Community Composition and Abundance of Ammonia-Oxidizing Archaea in Sediments from the Changjiang Estuary and its Adjacent Area in the East China Sea

Community composition and abundance of ammonia-oxidizing archaea (AOA) were investigated using ammonia monooxygenase α subunit (amoA) in sediments from the Changjiang estuary and its adjacent area in the East China Sea (ECS). Real-time quantitative polymerase chain reaction (qPCR), clone libraries and sequencing were performed to characterize the AOA community. Clone libraries analysis showed that the majority of amoA sequences fell within the Nitrosopumilus cluster. Correlation analysis showed that AOA diversity was closely related to the nitrite concentration, which was consistent with the canonical correspondence analysis (CCA) where a significant association between nitrite and AOA community composition was observed. The qPCR results were found to be significantly correlated with the environmental parameters. In the gravity cores, a significant positive correlation was found between ammonium concentrations and amoA gene copy numbers from different sediment depths at station S31. At station S33, however, ammonium concentration had a negative correlation and nitrite concentration had a positive correlation with amoA gene copy numbers. In the surface sediments, chlorophyll a concentration had a negative correlation and nitrate concentration had a positive correlation with amoA gene copy numbers. Compared amoA gene copy numbers from AOA with those from ammonia-oxidizing β-proteobacteria (β-AOB) in the same studied areas, the amoA gene copy ratio of β-AOB to AOA was negatively correlated with the phosphate concentration and dissolved oxygen concentration, but was not significantly correlated with either ammonium concentrations or salinity. Our data provided valuable information to achieve a better understanding of the potential role of ammonia oxidizers at the interface between terrestrial and marine environments.     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.     

Microbial Diversity in Uranium Mining-Impacted Soils as Revealed by High-Density 16S Microarray and Clone Library

Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.     

Microbial diversity and biogeochemistry of the Guaymas Basin deep‐sea hydrothermal plume

Hydrothermal plumes are hot spots of microbial biogeochemistry in the deep ocean, yet little is known about the diversity or ecology of microorganisms inhabiting plumes. Recent biogeochemical evidence shows that Mn(II) oxidation in the Guaymas Basin (GB) hydrothermal plume is microbially mediated and suggests that the plume microbial community is distinct from deep‐sea communities. Here we use a molecular approach to compare microbial diversity in the GB plume and in background deep seawater communities, and cultivation to identify Mn(II)‐oxidizing bacteria from plumes and sediments. Despite dramatic differences in Mn(II) oxidation rates between plumes and background seawater, microbial diversity and membership were remarkably similar. All bacterial clone libraries were dominated by Gammaproteobacteria and archaeal clone libraries were dominated by Crenarchaeota. Two lineages, both phylogenetically related to methanotrophs and/or methylotrophs, were consistently over‐represented in the plume. Eight Mn(II)‐oxidizing bacteria were isolated, but none of these or previously identified Mn(II) oxidizers were abundant in clone libraries. Taken together with Mn(II) oxidation rates measured in laboratory cultures and in the field, these results suggest that Mn(II) oxidation in the GB hydrothermal plume is mediated by genome‐level dynamics (gene content and/or expression) of microorganisms that are indigenous and abundant in the deep sea but have yet to be unidentified as Mn(II) oxidizers.     

Nitrogen‐cycling bacteria and archaea in the carbonate sediment of a coral reef

In the coarse‐grained carbonate sediments of coral reefs, advective porewater flow and the respiration of organic matter establish redox zones that are the scene of microbially mediated transformations of N compounds. To investigate the geobiology of N cycling in reef sediments, the benthic microbiota of Checker Reef in Kaneohe Bay, Hawaii, were surveyed for candidate nitrate reducers, ammonifying nitrite reducers, aerobic and anaerobic ammonia oxidizers (anammox) by identifying phylotypes of their key metabolic genes (napA, narG, nrfA, amoA) and ribotypes (unique RNA sequences) of anammox‐like 16S rRNA. Putative proteobacteria with the catalytic potential for nitrate reduction were identified in oxic, interfacial and anoxic habitats. The estimated richness of napA (≥202 in anoxic sediment) and narG (≥373 and ≥441 in oxic and interfacial sediment, respectively) indicates a diverse guild of nitrate reducers. The guild of nrfA hosts in interfacial reef sediment was dominated by Vibrio species. The identified members of the aerobic ammonium oxidizing guild (amoA hosts) were Crenarchaeota or close relatives of Nitrosomonadales. Putative anammox bacteria were detected in the RNA pool of Checker Reef sediment. More than half of these ribotypes show ≥90% identity with homologous sequences of Scalindua spp., while no evidence was found for members of the genera Brocadia or Kuenenia. In addition to exploring the diversity of these four nitrogen‐cycling microbial guilds in coral reef sediments, the abundances of aerobic ammonium oxidizers (amoA), nitrite oxidizers (nxrAB), ammonifying nitrite reducers (nrfA) and denitrifiers (nosZ) were estimated using real‐time PCR. Representatives of all targeted guilds were detected, suggesting that most processes of the biogeochemical N cycle can be catalyzed by the benthic microbiota of tropical coral reefs.     

Comparative analysis of genetic diversity and expression of<Emphasis Type="Italic"> amoA</Emphasis> in wastewater treatment processes

The genetic diversity and expression of amoA of autotrophic ammonia oxidizers in wastewater treatment processes were investigated by RT-PCR and denaturing gradient gel electrophoresis (DGGE) in order to identify active components of ammonia-oxidizer populations in a such processes. Ammonia oxidizers, evidenced by the presence of amoA mRNA, were regarded as metabolically active. The DGGE profiles derived from amoA mRNA and from its gene, which were amplified by RT-PCR or PCR using samples collected from a bench-scale reactor treating high concentration of inorganic ammonia, were similar. In contrast, RNA and DNA-derived DGGE profiles from three domestic wastewater treatment facilities were different from each other. These data indicate that the dominant ammonia oxidizers in the bench-scale reactor exhibited ammonia-oxidizing activity, whereas some ammonia oxidizers in the domestic wastewater treatment facilities apparently did not express high levels of amoA mRNA.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

The 16S rRNA and mcrA gene based comparative diversity of methanogens in cattle fed on high fibre based diet

In the present study, the diversity of rumen methanogens in crossbred Karan Fries cattle was determined by constructing 16S rRNA and mcrA (methyl coenzyme-M reductase α subunit) gene libraries using specific primers. All thirteen OTUs or phylotypes from 16S rRNA library clustered with order Methanobacteriales, twelve of which aligned with Methanobrevibacter spp., whereas one OTU resemble with Methanosphaera stadtmanae. Out of eighteen OTUs identified from mcrA gene library, fifteen clustered with order Methanobacteriales, two resemble with Methanomicrobiales and remaining one grouped with Methanosarcinales. These results revealed that Methanobrevibacter phylotype was predominantly present in Karan Fries crossbred cattle fed on high fibrous diet containing wheat straw. Compared to 16S rRNA gene, mcrA gene OTUs clustered in three orders providing better insights of rumen methanogens diversity in cattle.     

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH₄ cycling in rivers.     

Methanogen Diversity Evidenced by Molecular Characterization of Methyl Coenzyme M Reductase A (mcrA) Genes in Hydrothermal Sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H₂/CO₂- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H₂/CO₂, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Distribution and Diversity of Bacteria and Archaea in Marine Sediments Affected by Gas Hydrates at Mississippi Canyon in the Gulf of Mexico

The microbial community structures of gas hydrate-bearing (Core 9) and non-hydrate-bearing (Core 1) marine sediments were investigated at Mississippi Canyon (MC) 118 in the Gulf of Mexico. Quantification by quantitative competitive (QC)-PCR showed that bacterial abundance was 2–3 orders of magnitude higher than archaeal abundance in these cores. Sulfate-reducing bacteria (SRB) were present at 10³–10⁴ dsrAB gene copies/g in both cores; methanogens or anaerobic methanotrophs were only present in Core 9 (10²–10⁵ mcrA gene copies/g). Denaturing gradient gel electrophoresis (DGGE) showed distinct patterns of bacterial community structure between Core 9 and Core 1 with ε-Proteobacteria predominating in the former and γ-Proteobacteria in the latter. Clone libraries were successfully constructed for both Archaea and Bacteria using functional genes (mcrA and dsrAB, respectively). The mcrA gene was present in Core 9, suggesting enhanced abundance or activity of methanogens or methane-oxidizing archaea in the hydrate-impacted sediment. The mcrA gene sequences were dominated by group c-d and group e. The majority (80%) of the dsrAB gene sequences fell into Syntrophobacteraceae-related group. This study indicates that microbial community structures are considerably different between the hydrate-bearing and non-hydrate-bearing sediment at MC 118. Our study is among the initial steps toward a comprehensive and long-term monitoring of microbial dynamics associated with gas hydrates in the Gulf of Mexico.     

Bacterial Diversity of Weathered Terrestrial Icelandic Volcanic Glasses

The diversity of microbial communities inhabiting two terrestrial volcanic glasses of contrasting mineralogy and age was characterised. Basaltic glass from a <0.8 Ma hyaloclastite deposit (Valafell) harboured a more diverse Bacteria community than the younger rhyolitic glass from ～150-300 AD (D?madalshraun lava flow). Actinobacteria dominated 16S rRNA gene clone libraries from both sites, however, Proteobacteria, Acidobacteria and Cyanobacteria were also numerically abundant in each. A significant proportion (15-34%) of the sequenced clones displayed <85% sequence similarities with current database sequences, thus suggesting the presence of novel microbial diversity in each volcanic glass. The majority of clone sequences shared the greatest similarity to uncultured organisms, mainly from soil environments, among these clones from Antarctic environments and Hawaiian and Andean volcanic deposits. Additionally, a large number of clones within the Cyanobacteria and Proteobacteria were more similar to sequences from other lithic environments, included among these Icelandic clones from crystalline basalt and rhyolite, however, no similarities to sequences reported from marine volcanic glasses were observed. PhyloChip analysis detected substantially greater numbers of phylotypes at both sites than the corresponding clone libraries, but nonetheless also identified the basaltic glass community as the richer, containing approximately 29% unique phylotypes compared to rhyolitic glass.     

Diversity and abundance of ammonia-oxidizing prokaryotes in sediments from the coastal Pearl River estuary to the South China Sea

In the present study the diversity and abundance of nitrifying microbes including ammonia-oxidizing archaea (AOA) and betaproteobacteria (beta-AOB) were investigated, along with the physicochemical parameters potentially affecting them, in a transect of surface sediments from the coastal margin adjacent to the Pearl River estuary to the slope in the deep South China Sea. Nitrifying microbial diversity was determined by detecting the amoA (ammonia monooxygenase subunit A) gene. An obvious community structure shift for both AOA and beta-AOB from the coastal marginal areas to the slope in the deep-sea was detected, while the OTU numbers of AOA amoA were more stable than those of the beta-AOB. The OTUs of beta-AOB increased with the distance from the coastal margin areas to the slope in the deep-sea. Beta-AOB showed lower diversity with dominant strains in a polluted area but higher diversity without dominant strains in a clean area. Moreover, the diversity of beta-AOB was correlated with pH values, while no noticeable relationships were established between AOA and physicochemical parameters. Beta-AOB was more sensitive to transect environmental variability and might be a potential indicator for environmental changes. Additionally, the surface sediments surveyed in the South China Sea harboured diverse and distinct AOA and beta-AOB phylotypes different from other environments, suggesting the endemicity of some nitrifying prokaryotes in the South China Sea.     

Microbial Community Diversity in the Gut of the South American Termite Cornitermes cumulans (Isoptera: Termitidae)

Termites inhabit tropical and subtropical areas where they contribute to structure and composition of soils by efficiently degrading biomass with aid of resident gut microbiota. In this study, culture-independent molecular analysis was performed based on bacterial and archaeal 16S rRNA clone libraries to describe the gut microbial communities within Cornitermes cumulans, a South American litter-feeding termite. Our data reveal extensive bacterial diversity, mainly composed of organisms from the phyla Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and Fibrobacteres. In contrast, a low diversity of archaeal 16S rRNA sequences was found, comprising mainly members of the Crenarchaeota phylum. The diversity of archaeal methanogens was further analyzed by sequencing clones from a library for the mcrA gene, which encodes the enzyme methyl coenzyme reductase, responsible for catalyzing the last step in methane production, methane being an important greenhouse gas. The mcrA sequences were diverse and divided phylogenetically into three clades related to uncultured environmental archaea and methanogens found in different termite species. C. cumulans is a litter-feeding, mound-building termite considered a keystone species in natural ecosystems and also a pest in agriculture. Here, we describe the archaeal and bacterial communities within this termite, revealing for the first time its intriguing microbiota.     

Stratification of Archaeal communities in shallow sediments of the Pearl River Estuary,Southern China

Microorganisms are known to play fundamental roles in the biogeochemical cycling of carbon in the coastal environments. To get to know the composition and ecological roles of the archaeal communities within the sediments of the Pearl River Estuary, Southern China, the diversity and vertical distribution of archaea in a sediment core was reported based on the 16S rRNA and mcrA genes for the first time. Quantitative PCR analysis revealed that archaea were present at 10⁶–10⁷ 16S rRNA gene copies/g (wet weight) in the sediment core, and the proportion of mcrA versus 16S rRNA gene copies varied from 11 to 45%. 16S rRNA gene libraries were constructed and analyzed for the top layer (0–6 cm), middle layer (18–24 cm), sulfate-methane transition zone (SMTZ, 32–42 cm), and bottom layer (44–50 cm) sediments. The results indicated that Miscellaneous Crenarchaeotal Group (MCG) was the main component in the sediments. The MCG archaea could be further divided into six subgroups: MCG-A, B, C, D, E, and F. On the other hand, mcrA sequences from methanogens related to the order Methanomicrobiales and ANME-2 methanotrophs were detected in all sediment layers. Taken together, our data revealed a largely unknown archaeal community in which MCG dominated within the Pearl River estuarine sediments, while methanogens and methane-oxidizing archaea putatively involving in methane metabolism, were also found in the community. This is the first important step towards elucidating the biogeochemical roles of these archaea in the Pearl River Estuary.