Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan

A novel barophilic, extremely thermophilic bacterium was isolated from a deep-sea hydrothermal vent chimney at the Iheya Basin, in the Okinawa area, Japan. The cells were found to be rod shaped and surrounded by a sheath-like outer structure; the organism did not possess flagella and was not motile. Growth was observed between 45° and 80°C (optimum, 72°C, 45 min doubling time), pH 5.3 and 9.3 (optimum, pH 7.2–7.6), 6.6 and 79 g/l sea salts (optimum, 40 g/l), and 0.1 and 60 MPa (optimum, 20 MPa). Strain IHB1 was found to be a strictly anaerobic chemoorganotroph capable of utilizing yeast extract and proteinaceous substrates such as peptone and tryptone. Elemental sulfur or thiosulfate acted as electron acceptors improving growth. The isolate was able to utilize casein as a sole carbon and energy source in the presence of thiosulfate. The G + C content of the genomic DNA was 31.4 mol%. Phylogenetic analysis based on 16S rDNA sequences and DNA–DNA hybridization analysis indicated that the isolate is closely related to Thermosipho africanus; however, it represents a species distinct from the previously described members of the genus Thermosipho. On the basis of the physiological and molecular properties, we propose that the new isolate represents a new species, which we name Thermosipho japonicus sp. nov. (type strain: IHB1; JCM10495). Received: May 26, 1999 / Accepted: August 7, 1999     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides

An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T_1/2 values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2–7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.     

Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine,Lead, South Dakota,USA

The present study investigated the cultivable mesophilic (37°C) and thermophilic (60°C) cellulose-degrading bacterial diversity in a weathered soil-like sample collected from the deep subsurface (1.5 km depth) of the Homestake gold mine in Lead, South Dakota, USA. Chemical characterization of the sample by X-ray fluorescence spectroscopy revealed a high amount of toxic heavy metals such as Cu, Cr, Pb, Ni, and Zn. Molecular community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from enrichment cultures growing in presence of microcrystalline cellulose as the sole source of carbon. All phylotypes retrieved from enrichment cultures were affiliated to Firmicutes. Cellulose-degrading mesophilic and thermophilic pure cultures belonging to the genera Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and selected cultures were studied for enzyme activities. For a mesophilic isolate (DUSELG12), the optimum pH and temperature for carboxymethyl cellulase (CMCase) were 5.5 and 55°C, while for a thermophilic isolate (DUSELR7) they were 5.0 and 75°C, respectively. Furthermore, DUSELG12 retained about 40% CMCase activity after incubation at 60°C for 8 h. Most remarkably, thermophilic isolate, DUSELR7 retained 26% CMCase activity at 60°C up to a period of 300 h. Overall, the present work revealed the presence of different cellulose-degrading bacterial lineages in the unique deep subsurface environment of the mine. The results also have strong implications for biological conversion of cellulosic agricultural and forestry wastes to commodity chemicals including sugars.     

Thermoanaerobacterium thermostercus sp. nov., a new anaerobic thermophilic hydrogen-producing bacterium from buffalo-dung

A novel thermophilic, anaerobic, rod-shaped bacterium strain, designated Buff, was isolated from buffalo-dung samples collected from a buffalo-farm located in Caserta (Campania, south of Italy). Strain Buff was Gram-positive, motile and no spore-forming. The growth temperature range was 40–65°C with an optimum at 60°C, while pH growth range at 60°C was 5.5–8.0 with an optimum at about pH 6.5. NaCl growth concentration ranged from 0 to 2.0% with an optimum at 0.5% (w/v); no growth was observed with the presence of NaCl 3.0% (w/v). The strain produced ethanol, acetate, lactate, H₂, H₂S and CO₂ by glucose fermentation. The DNA G + C content was 34.4 mol%. As determined by 16S rRNA sequence analysis, this organism belonged to the genus Thermoanaerobacterium. On the basis of the physiological and molecular properties, we propose for strain Buff the new species designation Thermoanaerobacterium thermostercus sp. nov. This novel organism represents the first species of the genus Thermoanaerobacterium isolated from buffalo-dung. The type strain is Buff (=DSM 22141 = ATCC BAA-1776).     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.     

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90° C with an optimum around 85° C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H₂S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO₂, NH₃, and H₂S (in the presence of S°) were detected as end products. Growth was not inhibited by H₂. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322) Received: 7 July 1995 / Accepted: 25 August 1995     

Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6°C and a temperature range for growth between 22 and 63°C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain.     

<Emphasis Type="Italic">Larkinella arboricola</Emphasis> sp. nov., a new spiral-shaped bacterium of the phylum <Emphasis Type="Italic">Bacteroidetes</Emphasis> isolated from the microbial community of decomposing wood

An aerobic gram-negative bacterial strain Z-0532 with ring-shaped cells forming spirals in the course of growth was isolated from the humified solution produced by spruce wood decomposition. The new isolate was a chemoorganotrophic, mesophilic, moderately acidophilic organism with the temperature range of 6–32°C (optimum at 25–28°C) and pH range from 4.7 to 7.2 (optimum at pH 5.5–6.5). A broad range of substrates was used as carbon and energy sources, including sugars, some organic acids and polyalcohols, and soluble polymeric compounds (gelatin, esculin, starch, xylan, laminarin, dextrin, casein hydrolysate, and Tween-40). According to its physiological and biochemical characteristics, strain Z-0532 is a typical member of the trophic group of oligotrophic bacteria, which utilize the products of wood hydrolysis dissipated by xylotrophic microorganisms. The G+C base content of strain Z-0532 was 52.1 mol %. Sequencing of the 16S rRNA gene of the new isolate revealed 98% similarity to Larkinella insperata LMG 22510^T, which is a recently described species of the family Spirosomaceae of the phylum Bacteroidetes. The level of DNA: DNA homology between this species and strain Z-0532 was only 40%. The differences in the phenotypic and genotypic characteristics suggested classification of the isolate obtained from decomposing wood as a new species of the genus Larkinella, Larkinella arboricola sp. nov., with the type strain Z-0532^T (=VKM B-2528^T = DSM 21851^T).     

<Emphasis Type="Italic">Candida patagonica</Emphasis> sp. nov., a new species of yeast from cellar surfaces

A novel anamorphic yeast species belonging to the genus Candida has been isolated from cellar surfaces in North Patagonia. Morphological and physiological observation and phylogenetic analysis were performed. Pseudomycelium was plentifully produced. No sexual reproduction was observed. From sequence analysis of the 26S rDNA D1/D2 region, Candida bituminiphila and Zygoascus hellenicus were the closest species with 40 and 79 bp substitutions, respectively. C. bituminiphila differed physiologically from the novel species in its ability to assimilate sucrose and erythritol, in not fermenting any sugars, in growing without some vitamin compounds, and in growing at 40°C. All these data support the hypothesis that the new yeast, named Candida patagonica, is a novel species related to C. bituminiphila. The type strain is UNCOMA 159.5 (= CECT 12029 = CBS 10443).     

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus <Emphasis Type="Italic">Pyrococcus</Emphasis> from an Algerian hydrothermal hot spring

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80–85°C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.     

Desulforhopalus vacuolatus gen. nov., sp. nov., a new moderately psychrophilic sulfate-reducing bacterium with gas vacuoles isolated from a temperate estuary

A new type of gas-vacuolated, sulfate-reducing bacterium was isolated at 10° C from reduced mud (E₀ < 0) obtained from a temperate estuary with thiosulfate and lactate as substrates. The strain was moderately psychrophilic with optimum growth at 18–19° C and a maximum growth temperature of 24° C. Propionate, lactate, and alcohols served as electron donors and carbon sources. The organism grew heterotrophically only with hydrogen as electron donor. Propionate and lactate were incompletely oxidized to acetate; traces of lactate were fermented to propionate, CO₂, and possibly acetate in the presence of sulfate. Pyruvate was utilized both with and without an electron acceptor present. The strain did not contain desulfoviridin. The G+C content was 48.4 mol%. The differences in the 16S rRNA sequence of the isolate compared with that of its closest phylogenetic neighbors, bacteria of the genus Desulfobulbus, support the assignment of the isolate to a new genus. The isolate is described as the type strain of the new species and genus, Desulforhopalus vacuolatus. Received: 4 March 1996 / Accepted: 17 June 1996     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

The biotransformation of acrylonitrile was investigated using thermophilic nitrilase produced from a new isolate Streptomyces sp. MTCC 7546 in both the free and immobilized state. Under optimal conditions, the enzyme converts nitriles to acids without the formation of amides. The whole cells of the isolate were immobilized in agar-agar and the beads so formed were evaluated for 25 cycles at 50°C. The enzyme showed a little loss of activity during reuse. Seventy-one per cent of 0.5 M acrylonitrile was converted to acid at 6 h of incubation at a very low density of immobilized cells, while 100% conversion was observed at 3 h by free cells.     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.     

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.Lei Wang, Yun Tang and Shuo Wang contributed equally to this study.