Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Probing the archaeal diversity of a mixed thermophilic bioleaching culture by TGGE and FISH

The archaeal community present in a sample of Mixed Thermophilic Culture-B (MTC-B) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (TGGE) and fluorescence in situ hybridisation (FISH). Both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. Using the TGGE protocol developed, the relative species composition of the thermophilic bioleaching sample was analysed, and included four phylotypes belonging to the Sulfolobales, which were related to Stygiolobus azoricus, Metallosphaera sp. J1, Acidianus infernus and Sulfurisphaera ohwakuensis. However, the St. azoricus-like phylotype was difficult to resolve and some micro-heterogeneity was observed within this phylotype. Specific FISH probes were designed to qualitatively assess the presence of the phylotypes in MTC-B. The sample was dominated by Sf. ohwakuensis-like Archaea. In addition, the St. azoricus-like, Metallosphaera species-like and Acidianus species-like cells appeared in similar low abundance in the community. Most strikingly, FISH identified Sulfolobus shibatae-like cells present in low numbers in the sample even though these were not detected by PCR-dependent TGGE. These results highlight the importance of using more than one molecular technique when investigating the archaeal diversity of complex bioleaching reactor samples.     

Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor

The successful extraction and isolation of the hydrolysed tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus, a key thermophilic bioleaching archaeon, is described. The archaeal biomass was recovered directly from a thermophilic (68 degrees C) bioleaching tank reactor used to extract nickel from a pentlandite mineral concentrate. The initial Soxhlet extraction method employed was scaled to a bench-scale extraction procedure suitable for the preparation of gram-scale quantities of GDNT. The GDNT so obtained was analysed by 1D- and 2D-NMR techniques, providing the first complete 13C and 2D-NMR data-set for GDNT, including that for the intact underivatised calditol moiety. The study demonstrates the feasibility of recovering high-quality GNDT from thermophilic archaeal-mediated bioleaching reactors. The recovery of these lipids at relatively low cost, as a by-product from bioleaching reactors used in the metals processing industry, has important implications for future tetraether lipid availability and costs.     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

Sulfobacillus benefaciens sp. nov., an acidophilic facultative anaerobic Firmicute isolated from mineral bioleaching operations

Gram-positive bacteria found as the sole Firmicutes present in two mineral bioleaching stirred tanks, and a third bacterium isolated from a heap leaching operation, were shown to be closely related to each other but distinct from characterized acidophilic iron- and sulfur-oxidizing bacteria of the genus Sulfobacillus, to which they were affiliated. One of the isolates (BRGM2) was shown to be a thermo-tolerant (temperature optimum 38.5°C, and maximum 47°C) obligate acidophile (pH optimum 1.5, and minimum 0.8), and also noted to be a facultative anaerobe, growing via ferric iron respiration in the absence of oxygen. Although isolates BRGM2 and TVK8 were able to metabolize many monomeric organic substrates, their propensity for autotrophic growth was found to be greater than that of Sulfobacillus thermosulfidooxidans and the related acidophile, Sb. acidophilus. Faster growth rates of the novel isolates in the absence of organic carbon was considered to be a major reason why they, rather than Sb. thermosulfidooxidans (which shared many physiological characteristics) more successfully exploited conditions in the stirred tanks. Based on their phylogenetic and phenotypic characteristics, the isolates are designated strains of the proposed novel species, Sulfobacillus benefaciens, with isolate BRGM2 nominated as the type strain.     

Growth of a manganese oxidizing Pseudomonas sp. in continuous culture

Strain S-36, a marine Pseudomonas sp., was grown under manganese limitation in continuous culture. At dilution rates below a maximal growth rate of 0.066 h^-1, the rate at which the organism fixed CO₂ into macromolecules was equal to the cell carbon production rate. In addition, the total amount of cell carbon or CO₂ fixed at steady-state was in proportion to the amount of energy available from the oxidation of Mn²⁺ in the medium. These data suggest that the organism can grow by obtaining the energy for CO₂ fixation from manganese oxidation.     

The use of scanning electron microscopy and an electron microprobe in studies on oxidation of sulfur compounds by bacteria and bioleaching of heavy metals

Abstract: We present results obtained during studies on oxidation of reduced sulfur compounds by Thiobacillus neapolitanus and bioleaching of copper from limestone and sandstone ore. We have used simple and cheap methods for visualisation of chemical and biological phenomena in microscale involving examination with scanning electron microscope and electron microprobe.     

Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland

The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO₂, and H₂. The temperature optimum for growth was between 70 and 75° C, and growth occurred in the range of 50–75° C. The pH range for growth was 4.7–8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H₂ or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed. Received: 7 October 1996 / Accepted: 14 March 1997     

Thermococcus waiotapuensis sp. nov., an extremely thermophilic archaeon isolated from a freshwater hot spring

An extremely thermophilic, sulfur-dependent archaeon, strain WT1, was isolated from a freshwater hot spring in the Lake Taupo area of North Island, New Zealand. The cells are flagellated, strictly anaerobic cocci that grow optimally at 85 °C and 5.4 g NaCl l^–1. The strain grows heterotrophically on complex proteinaceous substrates or on appropriate salts plus amino acid mixtures and is also able to utilize maltose, starch, and pyruvate. Elemental sulfur could be replaced by cystine or thioglycollate. The range of temperatures allowing growth is from 60 to 90 °C; the pH supporting growth ranges from 5 to 8 (optimum, pH 7). Strain WT1 grew in a defined medium containing amino acids as the sole carbon and energy sources. The required amino acids were: Arg, His, Ile, Leu, Phe, Ser, Thr, Trp, Tyr, and Val. Strain WT1 showed sensitivity to rifampicin. DNA G+C content was 50.4 mol%. Phylogenetic analysis of the sequence encoding the 16S rRNA gene indicated that this isolate is a member of the Thermococcales. DNA/DNA hybridization studies revealed no similarity to several species of Thermococcus and Pyrococcus, with the exception of Thermococcus zilligii. Based on the reported results, we propose strain WT1 as a new species to be named Thermococcus waiotapuensis sp. nov. Received: 5 January 1999 / Accepted: 19 May 1999     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.     

Constitutive production of endoglucanase by a Bacillus sp. isolated from a Zimbabwean hot spring

A sporulating, aerobic Bacillus sp., isolated from Chimanimani hot springs, Zimbabwe, produced endoglucanase when cultured on medium with initial pH between 5.0 and 9.0 and at 30 to 60°C. Optimal production of endoglucanase was at pH 6.0. The enzyme was constitutively produced when the organism was cultured on starch, cellobiose, carboxymethylcellulose, sucrose, glucose, galactose, Avicel, lactose, mannose or maltose.The authors are with the Fermentation and Food Group, Department of Biochemistry, University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe     

Thermalkalibacillus uzonensis gen. nov. sp. nov, a novel aerobic alkali-tolerant thermophilic bacterium isolated from a hot spring in Uzon Caldera, Kamchatka

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58^T was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7–0.8 μm in width and 5.5–12 μm in length and produced terminal spherical spores of 1.2–1.6 μm in diameter with the mother cell swelling around 2 μm in diameter (drumstick-type morphology). Cells grew optimally at pH^25°C 8.2–8.4 and temperature 50–52°C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO₂ (both with or without H₂) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, d-galactose, d-mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58^T is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58^T (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.The Genbank accession number for the 16S rRNA gene sequence of strain JW/WZ-YB58^T is DQ221694.     

Isolation and characterization of a thermophilic benzoate-degrading,sulfate-reducing bacterium,Desulfotomaculum thermobenzoicum sp. nov.

A new thermophilic sulfate-reducing bacterium, strain TSB, that was spore-forming, rod-shaped, slightly motile and gram-positive, was isolated from a butyrate-containing enrichment culture inoculated with sludge of a thermophilic methane fermentation reactor. This isolate could oxidize benzoate completely. Strain TSB also oxidized some fatty acids and alcohols. SO _inf4 ^sup2- , SO _inf3 ^sup2- , S₂O _inf3 ^sup2- and NO _inf3 ^sup- were utilized as electron acceptors. With pyruvate or lactate the isolate grew without an external electron acceptor and produced acetate. The optimum temperature for growth was 62°C. The G+C content of DNA was 52.8 mol%. This isolate is described as a new species, Desulfotomaculum thermobenzoicum.     

Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> ATCC 35092

The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the -1,4-glucosidic linkage at the reducing end to an -1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.     

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50–70 °C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 °C for 12 h, only totally inactivated after more than 4-h incubation at 80 °C. It also showed much higher resistance to KCN, NaN₃ and H₂O₂ as compared to other SODs. Xin Wang and Haijie Yang contribute to this work equally.     

Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring

During screening for amylase-producing bacteria, a strain designated OnlT was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growth were 55 degrees C and pH 7. Strain On1(T) grew well in minimal medium containing starch as the sole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1(T) is a member of beta-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1(T) were 16:0 (about 30%), 18:1 omega 7c (about 20%) and summed feature 3 (16:1 omega 7c or 15:0 iso 2OH or both [about 31%]). Its DNA base ratio was 65.9 mol% G + C. We propose to classify strain On1(T) (= BCRC 17405T = LMG 22827T) as Caldimonas taiwanensis sp. nov.     

A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

A new isolate of Hydrogenobacter,an obligately chemolithoautotrophic,thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 m. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl⁷-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C_18:0, C_18:1 and C_20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.     

Tepidimonas aquatica sp. nov., a new slightly thermophilic beta-proteobacterium isolated from a hot water tank

A bacterial isolate, with an optimum growth temperature of about 50 degrees C, was recovered from a domestic hot water tank in Coimbra. Phylogenetic analysis using 16S rRNA gene sequence indicated that strain CLN-1T is a member of the beta-Proteobacteria and represents a new species of the genus Tepidimonas. The major fatty acids of strain CLN-1T are 16:0, 17:0 cyclo and 16:1 omega7c. Ubiquinone 8 is the major respiratory quinone, the major polar lipids are phosphatidylethanolamine, and phosphatidylglycerol. The new isolate is aerobic and facultatively chemolithoheterotrophic. Thiosulfate and tetrathionate are oxidized to sulfate in the presence of a metabolizable carbon source. Strain CLN-1T grows on amino acids and organic acids, but this organism does not assimilate carbohydrates. Glycerol is the only polyol assimilated. Resinic acids, namely abietic acid, dehydroabietic acid and isopimaric acid are not degraded. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strain CLN-1T represents a new species for which we offer the name Tepidimonas aquatica.     

Characterization of the smallest dimeric bile salt hydrolase from a thermophile Brevibacillus sp.

A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60°C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K _M and k _cat values of 3.08 μM and 6.32 × 10² s⁻¹, respectively, for glycodeoxycholic acid. The NH₂-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.