Continuous enrichment cultures : insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulftitator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO2 and H-2 produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments

Endolithic microbial communities in carbonate precipitates from serpentinite-hosted hyperalkaline springs of the Voltri Massif (Ligurian Alps, Northern Italy)

The Voltri Massif is an ophiolitic complex located in the Ligurian Alps close to the city of Genova (Northern Italy) where several springs discharge high pH (up to 11.7), low salinity waters produced by the active serpentinization of the ultramafic basement. Mixing of these hyperalkaline waters with the river waters along with the uptake of atmospheric carbon dioxide forms brownish carbonate precipitates covering the bedrock at the springs. Diverse archaeal and bacterial communities were detected in these carbonate precipitates using 454 pyrosequencing analyses of 16S ribosomal RNA (rRNA) genes. Archaeal communities were dominated by members of potential methane-producing and/or methane-oxidizing Methanobacteriales and Methanosarcinales (Euryarchaeota) together with ammonia-oxidizing Nitrososphaerales (Thaumarchaeota) similar to those found in other serpentinization-driven submarine and terrestrial ecosystems. Bacterial communities consisted of members of the Proteobacteria, Actinobacteria, Planctomycetes, Bacteroidetes, Chloroflexi, and Verrucomicrobia phyla, altogether accounting for 92.2 % of total retrieved bacterial 16S rRNA gene sequences. Amongst Bacteria, potential chemolithotrophy was mainly associated with Alpha- and Betaproteobacteria classes, including nitrogen-fixing, methane-oxidizing or hydrogen-oxidizing representatives of the genera Azospirillum, Methylosinus, and Hydrogenophaga/'Serpentinomonas', respectively. Besides, potential chemoorganotrophy was attributed mainly to representatives of Actinobacteria and Planctomycetales phyla. The reported 16S rRNA gene data strongly suggested that hydrogen, methane, and nitrogen-based chemolithotrophy can sustain growth of the microbial communities inhabiting the carbonate precipitates in the hyperalkaline springs of the Voltri Massif, similarly to what was previously observed in other serpentinite-hosted ecosystems

Fusibacter fontis sp. nov., a sulfur-reducing, anaerobic bacterium isolated from a mesothermic Tunisian spring

Strain KhalAKB1(T), a mesophilic, anaerobic, rod-shaped bacterium, was isolated from water collected from a mesothermic Tunisian spring. Cells were Gram-staining-Positive rods, occurring singly or in pairs and motile by one lateral flagellum. Strain KhalAKB1(T) grew at 15-45 degrees C (optimum 30 degrees C), at pH 5.5-8.5 (optimum pH 7.0) and in the presence of 0-35 g NaCl l(-1) (optimum 1 g NaCl l(-1)). It fermented yeast extract and a wide range of carbohydrates including cellobiose, D-glucose, D-ribose, sucrose, D-xylose, maltose, D-galactose and starch as electron donors. Acetate, ethanol, CO2 and H-2 were end products of glucose metabolism. It reduced elemental sulfur, but not sulfate, thiosulfate or sulfite, into sulfide. The DNA G+C content was 37.6 mol%. The predominant cellular fatty acids were C-14 : 0 and C-16 : 0. Phylogenetic analysis of the 16S rRNA gene sequence suggested Fusibacter bizertensis as the closest relative of this isolate (identity of 97.2 % to the type strain). Based on phenotypic, phylogenetic and genotypic taxonomic characteristics, strain KhalAKB1(T) is proposed to be assigned to a novel species within the genus Fusibacter, order Clostridiales, Fusibacter fontis sp. nov. The type strain is KhalAKB1(T) (=DSM 28450(T)=JCM 19912(T))

Vallitalea pronyensis sp nova, isolated from a marine alkaline hydrothermal chimney

A novel thermotolerant, anaerobic, Gram-stain-positive, spore-forming bacterium was isolated from a hydrothermal chimney in Prony Bay, New Caledonia. This strain, designated FatNl3(T), grew at 15-55 degrees C (optimum 30 degrees C) and at pH 5.8-8.9 (optimum 7.7). It was slightly halophilic, requiring at least 0.5% NaCl for growth (optimum 2.5-3.0 %), and was able to grow at up to 6% NaCl. Sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and nitrite were not used as terminal electron acceptors. Growth of strain FatNl3(T) was inhibited in the presence of sulfite (2 mM) or nitrite (2 mM). Strain FatNl3(T) fermented cellobiose, glucose, mannose, maltose, sucrose, galactose, lactose, ribose, fructose, rhannnose, raffinose, xylose, yeast extract, peptone and biotrypticase. The main fermentation products from glucose metabolism were acetate, ethanol, H-2 and CO2. The predominant cellular fatty acids were iso-C-15:0 and anteiso-C-15:0. The main polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, and unknown glycolipids and phospholipids. The G+C content of the genomic DNA was 36.6 mol%. On the basis of phylogenetic and physiological properties, strain FatNl3(T) (=DSM 25904=JCM 18391) belonging to the phylum Firmicutes, class Clostridia, order Clostridiales, is proposed as the type strain of a novel species of the genus Vallitalea, for which the name Vallitalea pronyensis sp. nov. is proposed

Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales

Cultivated members of the order Thermotogales comprise only thermophilic to hyperthermophilic anaerobic microorganisms. However, based on molecular studies, the existence of mesophilic members ("mesotoga") within this order has been postulated but has not been demonstrated by cultural approaches so far. A "mesotoga" (strain PhosAc3) that belonged to an uncultivated lineage distantly related to the thermophilic Kosmotoga genus has now been cultivated in axenic culture. It grew between 30 degrees C and 50 degrees C (optimum 40 degrees C) and oxidized lactate using elemental sulphur as a terminal electron acceptor. Further genomic and physiological characterization of strain PhosAc3 will be important not only for understanding bacterial adaptation to high and moderate temperatures at small evolutionary scales, but also because "mesotoga" might play a crucial ecological role in ecosystems polluted by aromatic compounds

Characterization of Alkaliphilus hydrothermalis sp nov., a novel alkaliphilic anaerobic bacterium, isolated from a carbonaceous chimney of the Prony hydrothermal field, New Caledonia

A novel anaerobic, alkaliphilic, Gram-positive staining bacterium was isolated from a hydrothermal chimney in the Prony Bay, New Caledonia. This strain designated FatMR1(T) grew at temperatures from 20 to 55 A degrees C (optimum 37 A degrees C) and at pH between 7.5 and 10.5 (optimum 8.8-9). NaCl is not required for growth (optimum 0.2-0.5 %), but is tolerated up to 3 %. Sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and nitrite are not used as terminal electron acceptors. Strain FatMR1(T) fermented pyruvate, yeast extract, peptone and biotrypcase and used fructose as the only sugar. The main fermentation products from fructose and proteinaceous compounds (e.g. peptone and biotrypcase) were acetate, H-2 and CO2. Crotonate was disproportionated to acetate and butyrate. The predominant cellular fatty acids were C-14:0 and C-16:0. The G + C content of the genomic DNA was 37.1 mol %. On the basis of phylogenetic, genetic, and physiological properties, strain FatMR1(T) (=DSM 25890(T), =JCM 18390(T)) belonging to the phylum Firmicutes, class Clostridia, order Clostridiales, is proposed as a novel species of the genus Alkaliphilus, A. hydrothermalis sp. nov