Nitrifiers Cooperate to Produce Nitrous Oxide in Plateau Wetland Sediments.

The thawing of dormant plateau permafrost emits nitrous oxide (N2O) through wetlands; however, the N2O production mechanism in plateau wetlands is still unclear. Here, we used the 15N-18O double tracer technique and metagenomic sequencing to analyze the N2O production mechanism in the Yunnan-Kweichow and Qinghai-Tibet plateau wetlands during the summer of 2020. N2O production activity was detected in all 16 sediment samples (elevation 1020-4601 m: 2.55 ± 0.42-26.38 ± 3.25 ng N g-1 d-1) and was promoted by nitrifier denitrification (ND). The key functional genes of ND (amoA, hao, and nirK) belonged to complete ammonia oxidizing (comammox) bacteria, and the key ND species was the comammox bacterium Nitrospira nitrificans. We found that the comammox bacterial species N. nitrificans and the ammonia oxidizing bacterial (AOB) species Nitrosomonas europaea cooperate to produce N2O in the plateau wetland sediments. Furthermore, we inferred that environmental factors (elevation and total organic matter (TOM)) influence the cooperation pattern via N. nitrificans, thus affecting the N2O production activity in the plateau wetland sediments. Our findings advance the mechanistic understanding of nitrifiers in biogeochemical cycles and global climate change.

Abundance, community and driving factor of nitrifiers in western China plateau.

Complete ammonia oxidation (comammox) is one of the most important biogeochemical processes, with recent studies showing that comammox process dominates nitrification in many ecosystems. However, the abundance, community and driving factor of comammox bacteria and other nitrifying microorganisms in plateau wetland is still unclear. Here, the abundances and community features of comammox bacteria, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the wetland sediments of western China plateaus were examined using qPCR and high-throughput sequencing. The results indicate that comammox bacteria were more abundant than AOA and AOB, and dominated the nitrification process. Compared with low-elevation samples (below 3000 m: samples 6-10, 12, 13, 15, 16), the abundance of comammox bacteria was much higher at high-elevation samples (above 3000 m: samples 1-5, 11, 14, 17, 18). The key species of AOA, AOB, and comammox bacteria were Nitrososphaera viennensis, Nitrosomonas europaea, and Nitrospira nitrificans, respectively. The key factor affecting comammox bacteria community was elevation. Elevation could increase the interaction links of key species Nitrospira nitrificans, resulting in high comammox bacterial abundance. The results of this study advance our knowledge of comammox bacteria in natural ecosystems.

Source, contribution and microbial N-cycle of N-compounds in China fresh snow.

The importance and contribution of nitrogen compounds and the related microbial nitrogen cycling processes in fresh snow are not well understood under the current research background. We collected fresh snow samples from 21 cities that 80% are from China during 2016 and 2017. Principal component analysis showed that SO42- were in the first principal component, and N-compounds were the second. Furthermore, the main pollutant ions SO42- and NO3- were from anthropogenic sources, and SO42- contributed (61%) more to the pollution load than NO3- (29%), which were confirmed through a series of precipitation mechanism analysis. We selected five N-cycle processes (consist of oxidation and reduction processes) for molecular biology experiments, including Ammonia-oxidation process, Nitrite-oxidation process, Denitrification process, Anaerobic-ammoxidation process (Anammox) and Dissimilatory nitrate reduction to ammonium process (DNRA). Except ammonia-oxidizing archaeal (AOA) and bacterial (AOB) amoA genes (above 107 copies g-1), molecular assays of key functional genes in various nitrogen conversion processes showed a belowed detection limit number, and AOB abundance was always higher than AOA. The determination of the microbial transformation rate using the 15N-isotope tracer technique showed that the potential rate of five N-conversion processes was very low, which is basically consistent with the results from molecular biology studies. Taken together, our results illustrated that microbial nitrogen cycle processes are not the primary biological processes causing the pollution in China fresh snow.

Nitrite-resistance mechanisms on wastewater treatment in denitrifying phosphorus removal process revealed by machine learning, co-occurrence, and metagenomics analysis.

Nitrite is a key intermediate in nitrogen metabolism that determines microbial transformations of N and P, greenhouse gas (N2O) emissions, and system nutrient removal efficiency. However, nitrite also exerts toxic effects on microorganisms. A lack of understanding of high nitrite-resistance mechanisms at community- and genome-scale resolutions hinders the optimization for robustness of wastewater treatment systems. Here, we established nitrite-dependent denitrifying and phosphorus removal (DPR) systems under a gradient concentration of nitrite (0, 5, 10, 15, 20, and 25 mg N/L), relying on 16S rRNA gene amplicon and metagenomics to explore high nitrite-resistance mechanism. The results demonstrated that specific taxa were adopted to change the metabolic relationship of the community through phenotypic evolution to resist toxic nitrite contributing to the enhancement of denitrification and inhibition of nitrification and phosphorus removal. The key specific species, Thauera enhanced denitrification, whereas Candidatus Nitrotoga decreased in abundance to maintain partial nitrification. The extinction of Candidatus Nitrotoga induced a simpler restructuring-community, forcing high nitrite-stimulating microbiome to establish a more focused denitrification rather than nitrification or P metabolism in response to nitrite toxicity. Our work provides insights for understanding microbiome adaptation to toxic nitrite and giving theoretical support for operation strategy of nitrite-based wastewater treatment technology.

Comparing with oxygen, nitrate simplifies microbial community assembly and improves function as an electron acceptor in wastewater treatment.

Biochemical oxidation and reduction are key processes in treating biological wastewater and they require the presence of electron acceptors. The functional impact of electron acceptors on microbiomes provides strategies for improving the treatment efficiency. This research focused on two of the most important electron acceptors, nitrate and oxygen. Molecule ecological network, null model, and functional prediction based on high-throughput sequencing were used to analyze the microbiomes features and assembly mechanism. The results revealed nitrate via the homogeneous selection (74.0%) decreased species diversity, while oxygen via the homogeneous selection (51.1%) and dispersal limitation (29.6%) increased the complexity of community structure. Microbes that were more strongly homogeneously selected for assembly included polyphosphate accumulating organisms (PAOs), such as Pseudomonas and variovorax in the nitrate impacted community; Pseudomonas, Candidatus_Accumulibacter, Thermomonas and Dechloromonas, in the oxygen impacted community. Nitrate simplified species interaction and increased the abundance of functional genes involving in tricarboxylic acid cycle (TCA cycle), electron transfer, nitrogen metabolism, and membrane transport. These findings contribute to our knowledge of assembly process and interactions among microorganisms and lay a theoretical basis for future microbial regulation strategies in wastewater treatment.

Towards a more labor-saving way in microbial ammonium oxidation: A review on complete ammonia oxidization (comammox).

In the Anthropocene, nitrogen pollution is becoming an increasing challenge for both mankind and the Earth system. Microbial nitrogen cycling begins with aerobic nitrification, which is also the key rate-limiting step. For over a century, it has been accepted that nitrification occurs sequentially involving ammonia oxidation, which produces nitrite followed by nitrite oxidation, generating nitrate. This perception was changed by the discovery of comammox Nitrospira bacteria and their metabolic pathway. In addition, this also provided us with new knowledge concerning the complex nitrogen cycle network. In the comammox process, ammonia can be completely oxidized to nitrate in one cell via the subsequent activity of the enzyme complexes, ammonia monooxygenase, hydroxylamine dehydrogenase, and nitrite oxidodreductase. Over the past five years, research on comammox made great progress. However, there still exist a lot of questions, including how much does comammox contribute to nitrification? How large is the diversity and are there new strains to be discovered? Do comammox bacteria produce the greenhouse gas N2O, and how or to which extent may they contribute to global climate change? The above four aspects are of great significance on the farmland nitrogen management, aquatic environment restoration, and mitigation of global climate change. As large number of comammox bacteria and pathways have been detected in various terrestrial and aquatic ecosystems, indicating that the comammox process may exert an important role in the global nitrogen cycle.

Ecological Insights Into Community Interactions, Assembly Processes and Function in the Denitrifying Phosphorus Removal Activated Sludge Driven by Phosphorus Sources.

The microbial characteristics in the wastewater treatment plants (WWTPs) strongly affect their optimal performance and functional stability. However, a cognitive gap remains regarding the characteristics of the microbial community driven by phosphorus sources, especially co-occurrence patterns and community assembly based on phylogenetic group. In this study, 59 denitrifying phosphorus removal (DPR) activated sludge samples were cultivated with phosphorus sources. The results suggested that homogeneous selection accounted for the largest proportion that ranged from 35.82 to 64.48%. Deterministic processes dominated in 12 microbial groups (bins): Candidatus_Accumulibacter and Pseudomonas in these bins belonged to phosphate-accumulating organisms (PAOs). Network analysis revealed that species interactions were intensive in cyclic nucleoside phosphate-influenced microbiota. Function prediction indicated that cyclic nucleoside phosphates increased the activity of enzymes related to denitrification and phosphorus metabolism and increased the α-diversity of microorganism but decreased the diversity of metabolic function. Based on these results, it was assumed that cyclic nucleoside phosphates, rather than inorganic phosphates, are the most available phosphorus source for majority microorganisms in DPR activated sludge. The study revealed the important role of phosphorus source in the construction and assembly of microbial communities and provided new insights about pollutant removal from WWTPs.

Comammox activity dominates nitrification process in the sediments of plateau wetland.

The recent discovery of complete ammonia oxidation (comammox) has increased our understanding of nitrification. Although comammox has been shown to play an important role in plain wetland ecosystems, studies of comammox contribution are still limited in plateau wetland ecosystems. Here, we analyzed the abundance, activity, community and biogeochemical mechanisms of the comammox bacteria in Yunnan-kweichow and Qinghai-Tibet plateau wetlands from elevations of 1000-5000 m. Comammox bacteria were widely distributed in all 16 sediment samples with abundances higher than 0.96 ± 0.26 × 107 copies g-1 (n = 16). Comammox showed high activity (1.18 ± 0.17 to 1.98 ± 0.08 mg N kg-1 d-1) at high-elevation (3000-5000 m) and dominated the nitrification process (activity contribution: 37.20 - 60.62%). The activity contribution of ammonia-oxidizing bacteria (1.07 ± 0.08 to 2.79 ± 0.35 mg N kg-1 d-1) dominated the nitrification process (44.55 - 64.15%) in low-elevation (1000-3000 m) samples. All detected comammox Nitrospira belonged to clade A, while clade B was not detected. Elevation always had a strongest effect on key comammox species. Thus, we infer that elevation may drive the high relative abundance of the species Candidatus Nitrospira nitrificans (avg. 12.40%) and the low relative abundance of the species Nitrospira sp. SG-bin2 (avg. 4.75%) in high-elevation samples that showed a high comammox activity (avg. 1.62 mg N kg-1 d-1) and high contribution (avg. 46.08%) to the nitrification process. These results indicate that comammox may be an important and currently underestimated microbial nitrification process in plateau wetland ecosystems.

Dissimilatory nitrate reduction to ammonium (DNRA) in traditional municipal wastewater treatment plants in China: Widespread but low contribution.

Recent reports on the occurrence and contribution of dissimilatory nitrate reduction to ammonium (DNRA) in marine, inland water, and soil systems have greatly improved our understanding of the global nitrogen (N) cycle. This also promoted the investigation of the role and ecological features of DNRA in anthropogenic ecosystems. However, so far, the use of DNRA in municipal wastewater treatment plants (WWTPs), which are one of the most common and largest biotechnologically artificial water ecosystems, has not been investigated. Accordingly, this study focused on the abundance, activity, community structure, and diversity of DNRA bacteria in full-scale WWTPs. DNRA bacteria were detected in all treatment units in six tested municipal WWTPs, even in aerobic zones (dissolved oxygen > 2 mg L-1). Although the relative abundance of DNRA bacteria (0.2-4.0%) was less than that of denitrifying bacteria (0.7-10.1%) among all investigated samples, the abundance of DNRA bacteria still reaches 109 gene copies g-1. However, 15N-isotope tracing indicated that the potential DNRA rates were significantly lower (0.4-2.1 nmol N g-1 h-1) than those of denitrification (9.5-15.7 nmol N g-1 h-1), but higher than anammox rate (0.3-1.3 nmol N g-1 h-1). The DNRA bacterial community structure was primarily affected by temperature gradient despite the treatment process. High-throughput sequencing analysis targeting the DNRA nrfA gene showed that Nitrospira accounted for the largest proportion of nrfA genes among all samples (6.2-36.3%), followed by Brocadia (5.9-22.1%). Network analysis further indicated that Nitrospira played an important role in both the DNRA bacterial community and entire bacterial community in municipal WWTPs. These results suggest that the ecological habitats of DNRA bacteria in anthropogenic ecosystems were far more abundant than previously assumed. However, the contribution to N transformation by the widespread DNRA was not significant in traditional municipal WWTPs.

Linking abundance and community of microbial N2O-producers and N2O-reducers with enzymatic N2O production potential in a riparian zone.

As aquatic-terrestrial ecotones, riparian zones are hotspots not only for denitrification but also for nitrous oxide (N2O) emission. Due to the potential role of nosZ II in N2O mitigation, emerging studies in terrestrial ecosystems have taken this newly reported N2O-reducer into account. However, our knowledge about the interactions between denitrification activities and both N2O-producers and reducers (especially for nosZ II) in aquatic ecosystems remains limited. In this study, we investigated spatiotemporal distributions of in situ N2O flux, potential N2O production rate, and potential denitrification rate, as well as of the related genes in a riparian zone of Baiyangdian Lake. Real-time quantitative PCR (qPCR) and high-throughput sequencing targeted functional genes were used to analyze the denitrifier communities. Results showed that great differences in microbial activities and abundances were observed between sites and seasons. Waterward sediments (constantly flooded area) had the lowest N2O production potential in both seasons. Not only the environmental factors (moisture content, NH4+ content and TOM) but also the community structure of N2O-producers and N2O-reducers (nirK/nirS and nosZ II/nosZ I ratios) could affect the potential N2O production rate. The abundance of the four functional genes in the winter was higher than in the summer, and the values all peaked at the occasionally flooded area in the winter. The dissimilarity in community composition was mainly driven by moisture content. Altogether, we propose that the N2O production potential was largely regulated by the community structure of N2O-producers and N2O-reducers in riparian zones. Increasing the constantly flooded area and reducing the occasionally flooded area of lake ecosystems may help reduce the level of denitrifier-produced N2O.

[Concentration of Nitrate in Main Anoxic Stage and PHA, TP Metabolism for Nitrogen and Phosphorus Removal in Single Sludge System with Continuous Flow].

Based on test results and mass balance, PHA, TP metabolic regularity was revealed under different nitrate nitrogen concentrations in main anoxic stage [c(NO3)] for nitrogen and phosphorus removal in single sludge system with continuous flow, then the effectiveness of using c(NO3) as control parameter was proved from the perspective of the reaction mechanism. During experiment period, the influent COD, total nitrogen (TN), and total phosphorus (TP) concentrations were stabilized at (285.78±18.19), (58.13±3.79), and(7.14±0.51) mg·L-1, respectively. The experiment was carried out under the condition that the c(NO3) values were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg·L-1 based on the feedback control structure using PLC automatic control system to control the nitrifying liquid flow with the water quality. The sludge load of COD was (0.253±0.071)kg·(kg·d)-1, the sludge load of TP in anaerobic stage was (0.006±0.001) kg·(kg·d)-1, the sludge load of TN in aerobic stage was (0.049±0.006) kg·(kg·d)-1, the hydraulic retention time (HRT) in bioreactor was 9h, the sludge recycle flow was 0.5, and the mixed liquor recycle was 1.0. The results showed that effect of c(NO3) value on PHA synthesis and storage rate in the ANS was conspicuous, and the percentage of PHA storage occupied 74% of COD removal when c(NO3) value was 2.5 mg·L-1.The impact of c(NO3) value on PHA degraded in the main anoxic stage was great, and the percentage of PHA degradation in the main anoxic stage occupied 55% of total PHA degradation when c(NO3) value was 2.5 mg·L-1. The phosphorus released in anaerobic stage changed along with increasing c(NO3), and the amount of phosphorus released obtained the maximum value 6.16 g·d-1 when c(NO3) value was 2.5 mg·L-1. In addition, under c(NO3) value of 2.5 mg·L-1, the amount of total phosphorus uptake and anoxic phosphorus uptake obtained the maximum values of 8.04 g·d-1 and 3.67 g·d-1, respectively. Then it was confirmed thatc(NO3) could serve as a run controlling parameter with the best value of 2.5 mg·L-1 from the perspective of PHA and TP metabolic mechanism.