Harvesting electricity with Geobacter bremensis isolated from compost.

Electrochemically active (EA) biofilms were formed on metallic dimensionally stable anode-type electrode (DSA), embedded in garden compost and polarized at +0.50 V/SCE. Analysis of 16S rRNA gene libraries revealed that biofilms were heavily enriched in Deltaproteobacteria in comparison to control biofilms formed on non-polarized electrodes, which were preferentially composed of Gammaproteobacteria and Firmicutes. Among Deltaproteobacteria, sequences affiliated with Pelobacter and Geobacter genera were identified. A bacterial consortium was cultivated, in which 25 isolates were identified as Geobacter bremensis. Pure cultures of 4 different G. bremensis isolates gave higher current densities (1400 mA/m(2) on DSA, 2490 mA/m(2) on graphite) than the original multi-species biofilms (in average 300 mA/m(2) on DSA) and the G. bremensis DSM type strain (100-300 A/m(2) on DSA; 2485 mA/m(2) on graphite). FISH analysis confirmed that G. bremensis represented a minor fraction in the original EA biofilm, in which species related to Pelobacter genus were predominant. The Pelobacter type strain did not show EA capacity, which can explain the lower performance of the multi-species biofilms. These results stressed the great interest of extracting and culturing pure EA strains from wild EA biofilms to improve the current density provided by microbial anodes.

Bacterial diversity of the cultivable fraction of a marine electroactive biofilm.

Stainless steel electrodes were cathodically polarized at -200 mV versus an Ag/AgCl reference electrode in natural seawater in order to produce current. The current increased and stabilized at 0.5 A/m(2) in less than 10 days. The cultivable fraction of the microbial biofilm population formed on the surface of the current harvesting cathodes was examined by culture dependent techniques. Three hundred fifty six isolates were obtained. They were primarily characterized by whole cell fatty acid methyl ester analysis followed by 16S rRNA gene sequencing. The results showed that the isolates represented different phylogenetic groups including members of the Alpha- and Gammaproteobacteria, the phylum Firmicutes, the family Flavobacteriacae and the phylum Actinobacteria. Denaturing gradient gel electrophoresis demonstrated that the microbial population of the biofilm formed on the cathode and of the surrounding seawater comprised the same dominant members. This study demonstrated that the cultivable microbial fraction of a marine electroactive biofilm is phylogenetically highly diverse.

Nautella italica gen. nov., sp. nov., isolated from a marine electroactive biofilm.

Five isolates obtained from a marine electroactive biofilm grown on a stainless steel cathode were investigated by using a polyphasic taxonomic approach. Analyses of whole-cell fatty acid methyl esters and 16S rRNA gene sequences showed that the isolates belonged to the Roseobacter lineage of the class Alphaproteobacteria. Both phenotypic and genotypic analyses demonstrated that the five new isolates constituted a single species that did not represent a recognized member of the Roseobacter lineage. Therefore the five isolates represent a novel genus and species, for which the name Nautella italica gen. nov., sp. nov. is proposed. The type strain is LMG 24365(T) (=CCUG 55857(T)). The DNA G+C content of the type strain is 61 mol%.

WITHDRAWN: Bacterial diversity of the cultivable fraction of a marine electroactive biofilm.

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Ruegeria scottomollicae sp. nov., isolated from a marine electroactive biofilm.

Seventy isolates were obtained from a marine electroactive biofilm that was generated on a cathodically polarized stainless steel electrode (Genoa, Italy). The genetic diversity was investigated by means of BOX-PCR fingerprinting and two clusters of isolates with similar BOX-PCR profiles were delineated. Whole-cell fatty acid methyl ester analysis and 16S rRNA gene sequence analysis showed that the isolates belonged to the Roseobacter lineage of the class Alphaproteobacteria. DNA-DNA hybridization experiments and a biochemical analysis demonstrated that four isolates belonged to the species Ruegeria mobilis. However, 66 isolates from the second BOX-PCR cluster constituted a novel species within the genus Ruegeria, for which the name Ruegeria scottomollicae sp. nov. is proposed. The DNA G+C content was 61.0+/-0.4 %. The type strain is LMG 24367(T) (=CCUG 55858(T)).

Alteromonas genovensis sp. nov., isolated from a marine electroactive biofilm and emended description of Alteromonas macleodii Baumann et al. 1972 (approved lists 1980).

Thirty-five isolates obtained from a marine electroactive biofilm grown on a stainless steel cathode (Genoa, Italy) were investigated by a polyphasic taxonomic approach. Whole-cell fatty acid methyl ester analysis indicated that the isolates belonged to the class Gammaproteobacteria and were closely related or belonging to the genus Alteromonas. Genomic fingerprinting using the BOX-PCR primer delineated five clusters of isolates with similar BOX-PCR fingerprints. This study demonstrated that isolates from four of the BOX-PCR clusters belonged to Alteromonas macleodii and that the 14 isolates representing BOX-PCR cluster 1 constituted a novel species, which shared 98.4 % 16S rRNA gene sequence similarity with its nearest phylogenetic neighbour, Alteromonas hispanica. Both phenotypic and genotypic analyses enabled this novel species, for which the name Alteromonas genovensis sp. nov. is proposed, to be differentiated from established species of the genus Alteromonas. The DNA G+C content of Alteromonas genovensis sp. nov. is 44.5 mol% and the type strain is LMG 24078(T) (=CCUG 55340(T)).

Microbial diversity in production waters of a low-temperature biodegraded oil reservoir.

Microbial communities in two production waters of a low-temperature and low-salinity petroleum reservoir in Canada were examined using cultural and molecular approaches. The predominant cultivated microorganisms were homoacetogens but sulfate-reducers, acetoclastic methanogens and denitrifiers also gave significant counts. The dominant members of the culturable population were affiliated with the Firmicutes, the "Deltaproteobacteria", the "Epsilonproteobacteria", the Spirochaetes and the Euryarchaeota. 16S rRNA gene clone libraries were also constructed from the total DNA collected from production waters. The bacterial library was entirely composed by a single phylotype related to Arcobacter. The archaeal phylotypes were generally very closely related to members of the orders Methanosarcinales and Methanomicrobiales. Consistent with earlier observations, our data suggest that methanogenesis is a dominant terminal process in the reservoir. Moreover, the cross-evaluation of culture-dependent and -independent techniques also indicates that, contrary to most studies, both acetoclastic and lithotrophic methanogens may be involved in this process. This first investigation of the microbial diversity in a non water-flooded low-temperature and low-salinity petroleum reservoir expands substantially our knowledge of the extent of microbial diversity and highlights the complexity of microbial communities involved in the oil field food chain.

Highly divergent genes for methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection.

The origins and the evolutionary history of tetrahydromethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.

Analysis of fae and fhcD genes in Mono Lake, California.

Genes for two enzymes of the tetrahydromethanopterin-linked C(1) transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).

Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake.

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C1 utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the 13C-labeled C1 substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae, was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae, allowed detection of alphaproteobacterial (Methylosinus) and gammaproteobacterial (Methylobacter, Methylomonas, and Methylomicrobium) methanotrophs, methylotrophs of the genus Methylobacterium, and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C1 compounds. Comparisons between 16S rRNA gene pools amplified from [13C]DNA and from [12C]DNA revealed that the proportion of Methylophilus-related sequences increased in the presence of [13C]methanol, [13C]methylamine, and [13C]formaldehyde; Novosphingobium-related sequences were enriched in the presence of [13C]methanol; Gemmatimonadaceae-related sequences were enriched in the presence of [13C]formaldehyde and [13C]formate; and Xanthomonadaceae-related sequences were enriched in the presence of [13C]formate. Analysis of fae genes amplified from [13C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C1 compound: Methylosinus sequences dominated the [13C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [13C]methylamine microcosm pool. The [13C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [13C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.

Fishing for biodiversity: novel methanopterin-linked C transfer genes deduced from the Sargasso Sea metagenome.

The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1800 major phylotypes (the Sargasso Sea metagenome-SSM) presents a great source for expanding local databases of genes indicative of a specific function. In this article we analyse the SSM for the presence of methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment. The sequences representative of these major phylotypes do not appear to belong to any known microbial group capable of methanopterin-linked C1 transfer. Instead, these sequences separate from all known sequences on phylogenetic trees, pointing toward their affiliation with novel microbial phyla. These data imply a broader distribution of methanopterin-linked functions in the microbial world than has been previously known.

Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin-linked C1-metabolism in bacteria.

In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats.

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5-4.0 and 60 or 85 degrees C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13 degrees N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60 degrees C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5-6.0 and the pH minimum at 3.0. At 85 degrees C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85 degrees C. From the 60 degrees C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0-2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

Diversity of Bacteria and Archaea associated with a carbonate-rich metalliferous sediment sample from the Rainbow vent field on the Mid-Atlantic Ridge.

Two sediment cores were collected in an inactive area of the deep-sea hydrothermal vent field Rainbow (36 degrees N on the Mid-Atlantic Ridge). Metals and carbonates were abundant throughout the cores; calcite (CaCO3) was found throughout the cores while dolomite [CaMg(CO3)2] and siderite (FeCO3) were only found in deeper layers. Using polymerase chain reaction (PCR)-amplified 16S rRNA gene sequence analysis, we examined the bacterial and archaeal diversity in a sediment layer that contained the three carbonates. The retrieved bacterial and archaeal sequences were new and less than 4% of the sequences exhibited 94% or more identity with that of cultured organisms. The analysis of the composition of the bacterial library revealed a high diversity of sequences. Half of the bacterial clones was affiliated to the gamma-Proteobacteria. Most of them had environmental sequences retrieved from deep-sea sediments as closest relatives, some of which being distantly related to free-living and symbiotic sulfur-oxidizers. Other sequences clustered in the alpha-, delta- and epsilon-Proteobacteria, the 'Bacteroidetes', the 'Planctomycetes', the 'Nitrospirae', the 'Actinobacteria', the 'Chlorobi ' and the 'Verrumicrobia'. Based on clonal abundance and sequence comparisons, phylotype groups putatively involved in the oxydation of sulfur compounds appeared to dominate in the studied sample. The majority of the archaeal sequences clustered in an euryarchaeotic lineage recently identified in the walls of black smokers suggesting a possible thermophilic way of life of these uncultured microorganisms. Oxygen isotopic composition of siderite and dolomite indicated that they were formed at 67 degrees C and 94 degrees C respectively. Together with chemical and microbiological data, this suggested that hydrothermal fluids may have circulated through this sediment.

Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments.

To contribute to the identification of methanogens, methanotrophs and sulfate-reducing bacteria (SRB) in microbial communities from the 13 degrees N (East Pacific Rise) and Rainbow (Mid-Atlantic Ridge) hydrothermal vent fields, we investigated the diversity of mcrA, pmoA and dsrAB genes sequences. Clone libraries were obtained using DNA isolated from fragments of diffuse vents, sediment and in situ samplers. The clones were categorized by restriction fragment length polymorphism, and representatives of each group were sequenced. Sequences were related to that of hyperthermophilic (order Methanopyrales and family Methanocaldococcaceae), thermophilic and mesophilic (family Methanococcaceae) methanogens, thermophilic (proposed genus 'Methylothermus') and mesophilic type I methanotrophs, and hyperthermophilic (order Archaeoglobales), thermophilic (order Thermodesulfobacteriales) and mesophilic (family Desulfobulbaceae) SRB. Several of the obtained sequences were distantly related to the genes of cultivated organisms, providing evidence of the existence of novel lineages in the three functional groups. This study provides for the first time an insight into the diversity of several functional genes of deep-sea hydrothermal system microorganisms.

Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal lineages in high-temperature environments.

In order to facilitate the evaluation of archaeal community diversity and distribution in high-temperature environments, 14 16S rRNA oligonucleotide probes were designed. Adequate hybridization and wash conditions of the probes encompassing most known hyperthermophilic Archaea, members of the orders Thermococcales, Desulfurococcales and Sulfolobales, of the families Methanocaldococcaceae, Pyrodictiaceae and Thermoproteaceae, of the genera Archaeoglobus, Methanopyrus and Ignicoccus, and of the as yet uncultured lineages Korarchaeota, Crenarchaeota marine group I, deep-sea hydrothermal vent euryarchaeotic group 2 (DHVE 2), and deep-sea hydrothermal vent euryarchaeotic group 8 (DHVE 8) were determined by dot-blot hybridization from target and non-target reference organisms and environmental clones. The oligonucleotide probes were also used to evaluate the archaeal community composition in nine deep-sea hydrothermal vent samples. All probes, except those targeting members of Sulfolobales, Thermoproteaceae, Pyrodictiaceae and Korarchaeota, gave positive hybridization signals when hybridized against 16S rDNA amplification products obtained from hydrothermal DNA extracts. The results confirmed the widespread occurrence of Thermococcales, Desulfurococcales, Methanocaldococcaceae and Archaeoglobus in deep-sea hydrothermal vents, and extended the known ecological habitats of uncultured lineages. Despite their wide coverage, the probes were unable to resolve the archaeal communities associated with hydrothermally influenced sediments, suggesting that these samples may contain novel lineages. This suite of oligonucleotide probes may represent an efficient tool for rapid qualitative and quantitative characterization of archaeal communities. Their application would help to provide new insights in the future into the composition, distribution and abundance of Archaea in high-temperature environments.

Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 degrees N).

To evaluate possible compositional changes in archaeal communities at a deep-sea hydrothermal vent field scale, we examined five different samples obtained after deploying in situ collectors for different times on three spatially separated venting sulphide structures on the East Pacific Rise (13 degrees N). Direct cell counts and whole-cell hybridizations with fluorescently labelled 16S rRNA-based oligonucleotide probes revealed that the relative abundance of archaeal populations represented from 14 to 33% of the prokaryotic community. 16S rDNA sequence analysis of the archaeal clone libraries indicated that a large percentage of clones were closely related to known archaeal isolates recovered from similar habitats. Among the 24 different phylotypes identified, Thermococcales-related sequences were dominant in all the libraries that also included representative genera of orders Methanopyrales, Methanococcales, Archaeoglobales and Desulfurococcales. The presence of most of these phylogenetic groups was confirmed in enrichment cultures performed at temperatures from 60 to 90 degrees C. Additional sequences with no known cultivated relatives grouped with the Marine group I Crenarchaeota, Korarchaeota and Deep-sea Hydrothermal Vent Euryarchaeota (DHVE) within which a novel lineage was identified. Furthermore, the archaeal community composition was distinct from vent to vent within the same vent field and varied within short time scales. This study provides new insights into microbial diversity and distribution at deep-sea hydrothermal vents.