New PCR primers targeting hydrazine synthase and cytochrome c biogenesis proteins in anammox bacteria

PCR primers targeting genes encoding the two proteins of anammox bacteria, hydrazine synthase and cytochrome c biogenesis protein, were designed and tested in this study. Three different ecotypes of samples, namely ocean sediments, coastal wetland sediments, and wastewater treatment plant (WWTP) samples, were used to assess the primer efficiency and the community structures of anammox bacteria retrieved by 16S ribosomal RNA (rRNA) and the functional genes. Abundances of hzsB gene of anammox bacteria in South China Sea (SCS) samples were significantly correlated with 16S rRNA gene by qPCR method. And hzsB and hzsC gene primer pair hzsB364f-hzsB640r and hzsC745f-hzsC862r in combination with anammox bacterial 16S rRNA gene primers were recommended for quantifying anammox bacteria. Congruent with 16S rRNA gene-based community study, functional gene hzsB could also delineate the coastal-ocean distributing pattern, and seawater depth was positively associated with the diversity and abundance of anammox bacteria from shallow- to deep-sea. Both hzsC and ccsA genes could differentiate marine samples between deep and shallow groups of the Scalindua sp. clades. As for WWTP samples, non-Scalindua anammox bacteria reflected by hzsB, hzsC, ccsA, and ccsB gene-based libraries showed a similar distribution pattern with that by 16S rRNA gene. NH₄ ⁺ and NH₄ ⁺/Σ(NO₃ ⁻ + NO₂ ⁻) positively correlated with anammox bacteria gene diversity, but organic matter contents correlated negatively with anammox bacteria gene diversity in SCS. Salinity was positively associated with diversity indices of hzsC and ccsB gene-harboring anammox bacteria communities and could potentially differentiate the distribution patterns between shallow- and deep-sea sediment samples. SCS surface sediments harbored considerably diverse community of Scalindua. A new Mai Po clade representing coastal estuary wetland anammox bacteria group based on 16S rRNA gene phylogeny is proposed. Existence of anammox bacteria within wider coverage of genera in Mai Po wetland indicates this unique niche is very complex, and species of anammox bacteria are niche-specific with different physiological properties towards substrates competing and chemical tolerance capability.

     

Further Analysis of Anammox Bacterial Community Structures Along an Anthropogenic Nitrogen-Input Gradient from the Riparian Sediments of the Pearl River Delta to the Deep-Ocean Sediments of the South China Sea

The community structures of anammox bacteria in sediments along an anthropogenic inorganic nitrogen input gradient were further delineated with the newly available information incorporated. Anammox bacterial 16S rRNA gene-amplified sequences retrieved from riparian sediments of the Pearl River, Mai Po coastal wetland, and the South China Sea (SCS) sediments were compiled, compared and analyzed. Results indicated that the community structures of anammox bacteria varied from the upstream of the Pearl River to deep-ocean sediment of the SCS along the anthropogenic input grandient. Mai Po wetland had the most diverse anammox bacteria, followed by the shallow SCS, deep SCS and the Pearl River. Genera of the anammox bacteria Kuenenia and Brocadia showed higher proportion in the riparian sediments of the Pearl River, while those of Kuenenia and Scalindua dominated the Mai Po coastal wetland. The Scalindua subclusters showed apparent segregation in coastal wetland (S. zhenghei-III and S. wagneri), shallow SCS (S. zhenghei-I and S3) and deep SCS (S. zhenghei-I, S2 and S. arabica). Pearson correlation analysis indicated nitrogen species [NH₄⁺ and ∑(NO₂^?+NO₃^? )] negatively correlated with the diversity indices of anammox bacteria. Canonical correspondence analysis (CCA) showed that salinity, inorganic nitrogen [NH₄+, ∑(NO₂^?+NO₃^?)], and ratio of NH₄⁺/∑(NO₂^? +NO₃^?) significantly affected the bacterial community compositions. Results collectively support that the community composition of anammox bacteria can serve as a bio-indicator to the anthropogenic terrestrial N input or pollution.     

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.     

A comparison of two 16S rRNA gene-based PCR primer sets in unraveling anammox bacteria from different environmental samples

Two 16S rRNA gene-based PCR primer sets (Brod541F/Amx820R and A438f/A684r) for detecting anammox bacteria were compared using sediments from Mai Po wetlands (MP), the South China Sea (SCS), a freshwater reservoir (R2), and sludge granules from a wastewater treatment plant (A2). By comparing their ability in profiling anammox bacteria, the recovered diversity, community structure, and abundance of anammox bacteria among all these diverse samples indicated that A438f/A684r performed better than Brod541F/Amx820R in retrieving anammox bacteria from these different environmental samples. Five Scalindua subclusters (zhenghei-I, SCS-I, SCS-III, arabica, and brodae) dominated in SCS whereas two Scalindua subclusters (zhenghei-II and wagneri) and one cluster of Kuenenia dominated in MP. R2 showed a higher diversity of anammox bacteria with two new retrieved clusters (R2-New-1 and R2-New-2), which deserves further detailed study. The dominance of Brocadia in sample A2 was supported by both of the primer sets used. Results collectively indicate strongly niche-specific community structures of anammox bacteria in different environments, and A438f/A684r is highly recommended for screening anammox bacteria from various environments when dealing with a collection of samples with diverse physiochemical characteristics.     

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples.     

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).     

Diversity and abundance of anammox bacterial community in the deep-ocean surface sediment from equatorial Pacific

The community structure and diversity of anaerobic ammonium oxidation (anammox) bacteria in the surface sediments of equatorial Pacific were investigated by phylogenic analysis of 16S rRNA and hydrazine oxidoreductase (hzo) genes and PCoA (principal coordinates analysis) statistical analysis. Results indicated that 16S rRNA and hzo sequences in the P2 (off the center of western Pacific warm pool) and P3 (in the eastern equatorial Pacific) sites all belong to the Candidatus “Scalindua”, the dominate anammox bacteria in the low-temperature marine environment proved by previous studies. However, in the P1 site (in center of warm pool of western Pacific), large part of 16S rRNA gene sequences formed a separated cluster. Meanwhile, hzo gene sequences from P1 sediment also grouped into a single cluster. PCoA analysis demonstrated that the anammox community structure in the P1 has significant geographical distributional difference from that of P2, P3, and other marine environments based on 16S rRNA and hzo genes. The abundances of anammox bacteria in surface sediments of equatorial Pacific were quantified by q-PCR analysis of hzo genes, which ranged from 3.98 × 10³ to 1.17 × 10⁴ copies g⁻¹ dry sediments. These results suggested that a special anammox bacteria phylotypes exist in the surface sediment of the western Pacific warm pool, which adapted to the specific habitat and maybe involved in the nitrogen loss process from the fixed inventory in the habitat.     

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus “Scalindua” group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.     

The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community

An anaerobic ammonium-oxidation (anammox) reactor was operated for more than 500 days and the anammox activity of the biomass in the reactor reached 0.58 kg N_total/kg VSS d. The removal ratios of NO₂⁻-N to NH₄⁺-N in both reactor and activity tests were nearly 1.1. The bacterial diversity in the reactor was investigated by analysis of 16S rRNA gene clone libraries and quantitative real-time PCR (qPCR). The analysis showed that more than half of the clones in the library were affiliated to recognized filamentous bacteria. The previously recognized anammox bacterium (AnAOB) Candidatus Kuenenia stuttgartiensis was only detected by using a Planctomycetes-specific 16S rRNA gene primer set. However, at least two different types of AnAOB were detected by the primer set targeting the hydrazine-oxidizing enzyme gene (hzo). The aerobic ammonium-oxidizing bacteria (AAOB) Nitrosomonas europaea–eutropha group, which is widely detected in oxygen-limited environments, was also found in this reactor. The result of qPCR indicated that AnAOB comprised 16% of the community population while AAOB comprised less than 1% in the reactor.     

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.     

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.     

Anammox Bacterial Diversity in Various Aquatic Ecosystems Based on the Detection of Hydrazine Oxidase Genes (<Emphasis Type="Italic">hzoA</Emphasis>/<Emphasis Type="Italic">hzoB</Emphasis>)

Anammox bacteria belonging to the phylum Planctomycetes are responsible for N removal through NH₄⁺ oxidation coupled with NO₂⁻ reduction. Microbial diversity and ecology of anammox bacteria have not yet been fully revealed due to limitations of 16S rRNA analysis. The hydrazine oxidase gene in cluster 1 (hereafter hzoA/hzoB) was suggested as a proper genetic marker due to its high expression and ubiquitous presence in anammox bacteria. We conducted a comparative analysis of 16S rRNA and hzoA/hzoB genes to reveal anammox bacterial diversity and distribution in various aquatic environments. Phylogenetic analyses of 16S rRNA and hzoA/hzoB genes showed the dominance of Scalindua organisms in marine ecosystems, but there was no congruence of 16S rRNA and hzoA/hzoB gene phylogenies among the freshwater anammox bacteria associated with Brocadia sp., Jettenia sp., and Anammoxoglobus sp. Higher diversity of anammox bacteria was revealed based on hzoA/hzoB genes than 16S rRNA genes in the examined environments. Multiple regression analysis showed that salinity had significant influence on differential distribution and diversity of anammox bacteria in different ecosystems. Thus, molecular detection and resulting phylogeny of the hzoA/hzoB gene generated a better understanding of anammox bacterial diversity and their ecological distribution in various aquatic ecosystems.     

Co-Occurring Anammox,Denitrification, and Codenitrification in Agricultural Soils

Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N₂ gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N₂ in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N₂ production, molecular and ¹⁵N isotope analyses were conducted with soil samples collected at six different agricultural fields in the United States. Denitrifying and anammox bacterial abundances were measured based on quantitative PCR (qPCR) of nitrous oxide reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed spacer (ITS) of Fusarium oxysporum was quantified to estimate the abundance of codenitrifying and denitrifying fungi. ¹⁵N tracer incubation experiments with ¹⁵NO₃⁻ or ¹⁵NH₄⁺ addition were conducted to measure the N₂ production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N₂ production rates in soil samples. Denitrifying bacteria were found to be the most abundant, followed by F. oxysporum based on the qPCR assays. The potential denitrification rates by bacteria and fungi ranged from 4.118 to 42.121 nmol N₂-N g⁻¹ day⁻¹, while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N₂-N g⁻¹ day⁻¹. Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N₂ production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.     

Effects of Environmental Factors on Anammox Bacterial Community Structure in Sediments of a Freshwater Aquaculture Farm,Yangcheng Lake

Previous studies have reported wide distribution of anaerobic ammonia oxidation (anammox) bacteria in various ecosystems. However, little is known about the distribution of anammox bacteria under varying environmental conditions in intensive aquaculture systems. In Yangcheng Lake, a famous crab farm situated in the Yangtze River Delta, sediment samples were collected in October (feeding period) and January (nonfeeding period) to analyze the distribution and diversity of anammox bacteria and their relationships with environmental factors. Based on the functional biomarker of Anammox bacteria, hzo gene, anammox bacterial clone libraries were constructed and their abundances were determined by quantitative PCR (qPCR). The Anammox bacteria were detected in the lake with the abundances ranging from 0.70 × 10⁵ to 6.05 × 10⁵ copies per gram of sediment. Sequences from eight clone libraries yielded seven unique operational taxonomic units (OTUs), distantly related to the Candidatus Jettenia genera with a similarity of about 91%. The Anammox bacterial community structures, diversities and abundances varied spatiotemporally with environmental conditions. In October, the level of the nitrogen compounds, the diversity, evenness and abundance of Anammox bacteria were higher than in January. The predominant OTU of samples changed from HZO-OTU-1 (34.25%) in January to HZO-OTU-2 (28.90%) in October. Moreover, the site (SW) nearing to sewage inlet was lack of HZO-OTU-7 in January. Canonical correspondence analysis (CCA) showed that the pore water NO₂^? concentration, ammonium to nitrogen oxides ratio (NH₄⁺/NO_x^?) and total organic carbon to total nitrogen ratio (TOC/TN) contributed most to Anammox bacterial community structures variances. Pearson correlations analysis revealed that the Anammox bacteria abundance had positive co-relationships with TN, NH₄⁺, NO₃^? concentrations, and negative correlation with TOC/TN in porewater.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.