Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.     

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B₁₂. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.Abbreviations 4ABS 4-aminobenzenesulfonate - CFU colony forming units - 4CS catechol-4-sulfonate - 4HB 4-hydroxybenzoate     

Production of raw cassava starch-digestive glucoamylase by a 2-deoxyglucose-resistant mutant of Rhizopus sp.

In order to improve the productivity of raw cassava starch-digestive glucoamylase of Rhizopus sp. MB46 in a liquid culture, a mutant strain, AF-1, which is resistant to 2-deoxyglucose, was derived. The mutant strain produced glucoamylase in the presence of 0.5% glucose though the parent strain did not. With a rice bran liquid medium the productivity was over 2-times that of the wild type strain. A rice bran liquid medium supplemented with β-cyclodextrin was also effective for glucoamylase production. Other maceration enzymes were also produced at a higher level with mutant strain AF-1 than with the wild type strain in a liquid culture as well as in a solid culture. The elution patterns of these enzymes on CM-cellulose column chromatography were principally the same with both strains except for glucoamylase. When 10% of raw cassava starch and cassava waste were digested with the culture filtrate of mutant strain AF-1, glucose was produced in 7% after 60-h incubation and 3.2% after 48-h incubation, respectively.     

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed. (c) 1993 John Wiley & Sons, Inc.     

Conditioned medium promotes the attachment ofAgrobacterium tumefaciens strain NT 1 to carrot cells

Summary Wild-typeAgrobacterium tumefaciens bind to carrot suspension culture cells. Avirulent strain NT 1 did not bind to carrot cells when they were incubated together in Murashige and Skoog medium. Conditioned medium was prepared by incubatingA. tumefaciens virulent strain C 58 with carrot cells and removing the bacteria and carrot cells using filter sterilization. This conditioned medium promoted the binding of NT 1 to carrot cells. Conditioned medium did not promote the nonspecific attachment ofEscherichia coli to carrot cells. These results suggest that when wild-typeA. tumefaciens are incubated with plant host cells, some substance(s) involved in bacterial attachment are released into the medium. Filter-sterilized medium from the incubation of the nonattachingchvB mutant A 1045 with carrot cells promoted the attachment of strain NT 1 even though A 1045 bacteria did not bind to the carrot cells. However, filter-sterilized medium from the incubation of the non-attachingatt mutant Att-B 123 with carrot cells was unable to promote the binding of strain NT 1. This suggests that nonattaching mutants ofA. tumefaciens can be divided into two groups on the basis of the properties of the substances released into the medium when the bacteria are incubated with carrot cells.Abbreviations MS Murashige and Skoog tissue culture medium Dedicated to the memory of Professor John G. Torrey     

产银杏内酯内生真菌的筛选及培养条件研究

通过对内生真菌的发酵提取物进行TLC和HPLC-UV分析,进行菌株筛选;对该菌株在不同培养基上的生长情况、产孢量、银杏内酯类物质产量的测定,确定最佳培养基;并用HPLC-ELSD测定了不同时间段的发酵液中银杏内酯类物质含量。结果,筛选出一株产量较高的烟曲霉原变种(Aspergillus fumigatusvar.fumigatus)FG052;对其培养条件的研究表明,PDA培养基、查氏培养基分别为其最佳传代和发酵培养基,菌丝最大生物量在发酵168 h,产银杏内酯类物质高峰在发酵144 h,此时总内酯产量可达0.13 mg/mL,pH值为4.86。本实验筛选的菌株稳定性较好,筛选的培养基价格低廉,碳氮比明确,且总内酯的产量高,可作为规模生产银杏内酯类物质的培养基。     

小球藻两个品系在自养与异养条件下的生长、能荷与色素差异

Chlorella protothecoides中国品系(C品系)与美国品系(A品系)在自养培养基中生长正常。在高浓度葡萄糖、低浓度有机氮的异养生长条件下,C品系仍为绿色,细胞生长与繁殖缓慢,指数期生长速率降低,细胞内腺苷酸能荷值(AEC)下降,但细胞仍保留叶绿素;A品系细胞转变成乳黄色,细胞生长与繁殖加快,指数期生长速率是自养细胞的2倍,细胞内腺苷酸能荷值仍保持高水平,向异养条件转换后约24h,细胞内叶绿素基本消失,重新向自养条件转换后细胞内叶绿素又逐渐恢复。AEC值是判别微藻在异养条件下能量代谢与生理状态的良好指标。同一种小球藻不同品系间的上述差异可能与细胞内基因调控的差异相关联。     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

一株产D-海因酶菌株的鉴定及离子束诱变选育

首次报道了产D-海因酶的巨大芽孢杆菌,通过对该菌进行离子束诱变,获得酶活最高增加3倍的突变株M5,对M5的产酶条件进行优化,得到最佳的发酵条件为玉米浆1.5%,葡萄糖1%,油酸1.5%,氯化钠0.5%,并添加50 mg.L-1的Mn2+、Zn2+及500 mg.L-1的Mg2+,pH8.0,30℃发酵24 h,酶活力可达到每毫升2.119 U,比优化前突变株提高了300%,比出发菌株提高了850%。     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

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 novel oligosaccharide oxidase by wheat bran solid-state fermentation

A search for oxidases that catalyze the oxidation of oligosaccharides has resulted in the isolation of several soil-derived fungus strains which produced novel oligosaccharide oxidases with different substrate specificity on wheat bran solid culture. One of these oxidases produced by Acremonium strictum T1 strain has been characterized. This enzyme showed high reactivity toward maltose, lactose, cellobiose and maltooligosaccharides composed of up to seven glucose units, and was named as glucooligosaccharide oxidase based on its substrate specificity. Strain T1 was subjected to a strain improvement program, and an enzyme hyper-producing mutant strain T1-38 was selected. This mutant strain produced glucooligosaccharide oxidase 75 times higher than the wild type strain T1. When cultivated in a solid medium comprised of 1 part of wheat bran and 1 part of water (w/w), enzyme activity reached a maximum level of 6 units per g of culture medium after 4 days cultivation. Characteristics of the enzyme including the substrate specificity were compared with two other novel oligosaccharide oxidases isolated in this laboratory. Batch type conversion of lactose to lactobionic acid using crude enzyme was also discussed.     

High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis

Summary A flocculent strain ofZymomonas mobilis (ZM4F JM1) was isolated in continuous culture. The parent strain, ZM4F, had lost its flocculating properties. The isolation was done in a conical fermentor at high dilution rate. Ethanol production by the new strain was then compared on a rich and mineral medium. The mineral medium showed high performance and could be used for industrial production of ethanol since it reduced one hundred fold the vitamin cost of the fermentation.     

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.     

A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain

Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1. Received: November 18, 1999 / Accepted: December 2, 1999     

苏云金杆菌在味精废水中的培养与发酵特性

该文讨论了苏云金杆菌HM－1菌株在以味精废水为原料的培养基上的生长,经多次摇瓶培养,确定了最适培养基成份,在此基础上,进行了30L通风搅拌罐的深层培养,研究了苏云金杆菌HM－1菌株在30升通风发酵罐深层培养的发酵特性。     

Isolation and characterization of a lipolytic bacterium capable of growing in a low-water-content oil-water emulsion

A unique lipolytic bacterium was isolated in a selective growth system consisting of 99% triglycerides and a 1% water phase. The bacterium, termed Pseudomonas aeruginosa YS-7, was able to grow in an environment of low water content and could also survive amphipathic, osmotic, and matrical water stress in a triglyceride-rich culture. The isolated strain was identified as P. aeruginosa on the basis of standard physiological, biochemical, and serological assays. The strain is a gram-negative motile rod, aerobic, pigment forming, and capable of growing at 42 degrees C. It is highly tolerant of high concentrations of the cationic detergent cetyltrimethylammonium bromide and of the fatty acid salts derived from bacterial hydrolysis of the oil. Growth of the bacterium in a pure culture in a 99% triglyceride medium lasted until most of the water was evaporated or consumed. Growth was accompanied by triglyceride hydrolysis, which continued to occur even after growth saturation until the water was totally depleted. No loss of viability was observed when the culture was maintained under water-depleted conditions for an additional 40 h. A second cycle of bacterial growth and triglyceride hydrolysis was immediately initiated upon the addition of 1% (vol/vol) water to the culture. Lipase activity was stable regardless of changes in culture conditions. The isolated strain is uniquely resistant to severe water stress in a triglyceride-rich medium or under cold acetone precipitation compared with 12 other microbial strains, including bacteria and yeasts. Among these 12, only the lipolytic strains grew in the 99% triglyceride medium, but they reached a cell mass fourfold smaller than that of P. aeruginosa YS-7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a lipolytic bacterium capable of growing in a low-water-content oil-water emulsion.

A unique lipolytic bacterium was isolated in a selective growth system consisting of 99% triglycerides and a 1% water phase. The bacterium, termed Pseudomonas aeruginosa YS-7, was able to grow in an environment of low water content and could also survive amphipathic, osmotic, and matrical water stress in a triglyceride-rich culture. The isolated strain was identified as P. aeruginosa on the basis of standard physiological, biochemical, and serological assays. The strain is a gram-negative motile rod, aerobic, pigment forming, and capable of growing at 42 degrees C. It is highly tolerant of high concentrations of the cationic detergent cetyltrimethylammonium bromide and of the fatty acid salts derived from bacterial hydrolysis of the oil. Growth of the bacterium in a pure culture in a 99% triglyceride medium lasted until most of the water was evaporated or consumed. Growth was accompanied by triglyceride hydrolysis, which continued to occur even after growth saturation until the water was totally depleted. No loss of viability was observed when the culture was maintained under water-depleted conditions for an additional 40 h. A second cycle of bacterial growth and triglyceride hydrolysis was immediately initiated upon the addition of 1% (vol/vol) water to the culture. Lipase activity was stable regardless of changes in culture conditions. The isolated strain is uniquely resistant to severe water stress in a triglyceride-rich medium or under cold acetone precipitation compared with 12 other microbial strains, including bacteria and yeasts. Among these 12, only the lipolytic strains grew in the 99% triglyceride medium, but they reached a cell mass fourfold smaller than that of P. aeruginosa YS-7.(ABSTRACT TRUNCATED AT 250 WORDS)     

氧化亚铁硫杆菌的分离鉴定及培养条件优化

[目的]获得可用于浸矿的菌株,对其培养条件进行优化.[方法]从成都热电厂采集土样中分离得到一株菌株,分析该菌株的形态学特征、培养特征及16S rDNA序列,确定菌株的分类地位.利用Design-Expert软件中的Box-Behnken法设计实验,通过响应面分析对初始pH值、温度、接种量和装液量4个因素进行优化,确定其最适培养条件.[结果]获得菌株Z1,该菌为革兰氏阴性菌,短杆状,经16S rDNA鉴定为嗜酸性氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans,简称At.f).确定菌株最适培养条件为:pH 1.8、温度30℃、接种量14％、装液量250 mL摇瓶装60 mL培养液.在此条件下,Z1的亚铁氧化率可达99.7％.[结论]Z1菌株适合于生物浸矿的应用.