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Organic carbon determines nitrous oxide consumption activity of clade I and II nosZ bacteria: Genomic and biokinetic insights
Abstract
Harnessing nitrous oxide (N2O)-reducing bacteria is a promising strategy to reduce the N2O footprint of engineered systems. Applying a preferred organic carbon source as an electron donor accelerates N2O consumption by these bacteria. However, their N2O consumption potential and activity when fed different organic carbon species remain unclear. Here, we systematically compared the effects of various organic carbon sources on the activity of N2O-reducing bacteria via investigation of their biokinetic properties and genomic potentials. Five organic carbon sources—acetate, succinate, glycerol, ethanol, and methanol—were fed to four N2O-reducing bacteria harboring either clade I or clade II nosZ gene. Respirometric analyses were performed with four N2O-reducing bacterial strains, identifying distinct shifts in DO- and N2O-consumption biokinetics in response to the different feeding schemes. Regardless of the N2O-reducing bacteria, higher N2O consumption rates, accompanied by higher biomass yields, were obtained with acetate and succinate. The biomass yield (15.45 ± 1.07 mg-biomass mmol-N2O−1) of Azospira sp. strain I13 (clade II nosZ) observed under acetate-fed condition was significantly higher than those of Paracoccus denitrificans and Pseudomonas stutzeri, exhibiting greater metabolic efficiency. However, the spectrum of the organic carbon species utilizable to Azospira sp. strain I13 was limited, as demonstrated by the highly variable N2O consumption rates observed with different substrates. The potential to metabolize the supplemented carbon sources was investigated by genomic analysis, the results of which corroborated the N2O consumption biokinetics results. Moreover, electron donor selection had a substantial impact on how N2O consumption activities were recovered after oxygen exposure. Collectively, our findings highlight the importance of choosing appropriate electron donor additives for increasing the N2O sink capability of biological nitrogen removal systems.
... External organic carbon is often supplemented for efficient denitrification, which increases the risk of GHG emissions and organic residues (Deng et al., 2020). In addition, the type of external carbon source also affect the properties of N 2 O-reducing bacteria and N 2 O emissions (Qi et al., 2022). Maximum N 2 O production, with a conversion ratio of 56%, was observed when glucose was used as the carbon source (Zhao et al., 2018). ...
... Second, low-intensity, intermittent, or AvN aeration modes are recommended to optimize the daily operation. Third, when external carbon source dosing is required, a distributed dosing mode can be adopted (Chai et al., 2019), and environmentally friendly carbon source additives can be selected (Qi et al., 2022). Moreover, modification of the energy-related system is vital to realize energy conservation and emission reduction in WWTPs. ...
Nitrous oxide (N2O), a potent greenhouse gas, significantly contributes to the carbon footprint of wastewater treatment plants (WWTPs) and contributes significantly to global climate change and to the deterioration of the natural environment. Our understanding of N2O generation mechanisms has significantly improved in the last decade, but the development of effective N2O emission mitigation strategies has lagged owing to the complexity of parameter regulation, substandard monitoring activities, and inadequate policy criteria. Based on critically screened published studies on N2O control in full-scale WWTPs, this review elucidates N2O generation pathway identifications and emission mechanisms and summarizes the impact of N2O on the total carbon footprint of WWTPs. In particular, a linear relationship was established between N2O emission factors and total nitrogen removal efficiencies in WWTPs located in China. Promising N2O mitigation options were proposed, which focus on optimizing operating conditions and implementation of innovative treatment processes. Furthermore, the sustainable operation of WWTPs has been anticipated to convert WWTPs into absolute greenhouse gas reducers as a result of the refinement and improvement of on-site monitoring activities, mitigation mechanisms, regulation of operational parameters, modeling, and policies.
... Recent studies confirmed that acetate can serve as selector for certain bacteria, particularly Acidovorax Comamonas, and Thauera spp. (Osaka et al., 2006;Lu et al., 2014), sometimes with higher N 2 O consumption capabilities (Qi et al., 2022) highlighting how important community composition and how shifts in community/predominant species can lead to lower N 2 O production (Di Capua, 2022;Kinh et al., 2017;Suenaga et al., 2018). Given that our research involved high concentrations of N 2 O, future studies should examine N 2 O reduction and microbial community structure at levels more consistent with observed N 2 O levels from wastewater treatment facilities. ...
... . In the NO 2 − only test, Azospira, which represented 10% of the entire community, was the dominant non-Comamonas/Acidovorax denitrifier, and similarly, Oxalobacteraceae family members represented 21% of the NO 2 − + N 2 O test. The occurrence of Azospira genus as the third most abundant genus in the NO 2 − sample could be associated with its ability to fully denitrify NO 2 −(Park et al., 2020) and to grow more favorably in presence of acetate(Qi et al., 2022), despite NO 2 reduction pathway being less favorable for biomass production than the NO 3 − reduction pathway. Another possible explanation is that partial DNB present in the NO 3 − batch could have prevented the growth of Azospira. ...
Nitrous oxide (N2O) is a potent greenhouse gas that can be produced by nitrifying and denitrifying bacteria. Yet the effects of N2O on microbial communities is not well understood. We used batch tests to explore the effects of N2O on mixed denitrifying communities. Batch tests were carried out with acetate as the electron donor and with the following electron acceptors: nitrate (NO3⁻), nitrite (NO2⁻), N2O, NO3⁻ + N2O, and NO2⁻ + N2O. Activated sludge from a municipal wastewater treatment plant was used as the inoculum. The bacteria grew readily with N2O as the sole acceptor. When N2O was provided along with NO3⁻ or NO2⁻, it was used concurrently and resulted in higher growth rates than the same acceptors without added N2O. The microbial communities resulting from N2O addition were significantly different at the genus level from those with just NO3⁻ or NO2⁻. Tests with N2O as the sole added acceptor revealed a reduced diversity. Analysis of inferred gene content using PICRUSt2 indicated a greater abundance of genera with a complete denitrification pathway when growing on N2O or NO2⁻, relative to all other tests. This suggests that specific N2O reduction rates are high, and that N2O alone selects for a low-diversity, fully denitrifying community. When N2O is present with NO2⁻ or NO3⁻, the microbial communities were more diverse and did not select exclusively for full denitrifiers. N2O alone appears to select for a “generalist” community with full denitrification pathways and lower diversity. In terms of denitrification genes, the combination of acceptors with N2O appeared to increase the number of microbes carrying nirK, while fully denitrifying bacteria appear more likely to carry nirS. Lastly, all the taxa in NO2⁻ and N2O samples were predicted to harbor nosZ. This suggests the potential for reduced N2O emissions in denitrifying systems.
... Recent studies confirmed that acetate can serve as selector for certain bacteria, particularly Acidovorax Comamonas, and Thauera spp. (Osaka et al., 2006;Lu et al., 2014), sometimes with higher N 2 O consumption capabilities (Qi et al., 2022) highlighting how important community composition and how shifts in community/predominant species can lead to lower N 2 O production (Di Capua, 2022;Kinh et al., 2017;Suenaga et al., 2018). Given that our research involved high concentrations of N 2 O, future studies should examine N 2 O reduction and microbial community structure at levels more consistent with observed N 2 O levels from wastewater treatment facilities. ...
... . In the NO 2 − only test, Azospira, which represented 10% of the entire community, was the dominant non-Comamonas/Acidovorax denitrifier, and similarly, Oxalobacteraceae family members represented 21% of the NO 2 − + N 2 O test. The occurrence of Azospira genus as the third most abundant genus in the NO 2 − sample could be associated with its ability to fully denitrify NO 2 −(Park et al., 2020) and to grow more favorably in presence of acetate(Qi et al., 2022), despite NO 2 reduction pathway being less favorable for biomass production than the NO 3 − reduction pathway. Another possible explanation is that partial DNB present in the NO 3 − batch could have prevented the growth of Azospira. ...
... Electrochemical sensing of oxygen gradients using Clark-type sensors has been well documented in microbial communities with many examples [42] spanning environmental microbiology [43,44], model systems [45], pathogen biology [15,46] and wastewater management [47]. The accuracy of this sensing method, both in terms of precision targeting of biofilm sub-populations using the micron-scale sensor tip and the sensitivity and dynamic range, provides a robust quantitative method for complex microbial communities [48]. ...
Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterized throughout the biofilm mass, but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilization throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualizations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.
... Functional genes related to CH 4 oxidation (pmoA) and gene related to N 2 O reduction (nosZ) exhibited a substantial sensitivity to variations in TOC concentration (p < 0.05). Genomic evidence has demonstrated a robust correlation between nosZ gene abundance and TOC concentration [59]. Conversely, both fdhB and mtaA genes exhibited notable susceptibility to shifts in pH levels (Fig. 6B). ...
Background
The conversion of natural wetlands to agricultural land through drainage contributes to 62% of the global wetland loss. Such conversion significantly alters greenhouse gas (GHG) fluxes, yet the underlying mechanisms of GHG fluxes resulting from drainage and long-term tillage practices remain highly uncertain. In this study, we measured GHG fluxes of a natural reed wetland (referred to as “Wetland”) and a drained wetland that used as farmland (referred to as “Dryland”).
Results
The results demonstrated that annual cumulative N2O and CO2 fluxes in Dryland were 282.77% and 53.79% higher than those in Wetland, respectively. However, CH4 annual cumulative fluxes decreased from 12,669.45 ± 564.69 kg·ha− 1 to 8,238.40 ± 207.72 kg·ha− 1 in Dryland compared to Wetland. The global warming potential (GWP) showed no significant difference between Dryland and Wetland, with comparable average rates of 427.50 ± 48.83 and 422.21 ± 73.59 mg·CO2-eq·m− 2·h− 1, respectively. Metagenomic analysis showed a decrease in the abundance of acetoclastic methanogens and their functional genes responsible for CH4 production. Functional genes related to CH4 oxidation (pmoA) and gene related to N2O reduction (nosZ) exhibited a substantial sensitivity to variations in TOC concentration (p < 0.05). Candidatus Methylomirabilis, belonging to the NC10 phylum, was identified as the dominant methanotroph and accounted for 49.26% of the methanotrophs. Its relative abundance was significantly higher in Dryland than in Wetland, as the nitrogenous fertilizer applied in Dryland acted as an electron acceptor, with the nearby Wetland produced CH4 serving as an electron donor. This suggests that Dryland may act as a CH4 sink, despite the significant enhancement in CO2 and N2O fluxes.
Conclusions
In conclusion, this study provides insights into the influence of drainage and long-term tillage on GHG fluxes in wetlands and their contribution to global warming.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40793-025-00682-w.
... The Environmental Protection Agency (EPA) and the Intergovernmental Panel on Climate Change (IPCC) have reported emission factors of 0.84% and 1.6% N 2 O-N/T-N inlet, respectively [9]. Furthermore, the proportion of nitrogen released as N 2 O can range from 0% to 14.6%, higher than previously estimated [10]. Reducing N 2 O emissions is crucial for mitigating global climate change and achieving sustainable development. ...
Nitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone depletion, with wastewater treatment plants (WWTPs) serving as significant sources of emissions due to biological processes involving bacteria. This study evaluates research on the role of bacteria in N2O emissions from WWTPs between 2000 and 2023 based on an analysis of the Web of Science Core Collection Database using keywords “bacteria”, “nitrous oxide”, “emission”, and “wastewater treatment plant”. The findings reveal substantial research growth in the past decade, with leading publications appearing in Water Research, Bioresource Technology, and Environmental Science & Technology. China, the United States, and Australia have been the most active contributors to this field. Key topics include denitrification, wastewater treatment, and N2O emissions. The microbial community composition significantly influences N2O emissions in WWTPs, with bacterial consortia playing a pivotal role. However, further research is needed to explore strain-specific genes, enzyme expressions, and the differentiation of processes contributing to N2O production and emission. System design and operation must also consider dissolved oxygen and nitrite concentration factors. Advances in genomics and artificial intelligence are expected to enhance strategies for reducing N2O emissions in WWTPs.
... The further degradation of fatty acids might be conducted by other members with genes for β-oxidation, including MAG118/214 Stutzerimonas and MAG10 Halomonas (Chen et al., 2020;Nguyen et al., 2008). Many microbes were present with multiple fermentative metabolism of ethanol (MAG118 Stutzerimonas), formate (MAG20 Acidaminobacter, MAG174 Fusibacter, MAG43 Magnetospirillum and MAG100 Shewanella) and acetate (MAG20 Acidaminobacter, MAG129 Halodesulfovibrio, MAG191 Pseudodesulfovibrio and MAG10 Halomonas) (Bell et al., 2018;Fredrickson et al., 2008;Imachi et al., 2020;Ke et al., 2018;Mauricio et al., 2022;Qi et al., 2022;Zhao et al., 2024). The methanogen MAG246 Methanosarcina might grow and produce methane through syntrophic interaction with the abovementioned microorganisms (Kouzuma et al., 2015). ...
CO2 enhanced oil recovery (CO2-EOR) is one of the common and effective ways for carbon capture, utilization and storage(CCUS) in China. The injection of CO2 into petroleum reservoirs may influence subsurface environments and further affect microorganisms in oil reservoirs. However, the current knowledge about the impact of CO2 flooding operation on microbial communities and their metabolic functions in oil reservoirs is still limited. In this study, the compositions and metabolic potential of microbial communities in production water from CO2-and water-flooded oil reservoirs in Jilin oilfield were investigated by using a metagenomic approach. Comparative analyses indicated that the microbial community compositions in CO2-flooded oil reservoir samples (GQ43 and GH46) were significantly different from those in water-flooded ones (WQ21 and WH71), with lower microbial diversity. The difference analysis (p<0.05) showed that Pseudomonas, Stutzerimonas, Marinobacterium,Pseudomonadaceae, Methanosarcina and Archaeoglobus were dominant in the former, while Azonexus, Sulfurospirillum, Candidatus Woesearchaeota, Candidatus Methanofastidiosa and Nanoarchaeota predominated in the latter. According to the high-quality metagenome-assembled genomes (MAGs) obtained, some members identified in the CO2-flooded oil reservoir samples might be involved in aerobic alkane biodegradation (Stutzerimonas and Hyphomonas), activated hydrocarbon utilization (Archaeoglobus and Magnetospirillum), fatty acid degradation (Stutzerimonas and Halomonas), fermentative metabolism (Stutzerimonas, Acidaminobacter, Fusibacter, Magnetospirillum, Shewanella, Halodesulfovibrio, Pseudodesulfovibrio and Halomonas), carbon fixation (Methanosarcina and Halodesulfovibrio)and syntrophic methanogenesis (Methanosarcina), simultaneously accompanied by dissimilatory sulfate reduction, thiosulfate reduction and denitrification. Whereas, a series of MAGs recovered from the water-flooded oil reservoir samples might be responsible for fumarate addition of aromatic hydrocarbons, activated hydrocarbon utilization, acetogenesis, reductive citrate cycle, dissimilatory nitrate reduction and sulfur metabolism (dissimilatory sulfate reduction, thiosulfate reduction and sulfur oxidation). These results contribute a broad and deep understanding of microbial communities and their roles in petroleum reservoirs especially affected by CO2 flooding operation, and provide the basic biological information for CCUS.
... ). N2O emission is a prominent pollutant of the agrarian sector and microbes like Azospira sp. and Bradyrhizobium diazoefficiens have high N 2 O reductase activities and thus minimise these emissions(Qi et al. 2022). Climate change can exacerbate ecosystem disturbances like wildfires, hurricanes, and deforestation. ...
Plant-microbial interactions are very crucial for the terrestrial ecosystem. These interactions occur through different signalling pathways at the Rhizospheric, Phyllospheric, and Endospheric regions. They are pivotal in strengthening the plant defence mechanism against biotic and abiotic stresses. A better knowledge of these relationships will help to develop novel solutions for sustainable agriculture and safeguarding the global food supply in the context of climate change and mounting environmental concerns. This review article emphasises the role of the microbiome in providing resistance to plants against various environmental stresses and aiding in their improved adaptation under changing climatic scenarios.
... Studies have already performed some enrichment and isolation of nosZ clade II carrying bacteria, such as Dechloromonas and Azospira which were proven to be N 2 O sinks (Conthe et al., 2018;Suenaga et al., 2019). Based on these results, nosZ clade II bacteria are more promising for use in agriculture to reduce N 2 O emissions because these microorganisms have higher growth yield, higher N 2 O affinity, and better resilience to oxygen exposure (Qi et al., 2021). In this study, nosZ clade II bacteria accounted for the majority of isolated nosZ-containing bacteria. ...
Nitrous oxide (N2O) emitted from agricultural soils destroys stratospheric ozone and contributes to global warming. A promising approach to reduce emissions is fertilizing the soil using organic wastes augmented by non-denitrifying N2O-reducing bacteria (NNRB). To realize this potential, we need a suite of NNRB strains that fulfill several criteria: efficient reduction of N2O, ability to grow in organic waste, and ability to survive in farmland soil. In this study, we enriched such organisms by sequential anaerobic batch incubations with N2O and reciprocating inoculation between the sterilized substrates of anaerobic manure digestate and soils. 16S rDNA amplicon sequencing and metagenomics analysis showed that a cluster of bacteria containing nosZ genes encoding N2O-reductase, was enriched during the incubation process. Strains of several dominant members were then isolated and characterized, and three of them were found to harbor the nosZ gene but none of the other denitrifying genes, thus qualifying as NNRB. The selected isolates were tested for their capacities to reduce N2O emissions from three different typical Chinese farmland soils. The results indicated the significant mitigation effect of these isolates, even in very acidic red soil. In conclusion, this study demonstrated a strategy to engineer the soil microbiome with promising NNRB with high adaptability to livestock manure digestate as well as different agricultural soils, which would be suitable for developing novel fertilizer for farmland application to efficiently mitigate the N2O emissions from agricultural soils.
... Besides N 2 O reduction rates, microorganisms with low K m values, such as P. denitrificans and D. aromatica, could be useful in scavenging low concentrations of dissolved N 2 O. It is important to note, however, that the kinetics and O 2 sensitivity of N 2 O reducers can be influenced by environmental factors, such as the type of organic carbons (30) and temperature (31). Therefore, when selecting appropriate N 2 O reducers for engineering applications, their N 2 O reduction kinetics and O 2 sensitivity should be measured under environmentally relevant conditions. ...
Some bacteria can reduce N 2 O in the presence of O 2 , whereas others cannot. It is unclear whether this trait of aerobic N 2 O reduction is related to the phylogeny and structure of N 2 O reductase.
... However, in a pure culture study comparing two Azospira sp. isolates subject to acetate, succinate, glycerol, methanol, and ethanol when using N 2 O as an electron acceptor demonstrate that they readily use all these compounds although the organic acids were preferred over the alcohols and the biokinetics for each compound vary between the two isolates (Qi et al., 2022). Thus, although our results indicate selection of betaproteobacterial N 2 O reducers with HEC as a C-substrate, variation within specific lineages are likely substantial. ...
Agricultural soils are a main source of nitrous oxide (N2O), a potent greenhouse gas and the dominant ozone-depleting substance emitted to the atmosphere. The only known sink of N2O in soil is the microbial reduction of N2O to N2. Carbon (C) availability is a key factor in determining microbial community composition in soil. However, its role in shaping the structure of N2O reducing communities in soil is unexplored. In this study, a microcosm experiment was set up in which two arable soils with contrasting edaphic properties were incubated anaerobically for 83 days with four different C substrates: glucose, acetate, hydroxyethylcellulose (HEC) and mixture of the three. We show that the effect of C addition on the abundance and diversity of clade I and clade II nosZ genes, encoding different variants of the N2O reductase, varies across the different C substrates differently in contrasting soil types, yet still plays an important role in selecting specific taxa of N2O reducers under denitrifying conditions. We observed an increase of betaproteobacterial clade I and II N2O reducing species with addition of HEC, whereas alphaproteobacterial clade I species and clade II species within other Proteobacteria and Bacteriodetes were associated with glucose and acetate. These results show that specific C-substrates select for certain lineages of nitrous oxide reducers and influence patterns of niche partitioning within clades of N2O reducers, whereas other soil factors drive differences between clade I and II nosZ communities.
... However, in a pure culture study comparing two Azospira sp. isolates subject to acetate, succinate, glycerol, methanol, and ethanol when using N 2 O as an electron acceptor demonstrate that they readily use all these compounds although the organic acids were preferred over the alcohols and the biokinetics for each compound vary between the two isolates (Qi et al., 2022). Thus, although our results indicate selection of betaproteobacterial N 2 O reducers with HEC as a C-substrate, variation within specific lineages are likely substantial. ...
... Also, the microaerophilic layer formed within a combined nitrifying and denitrifying biofilm can trigger more NO and N 2 O production by DNB due to the inhibition of the nitrous oxide reductase (NoS) enzyme in the presence of O 2 (Kinh et al., 2017a;Guo et al., 2017). Qi et al. (2022) found that N 2 O-reducing DNB can recover from exposure to DO and that this recovery is species-dependent. Zhou et al. (2022b) found that temperature can influence the abundance and composition of the N 2 O-reducing DNB. ...
Simultaneous nitrification-denitrification (SND) is an advantageous bioprocess that allows the complete removal of ammonia nitrogen through sequential redox reactions leading to nitrogen gas production. SND can govern nitrogen removal in single-stage biofilm systems, such as the moving bed biofilm reactor (MBBR) and aerobic granular sludge (AGS) system, as oxygen gradients allow the development of multilayered biofilms including nitrifying and denitrifying bacteria. Environmental and operational conditions can strongly influence SND performance, biofilm development and biochemical pathways. Recent advances have outlined the possibility to reduce the carbon and energy consumption of the process via the “shortcut pathway”, and simultaneously remove both N and phosphorus under specific operational conditions, opening new possibilities for wastewater treatment. This work critically reviews the factors influencing SND and its application in biofilm systems from laboratory to full scale. Operational strategies to enhance SND efficiency and hints to reduce nitrous oxide emission and operational costs are provided.
... N 2 O reduction). However, if such an organic-substrate dosing strategy were conducted over longer periods, and not just once, as in the case of this study, N 2 O reduction specialists might establish in the sludge helping to reduce the net N 2 O emissions (Orschler et al., 2021;Qi et al., 2022). ...
For two decades now, partial nitritation anammox (PNA) systems were suggested to more efficiently remove nitrogen (N) from mainstream municipal wastewater. Yet to date, only a few pilot-scale systems and even fewer full-scale implementations of this technology have been described. Process instability continues to restrict the broad application of PNA. Especially problematic are insufficient anammox biomass retention, the growth of undesired aerobic nitrite-oxidizers, and nitrous oxide (N2O) emissions. In this study, a two-stage mainstream pilot-scale PNA system, consisting of three reactors (carbon pre-treatment, nitritation, anammox - 8 m3 each), was operated over a year, treating municipal wastewater. The aim was to test whether both, robust autotrophic N removal and high effluent quality, can be achieved throughout the year. A second aim was to better understand rate limiting processes, potentially affecting the overall performance of PNA systems. In this pilot study, excellent effluent quality, in terms of inorganic nitrogen, was accomplished (average effluent concentrations: 0.4 mgNH4-N/L, 0.1 mgNO2-N/L, 0.9 mgNO3-N/L) even at wastewater temperatures previously considered problematic (as low as 8 °C). N removal was limited by nitritation rates (84 ± 43 mgNH4-N/L/d), while surplus anammox activity was observed at all times (178 ± 43 mgN/L/d). Throughout the study, nitrite-oxidation was maintained at a low level (
... and Dechloromonas spp. and/or higher specific affinity α N2O ¼ V m =K m; app À Á [23]. An often overlooked environmental factor that may also have caused such conflicting observations is the role of trace O 2 . ...
Microorganisms possessing N2O reductases (NosZ) are the only known environmental sink of N2O. While oxygen inhibition of NosZ activity is widely known, environments where N2O reduction occurs are often not devoid of O2. However, little is known regarding N2O reduction in microoxic systems. Here, 1.6-L chemostat cultures inoculated with activated sludge samples were sustained for ca. 100 days with low concentration (<2 ppmv) and feed rate (<1.44 µmoles h−1) of N2O, and the resulting microbial consortia were analyzed via quantitative PCR (qPCR) and metagenomic/metatranscriptomic analyses. Unintended but quantified intrusion of O2 sustained dissolved oxygen concentration above 4 µM; however, complete N2O reduction of influent N2O persisted throughout incubation. Metagenomic investigations indicated that the microbiomes were dominated by an uncultured taxon affiliated to Burkholderiales, and, along with the qPCR results, suggested coexistence of clade I and II N2O reducers. Contrastingly, metatranscriptomic nosZ pools were dominated by the Dechloromonas-like nosZ subclade, suggesting the importance of the microorganisms possessing this nosZ subclade in reduction of trace N2O. Further, co-expression of nosZ and ccoNO/cydAB genes found in the metagenome-assembled genomes representing these putative N2O-reducers implies a survival strategy to maximize utilization of scarcely available electron acceptors in microoxic environmental niches.
Nitrous oxide-reducing bacteria (N2ORB) are generally considered the only biological sink for the potent greenhouse gas N2O. Although N2O consumption activities by diverse heterotrophic N2ORB have been detected, knowledge gaps remain about the phylogenies, physiologies, and activities of N2ORB. Here, we successfully enriched a methylotrophic N2ORB consortium under intermittent oxygen and N2O supplies. 15N tracer analysis showed that the N2O consumption activity of the enriched consortium was higher than its N2O production activity in the presence of either a single or multiple electron acceptors (i.e., nitrogen oxides). The observed maximum N2O consumption was 80.7 μmol·g-biomass–1·h–1. Quantitative PCR results showed that clade I nosZ bacteria overwhelmed clade II nosZ bacteria at high (0.41 mmol·min–1) and low (0.08 mmol·min–1) N2O loading rates. The dilution rate and N2O loading rate affected the microbial community composition and activity. A higher N2O loading rate stimulated active and oxygen-tolerant N2ORB that boosted N2O consumption by approximately 50% in the presence of oxygen. Metagenomic analysis unraveled the predominance of a novel methylotrophic N2ORB, possessing entire denitrifying genes and high-affinity terminal oxidase genes, from the reactor with a high N2O loading rate. The unique physiological traits of the consortium enriched by methanol shed light on a novel function─aerobic N2O consumption by N2ORB─and pave the way for innovative N2O mitigation strategies applying powerful N2O sinks in engineered systems.
Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O and are central to global N2O mitigation efforts. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.
The reduction rate of nitrous oxide (N2O) is affected by the electron competition among the four denitrifying steps, limiting the mitigation of N2O emissions during wastewater treatment. We foresee essential to understand how combinations of electron acceptors (EAs) affect the microbial composition and reduction mechanisms of denitrifying communities. We enriched three denitrifying communities from activated sludge biomass with equivalent loads of different EAs: NO3– (R1), N2O (R2), and NO3– + N2O (R3). The resulting enrichments were compared in terms of (1) reduction of nitrogen oxides in absence/presence of other EAs (NO3–, NO2–, N2O), (2) their denitrification gene composition and (3) their microbial community composition. Batch results showed the presence of NO3– and NO2– suppressed N2O reduction rates in all three reactors. The effect was lower in the mixed-substrate feed community than in the single-substrate feed under infinite sludge retention time and chemostat operation modes. N2O-reducers of type nosZ II were enriched when N2O serves as the sole EA, whereas nosZ I type N2O-reducers were more prone to enrichment with NO3– as EA. The EA composition rather than the sludge retention mode differentiated the microbial communities. The genus Flavobacterium seems to play a significant role in alleviating the suppression of the N2O reduction rate caused by electron competition. Limited conditions of electron supply are the norm independently of high C/N levels, and a community co-enriched with NO3– and N2O alleviates more the competition for electrons in the nitrous oxide reductase enzyme than communities enriched with NO3– or N2O individually.
Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.
Farmed soils contribute substantially to global warming by emitting N2O (ref. ¹), and mitigation has proved difficult². Several microbial nitrogen transformations produce N2O, but the only biological sink for N2O is the enzyme NosZ, catalysing the reduction of N2O to N2 (ref. ³). Although strengthening the NosZ activity in soils would reduce N2O emissions, such bioengineering of the soil microbiota is considered challenging4,5. However, we have developed a technology to achieve this, using organic waste as a substrate and vector for N2O-respiring bacteria selected for their capacity to thrive in soil6–8. Here we have analysed the biokinetics of N2O reduction by our most promising N2O-respiring bacterium, Cloacibacterium sp. CB-01, its survival in soil and its effect on N2O emissions in field experiments. Fertilization with waste from biogas production, in which CB-01 had grown aerobically to about 6 × 10⁹ cells per millilitre, reduced N2O emissions by 50–95%, depending on soil type. The strong and long-lasting effect of CB-01 is ascribed to its tenacity in soil, rather than its biokinetic parameters, which were inferior to those of other strains of N2O-respiring bacteria. Scaling our data up to the European level, we find that national anthropogenic N2O emissions could be reduced by 5–20%, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N2O emissions for which other mitigation options are lacking at present.
Nitrogen is one of the key nutrients significant for the survival of all living organisms. This element is abundant in the atmosphere; however, its bioavailability to most of living organisms is dependent on its biogeochemical cycling where it is transformed into different usable forms. The microbe-mediated nitrogen transformation processes seen in most of the ecosystem are nitrogen fixation, nitrification, denitrification, anammox, and ammonification. The diversity, metabolism, and potential role in nitrogen biogeochemical cycling of microbes native to damaged ecosystems such as mining sites are less explored as compared with other terrestrial and aquatic ecosystem. The reduced pH, oliogotrophic soil, and inorganic contaminants present in mining effluents and tailings affect the microbial ammonia and nitrite oxidation rates. Mining-impacted sites do have toxicological consequences for such microbe-mediated environmental operations. Considering the global effect of mining wastes and the importance of planned reclamation of these sites, it is significant to understand the community structure and function of microbes relevant to nitrogen biogeochemistry.
Shallow lakes, recognized as hotspots for nitrogen cycling, contribute to the emission of the potent greenhouse gas nitrous oxide (N2O), but the current emission estimates for this gas have a high degree of uncertainty. However, the role of N2O-reducing bacteria (N2ORB) as N2O sinks and their contribution to N2O reduction in aquatic ecosystems in response to N2O dynamics have not been determined. Here, we investigated the N2O dynamics and microbial processes in the nitrogen cycle, which included both N2O production and consumption, in five shallow lakes spanning approximately 500 km. The investigated sites exhibited N2O oversaturation, with excess dissolved N2O concentrations (ΔN2O) ranging from 0.55 ± 0.61 to 53.17 ± 15.75 nM. Sediment-bound N2O (sN2O) was significantly positively correlated with the nitrate concentration in the overlying water (p < 0.05), suggesting that nitrate accumulation contributes to benthic N2O generation. High N2O consumption activity (RN2O) corresponded to low ΔN2O. In addition, a significant negative correlation was found between RN2O and nir/nosZ, showing that bacteria encoding nosZ contributed to N2O consumption in the benthic sediments. Redundancy analysis indicated that benthic functional genes effectively reflected the variations in RN2O and ∆N2O. qPCR analysis revealed that the clade II nosZ gene was more sensitive to ΔN2O than the clade I nosZ gene. Furthermore, four novel genera of potential nondenitrifying N2ORB were identified based on metagenome-assembled genome analysis. These genera, which are affiliated with clade II, lack genes responsible for N2O production. Collectively, benthic N2ORB, especially for clade II-type N2ORB, harnesses N2O consumption activity leading to low N2O emissions from shallow lakes. This study advances our knowledge of the role of benthic clade II-type N2ORB in regulating N2O emissions in shallow lakes.
Microorganisms encoding for the N 2 O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N 2 O, and are central to global N 2 O mitigation efforts. Yet, the ecological constraints selecting for different N 2 O-reducers strains and controlling the assembly of N 2 O-respiring communities remain largely unknown. Of particular biotechnological interest are clade II NosZ populations, which usually feature high N 2 O affinities and often lack other denitrification genes. Two planktonic N 2 O-respiring mixed cultures were enriched under limiting and excess dissolved N 2 O availability to assess the impact of substrate affinity and N 2 O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. We show that clade II N 2 O-reducers outcompete clade I affiliates for N 2 O at sufficiently low sludge dilution rates (0.006 h-1 ), a scenario previously only theorized based on pure-cultures. Under N 2 O limitation, all enriched N 2 O reducers encoded and expressed only clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture. We explain their coexistence with the genome-inferred metabolic exchange of cobalamin intermediates. Conversely, under excess N 2 O, clade I and II populations coexisted. Notably, the single dominant N 2 O reducer (genus Azonexus ) expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N 2 O concentrations (400 μM). Ultimately, we demonstrate that the solids dilution rate controls the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. Additionally, we suggest the significance of N 2 O-cobalamin interactions in shaping the composition of N 2 O-respiring microbiomes.
The effect of climate change on flora and fauna has been widely discussed for years. However, its consequences on microorganisms are generally poorly considered. The main effect of climate change on microbiota is related to biodiversity changes in different regions of the planet, mainly due to variations in temperature. These alterations are resulting in a worldwide (re)distribution of pathogens, which was not considered a few years ago. They mainly affect different food chain sectors (such as agriculture, livestock and fishing), as well as human health. Hence, the spread of numerous animal and plant pathogens has been observed in recent years from south to north (especially in America, Europe and Asia), leading to the spread of numerous plant and animal diseases, which results in economic and ecological losses. In addition, global warming that accompanies climate change could also be related to emerging antibiotic resistance. However, the mitigation of climate change goes hand in hand with microorganisms, which can help us through different natural and industrial processes. Thus, this manuscript presents the direct and indirect effects of climate change on microorganisms described up to date and how they act on this worldwide phenomenon.
Dam construction is regarded as the greatest anthropogenic disturbance in aquatic ecosystems, and it promotes denitrification, through which large N2O emissions occur. However, the effect of dams on N2O producers and other N2O-reducing microorganisms (especially for nosZ II), and the associated denitrification rates remain poorly understood. This study systematically investigated the spatial variation of potential denitrification rates in dammed river sediments in winter and summer and the microbial processes driving N2O production and reduction. Sediments in the transition zone of dammed rivers were found to be critical for N2O emission potential, with lower potential denitrification rate and N2O production rate in winter than in summer. In dammed river sediments, the dominant N2O-producing microorganisms and N2O-reducers were nirS-harboring bacteria and nosZ I-harboring bacteria, respectively. Diversity analysis showed that diversity of N2O-producing did not differ significantly between upstream and downstream sediments, whereas the population size and diversity of N2O-reducing microbial communities in upstream sediments significantly decreased, leading to biological homogenization. Further ecological network analysis revealed that the ecological network of nosZ II microbes was more complex than that of nosZ I microbes, and both exhibited more cooperation in the downstream sediments than in the upstream sediments. Mantel analysis showed that the potential N2O production rate was mainly influenced by electrical conductivity (EC), NH4+, and TC content, and that higher nosZ II/nosZ I ratios contributed to improved N2O sinks in dammed river sediments. Moreover, the Haliscomenobacter genus from the nosZ II-type community in the downstream sediments contributed significantly to N2O reduction. Collectively, this study elucidates the diversity and community distribution of nosZ-type denitrifying microorganisms influenced by dams, and also highlights the non-negligible role played by nosZ II-containing microbial groups in mitigating N2O emissions from dammed river sediments.
Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic-anoxic cycling increased the mobilization of organic C, increased nitrate (NO3 - ) removal rates, and attenuated production of nitrous oxide (N2 O). Here, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO3 - removal and attenuated N2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic-anoxic transition was strongly correlated with the N2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.
Aerobic environments exist widely in wastewater treatment plants (WWTP) and are unfavorable for greenhouse gas nitrous oxide (N2O) reduction. Here, a novel strain Pseudomonas sp. YR02, which can perform N2O reduction under aerobic conditions, was isolated. The successful amplification of four denitrifying genes proved its complete denitrifying ability. The inorganic nitrogen (IN) removal efficiencies (NRE) were >98.0% and intracellular nitrogen and gaseous nitrogen account for 52.6-58.4% and 41.6-47.4% of input nitrogen, respectively. The priority of IN utilization was TAN > NO3--N > NO2--N. The optimal conditions for IN and N2O removal were consistent, except for the C/N ratio, which is 15 and 5 for IN and N2O removal, respectively. The biokinetic constants analysis indicated strain YR02 had high potential to treat high ammonia and dissolved N2O wastewater. Strain YR02 bioaugmentation mitigated 98.7% of N2O emission and improved 32% NRE in WWTP, proving its application potential for N2O mitigation.
Wastewater treatment plants (WWTPs) are a major
source of N2O, a potent greenhouse gas with 300 times higher global warming potential than CO2. Several approaches have been proposed for mitigation of N2O emissions from WWTPs and have shown promising yet only site-specific results. Here, self-sustaining
biotrickling filtration, an end-of-the-pipe treatment technology, was
tested in situ at a full-scale WWTP under realistic operational conditions. Temporally varying untreated wastewater was used as
trickling medium, and no temperature control was applied. The off-gas from the covered WWTP aerated section was conveyed through the pilot-scale reactor, and an average removal efficiency of 57.9 ± 29.1% was achieved during 165 days of operation despite the generally low and largely fluctuating influent N2O concentrations (ranging between 4.8 and 96.4 ppmv). For the following 60-day period, the continuously operated reactor system removed 43.0 ± 21.2% of the periodically augmented N2O, exhibiting elimination capacities as high as 5.25 g N2O m−3·h−1. Additionally, the bench-scale experiments performed abreast corroborated the resilience of the system to short-term N2O starvations. Our results corroborate the feasibility of biotrickling filtration for mitigating N2O emitted from WWTPs and demonstrate its robustness toward suboptimal field operating conditions and N2O starvation, as also supported by analyses of the microbial compositions and nosZ gene profiles.
Iron-based ecological floating beds (EFBs) are often used to treat the secondary effluent from wastewater treatment plant to enhance the denitrification process. However, the impact and necessity of plants on iron-based EFBs have not been systematically studied. In this research, two iron-based EFBs with and without plants (EFB-P and EFB) were performed to investigate the response of plants on nutrient removal, GHG emissions, microbial communities and functional genes. Results showed the total nitrogen and total phosphorus removal in EFB-P was 45–79% and 48–72%, respectively, while that in EFB was 31–67% and 44–57%. Meanwhile, plants could decrease CH4 emission flux (0–3.89 mg m⁻² d⁻¹) and improve CO2 absorption (4704–22321 mg m⁻² d⁻¹). Plants could increase the abundance of Nitrosospira to 1.6% which was a kind of nitrifying bacteria dominant in plant rhizosphere. Among all denitrification related genera, Simplicispira (13.08%) and Novosphingobium (6.25%) accounted for the highest proportion of plant rhizosphere and iron scrap, respectively. Anammox bacteria such as Candidatus_Brocadia was more enriched on iron scraps with the highest proportion was 1.21% in EFB-P, and 2.20% in EFB. Principal co-ordinates analysis showed that plants were the critical factor determining microbial community composition. TN removal pathways were mixotrophic denitrification and anammox in EFB-P while TP removal pathways were plant uptake and phosphorus-iron coprecipitation. In general, plants play an important directly or indirectly role in iron-based EFBs systems, which could not only improve nutrients removal, but also minimize the global warming potential and alleviate the greenhouse effect to a certain extent.
Nitritation/denitritation is a promising strategy to treat sludge digester liquor but would be unstable and inefficient at extremely low C/N ratios. Here, a novel electrochemically assisted sequencing batch biofilm reactor (E-SBBR) was established to treat synthetic/real sludge digester liquor with decreasing C/N ratios. The results showed that the E-SBBR achieved stable nitritation and appreciable TN removal (>70%) even at C/N < 0.5. The high-strength free ammonium (FA) (91.1-132.8 mg NH3-N L⁻¹) and long inhibition time (>9h) magnified by electrolysis promoted the robustness of nitritation through efficient nitrite-oxidizing bacteria elimination. Meanwhile, mass balance denoted that heterotrophic denitritation dominated in the enhanced TN removal and relied on carbon supplementation from cell apoptosis/lysis stimulated by electrolysis and high-strength FA, further supported by the recovery of heterotrophic denitrifiers, fermentation bacteria, and relevant functional genes at extremely low C/N ratios. This study provides a novel nitrogen removal approach for the treatment of sludge digester liquor.
In denitrifying reactors, canonical complete denitrifying bacteria reduce nitrate (NO3-) to nitrogen via N2O. However, they can also produce N2O under certain conditions. We used a 15N tracer method, in which 15N-labeled NO3-/nitrite (NO2-) and nonlabeled N2O were simultaneously supplied with organic electron donors to five canonical complete denitrifying bacteria affiliated with either Clade I or Clade II nosZ. We calculated their NO3-, NO2-, and N2O consumption rates. The Clade II nosZ bacterium Azospira sp. strain I13 had the highest N2O consumption rate (3.47 ± 0.07 fmol/cell/h) and the second lowest NO3- consumption rate (0.20 ± 0.03 fmol/cell/h); hence, it is a N2O sink. A change from peptone- to acetate/citrate-based organic electron donors increased the NO3- consumption rate by 4.8 fold but barely affected the N2O consumption rate. Electron flow was directed to N2O rather than NO3- in Azospira sp. strain I13 and Az. oryzae strain PS only exerting a N2O sink but to NO3- in the Clade I nosZ N2O-reducing bacteria Pseudomonas stutzeri strain JCM 5965 and Alicycliphilus denitrificans strain I51. Transcriptome analyses revealed that the genotype could not fully describe the phenotype. The results show that N2O production and consumption differ among canonical denitrifying bacteria and will be useful for developing N2O mitigation strategies.
Nitrous oxide is a highly potent greenhouse gas and one of the main contributors to the greenhouse gas footprint of wastewater treatment plants (WWTP). Although nitrous oxide can be produced by abiotic reactions in these systems, biological N2O production resulting from the imbalance of nitrous oxide production and reduction by microbial populations is the dominant cause. The microbial populations responsible for the imbalance have not been clearly identified, yet are likely responsible for strong seasonal nitrous oxide concentration patterns. Here, we examined the seasonal nitrous oxide concentration pattern in Avedøre WWTP alongside abiotic parameters, and the microbial community composition based on 16S rRNA gene sequencing and already available metagenome-assembled genomes (MAGs). We found that the WWTP parameters could not explain the observed pattern. While no distinct community changes between periods of high and low dissolved nitrous oxide concentrations were determined, we found 26 and 28 species with positive and negative correlations to the seasonal N2O concentrations, respectively. MAGs were identified for 124 species (approximately 31% mean relative abundance of the community), and analysis of their genomic nitrogen transformation potential could explain this correlation for four of the negatively correlated species. Other abundant species were also analysed for their nitrogen transformation potential. Interestingly, only one full-denitrifier (Candidatus Dechloromonas phosphorivorans) was identified. 59 species had a nosZ gene predicted, with the majority identified as a clade II nosZ gene, mainly from the phylum Bacteriodota. A correlation of MAG-derived functional guilds with the N2O concentration pattern showed that there was a small but significant negative correlation with nitrite oxidizing bacteria and species with a nosZ gene (N2O reducers (DEN)). More research is required, specifically long-term activity measurements in relation to the N2O concentration to increase the resolution of these findings.
Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N 2 O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N 2 O. Gas-kinetics and meta-omic analyses showed that these NRB’s, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB’s were isolated, one of which matched the recovered MAG of a Dechloromonas , deemed by proteomics to be the dominant producer of N 2 O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N 2 O, their regulatory traits predicted that they act as N 2 O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N 2 O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.
Nitrous oxide (N2O), a potent greenhouse gas, is reduced to N2 gas by N2O‐reducing bacteria (N2ORB), a process which represents an N2O sink in natural and engineered ecosystems. The N2O sink activity by N2ORB depends on temperature and O2 exposure, yet the specifics are not yet understood. This study explores the effects of temperature and oxygen exposure on biokinetics of pure culture N2ORB. Four N2ORB, representing either clade I type nosZ (Pseudomonas stutzeri JCM5965 and Paracoccus denitrificans NBRC102528) or clade II type nosZ (Azospira sp. strains I09 and I13), were individually tested. The higher activation energy for N2O by Azospira sp. strain I13 (114.0 ± 22.6 kJ mol⁻¹) compared with the other tested N2ORB (38.3–60.1 kJ mol⁻¹) indicates that N2ORB can adapt to different temperatures. The O2 inhibition constants (KI) of Azospira sp. strain I09 and Ps. stutzeri JCM5965 increased from 0.06 ± 0.05 and 0.05 ± 0.02 μmol L⁻¹ to 0.92 ± 0.24 and 0.84 ± 0.31 μmol L⁻¹, respectively, as the temperature increased from 15°C to 35°C, while that of Azospira sp. strain I13 was temperature‐independent (p = 0.106). Within the range of temperatures examined, Azospira sp. strain I13 had a faster recovery after O2 exposure compared with Azospira sp. strain I09 and Ps. stutzeri JCM5965 (p < 0.05). These results suggest that temperature and O2 exposure result in the growth of ecophysiologically distinct N2ORB as N2O sinks. This knowledge can help develop a suitable N2O mitigation strategy according to the physiologies of the predominant N2ORB.
Nitrous oxide (N2O), like carbon dioxide, is a long-lived greenhouse gas that accumulates in the atmosphere. Over the past 150 years, increasing atmospheric N2O concentrations have contributed to stratospheric ozone depletion¹ and climate change², with the current rate of increase estimated at 2 per cent per decade. Existing national inventories do not provide a full picture of N2O emissions, owing to their omission of natural sources and limitations in methodology for attributing anthropogenic sources. Here we present a global N2O inventory that incorporates both natural and anthropogenic sources and accounts for the interaction between nitrogen additions and the biochemical processes that control N2O emissions. We use bottom-up (inventory, statistical extrapolation of flux measurements, process-based land and ocean modelling) and top-down (atmospheric inversion) approaches to provide a comprehensive quantification of global N2O sources and sinks resulting from 21 natural and human sectors between 1980 and 2016. Global N2O emissions were 17.0 (minimum–maximum estimates: 12.2–23.5) teragrams of nitrogen per year (bottom-up) and 16.9 (15.9–17.7) teragrams of nitrogen per year (top-down) between 2007 and 2016. Global human-induced emissions, which are dominated by nitrogen additions to croplands, increased by 30% over the past four decades to 7.3 (4.2–11.4) teragrams of nitrogen per year. This increase was mainly responsible for the growth in the atmospheric burden. Our findings point to growing N2O emissions in emerging economies—particularly Brazil, China and India. Analysis of process-based model estimates reveals an emerging N2O–climate feedback resulting from interactions between nitrogen additions and climate change. The recent growth in N2O emissions exceeds some of the highest projected emission scenarios3,4, underscoring the urgency to mitigate N2O emissions.
The substantial presence of denitrifiers has already been reported in partial nitritation anammox (PNA) systems using the 16S ribosomal RNA (rRNA) gene, but little is known about the phylogenetic diversity based on denitrification pathway functional genes. Therefore, we performed a metagenomic analysis to determine the distribution of denitrification genes and the associated phylogeny in PNA systems and whether a niche separation between PNA and conventional activated sludge (AS) systems exists. The results revealed a distinct abundance pattern of denitrification pathway genes and their association to the microbial species between PNA and AS systems. In contrast, the taxonomic analysis, based on the 16S rRNA gene, did not detect notable variability in denitrifying community composition across samples. In general, narG and nosZa2 genes were dominant in all samples. While the potential for different stages of denitrification was redundant, variation in species composition and lack of the complete denitrification gene pool in each species appears to confer niche separation between PNA and AS systems. This study suggests that targeted metagenomics can help to determine the denitrifying microbial composition at a fine‐scale resolution while overcoming current biases in quantitative polymerase chain reaction approaches due to a lack of appropriate primers.
Respiratory and catabolic genes are differentially distributed across microbial genomes. Thus, specific carbon sources may favor different respiratory processes. We profiled the influence of 94 carbon sources on the end products of nitrate respiration in microbial enrichment cultures from diverse terrestrial environments. We found that some carbon sources consistently favor dissimilatory nitrate reduction to ammonium (DNRA/nitrate ammonification) while other carbon sources favor nitrite accumulation or denitrification. For an enrichment culture from aquatic sediment, we sequenced the genomes of the most abundant strains, matched these genomes to 16S rDNA exact sequence variants (ESVs), and used 16S rDNA amplicon sequencing to track the differential enrichment of functionally distinct ESVs on different carbon sources. We found that changes in the abundances of strains with different genetic potentials for nitrite accumulation, DNRA or denitrification were correlated with the nitrite or ammonium concentrations in the enrichment cultures recovered on different carbon sources. Specifically, we found that either L-sorbose or D-cellobiose enriched for a Klebsiella nitrite accumulator, other sugars enriched for an Escherichia nitrate ammonifier, and citrate or formate enriched for a Pseudomonas denitrifier and a Sulfurospirillum nitrate ammonifier. Our results add important nuance to the current paradigm that higher concentrations of carbon will always favor DNRA over denitrification or nitrite accumulation, and we propose that, in some cases, carbon composition can be as important as carbon concentration in determining nitrate respiratory end products. Furthermore, our approach can be extended to other environments and metabolisms to characterize how selective parameters influence microbial community composition, gene content, and function.
Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes, but little is known about the microorganisms and metabolic pathways involved in this process. In this study, Pseudomonas stutzeri UFV5 was isolated from an activated sludge sample from oil wastewater treatment station and its ammonium removal via HN/AD was investigated by physicochemical and molecular approaches to better understand this process and optimize the biological ammonium removal in wastewater treatment plants. Results showed that P. stutzeri UFV5 removed all the ammonium in 48–72 hours using pyruvate, acetate, citrate or sodium succinate as carbon sources, C/N ratios 6, 8, 10 and 12, 3–6% salinities, pH 7–9 and temperatures of 20–40 °C. Comparative genomics and PCR revealed that genes encoding the enzymes involved in anaerobic denitrification process are present in P. stutzeri genome, but no gene that encodes enzymes involved in autotrophic nitrification was found. Furthermore, transcriptomics showed that none of the known enzymes of autotrophic nitrification and anaerobic denitrification had their expression differentiated and an upregulation of the biosynthesis machinery and protein translation was observed, besides several genes with unknown function, indicating a non-conventional mechanism involved in HN/AD process.
This study proposes a novel method to directly treat reject water with a high ammonium content, without relying on dilution. The originality of this method resides in leveraging the coordinated action of a methane- and methanol-dependent bacterial consortium and the biogas generated from wastewater treatment facilities. Specifically, ammonium is removed through autotrophic assimilation in the glutamate cycle of methanotrophs and Methylophilus while, simultaneously, methanol generated by methanotrophs is treated through formaldehyde assimilation as Methylophilus undergo the same ribulose monophosphate cycle as methanotrophs. Using this method, the backflow of high-concentration ammonium into the wastewater treatment process was reduced to 59% in a single operation using a sequencing batch reactor at a mean influent concentration of 877.3 mg L-1. However, the removal rate temporarily declined to an average of 37.6% at a concentration of 800 mg L-1 or above, which was imputed to the influence of toxic intermediates.
Purpose
Denitrification process in agricultural fields is a large source of nitrous oxide (N2O) emitted to the atmosphere. The rhizosphere soils tend to be the hotspots of denitrification in agricultural soils. Recent studies have reported the important role of nosZ II in eliminating N2O. However, little was known about how these more recently discovered N2O-reducing microorganisms together with other N2O-producers affected the N2O emission in agricultural rhizosphere soils.
Materials and methods
Here, we compared the potential N2O production rate, the denitrification end-product ratio, and the denitrifier communities between rhizosphere and non-rhizosphere soils of two types of crops in winter and summer. The potential activities were measured by acetylene inhibition technique. QPCR analysis was used to quantify the functional genes. High-throughput sequencing and clone library were conducted to analyze the community structure of denitrifiers.
Results and discussion
The rhizosphere soils had a higher N2O production potential but lower denitrification end-product ratio (N2O/(N2O+N2)) than the non-rhizosphere soils. The potential N2O production rate was correlated to the nirS-bacteria abundance, especially in terms of the genus Azospirillum. The N2O/(N2O+N2) ratio showed a negative correlation with both the diversity and abundance of the nosZ II-type N2O-reducers.
Conclusions
Altogether, we propose that nirS-type N2O-producers and nosZ II-type N2O-reducers can affect the N2O emission in agricultural rhizosphere soils, and enhancement of diversity and abundance of nosZ II-type N2O-reducers may help with the N2O mitigation from upland crops.
Background:
Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here, we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rule consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly by additional signal-level analysis with Nanopolish.
Results:
Training basecallers on taxon-specific data results in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network is able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences ('polishing') with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy.
Conclusions:
Basecalling accuracy has seen significant improvements over the last 2 years. The current version of ONT's Guppy basecaller performs well overall, with good accuracy and fast performance. If higher accuracy is required, users should consider producing a custom model using a larger neural network and/or training data from the same species.
We report here a draft genome sequence of Azospira sp. strain I13 in the class Betaproteobacteria , a facultative anaerobic bacterium responsible for nitrous oxide (N 2 O) reduction. Deciphering this genome would pave the way for the use of Azospira sp. strain I13 to facilitate N 2 O consumption in a nitrogen-removing bioreactor emitting N 2 O.
Nitrous oxide (N2O)-reducing bacteria, which reduce N2O to nitrogen in the absence of oxygen, are phylogenetically spread throughout various taxa and have a potential role as N2O sinks in the environment. However, research on their physiological traits has been limited. In particular, their activities under microaerophilic and aerobic conditions, which severely inhibit N2O reduction, remain poorly understood. We used an O2 and N2O micro-respirometric system to compare the N2O reduction kinetics of four strains, i.e., two strains of an Azospira sp., harboring clade II type nosZ, and Pseudomonas stutzeri and Paracoccus denitrificans, harboring clade I type nosZ, in the presence and absence of oxygen. In the absence of oxygen, the highest N2O-reducing activity, Vm,N2O, was 5.80 ± 1.78 × 10⁻³ pmol/h/cell of Azospira sp. I13, and the highest and lowest half-saturation constants were 34.8 ± 10.2 μM for Pa. denitirificans and 0.866 ± 0.29 μM for Azospira sp. I09. Only Azospira sp. I09 showed N2O-reducing activity under microaerophilic conditions at oxygen concentrations below 110 μM, although the activity was low (10% of Vm,N2O). This trait is represented by the higher O2 inhibition coefficient than those of the other strains. The activation rates of N2O reductase, which describe the resilience of the N2O reduction activity after O2 exposure, differ for the two strains of Azospira sp. (0.319 ± 0.028 h⁻¹ for strain I09 and 0.397 ± 0.064 h⁻¹ for strain I13) and Ps. stutzeri (0.200 ± 0.013 h⁻¹), suggesting that Azospira sp. has a potential for rapid recovery of N2O reduction and tolerance against O2 inhibition. These physiological characteristics of Azospira sp. can be of promise for mitigation of N2O emission in industrial applications.
Reduction of the greenhouse gas N2O to N2 is a trait among denitrifying and non-denitrifying microorganisms having an N2O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N2O emissions from ecosystems. To increase our understanding of the ecophysiology of N2O reducers, we determined the thermodynamic growth efficiency of N2O reduction and the selection of N2O reducers under N2O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N2O as electron acceptor and acetate as electron donor. The biomass yields were higher during N2O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N2O. Denitrifiers were favored over non-denitrifying N2O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N2O, as defined by μmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.
The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.
Bacteria capable of reduction of nitrous oxide (N2O) to N2 separate into clade I and clade II organisms on the basis of nos operon structures and nosZ sequence features. To explore the possible ecological consequences of distinct nos clusters, the growth of bacterial isolates with either clade I (Pseudomonas stutzeri strain DCP-Ps1, Shewanella loihica strain PV-4) or clade II (Dechloromonas aromatica strain RCB, Anaeromyxobacter dehalogenans strain 2CP-C) nosZ with N2O was examined. Growth curves did not reveal trends distinguishing the clade I and clade II organisms tested; however, the growth yields of clade II organisms exceeded those of clade I organisms by 1.5- to 1.8-fold. Further, whole-cell half-saturation constants (Kss) for N2O distinguished clade I from clade II organisms. The apparent Ks values of 0.324 ± 0.078 μM for D. aromatica and 1.34 ± 0.35 μM for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 μM) and S. loihica (7.07 ± 1.13 μM). Gen
BlastKOALA and GhostKOALA are automatic annotation servers for genome and metagenome sequences, which perform KEGG Orthology (KO) assignments to characterize individual gene functions and reconstruct KEGG pathways, BRITE hierarchies and KEGG modules to infer high-level functions of the organism or the ecosystem. Both servers are made freely available at the KEGG website (http://www.kegg.jp/blastkoala/). In BlastKOALA the KO assignment is done by a modified version of the internally used KOALA algorithm after the BLAST search against a non-redundant dataset of pangenome sequences at the species, genus or family level, which is generated from the KEGG GENES database by retaining the KO content of each taxonomic category. In GhostKOALA, which utilizes more rapid GHOSTX for database search and is suitable for metagenome annotation, the pangenome dataset is supplemented with CD-HIT clusters including those for viral genes. The result files may be downloaded and manipulated for further KEGG Mapper analysis, such as comparative pathway analysis using multiple BlastKOALA results.
Nitrous oxide (N2O) is an important anthropogenic greenhouse
gas and agriculture represents its largest source. It is at the heart of
debates over the efficacy of biofuels, the climate-forcing impact of
population growth, and the extent to which mitigation of
non-CO2 emissions can help avoid dangerous climate change.
Here we examine some of the major debates surrounding estimation of
agricultural N2O sources, and the challenges of projecting
and mitigating emissions in coming decades. We find that current flux
estimates -- using either top-down or bottom-up methods -- are
reasonably consistent at the global scale, but that a dearth of direct
measurements in some areas makes national and sub-national estimates
highly uncertain. We also highlight key uncertainties in projected
emissions and demonstrate the potential for dietary choice and
supply-chain mitigation.
Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.
Nitrous oxide (N(2)O) emissions from wastewater treatment plants vary substantially between plants, ranging from negligible to substantial (a few per cent of the total nitrogen load), probably because of different designs and operational conditions. In general, plants that achieve high levels of nitrogen removal emit less N(2)O, indicating that no compromise is required between high water quality and lower N(2)O emissions. N(2)O emissions primarily occur in aerated zones/compartments/periods owing to active stripping, and ammonia-oxidizing bacteria, rather than heterotrophic denitrifiers, are the main contributors. However, the detailed mechanisms remain to be fully elucidated, despite strong evidence suggesting that both nitrifier denitrification and the chemical breakdown of intermediates of hydroxylamine oxidation are probably involved. With increased understanding of the fundamental reactions responsible for N(2)O production in wastewater treatment systems and the conditions that stimulate their occurrence, reduction of N(2)O emissions from wastewater treatment systems through improved plant design and operation will be achieved in the near future.
Nitrous oxide (N(2)O) is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Global emissions continue to rise. More than two-thirds of these emissions arise from bacterial and fungal denitrification and nitrification processes in soils, largely as a result of the application of nitrogenous fertilizers. This article summarizes the outcomes of an interdisciplinary meeting, 'Nitrous oxide (N(2)O) the forgotten greenhouse gas', held at the Kavli Royal Society International Centre, from 23 to 24 May 2011. It provides an introduction and background to the nature of the problem, and summarizes the conclusions reached regarding the biological sources and sinks of N(2)O in oceans, soils and wastewaters, and discusses the genetic regulation and molecular details of the enzymes responsible. Techniques for providing global and local N(2)O budgets are discussed. The findings of the meeting are drawn together in a review of strategies for mitigating N(2)O emissions, under three headings, namely: (i) managing soil chemistry and microbiology, (ii) engineering crop plants to fix nitrogen, and (iii) sustainable agricultural intensification.
The emissions of nitrous oxide (N(2)O) and nitric oxide (NO) from biological nitrogen removal (BNR) operations via nitrification and denitrification is gaining increased prominence. While many factors relevant to the operation of denitrifying reactors can influence N(2)O and NO emissions from them, the role of different organic carbon sources on these emissions has not been systematically addressed or interpreted. The overall goal of this study was to evaluate the impact of three factors, organic carbon limitation, nitrite concentrations, and dissolved oxygen concentrations on gaseous N(2)O and NO emissions from two sequencing batch reactors (SBRs), operated, respectively, with methanol and ethanol as electron donors. During undisturbed ultimate-state operation, emissions of both N(2)O and NO from either reactor were minimal and in the range of <0.2% of influent nitrate-N load. Subsequently, the two reactors were challenged with transient organic carbon limitation and nitrite pulses, both of which had little impact on N(2)O or NO emissions for either electron donor. In contrast, transient exposure to oxygen led to increased production of N(2)O (up to 7.1% of influent nitrate-N load) from ethanol grown cultures, owing to their higher kinetics and potentially lower susceptibility to oxygen inhibition. A similar increase in N(2)O production was not observed from methanol grown cultures. These results suggest that for dissolved oxygen, but not for carbon limitation or nitrite exposure, N(2)O emission from heterotrophic denitrification reactors can vary as a function of the electron donor used.
Nitrous oxide (N2O) is a highly potent greenhouse gas and ozone-depleting substance, produced and consumed during denitrification. Evaluation of the N2O production and consumption activities of complete denitrifying bacteria is essential for understanding their capacity to act as N2O sinks in engineered systems for cost-effective nitrogen removal via nitrite (NO2−). However, the physiologies of these N2O-reducing bacteria (N2ORB) are poorly understood. This study aimed to evaluate the physiologies of two N2ORB, Azospira sp. strain I13 and Alicycliphilus denitrificans strain I51. A 15N tracer method was applied to determine N2O production and consumption activities in the co-presence of NO2− and N2O. Both N2ORB displayed a higher N2O consumption rate (RN2O by Azospira sp. strain I13 and Alicycliphilus denitrificans strain I51, 23.85 and 7.60 μmol-N mg-biomass−1 h−1, respectively) than N2O production rate (PN2O, 5.88 and 1.32 μmol-N mg-biomass−1 h−1, respectively) at an initial NO2− concentration of 2.14 mmol-N L−1 with exogenous addition of N2O, indicating that these N2ORB acted as N2O sinks. On increasing the NO2− concentration from 0.36 to 7.14 mmol-N L−1, the net N2O consumption rate RO_N2O (= RN2O – PN2O) decreased for both N2ORB; the magnitude of the decrease was greater for Azospira sp. strain I13 than for Alicycliphilus denitrificans strain I51. The formation of free nitrous acid (FNA) from NO2− in acidic conditions noticeably affected the N2O sink activities of the N2ORB. A higher FNA concentration decreased RO_N2O for both N2ORB, creating the risk of N2O emission at pH 6 and high NO2− concentration. Our results show the ranges of pH and NO2− concentration where Azospira sp. strain I13 is promising for use as an N2O sink in shortcut nitrogen removal via NO2−.
Biomass formation represents one of the most basic aspects of bacterial metabolism. While there is an abundance of information concerning individual reactions that result in cell duplication, there has been surprisingly little information on the bioenergetics of growth. For many years, it was assumed that biomass production (anabolism) was proportional to the amount of ATP which could be derived from energy-yielding pathways (catabolism), but later work showed that the ATP yield (YATP) was not necessarily a constant. Continuous-culture experiments indicated that bacteria utilized ATP for metabolic reactions that were not directly related to growth (maintenance functions). Mathematical derivations showed that maintenance energy appeared to be a growth rate-independent function of the cell mass and time. Later work, however, showed that maintenance energy alone could not account for all the variations in yield. Because only some of the discrepancy could be explained by the secretion of metabolites (overflow metabolism) or the diversion of catabolism to metabolic pathways which produced less ATP, it appeared that energy-excess cultures had mechanisms of spilling energy. Bacteria have the potential to spill excess ATP in futile enzyme cycles, but there has been little proof that such cycles are significant. Recent work indicated that bacteria can also use futile cycles of potassium, ammonia, and protons through the cell membrane to dissipate ATP either directly or indirectly. The utility of energy spilling in bacteria has been a curiosity. The deprivation of energy from potential competitors is at best a teleological explanation that cannot be easily supported by standard theories of natural selection. The priming of intracellular intermediates for future growth or protection of cells from potentially toxic end products (e.g., methylglyoxal) seems a more plausible explanation.
Substantial N2O emission results from activated sludge nitrogen removal processes. N2O-reducing organisms possessing NosZ-type N2O reductases have been recognized to play crucial roles in suppressing emission of N2O produced in anoxic activated sludge via denitrification; however, which of the diverse nosZ-possessing organisms function as the major N2O sink in situ remains largely unknown. Here, nosZ genes and transcripts in wastewater microbiomes were analyzed with the group-specific qPCR assays designed de novo combining culture-based and computational approaches. A sewage sample was enriched in a batch reactor fed continuous stream of N2 containing 20-10,000 ppmv N2O with excess amount (10 mM) of acetate as the source of carbon and electrons, where 14 genera of potential N2O-reducers were identified. All available amino acid sequences of NosZ affiliated to these taxa were grouped into five subgroups (two clade I and three clade II groups), and primer/probe sets exclusively and comprehensively targeting the subgroups were designed and validated with in silico PCR. Four distinct activated sludge samples from three different wastewater treatment plants in Korea were analyzed with the qPCR assays and the results were validated with the shotgun metagenome analysis results. With these group-specific qPCR assays, the nosZ genes and transcripts of six additional activated sludge samples were analyzed and the results of the analyses clearly indicated the dominance of two clade II nosZ subgroups (Flavobacterium-like and Dechloromonas-like) among both nosZ gene and transcript pools.
Partial denitrification (PD), which could provide sufficient nitrite for subsequent anaerobic ammonium oxidation, is a novel strategy for mainstream nitrogen removal. In this study, the performance of using glycerol as electron donor for nitrite accumulation in PD process was evaluated. Results showed that a C/N of 4.5 was effective for nitrite production (average nitrite accumulation rate: 34.32 mg N h⁻¹ gMLVSS⁻¹; average nitrate-to-nitrite transformation ratio (NTR): 91.1%) with pH ranging from 6.0 to 9.0. Also, a stable nitrite accumulation was achieved in long-term operation with the average NTR of 80.1%. Mechanism investigation found that the denitrifying bacteria Saccharibacteria (77.9%) was enriched in glycerol-driven reactors. Moreover, the enzymatic activity as well as the encoding genes (i.e. narG, narH and napA) involved in nitrate reduction were much higher than that for nitrite reduction (i.e. nirK), and this disparity was responsible for the efficient nitrite accumulation in glycerol-driven PD system.
Nitrous oxide (N2O) emitted from wastewater treatment plants has caused widespread concern. Over the past decade, people have made tremendous efforts to discover the microorganisms responsible for N2O production, elucidate metabolic pathways, establish production models and formulate mitigation strategies. The ultimate goal of all these efforts is to shed new light on how N2O is produced and how to reduce it, and one of the best ways is to find key opportunities by integrating the information obtained. This review article critically evaluates the knowledge gained in the field within a decade, especially in N2O production microbiology, biochemistry, models and mitigation strategies, with a focus on denitrification. Previous research has greatly deepened the understanding of the N2O generation mechanism, but further efforts are still needed due to the lack of standardized methodology for establishing N2O mitigation strategies in full-scale systems. One of the challenges seems to be to convert the denitrification process from a net N2O source into an effective sink, which is recommended as a key opportunity to reduce N2O production in this review.
The contamination of water resources by nitrate is a global problem. Indeed, traditional treatment technologies are not able to remove this ion from water. Alternatively, biological denitrification is a useful technique for natural water nitrate removal. This study aimed to evaluate the use of glycerol as a carbon source for drinking water nitrate removal via denitrification in a reactor using microorganisms from natural biomass. The experiment was carried out in a continuous fixed bed reactor using immobilised microorganisms from the vegetal Phyllostachys aurea. The tests were started in batch mode to provide cells growth and further immobilisation on the support. Then, the treatment experiments were accomplished in an up-flow continuous reactor. Ethanol was used as the primary carbon source, and it was gradually replaced by glycerol. The C:N (carbon to nitrogen) ratio and the hydraulic residence time (HRT) were evaluated. It was possible to remove 98.14% of nitrate using a C:N ratio and HRT of 3:1 and 1.51 days, respectively. The results have demonstrated that glycerol is a potential carbon source for denitrification in a continuous reactor using immobilised cells from natural biomass.
The recent discovery of nitrous oxide (N2O)-reducing bacteria suggests a potential biological sink for the potent greenhouse gas N2O. While some N2O-reducing bacteria have been described, characterization of more isolates will be required for an application towards N2O mitigation. Here, we describe the successful enrichment and isolation of high-affinity N2O-reducing bacteria using an N2O-fed reactor (N2OFR). Two N2OFRs, in which N2O was continuously and directly supplied as the sole electron acceptor to a biofilm grown on a gas-permeable membrane, were operated with acetate or a mixture of peptone-based organic substrates as an electron donor. In parallel, a NO3--fed reactor (NO3FR), filled with a non-woven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N2O vs. NO3- would enhance the dominance of distinct N2O-reducing bacteria. Clade II type nosZ bacteria became rapidly enriched over clade I type nosZ bacteria in the N2OFRs whereas the opposite held in the NO3FR. High-throughput sequencing of 16S rRNA gene amplicons revealed the dominance of Rhodocyclaceae in the N2OFRs. Strains of the Azospira and Dechloromonas genera, canonical denitrifiers harboring clade II type nosZ, were isolated with high frequency from the N2OFRs (132 out of 152 isolates). The isolates from the N2OFR demonstrated higher N2O uptake rates (Vmax: 4.23 ± 10-3-1.80 ± 10-2 pmol/h/cell) and lower N2O half-saturation coefficients (Km, N2O: 1.55-2.10 µM) than a clade I type nosZ isolate from the NO3FR. Furthermore, the clade II type nosZ isolates had higher specific growth rates on N2O than nitrite as an electron acceptor. Hence, continuously and exclusively supplying N2O in an N2OFR allows the enrichment and isolation of high-affinity N2O-reducing strains, which may be used as N2O sinks in bioaugmentation efforts.
Nitrous oxide (N2O) emissions from wastewater treatment contribute significantly to greenhouse gas emissions. They have been shown to exhibit a strong seasonal and daily profile in previously conducted monitoring campaigns. However, only two year-long online monitoring campaigns have been published to date. Based on three monitoring campaigns on three full-scale wastewater treatment plants (WWTPs) with different activated sludge configurations, each of which lasted at least one year, we propose a refined monitoring strategy for long-term emission monitoring with multiple flux chambers on open tanks. Our monitoring campaigns confirm that the N2O emissions exhibited a strong seasonal profile and were substantial on all three plants (1 - 2.4% of the total nitrogen load). These results confirm that N2O is the most important greenhouse gas emission from wastewater treatment. The temporal variation was more distinct than the spatial variation within aeration tanks. Nevertheless, multiple monitoring spots along a single lane are crucial to assess representative emission factors in flow-through systems. Sequencing batch reactor systems were shown to exhibit comparable emissions within one reactor but significant variation between parallel reactors. The results indicate that considerable emission differences between lanes are to be expected in cases of inhomogeneous loading and discontinuous feeding. For example, N2O emission could be shown to depend on the amount of treated reject water: lanes without emitted less than 1% of the influent load, while parallel lanes emitted around 3%. In case of inhomogeneous loading, monitoring of multiple lanes is required. Our study enables robust planning of monitoring campaigns on WWTPs with open tanks. Extensive full-scale emission monitoring campaigns are important as a basis for reliable decisions about reducing the climate impact of wastewater treatment. More specifically, such data sets help us to define general emission factors for wastewater treatment plants and to construct and critically evaluate N2O emission models.
N2O is a potent greenhouse gas and ozone-depletion agent. In this study, a biofiltration system was designed for removal of N2O emitted at low concentrations (<200 ppmv) from wastewater treatment plants. A biofiltration system was designed to utilize untreated wastewater from the primary sedimentation basin as the source of electron donor and nutrients and minimize the energy requirement by utilizing gravitational force and pressure differential to direct liquid medium and gas through the biofilter. The experiments performed with laboratory-scale biofilter in two different configurations confirmed the feasibility of the biofiltration system. The biofilter operated with cycling of raw wastewater exhibited up to 94% and 53% removal efficiency with 100 ppmv N2O in N2 and air background, respectively, as the feed gas, corroborating that untreated wastewater can serve as a robust source of electron donor and nutrients. The laboratory-scale biofilter operated with a continuous flow-through of synthetic wastewater attained >99.9% removal of N2O from N2 background at the gas flowrate up to 2,000 mL·min-1 and >50% N2O removal from air background at the gas flowrate of 200 mL·min-1. nosZ-containing genus including Flavobacterium (5.92%), Pseudomonas (4.26%) and Bosea (2.39%) were identified from the biofilm samples collected from the oxic biofilter, indicating these organisms were responsible for N2O removal.
Microorganisms withthecapacitytoreducethegreenhousegasnitrousoxide
(N2O) toharmlessdinitrogengasarereceivingincreasedattentiondueto
increasing N2O emissions(andourneedtomitigateclimatechange)andto
recent discoveriesofnovelN2O-reducing bacteriaandarchaea.Thediversityof
denitrifying andnondenitrifyingmicroorganismswithcapacityforN2O reduc-
tion wasrecentlyshowntobegreaterthanpreviouslyexpected.Aformerly
overlooked group(cladeII)intheenvironmentincludealargefractionofnon-
denitrifying N2O reducers,whichcouldbeN2O sinkswithoutmajorcontribution
to N2O formation.Wereviewtherecentadvancesaboutfundamentalunder-
standing ofthegenomics,physiology,andecologyofN2O reducersandthe
importance ofthese findings forcurbingN2O emissions.
Intended as a companion to the Fundamentals of Forensic DNA Typing volume published in 2009, Advanced Topics in Forensic DNA Typing: Methodology contains 18 chapters with 4 appendices providing up-to-date coverage of essential topics in this important field and citation to more than 2800 articles and internet resources. The book builds upon the previous two editions of John Butler's internationally acclaimed Forensic DNA Typing textbook with forensic DNA analysts as its primary audience. This book provides the most detailed information written to-date on DNA databases, low-level DNA, validation, and numerous other topics including a new chapter on legal aspects of DNA testing to prepare scientists for expert witness testimony. Over half of the content is new compared to previous editions. A forthcoming companion volume will cover interpretation issues. - Contains the latest information - hot-topics and new technologies - Well edited, attractively laid out, and makes productive use of its four-color format.
Nitrous oxide (N2O) is an important pollutant which is emitted during the biological nutrient removal (BNR) processes of wastewater treatment. Since it has a greenhouse effect which is 265 times higher than carbon dioxide, even relatively small amounts can result in a significant carbon footprint. Biological nitrogen (N) removal conventionally occurs with nitrification/denitrification, yet also through advanced processes such as nitritation/denitritation and completely autotrophic N-removal. The microbial pathways leading to the N2O emission include hydroxylamine oxidation and nitrifier denitrification, both activated by ammonia oxidizing bacteria, and heterotrophic denitrification. In this work, a critical review of the existing literature on N2O emissions during BNR is presented focusing on the most contributing parameters. Various factors increasing the N2O emissions either per se or combined are identified: low dissolved oxygen, high nitrite accumulation, low chemical oxygen demand to nitrogen ratio, slow growth of denitrifying bacteria, uncontrolled pH and temperature. However, there is no common pattern in reporting the N2O generation amongst the cited studies, a fact that complicates its evaluation. When simulating N2O emissions, all microbial pathways along with the potential contribution of abiotic N2O production during wastewater treatment at different dissolved oxygen/nitrite levels should be considered. The undeniable validation of the robustness of such models calls for reliable quantification techniques which simultaneously describe dissolved and gaseous N2O dynamics. Thus, the choice of the N-removal process, the optimal selection of operational parameters and the establishment of validated dynamic models combining multiple N2O pathways are essential for studying the emissions mitigation.
The purpose of this study was to evaluate the effect of mannitol as carbon source on nitrogen removal and nitrous oxide (N2O) emission during partial nitrification (PN) process. Laboratory-scale PN sequencing batch reactors (SBRs) were operated with mannitol and sodium acetate as carbon sources, respectively. Results showed that mannitol could remarkably reduce N2O-N emission by 41.03%, without influencing the removal efficiency of NH4(+)-N. However, it has a significant influence on nitrite accumulation ratio (NAR) and TN removal, which were 19.97% and 13.59% lower than that in PN with sodium acetate, respectively. Microbial analysis showed that the introduction of mannitol could increase the abundance of bacteria encoding nosZ genes. In addition, anti-oxidant enzymes (T-SOD, POD and CAT) activities were significantly reduced and the dehydrogenase activity had an obvious increase in mannitol system, indicating that mannitol could alleviate the inhibition of N2O reductase (N2OR) activities caused by high NO2(-)-N concentration.
The carbon source used for denitrification is a key factor in the reduction of nitrous oxide (N2O) produced from wastewater treatment plants because it affects denitrification activity. In this study, two laboratory-scale Modified Ludzak Ettinger (MLE) processes were operated with methanol and sodium acetate as the sole carbon sources. Removal efficiency of soluble nitrogen was not affected by carbon source, but the N2O emission rate from the acetate-fed MLE process (1.6 ± 0.6 μg N–N2O/min) was lower than that from the methanol-fed process (3.0 ± 0.7 μg N–N2O/min). This is supported by the batch experiment data showing the acetate-fed biomass had a higher N2O reduction rate of 10.3 mg/gVSS/h than that of the methanol-fed biomass (3.3 mg/gVSS/h). In the methanol-fed process, 34.9 % of the total bacteria was the genus Methylotenera, which is reportedly incapable of N2O reduction. The acetate-fed process enriched the genera Dechloromonas and Rubrivivax, potential N2O reducers, accounting for 12.2 and 15.9 % of the total bacteria, respectively. The results indicated that acetate is a suitable replacement for methanol for wastewater treatment plants interested in mitigating N2O emission from the MLE process.
The carbon source used for denitrification is a key factor in the reduction of nitrous oxide (N2O) produced from wastewater treatment plants because it affects denitrification activity. In this study, two laboratory-scale Modified Ludzak Ettinger (MLE) processes were operated with methanol and sodium acetate as the sole carbon sources. Removal efficiency of soluble nitrogen was not affected by carbon source, but the N2O emission rate from the acetate-fed MLE process (1.6 ± 0.6 μg N–N2O/min) was lower than that from the methanol-fed process (3.0 ± 0.7 μg N–N2O/min). This is supported by the batch experiment data showing the acetate-fed biomass had a higher N2O reduction rate of 10.3 mg/gVSS/h than that of the methanol-fed biomass (3.3 mg/gVSS/h). In the methanol-fed process, 34.9 % of the total bacteria was the genus Methylotenera, which is reportedly incapable of N2O reduction. The acetate-fed process enriched the genera Dechloromonas and Rubrivivax, potential N2O reducers, accounting for 12.2 and 15.9 % of the total bacteria, respectively. The results indicated that acetate is a suitable replacement for methanol for wastewater treatment plants interested in mitigating N2O emission from the MLE process.
Genomics has revolutionised biological research, but quality assessment of the resulting assembled sequences is complicated and remains mostly limited to technical measures like N50.
We propose a measure for quantitative assessment of genome assembly and annotation completeness based on evolutionarily informed expectations of gene content. We implemented the assessment procedure in open-source software, with sets of Benchmarking Universal Single-Copy Orthologs, named BUSCO.
Software implemented in Python and datasets available for download from http://busco.ezlab.org.
Evgeny.Zdobnov@unige.ch.
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Chemical absorption-biological reduction (BioDeNOx), which uses Fe(II)(EDTA) as a complexing agent for promoting the mass transfer efficiency of NO from gas to water, is a promising technology for removing nitric oxide (NO) from flue gases. The carbon source and pH are important parameters for Fe(II)(EDTA)-NO (the production of absorption) reduction and N2O emissions from BioDeNOx systems. Batch tests were performed to evaluate the effects of four different carbon sources (i.e., methanol, ethanol, sodium acetate, and glucose) on Fe(II)(EDTA)-NO reduction and N2O emissions at an initial pH of 7.2 ± 0.2. The removal efficiency of Fe(II)(EDTA)-NO was 93.9 %, with a theoretical rate of 0.77 mmol L(-1) h(-1) after 24 h of operation. The highest N2O production was 0.025 mmol L(-1) after 3 h when glucose was used as the carbon source. The capacities of the carbon sources to enhance the activity of the Fe(II)(EDTA)-NO reductase enzyme decreased in the following order based on the C/N ratio: glucose > ethanol > sodium acetate > methanol. Over the investigated pH range of 5.5-8.5, the Fe(II)(EDTA)-NO removal efficiency was highest at a pH of 7.5, with a theoretical rate of 0.88 mmol L(-1) h(-1). However, the N2O production was lowest at a pH of 8.5. The primary effect of pH on denitrification resulted from the inhibition of nosZ in acidic conditions.
The competition for electrons has been recently demonstrated to affect the reduction rates of the nitrogen oxides in a methanol enriched denitrifying community. The aim of this study was to test if electron competition also occurred when other substrates were used for denitrification and if that could have an effect on the potential nitrous oxide (N2O) production and subsequent consumption. A denitrifying culture was developed in a sequencing batch reactor using nitrate as electron acceptor and a combination of acetate, ethanol and methanol as carbon sources. Four sets of batch tests were conducted using acetate, ethanol, methanol and a combination of the three carbon sources respectively. For each set the effect of nitrate, nitrite and nitrous oxide on each other reduction rates when present individually or in combination was assessed. Results show that reduction rates are affected by the type of substrate added, probably due to different microbial populations specialized with consuming a particular substrate. Also, N2O reduction rate is the most reduced under the different electron competition scenarios tested, which results in N2O accumulation in some cases. The effect of substrate limitation on N2O reduction was also assessed.
Globally, denitrification is commonly employed in biological nitrogen removal processes to enhance water quality. However, substantial knowledge gaps remain concerning the overall community structure, population dynamics and metabolism of different organic carbon sources. This systematic review provides a summary of current findings pertaining to the microbial ecology of denitrification in biological wastewater treatment processes. DNA fingerprinting-based analysis has revealed a high level of microbial diversity in denitrification reactors and highlighted the impacts of carbon sources in determining overall denitrifying community composition. Stable isotope probing, fluorescence in situ hybridization, microarrays and meta-omics further link community structure with function by identifying the functional populations and their gene regulatory patterns at the transcriptional and translational levels. This review stresses the need to integrate microbial ecology information into conventional denitrification design and operation at full-scale. Some emerging questions, from physiological mechanisms to practical solutions, for example, eliminating nitrous oxide emissions and supplementing more sustainable carbon sources than methanol, are also discussed. A combination of high-throughput approaches is next in line for thorough assessment of wastewater denitrifying community structure and function. Though denitrification is used as an example here, this synergy between microbial ecology and process engineering is applicable to other biological wastewater treatment processes.
Nitrous oxide (N2O) production and expression of genes capable of its reduction were investigated in two full-scale parallel plug-flow activated sludge systems. These two systems continuously received wastewater with the same constituents, but operated under distinct nitrification efficiencies due to mixed liquor suspended solid (MLSS) concentration and the different hydraulic retention times (HRTs). A shorter HRT in system 2 resulted in a lower nitrification efficiency (40-60%) in conjunction with a high N2O emission (50.6 mg-N/L/day), whereas there was a higher nitrification efficiency (>99%) in system 1 with low N2O emission (22.6 mg-N/L/day). The DNA abundance of functional genes responsible for nitrification and denitrification were comparable in both systems, but transcription of nosZ mRNA in the lower N2O emission system (system 1) was one order of magnitude higher than that in the higher N2O emission system (system 2). The diversity and evenness of the nosZ gene were nearly identical; however, the predominant N2O reducing bacteria were phylogenetically distinct. Phylogenetic analysis indicated that N2O-reducing strains only retrieved in system 1 were close to the genera Rhodobacter, Oligotropha and Shinella, whereas they were close to the genera Mesorhizobium only in system 2. The distinct predominant N2O reducers may directly or indirectly influence N2O emissions.
Two acclimatized biomasses exposed to ammonium (NH 4 +) concentration of 600 mg N L −1 , one from a completely stirred tank reactor (CSTR), the other from a sequencing batch reactor (SBR), were assayed for nitritation performance, predominant nitrifying bacterial population and nitrous oxide (N 2 O) production. By virtue of fluctuating and constant NH 4 + concentrations respectively, the SBR and CSTR wastewater supply regimes were hypothesized to support different predominant ammonia-oxidizing bacteria (AOB) exhibiting distinct biokinetic properties. Nitritation efficiency (NO 2 − -N/NO 2+3 − -N) was higher in the SBR (89%) than the CSTR (30%) likely due to free ammonia and dissolved oxygen concentration. Quantita-tive fluorescence in situ hybridization (FISH) analyses revealed that fast-growing (r-strategist) AOB of halophilic and halotolerant Nitrosomonas lineage were more highly enriched in the SBR (76 ± 4.2%) than the CSTR (38 ± 6.0%). The CSTR predominantly enriched slow-growing (K-strategist) AOB Nitrosospira spp. (42 ± 1.9% versus 1.4 ± 0.8% in the SBR). Biokinetic parameter estimation consolidated the FISH result: the maximum growth rate and half-saturation coefficients for NH 4 + were higher in the SBR (max = 0.92 day, K NH4+ = 28.9 mg N L −1) relative to the CSTR (max = 0.42 day, K NH4+ = 3.47 mg N L −1), suggesting that the extent of nitritation may be controlled by choice of wastewater influent operational regime, which itself determines predominant AOB. N 2 O production was a maximum of 25 times higher (10.2 mg N-N 2 O h −1 at 0.5 mg O 2 L −1) in CSTR-enriched biomass than in SBR-enriched biomass (0.41 mg N-N 2 O h −1 at 0.5 mg O 2 L −1).
Limited availability of carbon sources has been regarded as an important factor leading to N2O accumulation during denitrification in wastewater treatment. By varying the carbon (methanol) loading rate to a methanol utilizing denitrifying culture in the presence of various electron acceptors (nitrate, nitrite, N2O and their combinations), this study quantitatively investigated the electron distribution among different nitrogen oxide reductases during denitrification. The results showed that electron competition occurs under not only carbon limiting but also carbon abundant conditions. The electron distribution among the nitrogen oxide reductases is affected by the carbon loading rate, with a lower fraction of electrons distributed to the N2O reductase with reduced carbon loading rate. N2O accumulation occurs when the electron flux going to nitrite reduction is higher than that going to N2O reduction. The study also showed that, for the culture investigated, the carbon to nitrogen ratio is not a key factor leading to N2O accumulation.
Nitrous oxide (N(2)O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N(2)) by bacteria and archaea harboring the N(2)O reductase (N(2)OR), which is the only known N(2)O sink in the biosphere. Despite its crucial role in mitigating N(2)O emissions, knowledge of the N(2)OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N(2)OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N(2)O-reducing communities. Examination of N(2)OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N(2)OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N(2)O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N(2)O-reducing microorganisms, which might be of importance for understanding the relationship between the diversity of N(2)O-reducing microorganisms and N(2)O reduction in different ecosystems.
Acidic pH has previously been found to increase nitrous oxide (N₂O) accumulation during heterotrophic denitrification in biological wastewater treatment. However, the mechanism of this phenomenon still needs to be clarified. By using an enriched methanol utilizing denitrifying culture as an example, this paper presents a comprehensive study on the effect of pH (6.0-9.0) on N₂O reduction kinetics with N₂O as the sole electron acceptor, as well as the effect of pH on N₂O accumulation with N₂O as an intermediate of nitrate reduction. The pH dependency of nitrate and nitrite reduction was also investigated. The maximum biomass-specificN₂O reduction rate is higher than the corresponding maximum nitrate and nitrite reduction rates in the entire pH range studied. However, the maximum biomass-specific N₂O reduction rate is much more sensitive to pH variation outside of the optimal range (pH 7.5 to pH 8.0) than the maximum biomass-specific nitrate and nitrite reduction rates. The half-saturation coefficient of the N₂O reductase increased from 0.10 mg N₂O-N/L to 0.92 mg N₂O-N/L as pH increased from pH 6.0 to 9.0. At pH 6.0, approximately 20% and 40% of the denitrified nitrate accumulated as N₂O in the presence and absence of methanol (as an exogenous carbon source), respectively. However, at pH 6.5, these fractions decreased to 0% and 30%, respectively. No N₂O accumulation occurred at pH 7.0 to 9.0 independent of the availability of methanol. These results suggest that the competition for electrons among different nitrogen oxides reductases likely plays a role in N₂O accumulation at low pH conditions.
During biological denitrification in Waste Water Treatment Plants (WWTPs), many parameters (chemical, physical and biological) are responsible for greenhouse gas emissions such as nitrous oxide (N2O) and nitric oxide (NO). The present study intends to investigate the impact of the carbon source more specifically on N2O emissions, but also on NO emissions. The experiments were done in a bioreactor performing batch denitrification at a laboratory scale. Three sources of carbon were tested: ethanol and acetate as short carbon chain compounds and a mixture composed of ethanol and acetate and two long carbon chain compounds: casein extract and meat extract. The nitrogen source was always nitrates (NO3−) and the ratio COD/N was set to three. Current nitrite and nitrate ions, nitric and nitrous oxide levels were monitored during experimentation. The results principally show that the acetate carbon source generates the highest N2O and NO emissions (74% and 19% of denitrified N-NO3−, respectively).The results of this work suggest that the type and length of the carbon source used are responsible for nitrogen emissions but not in the expected way. While the literature always focuses on the inhibitory effect of nitrites on N2O emissions, this work has singled out that NO may also exert inhibitory effects on the N2O reductase enzyme. These results may be explained by the diversity of denitrifying bacteria and their distinct metabolic pathways towards the added carbon substrates (influents).
Carbon sources such as methanol and glycerol are used for enhancing denitrification at wastewater treatment plants, which are required to meet increasingly stringent effluent nitrogen limits. Consequently, dosing strategies for these compounds could benefit from the development and application of molecular activity biomarkers to infer and distinguish between methanol- or glycerol-based denitrification in activated sludge. In this study, the applicability of genes coding for methanol dehydrogenase (mdh2 and mxaF) and glycerol dehydrogenase (dhaD) as potential biomarkers of denitrification activity using these specific substrates was explored and confirmed using a two-pronged approach. First, during short-term spikes of activated sludge biomass with glycerol, the ability of dhaD mRNA concentrations to closely track nitrate depletion profiles was demonstrated. Second, a high-degree of correlation of the mRNA concentrations of mdh2, mxaF and dhaD with methanol- and glycerol-based denitrification kinetics during long-term bioreactor operation using these substrates was also shown. Based on these results, expression of mdh2, mxaF and dhaD genes are promising biomarkers of in situ denitrification activity on methanol and glycerol, respectively, in mixed-culture engineered wastewater treatment processes.