Marinobacter alkaliphilus sp. nov., a novel alkaliphilic bacterium isolated from subseafloor alkaline serpentine mud from Ocean Drilling Program Site 1200 at South Chamorro Seamount, Mariana Forearc

Novel alkaliphilic, mesophilic bacteria were isolated from subseafloor alkaline serpentine mud from the Ocean Drilling Program (ODP) Hole 1200D at a serpentine mud volcano, South Chamorro Seamount in the Mariana Forearc. The cells of type strain ODP1200D-1.5^T were motile rods with a single polar flagellum. Growth was observed between 10 and 45–50°C (optimum temperature: 30–35°C, 45-min doubling time), between pH 6.5 and 10.8–11.4 (optimum: pH 8.5–9.0), and between NaCl concentrations of 0 and 21% (w/v) (optimum NaCl concentration: 2.5–3.5%). The isolate was a facultatively anaerobic heterotroph utilizing various complex substrates, hydrocarbons, carbohydrates, organic acids, and amino acids. Nitrate or fumarate could serve as an electron acceptor to support growth under anaerobic conditions. The G+C content of the genomic DNA was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Marinobacter and was the most closely related to M. aquaeolei strain VT8^T and M. hydrocarbonoclasticus strain SP.17^T, while DNA–DNA hybridization demonstrated that the new isolate could be genetically differentiated from the previously described species of Marinobacter. Based on the physiological and molecular properties of the new isolate, we propose the name Marinobacter alkaliphilus sp. nov., type strain: ODP1200D-1.5^T (JCM12291^T and ATCC BAA-889^T).     

Impact of Virioplankton on Archaeal and Bacterial Community Richness as Assessed in Seawater Batch Cultures

During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and Bacteria. One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.     

Archaeal Diversity in Waters from Deep South African Gold Mines

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.     

Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.     

Archaeal Diversity and Spatial Distribution in the Surface Sediment of the South China Sea

Archaea represent a significant portion of biomass in the marine sediments and may play an important role in global carbon cycle. However, the identity and composition of deep sea sediment Archaea are unclear. Here, we used the archaeal 16S rRNA gene primers to determine the diversity and community structure of Archaea from shallow water (<100 m) and deep water (>1500 m) sediments in the South China Sea. Phylogenetically the archaeal community is separated between the shallow- and deep sea sediments, with the former being dominated by the Thaumarchaeota and the latter by the Marine Benthic Group B, E and the South African GoldMine Euryarchaeotal Group as well as Thaumarchaeota. Sand content showed significant correlation with Thaumarchaeota, suggesting that the porous media may create an oxic environment that allowed these aerobic organisms to thrive in the surface sediments. The carbon isotope composition of total organic carbon was significantly correlated to the distribution of archaeal groups, suggesting that Archaea overall may be constrained by the availability or sources of organic carbon in the sediments of the South China Sea.     

Microbial community in a geothermal aquifer associated with the subsurface of the Great Artesian Basin, Australia

To investigate the biomass and phylogenetic diversity of the microbial community inhabiting the deep aquifer of the Great Artesian Basin (GAB), geothermal groundwater gushing out from the aquifer was sampled and analyzed. Microbial cells in the groundwater were stained with acridine orange and directly counted by epifluorescence microscopy. Microbial cells were present at a density of 10⁸–10⁹ cells per liter of groundwater. Archaeal and bacterial small-subunit rRNA genes (rDNAs) were amplified by PCR with Archaea- and Bacteria-specific primer sets, and clone libraries were constructed separately. A total of 59 clones were analyzed in archaeal and bacterial 16S rDNA libraries, respectively. The archaeal 16S rDNA clones were divided into nine operated taxonomic units (OTUs) by restriction fragment length polymorphism. These OTUs were closely related to the methanogenic genera Methanospirillum and Methanosaeta, the heterotrophic genus Thermoplasma, or miscellaneous crenarchaeota group. More than one-half of the archaeal clones (59% of total 59 clones) were placed beside phylogenetic clusters of methanogens. The majority of the methanogen-related clones (83%) was closely related to a group of hydrogenotrophic methanogens (genus Methanospirillum). The bacterial OTUs branched into seven phylogenetic clusters related to hydrogen-oxidizing thermophiles in the genera Hydrogenobacter and Hydrogenophilus, a sulfate-reducing thermophile in the genus Thermodesulfovibrio, chemoheterotropic bacteria in the genera Thermus and Aquaspirillum, or the candidate division OP10. Clones closely related to the thermophilic hydrogen-oxidizers in the genera Hydrogenobacter and Hydrogenophilus were dominant in the bacterial clone library (37% of a total of 59 clones). The dominancy of hydrogen-users strongly suggested that H₂ plays an important role as a primary substrate in the microbial ecosystem of this deep geothermal aquifer.     

Archaeal Population Dynamics during Sequential Reduction Processes in Rice Field Soil

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe³⁺, and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.     

Diversity and Community Structure of Archaea Inhabiting the Rhizoplane of Two Contrasting Plants from an Acidic Bog

Plant root exudates increase nutrient availability and influence microbial communities including archaeal members. We examined the archaeal community inhabiting the rhizoplane of two contrasting vascular plants, Dulichium arundinaceum and Sarracenia purpurea, from an acidic bog in upstate NY. Multiple archaeal 16S rRNA gene libraries showed that methanogenic Archaea were dominant in the rhizoplane of both plants. In addition, the community structure (evenness) of the rhizoplane was found markedly different from the bulk peat. The archaeal community in peat from the same site has been found dominated by the E2 group, meanwhile the rhizoplane communities on both plants were co-dominated by Methanosarcinaceae (MS), rice cluster (RC)-I, and E2. Complementary T-RFLP analysis confirmed the difference between bulk peat and rhizoplane, and further characterized the dominance pattern of MS, RC-I, and E2. In the rhizoplane, MS was dominant on both plants although as a less variable fraction in S. purpurea. RC-I was significantly more abundant than E2 on S. purpurea, while the opposite was observed on D. arundinaceum, suggesting a plant-specific enrichment. Also, the statistical analyses of T-RFLP data showed that although both plants overlap in their community structure, factors such as plant type, patch location, and time could explain nearly a third of the variability in the dataset. Other factors such as water table, plant replicate, and root depth had a low contribution to the observed variance. The results of this study illustrate the general effects of roots and the specific effects of plant types on their nearby archaeal communities which in bog-inhabiting plants were mainly composed by methanogenic groups.     

Prokaryotic Abundance and Community Composition in a Freshwater Iron-Rich Microbial Mat at Circumneutral pH

The abundance, diversity and composition of bacterial and archaeal communities in a freshwater iron-rich microbial mat were investigated using culture-dependent and culture-independent methods. The sampling site is a mixing zone where ferrous-iron-rich fluids encounter oxygen-rich environments. Quantitative PCR analysis shows that Bacteria dominated the mat community (>99% of the total cell numbers). Phylotypes related to iron-oxidizers in Gallionellaceae, methano/methylotrophs in Methylophilaceae and Methylococcaceae, sulfide-oxidizers in Sulfuricurvum and an uncultured clone group, called Terrestrial group I or the 1068 group, in the Epsilonproteobacteria were detected in the clone library from the original sample and/or the enrichment cultures. This result suggests that these members may play a role in Fe, S and C cycling in the mixing zone. Although Archaea were minor constituents numerically, phylogenetic analysis indicates that unique and diverse yet-uncultivated Archaea are present in the iron-rich mat. The phylotypes of these yet-uncultivated Archaea belong to environmental clone groups that have been recovered from other mixing zones in terrestrial and marine environments, and some of our phylotypes have significantly low similarity (80% or lower) with the archaeal clones reported previously. Our results provide further insights into the bacterial and archaeal communities in a microaerobic iron-rich freshwater environment in mixing zones.     

Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

T-RFLP clone characterization (screening) was optimized for a fast and basepair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP fingerprinting, a protocol was developed where 10 initial PCR cycles gave detectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to assess the suitability of the forward and reverse fragments for T-RFLP screening. Based on independent restriction digests with the tetrameric restriction enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic units (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing representative clones. Closely related clones ( approximately 97% similarity) could only be resolved with multiple restriction digests where forward and reverse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could be clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library ( approximately 12%). Therefore, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment.     

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.     

Crenarchaeota affiliated with group 1.1b are prevalent in coastal mineral soils of the Ross Sea region of Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (> 99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

How Stable Is Stable? Function versus Community Composition

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.     

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.     

Molecular Analyses of the Microbial Community Composition of an Anoxic Basin of a Municipal Wastewater Treatment Plant Reveal a Novel Lineage of Proteobacteria

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anoxic activated sludge from a municipal wastewater treatment plant. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by polymerase chain reaction using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 132 and 249 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was done using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota (93.8% of the operational taxonomic units [OTUs]) and Crenarchaeota (6.2% of the OTUs). Within the bacterial library, 84.8% of the OTUs represent novel putative phylotypes never described before and affiliated with ten divisions. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 60.4% of the OTUs, followed by Bacteroidetes (22.1%) and gram-positives (6.1%). Interestingly, we detected a novel Proteobacteria monophyletic group distinct from the five known subclasses, which we named New Lineage of Proteobacteria (NLP) lineage, and it is composed of eight clones representing 4.6% of the Proteobacteria. A new 16S rRNA-targeted hybridization probe was designed and fluorescent in situ hybridization analyses shows representatives of NLP as cocci-shaped microorganisms. The Chloroflexi, Acidobacterium, and Nitrospira phyla and TM7 candidate division are each represented by ≤3% of clone sequences. A comprehensive set of eight 16S and 23S rRNA-targeted oligonucleotide probes was used to quantify these major groups by dot blot hybridization within 12 samples. The Proteobacteria accounted for 82.5 ± 4.9%, representing the most abundant phyla. The Bacteroidetes and Planctomycetales groups accounted for 4.9 ± 1.3% and 4 ± 1.7%, respectively. Firmicutes and Actinobacteria together accounted for only 1.9 ± 0.5%. The set of probes covers 93.4 ± 14% of the total bacterial population rRNA within the anoxic basin.     

Molecular identification and dynamics of microbial communities in reactor treating organic household waste

The prokaryotic diversity associated with organic household waste (OHW), leachate (start-up inoculum), and mesophilic anaerobic digestion processes in the degradation of OHW for 44 and 90 days was investigated using a culture-independent approach. Bacterial and archaeal 16S rRNA and mcrA gene clone libraries were constructed from community DNA preparations. Bacterial clones were affiliated with 13 phyla, of which Firmicutes, Proteobacteria, and Bacteroidetes were represented in all libraries, whereas Actinobacteria, Thermotogae, Lentisphaerae, Acidobacteria, Chloroflexi, Cyanobacteria, Synergistetes, Spirochaetes, Deferribacteres, and Deinococcus-Thermus were exclusively identified in a single library. Within the Archaea domain, the Euryarchaeota phylum was the only one represented. Corresponding sequences were associated with the following orders of hydrogenotrophic methanogens: Methanomicrobiales (Methanoculleus genus) and Methanobacteriales (Methanosphaera and Methanobacterium genera). One archaeal clone was not affiliated with any order and may represent a novel taxon. Diversity indices showed greater diversity of Bacteria when compared to methanogenic Archaea.     

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.