Halanaerobacter jeridensis sp. nov., isolated from a hypersaline lake.

An obligatory anaerobic, moderately halophilic bacterium, designated strain CEJFG43(T), was isolated from a sample of sediment collected below the salt crust on the hypersaline El Jerid lake, in southern Tunisia. The cells of this novel strain were Gram-staining-negative, non-sporulating, motile, short rods. They grew in media with 6-30% (w/v) NaCl (optimum 15%), at 20-60 °C (optimum 45 °C) and at pH 5.5-9.5 (optimum pH 8.3). The micro-organism fermented glucose, fructose, ribose, raffinose, galactose, mannose, sucrose, maltose, xylose, mannitol, pyruvate and glycerol. The products of glucose fermentation were lactate, ethanol, acetate, H(2) and CO(2). The genomic G+C DNA content of strain CEJFG43(T) was 33.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFG43(T) belonged in the genus Halanaerobacter and was most closely related to Halanaerobacter lacunarum DSM 6640(T) (95.3% gene sequence similarity) and Halanaerobacter chitinivorans DSM 9569(T) (95.3%). The predominant cellular fatty acids were non-branched (C(16:0) and C(16:1)). Based on the phylogenetic and phenotypic evidence, strain CEJFG43(T) represents a novel species in the genus Halanaerobacter for which the name Halanaerobacter jeridensis sp. nov. is proposed. The type strain is CEJFG43(T) ( = DSM 23230(T) = JCM 16696(T)).

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir : New taxa: Firmicutes (Class Clostridia, Order Halanaerobiales, Halobacteroidaceae, Halobacteroides).

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).

Biological control of grey mould in strawberry fruits by halophilic bacteria.

AIMS: Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti-Botrytis metabolites and volatiles. METHODS AND RESULTS: Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea. Thirty strains (20.2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20 degrees C, from 50% to 91.66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui, B. subtilis, B. pumilus, B. licheniformis, Terribacillus halophilus, Halomonas elongata, Planococcus rifietoensis, Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites. CONCLUSIONS: Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.

Propionibacterium microaerophilum sp. nov., a microaerophilic bacterium isolated from olive mill wastewater.

A new gram-positive, facultative anaerobic, microaerophilic bacterium, designated strain M5T, was isolated from a decantation reservoir of olive mill wastewater. The cells were rod-shaped, non-motile, non-spore-forming and catalase-negative. Growth occurred at pH ranging from 4.5 to 9.5, with optimum growth at 7.0. The optimum temperature for growth was around 30 degrees C. Although growth occurred under anaerobic and aerobic conditions, the optimum O2 concentration for growth was determined as 5% in the gas phase of the culture. During anaerobic growth, glucose or lactate were mainly fermented to propionate, acetate and CO2. In the presence of O2 (more than 2%), glucose was oxidized completely to CO2. The G+C content of the DNA was 67.7+/-0.6 mol% and 16S rRNA gene analysis revealed that the new isolate belonged to the cluster of 'dairy' propionibacteria, Propionibacterium acidipropionici being its closest phylogenic relative (97.5% similarity). However, the level of DNA relatedness between strain M5T and P. acidipropionici was 56.2%. Consequently, both the phenotypic (range of substrates used) and genotypic characteristics of strain M5T allow it to be assigned as a new species of the genus Propionibacterium, Propionibacterium microaerophilum sp. nov. The type strain is strain M5T (= CNCM I-2360T = DSM 13435T).

Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern.

A new thermophilic, strictly halophilic, anaerobic, non-sporulating rod-shaped bacterium, measuring 0.5 x 3.0-8.0 microns and designated strain CTT3T, was isolated from a solar saltern. Strain CTT3T stained Gram-negative, was motile by means of laterally inserted flagella, had a genome G + C content of 33 mol% and grew optimally at 65 degrees C and pH 7.0 with 5% NaCl. The strain also grew readily at 70 degrees C in the presence of 15% NaCl. Strain CTT3T fermented cellobiose, fructose, glucose, maltose, mannitol, mannose, sucrose, glycerol, N-acetylglucosamine, starch, pyruvate and bio-Trypticase. It produced acetate, ethanol, H2 and presumably CO2 from glucose. 16S rRNA gene sequence analysis indicated that it is a member of cluster XII of the Clostridiales and related genera of the subphylum of the Gram-positive bacteria containing genomes of low G + C content. Its phenotypic and phylogenetic characteristics clearly differentiated it from all other members of this cluster. Based on the findings it is proposed that strain CTT3T be designated as a new species of a new genus, Thermohalobacter berrensis gen. nov., sp. nov. The type strain is CTT3T (= CNCM 105955T).

Salinivibrio costicola subsp. vallismortis subsp. nov., a halotolerant facultative anaerobe from Death Valley, and emended description of Salinivibrio costicola.

Strain DVT, a halotolerant, Gram-negative, facultatively anaerobic bacterium, was isolated from a hypersaline pond located in Death Valley, California. The cells were non-spore-forming, motile, curved rods (1.0-1.8 x 0.5-0.6 microns) and occurred singly, in pairs or rarely in chains. Strain DVT was oxidase-, catalase-, Voges-Proskauer-, amylase-, gelatinase- and lipase-positive and indole-negative. Nitrate, sulfate and fumarate were not used as electron acceptors. Carbohydrates served as energy sources both aerobically and anaerobically. Strain DVT grew optimally at 37 degrees C (temperature range 20-50 degrees C) with 2.5% NaCl (NaCl range 0-12.5%) and pH 7.3 (pH range of 5.5-8.5) in a glucose/yeast extract medium with a doubling time of 20 min (aerobically) or 41 min (anaerobically). The end products of glucose fermentation were ethanol, isobutyrate, propionate, lactate, formate and CO2. Strain DVT was resistant to penicillin, D-cycloserine, streptomycin and tetracycline (200 micrograms ml-1). The G + C content was 50 mol%. 16S rRNA gene sequence analysis indicated that it was closely related to Salinivibrio costicola (97.7%) and this was confirmed by DNA-DNA hybridization (93% relatedness). However, phenotypic characteristics such as halotolerance, gas production, growth at 50 degrees C, antibiotic resistance, sugar-utilization spectrum and phylogenetic signatures are sufficiently different from Salinivibrio costicola to warrant designating strain DVT as a new subspecies of Salinivibrio costicola, Salinivibrio costicola subsp. vallismortis subsp. nov. (= DSM 8285T).

Isolation and characterization of Desulfovibrio burkinensis sp. nov. from an African ricefield, and phylogeny of Desulfovibrio alcoholivorans.

A sulfate-reducing bacterium, strain HDvT (T = type strain), was isolated from an anoxic ricefield soil. Cells were Gram-negative, non-sporulating curved rods motile by means of a single polar flagellum. Cytochrome c3 and desulfoviridin were present. In the presence of sulfate, glycerol, 1,2- and 1,3-propanediol, dihydroxyacetone, pyruvate, lactate, fumarate, maleate, malate and succinate were incompletely oxidized mainly to acetate. Sulfite, thiosulfate, elemental sulfur, fumarate, maleate and malate were utilized as alternative electron acceptors. In the absence of added electron acceptors, pyruvate, fumarate, maleate, malate and dihydroxyacetone were fermented. The DNA base composition was 67 mol% G + C. The phylogenetic, phenotypic and physiological characteristics of strain HDvT indicate that it is a new species of the genus Desulfovibrio, for which the name Desulfovibrio burkinensis sp. nov. is proposed; the type strain is HDvT (= DSM 6830T). Phylogenetic analysis confirmed that Desulfovibrio alcoholivorans was a distinct species supporting the previously published phenotypic data.

Methanocalculus halotolerans gen. nov., sp. nov., isolated from an oil-producing well.

Two irregular coccoid methanogens designated SEBR 4845T and FR1T were isolated from an oilfield in Alsace, France. Strain SEBR 4845T (T = type strain) is a hydrogenotrophic halotolerant methanogen, which grows optimally at 5% NaCI (w/v) and tolerates up to 12% NaCI. It does not use methylated compounds and therefore cannot be ascribed to any of the known genera of the halophilic methylotrophic methanogens. It differs from hydrogenotrophic members of the orders Methanococcales and Methanomicrobia les in the NaCI growth range (0-12% NaCI), which is the widest reported to data for any hydrogenotrophic methanogen. 16S rRNA gene sequence analysis indicated that strain SEBR 4845T is a novel isolate for which a new genus is proposed, Methanocalculus halotolerans gen. nov., sp. nov. (= OCM470T) that might be indigenous to the oilfield ecosystem. Strain FR1T (=OCM 471) is a moderately halophilic methanogen which growths optimally at 10% NaCI and tolerates up to 20% NaCI. It grows on trimethylamine and methanol as carbon and energy sources. The G+C content of its DNA is 43 mol%. It is therefore phenotypically and genotypically related to members of the genus Methanohalophilus. This report provides evidence that methylotrophic and hydrogenotrophic, but not aceticlastic methanogens are present in a saline subsurface oilfield environment, as already observed in surface saline to hypersaline environments.

Methanoplanus petrolearius sp. nov., a novel methanogenic bacterium from an oil-producing well.

A disc-shaped methanogenic bacterium designated strain SEBR 4847T (T = type strain) was isolated from a sample collected from an African offshore oil field. Strain SEBR 4847T was non-motile, had a G + C content of 50 mol% and produced methane from H2 + CO2, formate, and CO2 + propanol. Strain SEBR 4847T grew optimally at 37 degrees C; no growth was observed at 25 degrees C or 45 degrees C. It grew in the presence of up to 50 g/l NaCl; 10-30 g/l was required for optimal growth. The optimum pH for growth was 7.0. Doubling time was about 10 h under optimal conditions. Based on 16S rRNA sequence analysis, the isolate was identified as a new species of the genus Methanoplanus and designated Methanoplanus petrolearius sp. nov. The type strain is SEBR 4847T (= OCM 486).

Effect of thiosulphate as electron acceptor on glucose and xylose oxidation by Thermoanaerobacter finnii and a Thermoanaerobacter sp. isolated from oil field water.

During glucose and xylose fermentation, Thermoanaerobacter finnii was observed to produce lactate, acetate, H2 and CO2, with ethanol being the major end product. Thermoanaerobacter strain SEBR 5268, an isolate from an oil field, also produced a similar range of end products from glucose and xylose fermentation, with the exception that both ethanol and lactate were the major products of sugar metabolism. Both these strains were able to reduce thiosulphate to sulphide in the presence of these two substrates, with acetate being the dominant metabolite in that case. In addition, a faster growth rate and increased cell yield were obtained in the presence of thiosulphate, than in its absence. The higher concentrations of acetate produced in the presence of thiosulphate rather than without any electron acceptor indicated that more ATP was generated from substrate-level phosphorylation. These results have implications for our understanding of the breakdown of carbohydrates present in organic matter found in the natural ecological niches of Thermoanaerobacter species (sulphide-, elemental sulphur- or sulphate-rich thermal hot springs and oil fields).

Description of Thermoanaerobacter brockii subsp. lactiethylicus subsp. nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp. finnii comb. nov., and an emended description of Thermoanaerobacter brockii.

A strictly anaerobic, thermophilic, gram-positive, spore-forming cubacterium designated strain SERB 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92 degrees C. The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 microns) with peritrichous flagella. The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomporphic and filamentous (length, 15 microns) in old cultures. Growth occurred at temperatures of 40 to 75 degrees C, and optimum growth occurred at temperatures between 55 and 60 degrees C. The fermentable substrates included glucose, fructose, galactose, mannose, cellobiose, maltose, sucrose, lactose, D-xylose, D-ribose, mannitol, pyruvate, and starch. The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2. The DNA base composition was 35 mol% G+C. The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99%. The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of > 70% were obtained, these organisms belong to the same species. Therefore, we propose that T. finnii should be reclassified as a subspecies of T. brockii, Thermoanaerobacter brockii subsp. finnii comb. nov. This automatically creates Thermoanaerobacter brockii subsp. brockii. We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T. brockii, Thermoanaerobacter brockii subsp. lactiehylicus.(ABSTRACT TRUNCATED AT 250 WORDS)

Haloanaerobium lacusroseus sp. nov., an extremely halophilic fermentative bacterium from the sediments of a hypersaline lake.

A new extremely halophilic chemoorganotrophic bacterium (strain H200T [T = type strain]) was isolated from the hypersaline sediments of Retba Lake in Senegal. This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grew optimally at 40 degrees C in the presence of 180 to 200 g of NaCl per liter. The DNA base composition was 32 mol% guanine plus cytosine. The fermentation products from glucose were ethanol, acetate, H2, and CO2. Yeast extract was required for growth. The fermentable substrates included D-fructose, galactose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, D-mannitol, glycerol, and Casamino Acids. On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species. The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species. We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus.

Haloanaerobium alcaliphilum sp. nov., an anaerobic moderate halophile from the sediments of Great Salt Lake, Utah.

A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST (T = type strain). Strain GSLST grew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0). The optimum temperature for growth was 37 degrees C, and no growth occurred at 15 or 55 degrees C. The optimum salt concentration for growth was 10%. Strain GSLST required yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine. Strain GSLST was resistant to penicillin, D-cycloserine, tetracycline, and streptomycin. The G + C content of this isolate was 31 mol%. The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLST fermented glycine betaine to acetate and trimethylamine. All of these traits distinguish this organism from all previously described halophilic anaerobes. The 16S rRNA gene sequence of strain GSLST was found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens. Therefore, strain GSLST (= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum.

Isolation and characterization of Halothermothrix orenii gen. nov., sp. nov., a halophilic, thermophilic, fermentative, strictly anaerobic bacterium.

The occurrence of thermophilic, halophilic anaerobic bacteria in the sediment of a Tunisian salted lake was tested in samples collected at 20-cm intervals down to a depth of 1.20 m. A long rod, present only in the 40- to 60-cm layer, was isolated at 60 degrees C in a medium containing 100 g of NaCl per liter and designated strain H168. This strain produced acetate, ethanol, H2, and CO2 from glucose metabolism. Fructose, xylose, ribose, cellobiose, and starch were also oxidized. The optimum temperature for growth was 60 degrees C. No growth was obtained at 42 or 70 degrees C. Strain H168 grew optimally in NaCl concentrations ranging from 50 to 100 g per liter, with the upper and lower limits of growth around 200 and 40 g per liter, respectively. The G+C ratio of the DNA was 39.6 mol%. Although halophilic, moderately thermophilic bacteria have been characterized among anaerobes, particularly within methanogens, strain H168 is the first true thermophilic (growing above 60 degrees C) halophilic anaerobic bacterium described so far. The phylogeny, physiology, morphology, lipid content, and high G+C content of strain H168 are sufficiently different from those of genera belonging to the family Haloanaerobiaceae to justify the definition of a new genus.