Anammox Bacterial 16S rRNA Gene Research Articles

A comprehensive insight into the abundance and community of anammox bacteria in sediments of Hangzhou Bay, China

A total of 13 surface sediments were collected from Hangzhou Bay (HZB) for an investigation into the distribution and influencing factors of anammox bacterial community. The anammox bacterial 16S rRNA and hzo genes ranged between 2.34 × 105 to 9.22 × 105 copies/g and 3.68 × 105 to 1.70 × 106 copies/g, respectively. The results of high throughput sequencing (HTS) revealed that the obtained OTUs were affiliated with five known genera, named Ca. Scalindua, Ca. Jettenia, Ca. Brocadia, Ca. Kuenenia and Ca. Anammoxoglobus. RDA analysis indicated that salinity, pH, and water depth influenced the anammox bacterial community. Furthermore, network analysis identified Ca. Scalindua as a key genus. Neutral community model (NCM) and modified stochasticity ratio (MST) indicated that the deterministic process dominated the anammox bacterial community assembly. Overall, this study offers a more comprehensive understanding of the abundance and community of anammox bacteria in the sediments of HZB.

Nitrogen removal in freshwater sediments of riparian zone: N-loss pathways and environmental controls.

The riparian zone is an important location of nitrogen removal in the terrestrial and aquatic ecosystems. Many studies have focused on the nitrogen removal efficiency and one or two nitrogen removal processes in the riparian zone, and less attention has been paid to the interaction of different nitrogen transformation processes and the impact of in situ environmental conditions. The molecular biotechnology, microcosm culture experiments and 15N stable isotope tracing techniques were used in this research at the riparian zone in Weinan section of the Wei River, to reveal the nitrogen removal mechanism of riparian zone with multi-layer lithologic structure. The results showed that the nitrogen removal rate in the riparian zone was 4.14-35.19 μmol·N·kg-1·h-1. Denitrification, dissimilatory reduction to ammonium (DNRA) and anaerobic ammonium oxidation (anammox) jointly achieved the natural attenuation process of nitrogen in the riparian zone, and denitrification was the dominant process (accounting for 59.6%). High dissolved organic nitrogen and nitrate ratio (DOC:NO3-) would promote denitrification, but when the NO3- content was less than 0.06 mg/kg, DNRA would occur in preference to denitrification. Furthermore, the abundances of functional genes (norB, nirS, nrfA) and anammox bacterial 16S rRNA gene showed similar distribution patterns with the corresponding nitrogen transformation rates. Sedimentary NOX-, Fe(II), dissolved organic carbon (DOC) and the nitrogen transformation functional microbial abundance were the main factors affecting nitrogen removal in the riparian zone. Fe (II) promoted NO3- attenuation through nitrate dependent ferrous oxidation process under microbial mediation, and DOC promotes NO3- attenuation through enhancing DNRA effect. The results of this study can be used for the management of the riparian zone and the prevention and control of global nitrogen pollution.

Overlooked contribution of water column to nitrogen removal in estuarine turbidity maximum zone (TMZ)

Estuarine systems are important sites of eliminating reactive nitrogen (N) delivered from land to sea. Numerous studies have focused on N cycling in estuarine sediment. However, the N elimination role of suspended sediments in estuarine turbid water column, which might provide anaerobic microenvironment for N loss, is rarely considered. This study examined the community dynamics and activities of denitrifying and anaerobic ammonium oxidation (anammox) bacteria in the water column of the turbidity maximum zone (TMZ) of the Yangtze Estuary, using molecular and 15N isotope-tracing techniques. Results showed that the anammox bacterial community was dominated by Candidatus Kuenenia and Candidatus Brocadia in the TMZ water column, while the main nirS-harboring denitrifiers were affiliated with Rhodobacterales. The denitrifying nirS gene was two orders of magnitude more abundant than anammox bacterial 16S rRNA gene, ranging from 1.77 × 105 to 1.42 × 108 copies l−1 and from 7.68 × 104 to 4.27 × 106 copies l−1, respectively. Compared with anammox, denitrification, with rates of 0.88 to 20.83 μmol N l−1 d−1, overwhelmingly dominated the N removal in the TMZ water column and was significantly correlated to suspended sediment concentrations (SSC). Based on the measured N removal rates, it was estimated that about 2.5 × 105 ton N was annually removed from the TMZ water column, accounting for approximately 18.5% of the total inorganic N (TIN) discharged from the Yangtze River. Overall, this study implies the importance of estuarine turbid water column in controlling N budget, and also improves the understanding of N loss mechanisms in estuarine TMZ systems.

Abundance, diversity, and community composition of anammox bacteria in sediments of Xiangshan Bay, China

Xiangshan Bay (XSB) is a typical semi-closed bay rich in dissolved inorganic nitrogen and anammox is one of the important processes of nitrogen cycle. The anammox bacterial abundance and diversity in XSB sediments was investigated. Five known genera were detected based on the anammox bacterial 16S rRNA gene, namely, Ca. Scalindua, Ca. Brocadia, Ca. Kuenenia, Ca. Jettenia, and Ca. Anammoxoglobus, and hzo genes affiliating with Ca. Scalindua were also discovered. The anammox bacterial 16S rRNA and hzo genes in the sediments varied from 2.10 × 106 to 2.18 × 107 and from 2.90 × 106 to 5.48 × 108 copies/g (fresh weight), respectively. Pearson correlation analysis demonstrated that the abundance of the hzo gene was positively correlated with the concentration of NH4+ but negatively correlated with salinity and pH. Redundancy analysis showed that salinity, dissolved oxygen, and temperature were the three main factors influencing the community composition of anammox bacteria obtained by the anammox bacterial 16S rRNA gene. These findings help us to further understand the distribution and community composition of anammox bacteria in estuary sediments.

Distribution and Community Composition of Anammox Bacteria in Shallow Groundwater of the Kathmandu Valley, Nepal.

The abundance and diversity of anaerobic ammonium oxidation (anammox) bacteria were assessed in 152 groundwater samples in the Kathmandu Valley, Nepal. Anammox bacterial 16S rRNA genes were detected in 54% (37/68) of samples collected in the dry season at 1.6×105–8.8×106 copies L–1, and in 60% (50/84) of samples collected in the wet season at 4.3×104–1.2×107 copies L–1. The 16S rRNA genes of “Candidatus Brocadia”, “Candidatus Anammoxoglobus”, and five new deduced anammox bacterial phylotypes were detected in the shallow groundwater samples. Diverse anammox bacteria were broadly distributed in the shallow groundwater aquifer of the Kathmandu Valley.

Spatial variability pattern of the anaerobic ammonia-oxidizing bacterial community across a salinity gradient from river to ocean

In natural habitats, the diversity of anaerobic ammonia-oxidizing (anammox) bacteria could be affected by multiple environmental variables. In this study, we investigated the distribution of the anammox bacterial community in surface sediment from the Dongjiang River (riverine sediment, DJ) to the Pearl River Estuary (estuarine sediment, PRE) and then to the South China Sea (coastal sediment, SCS). The results revealed evident differences in the structural diversity of anammox bacteria in three different habitats. Candidatus Brocadia accounted for approximately 90% of the total anammox bacteria in DJ, conversely, Ca. Scalindua dominated in the SCS. Nevertheless, Ca. Scalindua, Ca. Brocadia and Ca. Kuenenia coexisted in the PRE. The qPCR results indicated that anammox bacterial 16S rRNA gene abundance ranged from 2.23 × 105 to 1.19 × 107 copies g-1 of wet weight, but no significant correlation was found between the abundances and environmental variables (p > 0.05). The relative abundances of Ca. Brocadia gradually decreased with increasing salinity, and Ca. Scalindua showed the opposite trend, suggesting that salinity was a crucial factor in sculpturing the community composition of anammox bacteria in natural environments. Ca. Brocadia should be able to live in freshwater ecosystems, but it can also tolerate a certain level of salinity. Ca. Scalindua was halophilic anammox bacterium and exists only in saline environments. Ca. Kuenenia could adapt to a wide range of salinity and preferred to live in high DIN level conditions according to our search. The distribution pattern of anammox bacteria may be the result of microbial migration and long-term adaptation to salinity.

Earthworm gut: An overlooked niche for anaerobic ammonium oxidation in agricultural soil

Soil fauna takes an active part in accelerating turnover of nutrients in terrestrial ecosystems. Anaerobic ammonium oxidation (anammox) has been widely characterized, however, whether anammox is active in earthworm gut and the effect of earthworm on anammox in soil remain unknown. In this study, the activity, abundance and community of anammox bacteria in earthworm guts and soils from microcosms were determined using a 15N-tracing technique, quantitative PCR, and anammox bacterial 16S rRNA gene amplicon sequencing. Results showed that anammox rates in guts ranged between 5.81 and 14.19 nmol N g−1 dw gut content h−1, which were significantly (P < 0.01) higher than that in their surrounding soils during 30 day incubation. On the contrary, abundances of hzsB genes encoding subunit B hydrazine synthase in guts were significantly (P < 0.05) lower than those in their surrounding soils. Anammox rates, denitrification N2 production rates and hzsB genes in soils with earthworms were significantly (P < 0.05) lower than those in control soils. Anammox bacterial compositions differed significantly (P < 0.05) between gut and soil, and earthworm altered anammox bacterial communities in soils. Brocadia, Kuenenia and abundant unclassified anammox bacteria were detected in collected soils and gut contents, in which Brocadia was only detected in guts. These results suggested that microbes in earthworm gut increase, but present of earthworm reduces anammox and denitrification associated N loss by altering the anammox bacterial community compositions in soils.

Nitrogen loss by anaerobic ammonium oxidation in a mangrove wetland of the Zhangjiang Estuary, China

Anaerobic ammonium oxidation (anammox), a microbial process in which NH4+ is oxidized to N2 gas, is considered a significant nitrogen cycle process, but its significance in mangrove wetland sediments, particularly its depth- and genus-specific distribution and activity have remained uncertain. Here we report the vertical distribution, abundance, activity and role of anammox bacteria in mangrove sediments of Zhangjiang Estuary, China. We used stable isotope-tracer techniques, 16S rRNA and anammox bacterial functional gene (Hydrazine synthase B: hzsB) clone libraries and quantitative polymerase chain reaction (qPCR) assays, along with an assessment of nutrient profiles of sediment core samples. We observed a widespread occurrence of anammox bacteria at different depths of mangrove sediments. The abundance of anammox bacterial 16S rRNA and hzsB genes ranged from 0.41×107 to 9.74×107 and from 0.42×106 to 6.44×106 copies per gram of dry soil and peaked in the upper layer of mangrove sediments. We also verified the co-occurrence of different genera of anammox microorganisms in mangrove sediments, with Candidatus Scalindua and Candidatus Kuenenia being the dominant genera. Potential anammox rates ranged from 4.83 to 277.36 nmolN2·g−1·d−1 at different depths of sediment cores, and the highest rates were found in the deeper layer (70–100cm) of mangrove sediments. Scaling our findings up to the entire mangrove system, we estimated that anammox hotspots accounted for a loss of 751 gN·m−2·y−1, and contributed to over 12% of the nitrogen lost from the deeper layer of mangrove sediments in this region.

Evaluating the response of anaerobic ammonium-oxidizing bacteria to heavy metal spill in freshwater sediment.

Anaerobic ammonium-oxidizing (anammox) bacteria can play an important role in nitrogen elimination in the environment. However, the effect of heavy metals on anammox bacteria in aquatic ecosystem remains largely unknown. The present study investigated the variability of anammox bacterial community in a freshwater reservoir after a severe heavy metal spill. The richness (Chao1 richness estimator = 2-18), diversity (Shannon index = 0.26-2.04) and community structure of anammox bacteria changed considerably with sampling date, while anammox bacterial abundance (from 1.38 × 105 to 3.09 × 105 anammox bacterial 16S rRNA gene copies per gram dry sediment) was less responsive to metal spill. Anammox bacterial communities were mainly composed of Brocadia- and Anammoxoglobus-like bacteria as well as novel phylotype, however, there relative abundance varied among sampling dates. This work could add the knowledge of the response of anammox bacteria to heavy metal contamination.

Soil dissimilatory nitrate reduction processes in the Spartina alterniflora invasion chronosequences of a coastal wetland of southeastern China: Dynamics and environmental implications

The invasion of Spartina alterniflora has a significant influence on soil biogeochemistry cycling in coastal wetlands. However, the roles of the S. alterniflora invasion chronosequence in regulating soil dissimilatory NO3 − reduction processes (denitrification (DNF), anaerobic ammonium oxidation (ANA) and dissimilatory nitrate reduction to ammonium (DNRA)) remains unclear. The objective of this study was therefore to reveal the effects of S. alterniflora invasion on the soil NO3 − reduction processes and associated gene abundance. We investigated plant biomass, soil properties, NO3 − reduction processes and associated gene abundance of NO3 − reduction pathways following S. alterniflora invasion chronosequences of 6, 10, and 14 years compared to Cyperus malaccensis in a coastal wetland of southeastern China. The S. alterniflora invasion generally increased plant biomass, soil water content, available substrates, nirS, anammox bacterial 16S rRNA and nrfA gene abundance, but it decreased soil bulk density. Soil DNF, ANA and DNRA rates in stands of S. alterniflora ranged from 1.52 to 17.58, 0.31 to 1.27 and 0.14 to 2.01 nmol N g−1 h−1, respectively, which were generally higher than the values in stands of C. malaccensis. The soil NO3 − reduction rates generally increased with the increasing chronosequence of invasion by S. alterniflora, while the changes in DNF and ANA rates were less pronounced than changes in DNRA. DNF was the dominant pathway (70.00–92.41%), and the ANA and DNRA contributed 2.49–15.27% and 5.10–20.75% to the total NO3 − reduction, respectively. The contributions of DNF and ANA to the total NO3 − reduction decreased slightly, while the contribution of DNRA increased remarkably after S. alterniflora invasion. Soil NO3 − reduction processes were influenced by available substrates and associated microbial activities. It is estimated that an N loss of approximately 520.97 g N m−2 yr.−1 in C. malaccensis and 794.46 g N m−2 yr.−1 in S. alterniflora were linked to both DNF and ANA processes. The S. alterniflora invasion altered soil NO3 − reduction processes by increasing soil microbial activities and available substrates and thus may further mediate the soil N availability in the coastal wetlands.

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.

Effects of polybrominated diphenyl ethers and plant species on nitrification, denitrification and anammox in mangrove soils

Little is known about polybrominated diphenyl ethers (PBDEs) and planting affect biogeochemical processes, and their impact on microbial nitrogen (N) transformation in soil. A 12-month microcosm experiment was conducted to understand the effects of a mixture of PBDEs at two contamination levels, 2 and 20mgkg−1dry weight representing low and high soil contamination, respectively, using two mangrove plant species, namely Kandelia obovata (Ko) and Bruguiera gymnorrhiza (Bg), on nitrification, denitrification and anammox in mangrove soils. No significant changes in these N transformation processes were found at month 3 and at a low level of PBDEs in both plant species, suggesting that short-term exposure to 2mgkg−1 contamination did not affect microbial N transformation. At month 12, a high level of PBDE contamination significantly decreased the nitrification potential activity and the copy numbers of archaeal amoA and bacterial amoA gene in Ko soil, but such inhibitory effect was not significant in Bg soil. On the contrary, the denitrification-related parameters, including the activities of nitrate reductase and nitrite reductase, potential denitrification activity and copy numbers of nirK, nirS and nosZ gene, were stimulated by a high level of PBDE contamination in both Ko and Bg soils, and the stimulation was higher in the more anaerobic Bg soil. Different from denitrification, a high level of PBDE contamination decreased the copy numbers of anammox bacterial 16S rRNA gene in Bg soil but not in Ko soil; this was possibly related to the lower nitrate concentration in Bg soil that might inhibit the growth of anammox bacteria. These results indicated that the effects of PBDEs on microbial N transformation were plant species-specific, with the nitrifying microorganisms in Ko soil more susceptible to PBDE contamination, while denitrification and anammox in Bg soil were more sensitive.

Diversity, Community Composition and Abundance of Anammox Bacteria in Sediments of the North Marginal Seas of China.

Over the past few decades, anammox bacteria have been recognized as key players that contribute significantly to the release of large amounts of nitrogen in the global marine nitrogen cycle. In the present study, the diversity, community composition, and abundance of anammox bacteria from the sediments of four diverse regions in the north marginal seas in China were determined via clone library construction and a quantitative PCR analysis. The clone libraries retrieved by the 16S rRNA gene and Hzo gene markers indicated that “Candidatus Scalindua” was the predominant group throughout the sites examined. The 16S rRNA gene clone libraries revealed exceptional diversity by identifying two potential novel anammox clades, as evidenced by the high sequence similarities between these two clades and known anammox genera, and their unique phylogenetic positions with high bootstrap values. However, their potential roles in the anammox reaction need to be validated. Six novel members of Planctomycetes, divergent from the known genera of anammox bacteria, were also detected. A phylogenetic analysis by Hzo protein sequences revealed the existence of two known genera, i.e., “Candidatus Jettenia” and “Candidatus Anammoxoglobus”, which are rarely captured from marine sediments. Among all ecological parameters investigated, the distribution patterns and composition of anammox bacteria were found to be influenced by salinity, total organic matter, and temperature. The abundance of the anammox bacterial 16S rRNA gene from the sites examined ranged between 3.95×105 and 9.21×105 copies g−1 wet sediment and positively correlated with the median size of the sediment sample.

Activity, abundance and community structure of anammox bacteria along depth profiles in three different paddy soils

Anaerobic ammonium oxidation (anammox) is a globally important nitrogen-cycling process mediated by specialized microbes, and has been demonstrated to be ubiquitous in anoxic natural settings and bioreactors. However, our knowledge of its prevalence in different paddy soil types and along the depth profiles remains largely undocumented. Here, mesocosm incubations were constructed to investigate the activity and community compositions of anammox bacteria across different depth layers of three different paddy soils. 15N tracer experiments showed that anammox rates were prevalent in almost all depth layers of an inceptisol (Binhai, BH) and an oxisol (Leizhou, LZ), but was absent in an ultisol (Taoyuan, TY). The functional gene hzsB of anammox bacteria was only detected in BH and LZ soil profiles with detected anammox activity. Anammox accounted for 0.4%–12.2% of the total N2 production, and anammox rates were 0.02–0.77 nmol N g−1 dry soil h−1 and significantly correlated with soil pH and electric conductivity. Anammox activity and abundance of hzsB genes were significantly lower in the surface soil layers (0–5 cm) than in the sub-surface soil layers (20–60 cm). Candidatus Brocadia, Candidatus Kuenenia and Candidatus Jettenia were detected by cloning and sequencing, with Candidatus Brocadia being dominant in all the three soils and Candidatus Jettenia being more frequently detected in the LZ soil. The operational taxonomic unit number and diversity indices of anammox bacterial 16S rRNA gene increased with soil depth. Our results demonstrate that anammox is more common and active in alkaline soils than in acidic soils, in deep paddy soil layers than in surface soil layers, and that anammox activities are likely to be regulated by soil chemical properties such as pH, salinity and redox potential.

Distribution of anammox bacteria in a free-water-surface constructed wetland with wild rice (Zizania latifolia)

We investigated the distribution of anaerobic ammonium oxidation (anammox) bacteria under heterogeneous environmental conditions in a free-water-surface constructed wetland planted with wild rice (Zizania latifolia). The wetland receives effluent from a domestic wastewater treatment facility. Potential anammox activity was estimated using a nitrogen isotope pairing technique: activity was highest in the upper soil layer (0–2cm). We also surveyed the horizontal distribution of potential anammox activity at seven sites over the wetland under different environmental conditions, along with copy numbers of anammox genes, which were estimated using quantitative PCR analysis. Three submerged sites showed substantial potential anammox activity and relatively high anammox copy numbers. The highest activity and highest copy number were 50.3nmol N2g-drymatter−1h−1 and 1.42×1013copyg-DNA−1, respectively, observed at a site that was submerged, vegetated, and immediately adjacent to the inflow from the domestic wastewater treatment facility. Phylogenetic analysis of anammox bacterial 16S rRNA genes revealed anammox bacteria with high similarity to uncultured anammox bacteria from freshwater environments such as paddy fields, and close to species in genera including “Candidatus Brocadia” and “Ca. Jettenia”. The highest contribution of potential anammox activity to potential total N2 gas production was 3.1%. This study provides new and useful information about the distribution of anammox in a constructed wetland treating actual wastewater.

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere.

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N2 production with activities of 0.33–0.64 nmol N2 g−1 soil h−1, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N2 production with lower activities of 0.08–0.26 nmol N2 g−1 soil h−1. Higher anammox bacterial cells were observed (0.75–1.4 × 107 copies g−1 soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 106 copies g−1 soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia' and ‘Candidatus Brocadia' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.

Nitrogen removal and spatial distribution of denitrifier and anammox communities in a bioreactor for mine drainage treatment

Mine drainage water may contain high levels of nitrate (NO3−) due to undetonated nitrogen-based explosives. The removal of NO3− and nitrite (NO2−) in cold climates through the microbial process of denitrification was evaluated using a pilot-scale fixed-bed bioreactor (27 m3). Surface water was diverted into the above-ground bioreactor filled with sawdust, crushed rock, and sewage sludge. At hydraulic residence times of ca.15 h and with the addition of acetate, NO3− and NO2− were removed to below detection levels at a NO3− removal rate of 5–10 g N m−3 (bioreactor material) d−1. The functional groups contributing to nitrogen removal in the bioreactor were studied by quantifying nirS and nirK present in denitrifying bacteria, nosZI and nosZII genes from the nitrous oxide – reducing community, and a taxa-specific part of the16S rRNA gene for the anammox community. The abundances of nirS and nirK were almost 2 orders of magnitude greater than the anammox specific 16S rRNA gene, indicating that denitrification was the main process involved in nitrogen removal. The spatial distribution of the quantified genes was heterogeneous in the bioreactor, with trends observed in gene abundance as a function of depth, distance from the bioreactor inlet, and along specific flowpaths. There was a significant relationship between the abundance of nirS, nirK, and nosZI genes and depth in the bioreactor, such that the abundance of organisms containing these genes may be controlled by oxygen diffusion and substrate supply in the partially or completely water-saturated material. Among the investigated microbial functional groups, nirS and anammox bacterial 16S rRNA genes exhibited a systematic trend of decreasing and increasing abundance, respectively, with distance from the inlet, which suggested that the functional groups respond differently to changing environmental conditions. The greater abundance of nirK along central flowpaths may indicate that the bioreactor design favored preferential flow along these flowpaths, away from the sides of the bioreactor. An improved bioreactor design should consider the role of preferential flowpaths and the heterogeneous distribution of the genetic potential for denitrification, nitrous oxide reduction and anammox on bioreactor function.

Relative Abundance of Ammonia Oxidizers, Denitrifiers, and Anammox Bacteria in Sediments of Hyper‐Nutrified Estuarine Tidal Flats and in Relation to Environmental Conditions

The relative abundances of nitrifying, denitrifying and anammox prokaryotes in sediments of three hyper‐nutrified estuarine tidal flats of Laizhou Bay, Bohai Sea, China were investigated. Quantitative PCR estimates indicated that in most cases archaeal (AOA) amoA genes were more abundant than bacterial (AOB) amoA genes, and ratio of AOA/AOB was correlated with pH, Cd, and Cu. Of the denitrifiers, nirK‐type outnumbered nirS‐type, with nosZ‐type being the lowest. Variation of the ratio between nirK and nirS abundance depends on pH, nitrite, nitrate, and Cd. The combination of (nirS + nirK‐nosZ), an indicator of genetic potentials for N2O emission, was only related to the temperature. Anammox bacterial 16S rRNA gene abundances were correlated with salinity, pH, nitrite, and Cu. In contrast, the contribution of anammox to N2 production, by using anammox bacterial 16S rRNA/nosZ ratio as a proxy, was correlated to temperature, ammonium and dissolved oxygen in the overlying water, ratio of organic carbon to nitrogen, and arsenic in sediments. Our study stresses that abundances of N‐cycling functional groups respond differently to variations of environmental conditions, and multiple factors including heavy metals with relatively low concentrations may play a role in shaping nitrogen cycling processes in these estuarine tidal flats.

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.

Evaluating Primers for Profiling Anaerobic Ammonia Oxidizing Bacteria within Freshwater Environments

Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r was the most suitable primer set for multiple molecular assessments of anammox bacteria in freshwater environments.