Microbial pathways for nitrogen loss in an upland soil.

The distribution and importance of anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) have been identified in aquatic ecosystems; their role in agricultural upland soils however has not yet been well investigated. In this study, we examined spatio-temporal distributions of anammox and n-damo bacteria in soil profiles (300 cm depth) from an agricultural upland. Monitoring nitrogen (N) conversion activity using isotope-tracing techniques over the course of one year showed denitrification (99.0% N-loss in the winter and 85.0% N-loss in the summer) predominated over anammox (1.0% N-loss in the winter and 14.4% N-loss in the summer) and n-damo (0.6% N-loss in the winter) in surface soils (0-20 cm). While below 20 cm depth, N-loss was dominated by anammox (79.4 ± 14.3% in the winter and 65.4 ± 12.5% in the summer) and n-damo was not detected. Phylogenetic analysis showed that Candidatus Brocadia anammoxidans dominated the anammox community in the surface soil and Candidatus Brocadia fulgida dominated below 20 cm depth. Dissimilatory nitrate reduction to ammonium (DNRA), another nitrite reduction process, was found to play a limited role (4.9 ± 3.5%) in the surface soil compared with denitrification; below 80 cm DNRA rates were much higher than rates of anammox and denitrification. Ammonium oxidation was the main source of NO2- above 80 cm (70.9 ± 23.3%), the key influencing factor on anammox rates, and nitrate reduction (100%) was the main NO2- source below 80 cm. Considering the anammox, n-damo and denitrification rates as a whole in the sampled soil profile, denitrification is still the main N-loss process in upland soils.

Microbial Nitrogen Cycle Hotspots in the Plant-Bed/Ditch System of a Constructed Wetland with N2O Mitigation.

Artificial microbial nitrogen (N) cycle hotspots in the plant-bed/ditch system were developed and investigated based on intact core and slurry assays measurement using isotopic tracing technology, quantitative PCR and high-throughput sequencing. By increasing hydraulic retention time and periodically fluctuating water level in heterogeneous riparian zones, hotspots of anammox, nitrification, denitrification, ammonium (NH4+) oxidation, nitrite (NO2-) oxidation, nitrate (NO3-) reduction and DNRA were all stimulated at the interface sediments, with the abundance and activity being about 1-3 orders of magnitude higher than those in nonhotspots. Isotopic pairing experiments revealed that in microbial hotspots, nitrite sources were higher than the sinks, and both NH4+ oxidation (55.8%) and NO3- reduction (44.2%) provided nitrite for anammox, which accounted for 43.0% of N-loss and 44.4% of NH4+ removal in riparian zones but did not involve nitrous oxide (N2O) emission risks. High-throughput analysis identified that bacterial quorum sensing mediated this anammox hotspot with B.fulgida dominating the anammox community, but it was B. anammoxidans and Jettenia sp. that contributed more to anammox activity. In the nonhotspot zones, the NO2- source (NO3- reduction dominated) was lower than the sink, limiting the effects on anammox. The in situ N2O flux measurement showed that the microbial hotspot had a 27.1% reduced N2O emission flux compared with the nonhotspot zones.

Anaerobic ammonium oxidation is a major N-sink in aquifer systems around the world.

Global-scale N-oxide contamination of groundwater within aquifers occurs due to the widespread use of N-bearing fertilizers and chemicals, threatening both human and environmental health. However, the conversion of these pollutants in active nitrogen (N) cycling processes in the subsurface biosphere still remains unclear. This study investigates the global occurrence of anaerobic ammonium oxidation (anammox) in aquifers, where anammox was found to be turned on and off between saturated and unsaturated soil horizons, and contributed 36.8-79.5% to N loss in saturated soil horizons, the remainder being due to denitrification which has traditionally been considered the main pathway for removal of N-pollutants from aquifers. Although anammox activity was undetectable in the unsaturated soil horizons, it could potentially be activated by contact with ascending groundwater. High-throughput pyrosequencing analysis identified Candidatus Brocadia anammoxidans as being the most abundant anammox bacterium in the saturated soils investigated. However, the anammox bacterial abundance was determined by the relative richness of Candidatus Jettenia asiatica. Isotopic pairing experiments revealed that coupling anammox with ammonium oxidation and respiratory ammonification enabled the formation of a revised N cycle in aquifer systems, in which respiratory ammonification acted as an important coordinator. Anammox can therefore contribute substantially to aquifer N cycling and its role in remediation of aquifers contaminated with N-oxides may be of global importance.

Spatiotemporal shifts of ammonia-oxidizing archaea abundance and structure during the restoration of a multiple pond and plant-bed/ditch wetland.

Ammonia-oxidizing archaea (AOA) microorganisms have been increasingly found in aquatic and terrestrial environments. These microorganisms make vital contributions to ammonia oxidation in such systems. However, their community succession characteristics in man-made wetland ecosystems have scarcely been reported. We assessed the AOA's spatiotemporal shifts in the sediments of a constructed wetland (CW) - the Shijiuyang constructed wetland (SJY-CW) - in China from the third year (2011) to the fifth year (2013) of the CW operation. The SJY-CW is composed of a pretreatment pond, a multiple plant-bed/ditch system, and a post-treatment pond. Results showed that AOA abundance in the pre- and post-treatment ponds remained invariant through 2011-2012 and decreased in 2013, while the abundance in the plant-bed/ditch system decreased gradually with wetland operation. The AOA abundance in 2013 was one order of magnitude lower than that through 2011-2012, and the AOA abundance in the plant-bed/ditch system was generally higher than that in the pre- and post-treatment ponds from 2011 to 2013. AOA diversity showed little temporal differentiation with a slightly decreasing trend for community richness index Chao1 and diversity index Shannon H' from 2011 to 2013. The AOA community was dominated by the Nitrososphaera cluster accompanied by an increasing Nitrosopumilus cluster and Nitrososphaera sister cluster within the wetland operation. Hierarchical clustering and redundancy analysis verified the horizontal shifts of AOA communities. The shifts occurred preferentially in the central plant-bed/ditch system. The operational duration of the wetland became a key factor influencing AOA abundance and community shift in SJY-CW sediments.

Denitrification is the main microbial N loss pathway on the Qinghai-Tibet Plateau above an elevation of 5000 m.

Soil nitrogen (N) deficiency is the major factor contributing to low primary productivity on the Qinghai-Tibet Plateau. However, most of our understanding of N cycling is still based on human disturbed environments, and the microbial mechanisms governing N loss in low primary productivity environment remain unclear. This study explores three microbial N loss pathways in eight wetland and dryland soil profiles from the Qinghai-Tibet Plateau, at an elevation of above 5000 m with little human activity, using 15N isotopic tracing slurry technology, quantitative PCR, and high-throughput sequencing. No denitrifying anaerobic methane oxidation was detected. Anammox occurred in two of the wetland (n = 4) and dryland (n = 4) soil profiles, while denitrification widely occurred and was the dominant N loss pathway in all samples. Where denitrification and anammox co-occurred, both abundance and activity were higher in wetland than in dryland soils and higher in surface than in subsurface soils. In comparison with non-anammox sites, nitrate levels initiate anammox-related N cycling. High-throughput sequencing and network analysis of nirK, nirS, nosZ, and hzsB gene communities showed that Bradyrhizobiaceae (a family of rhizobia) may play a dominant role in N loss pathways in this region. Given the geological evolution and relatively undisturbed habitat, these findings strongly suggest that denitrification is the dominant N loss pathway in terrestrial habitats of the Qing-Tibet Plateau with minimal anthropogenic activity.

High-throughput analysis of anammox bacteria in wetland and dryland soils along the altitudinal gradient in Qinghai-Tibet Plateau.

This study investigated the diversity, community composition, and abundance of anaerobic ammonium oxidation (anammox) bacteria along the altitudinal gradient in Qinghai-Tibet Plateau. Two types of soil samples (wetland and dryland soils, n = 123) were collected from 641 m to 5,033 m altitudes. Polymerase chain reaction (PCR) screening showed that anammox were not widespread, and were only detected in 9 sampling sites of the 50 sites tested by amplifying the 16S rRNA genes. Then, only samples collected from Linzhi (2,715 m), Rikaze (4,030 m), and Naqu (5,011 m), which were positive for the presence of anammox, were further processed to explore the biogeography of anammox bacteria in Qinghai-Tibet Plateau. Results of high-throughput sequencing targeting the hydrazine synthesis ß-subunit (hzsB) gene revealed the presence of three known anammox genera (Candidatus Brocadia, Candidatus Jettenia, and Candidatus Kuenenia) in both soil types. Their diversity, community composition, and abundance did not show significant variation with altitude at large scale. However, it was the small-scale environmental heterogeneities between wetland and dryland soils that determined their biogeographical distribution. Specifically, the dryland soils had higher diversity of anammox bacteria than the wetland soils, but their abundance patterns varied. The community composition of anammox bacteria were found to be influenced by soil nitrate content.

Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal.

Under decreasing C/N (from 8.8 to 3.5) conditions, an alternating anaerobic/aerobic biofilter (AABF) was used to remove nitrogen and accumulate/recover phosphorus (P) from synthetic wastewater. The AABF was periodically (every 10 days) fed with an additional carbon source (10 L, chemical oxygen demand (COD) = 900 mg L-1 sodium acetate (NaAC) solution) in the anaerobic phase to induce the release of P sequestered in the biofilm. An increase in PHA storage in the biofilm was observed and characterized with TEM and a GC-MS method. The accumulation of P and removal of total nitrogen occurred primarily in the aerobic phase. As the NH4+-N loading rate increased from 0.095 to 0.238 kg m-3 d-1 at a total empty bed retention time (EBRT) of 4.6 h, the TN removal in AABF was reduced from 91.2% to 43.4%, while the P removal or recovery rate remained unaffected. The high-throughput community sequencing analysis indicated that the relative abundance of Candidatus Competibacter, Nitrospira and Arcobacter increased while the Accumulibacter phosphatis decreased with an increase of ammonium loading rate within a short operational period (30 days). A putative N and P removal pattern via simultaneous nitrification and PHA-based denitrification, as well as P accumulation in the biofilm was proposed. The research demonstrated that an efficient N removal and P recovery process, i.e., simultaneous nitrification and denitrification, P accumulation and carbon source-regulated P recovery can be achieved by the symbiotic functional groups in a single biofilm reactor.

Nitrogen loss by anaerobic ammonium oxidation in unconfined aquifer soils.

Anaerobic ammonium oxidation (anammox) is recognized as an important process for nitrogen cycling, yet little is known about its role in the subsurface biosphere. In this study, we investigated the presence, abundance, and role of anammox bacteria in upland soil cores from Tianjin, China (20 m depth) and Basel, Switzerland (10 m depth), using isotope-tracing techniques, (q)PCR assays, and 16 S rRNA &hzsB gene clone libraries, along with nutrient profiles of soil core samples. Anammox in the phreatic (water-saturated) zone contributed to 37.5-67.6% of the N-loss (up to 0.675 gN m-2 d-1), with anammox activities of 0.005-0.74 nmolN g-1 soil h-1, which were even higher than the denitrification rates. By contrast, no significant anammox was measured in the vadose zone. Higher anammox bacterial cell densities were observed (0.75-1.4 × 107 copies g-1 soil) in the phreatic zone, where ammonia-oxidizing bacteria (AOB) maybe the major source of nitrite for anammox bacteria. The anammox bacterial cells in soils of the vadose zone were all <103 copies g-1 soil. We suggest that the subsurface provides a favorable niche for anammox bacteria whose contribution to N cycling and groundwater nitrate removal seems considerably larger than previously known.