V. V. Kevbrin

Telmatobacter bradus gen. nov., sp. nov., a cellulolytic facultative anaerobe from subdivision 1 of the Acidobacteria, and emended description of Acidobacterium capsulatum Kishimoto et al. 1991

A gram-negative, facultatively anaerobic, chemo-organotrophic, non-pigmented, slow-growing bacterium was isolated from acidic Sphagnum peat and designated strain TPB6017(T). Cells of this strain were long rods that multiplied by normal cell division and were motile by means of a single flagellum. Cells grew under reduced oxygen tension and under anoxic conditions and were able to ferment sugars and several polysaccharides, including amorphous and crystalline cellulose. Strain TPB6017(T) was a psychrotolerant acidophile capable of growth between pH 3.0 and 7.5 (optimum 4.5-5.0) and at 4-35 °C (optimum 20-28 °C). It was extremely sensitive to salt stress; growth was inhibited at NaCl concentrations above 0.1 % (w/v). The major fatty acids were iso-C(15 : 0) and iso-C(17 : 1)ω9c; the polar lipids were phosphatidylethanolamine and a number of phospholipids and aminophospholipids with an unknown structure. The quinone was MK-8. The DNA G+C content was 57.6 mol%. Comparative 16S rRNA gene sequence analysis revealed that strain TPB6017(T) was a member of subdivision 1 of the phylum Acidobacteria and belonged to a phylogenetic lineage defined by the acidophilic aerobic chemo-organotroph Acidobacterium capsulatum (92.3 % sequence similarity). However, cell morphology, type of flagellation, the absence of pigment, differences in fatty acid and polar lipid composition, possession of a cellulolytic capability, inability to grow under fully oxic conditions and good growth in anoxic conditions distinguished strain TPB6017(T) from A. capsulatum. Therefore, it is proposed that strain TPB6017(T) represents a novel acidobacterium species in a new genus, Telmatobacter bradus gen. nov., sp. nov.; strain TPB6017(T) ( = DSM 23630(T) = VKM B-2570(T)) is the type strain.

Spirochaeta alkalica sp. nov., Spirochaeta africana sp. nov., and Spirochaeta asiatica sp. nov., Alkaliphilic Anaerobes from the Continental Soda Lakes in Central Asia and the East African Rift

During a study of microbial communities in athalassic bodies of water, three new species within the genus Spirochaeta were described. These are alkaliphilic Spirochaeta alkalica sp. nov. Z-7491 (DSM 8900) and halophilic S. africana sp. nov. Z-7692 (DSM 8902) from the soda-depositing Lake Magadi in Central Africa and haloalkaliphilic S. asiatica sp. nov. Z-7591 (DSM 8901) from Lake Khatyn, Central Asia. These mesophilic spirochetes develop at pHs of > 9 as anaerobic saccharolytic dissipotrophs. The DNA base compositions (moles percent G+C) of the strains were as follows: S. alkalica Z-7491, 57.1; S. africana Z-7692, 56.1; and S. asiatica Z-7591, 49.2. The optimum growth parameters (temperature, pH, and NaCl concentration [percent, wt/vol], respectively) were as follows: for S. alkalica Z-7491, 35 degrees C, 9.2, and 5 to 7%; for S. africana Z-7692, 35 degrees C, 9.3, and 5 to 7%; and for S. asiatica Z-7591, 35 degrees C, 8.9, and 3 to 6%. The products of glucose fermentation were acetate, hydrogen, ethanol, and lactate, in different proportions, for S. alkalica and S. africana; for S. asiatica, they were acetate, ethanol, and lactate. S. asiatica is strictly anaerobic, while S. alkalica and S. africana are rather aerotolerant. All three species group within the radiation of the majority of the species of the genus Spirochaeta. Studies of the genes encoding 16S rRNA indicate a possible fanning out of the phylogenetic tree of spirochetes.

Clostridium alkalicellum sp. nov., an Obligately Alkaliphilic Cellulolytic Bacterium from a Soda Lake in the Baikal Region

The first anaerobic alkaliphilic cellulolytic microorganism has been isolated from the Verkhnee Beloe soda lake (Buryatiya, Russia) with pH 10.2 and a salt content of up to 24 g/l. Five strains were characterized. Strain Z-7026 was chosen as the type strain. The cells of the isolate are gram-positive spore-forming rods. A mucous external capsule is produced. The microorganism is obligately alkaliphilic, growing in a pH range of 8.0–10.2, with an optimum at pH 9.0. Sodium ions and, in carbonate-buffered media, sodium chloride are obligately required. The microorganism is slightly halophilic; it grows at 0.017–0.4 M Na+ with an optimum at 0.15–0.3 M Na+. The metabolism is fermentative and strictly anaerobic. Cellulose, cellobiose, and xylan can be used as growth substrates. Plant and algal debris can be fermented. Lactate, ethanol, acetate, hydrogen, and traces of formate are produced during cellulose or cellobiose fermentation. Yeast extract or vitamins are required for anabolic purposes. The microorganism fixes dinitrogen and is nitrogenase-positive. It is tolerant to up to 48 mM Na2S. Growth is not inhibited by kanamycin or neomycin. Chloramphenicol, streptomycin, penicillin, ampicillin, ampiox, bacillin, novobiocin, and bacitracin suppress growth. The DNA G+C content is 29.9 mol %. According to the nucleotide sequence of its 16S rRNA gene, strain Z-7026 is phylogenetically close to the neutrophilic cellulolytic bacteria Clostridium thermocellum (95.5%), C. aldrichii (94.9%), and Acetivibrio cellulolyticus (94.8%). It is proposed as a new species: Clostridium alkalicellum sp. nov.

Tindallia magadii gen. nov., sp. nov.: An Alkaliphilic Anaerobic Ammonifier from Soda Lake Deposits

Abstract. Strain Z-7934, an alkaliphilic, obligately anaerobic, fermentative, asporogenous bacterium with Gram-positive cell wall structure, was isolated from soda deposits in Lake Magadi, Kenya. The organism ferments only a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonia. It is a true alkaliphile, with pH range for growth ranging from 7.5 to 10.5 (optimum pH 8.5), and growth is dependent on the presence of sodium ions. The G+C content of the genomic DNA is 37.6 mol%. 16S rDNA sequence analysis of strain Z-7934 shows that it belongs phylogenetically to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and unique physiological properties, we propose a new genus and new species, Tindallia magadii, for this strain. The type strain is Z-7934T (=DSM 10318).

Analysis of the bacterial community developing in the course of Sphagnum moss decomposition

Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 × 107 cells ml−1. About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Planctomycetes. The population sizes of the Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of the Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of the Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.

Halomonas mongoliensis sp. nov. and Halomonas kenyensis sp. nov., new haloalkaliphilic denitrifiers capable of N2O reduction, isolated from soda lakes

In the course of the search for N2O-utilizing microorganisms, two novel strains of haloalkaliphilic denitrifying bacteria, Z-7009 and AIR-2, were isolated from soda lakes of Mongolia and Kenya. These microorganisms are true alkaliphiles and grow in the pH ranges of 8.0–10.5 and 7.5–10.6, respectively. They are facultative anaerobes with an oxidative type of metabolism, able to utilize a wide range of organic substrates and reduce nitrate, nitrous oxide, and, to a lesser extent, nitrite to gaseous nitrogen. They can oxidize sulfide in the presence of acetate as the carbon source and nitrous oxide (strain Z-7009) or nitrate (strain AIR-2) as the electron acceptor. The strains require Na+ ions. They grow at 0.16–2.2 M Na+ (Z-7009) and 0.04–2.2 M Na+ (AIR-2) in the medium. The G+C contents of the DNA of strains Z-7009 and AIR-2 are 67.9 and 65.5 mol %, respectively. According to the results of 16S rRNA gene sequencing and DNA-DNA hybridization, as well as on the basis of physiological properties, the strains were classified as new species of the genus Halomonas: Halomonas mongoliensis, with the type strain Z-7009T (=DSM 17332, =VKM B2353), and Halomonas kenyensis, with the type strain AIR-2T (=DSM 17331, =VKM B2354).

[Methanocalculus natronophilus sp. nov., a new alkaliphilic hydrogenotrophic methanogenic archaeon from a soda lake, and proposal of the new family Methanocalculaceae].

A mesophilic hydrogenotrophic methanogenic archaeon, strain Z-7105T, was isolated from the bottom sediments of a collector in the vicinity of a soda lake Tanatar II (Altai, Russia). The cells were motile, irregular cocci 0.2–1.2 μm in diameter. The organism was an obligate alkaliphile, growing within a pH range from 8.0 to 10.2, with the optimum at pH 9.0–9.5. It was obligately dependent on carbonates, growing at 0.5 to 1.6 M total carbonates with the optimum at 0.7–0.9 M. Sodium ions were also obligately required at concentrations from 0.9 to 3.3 M Na+ (optimum at 1.4–1.9 M). The organism was halotolerant, but Clions were not required. Hydrogen and formate were used as electron donors. Acetate was required for anabolism. The DNA G+C content was 50.2 mol %. According to the results of its 16S rRNA gene sequence analysis, the isolate belonged to the genus Methanocalculus, being the first known alkaliphilic member of this genus. Its similarity to the neutrophilic and halotolerant Methanocalculus species (M. halotolerans, M. taiwanensis, M. pumilus, and M. chunghsingensis) was 98.2–97.1%, which is within the interspecific range for this genus. The level of DNA-DNA hybridization between strain Z-7105T and the Methanocalculus type species M. halotolerans DSM 14092T was 32%. The genus Methanocalculus, including the new isolate and the previously described species, is distant from other genera of methanogens (<90% 16S rRNA gene similarity). Based on significant phenotypic differences and the results of phylogenetic analysis, including DNA-DNA hybridization, it is proposed to assign strain Z-7105T (=DSM 25006T, =VKM B-2765T) to the new species Methanocalculus natronophilus sp. nov., and to incorporate the genus into the new family Methanocalculaceae fam. nov.

Descriptions of Roseiarcus fermentans gen. nov., sp. nov., a bacteriochlorophyll a-containing fermentative bacterium related phylogenetically to alphaproteobacterial methanotrophs, and of the family Roseiarcaceae fam. nov.

A light-pink-pigmented, microaerophilic bacterium was obtained from a methanotrophic consortium enriched from acidic Sphagnum peat and designated strain Pf56(T). Cells of this bacterium were Gram-negative, non-motile, thick curved rods that contained a vesicular intracytoplasmic membrane system characteristic of some purple non-sulfur alphaproteobacteria. The absorption spectrum of acetone/methanol extracts of cells grown in the light showed maxima at 363, 475, 505, 601 and 770 nm; the peaks at 363 and 770 nm are characteristic of bacteriochlorophyll a. However, in contrast to purple non-sulfur bacteria, strain Pf56(T) was unable to grow phototrophically under anoxic conditions in the light. Best growth occurred on some sugars and organic acids under micro-oxic conditions by means of fermentation. The fermentation products were propionate, acetate and hydrogen. Slow chemo-organotrophic growth was also observed under fully oxic conditions. Light stimulated growth. C1 substrates were not utilized. Strain Pf56(T) grew at pH 4.0-7.0 (optimum pH 5.5-6.5) and at 15-30 °C (optimum 22-28 °C). The major cellular fatty acids were 19 : 0 cyclo ω8c and 18 : 1ω7c; quinones were represented by ubiquinone Q-10. The G+C content of the DNA was 70.0 mol%. Strain Pf56 displays 93.6-94.7 and 92.7-93.7% 16S rRNA gene sequence similarity to members of the families Methylocystaceae and Beijerinckiaceae, respectively, and belongs to a large cluster of environmental sequences retrieved from various wetlands and forest soils in cultivation-independent studies. Phenotypic, genotypic and chemotaxonomic characteristics of strain Pf56(T) suggest that it represents a novel genus and species of bacteriochlorophyll a-containing fermentative bacteria, for which the name Roseiarcus fermentans gen. nov., sp. nov. is proposed. Strain Pf56(T) ( = DSM 24875(T) = VKM B-2876(T)) is the type strain of Roseiarcus fermentans, and is also the first characterized member of a novel family within the class Alphaproteobacteria, Roseiarcaceae fam. nov.

Reduction of synthetic ferrihydrite by a binary anaerobic culture of Anaerobacillus alkalilacustris and Geoalkalibacter ferrihydriticus grown on mannitol at pH 9.5

In the course of an investigation of alkaliphilic iron reduction, metabiotic interactions in a binary culture reducing synthetic ferrihydrite (SF) have been studied. The binary culture contained two anaerobic bacteria: the alkaliphilic organotrophic bacillus Anaerobacillus alkalilacustris, which ferments sugars and sugar alcohols and is incapable of iron reduction, and the dissimilatory iron-reducing bacterium Geoalkalibacter ferrihydriticus, which is able to grow on acetate at the expense of anaerobic respiration. The experiments were performed under conditions of SF excess and deficiency. It was expected that G. ferrihydriticus would oxidize the acetate formed in the course of mannitol fermentation by A. alkalilacustris. The results were different from the expected ones: in the binary culture, fermentation products other than acetate were used for iron reduction; these were primarily formate and ethanol, which led to acetate accumulation rather than consumption. The reduction of SF to magnetite and/or siderite followed the earlier established regularities. The preferential order of donor utilization by G. ferrihydriticus did not conform to the energy yields of the corresponding reactions. Thus, it has been shown that there may be interactions in microbial communities that cannot be predicted from the characteristics of pure cultures. The degradation pathways of organic matter in communities may differ considerably from those observed in pure cultures, even in pure cultures of highly specialized organisms.

Anaerobranca zavarzinii sp. nov., an anaerobic, alkalithermophilic bacterium isolated from Kamchatka thermal fields

A novel obligately anaerobic, alkalithermophilic, chemo-organotrophic bacterium was isolated from a small and very shallow geothermally heated pool at Pushino (Kamchatka, Far East Russia). The bacterium, designated strain JW/VK-KS5Y(T), was a Gram staining negative, Gram type positive rod. The cells were sometimes branched, with a tendency to grow in long chains, and were non-sporulating and non-motile. The shortest observed doubling time was 28 min when the novel strain was grown at 54-60 degrees C in 120 mM sodium carbonate-containing medium at pH(25 degrees C) 8.5-9.0. The novel bacterium grew on yeast extract and soytone as sole carbon and energy sources but could also use fumarate, thiosulfate and sulfur as electron acceptors. The DNA G+C content was 32.5 mol%. Based on phylogenetic, DNA-DNA hybridization and phenotypic data, it was concluded that isolate JW/VK-KS5Y(T) (=VKM B-2436(T)=DSM 18970(T)) represents the type strain of a novel species, Anaerobranca zavarzinii sp. nov.