A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only 相似文献   

Induced cooperation between marine nitrifiers and anaerobic ammonium-oxidizing bacteria by incremental exposure to oxygen

In oxygen-limited marine ecosystems cooperation between marine nitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria is of importance to nitrogen cycling. Strong evidence for cooperation between anammox bacteria and nitrifiers has been provided by environmental studies but little is known about the development of such communities, the effects of environmental parameters and the physiological traits of their constituents. In this study, a marine laboratory model system was developed. Cooperation between marine nitrifiers and anammox bacteria was induced by incremental exposure of a marine anammox community dominated by Scalindua species to oxygen in a bioreactor set-up under high ammonium (40 mM influent) conditions. Changes in the activities of the relevant functional groups (anammox bacteria, aerobic ammonia oxidizers and nitrite oxidizers) were monitored by batch tests. Changes in community composition were followed by Fluorescence in situ Hybridization (FISH) and by amplification and sequencing of 16S rRNA and amoA genes. A co-culture of Scalindua sp., an aerobic ammonia-oxidizing Nitrosomonas-like species, and an aerobic (most likely Nitrospira sp.) nitrite oxidizer was obtained. Aerobic ammonia oxidizers became active immediately upon exposure to oxygen and their numbers increased 60-fold. Crenarchaea closely related to the ammonia-oxidizer Candidatus 'Nitrosopumilus maritimus' were detected in very low numbers and their contribution to nitrification was assumed negligible. Activity of anammox bacteria was not inhibited by the increased oxygen availability. The developed marine model system proved an effective tool to study the interactions between marine anammox bacteria and nitrifiers and their responses to changes in environmentally relevant conditions.     

Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of approximately 700-bp 16S rRNA gene sequences with >96% homology to the "Candidatus Scalindua" group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to "Ca. Scalindua," "Candidatus Brocadia," and "Candidatus Kuenenia." This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Dominance of Candidatus Scalindua species in anammox community revealed in soils with different duration of rice paddy cultivation in Northeast China

The anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the oxygen-limited zone for nitrogen cycling, but their roles in agricultural ecosystems are still poorly understood. In this study, soil samples were taken from the rhizosphere and non-rhizosphere and from surface (0–5 cm) and subsurface (20–25 cm) layers with 1, 4, and 9 years of rice cultivation history on the typical albic soil of Northeast China to examine the diversity and distribution of anammox bacteria based on 16S rRNA gene and hydrazine oxidoreductase encoding gene (hzo). By comparing these soil samples, no obvious difference was observed in community composition between the rhizosphere and non-rhizosphere or the surface and subsurface layers. Surprisingly, anammox bacterial communities of these rice paddy soils were consisted of mainly Candidatus Scalindua species, which are best known to be dominant in marine and pristine environments. The highest diversity was revealed in the 4-year paddy soil based on clone library analysis. Phylogenetic analysis of 16S rRNA gene and deduced HZO from the corresponding encoding gene showed that most of the obtained clones are grouped together with Candidatus Scalindua sorokinii, Candidatus Scalindua brodae, and Candidatus Scalindua spp. of seawater. The obtained clone sequences from all samples are distributed in two subclusters that contain sequences from environmental samples only. Tentative new species were also discovered in this paddy soil. This study provides the first evidence on the existence of anammox bacteria with limited diversity in agricultural ecosystems in Northern China.     

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, "Candidatus Brocadia anammoxidans" and "Candidatus Kuenenia stuttgartiensis", and one marine species, "Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. "Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle—the anammoxosome—in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.     

More refined diversity of anammox bacteria recovered and distribution in different ecosystems

A newly reported 16S rRNA gene-based PCR primer set was successfully applied to detect anammox bacteria from four ecosystem samples, including sediments from marine, reservoir, mangrove wetland, and wastewater treatment plant sludge. This primer set showed ability to amplify a much wider coverage of all reported anammox bacterial genera. Based on the phylogenetic analyses of 16S rRNA gene of anammox bacteria, two new clusters were obtained, one closely related to Candidatus Scalindua, and the other in a previously reported novel genus related to Candidatus Brocadia. In the Scalindua cluster, four new subclusters were also found in this study, mainly by sequences of the South China Sea sediments, presenting a higher diversity of Candidatus Scalindua in marine environment. Community structure analyses indicated that samples were grouped together based on ecosystems, showing a niche-specific distribution. Phylogenetic analyses of anammox bacteria in samples from the South China Sea also indicated distinguished community structure along the depth. Pearson correlation analysis showed that the amount of anammox bacteria in the detected samples was positively correlated with the nitrate concentration. According to Canonical Correspondence Analysis, pH, temperature, nitrite, and nitrate concentration strongly affected the diversity and distribution of anammox bacteria in South China Sea sediments. Results collectively indicated a promising application of this new primer set and higher anammox bacteria diversity in the marine environment.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia‐oxidizing archaea (AOA) rather than marine ammonia‐oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium‐oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory‐scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C₁₈ anammox [3]‐ladderane fatty acid was highly abundant in the oxygen‐limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.     

Enrichment of anammox bacteria from marine environment for the construction of a bioremediation reactor

In the global ocean nitrogen cycle, the anaerobic ammonium-oxidizing (anammox) process is recognized as important. In this study, we established an enrichment culture of marine anammox bacteria (MAB) in a column-type reactor. The reactor, which included a porous polyester non-woven fabric that had been placed at the sea floor in advance for enrichment, was continuously fed with NH₄Cl and NaNO₂ for more than 1 year. Anammox activity in the MAB reactor was confirmed by ¹⁵N tracer analysis using ¹⁵NH₄Cl and Na¹⁴NO₂. We identified two 16S rRNA genes in the amplified DNA fragments derived from MAB, which were highly homologous with those from Candidatus “Scalindua wagneri” and an uncultured planctomycete clone. Fluorescence in situ hybridization analysis using an anammox-specific probe also confirmed that MAB predominated in the reactor. To our knowledge, this is the first report on the establishment of an enrichment culture of anammox bacteria from the marine environment using a continuous culture system.     

Residence of Habitat-Specific Anammox Bacteria in the Deep-Sea Subsurface Sediments of the South China Sea: Analyses of Marker Gene Abundance with Physical Chemical Parameters

Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS) were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S rRNA gene and hydrazine oxidoreductase encoding hzo gene were all closely related to the Candidatus Scalindua genus. A total of 96 16S rRNA gene sequences from 346 clones were grouped into five subclusters: two subclusters affiliated with the brodae and arabica species, while three new subclusters named zhenghei-I, -II, and -III showed ≤97.4% nucleic acid sequence identity with other known Candidatus Scalindua species. Meanwhile, 88 hzo gene sequences from the sediments also formed five distant subclusters within hzo cluster 1c. Through fluorescent real-time PCR analysis, the abundance of anammox bacteria in deep-sea subsurface sediment was quantified by hzo genes, which ranged from 1.19 × 10⁴ to 7.17 × 10⁴ copies per gram of dry sediments. Combining all the information from this study, diverse Candidatus Scalindua anammox bacteria were found in the deep-sea subsurface sediments of the SCS, and they could be involved in the nitrogen loss from the fixed inventory in the habitat.     

高通量测序分析多种典型生境中厌氧氨氧化细菌的多样性分布特征

【背景】厌氧氨氧化过程是氮素循环过程的重要途径之一,在氮素循环中发挥重要作用。先前的研究已经证实了厌氧氨氧化细菌存在于多种生境中,但对其多样性分布还没有系统的研究。【目的】对厌氧氨氧化细菌在不同类型生境中的多样性分布规律进行深入分析,充分展示其在不同生境中的群落结构特点,并揭示多样性分布与环境因素之间的关系。【方法】在建立厌氧氨氧化细菌16S rRNA基因序列数据库的基础上,运用高通量测序技术分析其在不同生境中的多样性分布特征。【结果】厌氧氨氧化细菌在红树林、海湾和河口生境中的多样性水平较高,而污泥和红壤的多样性水平明显较低。系统发育分析表明,这些生境中的厌氧氨氧化细菌主要由Candidatus Brocadia、Ca.Scalindua和未明确分类地位的菌属组成;从河流到红树林生态系统,随着盐度的增加,厌氧氨氧化细菌的优势种属由Ca. Brocadia转变到Ca. Scalindua,相关性分析也表明了盐度是导致不同生境中厌氧氨氧化细菌群落结构差异的主要因素。【结论】不同生境中存在不同的厌氧氨氧化细菌种群结构,环境条件的差异影响了厌氧氨氧化细菌的种群分布和系统演化。     

Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tanganyika)

Here we provide the first direct evidence for the anammox process (anaerobic ammonium oxidation) in a lacustrine system, Lake Tanganyika, the second largest lake in the world. Incubations with (15)N labelled nitrate showed that anammox occurred in the suboxic water layer at 100-110 m water depth. Anammox rates up to 10 nM N(2) h(-1) are comparable to those reported for the marine water column. Up to approximately 13% of produced N(2) could be attributed to the anammox process whereas the remainder was related to denitrification. Typical lipid biomarkers characteristic of anammox bacteria were found in filtered water from the depths where anammox occurred, thus supporting the presence of anammox bacteria. Further evidence is provided by fluorescence in situ hybridization (FISH), revealing up to 13 000 anammox bacteria cells per ml or 1.4% of all DAPI (4'-6-Diamidino-2-phenylindole)-stained cells. Phylogenetic analyses of partial 16S rRNA genes indicated the presence of sequences most closely related to the known anammox bacterium Candidatus "Scalindua brodae" (95.7% similarity). Using the incubation results, a total loss of 0.2 Tg N(2) per year linked to anammox was estimated for the Northern basin of Lake Tanganyika.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.     

Biogeographical distribution of diverse anaerobic ammonium oxidizing (anammox) bacteria in Cape Fear River Estuary

Summary Anaerobic ammonium oxidation (anammox) specific PCR method was developed to examine diversity and distribution of anammox bacteria in sediments collected from three different sites at Cape Fear River Estuary, North Carolina, where environmental parameters vary greatly over the year. Abundance and activities of anammox bacteria in these sediments were measured using the quantitative PCR (Q-PCR) method and ¹⁵N isotope tracer incubations. Different anammox bacterial communities composed with Brocadia , Kuenenia , Jettenia or Scalindua were found among sites along the estuarine gradient. Seasonal variations of anammox community structures were observed along the estuary based on terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Correlation analysis suggested that salinity variation influenced the diversity and distribution of different anammox bacteria in the estuary. Q-PCR assays of anammox bacteria showed temporal and spatial variations of their abundances, which were highly correlated to salinity variation. ¹⁵N isotope tracer incubations measured different anammox rates and its per cent contribution to total N₂ production among sites. The highest anammox rate was found at the site where Scalindua organisms dominated with the highest anammox bacterial abundance. Thus, we demonstrated a biogeographical distribution of diverse anammox bacteria influenced by salinity, and provide evidence to link anammox abundance and activities in estuarine sediments.     

Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system

Recent studies have shown that the anaerobic oxidation of ammonium by anammox bacteria plays an important role in catalyzing the loss of nitrogen from marine oxygen minimum zones (OMZ). However, in situ oxygen concentrations of up to 25 microM and ammonium concentrations close to or below the detection limit in the layer of anammox activity are hard to reconcile with the current knowledge of the physiology of anammox bacteria. We therefore investigated samples from the Namibian OMZ by comparative 16S rRNA gene analysis and fluorescence in situ hybridization. Our results showed that "Candidatus Scalindua" spp., the typical marine anammox bacteria, colonized microscopic particles that were likely the remains of either macroscopic marine snow particles or resuspended particles. These particles were slightly but significantly (P < 0.01) enriched in Gammaproteobacteria (11.8% +/- 5.0%) compared to the free-water phase (8.1% +/- 1.8%). No preference for the attachment to particles could be observed for members of the Alphaproteobacteria and Bacteroidetes, which were abundant (12 to 17%) in both habitats. The alphaproteobacterial SAR11 clade, the Euryarchaeota, and group I Crenarchaeota, were all significantly depleted in particles compared to their presence in the free-water phase (16.5% +/- 3.5% versus 2.6% +/- 1.7%, 2.7% +/- 1.9% versus <1%, and 14.9% +/- 4.6% versus 2.2% +/- 1.8%, respectively, all P < 0.001). Sequence analysis of the crenarchaeotal 16S rRNA genes showed a 99% sequence identity to the nitrifying "Nitrosopumilus maritimus." Even though we could not observe conspicuous consortium-like structures of anammox bacteria with particle-enriched bacterioplankton groups, we hypothesize that members of Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes play a critical role in extending the anammox reaction to nutrient-depleted suboxic water layers in the Namibian upwelling system by creating anoxic, nutrient-enriched microniches.     

Molecular Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in High-Temperature Petroleum Reservoirs

Anaerobic ammonium-oxidizing (anammox) process plays an important role in the nitrogen cycle of the worldwide anoxic and mesophilic habitats. Recently, the existence and activity of anammox bacteria have been detected in some thermophilic environments, but their existence in the geothermal subterranean oil reservoirs is still not reported. This study investigated the abundance, distribution and functional diversity of anammox bacteria in nine out of 17 high-temperature oil reservoirs by molecular ecology analysis. High concentration (5.31–39.2 mg l^?1) of ammonium was detected in the production water from these oilfields with temperatures between 55°C and 75°C. Both 16S rRNA and hzo molecular biomarkers indicated the occurrence of anammox bacteria in nine out of 17 samples. Most of 16S rRNA gene phylotypes are closely related to the known anammox bacterial genera Candidatus Brocadia, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia, while hzo gene phylotypes are closely related to the genera Candidatus Anammoxoglobus, Candidatus Kuenenia, Candidatus Scalindua, and Candidatus Jettenia. The total bacterial and anammox bacterial densities were 6.4?±?0.5?×?10³ to 2.0?±?0.18?×?10⁶ cells ml^?1 and 6.6?±?0.51?×?10² to 4.9?±?0.36?×?10⁴ cell ml^?1, respectively. The cluster I of 16S rRNA gene sequences showed distant identity (<92%) to the known Candidatus Scalindua species, inferring this cluster of anammox bacteria to be a new species, and a tentative name Candidatus “Scalindua sinooilfield” was proposed. The results extended the existence of anammox bacteria to the high-temperature oil reservoirs.     

Molecular Evidence for the Broad Distribution of Anaerobic Ammonium-Oxidizing Bacteria in Freshwater and Marine Sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of ~700-bp 16S rRNA gene sequences with >96% homology to the “Candidatus Scalindua” group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to “Ca. Scalindua,” “Candidatus Brocadia,” and “Candidatus Kuenenia.” This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Diversity,abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface sediments of the South China Sea

In marine ecosystems, both nitrite‐reducing bacteria and anaerobic ammonium‐oxidizing (anammox) bacteria, containing different types of NO‐forming nitrite reductase–encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua‐nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO‐forming nitrite‐reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua‐nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua‐nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua‐nirS genes share similar niche in deep‐sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite‐reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).     

Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.     

Anammox bacterial populations in deep marine hypersaline gradient systems

To extend the knowledge of anaerobic ammonium oxidation (anammox) habitats, bacterial communities were examined in two hypersaline sulphidic basins in Eastern Mediterranean Sea. The 2 m thick seawater–brine haloclines of the deep anoxic hypersaline basins Bannock and L’Atalante were sampled in intervals of 10 cm with increasing salinity. ¹⁵N isotope pairing incubation experiments showed the production of ²⁹N₂ and ³⁰N₂ gases in the chemoclines, ranging from 6.0 to 9.2 % salinity of the L’Atalante basin. Potential anammox rates ranged from 2.52 to 49.65 nmol N₂ L^?1 day^?1 while denitrification was a major N₂ production pathway, accounting for more than 85.5 % of total N₂ production. Anammox-related 16S rRNA genes were detected along the L’Atalante and Bannock haloclines up to 24 % salinity, and the amplification of the hydrazine synthase genes (hzsA) further confirmed the presence of anammox bacteria in Bannock. Fluorescence in situ hybridisation and sequence analysis of 16S rRNA genes identified representatives of the marine anammox genus ‘Candidatus Scalindua’ and putatively new operational taxonomic units closely affiliated to sequences retrieved in marine environments that have documented anammox activity. ‘Scalindua brodae’ like sequences constituted up to 84.4 % of the sequences retrieved from Bannock. The anammox community in L’Atalante was different than in Bannock and was stratified according to salinity increase. This study putatively extends anammox bacterial habitats to extremely saline sulphidic ecosystems.