Jean-Luc Cayol

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.

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.

Stenotrophomonas acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor.

Two of several strictly aerobic, mesophilic bacteria isolated from a lab-scale upflow anaerobic sludge blanket (UASB) reactor treating a petrochemical wastewater, strains AMX 17 and AMX 19T, were subjected to detailed taxonomic study. Cells were gram-negative, motile, non-sporulating, straight to curved rods with a polar flagellum. The isolates exhibited phenotypic traits of members of the genus Stenotrophomonas, including cellular fatty acid composition and the limited range of substrates that could be used. Sugars and many amino acids were utilized. Antibiotic susceptibility and physiological characteristics were determined. The DNA base composition was 66.9 mol% G+C. Phylogenetic analysis revealed that the nearest relatives were Stenotrophomonas maltophilia LMG 11114, Stenotrophomonas nitritireducens DSM 12575T and Pseudomonas pictorum ATCC 23328T (similarity of 98.1-98.8%). Xanthomonas species, S. maltophilia LMG 958T and Stenotrophomonas africana CIP 104854T showed high 16S rRNA sequence similarities (96.4-97.3%). The high similarity found in cellular fatty acid profiles and identical partial 16S rRNA sequences (500 bp) for strains AMX 17 and AMX 19T indicate that they belong to the same species. DNA-DNA hybridizations revealed respectively 26.7, 31, 65.8 and 43.6% homology between isolate AMX 19T and S. africana CIP 104854T, S. maltophilia CIP 60.77T, S. nitritireducens DSM 12575T and P. pictorum ATCC 23328T. These results allow the proposal of strain AMX 19T (= DSM 13117T = ATCC 700916T = CIP 106456T) as representative of a novel species of the genus Stenotrophomonas, with the name Stenotrophomonas acidaminiphila sp. nov.

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)

Sporanaerobacter acetigenes gen. nov., sp. nov., a novel acetogenic, facultatively sulfur- reducing bacterium

A strictly anaerobic, moderately thermophilic, sporulating rod, designated strain Lup 33T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor in Mexico. Strain Lup 33T possessed a few laterally inserted flagella, had a DNA G+C content of 32.2 mol % and grew optimally at pH 7.4 and 40 degrees C. Growth was observed at temperatures of up to 50 degrees C and was inhibited in the presence of 5% NaCl. Strain Lup 33T is heterotrophic and utilized some sugars, peptides and various single amino acids. Gelatin and casein were not used as energy sources. It performed the Stickland reaction and reduced elemental sulfur to sulfide. Acetate was the only fatty acid detected from glucose fermentation, whereas acetate together with isobutyrate and isovalerate were found as end products from peptone fermentation. Phylogenetically, strain Lup 33T branched with members of cluster XII of the order Clostridiales, with Clostridium hastiforme as the closest relative (similarity of 93%). On the basis of the phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed as a novel species of a new genus, Sporanaerobacter acetigenes gen. nov., sp. nov. The type strain is strain Lup 33T (= DSM 13106T = CIP 106730T).

Clostridium thiosulfatireducens sp. nov., a proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from an upflow anaerobic sludge blanket (UASB) reactor

A strictly anaerobic, gram-positive, sporulating rod (0.5-0.6 x 2.0-4.0 microm), designated strain Lup 21T, was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating cheese-factory wastewater. Strain Lup 21T was motile by means of peritrichous flagella, had a G+C content of 31.4 mol% and grew optimally at 37 degrees C, pH 7.4, in the absence of NaCl. It is a heterotrophic micro-organism, utilizing proteinaceous compounds (gelatin, peptides, Casamino acids and various single amino acids) but unable to use any of the carbohydrates tested as a carbon and energy source. It reduced thiosulfate and elemental sulfur to sulfide in the presence of Casamino acids as carbon and energy sources. Acetate, butyrate, isobutyrate, isovalerate, CO2 and sulfide were end products from oxidation of gelatin and Casamino acids in the presence of thiosulfate as an electron acceptor. In the absence of thiosulfate, serine, lysine, methionine and histidine were fermented. On the basis of 16S rRNA similarity, strain Lup 21T was related to members of the low-G+C Clostridiales group, Clostridium subterminale DSM 6970T being the closest relative (with a sequence similarity of 99.4%). DNA-DNA hybridization was 56% with this species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the isolate was designated as a novel species of the genus Clostridium, Clostridium thiosulfatireducens sp. nov. The type strain is strain Lup 21T (= DSM 13105T = CIP 106908T).

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).

Thermodesulfovibrio hydrogeniphilus sp. nov., a new thermophilic sulphate-reducing bacterium isolated from a Tunisian hot spring

A new thermophilic sulphate-reducing bacterium (strain Hbr5T) was enriched and isolated from a terrestrial Tunisian hot spring. It was a non-spore-forming Gram-negative curved or vibrio-shaped bacterium. It appeared singly or in long chains and was actively motile by a polar flagellum. It possessed c-type cytochromes and desulfofuscidin. Growth occurred between 50 and 70 degrees C, with an optimum of 65 degrees C at pH 7.1. In the presence of sulphate as a terminal electron acceptor, this strain readily used H2 but formate only poorly. It could use sulphate, thiosulphate, sulphite or arsenate as electron acceptors. Its DNA G+C content was 36.1 mol%. Based on phenotypic, genomic, and phylogenetic characteristics, strain Hbr5T (=DSM 18151T, =JCM 13991T) is proposed to be assigned to a novel species of genus Thermodesulfovibrio, T. hydrogeniphilus sp. nov.

Molecular clustering of Pycnoporus strains from various geographic origins and isolation of monokaryotic strains for laccase hyperproduction

The production of laccase, an enzyme of industrial interest, was screened among species of the genus Pycnoporus, in particular P. sanguineus. Strains were isolated from various tropical Chinese environments and phylogenetically compared to ones deposited in international collections. Molecular clustering, based on ribosomal ITS1–5.8S-ITS2 genomic sequence analysis, showed that the Chinese strains of P. sanguineus formed an homogeneous phylogenetic group distinguished by its laccase-overproducing character. The dikaryotic strain P. sanguineus G05 was selected for its ability to produce up to 40000 U 1 −1 laccase in the presence of 2,5-xylidine, Tween 80 and maize bran. Since fruit bodies of P. sanguineus could be formed in the laboratory, monokaryotic laccase-hyperproducing strains were isolated using classic genetical methods. Among these isolates, strain G05.10 synthesized up to 71 000 U 1 −1 laccase, with a productivity of 5069 U 1 −1 d −1 . The laccase was purified and identified as a 70 kDa protein with an acidic pI, and was very stable at high temperatures.

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.