A new symbiotic nanoarchaeote (Candidatus Nanoclepta minutus) and its host (Zestosphaera tikiterensis gen. nov., sp. nov.) from a New Zealand hot spring

Three thermophilic Nanoarchaeota-Crenarchaeota symbiotic systems have been described. We obtained another stable anaerobic enrichment culture at 80 °C, pH 6.0 from a New Zealand hot spring. The nanoarchaeote (Ncl-1) and its host (NZ3^T) were isolated in co-culture and their genomes assembled. The small (∼200 nm) flagellated cocci were often attached to larger cocci. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi. Their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Ncl-1 also relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3^T was isolated and grows on proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3^T requires thiosulfate and grows best at 82 °C, pH 6.0. NZ3^T is most closely related to the Desulfurococcaceae, Ignisphaera aggregans (∼92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3^T represent novel genera in the Nanoarchaeota and the Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3^T (=DSMZ 107634^T = OCM 1213^T).     

Non-diauxic fermentation of acid hydrolysed pine by a strain of Clostridium thermohydrosulfuricum isolated from a New Zealand hot spring

Abstract Fermentation of dilute acid hydrolysed pine could be achieved by all of 18 thermophilic glycolytic anaerobic bacteria tested, while only seven isolates representing the species Clostridium thermohydrosulfuricum, Thermoanaerobium brockii and Thermoanaerobacter ethanolicus were able to ferment undiluted hydrolysate. A strain of C. thermohydrosulfuricum , designated Rt8.B1, was notably better at fermenting the hydrolysate and was studied further. Studies showed that glucose and xylose present in the hydrolysate were utilized simultaneously, a phenomenon which also occurred in a medium containing glucose and xylose as the added carbohydrates.     

Thermoanaerobium lactoethylicum spec. nov. a new anaerobic bacterium from a hot spring of Kamchatka

A new anaerobic thermophilic Gram-positive, nonsporeforming bacterium strain ZE-1 was isolated from a hot spring of Kamchatka (USSR). The cells are rod-shaped, (0.5–0.8 · 2.0–20 m), non-motile. The bacterium can grow between 42 and 75°C; the optimal temperature is 65°C. The growth is possible between pH values 5.0 and 8.5; optimal pH is 7.0. The cultures grow on the media containing peptone, yeast extract, or casein hydrolysate as nitrogen sources in the presence of glucose or some other sugars, mannitol or starch. The main fermentation products of glucose are ethanol, acetate, lactate, H₂, CO₂; byproducts are propionic, butyric and isovaleric acids. Glucose is metabolized via Embden-Meyerhoff-Parnas pathway. Molecular hydrogen does not inhibit growth. The bacterium does not reduce aceton to isopropanol, but is able to form H₂S from elemental sulfur. The bacterium contains a soluble hydrogenase. This enzyme catalyzes both evolution and uptake of H₂ and is active in the presence of methyl viologen. The DNA-base composition is 34.6 mol%; the genome size 2.08x10⁹ D. The name proposed for the isolated bacterium strain ZE-1 is Thermoanaerobium lactoethylicum spec. nov.     

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     

Thermoanaerobium brockii gen. nov. and sp. nov., a new chemoorganotrophic,caldoactive, anaerobic bacterium

The isolation of a new anaerobic thermophilic bacterium, Thermoanaerobium brockii, from volcanic features is described. Successful enrichment required a complex medium containing glucose or other fermentable sugars and incubation temperatures of 55–80° C. Strains of T. brockii were gram positive, rods of uneven length that existed singly, in pairs, chains or filaments. Electron micrographs of thin sections of cell revealed a monolayered cell wall and a constrictive or pinching off cell division process. The organism was nonsporeforming, obligately anaerobic and chemoorganotrophic. The optimal temperature for growth was 65–70° C, the maxium was below 85° C and the minimum above 35° C. The doubling time at the optimal temperature for growth was about 1 h. The DNA base composition of three strains of T. brockii varied from 30.0–31.4 mol % guanosine plus cytosine. Fermentable carbohydrates included glucose, sucrose, maltose, lactose, cellobiose and insoluble starch. The fermentation products of cells grown on glucose were ethanol, lactic acid, acetic acid, hydrogen and carbon dioxide. Growth of all strains tested was inhibited by fairly low concentrations of cycloserine, penicillin, streptomycin, tetracycline and chloramphenicol. The possible ecological, evolutionary, and industrial significance, and taxonomic relationships of Thermoanaerobium are discussed.Abbreviations TYEG complex medium containing mineral salts, 0.3% yeast extract, 1.0% tryptone and 0.5% glucose - O.D. optical density - G+C guanosine plus cytosine     

Characterization of a thermophilic sulfur oxidizing enrichment culture dominated by a Sulfolobus sp. obtained from an underground hot spring for use in extreme bioleaching conditions

A thermoacidophilic elemental sulfur and chalcopyrite oxidizing enrichment culture VS2 was obtained from hot spring run-off sediments of an underground mine. It contained only archaeal species, namely a Sulfolobus metallicus-related organism (96% similarity in partial 16S rRNA gene) and Thermoplasma acidophilum (98% similarity in partial 16S rRNA gene). The VS2 culture grew in a temperature range of 35–76°C. Sulfur oxidation by VS2 was optimal at 70°C, with the highest oxidation rate being 99 mg S⁰ l⁻¹ day⁻¹. At 50°C, the highest sulfur oxidation rate was 89 mg l⁻¹ day⁻¹ (in the presence of 5 g Cl⁻ l⁻¹). Sulfur oxidation was not significantly affected by 0.02–0.1 g l⁻¹ yeast extract or saline water (total salinity of 0.6 M) that simulated mine water at field application sites with availability of only saline water. Chloride ions at a concentration above 10 g l⁻¹ inhibited sulfur oxidation. Both granular and powdered forms of sulfur were bioavailable, but the oxidation rate of granular sulfur was less than 50% of the powdered form. Chalcopyrite concentrate oxidation (1% w/v) by the VS2 resulted in a 90% Cu yield in 30 days.     

Tenuifilum thalassicum gen. nov., sp. nov., a novel moderate thermophilic anaerobic bacterium from a Kunashir Island shallow hot spring representing a new family Tenuifilaceae fam. nov. in the class Bacteroidia

A novel anaerobic moderately thermophilic bacterium, designated strain 38H-str^T, was isolated from a 12 m deep hot spring of the Kunashir Island shore. Gram-negative cells were non-spore-forming, motile, straight or curved filamentous rods, occasionally forming loops and knots. The strain grew at 20–65 °C and pH range of 4.0–9.0 with an optimum at 50 °C and pH 6.5–7.0. Strain 38H-str^T required 0.5–2.5% NaCl (1.5% is an optimum) for growth. It was a chemoorganoheterotroph, growing on carbohydrates (starch, pullulan, alginate, laminarin, beta-glucan) or peptide mixtures and proteins (peptone, tryptone, gelatin, and α- or β- keratins). Major products of glucose fermentation were acetate, hydrogen, and carbon dioxide. Major cellular fatty acids were iso- and anteiso-C_15:0. Phosphatidylethanolamine, an unidentified phospholipid, and three unidentified polar lipids were detected in cellular lipids fractions. The quinone was MK-7. The size of complete genome of strain 38H-str^T was 3.2 Mb; DNA G+C content was 38.3 mol%. According to 16S rRNA gene sequence and conserved protein sequences phylogenies, strain 38H-str^T represented a deeply branched lineage near the root of the class Bacteroidia. Based on phylogenetic analysis and phenotypic features the novel isolate was assigned to a novel family within the order Bacteroidales for which the name Tenuifilaceae fam. nov. is proposed. Strain 38H-str^T (=DSM 100343^T =VKM B-2964^T) represents the first genus and species Tenuifilum thalassicum gen. nov., sp. nov.     

<Emphasis Type="Italic">Desulfomicrobium thermophilum</Emphasis> sp. nov., a novel thermophilic sulphate-reducing bacterium isolated from a terrestrial hot spring in Colombia

A moderately thermophilic, sulphate-reducing bacterium, designated strain P6-2(T), was isolated from a terrestrial hot spring located at a height of 2,500 m in the Andean region, Colombia (5 degrees 43'69'N, 73 degrees 6'10'W). Cells of strain P6-2(T) were rod-shaped, stained Gram-negative and were motile by means of a single polar flagellum. The strain grew lithotrophically with H(2) as the electron donor and organotrophically on lactate, pyruvate, ethanol, malate, fumarate, n-propanol and succinate in the presence of sulphate as the terminal electron acceptor. Fumarate and pyruvate was fermented. Strain P6-2(T) grew optimally at 55 degrees C (range 37-60 degrees C), pH 6.6 (range 5.8-8.8) in the presence of 0.5% NaCl (range 0-4.5%) with lactate and sulphate and produced acetate, CO(2) and H(2)S as the major end-products. Sulphate, sulphite and thiosulphate could be used as electron acceptors but not elemental sulphur or nitrate. The G + C content of the genomic DNA was 58.7 mol%. The 16S rRNA sequence analysis indicated that strain P6-2(T) was a member of the class Deltaproteobacteria, domain Bacteria with Desulfomicrobium baculatum being the closest relative (similarity value of 94%). Phylogeny of genes encoding alpha- and beta-subunits of the dissimilatory sulphite reductase (dsrAB genes) supported its affiliation to members of the genus Desulfomicrobium. On the basis of this evidence, we propose to assign strain P6-2(T) as new species of the genus Desulfomicrobium, D. thermophilum sp. nov., with strain P6-2(T) as the type strain (= DSM 16697(T) = CCUG 49732(T)).     

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.     

Streptomyces calidiresistens sp. nov., isolated from a hot spring sediment

A Streptomyces-like actinomycete strain, designated as YIM 78087^T, was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic position of strain YIM 78087^T was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YIM 78087^T belongs to the genus Streptomyces and is closely related to Streptomyces fimbriatus DSM 40942^T, Streptomyces marinus DSM 41968^T and Streptomyces qinglanensis DSM 42035^T (97.18, 97.05 and 97.1 % similarity, respectively). Combined with the low values of DNA–DNA hybridization between strain YIM 78087^T and its closest neighbours, these analyses indicated that this new isolate represents a different genomic species in the genus Streptomyces. The predominant menaquinones of strain YIM 78087^T were identified as MK-9 (H₄) and MK-9 (H₆). The major fatty acids were identified as anteiso-C_15:0 (28.4 %), anteiso-C_17:0 (23.0 %) and iso-C_16:0 (15.1 %). The whole-cell hydrolysates found to contain glucose, mannose and ribose. The DNA G+C content was determined to be 73.0 mol%. Based on the comparative analysis of phenotypic and genotypic characteristics, it is proposed that strain YIM 78087^T represents a novel species of the genus Streptomyces, for which the name Streptomyces calidiresistens sp. nov., is proposed. The type strain is YIM 78087^T (=BCRC 16955^T=DSM 42108^T=JCM 19629^T).     

Azoarcus taiwanensis sp. nov., a denitrifying species isolated from a hot spring

The strain NSC3^T, a novel, facultative, chemolithotrophic, denitrifying, alkaliphilic, sulfide-oxidizing bacterium isolated from a hot spring in Yang-Ming Mountain, Taiwan, was Gram negative, rod shaped, and motile by single polar flagella and grew facultatively by adopting a denitrifying metabolism. The 16S rRNA sequence analysis revealed that strain NSC3^T belongs to beta subclass of the Proteobacteria and most closely related to Azoarcus evansii KB740^T (95.44 %), Azoarcus toluvorans Td-21^T (95.21 %), Azoarcus tolulyticus Tol-4^T (95.08 %), and Azoarcus toluclasticus MF63^T (94.94 %). The phylogenetic analyses based on 16S rRNA gene sequences indicated that the strain NSC3^T formed a distinct lineage in the Betaproteobacteria and that it exhibited the highest level of sequence similarity with species of the genera Azoarcus (95.28–93.13 %). The major fatty acids of the type strain were C_16:0 (26.9 %), C_16:1w7c (28.9 %), C_18:0 (9.6 %), and C_18:1w7c/w6c (29.9 %). The DNA G+C content of genomic DNA was 63.7 mol%. On the basis of the 16S rRNA sequence similarity, phenotypic and genotypic characteristics, and chemotaxonomic data, the strain NSC3^T could be differentiated from other species of the genus Azoarcus. Therefore, strain NSC3^T (equal to BCRC 80111^T and DSM 24109^T) is proposed as a novel species in genus Azoarcus, for which the name Azoarcus taiwanensis sp. nov. is proposed. The strain NSC3^T is deposited in Bioresource Collection and Research Center, Taiwan, under the reference number BCRC 80111^T, and German Collection of Microorganisms and Cell Cultures, Germany (DSMZ), with DSM 24109^T.     

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     

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     

Tepidimonas taiwanensis sp. nov., a novel alkaline-protease-producing bacterium isolated from a hot spring

The bacterial strain designated I1-1^T was isolated from a hot spring located in the Pingtung area, southern Taiwan. Cells of this organism were Gram reaction negative rods, motile by a single polar flagellum. Optimum conditions for growth were 55°C and pH 7. Strain I1-1^T grew well in lower nutrient media such as 5–10% Luria–Bertani broth, and its extracellular products expressed alkaline protease activity. The 16S rRNA gene sequence analysis indicates that strain I1-1^T is a member of -Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA–DNA similarity data, whole-cell protein analysis, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Tepidimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain I1-1^T were 16:0 (about 41%), 18:1 7c (about 13%), and summed feature 3 [16:1 7c or 15:0 iso 2OH or both (about 26%)]. Its DNA base ratio was 68.1 mol%. We propose to classify strain I1-1^T (=BCRC 17406^T=LMG 22826^T) as Tepidimonas taiwanensis sp. nov.     

Thermoanaerobium acetigenum spec. nov., a new anaerobic,extremely thermophilic,xylanolytic non-spore-forming bacterium isolated from an Icelandic hot spring

An anaerobic, extremely thermophilic, xylanolytic nonspore-forming bacterium, strain X6B, was isolated from a 70°C Icelandic hot spring sediment. The bacterium was rod-shaped, 3.6–5.9 m long and 0.7 to 1.0 m wide, and cells grew singly, in pairs, and occasionally formed chains. The bacterium was nonmotile with no flagella. Cells from mid-to late exponential gowth-phase cultures stained gram-negative but had a gram-positive like cell wall structure in transmission electron photomicrographs. The bacterium grew between 50°C and 78°C with an optimum temperature at about 65°C to 68°C. Growth occurred between pH 5.2 and 8.5 with an optimum pH close to 7. During growth on beech wood xylan, glucose and d-xylose, the isolate produced CO₂, acetate and H₂ as major fermentation products, and a small amounts of ethanol; lactate was not produced. X6B did not reduce acetone to isopropanol or sulphate or thiosulfate to sulfide. The base composition of X6B's cellular DNA was 35.7 mol% guanine + cytosine. The properties of this strain do not fit any previously described species. The name proposed for the isolated bacterium was Thermoanaerobium acetigenum, spec. nov.     

Alicyclobacillus tengchongensis sp. nov., a thermo-acidophilic bacterium isolated from hot spring soil

A thermo-acidophilic bacterium, designated strain ACK006^T, was isolated from the soil of a hot spring at Tengchong in China. Cells were Gram-staining-positive, motile, catalase-positive and oxidase-negative, spore-forming rods. The isolate grew aerobically at 30–50°C (optimum at 45°C), pH 2.0–6.0 (optimum pH 3.2) and 0–5.0% (w/v) NaCl (optimum 1% NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain ACK006^T belongs to the genus Alicyclobacillus with the sequence similarity of 92.3, 92.4, 92.5, and 92.8% to Alicyclobacillus cycloheptanicus SCH^T, Alicyclobacillus ferrooxydans TC-34^T, Alicyclobacillus contaminans 3-A191^T and Alicyclobacillus disulfidooxidans SD-11^T, respectively. Similarity to other species of the genus Alicyclobacillus was 90.3–92.8% and similarity to species of the genus Tumebacillus was 85.9–87.8%. The genomic DNA G+C content was 53.7 mol%. The predominant menaquinone was MK-7. Major fatty acids were ω-cycloheptane C_18:0, iso-C_17:0 and anteiso-C_17:0. The cell-wall peptidoglycan was the A1γ type; containing meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic analysis from this study, strain ACK006^T represents a novel species of the genus Alicyclobacillus for which the name Alicyclobacillus tengchongensis sp. nov. is proposed. The type strain is ACK006^T (=KCTC 33022^T =DSM 25924^T).     

Thermodesulfovibrio hydrogeniphilus sp. nov., a new thermophilic sulphate-reducing bacterium isolated from a Tunisian hot spring

A new thermophilic sulphate-reducing bacterium (strain Hbr5^T) was enriched and isolated from a terrestrial Tunisian hot spring. It was a non-spore-forming Gram-negative curved or vibrio-shaped bacterium. It appeared singly or in long chains and was actively motile by a polar flagellum. It possessed c-type cytochromes and desulfofuscidin. Growth occurred between 50 and 70 °C, with an optimum of 65 °C at pH 7.1. In the presence of sulphate as a terminal electron acceptor, this strain readily used H₂ but formate only poorly. It could use sulphate, thiosulphate, sulphite or arsenate as electron acceptors. Its DNA G+C content was 36.1 mol%. Based on phenotypic, genomic, and phylogenetic characteristics, strain Hbr5^T (=DSM 18151^T, =JCM 13991^T) is proposed to be assigned to a novel species of genus Thermodesulfovibrio, T. hydrogeniphilus sp. nov.