Studies on the central metabolism of Thermus aquaticus,an extreme thermophilic bacterium: Anaplerotic reactions and their regulation

The physiology of Thermus aquaticus strain Z05 was investigated. Substantial evidence for gene and enzyme regulation in the central metabolism of this extreme thermophile was found.Two anaplerotic pathways were detected: (1) phosphoenolpyruvate carboxylase; (2) a glyoxylate shunt which proved to be essential for growth on pyruvate as well as acetate. The synthesis of isocitrate lyase and malate synthase were found to depend on a common control mechanism. Pronounced regulatory effects were observed on the activity of malic enzyme, pyruvate kinase and phosphoenolpyruvate carboxylase. The data could be fitted together into a picture of the metabolism during glycolysis and gluconeogenesis which shows how variations of enzyme levels and activities correlate with the apparent needs of the cell.Our results call attention to a peculiar metabolic analogy between T. aquaticus and Acinetobacter Abbreviations ace acetate nonutilizing - Acetyl-CoA acetyl-coenzyme A - I.U. international unit - PEP phosphoenolpyruvate - T Thermus     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162).All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin.The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates.The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.Abbreviations GC guanosine cytosine - ATCC American Type Culture Collection - DSM Deutsche Sammlung von Mikroorganismen     

Thermovorax subterraneus, gen. nov., sp. nov., a thermophilic hydrogen-producing bacterium isolated from geothermally active underground mine

A thermophilic, rod-shaped, motile, Gram-positive, spore-forming bacterium strain 70B^T was isolated from a geothermally active underground mine in Japan. The temperature and pH range for growth was 50–81°C (optimum 71°C) and 6.2–9.8 (optimum pH 7–7.5), respectively. Growth occurred in the presence 0–2% NaCl (optimum 1% NaCl). Strain 70B^T could utilize glucose, fructose, mannose, mannitol, pyruvate, cellobiose and tryptone as substrates. Thiosulfate was used as electron acceptor. Major whole-cell fatty acids were iso-C_15:0, C_16:0 DMA (dimethyl acetal), C_16:0 and anteiso-C_15:0. The G+C mol% of the DNA was 44.2%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the closest relatives of strain 70B^T were Thermosediminibacter oceani DSM 16646^T (94% similarity) and Thermosediminibacter litoriperuensis DSM 16647 (93% similarity). The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain 70B^T represents a novel species in a new genus, for which the name Thermovorax subterraneus gen. nov., sp. nov. is proposed. The type strain of Thermovorax subterraneus is 70B^T (=DSM 21563 = JCM 15541).     

Thermotoga neapolitana sp. nov. of the extremely thermophilic,eubacterial genus Thermotoga

A second species of the extremely thermophilic, eubacterial genus Thermotoga is described as clearly distinguished from the type species Thermotoga maritima by physiological and phylogenetic criteria. It is named Thermotoga neapolitana.     

Thermococcus litoralis sp. nov.: A new species of extremely thermophilic marine archaebacteria

We describe a new species, Thermococcus litoralis, which is different from the type species Thermococcus celer in molecular, morphological and physiological characteristics.Abbreviations 3 x SSC (standard saline citrate) - 0.45 M NaCl 0.045 M Na₃-citrate     

Effect of fermentation temperature on hydrogen production from cow waste slurry by using anaerobic microflora within the slurry

We examined hydrogen production from a dairy cow waste slurry (13.4 g of volatile solids per liter) by batch cultures in a temperature range from 37 to 85°C, using microflora naturally present within the slurry. Without the addition of seed bacteria, hydrogen was produced by simply incubating the slurry, using the microflora within the slurry. Interestingly, two peaks of fermentation temperatures for hydrogen production from the slurry were observed at 60 and 75°C (392 and 248 ml H₂ per liter of slurry, respectively). After the termination of the hydrogen evolution, the microflora cultured at 60°C displayed hydrogen-consuming activity, but hydrogen-consuming activity of the microflora cultured at 75°C was not detected, at least for 24 days. At both 60 and 75°C, the main by-product was acetate, and the optimum pH of the slurry for hydrogen production was around neutral. Bacteria related to hydrogen-producing moderate and extreme thermophiles, Clostridium thermocellum and Caldanaerobacter subterraneus, were detected in the slurries cultured at 60 and 75°C, respectively, by denaturing gradient gel electrophoresis analyses, using the V3 region of 16S rDNA.     

Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new,extreme thermophilic,anaerobic bacterium

Two strains, JW 200 and JW 201, of an extreme thermophilic, non-spore-forming anaerobic bacterium were isolated from alkaline and slightly acidic hot springs located in Yellowstone National Park. Both strains were peritrichously flagellated rods. Cell size varied from 0.5–0.8 by 4–100 m; coccoid-shaped cells of about 1 m in diameter frequently occurred. Division was often unequal. Spheroplast-like forms were visible at the late logarithmic growth phase. The Gram reaction was variable. The DNA base composition of the two strains was between 37 and 39 mol% guanine plus cytosine as determined by buoyant density measurements and approximately 32% by the thermal denaturation method. The main fermentation products from hexoses were ethanol and CO₂. Growth occurred between 37 and 78°C and from pH 4.4 to 9.8. The name Thermoanaerobacter ethanolicus gen. nov., spec. nov. was proposed for the two, new isolates. Strain JW 200 was designated as the type strain.A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Los Angeles, CA, 1979 (J. Wiegel and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol., 1979, 163, p. 105) and at the 27th IUPAC Congress Helsinki, 1979 (L. G. Ljungdahl and J. Wiegel, Abstracts p. 546)     

Production of hydrogen from marine macro-algae biomass using anaerobic sewage sludge microflora

Hydrogen was produced from various marine macro-algae (seaweeds) through anaerobic fermentation using an undefined bacterial consortium. In this study, anaerobic fermentation from various marine macro-algae for Ulva lactuca, Porphyra tenera, Undaria pinnatifida, and Laminaria japonica was studied. From this analysis Laminaria japorica was determined to be the optimum substrate for hydrogen production. When L. japornica was used as the carbon source for enhanced hydrogen production, the optimum fermentation temperature, substrate concentration, initial pH, and pretreatment condition were determined to be 35°C, 5%, 7.5, and BT120 (Ball mill and thermal treatments at 120°C for 30 min), respectively. In addition, hydrogen production was improved when the sludge was heat-treated at 65°C for 20 min. Under these conditions, about 4,164 mL of hydrogen was produced from 50 g/L of dry algae (L. japonica) for 50 h, with a hydrogen concentration around 34.4%. And the maximum hydrogen production rate and yield were found to be 70 mL/L·h and 28 mL/g dry algae, respectively.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines and Thailand

Most community molecular studies of thermophilic cyanobacterial mats to date have focused on Synechococcus occurring at temperatures of ~50–65°C. These reveal that molecular diversity exceeds that indicated by morphology, and that phylogeographic lineages exist. The moderately thermophilic and generally filamentous cyanobacterial mat communities occurring at lower temperatures have not previously been investigated at the community molecular level. Here we report community diversity in mats of 42–53°C recovered from previously unstudied geothermal locations. Separation of 16S rRNA gene-defined genotypes from community DNA was achieved by DGGE. Genotypic diversity was greater than morphotype diversity in all mats sampled, although genotypes generally corresponded to observed morphotypes. Thirty-six sequences were recovered from DGGE bands. Phylogenetic analyses revealed these to form novel thermophilic lineages distinct from their mesophilic counterparts, within Calothrix, Cyanothece, Fischerella, Phormidium, Pleurocapsa, Oscillatoria and Synechococcus. Where filamentous cyanobacterial sequences belonging to the same genus were recovered from the same site, these were generally closely affiliated. Location-specific sequences were observed for some genotypes recovered from geochemically similar yet spatially separated sites, thus providing evidence for phylogeographic lineages that evolve in isolation. Other genotypes were more closely affiliated to geographically remote counterparts from similar habitats suggesting that adaptation to certain niches is also important.     

牛粪堆肥中纤维素高效降解菌的筛选与产酶条件优化

【背景】纤维素是一种有待开发利用的生物质资源,对于能源危机、环境污染问题的解决具有重要作用。【目的】从牛粪堆肥中分离出产纤维素酶的细菌,研究该菌株的纤维素降解能力。【方法】采用纤维素固体平板刚果红染色法进行初筛、液体发酵纤维素酶活测定法进行复筛。【结果】筛选获得一株具有高产纤维素酶活性的解淀粉芽孢杆菌(Bacillusamyloliquefaciens),命名为N5。单因素分析试验结果显示,菌株N5具有较好的pH、温度和盐度耐受性,正交优化试验结果表明,菌株N5产纤维素酶的最佳条件为：发酵初始pH 5.0,发酵时间96 h,发酵温度40℃。在此条件下,羧甲基纤维素(carboxymethyl cellulose, CMC)酶活为189.27 U/mL。此外,菌株N5能够在7 d内使水稻秸秆减重率达到19.35%。扫描电镜结果表明菌株N5能够有效促进水稻秸秆降解。【结论】菌株N5具有较高的纤维素酶活力,具有开发成高效好氧堆肥菌剂的潜质,这为固体废弃物中纤维素的生物转化提供了优质菌种资源。     

Isolation and characterization of a strictly xylan-degrading Dictyoglomus from a man-made,thermophilic anaerobic environment

A thermophilic, strictly anaerobic eubacterium which utilized an unusually limited range of substrates was isolated from a sludge and pulp sample from a paperpulp cooling tank at a paper-board factory in Finland. The organism grew only with beech wood or oat spelt xylan; no growth occurred with soluble sugars, other polysaccharides, peptone, or yeast extract. The organism was rod-shaped, long (up to 20 m), thin (0.3 m), gramnegative, and in late-exponential and stationary phase cultures formed ball of yarn like structures; endospores were not observed and the organism was not motile. The organism grew fastest (=0.08 – 0.09 h^-1) at 65 to 75°C and pH 6.5 to 8.4, with a maximum growth temperature between 75 and 80°C and an upper pH limit near 9. During growth on beech xylan the isolate produced only acetate, H₂, and CO₂ as fermentation products. The guanine + cytosine (G+C) content of the isolates cellular DNA was 34%. The unusual morphology of the isolate is characteristic of the genus Dictyoglomus, and the limited substrate range, higher G+C ratio, and different fermentation products indicated that the isolate was a new species in that genus.     

Ultrastructure of an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum

Calderobacterium hydrogenophilum is an extreme thermophilic, obligately chemoautotrophic, hydrogen-oxidizing bacterium. The cells were shown to be nonmotile straight rods of average size 0.4x2.5 m. After negative-staining of the whole cells, no flagella were observed. The multilayered cell wall was of type 1 and possessed a crystalline proteinaceous surface layer exhibiting p4 symmetry. The square unit cells had a lattice constant of approximately 11 nm. Cell division occurred by a constriction mechanism. C. hydrogenophilum differred from a similar hydrogen-oxidizing eubacterium, Hydrogenobacter thermophilus, by the absence of intracytoplasmic membrane structures in chemically fixed cells. However, an electron-dense intracytoplasmic hemispherical structure adhering to the inner membrane was frequently observed.     

Performance and microbial community analysis of two-stage process with extreme thermophilic hydrogen and thermophilic methane production from hydrolysate in UASB reactors

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H₂/g-VS (190 ml-H₂/g-sugars) and 307 ml-CH₄/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H₂-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH₄-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.     

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg²⁺ enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co²⁺.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, IndiaNCL Communication No. 5813     

Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet

Despite detailed study of selected thermophilic taxa, overall community diversity of bacteria in thermophilic mats remains relatively poorly understood. A sequence-based survey of bacterial communities from several hot spring locations in central Tibet was undertaken. Diversity and frequency of occurrence for 140 unique 16S rRNA gene phylotypes were identified in clone libraries constructed from environmental samples. A lineage-per-time plot revealed that individual locations have evolved to support relatively large numbers of phylogenetically closely related phylotypes. Application of the F _ST statistic and P test to community data was used to demonstrate that phylogenetic divergence between locations was significant, thus emphasizing the status of hot springs as isolated habitats. Among phylotypes, only the Chlorobi were ubiquitous to all mats, other phototrophs (Cyanobacteria and Chloroflexi) occurred in most but not all samples and generally accounted for a large number of recovered phylotypes. Phylogenetic analyses of phototrophic phylotypes revealed support for location-specific lineages. The alpha, beta and gamma proteobacteria were also frequently recovered phyla, suggesting they may be abundant phylotypes in mats, a hitherto unappreciated aspect of thermophilic mat biodiversity. Samples from one location indicated that where phototrophic bacteria were rare or absent due to niche disturbance, the relative frequency of proteobacterial phylotypes increased. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution of clinically important thermophilic actinomycetes in vegetable substrates and soil in north-western India

Medically important thermophilic actinomycetes were isolated from 218 (64%) of 341 samples of vegetable substrates and soil examined from sites in north-western India. Thermoactinomyces vulgaris (T. candidus) was the commonest species, occurring in 56% of samples, followed by Saccharomonospora viridis in 29%, Thermoactinomyces thalpophilus in 27%, Faenia rectivirgula (Micropolyspora faeni) in 21% and Thermoactinomyces sacchari in 14%. T. vulgaris and T. thalpophilus were isolated from all types of substrate examined, with T. vulgaris always more common than T. thalpophilus. Of the other thermophilic actinomycetes, F. rectivirgula was isolated predominantly from hay (44%) and S. viridis (56%) and T. sacchari (44%) from sugarcane bagasse. The largest populations of T. vulgaris and T. thalpophilus were found in paddy straw, followed by T. sacchari, S. viridis and F. rectivirgula in sugar-cane bagasse.The widespread occurrence of these clinically important thermophilic actinomycetes suggests that exposure of humans and animals to them may be frequent in north-western India. Studies are required to determine the prevalence of extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by thermophilic actinomycetes in the local population.     

木质纤维素燃料乙醇蒸馏废水的高温厌氧处理及菌群结构分析

【目的】为开发高效的高浓度木质纤维素燃料乙醇蒸馏废水厌氧处理及资源化利用工艺,以活性炭为载体,在实验室规模上对高温厌氧流化床反应器处理木质纤维素燃料乙醇蒸馏废水进行研究。【方法】反应器经65 d梯度驯化后启动,对工艺参数进行一系列优化,并通过基于16S rRNA基因的分子生态学技术分析厌氧污泥中的优势菌群。【结果】实验获得了最优的反应条件和处理效果:厌氧流化床反应器(Anaerobic fluidized bed reactor,AFBR)在温度55±1°C、有机负荷率(OLR)13.8 g COD/(L·d)及水力停留时间(HRT)48 h操作时,COD去除率达到90%以上,同时甲烷产率达到290 mL/g COD;菌群鉴定分析结果显示高温厌氧活性污泥中Clostridia所占比例最大,产甲烷菌属以Methanoculleus和Methanosarcina为主,其它功能菌群主要为Alphaproteobacteria等。【结论】AFBR反应器可高效降解木质纤维素燃料乙醇蒸馏废水并产生生物能源甲烷,其反应体系内微生物种类丰富。