Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.     

Bacillus tusciae,a new species of thermoacidophilic,facultatively chemolithoautotrophic hydrogen oxidizing sporeformer from a geothermal area

A thermophilic, chemolithoautotrophic, hydrogen-oxidizing sporeformer has been isolated from ponds in a solfatara in the geothermal area of Tuscany (Italy). Some physicochemical parameters of the habitat were determined. The habitat was characterized by the presence of molecular hydrogen in the escaping gases, a very low content of phosphate and organic matter. Temperature and water level in the ponds varied widely. The organism formed oval, subterminal spores, which swelled distinctly the sporangium. Optimal growth occured between pH 4.2 and 4.8 at 55°C. It grew best under autotrophic conditions, but organic substrates including short chain fatty acids, amino acids and alcohols could also support heterotrophic growth. Sugars were not metabolized. The hydrogenase was soluble but did not reduce pyridine nucleotides.Based on its morphological and biochemical features, the organism belongs to the genus Bacillus, but differs from all the previously described species. It is therefore proposed as constituting a new species, Bacillus tusciae.Abbreviations SCC standard saline citrate buffer - 0.15 M NaCl 0.015 M trisodium citrate, pH 7.0 Dedicated to Prof. H.-G. Schlegel on the occasion of his 60th birthday     

Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas

Microbial fermentations are potential producers of sustainable energy carriers. In this study, ethanol and hydrogen production was studied by two thermophilic bacteria (strain AK15 and AK17) isolated from geothermal springs in Iceland. Strain AK15 was affiliated with Clostridium uzonii (98.8%), while AK17 was affiliated with Thermoanaerobacterium aciditolerans (99.2%) based on the 16S rRNA gene sequence analysis. Both strains fermented a wide variety of sugar residues typically found in lignocellulosic materials, and some polysaccharides. In the batch cultivations, strain AK17 produced ethanol from glucose and xylose fermentations of up to 1.6 mol-EtOH/mol-glucose (80% of the theoretical maximum) and 1.1 mol-EtOH/mol-xylose (66%), respectively. The hydrogen yields by AK17 were up to 1.2 mol-H2/ mol-glucose (30% of the theoretical maximum) and 1.0 mol-H2/mol-xylose (30%). The strain AK15 produced hydrogen as the main fermentation product from glucose (up to 1.9 mol-H2/mol-glucose [48%]) and xylose (1.1 mol-H2/mol-xylose [33%]). The strain AK17 tolerated exogenously added ethanol up to 4% (v/v). The ethanol and hydrogen production performance from glucose by a co-culture of the strains AK15 and AK17 was studied in a continuous-flow bioreactor at 60 degrees C. Stable and continuous ethanol and hydrogen co-production was achieved with ethanol yield of 1.35 mol-EtOH/mol-glucose, and with the hydrogen production rate of 6.1 mmol/h/L (H2 yield of 0.80 mol-H2/mol-glucose). PCR-DGGE analysis revealed that the AK17 became the dominant bacterium in the bioreactor. In conclusion, strain AK17 is a promising strain for the co-production of ethanol and hydrogen with a wide substrate utilization spectrum, relatively high ethanol tolerance, and ethanol yields among the highest reported for thermoanaerobes.     

Isolation and identification of a thermophilic strain producing trehalose synthase from geothermal water in China

A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.     

Thiobacillus sp. W5, the dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes

The floating filter technique was successfully adapted for the isolation of the dominant, chemolithoautotrophic, sulfide-oxidizing bacterium from a sulfur-producing reactor after conventional isolation techniques had failed. The inoculated polycarbonate filters, floating on mineral medium, were incubated under gaseous hydrogen sulfide at non-toxic levels. This technique gave 200-fold higher recoveries than conventional isolation techniques. Viable counts on the filters, making up 15% of the total count, appeared to be all of the same species. Chemostat cultures of the new isolate had a very high sulfur-forming capacity, converting almost all hydrogen sulfide in the medium to elemental sulfur under high sulfide loads (27.5 mmol l^-1 h^-1) and fully aerobic conditions. This behaviour closely resembled that of the microbial community in the sulfur-producing reactor. Moreover, similar protein patterns were obtained by electrophoresis of cell-free extracts from the isolate and the mixed culture. It has therefore been concluded that this isolate represents the dominant sulfide-oxidizing population in the reactor. The isolate has been shown to be a new Thiobacillus species, related to Thiobacillus neapolitanus. In view of the general confusion currently surrounding the taxonomy of the thiobacilli, a new species has not been formally created. Instead, the isolate has been given the working name Thiobacillus sp. W5.     

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.     

Isolation and characterization of Methylophaga sulfidovorans sp. nov.: an obligately methylotrophic, aerobic, dimethylsulfide oxidizing bacterium from a microbial mat

Abstract: Sediment from a microbial mat from the South-West coast of the Netherlands consumed dimethylsulfide (DMS) under oxic and anoxic conditions. From this sediment, a Gram-negative, oval DMS oxidizing bacterium, strain RB-1, was isolated. Its substrate range is typical of an obligately methylotrophic organism. Enzyme analysis revealed the presence of the ribulose monophosphate pathway for carbon assimilation, and the ability to use the linear dissimilatory pathway via formate to carbon dioxide, as well as the cyclic pathway via the ribulose monophosphate route for carbon dissimilation. 16S rRNA sequence analysis showed high similarity with species belonging to the genus Methylophaga . Because of the specific dimethylsulfide and hydrogen sulfide oxidizing capacity, the new isolate was named Methylophaga sulfidovorans .     

Isolation of a strain of Bacillus schlegelii from geothermally heated antarctic soil

Abstract A bacterium capable of growth from 59 to 72° C was isolated from geothermal soil collected from Mount Erebus, Ross Island, Antarctica. The isolate was enriched in medium containing thiosulphate and bicarbonate. Subsequently the organism was found also to be capable of heterotrophic growth and autotrophic growth in the presence of hydrogen and carbon dioxide. In a comparison with Bacillus schlegelii and Bacillus tusciae the isolate most closely resembled B. schlegelii . This conclusion was supported by the finding that B. schlegelii is also capable of autotrophic growth using thiosulphate. The new isolate had a characteristic subunit layer on the cell wall which is typical of B. schlegelii .     

Thermostable amylase from an aerobic,gram-negative,non-spore forming thermophilic bacterium

Summary An obligate aerobic, rod-shaped, Gram-negative, non-spore forming thermophilic bacterium was isolated from soil (Jordan) on starch nutrient agar at 60°C. Starch, dextrin, maltose and pullalan induced the synthesis of amylase, while glucose, lactose and fructose did not. The formation of heat stable amylase started in the early exponential phase, while maximal extracellular enzyme activity (21.75 U/ml) was detected at the end of the decline phase when most of the cells appeared as spheres.     

Isolation,identification of sludge-lysing strain and its utilization in thermophilic aerobic digestion for waste activated sludge

A strain of sludge-lysing bacteria was isolated from waste activated sludge (WAS) in this study. The result of 16S rRNA gene analysis demonstrated that it was a species of new genus Brevibacillus (named Brevibacillus sp. KH3). The strain could release the protease with molecule weight of about 40 kDa which could enhance the efficiency of sludge thermophilic aerobic digestion. During the sterilized sludge digestion experiment inoculated with Brevibacillus sp. KH3, the maximum protease activity was 0.41 U/ml at pH 8 and 50 °C, and maximum TSS removal ratio achieved 32.8% after 120 h digestion at pH 8 and 50 °C. In the case of un-sterilized sludge digestion inoculated with Brevibacillus sp. KH3, TSS removal ratio in inoculated-group was 54.8%, increasing at 11.86% compared with un-inoculation (46.2%). The result demonstrated that inoculation of Brevibacillus sp. KH3 could help to degrade the EPS and promote the collapse of cells and inhibit the growth of certain kinds of microorganisms. It indicated that Brevibacillus sp. KH3 strain had a high potential to enhance WAS-degradation efficiency in thermophilic aerobic digestion.     

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.     

Isolation of succinate dehydrogenase from Desulfobulbus elongatus, a propionate oxidizing, sulfate reducing bacterium

Succinate dehydrogenase was purified from the particulate fraction of Desulfobulbus. The enzyme catalyzed both fumarate reduction and succinate oxidation but the rate of fumarate reduction was 8-times less than that of succinate oxidation. Quantitative analysis showed the presence of 1 mol of covalently bound flavin and 1 mol of cytochrome b per mol of succinate dehydrogenase. The enzyme contained three subunits with molecular mass 68.5, 27.5 and 22 kDa. EPR spectroscopy indicated the presence of at least two iron sulfur clusters. 2-Heptyl-4-hydroxy-quinoline-N-oxide inhibited the electron-transfer between succinate dehydrogenase and a high redox potential cytochrome c3 from Desulfobulbus elongatus.     

Biodegradation of potato slops from a rural distillery by thermophilic aerobic bacteria

Livestock manure may contain pathogenic organisms which pose a risk to the health of animals or humans if the manure is not adequately treated or disposed of. One possible treatment method is composting. However to ensure that pathogen destruction occurs, temperatures need to be sufficiently high throughout the heap to ensure that pathogens are inactivated. The temperature required to inactivate a marker organism, Escherichia coli 11943, has been investigated, and found to depend on substrate composition, moisture content and duration of incubation. Results show that temperatures in excess of 55 degrees C for 2 h are required for inactivation. Data are presented showing the levels of faecal coliforms in compost heaps where temperatures did not rise above mesophilic levels (35 degrees C where samples were taken).     

A new aerobic chemolithoautotrophic arsenic oxidizing microorganism isolated from a high Andean watershed

Biological arsenic oxidation has been suggested as a key biogeochemical process that controls the mobilization and fate of this metalloid in aqueous environments. To the best of our knowledge, only four aerobic chemolithoautotrophic arsenite-oxidizing (CAO) bacteria have been shown to grow via direct arsenic oxidation and to have the essential genes for chemolithoautotrophic arsenite oxidation. In this study, a new CAO bacterium was isolated from a high Andean watershed evidencing natural dissolved arsenic attenuation. The bacterial isolate, designated TS-1, is closely related to the Ancylobacter genus, in the Alphaproteobacteria class. Results showed that TS-1 has genes for arsenite oxidation and carbon fixation. The dependence of bacterial growth from arsenite oxidation was demonstrated. In addition, a mathematical model was suggested and the kinetic parameters were obtained by simultaneously fitting the biomass growth, arsenite depletion curves, and arsenate production. This research increases the knowledge of chemolithoautotrophic arsenic oxidizing microorganisms and its potential role as a driver for natural arsenic attenuation.     

Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract A thermophilic acetate-decarboxylating methanogenic bacterium was isolated from a laboratory-scale 60°C sludge digestor. Cells form straight filaments with flat to blunted ends normally consisting of 2–3 cells held together by a sheath-like outer cell wall. The organism uses acetate, H₂-CO₂ and formate for methanogenesis and growth. With acetate as the sole methanogenic substrate, almost all of the radioactivity from methyl-labelled acetate appeared as methane. Acetate was converted to methane in equimolar amounts with a doubling time of 3 days.     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.