Suggested role of NosZ in preventing N2O inhibition of dissimilatory nitrite reduction to ammonium.

IMPORTANCE: Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3 - and/or NO2 - to NH4 +. Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.

Comparative genomic analysis of Methylocystis sp. MJC1 as a platform strain for polyhydroxybutyrate biosynthesis.

Biodegradable polyhydroxybutyrate (PHB) can be produced from methane by some type II methanotroph such as the genus Methylocystis. This study presents the comparative genomic analysis of a newly isolated methanotroph, Methylocystis sp. MJC1 as a biodegradable PHB-producing platform strain. Methylocystis sp. MJC1 accumulates up to 44.5% of PHB based on dry cell weight under nitrogen-limiting conditions. To facilitate its development as a PHB-producing platform strain, the complete genome sequence of Methylocystis sp. MJC1 was assembled, functionally annotated, and compared with genomes of other Methylocystis species. Phylogenetic analysis has shown that Methylocystis parvus to be the closest species to Methylocystis sp. MJC1. Genome functional annotation revealed that Methylocystis sp. MJC1 contains all major type II methanotroph biochemical pathways such as the serine cycle, EMC pathway, and Krebs cycle. Interestingly, Methylocystis sp. MJC1 has both particulate and soluble methane monooxygenases, which are not commonly found among Methylocystis species. In addition, this species also possesses most of the RuMP pathway reactions, a characteristic of type I methanotrophs, and all PHB biosynthetic genes. These comparative analysis would open the possibility of future practical applications such as the development of organism-specific genome-scale models and application of metabolic engineering strategies to Methylocystis sp. MJC1.

Biotrickling Filtration for the Reduction of N2O Emitted during Wastewater Treatment: Results from a Long-Term In Situ Pilot-Scale Testing.

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.

Identification of nosZ-expressing microorganisms consuming trace N2O in microaerobic chemostat consortia dominated by an uncultured Burkholderiales.

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.

Organic carbon determines nitrous oxide consumption activity of clade I and II nosZ bacteria: Genomic and biokinetic insights.

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.

Metagenomic insights into co-proliferation of Vibrio spp. and dinoflagellates Prorocentrum during a spring algal bloom in the coastal East China Sea.

Coastal harmful algal blooms (HABs), commonly termed 'red tides', have severe undesirable consequences to the marine ecosystems and local fishery and tourism industries. Increase in nitrogen and/or phosphorus loading is often regarded as the major culprits of increasing frequency and intensity of the coastal HAB; however, fundamental understanding is lacking as to the causes and mechanism of bloom formation despite decades of intensive investigation. In this study, we interrogated the prokaryotic microbiomes of surface water samples collected at two neighboring segments of East China Sea that contrast greatly in terms of the intensity and frequency of Prorocentrum-dominated HAB. Mantel tests identified significant correlations between the structural and functional composition of the microbiomes and the physicochemical state and the algal biomass density of the surface seawater, implying the possibility that prokaryotic microbiota may play key roles in the coastal HAB. A conspicuous feature of the microbiomes at the sites characterized with high trophic state index and eukaryotic algal cell counts was disproportionate proliferation of Vibrio spp., and their complete domination of the functional genes attributable to the dissimilatory nitrate reduction to ammonia (DNRA) pathway substantially enriched at these sites. The genes attributed to phosphorus uptake function were significantly enriched at these sites, presumably due to the Pi-deficiency induced by algal growth; however, the profiles of the phosphorus mineralization genes lacked consistency, barring any conclusive evidence with regard to contribution of prokaryotic microbiota to phosphorus bioavailability. The results of the co-occurrence network analysis performed with the core prokaryotic microbiome supported that the observed proliferation of Vibrio and HAB may be causally associated. The findings of this study suggest a previously unidentified association between Vibrio proliferation and the Prorocentrum-dominated HAB in the subtropical East China Sea, and opens a discussion regarding a theoretically unlikely, but still possible, involvement of Vibrio-mediated DNRA in Vibrio-Prorocentrum symbiosis. Further experimental substantiation of this supposed symbiotic mechanism may prove crucial in understanding the dynamics of explosive local algal growth in the region during spring algal blooms.

Cometabolic Vinyl Chloride Degradation at Acidic pH Catalyzed by Acidophilic Methanotrophs Isolated from Alpine Peat Bogs.

Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 µM) or absence (<0.01 µM) of copper, suggesting that VC removal had little dependence on copper availability, which regulates expression and activity of soluble and particulate methane monooxygenases in methanotrophs. The model neutrophilic methanotroph Methylosinus trichosporium strain OB3b also grew and cometabolized VC at pH 5.0 regardless of copper availability. Bioaugmentation of acidic peat soil slurries with methanotroph isolates demonstrated enhanced VC degradation and VC consumption below the maximum concentration level of 2 µg L-1. Community profiling of the microcosms suggested species-specific differences, indicating that robust bioaugmentation with methanotroph cultures requires further research.

Enhancement of Nitrous Oxide Emissions in Soil Microbial Consortia via Copper Competition between Proteobacterial Methanotrophs and Denitrifiers.

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized, post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soil's native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Finally, with microcosms amended with various cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration, while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit. IMPORTANCE Proteobacterial methanotrophs-groups of microorganisms that utilize methane as a source of energy and carbon-have been known to employ unique mechanisms to scavenge copper, namely, utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and coculture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments cohabited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allows for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for a significant proportion of N2O efflux from agricultural soils.

Effect of Fluid-Rock Interactions on In Situ Bacterial Alteration of Interfacial Properties and Wettability of CO2-Brine-Mineral Systems for Geologic Carbon Storage.

This study explored the feasibility of biosurfactant amendment in modifying the interfacial characteristics of carbon dioxide (CO2) with rock minerals under high-pressure conditions for GCS. In particular, while varying the CO2 phase and the rock mineral, we quantitatively examined the production of biosurfactants by Bacillus subtilis and their effects on interfacial tension (IFT) and wettability in CO2-brine-mineral systems. The results demonstrated that surfactin produced by B. subtilis caused the reduction of CO2-brine IFT and modified the wettability of both quartz and calcite minerals to be more CO2-wet. The production yield of surfactin was substantially greater with the calcite mineral than with the quartz mineral. The calcite played the role of a pH buffer, consistently maintaining the brine pH above 6. By contrast, an acidic condition in CO2-brine-quartz systems caused the precipitation of surfactin, and hence surfactin lost its ability as a surface-active agent. Meanwhile, the CO2-driven mineral dissolution and precipitation in CO2-brine-calcite systems under a non-equilibrium system altered the solid substrates, produced surface roughness, and caused contact angle variations. These results provide unique experimental data on biosurfactant-mediated interfacial properties and wettability in GCS-relevant conditions, which support the exploitation of in situ biosurfactant production for biosurfactant-aided CO2 injection.

Quantification of nosZ genes and transcripts in activated sludge microbiomes with novel group-specific qPCR methods validated with metagenomic analyses.

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 primers/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.

Involvement of NO3- in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3--reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2- accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2--to-NH4+ reduction at submillimolar NO3- concentrations and downregulation of nrfA or nirB transcription when NO3- was being reduced to NO2- In batch and chemostat experiments, both isolates produced NH4+ from NO3- reduction when incubated with excess organic electron donors, while incubation with excess NO3- resulted in NO2- buildup but no substantial NH4+ production, presumably due to inhibitory NO3- concentrations. This previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3- and NO2- Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3- and NO2- to NH4+, a reactive nitrogen compound with a higher tendency to be retained in the soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA bacterial ecophysiology. Here, we have developed a new screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils.

Identification of primary effecters of N2O emissions from full-scale biological nitrogen removal systems using random forest approach.

Wastewater treatment plants (WWTPs) have long been recognized as point sources of N2O, a potent greenhouse gas and ozone-depleting agent. Multiple mechanisms, both biotic and abiotic, have been suggested to be responsible for N2O production from WWTPs, with basis on extrapolation from laboratory results and statistical analyses of metadata collected from operational full-scale plants. In this study, random forest (RF) analysis, a machine-learning approach for feature selection from highly multivariate datasets, was adopted to investigate N2O production mechanism in activated sludge tanks of WWTPs from a novel perspective. Standardized measurements of N2O effluxes coupled with exhaustive metadata collection were performed at activated sludge tanks of three biological nitrogen removal WWTPs at different times of the year. The multivariate datasets were used as inputs for RF analyses. Computation of the permutation variable importance measures returned biomass-normalized dissolved inorganic carbon concentration (DIC·VSS-1) and specific ammonia oxidation activity (sOURAOB) as the most influential parameters determining N2O emissions from the aerated zones (or phases) of activated sludge bioreactors. For the anoxic tanks, dissolved-organic-carbon-to-NO2-/NO3- ratio (DOC·(NO2--N + NO3--N)-1) was singled out as the most influential. These data analysis results clearly indicate disparate mechanisms for N2O generation in the oxic and anoxic activated sludge bioreactors, and provide evidences against significant contributions of N2O carryover across different zones or phases or niche-specific microbial reactions, with aerobic NH3/NH4+ oxidation to NO2- and anoxic denitrification predominantly responsible from aerated and anoxic zones or phases of activated sludge bioreactors, respectively.

Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.

Enhanced Nitrous Oxide Production in Denitrifying Dechloromonas aromatica Strain RCB Under Salt or Alkaline Stress Conditions.

Salinity and pH have direct and indirect impacts on the growth and metabolic activities of microorganisms. In this study, the effects of salt and alkaline stresses on the kinetic balance between nitrous oxide (N2O) production and consumption in the denitrification pathway of Dechloromonas aromatica strain RCB were examined. N2O accumulated transiently only in insignificant amounts at low salinity (≤0.5% NaCl) and circumneutral pH (7.0 and 7.5). As compared to these control conditions, incubation at 0.7% salinity resulted in substantially longer lag phase and slower growth rate, along with the increase in the amounts of transiently accumulated N2O (15.8 ± 2.8 µmoles N2O-N/vessel). Incubation at pH 8.0 severely inhibited growth and resulted in permanent accumulation of 29.9 ± 1.3 µmoles N2O-N/vessel from reduction of 151 ± 20 µmoles NO3 -/vessel. Monitoring of temporal changes in nirS 1, nirS 2, and nosZ transcription suggested that the nosZ/(nirS 1+nirS 2) ratios were indicative of whether N2O was produced or consumed at the time points where measurements were taken. The salt and alkaline stresses altered the N2O consumption kinetics of the resting D. aromatica cells with expressed nitrous oxide reductases. The N2O consumption rates of the cells subjected to the salt and alkaline stress conditions were significantly reduced from 0.84 ± 0.007 µmoles min-1 mg protein-1 of the control to 0.27 ± 0.02 µmoles min-1 mg protein-1 and 0.31 ± 0.03 µmoles min-1 mg protein-1, respectively, when the initial dissolved N2O concentration was 0.1 mM. As the rates of N2O production from NO2 - reduction was not significantly affected by the stresses (0.45-0.55 µmoles min-1 mg protein-1), the N2O consumption rate was lower than the N2O production rate at the stress conditions, but not at the control condition. These results clearly indicate that the altered kinetics of expressed nitrous oxide reductase and the resultant disruption of kinetic balance between N2O production and consumption was another cause of enhanced N2O emission observed under the salt and alkaline stress conditions. These findings suggest that canonical denitrifiers may become a significant N2O source when faced with abrupt environmental changes.

Ecological and physiological implications of nitrogen oxide reduction pathways on greenhouse gas emissions in agroecosystems.

Microbial reductive pathways of nitrogen (N) oxides are highly relevant to net emissions of greenhouse gases (GHG) from agroecosystems. Several biotic and abiotic N-oxide reductive pathways influence the N budget and net GHG production in soil. This review summarizes the recent findings of N-oxide reduction pathways and their implications to GHG emissions in agroecosystems and proposes several mitigation strategies. Denitrification is the primary N-oxide reductive pathway that results in direct N2O emissions and fixed N losses, which add to the net carbon footprint. We highlight how dissimilatory nitrate reduction to ammonium (DNRA), an alternative N-oxide reduction pathway, may be used to reduce N2O production and N losses via denitrification. Implications of nosZ abundance and diversity and expressed N2O reductase activity to soil N2O emissions are reviewed with focus on the role of the N2O-reducers as an important N2O sink. Non-prokaryotic N2O sources, e.g. fungal denitrification, codenitrification and chemodenitrification, are also summarized to emphasize their potential significance as modulators of soil N2O emissions. Through the extensive review of these recent scientific advancements, this study posits opportunities for GHG mitigation through manipulation of microbial N-oxide reductive pathways in soil.

A Serial Biofiltration System for Effective Removal of Low-Concentration Nitrous Oxide in Oxic Gas Streams: Mathematical Modeling of Reactor Performance and Experimental Validation.

Wastewater treatment plants (WWTPs) are among the major anthropogenic sources of N2O, a major greenhouse gas and ozone-depleting agent. We recently devised a zero-energy zero-carbon biofiltration system easily applicable to activated sludge-type WWTPs and performed lab-scale proof-of-concept experiments. The major drawback of the system was the diminished performance observed when fully oxic gas streams were treated. Here, a serial biofiltration system was tested as a potential improvement. A laboratory system with three serially positioned biofilters, each receiving a separate feed of artificial wastewater, was fed N2O-containing gas streams of varied flow rates (200-2000 mL·min-1) and O2 concentrations (0-21%). Use of the serial setup substantially improved the reactor performance. Fed fully oxic gas at a flow rate of 1000 mL·min-1, the system removed N2O at an elimination capacity of 0.402 ± 0.009 g N2O·m-3·h-1 (52.5% removal), which was approximately 2.4-fold higher than that achieved with a single biofilter, 0.171 ± 0.024 g N2O·m-3·h-1. These data were used to validate the mathematical model developed to estimate the performance of the N2O biofiltration system. The Nash-Sutcliffe efficiency indices ranged from 0.78 to 0.93, confirming high predictability, and the model provided mechanistic insights into aerobic N2O removal and the performance enhancement achieved with the serial configuration.

Novel approaches and reasons to isolate methanotrophic bacteria with biotechnological potentials: recent achievements and perspectives.

The recent drop in the price of natural gas has rekindled the interests in methanotrophs, the organisms capable of utilizing methane as the sole electron donor and carbon source, as biocatalysts for various industrial applications. As heterologous expression of the methane monooxygenases in more amenable hosts has been proven to be nearly impossible, future success in methanotroph biotechnology largely depends on securing phylogenetically and phenotypically diverse methanotrophs with relatively high growth rates. For long, isolation of methanotrophs have relied on repeated single colony picking after initial batch enrichment with methane, which is a very rigorous and time-consuming process. In this review, three unconventional isolation methods devised for facilitation of the isolation process, diversification of targeted methanotrophs, and/or screening of rapid growers are summarized. The soil substrate membrane method allowed for isolation of previously elusive methanotrophs and application of high-throughput extinction plating technique facilitated the isolation procedure. Use of a chemostat with gradually increased dilution rates proved effective in screening for the fastest-growing methanotrophs from environmental samples. Development of new isolation technologies incorporating microfluidics and single-cell techniques may lead to discovery of previously unculturable methanotrophs with unexpected metabolic potentials and thus, certainly warrant future investigation.

Rapid isolation of fast-growing methanotrophs from environmental samples using continuous cultivation with gradually increased dilution rates.

Methanotrophs have recently gained interest as biocatalysts for mitigation of greenhouse gas emission and conversion of methane to value-added products; however, their slow growth has, at least partially, hindered their industrial application. A rapid isolation technique that specifically screens for the fastest-growing methanotrophs was developed using continuous cultivation with gradually increased dilution rates. Environmental samples collected from methane-rich environments were enriched in continuously stirred tank reactors with unrestricted supply of methane and air. The reactor was started at the dilution rate of 0.1 h-1, and the dilution rates were increased with an increment of 0.05 h-1 until the reactor was completely washed out. The shifts in the overall microbial population and methanotrophic community at each step of the isolation procedure were monitored with 16S rRNA amplicon sequencing. The predominant methanotrophic groups recovered after reactor operations were affiliated to the gammaproteobacterial genera Methylomonas and Methylosarcina. The methanotrophic strains isolated from the reactor samples collected at their respective highest dilution rates exhibited specific growth rates up to 0.40 h-1; the highest value reported for methanotrophs. The novel isolation method developed in this study significantly shortened the time and efforts needed for isolation of methanotrophs from environmental samples and was capable of screening for the methanotrophs with the fastest growth rates.

Microbial community analyses of produced waters from high-temperature oil reservoirs reveal unexpected similarity between geographically distant oil reservoirs.

As a preliminary investigation for the development of microbial-enhanced oil recovery strategies for high-temperature oil reservoirs (~70 to 90°C), we have investigated the indigenous microbial community compositions of produced waters from five different high-temperature oil reservoirs near Segno, Texas, U.S. (~80 to 85°C) and Crossfield, Alberta, Canada (~75°C). The DNA extracted from these low-biomass-produced water samples were analysed with MiSeq amplicon sequencing of partial 16S rRNA genes. These sequences were analysed along with additional sequence data sets available from existing databases. Despite the geographical distance and difference in the physicochemical properties, the microbial compositions of the Segno and Crossfield produced waters exhibited unexpectedly high similarity, as indicated by the results of beta diversity analyses. The major operational taxonomic units included acetoclastic and hydrogenotrophic methanogens (Methanosaetaceae, Methanobacterium and Methanoculleus), as well as bacteria belonging to the families Clostridiaceae and Thermotogaceae, which have been recognized to include thermophilic, thermotolerant, and/or spore-forming subtaxa. The sequence data retrieved from the databases exhibited different clustering patterns, as the communities from close geographical locations invariably had low beta diversity and the physicochemical properties and conditions of the reservoirs apparently did not have a substantial role in shaping of microbial communities.