Bacterial diversity of an acid mine drainage beside the Xichú River (Mexico) accessed by culture-dependent and culture-independent approaches.

Xichú River is a Mexican river located in an environmental preservation area called Sierra Gorda Biosphere Reserve. Around it, there are tons of abandoned mine residues that represent a serious environmental issue. Sediment samples of Xichú River, visibly contaminated by flows of an acid mine drainage, were collected to study their prokaryotic diversity. The study was based on both cultural and non-cultural approaches. The analysis of total 16S rRNA gene by MiSEQ sequencing allowed to identify 182 Operational Taxonomic Units. The community was dominated by Pseudomonadota, Bacteroidota, "Desulfobacterota" and Acidobacteriota (27, 21, 19 and 16%, respectively). Different culture conditions were used focusing on the isolation of anaerobic bacteria, including sulfate-reducing bacteria (SRB) and arsenate-reducing bacteria (ARB). Finally, 16 strains were isolated. Among them, 12 were phylogenetically identified, with two strains being SRB, belonging to the genus Solidesulfovibrio ("Desulfobacterota"), while ten are ARB belonging to the genera Azospira (Pseudomonadota), Peribacillus (Bacillota), Raineyella and Propionicimonas (Actinomycetota). The isolate representative of Raineyella genus probably corresponds to a new species, which, besides arsenate, also reduces nitrate, nitrite, and fumarate.

Desulfobotulus mexicanus sp. nov., a novel sulfate-reducing bacterium isolated from the sediment of an alkaline crater lake in Mexico.

A novel Gram-negative, non-spore-forming, vibrio-shaped, anaerobic, alkaliphilic, sulfate-reducing bacterium, designated strain PAR22NT, was isolated from sediment samples collected at an alkaline crater lake in Guanajuato (Mexico). Strain PAR22NT grew at temperatures between 15 and 37 °C (optimum, 32 °C), at pH between pH 8.3 and 10.1 (optimum, pH 9.0-9.6), and in the presence of NaCl up to 10 %. Pyruvate, 2-methylbutyrate and fatty acids (4-18 carbon atoms) were used as electron donors in the presence of sulfate as a terminal electron acceptor and were incompletely oxidized to acetate and CO2. Besides sulfate, both sulfite and elemental sulfur were also used as terminal electron acceptors and were reduced to sulfide. The predominant fatty acids were summed feature 10 (C18 : 1 ω7c and/or C18 : 1 ω9t and/or C18 : 1 ω12t), C18 : 1 ω9c and C16 : 0. The genome size of strain PAR22NT was 3.8 Mb including 3391 predicted genes. The genomic DNA G+C content was 49.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belongs to the genus Desulfobotulus within the class Deltaproteobacteria. Its closest phylogenetic relatives are Desulfobotulus alkaliphilus (98.4 % similarity) and Desulfobotulus sapovorans (97.9 % similarity). Based on phylogenetic, phenotypic and chemotaxonomic characteristics, we propose that the isolate represents a novel species of the genus Desulfobotulus with the name Desulfobotulus mexicanus sp. nov. The type strain is PAR22NT (=DSM 105758T=JCM 32146T).

Desulfonatronum parangueonense sp. nov., a sulfate-reducing bacterium isolated from sediment of an alkaline crater lake.

Novel Gram-stain-negative, non-spore-forming, vibrio-shaped, anaerobic, alkaliphilic, sulfate-reducing bacteria, designated strains PAR180T and PAR190, were isolated from sediments collected at an alkaline crater lake in Guanajuato (Mexico). Strain PAR180T grew at temperatures between 15 and 40 °C (optimum 35 °C), and at pH between 8.3 and 10.4 (optimum 9). It was halotolerant, growing with up to 8 % (w/v) NaCl. Lactate, formate, pyruvate and ethanol were used as electron donors in the presence of sulfate and were incompletely oxidized to acetate and CO2. The isolate was able to grow with hydrogen and with CO2 as a carbon source. Beside sulfate, sulfite and thiosulfate were used as terminal electron acceptors. The isolate was able to grow by disproportionation of sulfite and thiosulfate, but not elemental sulfur, using acetate as a carbon source. The predominant fatty acids were C16 : 0, C16 : 1ω7c and summed feature 10 (C18 : 1ω7c and/or C18 : 1ω9t and/or C18 : 1ω12t). The DNA G+C content was 56.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belongs to the genus Desulfonatronum, class Deltaproteobacteria. Its closest relative is Desulfonatronum thiosulfatophilum (98.7 % 16S rRNA gene sequence similarity). The DNA-DNA relatedness value between strain PAR180T and the type strain of D. thiosulfatophilum was 37.1±2.5 %. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, the isolates is considered to represent a novel species of the genus Desulfonatronum, for which the name Desulfonatronum parangueonense sp. nov. is proposed. The type strain is PAR180T (=DSM 103602T=JCM 31598T).

Crassaminicella profunda gen. nov., sp. nov., an anaerobic marine bacterium isolated from deep-sea sediments.

A novel, anaerobic, chemo-organotrophic bacterium, designated strain Ra1766H(T), was isolated from sediments of the Guaymas basin (Gulf of California, Mexico) taken from a depth of 2002  m. Cells were thin, motile, Gram-stain-positive, flexible rods forming terminal endospores. Strain Ra1766H(T) grew at temperatures of 25-45 °C (optimum 30 °C), pH 6.7-8.1 (optimum 7.5) and in a salinity of 5-60 g l(-1) NaCl (optimum 30 g l(-1)). It was an obligate heterotrophic bacterium fermenting carbohydrates (glucose and mannose) and organic acids (pyruvate and succinate). Casamino acids and amino acids (glutamate, aspartate and glycine) were also fermented. The main end products from glucose fermentation were acetate, butyrate, ethanol, H2 and CO2. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, nitrate, nitrite and Fe(III) were not used as terminal electron acceptors. The predominant cellular fatty acids were C14  : 0, C16 : 1ω7, C16 : 1ω7 DMA and C16 : 0. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipids. The G+C content of the genomic DNA was 33.7 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Ra1766H(T) was affiliated to cluster XI of the order Clostridiales, phylum Firmicutes. The closest phylogenetic relative of Ra1766H(T) was Geosporobacter subterraneus (94.2% 16S rRNA gene sequence similarity). On the basis of phylogenetic inference and phenotypic properties, strain Ra1766H(T) ( = DSM 27501(T) = JCM 19377(T)) is proposed to be the type strain of a novel species of a novel genus, named Crassaminicella profunda.

Petrotoga halophila sp. nov., a thermophilic, moderately halophilic, fermentative bacterium isolated from an offshore oil well in Congo.

A novel thermophilic, moderately halophilic, rod-shaped bacterium, strain MET-B(T), with a sheath-like outer structure (toga) was isolated from an offshore oil-producing well in Congo, West Africa. Strain MET-B(T) was a Gram-negative bacterium with the ability to reduce elemental sulfur, but not sulfate, thiosulfate or sulfite into sulfide. The optimum growth conditions were 60 degrees C, pH 6.7-7.2 and 4-6 % NaCl. The DNA G+C content was 34.6 mol%. Strain MET-B(T) was phylogenetically related to members of the genus Petrotoga; Petrotoga miotherma, Petrotoga olearia and Petrotoga mexicana were the closest relatives, with type strains exhibiting more than 99 % identity in an analysis of small-subunit rRNA gene sequences. The values for DNA-DNA relatedness between the type strains of these three species and strain MET-B(T) were less than 42 %. As MET-B(T) was found to be genetically and physiologically different from other species of the genus Petrotoga, this strain is proposed as representing a novel species, for which the name Petrotoga halophila sp. nov. is proposed. The type strain is MET-B(T) (=DSM 16923(T)=CCUG 50214(T)).

Tindallia texcoconensis sp. nov., a new haloalkaliphilic bacterium isolated from lake Texcoco, Mexico.

A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300(T)) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300(T) was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35 degrees C with absence of growth above 45 degrees C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300(T) has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA-DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300(T) is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300(T ) = DSM 18041(T) = JCM 13990(T)).

Desulfomicrobium thermophilum sp. nov., a novel thermophilic sulphate-reducing bacterium isolated from a terrestrial hot spring in Colombia.

A moderately thermophilic, sulphate-reducing bacterium, designated strain P6-2(T), was isolated from a terrestrial hot spring located at a height of 2,500 m in the Andean region, Colombia (5 degrees 43'69''N, 73 degrees 6'10''W). Cells of strain P6-2(T) were rod-shaped, stained Gram-negative and were motile by means of a single polar flagellum. The strain grew lithotrophically with H(2) as the electron donor and organotrophically on lactate, pyruvate, ethanol, malate, fumarate, n-propanol and succinate in the presence of sulphate as the terminal electron acceptor. Fumarate and pyruvate was fermented. Strain P6-2(T) grew optimally at 55 degrees C (range 37-60 degrees C), pH 6.6 (range 5.8-8.8) in the presence of 0.5% NaCl (range 0-4.5%) with lactate and sulphate and produced acetate, CO(2) and H(2)S as the major end-products. Sulphate, sulphite and thiosulphate could be used as electron acceptors but not elemental sulphur or nitrate. The G + C content of the genomic DNA was 58.7 mol%. The 16S rRNA sequence analysis indicated that strain P6-2(T) was a member of the class Deltaproteobacteria, domain Bacteria with Desulfomicrobium baculatum being the closest relative (similarity value of 94%). Phylogeny of genes encoding alpha- and beta-subunits of the dissimilatory sulphite reductase (dsrAB genes) supported its affiliation to members of the genus Desulfomicrobium. On the basis of this evidence, we propose to assign strain P6-2(T) as new species of the genus Desulfomicrobium, D. thermophilum sp. nov., with strain P6-2(T) as the type strain (= DSM 16697(T) = CCUG 49732(T)).

Reclassification of the sulfate- and nitrate-reducing bacterium Desulfovibrio vulgaris subsp. oxamicus as Desulfovibrio oxamicus sp. nov., comb. nov.

Desulfovibrio vulgaris subsp. oxamicus (type strain, DSM 1925(T)) was found to use nitrate as a terminal electron acceptor, the latter being reduced to ammonium. Phylogenetic studies indicated that strain DSM 1925(T) was distantly related to the type strain of Desulfovibrio vulgaris (95.4 % similarity of the small-subunit rRNA gene) and had as its closest phylogenetic relatives two other nitrate- and sulfate-reducing bacteria, namely Desulfovibrio termitidis (99.4 % similarity) and Desulfovibrio longreachensis (98.4 % similarity). Additional experiments were conducted to characterize better strain DSM 1925(T). This strain incompletely oxidized lactate and ethanol to acetate. It also oxidized butanol, pyruvate and citrate, but not glucose, fructose, acetate, propionate, butyrate, methanol, glycerol or peptone. The optimum temperature for growth was 37 degrees C (range 16-50 degrees C) and the optimum NaCl concentration for growth was 0.1 % (range 0-5 %). Because of significant genotypic and phenotypic differences from Desulfovibrio termitidis and Desulfovibrio longreachensis, reclassification of Desulfovibrio vulgaris subsp. oxamicus as Desulfovibrio oxamicus sp. nov., comb. nov., is proposed. The type strain is strain Monticello 2(T) (=DSM 1925(T)=NCIMB 9442(T)=ATCC 33405(T)).

Purification and preliminary characterization of tetraheme cytochrome c3 and adenylylsulfate reductase from the peptidolytic sulfate-reducing bacterium Desulfovibrio aminophilus DSM 12254.

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).

Petrotoga mexicana sp. nov., a novel thermophilic, anaerobic and xylanolytic bacterium isolated from an oil-producing well in the Gulf of Mexico.

A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O. The DNA G+C content was 36.1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8%. However DNA-DNA relatedness values between these species and strain MET12T were lower than 70%. As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.

Garciella nitratireducens gen. nov., sp. nov., an anaerobic, thermophilic, nitrate- and thiosulfate-reducing bacterium isolated from an oilfield separator in the Gulf of Mexico.

A novel Gram-positive, anaerobic and thermophilic bacterium, strain MET79(T), was isolated from an oil well located in the Gulf of Mexico. Cells were straight rods, motile by a subpolar flagellum. Spores were formed in old cultures. Inner gas vacuoles swelled the cells when exposed to air. The optimum growth conditions were 55 degrees C, pH 7.5 and 1 % NaCl. Yeast extract was required for growth. Strain MET79(T) fermented several sugars, some organic acids and Casamino acids. Glucose was fermented into lactate, acetate, butyrate, H(2) and CO(2). Strain MET79(T) reduced thiosulfate to hydrogen sulfide and nitrate to ammonium. The DNA G+C content was 30.9 mol%. The closest phylogenetic relative of strain MET79(T) was Caloranaerobacter azorensis (88.7 % 16S rDNA sequence similarity). As strain MET79(T) (=DSM 15102(T)=CIP 107615(T)) was physiologically and phylogenetically different from its closest relatives, it is assigned as the type strain of a novel species of a new genus, Garciella nitratireducens gen. nov., sp. nov.

Desulfovibrio capillatus sp. nov., a novel sulfate-reducing bacterium isolated from an oil field separator located in the Gulf of Mexico.

A new spirilloid sulfate-reducing bacterium designated strain MET2(T) (T=type strain), was isolated from a Mexican oil field separator. Electron microscopy revealed a Gram-negative cell wall consisting of a 150nm thick undulating outer membrane. Strain MET2(T) appeared singly or in long chains and was actively motile with a corkscrew-like motion. The isolate grew optimally at 40 degrees C, pH 7.4 and 3% NaCl in a medium containing lactate, thiosulfate and yeast extract. Sulfate, sulfite, thiosulfate, and elemental sulfur served as electron acceptors but not nitrate or fumarate. Lactate, pyruvate and H(2) (with acetate as carbon source) were used as electron donors. Pyruvate was fermented. Desulfoviridin and cyt c were present. The G+C content of the DNA was 58.7mol%. Phylogenetic analysis based on 16S rDNA sequencing showed that strain MET2(T) was a member of the genus Desulfovibrio with "D. gracilis" and D. longus being its closest relatives (similarities of 98.3% and 97.1%, respectively). However, DNA-DNA hybridization studies indicated poor homologies (values <70%) with both species. On the basis of genotypic, phenotypic, and phylogenetic characteristics, strain MET2(T) (=DSM14982(T)=CIP107483(T)) is proposed as the type strain of a new species, Desulfovibrio capillatus sp. nov. GenBank accession number for the 16S rDNA sequence for MET2(T) is AY176773.

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).