Isolation and Characterization of Acetate-Utilizing Anaerobes from a Freshwater Sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5–1 μm × 3–4 μm long). Doubling times with acetate at 30–35°C were 5.6–8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related toMethanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5–0.7 μm × 1.5–3.5 μm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30°C the doubling times with acetate were 3.9–5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H₂, CO₂, and sulfate. The closest relative of strain ASRB-Zg isDesulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5–0.7 μm × 0.7–1 μm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20–25°C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H₂, CO₂, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg isVariovorax paradoxus.     

Methanosarcina mazei Strain O1M9704, Methanogen with Novel Tubule Isolated from Estuarine Environment

A new methanogenic isolate, designated as strain O1M9704 (=OCM 667), was isolated from the sediment of the estuarine environment in Eriln Shi, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. Cells grew optimally at 37°C with 0.5% NaCl in neutral pH. The cells were stained Gram-negative, nonmotile, irregular coccus 0.3–0.6 μm in diameter. A comparison of 16S rDNA sequences phylogenetically related strain O1M9704 to Methanosarcina mazei. Gas vacuoles were observed both under phase contrast microscope and in thin sections in the electron microscope. Negative stain of electron micrographs showed a novel character of strain O1M9704 with tubule structure extended out of the cells. The tubule structure and gas vacuoles may benefit the adaptation of methanoarchaea in estuarine environment. Received: 10 December 1999 / Accepted: 10 January 2000     

Characterization of Microaerobacter geothermalis gen. nov., sp. nov., a novel microaerophilic, nitrate- and nitrite-reducing thermophilic bacterium isolated from a terrestrial hot spring in Tunisia

A novel thermophilic anaerobic and microaerophilic bacterium (optimal growth in the presence of 5–10% O₂), strain Nad S1^T was isolated from the terrestrial hot spring of Hammam Sidi Jdidi, Nabeul, Tunisia. Cells were motile rods having a Gram-positive cell wall structure. Strain Nad S1^T grew optimally at 55°C (range 37–70°C). Optimum pH for growth was 6.5–7.0. It was halotolerant growing with NaCl up to 7% (optimum concentration 1.5–3.0%). It grew chemoorganotrophically on various carbohydrates, organic-acids and amino-acids as energy sources, or chemolithotrophically on H₂ using nitrate, as terminal electron acceptor. Beside oxygen (under microaerobic conditions) and nitrate, nitrite was also used. Nitrate was completely reduced to N₂. No fermentation occurred. The genomic DNA G + C content was 41.8 mol%. Based on 16S rRNA gene sequence analysis, strain Nad S1^T belongs to the Bacillaceae family within the class ‘Bacilli’. Because of its phylogenetic and phenotypic characteristics, we propose this isolate to be assigned as a novel genus and a novel species within the domain Bacteria, Microaerobacter geothermalis gen. nov., sp. nov. The type strain is Nad S1^T (=DSM 22679^T =JCM 16213^T).     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of Halanaerobaculum tunisiense gen. nov., sp. nov., a new halophilic fermentative, strictly anaerobic bacterium isolated from a hypersaline lake in Tunisia

A new halophilic anaerobe was isolated from the hypersaline surface sediments of El-Djerid Chott, Tunisia. The isolate, designated as strain 6SANG, grew at NaCl concentrations ranging from 14 to 30%, with an optimum at 20–22%. Strain 6SANG was a non-spore-forming, non-motile, rod-shaped bacterium, appearing singly, in pairs, or occasionally as long chains (0.7–1 × 4–13 μm) and showed a Gram-negative-like cell wall pattern. It grew optimally at pH values between 7.2 and 7.4, but had a very broad pH range for growth (5.9–8.4). Optimum temperature for growth was 42°C (range 30–50°C). Strain 6SANG required yeast extract for growth on sugars. Glucose, sucrose, galactose, mannose, maltose, cellobiose, pyruvate, and starch were fermented. The end products from glucose fermentation were acetate, butyrate, lactate, H₂, and CO₂. The G + C ratio of the DNA was 34.3 mol%. Strain 6SANG exhibited 16S rRNA gene sequence similarity values of 91–92% with members of the genus Halobacteroides, H. halobius being its closest phylogenetic relative. Based on phenotypic and phylogenetic characteristics, we propose that this bacterium be classified as a novel species of a novel genus, Halanaerobaculum tunisiense gen. nov., sp. nov. The type strain is 6SANG^T (=DSM 19997^T = JCM 15060^T).     

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     

Oleispira lenta sp. nov., a novel marine bacterium isolated from Yellow sea coastal seawater in Qingdao, China

The taxonomic position of strain DFH11^T, which was isolated from coastal seawater off Qingdao, People’s Republic of China in 2007, was determined. Strain DFH11^T comprised Gram-negative, motile, strictly aerobic spirilli that did not produce catalase. Comparative 16S rRNA gene sequence analysis revealed that strain DFH11^T shared ~97.2, 93.3, 91.8, 91.7 and 91.5% sequence similarities with Oleispira antarctica, Spongiispira norvegica, Bermanella marisrubri, Oceaniserpentilla haliotis and Reinekea aestuarii, respectively. DNA–DNA hybridization experiments indicated that the strain was distinct from its closest phylogenetic neighbour, O. antarctica. The strain grew optimally in 2–3% (w/v) NaCl, at pH 5.0–10.0 (optimally at pH 7.0) and between 0 and 30°C (optimum growth temperature 28°C). The strain exhibited a restricted substrate profile, with a preference for aliphatic hydrocarbons, that is consistent with its closest phylogenetic neighbour O. antarctica. Growth of the isolate at different temperatures affected the cellular fatty acid profile. 28°C cultured cells contained C_16:1ω7c and/or iso-C_15:0 2-OH (50.4%) and C_16:0 (19.2%) as the major fatty acids. However, the major fatty acids of the cells cultured at 4°C were C_16:1ω7c and/or C_16:1ω6c (40.2%), C_16:0 (17.2%) and C_17:1ω8c (10.1%). The G+C content of the genomic DNA was 42.7 mol%. Phylogeny based on 16S rRNA gene sequences together with data from DNA–DNA hybridization, phenotypic and chemotaxonomic characterization revealed that DFH11^T should be classified as a novel species of the genus Oleispira, for which the name Oleispira lenta sp. nov. is proposed, with the type strain DFH11^T (=NCIMB 14529^T = LMG 24829^T).     

<Emphasis Type="Italic">Arhodomonas recens</Emphasis> sp. nov., a halophilic alkane-utilizing hydrogen-oxidizing bacterium from the brines of flotation enrichment of potassium minerals

A halophilic nonpigmented rod-shaped (0.8–1.0 × 2.0–2.5 μm), gram-negative bacterium with a single polar flagellum (strain RS91) was isolated from acidic brines of flotation enrichment of potassium minerals (Silvinit Co., Solikamsk, Russia). The strain grew in the media with 2 to 25% NaCl (optimum at 10–12%), 20–45°C (optimum at 37°C), and pH 5.5–8.5 (optimum 6.5–7.5). It was an aerobe or facultative anaerobe incapable of fermentation. The strain was characterized by the absence of growth on glucose, fructose, and citrate, extensive aerobic growth on n-hexadecane and in the mineral medium with H₂ + O₂ + CO₂ in the gas phase, anaerobic nitrate reduction with acetate or hydrogen (under H₂ + CO₂ + N₂), and variable fatty acid composition. The DNA G+C content was 68.2 mol %. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that while strain RS91 was most closely related to Arhodomonas aquaeolei HA-1^T (98.3%) and Nitrococcus mobilis (98.1%), it was only remotely related to the halophilic phototroph Halorhodospira halophila (90.6%). Based on the combination of its phenotypic and genotypic characteristics, the organism was classified as a new species of the genus Arhodomonas, family Ectothiorhodospiraceae with the proposed name Arhodomonas recens sp. nov. The type strain is RS91^T (= IEGM 796^T = VKPM B-11280^T).     

Salinicoccus salitudinis sp. nov., a new moderately halophilic bacterium isolated from a saline soil sample

A novel pale-yellow-pigmented, moderately halophilic, facultatively alkaliphilic, non-motile, non-spore-forming, catalase- and oxidase-positive, obligately aerobic Gram-positive coccus, strain YIM-C678^T was isolated from a saline soil sample collected from a hypersaline habitat in the Qaidam basin, northwest China. The organism grew at 4–37°C and pH 6.0–11.0, with optimum growth at 25°C and pH 8.0. Strain YIM-C678^T grew optimally in the presence of 10–12% (w/v) NaCl and growth was observed in 1–25% (w/v) NaCl. The cell wall murein type was l-Lys-Gly₅. Major cellular fatty acids were anteiso-C_15:0, iso-C_15:0, iso-C_16:0 and C_16:0. Menaquinone 6 (MK-6) was the major respiratory quinone. The DNA G + C content was 46.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM-C678^T belonged to the family Staphylococcaceae and was most closely related to the eight described species of the genus Salinicoccus with sequence similarities from 92.2 (S. luteus YIM 70202^T) to 97.5% (S. kunmingensis YIM Y15^T). The DNA–DNA relatedness between strain YIM-C678^T and S. kunmingensis YIM Y15^T was 35.4%. Chemotaxonomic data and 16S rRNA gene sequence analysis supported the affiliation of strain YIM-C678^T with the genus Salinicoccus. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic differences and DNA–DNA hybridization data supported the view that the bacterium represents a novel species of the genus Salinicoccus, for which the name Salinicoccus salitudinis sp. nov. is proposed, with YIM-C678^T (=DSM 17846 = CGMCC 1.6299) as the type strain.     

Specific characteristics of the strains isolated from a thermoacidophilic microbial community oxidizing antimony sulfide ore

Investigation of the phenotypic properties of three mixotrophic bacteria, strains Sb-K, Sb-F, and Sb-S, isolated from an aboriginal thermoacidophilic microbial community participating in biooxidation of ore with high antimony content (26%) and ore concentrates from the Olympiadinskoe deposit under semicontinuous cultivation conditions at 46 ± 1°C, revealed the differentiating characteristics of these strains. The isolated cultures grew lithotrophically through different numbers of transfers: strains Sb-F and Sb-K grew through seven and eight transfers, respectively, and strain Sb-S grew through two or three transfers. Strains Sb-K and Sb-S utilized a wide range of organic substrates for active organotrophic growth during nine or ten transfers, while strain Sb-F was less tolerant to organic compounds. Strain Sb-K grew on a medium with the ore and sulfide ore concentrates in the pH range of 1.0–3.0. Growth of strains Sb-F and Sb-S occurred in the pH ranges of 1.0–2.5 and 1.5–5.5 on media with Fe²⁺ and S⁰, respectively.. The optimal initial pH values of the media, corresponding to the maximum specific growth rates, were 1.6–1.7, 1.9, and 2.0–3.0 for strains Sb-K, Sb-F, and Sb-S, respectively. All three strains were able to grow within a broad temperature range, 20–65°C, with an optimum at 46°C (Sb-K), 40–46°C (Sb-F), and 48–50°C (Sb-S). According to the results of DNA-DNA hybridization and phylogenetic analysis, as well as their phenotypic characteristics, the isolates can be classified as novel strains of species of the genus Sulfobacillus. Strains Sb-K, Sb-F, and Sb-S, isolated as predominant cultures on the media with sulfide compounds, iron, or sulfur, respectively, were affiliated to the species S. thermotolerans, S. sibiricus, and S. thermosulfidooxidans.     

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 lactic acid bacteria strains with ornithine producing capacity from natural sea salt

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO₂ production. The isolates grew well on MRS broth at 30–37°C and a pH of 6.5–8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0–6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.     

Isolation and Identification of Ruminal Methanogens from Grazing Cattle

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H₂/CO₂ and formate, and optimally at 38°C and pH 6.5–7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H₂/CO₂, acetate, and methyl-containing compounds, with optimal growth at 40°C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management. Received: 26 October 1999 / Accepted: 22 November 1999     

Identification of the dominant bacterium of two-stage biooxidation of gold-arsenic concentrate

In the process of biooxidation at 39°C in a continuous mode of the gold-arsenic concentrate from the Olympiadinskoe deposit, which was pretreated by chemical leaching with ferric ions, by a microbial association from the BIO department reactors of the Polyus gold mining company, a bacterial culture designated as strain HT-4 was isolated. The bacterium was a spore-forming rod 0.5–0.6 × 1.4–2.0 μm with a flagellum. The optimal temperature for growth and Fe²⁺ oxidation was 55°C. The strain grew in the pH range from 1.21 to 2.10 with the optimum at pH 1.6. The organism was incapable of lithotrophic and organotrophic growth. It grew mixotrophically by Fe²⁺ oxidation in the presence of 0.02% yeast extract. The DNA G+C base content was 48.6 mol %. Based on comparative phylogenetic analysis of 1472-bp nucleotide sequences of 16S rRNA genes, strain HT-4 was classified as Sulfobacillus thermosulfidooxidans. Analysis by pulse-field gel electrophoresis revealed a unique profile of the NotI fragments of the chromosomal DNA. These results demonstrate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of concentrates in different technological conditions. The strain “S. olympiadicus S-5” dominated in the process of biooxidation of original concentrate not treated with ferric iron, while S. thermosulfidooxidans HT-4 was predominant in biooxidation of the chemically leached concentrate.     

Thermococcus chitonophagus sp. nov., a novel,chitin-degrading,hyperthermophilic archaeum from a deep-sea hydrothermal vent environment

From a hydrothermal vent site off the Mexican west coast (20°50′N, 109°06′W) at a depth of 2,600 m, a novel, hyperthermophilic, anaerobic archaeum was isolated. Cells were round to slightly irregular cocci, 1.2–2.5 μm in diameter and were motile by means of a tuft of flagella. The new isolate grew between 60 and 93°C (optimum: 85°C), from pH 3.5 to 9 (optimum: pH 6.7), and from 0.8 to 8% NaCl (optimum: 2%). The isolate was an obligate organotroph, using chitin, yeast extract, meat extract, and peptone for growth. Chitin was fermented to H₂, CO₂, NH₃, acetate, and formate. H₂S was formed in the presence of sulfur. The chitinoclastic enzyme system was oxygen-stable, cell-associated, and inducible by chitin. The cell wall was composed of a surface layer of hex- americ protein complexes arranged on a p6 lattice. The core lipids consisted of glycerol diphytanyl diethers and acyclic and cyclic glycerol diphytanyl tetraethers. The G+C content was 46.5 mol%. DNA/DNA hybridization and 16S rRNA sequencing indicated that the new isolate belongs to the genus Thermococcus, representing a new species, Thermococcus chitonophagus. The type strain is isolate GC74, DSM 10152. Received: 8 May 1995 / Accepted: 26 June 1995     

Selective enrichment and characterization of Roseospirillum parvum, gen. nov. and sp. nov., a new purple nonsulfur bacterium with unusual light absorption properties

A new type of phototrophic purple bacterium, strain 930I, was isolated from a microbial mat covering intertidal sandy sediments of Great Sippewissett Salt Marsh (Woods Hole, Mass., USA). The bacterium could only be enriched at a wavelength of 932 (± 10) nm. Cells were vibrioid- to spirilloid-shaped and motile by means of bipolar monotrichous flagellation. The intracytoplasmic membranes were of the lamellar type. Photosynthetic pigments comprised bacteriochlorophyll a and the carotenoids spirilloxanthin and lycopenal. The isolated strain exhibited an unusual, long-wavelength absorption maximum at 911 nm. Sulfide or thiosulfate served as electron donor for anoxygenic phototrophic growth. During growth on sulfide, elemental sulfur globules formed outside the cells. Elemental sulfur could not be further oxidized to sulfate. In the presence of sulfide plus bicarbonate, fructose, acetate, propionate, butyrate, valerate, 2-oxoglutarate, pyruvate, lactate, malate, succinate, fumarate, malonate, casamino acids, yeast extract, L(+)-alanine, and L(+)-glutamate were assimilated. Sulfide, thiosulfate, or elemental sulfur served as a reduced sulfur source for photosynthetic growth. Maximum growth rates were obtained at pH 7.9, 30 °C, 50 μmol quanta m^–2 s^–1 of daylight fluorescent tubes, and a salinity of 1–2% NaCl. The strain grew microaerophilically in the dark at a partial pressure of 1 kPa O₂. The DNA base composition was 71.2 mol% G + C. Sequence comparison of 16S rRNA genes indicated that the isolate is a member of the α-Proteobacteria and is most closely related to Rhodobium orientis at a similarity level of 93.5%. Because of the large phylogenetic distance to known phototrophic species of the α-Proteobacteria and of its unique absorption spectrum, strain 930I is described as a new genus and species, Roseospirillum parvum gen. nov. and sp. nov. Received: 29 December 1998 / Accepted: 17 March 1999     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Description of <Emphasis Type="Italic">Tessaracoccus profundi</Emphasis> sp.nov., a deep-subsurface actinobacterium isolated from a Chesapeake impact crater drill core (940 m depth)

A novel actinobacterium, designated CB31^T, was isolated from a 940 m depth sample of a drilling core obtained from the Chesapeake meteor impact crater. The strain was isolated aerobically on R2A medium agar plates supplemented with NaCl (20 g l⁻¹) and MgCl₂·6H₂O (3 g l⁻¹). The colonies were circular, convex, smooth and orange. Cells were slightly curved, rod-shaped in young cultures and often appeared in pairs. In older cultures cells were coccoid. Cells stained Gram-positive, were non-motile and did not form endospores. The diagnostic diamino acid of the peptidoglycan was ll-diaminopimelic acid. The polar lipids included phosphatidylglycerol, diphosphatidglycerol, four different glycolipids, two further phospholipids and one unidentified lipid. The dominant menaquinone was MK-9(H₄) (70%). The major cellular fatty acid was anteiso C15:0 (83%). The DNA G + C content was 68 mol%. The strain grew anaerobically by reducing nitrate to nitrite or by fermenting glucose. It was catalase positive and oxidase negative. It grew between 10 and 45°C, with an optimum between 35 and 40°C. The pH range for growth was 5.7–9.3, with an optimum at pH 7.5. The closest phylogenetic neighbors based on 16S rRNA gene sequence identity were members of the genus Tessaracoccus (95–96% identity). On the basis of phenotypic and phylogenetic distinctiveness, strain CB31^T is considered to represent a novel species of the genus Tessaracoccus, for which we propose the name Tessaracoccus profundi sp. nov.. It is the first member of this genus that has been isolated from a deep subsurface environment. The type strain is CB31^T (=NCIMB 14440^T = DSM 21240^T).     

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     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.