不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

Photoheterotrophic utilization of acetate by the wild type and an acetate-adapted mutant of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata strain St. Louis can grow anaerobically in the light-with acetate as the carbon source. The organism is sensitive to acetate, however, initial concentrations exceeding 25 mM resulting in an extensive growth lag. Bicarbonate is not required for growth of this strain on acetate, but addition of bicarbonate shortens the lag phase in media with high initial acetate concentration. A spontaneous mutant which exhibited a minimal lag phase and rapid growth rates on acetate media was derived from strain St. Louis. This mutant possessed elevated levels of the glyoxylate cycle enzyme, isocitrate lyase.     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

Characteristics of Egyptian boza and a fermentable yeast strain isolated from the wheat bread

A sample of indigenous beer, boza was collected at Cairo, Egypt and analysed. Boza was an off-white porridge-like slurry containing 3.8% (v/v) ethanol. Volatile esters and higher alcohols such as ethyl acetate and isoamyl alcohol were detected in the boza by gas chromatography. The pH of the boza was 3.7. Organoleptically, this alcoholic beverage had an estery flavour and a sour taste. A fermentable yeast strain EG1 was isolated from the material wheat bread and identified, and was considered to resemble Candida krusei. The rice sake made with the yeast strain C. krusei EG1 at 30 °C contained 11.7% ethanol, 74.1 mg/l ethyl acetate and its pH value was 4.2.     

Fermentation of cinnamate by a mesophilic strict anaerobe,Acetivibrio multivorans sp. nov.

A cinnamate-fermenting bacterium (strain PeC1) was isolated in pure culture from anoxic sludge of an oil refinery wastewater treatment facility. It was a mesophilic gram-negative non-sporing actively motile rod. It did not reduce nitrate, sulfte, or other sulfur compounds as electron acceptors. It fermented cinnamate to 3-phenylpropionate, benzoate, and acetate; crotonate to butyrate and acetate; pyruvate to lactate and acetate; acetoin to ethanol and acetate; and carbohydrates to ethanol, formate, and acetate. The DNA base ratio of the strain was 44 mol% guanine plus cytosine. It is described as a new species of the genus Acetivibrio, A. multivorans sp. nov.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

A low-sodium-salt formulation for the fermentation of salinosporamides by <Emphasis Type="Italic">Salinispora tropica</Emphasis> strain NPS21184

In this paper, we described the development of a potassium-chloride-based-salt formulation containing low sodium concentrations (5.0 to 11 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). The sodium present in the media was essentially derived from the complex nitrogen sources Hy Soy, yeast extract, and peptone used in the media. We demonstrated that good growth rate and yield of S. tropica strain NPS21184 were detected in both agar and liquid media containing the potassium-chloride-based-salt formulation with sodium concentration as low as 5.0 mM, significantly less than the critical seawater-growth requirement concentration of 50 mM sodium for a marine microorganism. We also observed good production of NPI-0052 (176 to 243 mg/l) by S. tropica strain NPS21184 grown in production media containing the potassium chloride-based-salt formulation. The production of deschloro analog, salinosporamide B (NPI-0047), was significantly lower in the low-sodium-salt-formulation medium than in the high-sodium-salt-formulation media. We demonstrated that while S. tropica strain NPS21184 is a novel marine actinomycete that requires high salt content for growth, it does not require sodium-chloride-based seawater-type media for growth and production of NPI-0052.     

Analyses of the acetate-producing pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen-deprived conditions

Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions. An erratum to this article can be found at     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais     

Sulfobacillus sibiricus sp. nov., a New Moderately Thermophilic Bacterium

In the course of pilot industrial testing of a biohydrometallurgical technology for processing gold-arsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha (Yakutiya)), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe²⁺, S⁰, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55°C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on a medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow in the absence of an inorganic energy source on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G+C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA–DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis), which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9–98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.     

The effect of calcium ion on the biodegradation of octylphenol polyethoxylates,and the antiandrogenic activity of their biodegradates

Because limes have been used as important fertilizers to neutralize acidified farmland in Japan, our interest in this study was focused on the effect of calcium ion on the biodegradation of octylphenol polyethoxylates (OPEOn) by a pure culture of Pseudomonas putida S5 isolated from a rice paddy field in Japan. In the presence of calcium ion, P. putida S5 accelerated the formation of octylphenol oligoethoxy carboxylates (OPECn) rather than that of octylphenol oligoethoxylates under an aerobic condition, indicating that more soluble biodegradates with terminal carboxyl group may liquate out easily to surface and ground water rather than more hydrophobic biodegradates with shorter ethylene oxide residues. Therefore, the androgen receptor (AR) activity of their degradation products was characterized using an in vitro reporter gene assay. As ethylene oxide chain length decreased, the biodegradates, OPEOn (n < 3), increased their AR antagonist activity. However, OPECn (n < 3) were unable to determine their AR activity because of their cytotoxicity in our reporter gene assay system.     

Isolation and characterization of a carbon monoxide utilizing strain of the acetogen Peptostreptococcus productus

From sludge obtained from the sewage digester plant in Marburg-Cappel a strictly anaerobic bacterium was enriched and isolated with carbon monoxide as the sole energy source. Based on morphological and physiological characteristics the isolate was identified as a strain of Peptostreptococcus productus, which was called strain Marburg. The organism was able to grow on CO (50% at 200 kPa) as the sole energy source at a doubling time of 3 h and converted this substrate to acetate and CO₂. The type strain of P. productus was not able to grow at the expense of CO. Electron microscopic investigations of strain Marburg cells revealed a cell wall which was different from that of other Gram-positive prokaryotes. DNA:DNA hybridization studies of the DNA isolated from strain Marburg and the type strain as well as some morphological and physiological properties of both strains confirmed the low degree or relatedness between the two strains.     

Identification of 4-O-methyl-D-xylose as a constituent of the cell wall of Chlorella vulgaris

From the cell wall of a strain of Chlorella vulgaris a sugar was isolated after acid hydrolysis and was identified as 4-O-methyl-D-xylose by the following criteria: (i) mass spectroscopy of its alditol acetate revealed characteristic primary fragments with m/e 117 and m/e 261, and, when one deuterium atom was substituted at C-1, with m/e 262 instead of m/e 261; (ii) after demethylation with BCl₃, xylose was identified as its parent sugar by chromatographic methods; (iii) L-iditol: NAD 5-oxidoreductase (sorbitol dehydrogenase) catalyzed the oxidation of its alditol, but not of 4-O-methyl-L-xylitol. 4-O-Methyl-D-xylose amounted to approx. 10% of the cell walls' dry weight or 1.6% of the cells' dry weight.     

Glucose and acetate influences on the behavior of the recombinant strainEscherichia coli HB 101 (GAPDH)

Summary This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) byE. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L^–1 of biomass and 6 g L^–1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L^–1, and in fed-batch experiments for glucose availability of 10 g h^–1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.     

Citric acid wastewater as electron donor for biological sulfate reduction

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range.     

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain ofEscherichia coli

Summary The influence of complex compounds on the growth of a recombinant strain ofEscherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.     

Hydrogen oxidation by membranes from autotrophically grown Alcaligenes eutrophus H16: role of the cyanide-resistant pathway in energy transduction

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, aK _m for acetate of 160 mM, and ak _cat of 16,800 min^-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.Abbreviations Acetyl-P Acetyl-phosphate - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide     

Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka

A facultative aerobic, moderately thermophilic, spore forming bacterium, strain JW/VK-KG4 was isolated from an enrichment culture obtained from the Geyser valley, a geothermally heated environment located in the Kamchatka peninsula (Far East region of Russia). The cells were rod shaped, motile, peritrichous flagellated stained Gram positive and had a Gram positive type cell wall. Aerobically, the strain utilized a range of carbohydrates including glucose, fructose, trehalose, proteinuous substrates, and pectin as well. Anaerobically, only carbohydrates are utilized. When growing on carbohydrates, the strain required yeast extract and vitamin B₁₂. Anaerobically, glucose was fermented to lactate as main product and acetate, formate, ethanol as minor products. Aerobically, even in well-aerated cultures (agitated at 500 rpm), glucose oxidation was incomplete and lactate and acetate were found in culture supernatants as by-products. Optimal growth of the isolate was observed at pH^{25 C} 6.8–8.5 and 60°C. The doubling times on glucose at optimal growth conditions were 34 min (aerobically) and 40 min (anaerobically). The G+C content was 42.3 mol% as determined by T_m assay. Sequence analysis of the 16S rRNA gene indicated an affiliation of strain JW/VK-KG4 with Anoxybacillus species. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA homology with validly published species of Anoxybacillus, it is proposed that strain JW/VK-KG4 represents a new species in the genus Anoxybacillus as A. kamchatkensis sp. nov. The type strain for the novel species is JW/VK-KG4^T (=DSM 14988, =ATCC BAA-549). The GenBank accession number for the 16S rDNA sequence is AF510985.     

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.