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.     

Habitat Segregation of a Functional Gene Encoding Nitrate Ammonification in Estuarine Sediments

Distribution and diversity of nrfA gene encoding dissimilatory nitrite reduction to ammonium (DNRA) in the sediments of the Colne River, North Essex, UK, were investigated. Sequencing cloned nrfA fragments amplified from environmental DNA enabled structure analysis of the bacterial community responsible for this pathway. The DNA was extracted from the sediment samples at different depths from the estuary ranging from freshwater to seawater regions, and amplified using specific PCR primer pairs targeting for the nrfA gene. Analysis of the nrfA clones showed two distinct clusters corresponding to their origins, namely, divided into the stable sites (marine and freshwater regions) and the unstable sites (brackish water region), where the tidal rise and fall constantly disturbs the environmental conditions. In addition, the nrfA clones from the deeper layer of the sediment formed a more homogenous community than those from the surface layer of the sediment. This may be due to more isolated and anaerobic conditions kept in the deeper sediment less influenced by the overlying water and other environmental factors. Most of the nrfA clones from the Colne estuarine sediments formed several distinct clusters including known nitrate ammonifiers such as Aeromonas, Shewanella, Desulfovibrio and Sulfurospillum. One of which was, however, related to Bacteroides but still quite divergent (～70% identity) and the rest forming unknown clusters of supposedly uncultured members of bacteria. This is the first trial to describe the nrfA partial sequences derived from a natural environment, with reference to their habitat-specific community structure.     

Ribosomal tag pyrosequencing of DNA and RNA from benthic coral reef microbiota: community spatial structure, rare members and nitrogen-cycling guilds

Ribosomal tag libraries based on DNA and RNA in coral reef sediment from Hawaii show the microbial community to be dominated by the bacterial phyla Proteobacteria, Firmicutes and Actinobacteria, the archaeal order Nitrosopumilales and the uncultivated divisions Marine Group III (Euryarchaeota) and Marine Benthic Group C (Crenarchaeota). Operational taxonomic units (OTUs) number in the high thousands, and richness varies with site, presence or absence of porewater sulfide (sediment depth), and nucleotide pool. Rank abundance curves of DNA-based libraries, but not RNA-based libraries, possess a tail of low abundance taxa, supporting the existence of an inactive 'rare' biosphere. While bacterial libraries from two oxic samples differ markedly, those from two anoxic (sulfidic) samples are similar. The four dominant bacterial OTUs are members of genera that include pathogens, but are found in marine environments, and include facultative anaerobes, i.e. dissimilatory nitrate reducers and denitrifiers. This may explain their abundance in both oxic and anoxic samples. A numerous archaeon is closely related to the lithoautotrophic ammonia oxidizer Nitrosopumilus maritimus. Known bacterial ammonia oxidizers are essentially absent, but bacterial nitrite oxidizers are abundant. Although other studies of this reef found evidence for anaerobic ammonia oxidizers, primer bias rendered that clade invisible to this study.     

Relative Abundances of Proteobacterial Membrane-Bound and Periplasmic Nitrate Reductases in Selected Environments

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 × 10² to 6.8 × 10⁴ copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.     

Respiratory and dissimilatory nitrate-reducing communities from an extreme saline alkaline soil of the former lake Texcoco (Mexico)

The diversity of the dissimilatory and respiratory nitrate-reducing communities was studied in two soils of the former lake Texcoco (Mexico). Genes encoding the membrane-bound nitrate reductase (narG) and the periplasmic nitrate reductase (napA) were used as functional markers. To investigate bacterial communities containing napA and narG in saline alkaline soils of the former lake Texcoco, libraries of the two sites were constructed (soil T3 with pH 11 and electrolytic conductivity in saturated extract (EC_SE) 160 dS m⁻¹ and soil T1 with pH 8.5 and EC_SE 0.8 dS m⁻¹). Phylogenetic analysis of napA sequences separated the clone families into two main groups: dependent or independent of NapB. Most of napA sequences from site T1 were grouped in the NapB-dependent clade, meanwhile most of the napA sequences from the extreme soil T3 were affiliated to the NapB-independent group. For both sites, partial narG sequences were associated with representatives of the Proteobacteria, Firmicutes and Actinobacteria phyla, but the proportions of the clones were different. Our results support the concept of a specific and complex nitrate-reducing community for each soil of the former lake Texcoco.     

Ammonia oxidation and nitrate reduction marker genes are key indicators of nitrogen losses in temperate forest catchments

Chronic nitrogen inputs can alleviate N limitation and potentially impose N losses in forests, indicated by soil enrichment in ¹⁵N over ¹⁴N. However, the complexity of the nitrogen cycle hinders accurate quantification of N fluxes. Simultaneously, soil ecologists are striving to find meaningful indicators to characterise the “openness” of the nitrogen cycle. We integrate soil δ¹⁵N with constrained ecosystem N losses and the functional gene potential of the soil microbiome in 14 temperate forest catchments. We show that N losses are associated with soil δ¹⁵N and that δ¹⁵N scales with the abundance of soil bacteria. The abundance of the archaeal amoA gene, representing the first step in nitrification (ammonia oxidation to nitrite), followed by the abundance of narG and napA genes, associated with the first step in denitrification (nitrate reduction to nitrite), explains most of the variability in soil δ¹⁵N. These genes are more informative than the denitrification genes nirS and nirK, which are directly linked to N₂O production. Nitrite formation thus appears to be the critical step associated with N losses. Furthermore, we show that the genetic potential for ammonia oxidation and nitrate reduction is representative of forest soil ¹⁵N enrichment and thus indicative of ecosystem N losses.     

Diverse communities of active Bacteria and Archaea along oxygen gradients in coral reef sediments

Microbial communities inhabiting highly permeable sediments of Checker Reef in Kaneohe Bay, Hawaii, were characterized in relation to porewater geochemistry (O₂, NO₃ ⁻, NO₂ ⁻, NH₄ ⁺, phosphate). The physiologically active part of the population, assessed by sequencing cDNA libraries of 16S rRNA amplicons, was very diverse, with an estimated ribotype richness ≥1,380 in anoxic sediment. Quantitative analysis of community structure by rRNA-targeted fluorescence in situ hybridization (FISH) indicated that the archaeal population (9–18%) was dominated by marine Crenarchaeota (5–9%). Planctomycetales were the most abundant group in the oxic and interfacial habitat (17–19%) but were a minority (<5%) in anoxic reef sediment, where γ-Proteobacteria were numerically dominant (18%). Another 9–14% of the microbial benthos belonged to β-Proteobacteria, predominantly within the order Nitrosomonadales, many cultured representatives of which are NH₄ ⁺ oxidizers. The results of this study contribute to the phylogenetic characterization of benthic microbial communities that are important in organic matter degradation and nutrient recycling in coral reef ecosystems.     

Higher nitrate-reducer diversity in macrophyte-colonized compared to unvegetated freshwater sediment

Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification–denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.     

Patterns and drivers of anaerobic nitrogen transformations in sediments of thermokarst lakes

Significant attention has been given to the way in which the soil nitrogen (N) cycle responds to permafrost thaw in recent years, yet little is known about anaerobic N transformations in thermokarst lakes, which account for more than one-third of thermokarst landforms across permafrost regions. Based on the N isotope dilution and tracing technique, combined with qPCR and high-throughput sequencing, we presented large-scale measurements of anaerobic N transformations of sediments across 30 thermokarst lakes over the Tibetan alpine permafrost region. Our results showed that gross N mineralization, ammonium immobilization, and dissimilatory nitrate reduction rates in thermokarst lakes were higher in the eastern part of our study area than in the west. Denitrification dominated in the dissimilatory nitrate reduction processes, being two and one orders of magnitude higher than anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA), respectively. The abundances of the dissimilatory nitrate reduction genes (nirK, nirS, hzsB, and nrfA) exhibited patterns consistent with sediment N transformation rates, while α diversity did not. The inter-lake variability in gross N mineralization and ammonium immobilization was dominantly driven by microbial biomass, while the variability in anammox and DNRA was driven by substrate supply and organic carbon content, respectively. Denitrification was jointly affected by nirS abundance and organic carbon content. Overall, the patterns and drivers of anaerobic N transformation rates detected in this study provide a new perspective on potential N release, retention, and removal upon the formation and development of thermokarst lakes.     

Microbial carbon monoxide consumption in salt marsh sediments

We have examined sediments from a fringing salt marsh in Maine to further understand marine CO metabolism, about which relatively little is known. Intact cores from the marsh emitted CO during dark oxic incubations, but emission rates were significantly higher during anoxic incubations, which provided evidence for simultaneous production and aerobic consumption in surface sediments. CO emission rates were also elevated when cores were exposed to light, which indicated that photochemical reactions play a role in CO production. A kinetic analysis of marsh surface sediments yielded an apparent K(m) of about 82 ppm, which exceeded values reported for well-aerated soils that consume atmospheric CO (65nM). Surface (0-0.2 cm depth interval) sediment slurries incubated under oxic conditions rapidly consumed CO, and methyl fluoride did not inhibit uptake, which indicated that neither ammonia nor methane oxidizers contributed to the observed activity. In contrast, aerobic CO uptake was inhibited by additions of readily available organic substrates (pyruvate, glucose and glycine), but not by cellulose. CO was also consumed by surface and sub-surface sediment slurries incubated under anaerobic conditions, but rates were less than during aerobic incubations. Molybdate and nitrate or nitrite, but not 2-bromoethanesulfonic acid, partially inhibited anaerobic uptake. These results suggest that sulfidogens and acetogens, but not dissimilatory nitrate reducers or methanogens, actively consume CO. Sediment-free plant roots also oxidized CO aerobically; rates for Spartina patens and Limonium carolinianum roots were significantly higher than rates for Spartina alterniflora roots. Thus plants may also impact CO cycling in estuarine environments.     

Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida

The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N₂O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C₂H₂, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Received: 9 April 1998 / Accepted: 15 May 1998     

Complex nitrogen cycling in the sponge Geodia barretti

Marine sponges constitute major parts of coral reefs and deep‐water communities. They often harbour high amounts of phylogenetically and physiologically diverse microbes, which are so far poorly characterized. Many of these sponges regulate their internal oxygen concentration by modulating their ventilation behaviour providing a suitable habitat for both aerobic and anaerobic microbes. In the present study, both aerobic (nitrification) and anaerobic (denitrification, anammox) microbial processes of the nitrogen cycle were quantified in the sponge Geodia barretti and possible involved microbes were identified by molecular techniques. Nitrification rates of 566 nmol N cm^?3 sponge day^?1 were obtained when monitoring the production of nitrite and nitrate. In support of this finding, ammonia‐oxidizing Archaea (crenarchaeotes) were found by amplification of the amoA gene, and nitrite‐oxidizing bacteria of the genus Nitrospira were detected based on rRNA gene analyses. Incubation experiments with stable isotopes (¹⁵NO₃^‐ and ¹⁵NH₄⁺) revealed denitrification and anaerobic ammonium oxidation (anammox) rates of 92 nmol N cm^?3 sponge day^?1 and 3 nmol N cm^?3 sponge day^?1 respectively. Accordingly, sequences closely related to ‘Candidatus Scalindua sorokinii’ and ‘Candidatus Scalindua brodae’ were detected in 16S rRNA gene libraries. The amplification of the nirS gene revealed the presence of denitrifiers, likely belonging to the Betaproteobacteria. This is the first proof of anammox and denitrification in the same animal host, and the first proof of anammox and denitrification in sponges. The close and complex interactions of aerobic, anaerobic, autotrophic and heterotrophic microbial processes are fuelled by metabolic waste products of the sponge host, and enable efficient utilization and recirculation of nutrients within the sponge–microbe system. Since denitrification and anammox remove inorganic nitrogen from the environment, sponges may function as so far unrecognized nitrogen sinks in the ocean. In certain marine environments with high sponge cover, sponge‐mediated nitrogen mineralization processes might even be more important than sediment processes.     

Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0–5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20–37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.     

Seasonal dynamics of ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wetland sediments of subtropical coastal mangrove

Mangrove wetlands are an important ecosystem in tropical and subtropical regions, and the sediments may contain both oxic and anoxic zones. In this study, ammonia/ammonium-oxidizing prokaryotes (AOPs) in yellow and black sediments with vegetation and non-vegetated sediments in a mangrove wetland of subtropical Hong Kong were investigated in winter and summer. The phylogenetic diversity of anammox bacterial 16S rRNA genes and archaeal and bacterial amoA genes (encoding ammonia monooxygenase alpha-subunit) were analyzed using PCR amplification and denaturing gradient gel electrophoresis to reveal their community structures. Quantitative PCR was also used to detect their gene abundances. The results showed that seasonality had little effect, but sediment type had a noticeable influence on the community structures and abundances of anammox bacteria. For ammonia-oxidizing archaea (AOA), seasonality had a small effect on their community structures, but a significant effect on their abundances: AOA amoA genes were significantly higher in winter than in summer. In winter, the vegetated yellow sediments had lower AOA amoA genes than the other types of sediments, but in summer, the vegetated yellow sediments had higher AOA amoA genes than the other types of sediments. Sediment type had no apparent effect on AOA community structures in winter. In summer, however, the vegetated yellow sediments showed obviously different AOA community structures from the other types of sediments. For ammonia-oxidizing bacteria (AOB), seasonality had a significant effect on their community structures and abundances: AOB amoA genes in winter were apparently higher than in summer, and AOB community structures were different between winter and summer. Sediment type had little effect on AOB community structures, but had a noticeable effect on the abundances: AOB amoA genes of the vegetated yellow sediments were obviously lower than the black ones in both seasons. This study has demonstrated that seasonality and sediment type affected community structures and abundances of AOPs differently in oxic and anoxic sediments of the mangrove wetland.     

Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions

A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO₂ content (pCO₂) at the Swiss FACE (Free Air CO₂ Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO₂ in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO₂ treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO₂. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO₂ treatment.     

Holocene growth history of an eastern Pacific fringing reef,Punta Islotes,Costa Rica

Rock and sediment cores reveal that a well-developed fringing reef in Golfo Dulce, Pacific Costa Rica, up to 9 m thick was established on Cretaceous basalt about 5500 y BP. It is presently being smothered with fine sediments and is almost completely dead. This reef is made up of three main facies that are represented by comparable extant reef zones: reef-flat branching coral, fore-reef slope massive coral, and fore-reef talus sediment facies. Reef growth began with the establishment of small patch reefs dominantly formed by the branching coral Pocillopora damicornis. P. damicornis spread across the basalt bench and massive colonies of Porites lobata grew on the outer slopes, eventually blocking the seaward transport of Pocillopora fragments to the fore-reef talus sediments. The reef flourished until 500 years ago. Lower accumulation rates during the past 500 years may be due to deteriorating environmental conditions rather than slower growth after the reef reached sea level. Present-day reef communities are severely degraded with less than 2% living coral cover. The increased turbidity associated with the final stage of degradation of this reef is probably related to human activity on the adjacent shores, including deforestation, mining, and road construction.     

Distribution and Diversity of Archaeal Ammonia Monooxygenase Genes Associated with Corals

Corals are known to harbor diverse microbial communities of Bacteria and Archaea, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A (amoA) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial amoA genes, and archaeal amoA gene sequences were obtained from five different species of coral collected in Bocas del Toro, Panama. The 210 coral-associated archaeal amoA sequences recovered in this study were broadly distributed phylogenetically, with most only distantly related to previously reported sequences from coastal/estuarine sediments and oceanic water columns. In contrast, the bacterial amoA gene could not be amplified from any of these samples. These results offer further evidence for the widespread presence of the archaeal amoA gene in marine ecosystems, including coral reefs.