Shanyun Wang

Anaerobic ammonia oxidation in a fertilized paddy soil

Evidence for anaerobic ammonium oxidation in a paddy field was obtained in Southern China using an isotope-pairing technique, quantitative PCR assays and 16S rRNA gene clone libraries, along with nutrient profiles of soil cores. A paddy field with a high load of slurry manure as fertilizer was selected for this study and was shown to contain a high amount of ammonium (6.2–178.8 mg kg−1). The anaerobic oxidation of ammonium (anammox) rates in this paddy soil ranged between 0.5 and 2.9 nmolN per gram of soil per hour in different depths of the soil core, and the specific cellular anammox activity observed in batch tests ranged from 2.9 to 21 fmol per cell per day. Anammox contributed 4–37% to soil N2 production, the remainder being due to denitrification. The 16S rRNA gene sequences of surface soil were closely related to the anammox bacteria ‘Kuenenia’, ‘Anammoxoglobus’ and ‘Jettenia’. Most of the anammox 16S rRNA genes retrieved from the deeper soil were affiliated to ‘Brocadia’. The retrieval of mainly bacterial amoA sequences in the upper part of the paddy soil indicated that nitrifying bacteria may be the major source of nitrite for anammox bacteria in the cultivated horizon. In the deeper oxygen-limited parts, only archaeal amoA sequences were found, indicating that archaea may produce nitrite in this part of the soil. It is estimated that a total loss of 76 g N m−2 per year is linked to anammox in the paddy field.

Quantitative analyses of ammonia-oxidizing Archaea and bacteria in the sediments of four nitrogen-rich wetlands in China

With the rapid development of ammonia-synthesizing industry, the ammonia-nitrogen pollution in wetlands acting as the sink of point and diffuse pollution has been increased dramatically. Most of ammonia-nitrogen is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Current research findings have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex wetlands environment, it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea or Bacteria as implied by specific high abundance. In this research, the abundance and composition of Archaea and Bacteria in sediments of four kinds of wetlands with different nitrogen concentration were investigated by using quantitative real-time polymerase chain reaction, cloning, and sequencing approaches based on amoA genes. The results indicated that AOA distributed widely in wetland sediments, and the phylogenetic tree revealed that archaeal amoA functional gene sequences from wetlands sediments cluster as two major evolutionary branches: soil/sediment and sediment/water. The bacteria functionally dominated microbial ammonia oxidation in different wetlands sediments on the basis of molecule analysis, potential nitrification rate, and soil chemistry. Moreover, the factors influencing AOA and AOB abundances with environmental indicator were also analyzed, and the results addressed the copy numbers of archaeal and bacterial amoA functional gene having the higher correlation with pH and ammonia concentration. The pH had relatively great negative impact on the abundance of AOA and AOB, while ammonia concentration showed positive impact on AOB abundance only. These findings could be fundamental to improve understanding of the importance of AOB and AOA in nitrogen and other nutrients cycle in wetland ecosystems.

Manure fertilization alters the population of ammonia-oxidizing bacteria rather than ammonia-oxidizing archaea in a paddy soil

Manure fertilizers are widely used in agriculture and highly impacted the soil microbial communities such as ammonia oxidizers. However, the knowledge on the communities of archaeal versus bacterial ammonia oxidizers in paddy soil affected by manure fertilization remains largely unknown, especially for a long‐term influence. In present work, the impact of manure fertilization on the population of ammonia oxidizers, related potential nitrification rates (PNRs) and the key factors manipulating the impact were investigated through studying two composite soil cores (long‐term fed with manure fertilization versus undisturbed). Moreover, soil incubated with NH4+ for 5 weeks was designed to verify the field research. The results showed that the copy numbers of bacterial amoA gene in the manure fed soil were significant higher than those in the unfed soil (p < 0.05), suggesting a clear stimulating effect of long‐term manure fertilization on the population of ammonia‐oxidizing bacteria (AOB). The detected PNRs in the manure fed soil core (14–218 nmol L−1 N g−1 h−1) were significant higher than those in the unfed soil core (5–72 nmol L−1 N g−1 h−1; p < 0.05). Highly correlations between the PNRs and the bacterial amoA gene copies rather than archaeal amoA gene were observed, indicating strong nitrification capacity related to bacterial ammonia oxidizers. The NH4+‐N significantly correlated to the abundance of AOB (p < 0.01) and explained 96.1% of the environmental variation, showing the NH4+‐N was the main factor impacting the population of AOB. The incubation experiment demonstrated a clear increase of the bacterial amoA gene abundance (2.0 × 106 to 8.4 × 106 g−1 d.w.s. and 1.6 × 104 to 4.8 × 105 g−1 d.w.s.) in both soil but not for the archaeal amoA gene, in agreement with the field observation. Overall, our results suggested that manure fertilization promoted the population size of bacterial ammonia oxidizers rather than their archaeal counterparts whether in long‐term or short‐term usage and the NH4+‐N was the key impact factor.

Ubiquitous anaerobic ammonium oxidation in inland waters of China: an overlooked nitrous oxide mitigation process

Denitrification has long been regarded as the only pathway for terrestrial nitrogen (N) loss to the atmosphere. Here we demonstrate that large-scale anaerobic ammonium oxidation (anammox), an overlooked N loss process alternative to denitrification which bypasses nitrous oxide (N2O), is ubiquitous in inland waters of China and contributes significantly to N loss. Anammox rates in aquatic systems show different levels (1.0–975.9 μmol N m−2 h−1, n = 256) with hotspots occurring at oxic-anoxic interfaces and harboring distinct biogeochemical and biogeographical features. Extrapolation of these results to the China-national level shows that anammox could contribute about 2.0 Tg N yr−1, which equals averagely 11.4% of the total N loss from China’s inland waters. Our results indicate that a significant amount of the nitrogen lost from inland waters bypasses denitrification, which is important for constructing more accurate climate models and may significantly reduce potential N2O emission risk at a large scale.

Nitrous oxide reductase gene (nosZ) and N2O reduction along the littoral gradient of a eutrophic freshwater lake

Lake littoral zones are characterized by heterogeneity in the biogeochemistry of nutrient elements. This study aimed to explore the relationship between the nitrous oxide reductase gene (nosZ)-encoding denitrifier community composition/abundance and N2O reduction. Five samples (deep sediment, near-transition sediment, transition site, near-transition land and land soil) were collected along a littoral gradient of eutrophic Baiyangdian Lake, North China. To investigate the relationship between the nosZ-encoding denitrifier community structure and N2O reduction, the nosZ-encoding denitrifier community composition/abundance, potential denitrification rate (DNR) and potential N2O production rate (pN2O) were investigated using molecular biological technologies and laboratory incubation experiments. The results showed that the average DNR of sediments was about 25 times higher than that of land soils, reaching 282.5 nmol N/(g dry weight (dw) x hr) and that the average pN2O of sediments was about 3.5 times higher than that of land soils, reaching 15.7 nmol N/(g dw x hr). In the land area, the nosZ gene abundance showed a negative correlation with the N2O/(N2O + N2) ratio, indicating that nosZ gene abundance dominated N2O reduction both in the surface soils of the land area and in the soil core of the transition site. Phylogenetic analysis showed that all the nosZ sequences recovered from sediment clustered closely with the isolates Azospirillum largimobile and Azospirillum irakense affiliated to Rhodospirillaceae in alpha-Proteobacteria, while about 92.3% (12/13) of the nosZ sequences recovered from land soil affiliated to Rhizobiaceae and Bradyrhizobiaceae in alpha-Proteobacteria. The community composition of nosZ gene-encoding denitrifiers appeared to be coupled with N2O reduction along the littoral gradient.

Species, Abundance and Function of Ammonia-oxidizing Archaea in Inland Waters across China

Ammonia oxidation is the first step in nitrification and was thought to be performed solely by specialized bacteria. The discovery of ammonia-oxidizing archaea (AOA) changed this view. We examined the large scale and spatio-temporal occurrence, abundance and role of AOA throughout Chinese inland waters (n = 28). Molecular survey showed that AOA was ubiquitous in inland waters. The existence of AOA in extreme acidic, alkaline, hot, cold, eutrophic and oligotrophic environments expanded the tolerance limits of AOA, especially their known temperature tolerance to −25 °C, and substrate load to 42.04 mM. There were spatio-temporal divergences of AOA community structure in inland waters, and the diversity of AOA in inland water ecosystems was high with 34 observed species-level operational taxonomic units (OTUs; based on a 15% cutoff) distributed widely in group I.1b, I.1a, and I.1a-associated. The abundance of AOA was quite high (8.5 × 104 to 8.5 × 109 copies g−1), and AOA outnumbered ammonia-oxidizing bacteria (AOB) in the inland waters where little human activities were involved. On the whole AOB predominate the ammonia oxidation rate over AOA in inland water ecosystems, and AOA play an indispensable role in global nitrogen cycle considering that AOA occupy a broader habitat range than AOB, especially in extreme environments.

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.

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 β‐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.