Ammonia Monooxygenase Research Articles

Effect of biodegradable PBAT microplastics on the C and N accumulation of functional organic pools in tropical latosol

Microplastics (MPs) pollution is becoming an emerging global stressor for soil ecosystems. However, studies on the impacts of biodegradable MPs on soil C sequestration have been mainly based on bulk C quantity, without considering the storage form of C, its persistency and N demand. To address this issue, the common poly (butylene adipate-co-terephthalate) (PBAT) was used as the model, and its effects on soil functional organic pools, including mineral-associated (MAOM), particulate (POM) and dissolved organic matter (DOM), were investigated from the novel coupled perspective of C and N stocks. After adding PBAT-MPs, the contents of soil POM-C, DOM-C, and MAOM-C were increased by 546.9 %–697.8 %, 54.2 %–90.3 %, and 13.7 %–18.9 %, respectively. Accordingly, the total C increased by 116.0 %–191.1 %. Structural equation modeling showed that soil C pools were regulated by PBAT input and microbial metabolism associated with C and N enzymes. Specifically, PBAT debris could be disguised as soil C to promote POM formation, which was the main pathway for C accumulation. Inversely, the MAOM-C and DOM-C formation was attributed to the PBAT microbial product and the selective consumption in DOM-N. Random forest model confirmed that N-activated (e.g., Nitrospirae) and PBAT-degrading bacteria (e.g., Gemmatinadetes) were important taxa for soil C accumulation, and the key enzymes were rhizopus oryzae lipas, invertase, and ammonia monooxygenase. The soil N accumulation was mainly related to the oligotrophic taxa (e.g., Chloroflexi and Ascomycota) associated with aggregate formation, decreasing the DOM-N by 46.9 %–84.3 %, but did not significantly change the total N storage and other N pools. Collectively, the findings highlight the urgency to control the nutrient imbalance risk of labile N loss and recalcitrant C enrichment in POM to avoid the depressed turnover rate of organic matter in MPs-polluted soil.

Rapid start-up of the biofilter for simultaneous manganese and ammonia removal at low temperature: Effects of phosphate and copper

In low-temperature environments, low nutrients such as inorganic salts limiting microbial proliferation further extended the start-up period of biofilters for drinking water treatment. This study explored the ammonia and manganese (Mn) removal performance and the relevant mechanisms under single and combined PO43− at 30 μg/L and Cu2+ at 10 μg/L. The start-up periods of R0 (control), R1 (Cu2+ single), R2 (PO43− single) and R3 (combined Cu2+ and PO43−) were 98, 82, 32 and 24 days, respectively. PO43− increased the concentration of extracellular polymeric substances (EPS) by stimulating cellular metabolism, which promoted the formation of biofilm. Additionally, the combined Cu2+ and PO43− significantly promoted the ammonia monooxygenase (AMO) and nitrite oxidoreductase (NXR) activities, which further facilitated the removal of ammonia. The combined Cu2+ and PO43− accelerated the conversion of NH4+-N to NO2−-N and NO2−-N to NO3−-N processes thereby reducing the inhibition of Mn removal by NH4+-N and NO2−-N, and the kinetics of biological manganese oxidation showed that the combined Cu2+ and PO43− increased the rate of Mn removal, which both promoted the Mn removal in the biofilters. Furthermore, the combined Cu2+ and PO43− supported the proliferation of functional microorganisms. In summary, the synergistic effect of Cu2+ and PO43− enhanced the Mn and ammonia removal performance and achieved rapid start-up of the biofilter at low temperatures.

Nitrogen (N) “supplementation, slow release, and retention” strategy improves N use efficiency via the synergistic effect of biochar, nitrogen-fixing bacteria, and dicyandiamide

Irrational nitrogen (N) fertilizer management and application practices have led to a range of ecological and environmental problems that seriously threaten food security. In this study, an effective N fertilizer management strategy was established for improving N fertilizer utilization efficiency (NUE). Biochar, N2-fixing bacteria (Enterobacter cloacae), and a nitrification inhibitor (dicyandiamide, DCD) were simultaneously added to the soil during maize cultivation. The goal was to increase soil ammonium nitrogen content and NUE by regulating the relative abundance, enzyme activity, and functional gene expression of N conversion-related soil microbes. Biochar combined with E. cloacae and DCD significantly increased soil N content, and the NUE reached 46.69 %. The relative abundance of Burkholderia and Bradyrhizobium and the activity of nitrogenase increased significantly during biological N2 fixation. Further, the abundance of the nifH gene was significantly up-regulated. The relative abundance of Sphingomonas, Pseudomonas, Nitrospira, and Castellaniella and the activities of ammonia monooxygenase and nitrate reductase decreased significantly during nitrification and denitrification. Moreover, the abundance of the genes amoA and narG was significantly down-regulated. Correlation analyses showed that the increase in soil N2 fixation and the suppression of nitrification and denitrification reactions were the key contributors to the increase in soil N content and NUE. Biochar combined with E. cloacae and DCD synergistically enabled the supplementation, slow release, and retention of N, thus providing adequate N for maize growth. Thus, the combination of biochar, E. cloacae, and DCD is effective for mitigating the irrational application of N fertilizers and reducing N pollution.

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome

Nitrosomonas europaea, an aerobic ammonia oxidizing bacterium, is responsible for the first and rate-limiting step of the nitrification process, and their ammonia oxidation activities are critical for the biogeochemical cycling and the biological nitrogen removal of wastewater treatment. In the present study, N.europaea cells were cultivated in the inorganic or organic media (the NBRC829 and the nutrient-rich, NR, media, respectively), and the cells proliferated in the form of planktonic and biofilm in those media, respectively. The N.europaea cells in the biofilm growth mode produced larger amounts of the extracellular polymeric substances (EPS), and the composition of the EPS was characterized by the chemical analyses including Fourier transform infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (NMR) measurements. The RNA-Seq analysis of N.europaea in the biofilm or planktonic growth mode revealed that the following gene transcripts involved in central nitrogen metabolisms were abundant in the biofilm growth mode; amo encoding ammonia monooxygenase, hao encoding hydroxylamine dehydrogenase, the gene encoding nitrosocyanine, nirK encoding copper-containing nitrite reductase. Additionally, the transcripts of the pepA and wza involved in the bacterial floc formation and the translocation of EPS, respectively, were also abundant in the biofilm-growth mode. Our study was first to characterize the EPS production and transcriptome of N.europaea in the biofilm and planktonic growth mode.

Unraveling microbial characteristics of simultaneous nitrification, denitrification and phosphorus removal in a membrane-aerated biofilm reactor

This study describes the simultaneous removal of carbon, ammonium, and phosphate from domestic wastewater by a membrane-aerated biofilm reactor (MABR) which was operated for 360 days. During the operation, the maximum removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) reached 93.1%, 83.98%, and 96.41%, respectively. Statistical analysis showed that the MABR could potentially treat wastewater with a high ammonium concentration and a relatively low C/N ratio. Dissolved oxygen and multiple pollutants, including ammonium, carbon, phosphate, and sulfate, shaped the structure of the microbial community in the MABR. High throughput sequencing uncovered the crucial microbiome in ammonium transformation in MABR. Phylogenetic analysis of the ammonia monooxygenase (amoA) genes revealed an important role for comammox Nitrospira in the nitrification process. Diverse novel phosphate-accumulating organisms (Thauera, Bacillus, and Pseudomonas) and sulfur-oxidizing bacteria (Thiobacillus, Thiothrix and Sulfurimonas) were potentially involved in denitrification in MABR. The results from this study suggested that MABR could be a feasible system for the simultaneous removal of nitrogen, carbon, phosphorus, and sulfur from sewage water.

Nitrite improved nitrification efficiency and enriched ammonia-oxidizing archaea and bacteria in the simultaneous nitrification and denitrification process

Simultaneous nitrification and denitrification (SND) is effective and energy-saving for wastewater treatment. As an inevitable intermediate product in the SND process, nitrite affects the efficiency of ammonia oxidation and the composition of nitrifiers. To investigate the impact of nitrite on ammonia oxidation efficiency, two reactors performing SND were respectively operated without nitrite (R1 as control) and with 20 mg N/L nitrite addition (R2 as experimental). The total nitrogen removal efficiency was 74.5% in R1 while 99.0% in R2. With nitrite addition (i.e., 20 mg N/L), the ammonia removal rate in R2 increased to 4.5 times of that in R1. The ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) contributed to respective around 46.9% and 41.8% ammonia removal in R2 based on the results of experiments with specific inhibitors. The number of respective AOA and AOB ammonia monooxygenase gene (amoA) copies increased by 280 and 30 times due to nitrite addition, according to the qPCR results. The high-throughput sequencing results illustrated the increase of dominant AOB species from 0.40% in R1 to 1.59% in R2 and the phylogenetic tree analysis revealed a close link to Nitrosospira multiformis. These results indicated that the ammonia removal efficiency was improved and AOA/AOB were enriched by nitrite addition. The specific nitrite reductases in AOA and AOB boosted the adaptation of nitrite addition. This study demonstrated the positive impacts of nitrite addition on the ammonia removal efficiency and rate in the SND process.

Geochemical factors impacting nitrifying communities in sandy sediments.

Sandy sediment beaches covering 70% of non-ice-covered coastlines are important ecosystems for nutrient cycling along the land-ocean continuum. Subterranean estuaries (STEs), where groundwater and seawater meet, are hotspots for biogeochemical cycling within sandy beaches. The STE microbial community facilitates biogeochemical reactions, determining the fate of nutrients, including nitrogen (N), supplied by groundwater. Nitrification influences the fate of N, oxidising reduced dissolved inorganic nitrogen (DIN), making it available for N removal. We used metabarcoding of 16S rRNA genes and quantitative PCR (qPCR) of ammonia monooxygenase (amoA) genes to characterise spatial and temporal variation in STE microbial community structure and nitrifying organisms. We examined nitrifier diversity, distribution and abundance to determine how geochemical measurements influenced their distribution in STEs. Sediment microbial communities varied with depth (p-value = 0.001) and followed geochemical gradients in dissolved oxygen (DO), salinity, pH, dissolved inorganic carbon and DIN. Genetic potential for nitrification in the STE was evidenced by qPCR quantification of amoA genes. Ammonia oxidiser abundance was best explained by DIN, DO and pH. Our results suggest that geochemical gradients are tightly linked to STE community composition and nitrifier abundance, which are important to determine the fate and transport of groundwater-derived nutrients to coastal waters.

Sediment depth-related variations of comammox Nitrospira: Evidence in the Three Gorges Reservoir, China

The recent discovery of comammox Nitrospira as complete ammonia-oxidizing microorganism has fundamentally revolutionized our understanding of nitrogen cycling in sediment environments. However, knowledge regarding their abundance, biodiversity, community structure, and interactions is predominantly limited to the upper layers (0–20 cm). To address this gap, we collected sediment samples along profiles ranging from 0 to 300 cm in depth at three locations within the middle segment of the Three Gorges Reservoir (TGR), China. Quantitative real-time PCR (qPCR) analyses suggested that comammox bacteria were not only ubiquitous in deep sediments but also more abundant than ammonia-oxidizing bacteria (AOB). Ammonia monooxygenases subunit A (amoA) gene amplicon sequencing illuminated that comammox bacteria were more sensitive to sedimental depth compared to AOB and ammonia-oxidizing archaea (AOA), as evidenced by a more significant decline in community diversity and similarity over distance along sediment vertical profiles. Notably, we discovered that the amoA gene abundance, alpha- and beta-diversity of comammox bacteria exerted an essential contribution to potential nitrification rates according to random forest model. Phylogenetic analysis indicted that most comammox bacteria within sediment samples belonged to clade A.2. Intriguingly, the average relative abundance of comammox clade A.2 displayed a noteworthy rise with sediment depth, whereas clade A.1 demonstrated a converse pattern, unveiling distinct ecological niche adaptations of these two clades along the sediment profile. Ecological network analysis further revealed closer interactions between comammox bacteria and canonical ammonia oxidizers in the superficial layer (0–40 cm), with the network structure gradually simplifying from superficial to deep sediment (200–300 cm). Overall, these findings broaden the current recognition of the geographic distribution and niche segregation of comammox bacteria at the fine scale of the sediments ecosystems and provide insights into sediment depth-related variations of their coexistence network patterns in large freshwater reservoirs.

Evaluation of long-term operational and production performance of biofloc technology (BFT) system within various particle size

Biofloc technology (BFT) is a promising technique, but there has been ongoing debate between biofloc's properties and particle size. This study investigated the relationship between long-term particle size variation and the production performance of BFT aquaculture systems. External shearing force introduction could keep bioflocs at a smaller size level chronically, and 97% of smaller bioflocs were below 181.97 μm which was obviously lower than the normal size group (Control). No significant difference in the daily water treatment capacity for bioflocs within different particle size was detected (p > 0.05), but the activity of Soil-Dehydrogenase (S-DHA), Ammonia Monooxygenase (AMO), Hydroxylamine Oxidase (HAO) and Nitrite Oxidase (NOR) were all higher in smaller bioflocs. Smaller bioflocs were better enriched heavy metal elements and less damage to the gill lamellae was also detected in smaller size group. The survival rate and final mean weight of tilapia (Oreochromis niloticus) cultured in the BFT system based on smaller size bioflocs were significantly higher than the control (p < 0.05). The microbial compositions of varying particle sizes bioflocs demonstrated remarkable distinctions, and higher abundance of particle-attached bacteria (e.g., Flavobacteriaceae, Rhodobacteraceae) was detected in normal size bioflocs. Overall, this study comprehensively evaluated the long-term application of various particle size bioflocs and provided a practical basis for the sustainable BFT development.

Vertical segregation and phylogenetic characterization of archaea and archaeal ammonia monooxygenase gene in the water column of the western Arctic Ocean.

Archaea constitute a substantial fraction of marine microbial biomass and play critical roles in the biogeochemistry of oceans. However, studies on their distribution and ecology in the Arctic Ocean are relatively scarce. Here, we studied the distributions of archaea and archaeal ammonia monooxygenase (amoA) gene in the western Arctic Ocean, using the amplicon sequencing approach from the sea surface to deep waters up to 3040m depth. A total of five archaeal phyla, Nitrososphaerota, "Euryarchaeota", "Halobacteriota," "Nanoarchaeota", and Candidatus Thermoplasmatota, were detected. We observed a clear, depth-dependent vertical segregation among archaeal communities. Ca. Thermoplasmatota (66.8%) was the most dominant phylum in the surface waters. At the same time, Nitrososphaerota (55.9%) was dominant in the deep waters. Most of the amoA gene OTUs (99%) belonged to the Nitrosopumilales and were further clustered into five subclades ("NP-Alpha", "NP-Delta", "NP-Epsilon", "NP-Gamma", and "NP-Theta"). "NP-Epsilon" was the most dominant clade throughout the water column and "NP_Alpha" showed higher abundance only in the deeper water. Salinity and inorganic nutrient concentrations were the major factors that determined the vertical segregation of archaea. We anticipate that the observed differences in the vertical distribution of archaea might contribute to the compartmentalization of dark carbon fixation and nitrification in deeper water and organic matter degradation in surface waters of the Arctic Ocean.

Comammox dominate soil nitrification under different N fertilization regimes in semi-arid areas of Northeast China

The newly identified complete ammonia oxidation (Comammox), which is capable of oxidizing ammonia directly to nitrate, has complemented the knowledge of nitrification in the global nitrogen (N) cycle. Knowledge of the community compositions and contributions to nitrification of autotrophic ammonia-oxidizing archaea (AOA) and bacteria (AOB) and Comammox remain void. In this study, the abundance, community compositions, and contributions to nitrification of AOA, AOB, and Comammox were observed in five N gradients (0, 90, 150, 210, and 270 kg ha−1) in a semi-arid area of Northeast China. The results indicated that, compared with low N application rates, higher N application rates significantly improved the soil ammonia monooxygenase (AMO) and the hydroxylamine oxidase (HAO) activities while total nitrogen (TN) was noted to be the main factor driving AMO and HAO activities. Soil potential nitrification rate (PNR) significantly increased with the increase in N application rate, and soil PNR was positively correlated with TN, nitrate nitrogen (NO3−-N), soil organic matter (SOM), and ammonium nitrogen (NH4+-N). The structural equation model (SEM) showed that TN was the main factor driving the community composition of Comammox; SOM had a greatest effect on the community composition of AOA while NH4+-N had a greatest effect on the community composition of AOB. The soil PNR had a direct effect on the yield. Overall, the findings of this study highlighted that N application was more conducive to the reproduction of soil nitrifying microorganisms while Comammox was the dominant contributor to nitrification under N fertilization regimes in the semi-arid area of Northeast China.

Italian ryegrass (Lolium multiflorum L.)-rice (Oryza sativa L.) rotation promotes the nitrogen cycle in the rice rhizosphere through dominant ammonia-oxidizing bacteria

The dominant ammonium bacteria established in different crop rotations are directly related to the conversion and utilization of nitrogen (N) in paddy soil. This study investigated the ammonia oxidation process in the rice rhizosphere of Italian ryegrass (Lolium multiflorum L.)-rice (IRR), celery (Apium graveolens)-rice (CLR) and winter fallow-rice (WFR) rotations in South China through the amoA functional gene analysis based on Miseq high-throughput sequencing platform and qPCR methods. The abundance of amoA gene in IRR was increased by 116.67 % (P < 0.05) and 42.76 % (P > 0.05) compared with CLR and WFR, respectively. Nitrosomonas became the dominant genus in the rhizosphere of IRR, with the highest relative abundance (59.30 %). The contents of soil organic matter, total N and nitrate nitrogen (NO3−-N) in the rice rhizosphere soil in IRR were significantly higher than other two treatments (P < 0.05) by 12.47–14.31 %, 16.88–25.00 %, and 97.48–159.60 %, respectively. Although lower microbial diversity was observed in IRR, the soil fluorescein diacetate hydrolysis enzyme activities and ammonia monooxygenase activities in the rice rhizosphere were higher in IRR than those in CLR and WFR (P < 0.05). Also, the soluble sugar and crude protein of milled rice were highest in IRR among the three rotation treatments (P < 0.05). The ammonia oxidation efficiency of ammonium-oxidizing bacteria was largely dependent on key taxa, such as Nitrosomonas (dominant genus in IRR), rather than the dynamics of AOB community structure. The key amoA-nitrifier taxa of the AOB community developed in IRR are capable of promoting rice production by improving soil N cycle efficiency and reducing the loss of N. Our findings illustrate the regulatory mechanism of IRR on the rice rhizosphere AOB community and reveal the important role of winter ryegrass in promoting soil nitrogen cycling during nutrient transformation of agricultural ecosystems in paddy fields.

Ammonia monooxygenase-mediated transformation of 17α-ethinylestradiol: Underlying molecular mechanism

17α-ethinylestradiol (EE2) has received increasing attention as an emerging and difficult-to-remove emerging contaminant in recent years. Ammonia-oxidizing bacteria (AOB) have been reported to be effective in EE2 removal, and ammonia monooxygenase (AMO) is considered as the primary enzyme for EE2 removal. However, the molecular mechanism underlying the transformation of EE2 by AOB and AMO is still unclear. This study investigated the molecular mechanism of EE2 degradation using a combination of experimental and computational simulation methods. The results revealed that ammonia nitrogen was essential for the co-metabolism of EE2 by AOB, and that NH3 bound with CuC (one active site of AMO) to induce a conformational change in AMO, allowing EE2 to bind with the other active site (CuB), and then EE2 underwent biological transformation. These results provide a theoretical basis and a novel research perspective on the removal of ammonia nitrogen and emerging contaminants (e.g., EE2) in wastewater treatment.

Activity of ammonia-oxidizing bacteria in enriched cultures exposed to 3,4-dimethyl-1H-pyrazole dihydrogen phosphate nitrification inhibitor

The use of nitrification inhibitors is an interesting tool to achieve a higher N efficiency in plants while decreasing the environmental impact of N fertilization. However, an integrated evaluation of the efficiency of nitrification inhibitors over time, understood as the period in which the nitrifying activity is inhibited or slows down, is necessary to assess whether their use is ecofriendly and sustainable.To test the direct efficiency of 3,4-dimethyl-1H-pyrazole dihydrogen phosphate (DMPP) on nitrification, a study has been carried out in two cultures enriched with ammonia-oxidizing bacteria (AOB) obtained from a soil with continuous N fertilization (80 kg N ha−1 year−1 as NH4NO3) and from soil without N fertilization. In addition, Cu has been evaluated as a cofactor of ammonia monoxygenase, a key enzyme in the nitrifying activity of AOBs. On the other hand, the stability of DMP has been studied both in the cultivation system enriched in AOBs and in soil to assess the efficiency of the inhibitor due to its persistence over time.Our work reveals that nitrification rates observed in cultures enriched in AOBs from genus Nitrosospira isolated from soils with continuous N fertilization were not higher than those of cultures without N fertilization. In AOB cultures, DMPP was a very efficient inhibitor of nitrification (> 50 % inhibition of integrated AMO activity), mainly due to the stability of DMP (3,4-dimethyl-1 H-pyrazole) in the cultures. However, DMP stability was significantly lower under soil conditions (> 90 % of DMP was degraded in the first 30 days of incubation). Other metals are suggested as cofactors of the enzyme ammonia monooxygenase alternatively to Cu.

Nitrogen fertilization and irrigation types do not affect the overall N2O production potential of a sandy soil, but the microbial community structure and the quantity of functional genes related to the N cycle

Nitrous oxide (N2O), one of the major greenhouse gases, features a 273-fold higher global warming potential compared to carbon dioxide (CO2). Soil-born N2O emissions are based on microbial processes, while the microbial processes are affected by environmental factors and agronomic management measures. The objective of this study was the exploration of the effects of different irrigation and nitrogen (N) fertilization regimes on N2O emissions in relation to the microbial community structure and the quantities of genes involved in the N cycle. The following four treatments were investigated in a sandy soil during the potato cropping season in 2019 in Brandenburg, Germany: drip irrigation without N fertilization (DI-ZN), drip irrigation with broadcasted N fertilization (DI-N), simultaneous application of dissolved N in irrigation water, the so-called fertigation (F), and fertigation without crops (F-ZC). N2O fluxes were measured with the closed chamber method. Microbial biomass was analyzed using phospholipid fatty acids (PLFAs). The bacterial functional genes amoA (ammonium monooxygenase), nxrB (nitrite oxidoreductase), narG (nitrate reductase), nirS/nirK (nitrite reductase), and nosZ (N2O reductase) were determined using quantitative real-time polymerase chain reaction (qPCR). N2O fluxes varied over the season and could mainly be explained by temperature changes. The fertilized treatments showed slightly higher area-related cumulative N2O emissions (DI-ZN: 1.13 kg N2O-N ha−1, DI-N: 1.28 N2O-N ha−1, F: 1.26 N2O-N ha−1, median values). Additionally, N2O flux rates as well as the area- and yield-related N2O emissions of DI-N and F were approximately at the same level, even though the quantities and correlation patterns of the investigated functional genes differed. Highest seasonal median flux rates were detected under DI-N (15.0–100.1 μg N2O-N m−2 h−1), where a greater influence of a nirS/nosZ harboring microbial community was found. In contrast, under F more diverse correlation patterns were found, especially regarding a predominance of nirK and nirS/nosZ harboring microbial community. F-ZC showed the lowest N2O flux rates (−8.7–44.2 μg N2O-N m−2 h−1), which might be explained by the lack of plant carbon sources, particularly root exudates. To conclude, the applied treatments affected the microbial community structure and their genetically determined potential for N2O production, whereby the overall N2O flux rates and hence N2O emissions remained unaffected. Although fertigation is considered to be more demand-based particularly in terms of N fertilization, this study indicates that agronomic management measures according to good faming practice might feature an oversupply of N, neglecting the real N demand of cultivated crops and hence hindering the mitigation of N2O emissions.

Quantification of nitrogen cycle functional genes from viable archaea and bacteria in paddy soil.

One of the main challenges of culture-independent soil microbiology is distinguishing the microbial community's viable fraction from dead matter. Propidium monoazide (PMA) binds the DNA of dead cells, preventing its amplification. This dye could represent a robust means to overcome the drawbacks of other selective methods, such as ribonucleic acid-based analyses. We quantified functional genes from viable archaea and bacteria in soil by combining the use of PMA and quantitative polymerase chain reaction. Four N-cycle-related functional genes (bacterial and archaeal ammonia monooxygenase, nitrate reductase, and nitrite reductase) were successfully quantified from the living fraction of bacteria and archaea of a paddy soil. The protocol was also tested with pure bacterial cultures and soils with different physical and chemical properties. The experiment results revealed a contrasting impact of mineral and organic fertilizers on the abundance of microbial genes related to the N-cycle in paddy soil.

Effects of UV-B Radiation on the Chemical Composition of Azolla and Its Decomposition after Returning to the Field and Nitrogen Transformation in Soil

In the present research, the effects of UV-B radiation (5.00 kJ·m−2) on the chemical composition of Azolla were investigated, and the decomposition of Azolla residues after UV-B radiation, the nitrogen form, enzyme activity, and bacterial community in paddy soil were analyzed. Compared to the natural light treatment, the total nitrogen content of Azolla was significantly increased by 17.0% under UV-B radiation treatment. Compared to returned Azolla grown under natural light, the decomposition rate of cellulose, lignin, and total nitrogen of returned Azolla grown under UV-B radiation significantly increased, which led to an increase in the activities of nitrogen transformation enzymes, including neutral protease, ammonia monooxygenase, nitrogenase, nitrate reductase, and nitrite reductase, and the contents of different nitrogen forms (NH4+-N, NO3−-N, soluble organic nitrogen, and microbial biomass nitrogen) in paddy soil, while N2O emission flux was significantly reduced by 20–30%. The dominant bacteria in soil supplied with Azolla grown under natural light were Firmicutes, Clostridia, Clostridiales, and Lachnospiraceae. However, returning Azolla grown under UV-B radiation to the soil significantly changed the bacterial community structure in soil, resulting in a decrease in the number of ammonifying bacteria, nitrifying bacteria, and nitrogen-fixing bacteria and an increase in the number of denitrifying bacteria, inducing changes in the dominant bacteria to Methanomicrobiales, Methanoregulaceae, and Methanoregula. According to the structural equation model, returning Azolla to the field would reduce N2O emissions by increasing Azolla lignin decomposition and ammonia monooxygenase activity, reducing the number of nitrifying bacteria and reducing nitrite reductase activity in soil. Thus, UV-B radiation can directly change the phytochemical components and their decomposition in soil, thus indirectly affecting the bacterial community structure, enzyme activity, and nitrogen transformation, which play important ecological roles in regulating the nutrient transformation of terrestrial ecosystems.

The coexisted nitrate and nitrite as a driving force for the aerobic denitrification of Peribacillus sp. EM-C3

A heterotrophic nitrifying and aerobic denitrifying strain was newly isolated from a biogas slurry sample and assigned to be Peribacillus sp. EM-C3 based on phylogenetic and morphological analyses. This strain was capable of removing approximately 95.96% of ▪ , 93.63% of ▪ , and 99.71% of ▪ without nitrite accumulation, with maximum conversion rates of 4.98, 2.73, and 3.17 mg/L/h, respectively. Nitrogen balance analysis showed that high total nitrogen loss (> 50%) was achieved by utilizing ▪ , ▪ , or ▪ as a nitrogen source. Importantly, the maximum ▪ and ▪ removal rates increased to 3.78 and 4.42 mg/L/h when both ▪ and ▪ were present in the system, as this promoted aerobic denitrification by the strain. The key activities of enzymes involved in nitrogen metabolism (ammonia monooxygenase, and nitrate and nitrite reductases) were 0.45, 0.15, and 0.01 U/mg protein, respectively. Taken together, these results show that the coexistence of nitrate and nitrite may promote aerobic denitrification by Peribacillus sp. EM-C3.

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

The method of spiking synthetic internal standard genes (ISGs) to samples for amplicon sequencing, generating sequences and converting absolute gene numbers from read counts has been used only for phylogenetic markers and has not been applied to functional markers. In this study, we developed ISGs, including gene sequences of the 16S rRNA, pmoA, encoding a subunit of particulate methane monooxygenase and amoA, encoding a subunit of ammonia monooxygenase. We added ISGs to the samples, amplified the target genes and performed amplicon sequencing. For the mock community, the copy numbers converted from read counts using ISGs were equivalent to those obtained by the quantitative real-time polymerase chain reaction (4.0 × 104 versus 4.1 × 104 and 3.0 × 103 versus 4.0 × 103 copies μL-DNA-1 for 16S rRNA and pmoA genes, respectively), but we also identified underestimation, possibly due to primer coverage (7.8 × 102 versus 3.7 × 103 μL-DNA-1 for amoA gene). We then applied this method to environmental samples and analysed phylogeny, functional diversity and absolute quantities. One Methylocystis population was most abundant in the sludge samples [16S rRNA gene (3.8 × 109 copies g-1 ) and the pmoA gene (2.3 × 109 copies g-1 )] and were potentially interrelated. This study demonstrates that ISG spiking is useful for evaluating sequencing data processing and quantifying functional markers.