Isolation and characterization of "Methanosphaera cuniculi" sp. nov

A nonmotile, gram-positive, spherical organism was isolated from the intestinal tracts of rabbits. Both hydrogen and methanol were required for growth. No methane was produced from hydrogen-carbon dioxide, formate, acetate, methylamines, ethanol, or isopropanol. The optimum pH was 6.8, and the optimum temperature was 35 to 40 degrees C. The DNA G+C content is 23 mol%. The pseudomurein cell wall contained serine. These characteristics and the immunological fingerprinting results are consistent with its placement in the genus Methanosphaera as a new species.     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

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.     

Isolation and characterization of Cytophaga flevensis sp. nov., a new agarolytic flexibacterium

The occurrence of agarolytic microorganisms in a variety of natural environments was investigated using enrichment culture techniques. From samples taken from the IJsselmeer, and adjacent areas in the central part of The Netherlands a new agarolytic flexibacterium was isolated. Enrichments inoculated with samples from other sources were unsuccessful. The yellow-pigmented organism exhibits gliding motility, but spreading was only occasionally detected. Since neither fruiting bodies nor microcysts were observed, the organism was placed into the genus Cytophaga and designated as Cytophaga flevensis sp. nov. The occurrence of this species is probably restricted to the IJsselmeer, a fresh-water lake that was in open contact with the North Sea until 1932, and some other lakes which are in direct contact with the IJsselmeer. The organism utilizes only carbohydrates as a carbon and energy source for growth and is strictly aerobic. When the organism is grown on agar, on its components or on melibiose, an extracellular agarase is produced. This enzyme is highly specific for polysaccharides containing neoagarobiose units and hydrolyzes neoagaro-oligosaccharides with a degree of polymerization of more than four.     

Isolation and characterization of Bacillus thioparus sp. nov., chemolithoautotrophic, thiosulfate-oxidizing bacterium

A novel bacterium, strain BMP-1(T), was isolated from a continuous wastewater treatment culture system operating with a bacterial consortium. Cells of the isolate were Gram-variable, aerobic, moderately halotolerant, motile and endospore-forming rods. Strain BMP-1(T) grew chemolithoautotrophically by oxidation of thiosulfate to sulfate with a growth yield of 1.07 g protein mol(-1) of thiosulfate consumed. DNA G+C content was 43.8 mol%. Its cell wall had peptidoglycan based on m-diaminopimelic acid, and the major component of fatty acid was C(15 : 0). The 16S rRNA gene analysis showed that strain belongs to the genus Bacillus, sharing a 99.5% of sequence similarity with Bacillus jeotgali CCM 7133(T). DNA-DNA hybridization between the isolate of this study and this strain was 44%. Thus, the inclusion of strain BMP-1(T) in the genus Bacillus is suggested as a novel species and the name Bacillus thioparus sp. nov. (Type strain BMP-1(T)=BM-B-436(T)=CECT 7196(T)) is proposed. The sequence of the 16S rRNA gene has been deposited in GenBank with accession number DQ371431.     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

Isolation and characterization of two novel alkalitolerant sulfidogens from a Thiopaq bioreactor, Desulfonatronum alkalitolerans sp. nov., and Sulfurospirillum alkalitolerans sp. nov

Two obligately anaerobic sulfidogenic bacterial strains were isolated from the full-scale Thiopaq bioreactor in Lelystad (The Netherlands) removing H₂S from biogas under oxygen-limiting and moderately haloalkaline conditions. Strain HSRB-L represents a dominant culturable sulfate-reducing bacterium in the reactor. It utilizes formate, H₂ (with acetate as C-source) and lactate as e-donors, and sulfate, thiosulfate and sulfite as e-acceptors. It is haloalkalitolerant, with a pH range for lithotrophic growth from 7.5 to 9.7 (optimum at 8.5–9) and a salt range from 0.1 to 1.75 M total Na⁺ (optimum at 0.6 M). The strain is a member of the genus Desulfonatronum and is proposed as a novel species D. alkalitolerans. The second strain, strain HTRB-L1, represents a dominant thiosulfate/sulfur reducer in the reactor. It is an obligate anaerobe utilizing formate and H₂ (with acetate as C-source), lactate, pyruvate and fumarate as e-donors, and thiosulfate (incomplete reduction), sulfur, arsenate and fumarate as e-acceptors. With lactate as e-donor it also grows as an ammonifyer in the presence of nitrate and nitrite. HTRB-L1 is haloalkalitolerant, with a pH range for lithotrophic growth from 7.1 to 9.7 (optimum at 8.5) and a salt range from 0.6 to 1.5 M total Na⁺ (optimum at 0.6 M). Phylogenetic analysis showed that strain HTRB-L1 is a novel species within the genus Sulfurospirillum (Epsilonproteobacteria) for which a name Sulfurospirillum alkalitolerans is proposed.     

Isolation and characterization of Methanoculleus receptaculi sp. nov. from Shengli oil field, China

Three strictly anaerobic, thermophilic methanogens (ZC-2T, ZC-3 and ZC-6) were isolated from Shengli oil field, China. The 16S rRNA gene sequences of the three strains were nearly identical, possessing > 99.8% sequence similarity. They also possessed high sequence similarity, 97.4%, to Methanoculleus palmolei strain INSLUZ(T) (97.4% and 97.5%, respectively), indicating that they represented a novel species within the genus Methanoculleus. Cells of strain ZC-2T were nonmotile cocci, 0.8-1.7 microm in diameter, and always occurred singly or in pairs. The three strains used H2/CO2 or sodium formate as substrates for methanogenesis but not sodium acetate, trimethylamine, monomethylamine, ethanol, dimethyl sulfide, isopropanol, isobutanol, butan-2-ol or H2/CO. Optimum growth of strain ZC-2T occurred in the presence of 0.2 M NaCl, pH 7.5-7.8 and temperature 50-55 degrees C with a specific growth rate of 0.084 h(-1). The mol% G+C content of the genomic DNA was 55.2 mol%. Based on these phenotypic and phylogenetic characteristics, strains ZC-2T, ZC-3 and ZC-6 are proposed to represent a novel species in the genus Methanoculleus and named Methanoculleus receptaculi sp. nov. The type strain is ZC-2T (CGMCC 1.5087T=DSM 18860T).     

Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov.

Abstract Spore-forming sulfate-reducing bacteria (SRB) were enriched selectively from various kinds of aerobic soils with fatty acids as the sole carbon and energy source. A Gram-negative motile rod-shaped bacterium, which produced gas vacuoles during sporulation was isolated. It degraded alcohols, aromatic and n-fatty acids (up to C₁₈) except for propionate, completely to CO₂. Sulfate, sulfite, thiosulfate or elemental sulfur served as electron acceptors. Because of its sensitivity to H₂S, the isolate never produced more than 8 mM dissolved sulfide at pH 7.0. G + C-content of the DNA was 48.0 mol %. The isolated strain Pato is described as a new species Desulfotomaculum sapomandens .     

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir

Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799-2,287 m; temperature, 60 degrees-84 degrees C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%-98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40 degrees and 88 degrees C, with the optimum at 78 degrees C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%-2.0%. The doubling time at optimal growth conditions was about 43 min. The G+C content of the DNA was 38.4 mol%. The DNA-DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.     

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.     

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (≥ 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus. Received: 3 April 1998 / Accepted: 2 July 1998     

Isolation and characterization of a strictly anaerobic,cellulolytic spore former: Clostridium chartatabidum sp. nov.

Cellulolytic, strictly anaerobic spore-forming bacteria were isolated from chloroform treated rumen contents. They were different from previously described cellulolytic rumen clostridia in several characteristics. They formed subterminal rod-shaped spores approximately 0.7 m by 3.5 m. In broth cultures the growth rate was maximal at 39°C and after log growth extensive autolysis occurred. Fermentation products consisted of acetate, butyrate, hydrogen and ethanol. The GC content was 31%.     

浮霉菌门严格厌氧产氢细菌(Thermopirellula anaerolimosa)的分离及其生理特性

[目的]厌氧颗粒污泥中含有大量未知微生物资源,利用低浓度底物及添加抗生素的培养基进行厌氧发酵细菌的筛选,并对分离菌株进行生理生化特性研究.[方法]利用系列稀释法及亨盖特厌氧滚管技术从制糖废水厌氧处理反应器的颗粒污泥中分离到一株高温厌氧产氢细菌VM20-7T,通过16S rRNA基因序列同源性确定其系统发育地位.[结果]菌株VM20-7T为高温、严格厌氧、革兰氏阴性梨形细菌,细胞大小为(0.7-2.0)μm×(0.7-2.0) μm,不运动,不产芽胞.其生长温度范围为35℃-50℃(最适温度45℃),pH范围为6.0-8.3(最适pH7.0-7.5),NaCl耐受范围为0％-0.5％(w/v,最适浓度0％).菌株VM20-7T可利用葡萄糖、麦芽糖、核糖等多种糖类为唯一碳源生长,葡萄糖发酵终产物是乙酸和H2.该菌株不利用硝酸盐、硫酸盐等作为电子受体生长.G+C含量为60.9 mol％,16S rRNA基因序列同源性显示菌株属于浮霉菌门,但与已培养菌株的同源性较低,与梨形菌属一红小梨形菌属-芽殖小小梨形菌属(Pirellula-Rhodopirellul -Blastopirellula,PRB)分支的亲缘关系最近,但序列相似性也仅为82.7％-84.3％.[结论]利用低浓度糖类并添加抗生素分离厌氧颗粒污泥中的微生物,获得了浮霉菌门首例严格厌氧细菌VM20-7T.生理生化特性和系统发育分析显示,菌株VM20-7T为浮霉菌目的新属新种,命名为Thermopirellula anaerolimosa.该菌株的菌种保藏号为CGMCC 1.5169T=JCM 17478T=DSM 24165T.     

Isolation and characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete.

An actinomycete, isolated as a contaminant of a culture medium containing 25% NaCl, has been classified as Actinopolyspora halophila gen. et sp. nov. in the family Nocardiaceae. The morphology and biochemical characteristics of this organism distinguish it from other members of the family Nocardiaceae and other genera possessing a type IV cell wall. It requires high NaCl concentrations for growth and can grow in saturated NaCl. The lowest concentration permitting growth in liquid medium is 12%, and on solid medium, 10%. Colonies developing at lower salt concentrations contain holes resembling viral plaques. No growth occurred in a medium containing 30% KCl instead of NaCl. This organism can grow in simple media with NH4+ salts as nitrogen source and different sugars and other compounds as carbon source. Though it has a salt requirement almost as great as the extremely halophilic rods and cocci, it differs from these in containing diaminopimelic acid and in sensitivity to lysozyme; both properties suggest that it has a mucopeptide cell wall. It also contains some phospholipids common to other actinomycetes, but does not contain any phytanyl ether linked lipids characteristic of other extremely halophilic bacteria.     

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan

In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira‐positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re‐isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome‐to‐genome distances and average nucleotide identity in silico using whole genome sequences and DNA–DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 ^T, = JCM 32515 ^T = CIP111620 ^T), Leptospira ellinghausenii sp. nov., (type strain E18 ^T, = JCM 32516 ^T = CIP111618 ^T) and Leptospira ryugenii sp. nov., (type strain YH101 ^T, = JCM 32518 ^T = CIP111617 ^T) are proposed.     

Isolation and characterization of a novel violacein-like pigment producing psychrotrophic bacterial species Janthinobacterium svalbardensis sp. nov

A bacterial strain designated JA-1, related to Janthinobacterium lividum, was isolated from glacier ice samples from the island Spitsbergen in the Arctic. The strain was tested for phenotypic traits and the most prominent appeared to be the dark red brown to black pigmentation different from the violet pigment of Janthinobacterium, Chromobacterium and Iodobacter. Phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridization tests showed that strain JA-1 belongs to the genus Janthinobacterium but represents a novel lineage distinct from the two known species of this genus, J. lividum and Janthinobacterium agaricidamnosum. The DNA G + C content of strain JA-1 was determined to be 62.3 mol %. The isolate is a psychrotrophic Gram negative bacterium, rod-shaped with rounded ends, containing intracellular inclusions and one polar flagellum. On the basis of the presented results strain JA-1 is proposed as the type strain of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium svalbardensis sp. nov. is proposed (JA-1^T = DSM 25734, ZIM B637).     

Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov

Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.