Hydroxylamine Dehydrogenase Research Articles

Enhancing effects and mechanisms by reductive iron on constructed wetland with perfluorooctanoic acid exposure: Comparison between zero-valent and ferrous iron

Reductive iron caused efficient enhancement of constructed wetlands (CWs) under toxic stress like perfluorooctanoic acid (PFOA), while effects and mechanisms brought by different iron have not been illustrated. In this study, impacts of zero-valent (Fe(0)) and ferrous iron (Fe(II)) on CW systems with PFOA exposure were compared. Higher ammonium removal above 98 % was discovered with Fe(0) addition than Fe(II), according with more expression of hydroxylamine dehydrogenase [EC:1.7.2.6]. On the contrary, Fe(II) was more prone to stimulate activities of ammonia monooxygenase and nitrite oxidoreductase, consisting with higher abundance of Nitrosomonas and Nitrospira than Fe(0). Fe(II) had more potential to improve denitrification, with total nitrogen removal 2.64–6.83 % higher compared to Fe(0) group. It was aligned with boost of dominant denitrifying bacteria like Zoogloea (53.48 %), which probably resulted from promotion of metabolism pathways related to nitrite reduction. Phosphorus removal with Fe(II) addition was 4.59–19.04 % higher than Fe(0). Higher TP removal in Fe(II) group could result from more enhancement of physicochemical processes, compared with Fe(0). Moreover, decrease of reactive oxygen species production by citrate and activation of mitochondrial metabolism was explanation for alleviation of PFOA stress with reductive iron introduction, particularly Fe(II). As for plants, Fe(0) led to higher chlorophyll contents and lower activity of antioxidant enzymes like catalase and malondialdehyde. Current work offered new information about comparative effects of reductive iron on CWs under PFOA exposure.

Comparative Analysis of Bacterial Information of Biofilms and Activated Sludge in Full-Scale MBBR-IFAS Systems.

This study extensively analyzed the bacterial information of biofilms and activated sludge in oxic reactors of full-scale moving bed biofilm reactor-integrated fixed-film activated sludge (MBBR-IFAS) systems. The bacterial communities of biofilms and activated sludge differed statistically (R = 0.624, p < 0.01). The denitrifying genera Ignavibacterium, Phaeodactylibacter, Terrimonas, and Arcobacter were more abundant in activated sludge (p < 0.05), while comammox Nitrospira was more abundant in biofilms (p < 0.05), with an average relative abundance of 8.13%. Nitrospira and Nitrosomonas had weak co-occurrence relationships with other genera in the MBBR-IFAS systems. Potential function analysis revealed no differences in pathways at levels 1 and 2 based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) between biofilms and activated sludge. However, in terms of pathways at level 3, biofilms had more potential in 26 pathways, including various organic biodegradation and membrane and signal transportation pathways. In comparison, activated sludge had more potential in only five pathways, including glycan biosynthesis and metabolism. With respect to nitrogen metabolism, biofilms had greater potential for nitrification (ammonia oxidation) (M00528), and complete nitrification (comammox) (M00804) concretely accounted for methane/ammonia monooxygenase (K10944, K10945, and K10946) and hydroxylamine dehydrogenase (K10535). This study provides a theoretical basis for MBBR-IFAS systems from the perspective of microorganisms.

Innovative inbuilt moving bed biofilm reactor for nitrogen removal applied in household aquarium.

An innovative inbuilt moving bed biofilm reactor (MBBR) was created to protect fish from nitrogen in a household aquarium. During the 90 experimental days, the ammonia nitrogen (NH4+-N) concentration in the aquarium with the inbuilt MBBR was always below 0.5 mg/L, which would not threaten the fish. Concurrently, nitrite and nitrate nitrogen concentrations were always below 0.05 mg/L and 4.5 mg/L, respectively. However, the blank contrast aquarium accumulated 1.985 mg/L NH4+-N on the 16th day, which caused the fish to die. The suspended biofilms could achieve the specific NH4+-N removal rate of 45.43 g/m3/d. Biofilms presented sparsely with filamentous structures and showed certain degrees of roughness. The bacterial communities of the suspended biofilms and the sediment were statistically different (p < 0.05), reflected in denitrifying and nitrifying bacteria. In particular, the relative abundance of Nitrospira reached 1.4%, while the genus was barely found in sediments. The suspended biofilms showed potentials for nitrification function with the predicted sequence numbers of ammonia monooxygenase [1.14.99.39] and hydroxylamine dehydrogenase [EC:1.7.2.6] of 220 and 221, while the values of the sediment were only 5 and 1. This study created an efficient NH4+-N removal inbuilt MBBR for household aquariums and explored its mechanism to afford a basis for its utilization.

Effects of root-applied biochar on soil nitrogen transformation and root nitrogen metabolism of cucumber seedlings in facility continuous cropping soils.

The problem of soil barrier caused by excessive accumulation of nitrogen is common in continuous cropping soil of facility agriculture. To investigate the modulating effects of biochar amendment on soil nitrogen transformation in greenhouse continuous cropping systems, we conducted a pot experiment with two treatments, no biochar addition (CK) and 5% biochar addition (mass ratio). We analyzed the effects of biochar addition on soil microbial community structure, abundances of genes functioning in nitrogen cycling, root growth and nitrogen metabolism-related genes expressions of cucumber seedlings. The results showed that biochar addition significantly increased plant height, root dry mass, total root length, root surface area, and root volume of cucumber seedlings. Rhizosphere environment was improved, which enhanced root nitrogen absorption by inducing the up-regulation of genes expressions related to plant nitrogen metabolism. Biochar addition significantly increased soil microbial biomass nitrogen, nitrate nitrogen, and nitrite nitrogen contents. The abundances of bacteria that involved in nitrogen metabolism, including Proteobacteria, Cyanobacteria, and Rhizobiales (soil nitrogen-fixing bacteria), were also significantly improved in the soil. The abundances of genes functioning in soil nitrification and nitrogen assimilation reduction, and the activities of enzymes involved in nitrogen metabolisms such as hydroxylamine dehydrogenase, nitronate monooxygenase, carbonic anhydrase were increased. In summary, biochar addition improved soil physicochemical properties and microbial community, and affected soil nitrogen cycling through promoting nitrification and nitrogen assimilation. Finally, nitrogen adsorption capacity and growth of cucumber plant was increased.

Performance evaluation and metagenomic analysis of sequencing batch reactor under transient 2,4,6-trichlorophenol shock

The transient chlorophenol shock under some emergency conditions might directly affect the pollutant removal of bioreactor. Therefore, the recovery of bioreactor performance after transient chlorophenol shock is a noteworthy issue. In the present research, the performance, antioxidant response, microbial succession and functional genes of sequencing batch reactor (SBR) were evaluated under transient 2,4,6-trichlorophenol (2,4,6-TCP) shock. The chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) removal efficiencies decreased sharply in the first 4 days after 60 mg/L 2,4,6-TCP shock for 24 h and gradually recovered to normal in the subsequent 8 days. The nitrogen removal rates and their corresponding enzymatic activities rapidly decreased after transient 2,4,6-TCP shock and then gradually increased to normal. The increase of antioxidant enzymatic activity, Cu-Zn SOD genes and Fe-Mn SOD genes contributed to the recovery of SBR performance. The abundance of genes encoding ammonia monooxygenase and hydroxylamine dehydrogenase decreased after transient 2,4,6-TCP shock, including amoA, amoC and nxrA. Thauera, Dechloromonas and Candidatus_Competibacter played key roles in the restorative process, which provided stable abundances of narG, norB , norC and nosZ. The results will deeply understand into the effect of transient 2,4,6-TCP shock on bioreactor performance and provide theoretical basis to build promising recoveries strategy of bioreactor performance.

Metagenomic Insight Reveals the Microbial Structure and Function of the Full-Scale Coking Wastewater Treatment System: Gene-Based Nitrogen Removal

Microbial communities play crucial roles in pollutant removal and system stability in biological systems for coking wastewater (CWW) treatment, but a comprehensive understanding of their structure and functions is still lacking. A five month survey of four sequential bioreactors, anoxic 1/oxic 1/anoxic 2/oxic 2 (A1/O1/A2/O2), was carried out in a full-scale CWW treatment system in China to elucidate operational performance and microbial ecology. The results showed that A1/O1/A2/O2 had excellent and stable performance for nitrogen removal. Both total nitrogen (TN; (17.38 ± 6.89) mg·L−1) and ammonium–nitrogen (NH4+-N; (2.10 ± 1.34) mg·L−1) in the final biological effluent satisfied the Chinese national standards for CWW. Integrated analysis of 16S ribosome RNA (rRNA) sequencing and metagenomic sequencing showed that the bacterial communities and metagenomic function profiles of A1 and O1 shared similar functional structures, while those of A2 significantly varied from those of other bioreactors (p < 0.05). The results indicated that microbial activity was strongly connected with activated sludge function. Nitrosospira, Nitrosomonas, and SM1A02 were responsible for nitrification during the primary anoxic–oxic (AO) stage and Azoarcus and Thauera acted as important denitrifiers in A2. Nitrogen cycling-related enzymes and genes work in the A1/O1/A2/O2 system. Moreover, the hao genes catalyzing hydroxylamine dehydrogenase (EC 1.7.2.6) and the napA and napB genes catalyzing nitrate reductase (EC 1.9.6.1) played important roles in the nitrification and denitrification processes in the primary and secondary AO stages, respectively. The mixed liquor suspended solids (MLSS)/total solids (TS), TN removal rate (RR), total organic carbon (TOC) (RR), and NH4+-N (RR) were the most important environmental factors for regulating the structure of core bacterial genera and nitrogen-cycling genes. Proteobacteria were the potential main participants in nitrogen metabolism in the A1/O1/A2/O2 system for CWW treatment. This study provides an original and comprehensive understanding of the microbial community and functions at the gene level, which is crucial for the efficient and stable operation of the full-scale biological process for CWW treatment.

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.

Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

Hot moment of N2O emissions in seasonally frozen peatlands.

Since the start of the Anthropocene, northern seasonally frozen peatlands have been warming at a rate of 0.6 °C per decade, twice that of the Earth's average rate, thereby triggering increased nitrogen mineralization with subsequent potentially large losses of nitrous oxide (N2O) to the atmosphere. Here we provide evidence that seasonally frozen peatlands are important N2O emission sources in the Northern Hemisphere and the thawing periods are the hot moment of annual N2O emissions. The flux during the hot moment of thawing in spring was 1.20 ± 0.82 mg N2O m-2 d-1, significantly higher than that during the other periods (freezing, -0.12 ± 0.02 mg N2O m-2 d-1; frozen, 0.04 ± 0.04 mg N2O m-2 d-1; thawed, 0.09 ± 0.01 mg N2O m-2 d-1) or observed for other ecosystems at the same latitude in previous studies. The observed emission flux is even higher than those of tropical forests, the World's largest natural terrestrial N2O source. Furthermore, based on soil incubation with 15N and 18O isotope tracing and differential inhibitors, heterotrophic bacterial and fungal denitrification was revealed as the main source of N2O in peatland profiles (0-200 cm). Metagenomic, metatranscriptomic, and qPCR assays further revealed that seasonally frozen peatlands have high N2O emission potential, but thawing significantly stimulates expression of genes encoding N2O-producing protein complexes (hydroxylamine dehydrogenase (hao) and nitric oxide reductase (nor)), resulting in high N2O emissions during spring. This hot moment converts seasonally frozen peatlands into an important N2O emission source when it is otherwise a sink. Extrapolation of our data to all northern peatland areas reveals that the hot moment emissions could amount to approximately 0.17 Tg of N2O yr-1. However, these N2O emissions are still not routinely included in Earth system models and global IPCC assessments.

Succession and PICRUSt2-based Predicted Functional Analysis of Microbial Communities During the Sludge Bulking Occurrence and Restoration in One-stage Combined Partial Nitritation and ANAMMOX Process

This study was based on the pilot one-stage combined partial nitritation and ANAMMOX process (CPNA), using data mining and analysis of 16S rRNA high-throughput sequencing data of activated sludge in the process of sludge bulking and recovery, combined with PISCRUSt2. The function prediction analysis aimed to reveal the microbial community changes and the characteristics of nitrogen metabolism and carbon metabolism at different stages of sludge bulking and recovery of the one-stage CPNA process. The results of the study showed that the microbial α-diversity in the sludge bulking and recovery process first increased and then declined. The relative abundance of Nitrosomonas, Candidatus_Brocadia, and Thaurea decreased in the sludge-bulking stage from 12.36%, 11.86%, and 0.272% to 5.97%, 8.30%, and 0.061%, whereas the relative abundance of Candidatus Kuenenia remained stable. The relative abundance of Levilinea, Longilinea, and Turicibacter increased from 0.031%, 0.018%, and 0.009% to 0.055%, 0.025%, and 0.033%. The PICRUSt2 function prediction analysis results showed that there were a total of 47 functional enzyme genes involved in nitrogen metabolism, of which nitrification, denitrification, dissimilative nitrate reduction (DNRA), assimilation nitrate reduction (ANRA), and nitrogen fixation were relatively abundant. The degrees of each had changed. During the sludge-bulking stage, the relative abundance of the ammonia monooxygenase gene (pmoABC-amoABC) and the hydroxylamine dehydrogenase gene hao decreased, whereas the relative abundance of the nitrate-reducing gene increased at the initial stage and then showed a downward trend. Carbon metabolism analysis showed that sodium acetate had a promoting effect on the heterotrophic growth of the CPNA process, but the energy metabolism and glucose production of sodium acetate were not active.

Metagenomic analyses reveal nitrogen metabolism responses to copper and chromium contamination in sludge-based microbial communities

In this study, we used metagenomic sequencing to explore the effects of chromium (Cr) and copper (Cu) on microbial communities and function by analyzing pathway of nitrogen metabolism based on KEGG annotation. High concentrations of Cr (VI) and Cu (II) reduced the removal efficiency of total nitrogen and ammonium in the system. Metagenomic analyses showed that the taxonomic composition of nitrogen metabolism-associated microorganisms (NMAM) was mainly composed of Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Sphingobacteria, Deltaproteobacteria, Flavobacteria and Nitrospira. Different concentrations of Cr (VI) and Cu (II) led to great changes in NMAM in activated sludge and enriched dominant microbes, respectively, suggesting specific response strategies in microbial communities. NMDS (Non-metric multidimensional scaling) analyses revealed the impact of different Cr (VI) and Cu (II) concentrations on NMAM β diversity, and indicated that Cr (VI) treatment induced greater variations in NMAM communities than Cu (II). We also identified a significant association (p < 0.05) between enzymes of nitrogen metabolism pathway and corresponding NMAM. This study revealed that under Cu (II) and Cr (VI) stress, the sludge nitrification pathway was inhibited, especially for ammonia monooxygenase and hydroxylamine dehydrogenase. These combined effects led to a decrease in sludge efficiency for nitrogen removal. These findings underscore the influence of heavy metals in water treatment.

The enzymes-based intermediary model explains the influence mechanism of different aeration strategies on nitrogen removal in a sequencing batch biofilm reactor treating simulated aquaculture wastewater

Aquaculture wastewater can be efficiently treated by sequencing batch biofilm reactor (SBBR) via simultaneous nitrification and denitrification (SND), which is heavily influenced by different aeration strategies. The triangular associations among functional microbial abundance, key enzymes, and purification performance can be helpful to explain their influence mechanism on nitrogen removal from SBBR. For the present study, ten such systems for aquaculture wastewater polishing were operated under the pre-aeration (Pre-A) or post-aeration (Post-B) mode with different ratios of aeration to non-aeration or non-aeration to aeration. It was found that the average percent removals of ammonium (NH4+-N) and total inorganic nitrogen (TIN) were 84.72% and 61.98% in Pre-A, and were 93.84% and 55.29% in Post-B, with no obvious nitrite accumulation (0.13 ± 0.07 mg/L in Pre-A and 0.6 ± 0.2 mg/L in Post-B). The proteomic analysis illustrated that the aeration sequence affected the intermediary effect of ammonia monooxygenase (EC1.14.99.39) and hydroxylamine dehydrogenase (EC1.7.2.6). Post-aeration was beneficial to the consistency of the nitrifier abundance, the key nitrifying enzyme abundance, and NH4+-N removal. Meanwhile, the aeration sequence influenced the triangular associations among the heterotrophic denitrifier abundance, the denitrifying enzyme abundance, and TIN removal. In addition, nitrous-oxide reductase (EC1.7.2.4) was inhibited in the Post-B treatment. The different aeration strategies had no obvious impact on the taxonomic composition of autotrophic nitrifiers in these SBBR systems, but greatly influenced the taxonomic composition of heterotrophic denitrifiers at the genus level, especially affecting the proportion of denitrifying polyphosphate accumulating organisms (PAOs).

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.

Comprehensive metagenomic and enzyme activity analysis reveals the negatively influential and potentially toxic mechanism of polystyrene nanoparticles on nitrogen transformation in constructed wetlands

The widespread use of nanoplastics inevitably leads to their increasing emission into constructed wetlands (CWs). However, little is known about the impacts of nanoplastics on nitrogen transformation in CWs. In this study, the influence of polystyrene nanoparticles (PS NPs), one of the most widely used plastics, on the nitrogen transformation in CWs was comprehensively investigated, and the influential and toxic mechanism was evaluated through metagenomic analysis (DNA level) and key enzyme activities (protein level) related to N-transformation metabolism and antioxidant systems. The results showed that over 97% of PS NPs were retained in CWs, and the biofilm of sand was the main sink of PS NPs. Exposure to 1 and 10 mg/L PS NPs suppressed the nitrogen transformation, causing a certain degree of inhibition in TN removal, especially in the relatively short term of the exposure experiment (p < 0.05). At the protein level, 1 and 10 mg/L PS NPs negatively affect enzyme activities involved in denitrification (nitrate reductase and nitrite reductase) and electron transport system activity (ETSA). In contrast, 10 mg/L of PS NPs significantly suppressed the activities of nitrifying enzymes (ammonia monooxygenase, hydroxylamine dehydrogenase and nitrite oxidoreductase), whereas 1 mg/L PS NPs showed no impacts on nitrifying enzymes. Metagenomic analysis further certified that PS NPs restrained the relative abundances of genes involved in nitrogen transformation including nitrification and denitrification biochemical metabolisms (the electron production, electron transport and electron consumption processes). It also indicated that PS NPs could affect nitrogen transformation by reducing the abundance of genes for electron donor and ATP production involved in carbon metabolism (glycolysis and tricarboxylic acid cycle metabolism). In our study, the potential toxic mechanisms of PS NPs attributed to over production of reactive oxygen species and variations of antioxidant systems in macrophytes and microorganisms. These results provided valuable information for evaluating the impacts of PS NPs on CWs and arouse more attention to their impacts on the global geochemical nitrogen and carbon cycles.

In-situ expressions of comammox Nitrospira along the Yangtze River

The recent discovery of comammox Nitrospira as complete nitrifiers has significantly enriched our understanding on the nitrogen cycle, yet little is known about their metabolic transcripts in natural aquatic ecosystems. Using the genome-centric metatranscriptomics, we provided the first in-situ expression patterns of comammox Nitrospira along the Yangtze River. Our study confirmed widespread expressions of comammox Nitrospira, with the highest transcription accounting for 33.3% and 63.8% of amoA and nxrAB genes expressed in ammonia-oxidizing prokaryotes (AOPs) and Nitrospira sublineages I/II, respectively. Moreover, comammox two clades differed in nitrification, with clade A acting as the dominator to ammonia oxidation in comammox, and clade B contributing more transcripts to nitrite oxidation than to ammonia oxidation. Compared to canonical Nitrospira, comammox community had lower expressions of ammonia/nitrite transporters and nitrogen assimilatory genes, but far higher expressions in urea transport and hydrolysis, facilitating to derivation of ammonia and energy mainly through intracellular ureolytic metabolism. This suggests no need for “reciprocal-feeding” between canonical Nitrospira and AOPs in a natural river. Aerobic mixotrophy of comammox bacteria was suggested by expressions of genes coding for respiratory complexes I-V, oxidative/reductive TCA cycle, oxygen stress defenses, and transport/catabolism of simple carbohydrates and low-biosynthetic-cost amino acids. Intriguingly, significant positive correlations among expressions of ammonia monooxygenases, hydroxylamine dehydrogenase and copper-dependent nitrite reductase indicated that comammox Nitrospira had the potential of converting nitrite to nitric oxide accompanied by ammonia oxidation under low-C/N and aerobic conditions, while gene expressions in this pathway were significantly and positively associated with pH. Overall, this study illustrated novel transcriptional characteristics of comammox Nitrospira, and highlighted the necessity of reassessing their contributions to biogeochemical carbon and nitrogen cycling with perspective of in-situ meta-omics as well as culture experiments.

Transcriptomic and Metabolomic Responses to Carbon and Nitrogen Sources in Methylomicrobium album BG8.

Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled the phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated hydroxylamine dehydrogenase (haoAB) and overall central metabolic activity, whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCE Methanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism vary across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

Combined genome-centric metagenomics and stable isotope probing unveils the microbial pathways of aerobic methane oxidation coupled to denitrification process under hypoxic conditions

Obligate aerobic methanotrophs have been proven to oxidize methane and participate in denitrification under hypoxic conditions. However, this phenomenon and its metabolic mechanism have not been investigated in detail in aerobic methane oxidation coupled to denitrification (AME-D) process. In this study, a type of hypoxic AME-D consortium was enriched and operated for a long time in a CH4-cycling bioreactor with strict anaerobic control and the nitrite removal rate reached approximately 50 mg N/L/d. Metagenomics combined with DNA stable-isotope probing demonstrated that the genus Methylomonas, which constitutes type I aerobic methanotrophs, was the dominant member and contributed to methane oxidation and partial denitrification. Metagenomic binning recovered a near-complete (98%) draft genome affiliated with the family Methylococcaceae containing essential genes that encode nitrite reductase (nirK), nitric oxide reductase (norBC) and hydroxylamine dehydrogenase (hao). Metabolic reconstruction of the selected Methylococcaceae genomes also revealed a potential link between methanotrophy and partial denitrification.

Nitrosomonas europaea MazF Specifically Recognises the UGG Motif and Promotes Selective RNA Degradation.

Toxin-antitoxin (TA) systems are implicated in prokaryotic stress adaptation. Previously, bioinformatics analysis predicted that such systems are abundant in some slowly growing chemolithotrophs; e.g., Nitrosomonas europaea. Nevertheless, the molecular functions of these stress-response modules remain largely unclear, limiting insight regarding their physiological roles. Herein, we show that one of the putative MazF family members, encoded at the ALW85_RS04820 locus, constitutes a functional toxin that engenders a TA pair with its cognate MazE antitoxin. The coordinate application of a specialised RNA-Seq and a fluorescence quenching technique clarified that a unique triplet, UGG, serves as the determinant for MazF cleavage. Notably, statistical analysis predicted that two transcripts, which are unique in the autotroph, comprise the prime targets of the MazF endoribonuclease: hydroxylamine dehydrogenase (hao), which is essential for ammonia oxidation, and a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL), which plays an important role in carbon assimilation. Given that N. europaea obtains energy and reductants via ammonia oxidation and the carbon for its growth from carbon dioxide, the chemolithotroph might use the MazF endoribonuclease to modulate its translation profile and subsequent biochemical reactions.

The New Developments Made in the Autotrophic and Heterotrophic Ammonia Oxidation

Nitrification is defined as conversion of the ammonium(NH4+-N) to nitrate and it is generally divided into two categories: autotrophic nitrification and heterotrophic nitrification. With the metagenomes retrieved from the Nitrospira highly enriched biosamples, both the ammonium oxidation gens (full set of AMO and hydroxylamine dehydrogenase (HAO) genes) and nitrite oxidoreductase (NXR) coding genes necessary for nitrite oxidation were found in a completely nitrifying bacterium from the genus Nitrospira. Various species of Nitrospira (containing phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes) had been found in many engineered systems and were proved to be comammox bacterium. Some anammox bacteria were found to use mono or dimethylamine and even methanol as electron donors, but the enzymes the employed in the special metabolism were not known. Many heterotrophic nitrifying bacteria have been isolated and characterized from soil, activated sludge, wastewater treatment system. A denitrification process was developed with poly(3-hydroxybutyrate) (PHB) granulesas source of carbon for denitrification. It is possible from metabolic foundations that the heterotrophic nitrifier can consume intracellular stored PHB as the alternative carbon source. This direction still waiting for further exploration.

Comparison of the Effects of Phenylhydrazine Hydrochloride and Dicyandiamide on Ammonia-Oxidizing Bacteria and Archaea in Andosols.

Dicyandiamide, a routinely used commercial nitrification inhibitor (NI), inhibits ammonia oxidation catalyzed by ammonia monooxygenase (AMO). Phenylhydrazine hydrochloride has shown considerable potential for the development of next-generation NIs targeting hydroxylamine dehydrogenase (HAO). The effects of the AMO inhibitor and the HAO inhibitor on ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) present in agricultural soils have not been compared thus far. In the present study, the effects of the two inhibitors on soil nitrification and the abundance of AOA and AOB as well as their community structure were investigated in a soil microcosm using quantitative polymerase chain reaction and pyrosequencing. The net nitrification rates and the growth of AOA and AOB in this soil microcosm were inhibited by both NIs. Both NIs had limited effect on the community structure of AOB and no effect on that of AOA in this soil microcosm. The effects of phenylhydrazine hydrochloride were similar to those of dicyandiamide. These results indicated that organohydrazine-based NIs have potential for the development of next-generation NIs targeting HAO in the future.