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

Factors influencing the resistance of biological monitors to ethylene oxide

The resistance of bacterial spore monitors is markedly influenced by the environmental conditions existing during development of the spores and, subsequently, in the preparation and evaluation of the monitor. Sporulation medium, suspending medium, pasteurization and storage conditions influence resistance of spores of Bacillus subtilis var. niger to ethylene oxide, but incubation temperature and age of sporulating culture appear to be unimportant. The conditions under which the spore suspension is dried on the supporting medium of the monitor exerts a major influence on resistance. Spores exposed to ethylene oxide are abnormally susceptible to damage by shaking with Ballotini, a method frequently used to recover spores from monitors. Nutritional conditions, pH and temperature of incubation influence the ability of survivors to form colonies on solidified media.     

Effect of culture conditions on tremorgen production by some Penicillium species.

Four strains each of seven tremorgenic Penicillium species were grown under various conditions and tested for tremorgen production by intraperitoneal injection of mice and by chemical analysis. Half of the strains had previously been found to be tremorgenic on bioassay after growth on Czapek Dox yeast extract broth or potato-milk-sucrose broth for 3 weeks at 26 degrees C. In the tests reported here nearly all previously nontremorgenic strains were either tremorgenic to mice or produced tremorgens detectable by chemical analysis but did so after longer incubation periods than used in the original screening. Bioassay was not suitable for the estimation of absolute levels but was preferable to chemical analysis when the identity of the tremorgens was not known. Species and strains within species gave different responses to changes in culture medium, incubation temperature, light irradiation, and shaking. Overall, tremorgen production was maximal at 20 or 26 degrees C, increased with time, and was reduced in shaken culture.     

Effect of culture conditions on tremorgen production by some Penicillium species

Four strains each of seven tremorgenic Penicillium species were grown under various conditions and tested for tremorgen production by intraperitoneal injection of mice and by chemical analysis. Half of the strains had previously been found to be tremorgenic on bioassay after growth on Czapek Dox yeast extract broth or potato-milk-sucrose broth for 3 weeks at 26 degrees C. In the tests reported here nearly all previously nontremorgenic strains were either tremorgenic to mice or produced tremorgens detectable by chemical analysis but did so after longer incubation periods than used in the original screening. Bioassay was not suitable for the estimation of absolute levels but was preferable to chemical analysis when the identity of the tremorgens was not known. Species and strains within species gave different responses to changes in culture medium, incubation temperature, light irradiation, and shaking. Overall, tremorgen production was maximal at 20 or 26 degrees C, increased with time, and was reduced in shaken culture.     

Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

Growth and extracellular proteinase production byEnterococcus faecalis subsp.liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37°C. Proteinase production was not affected by temperature in the range studied (7–45°C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

Effect of glucose concentration on the biosynthesis of prodigiosin by serratia marcescens (author's transl)]

Serratia marcescens is an enterobacteria which produces a characteristic red pigment denominated prodigiosin. To study the effect of glucose on the kinetics of this secondary metabolite, cultures of Serratia marcescens S10 were incubated at 30 degrees C in the mineral medium GL, with glucose (2 g/l) as the carbon source. Prodigiosin production in relation to glucose consumption is studied, and parallel-wise, the effect of various concentrations of glucose on prodigiosin production. The kinetics data show the close correlation between glucose consumption and the synthesis of prodigiosin. This substrate inhibits the synthesis of pigment in cultures grown on solid medium GL with concentrations of glucose up to 15 g/l.     

Optimization of culture conditions for production of antimalarial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162

AIMS: The aim of this work was to optimize the production of a novel antimaralial menisporopsin A by the seed fungus Menisporopsis theobromae BCC 4162. METHODS AND RESULTS: Fungal cultures were grown in shake flasks at 25 degrees C in the basal medium with varying carbon and nitrogen sources, aeration rates and initial pH levels. The optimal carbon and nitrogen sources that improved the production of menisporopsin A were 1% fructose and 2.5% meat extract respectively. The production was further enhanced when the culture incubated on a shaker at 200 rev min(-1) with an initial pH of 8. The yield of menisporopsin A cultured under the optimized conditions was increased from 348.30 (obtained from basal medium) to 889.02 mg l(-1), and the cultivation time was reduced from 28 to only 4 days. As a result, the productivity of menisporopsin A was greatly enhanced to 222.26 mg l(-1) day(-1) which is 18-fold higher than that of basal conditions. Larger scale production in a fermenter was also achieved, yielding menisporopsin A at a maximal level of 594.32 mg l(-1) in 4 days. CONCLUSIONS: The optimized culture conditions for menisporopsin A production by M. theobromae BCC 4162 was the cultivation under shaking or agitation at 25 degrees C in fructose-meat extract medium with an initial pH of 8. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of menisporopsin A in a fermenter with a relatively short incubation period could be valuable for further utilization for chemical structure modification and derivatization.     

Cation transport in Serratia marcescens and Serratia marinorubra

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba, were determined by atomic-absorption spectrometry. The intracellular K(+) and Mg(2+) contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg(2+) content of S. marinoruba was generally greater than that of S. marcescens. The Na(+) content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K(+) by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra. The slower rate of K(+) transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K(+) transport was found to have a pH optimum of 5.5 to 6.1 for S. marcescens, and the K(m) for K(+) was approximately 1.6 mm. Na(+) and Mg(2+) were not taken up by the cells, although the presence of Mg(2+) tended to decrease rates of K(+) uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at pH >7.     

Optimal conditions for chitinase production by<Emphasis Type="Italic">Serratia marcescens</Emphasis>

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.