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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.

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 (

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.

Soils affected by animal wastes have simultaneously high N and P contents. Despite the reports of high nitrous oxide (N2O) emissions in such environments, the role that P plays in N2O dynamics has not yet been systematically investigated. Here, we report the enhancement effect of orthophosphate (PO43−-P) abundance on N2O yields from abiotic NH2OH decomposition, which may have substantial implications to N2O emissions from nitrification in such P-rich soils. The axenic cultures of Nitrosomonas europaea, an ammonia-oxidizing bacterium previously reported to leak NH2OH, exhibited significantly higher N2O yields when incubated at higher PO43−-P concentrations. As NH4+-to-NO2− turnover and growth rates were unaffected even at the highest PO43−-P concentration examined, the abiotic interaction between extracellularly released NH2OH and PO43−-P was the most plausible mechanism of enhanced N2O emission in these nitrifier cultures. This proposed mechanism was supported by the results of abiotic NH2OH incubation whereby higher PO43−-P concentration resulted in higher N2O yield. Orthophosphate enhancement of NH2OH-to-N2O turnover was then simulated with addition of 5 μmol NH2OH to an ornithogenic soil with high PO43−-P content (23.9 ± 6.7 g/kg wet soil) and active nitrification activity after sterilization. The N2O yield, 69.0 ± 4.6%, was significantly higher than the N2O yields for other examined soils with lower PO43−-P contents (0–1.94 g/kg wet soil), and the PO43−-P contents of the examined soils exhibited strong correlation with the N2O yields. These findings suggest that N2O production from nitrification via abiotic turnover of released NH2OH may be a consequential mechanism of N2O emissions in PO43−-P-rich soils.

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 (−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.

Microorganisms possessing N

Oil palm plantations, irreversibly claimed primarily from tropical forest, carpet the landscape in Malaysia and Indonesia, the largest global producers of palm oil. The impact of forest conversion to oil palm agriculture on the plant and animal diversity has gained worldwide attention, but knowledge on the effects on microbially mediated belowground soil processes which drive ecosystem-level responses such as greenhouse gas (GHG) fluxes, particularly methane and nitrous oxide, remain scarce and fragmented. Focusing on the soil microbiome, as well as environmental drivers of soil biogeochemical processes, we synthesize previous research works to provide an overview of the current state of scientific understanding on the effects of deforestation for oil palm agriculture. Forest conversion to oil palm plantations is associated with increased pH, and lowered C and N contents, as typically observed in agricultural soils. Interestingly, in contrast to plant and animal diversity, soil bacterial and functional diversity, as well as fungal abundance, were unaffected or increased. Furthermore, community composition was altered by the land transformation. This indicates the resilience of the microbial diversity to deforestation for oil palm agriculture. However, it remains to be determined whether and how such community resilience would translate to the resilience of soil microbial groups mediating methane- and N-cycling processes central to greenhouse gas turnover.

Modification of oil–brine–minerals interfacial properties with biosurfactant-producing microorganisms and their extracellular metabolites has been considered as one of the viable strategies for mic...

BackgroundCoastal harmful algal blooms (HAB), commonly termed ‘Red tides’, have severe undesirable consequences to the marine ecosystems and local fishery and tourism industries. Increase in nitrogen and/or phosphate loading is often attributed 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.ResultsIn 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 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 significantly enriched at these sites. The genes attributed to phosphorus uptake function were also 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 phosphorous bioavailability. The results of the co-occurrence network analysis performed with the core prokaryotic microbiome also supported that the observed proliferation of Vibrio and HAB may be causally associated.ConclusionsThe 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 an unlikely, but still possible, involvement of Vibrio-mediated DNRA in Vibrio-Prorocentrum symbiosis. Further substantiation of this interaction with culture-based experiments may prove crucial for understanding of the dynamics of explosive local algal growth during HAB events endemic to the region.

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.

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 N(2)O 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 N(2)O production, with more pronounced responses observed for soils with lower copper content. The N(2)O 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 CH(4) enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N(2)O 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 N(2)O 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 N(2)O 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 N(2)O production may account for a significant proportion of N(2)O efflux from agricultural soils.

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This study explored the feasibility of biosurfactant amendment in modifying the interfacial characteristics of carbon dioxide (CO

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. Lastly, with microcosms amended with varying 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.ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize 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 co-culture 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 co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow 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 significant proportion of N2O efflux from agricultural soils.

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.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently gained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, 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 microbial isolates capable of DNRA have been isolated from rice paddy soil with apparent dominance of denitrification using a novel high-throughput screening method. Six of these isolates, each assigned to a disparate genus, was examined to improve understanding of DNRA physiology. All isolates carriednrfAand/ornirB, and an isolate affiliated toBacilluspossessed a clade IInosZgene and was capable of N2O reduction. A common prominent physiological feature observed in all DNRA isolates was NO2−accumulation observed before NH4+production, which was further examined withCitrobactersp. DNRA3 (possessingnrfAandnirB)andEnterobactersp. DNRA5 (possessing onlynirB). In both organisms, NO2−-to-NH4+reduction was inhibited by submillimolar NO3−, andnrfAornirBtranscription was down-regulated when NO3−was being reduced to NO2−. Both batch and chemostat incubations of these isolates with excess organic electron donors produced NH4+from reduction of NO3−; however, incubation with excess NO3−resulted in NO2−buildup but no substantial NH4+production, presumably due to NO3−presence. 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-vs-DNRA competition in soil.IMPORTANCEDissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3−and NO2−. Unlike denitrification leading to nitrogen loss and N2O emission, DNRA reduces NO3−and NO2−to NH4+, a reactive nitrogen with higher tendency to be retained in 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 ecophysiology. Here, we have developed a novel high-throughput 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 key to understanding why significant DNRA activity is rarely observed in nitrogen-rich agricultural soils.

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 NO(3)(−)-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 N(2)O reduction. A common prominent physiological feature observed in the isolates was NO(2)(−) accumulation before NH(4)(+) 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 NO(2)(−)-to-NH(4)(+) reduction at submillimolar NO(3)(−) concentrations and downregulation of nrfA or nirB transcription when NO(3)(−) was being reduced to NO(2)(−). In batch and chemostat experiments, both isolates produced NH(4)(+) from NO(3)(−) reduction when incubated with excess organic electron donors, while incubation with excess NO(3)(−) resulted in NO(2)(−) buildup but no substantial NH(4)(+) production, presumably due to inhibitory NO(3)(−) concentrations. This previously overlooked link between NO(3)(−) repression of NO(2)(−)-to-NH(4)(+) 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 NO(3)(−) and NO(2)(−). Unlike denitrification, which leads to nitrogen loss and N(2)O emission, DNRA reduces NO(3)(−) and NO(2)(−) to NH(4)(+), 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 NO(3)(−) repression of NO(2)(−)-to-NH(4)(+) 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.

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.

Wastewater treatment plants (WWTPs) have long been recognized as point sources of N

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

Summary 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.

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.

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a "copper monopoly" that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N2O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N2O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N2O production was observed from NO3- reduction, whereas no significant N2O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N2O emissions, with the potential for increasing net greenhouse gas emissions.

Surface and ground water contamination with NO 2 − /NO 3 − is one of the most serious environmental issues due to their adverse effects on human health and ecosystem. Various technologies have been investigated for removal of NO 2 − /NO 3 − from surface and ground water; nevertheless, the idea of utilizing NO 2 − /NO 3 − contaminated water and wastewater to produce value-added products has not yet been much explored to date. Here, we have developed a novel method for utilizing NO 2 − /NO 3 − -contaminated water as a source of nitrogen for the synthesis of benzaldoxime from benzaldehyde using ecofriendly nanoscale zero-valent iron (nZVI) as the reductant. Control experiments with NH 4 + and NH 2 OH supported the proposed reaction mechanism that NH 2 OH was generated in situ as a reactive intermediate from NO 2 − /NO 3 − reduction and reacted with benzaldehyde to form benzaldoxime. The benzaldoxime yield was the largest at the highest temperature tested, 100 °C, and an nZVI-to-N ratio of ∼7 was optimal for benzaldoxime synthesis. At 100 °C, O 2 in the headspace did not have any negative effect on the reaction. Benzaldoxime yields up to 0.70 ± 0.04 mmoles and 0.67 ± 0.05 mmoles were observed from reaction of 1 mmole benzaldehyde with 2 mmoles NO 2 − and NO 3 − , respectively. Meanwhile, >95% of NO 2 − /NO 3 − were reduced to either benzaldoxime or NH 4 + . This novel method suggests a promising option for utilization of water contaminated with NO 2 − /NO 3 − for the production of a value-added product.

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 ( 99...

Water reuse systems have been widely implemented across the globe as a solution to water shortage and freshwater contamination. Ongoing controversies regarding supposedly high energy cost and the lack of sufficient data to support benefits of water reuse are delaying further expansion of implementation of water reuse systems in South Korea. In order to clarify the ambiguity regarding the energy demand and provide an unbiased comparison between the water reuse alternatives and the conventional water supply system, energy consumptions and greenhouse gas emissions in operation phases of different water reuse facilities and the conventional water supply system were examined. The average total energy consumption and the greenhouse gas emission of the conventional process were calculated to be 0.511 kWh/m3 and 0.43 kgCO2e/m3, respectively. Centralized wastewater reuse systems had prohibitively high energy consumptions (1.224–1.914 kWh/m3) and greenhouse gas emissions (0.72–0.83 kgCO2e/m3). The decentralized wastewater reuse systems, greywater reuse and rainwater harvesting systems, all used for non-potable purposes, had comparable or higher energy demands than the conventional process (0.246–0.970 kWh/m3 after adjustment), although estimated greenhouse gas emissions from these processes were lower than the conventional process (0–0.33 kgCO2e/m3 after adjustment). Considering the hidden environmental benefit (0.357 kWh/m3) from reduction of contaminant release, the energy demand of greywater reuse drops far below that of the conventional system, suggesting that decentralized water reuse is the key to an energy-efficient water management with minimal impact on climate change.

Bacteria capable of reduction of nitrous oxide (N 2 O) to N 2 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 N 2 O 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 ( K s s) for N 2 O distinguished clade I from clade II organisms. The apparent K s 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). Genome sequencing demonstrated that Dechloromonas denitrificans possessed a clade II nosZ gene, and a measured K s of 1.01 ± 0.18 μM for N 2 O was consistent with the values determined for the other clade II organisms tested. These observations provide a plausible mechanistic basis for why the relative activity of bacteria with clade I nos operons compared to that of bacteria with clade II nos operons may control N 2 O emissions and determine a soil's N 2 O sink capacity. IMPORTANCE Anthropogenic activities, in particular fertilizer application for agricultural production, increase N 2 O emissions to the atmosphere. N 2 O is a strong greenhouse gas with ozone destruction potential, and there is concern that nitrogen may become the major driver of climate change. Microbial N 2 O reductase (NosZ) catalyzes N 2 O reduction to environmentally benign dinitrogen gas and represents the major N 2 O sink process. The observation that bacterial groups with clade I nosZ versus those with clade II nosZ exhibit distinct affinities to N 2 O has implications for N 2 O flux models, and these distinct characteristics may provide opportunities to curb N 2 O emissions from relevant soil ecosystems.

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.

Methanotrophs are microorganisms that are able to utilize methane as the electron donor and carbon source. For long, methanotrophs have been widely studied for their application in environmental biotechnology, due mainly to the exclusive ownership of the unique enzymes that mediate oxidation of methane to methanol, namely the particulate methane monooxygenases (pMMO) and soluble methane monooxygenases (sMMO). Utilizing these methane monooxygenases, methanotrophs are capable of co-oxidizing a broad range of organic pollutants including chlorinated ethenes. Thus, methanotrophs have long been studied and utilized as biocatalysts for in situ bioremediation of soil and aquatic environments contaminated with these xenobiotic compounds. Due to the growing concerns in anthropogenically induced climate change and global warming, methanotrophs have increasingly gained attention also for greenhouse gas mitigation purposes. Active methane removal using methanotrophic biofilters of diverse configurations have proven to be effective for treatments of gases with relatively high methane concentrations, e.g., landfill gases and animal husbandry tank exhausts. Furthermore, improving the atmospheric methane sink capability of agricultural soils has been one of the foremost foci of climate-smart soil research. This chapter provides an extensive overview of scientific and engineering breakthroughs geared towards practical applications of methanotroph biotechnology in managing impending environmental problems.

Salinity and pH are important environmental parameters with direct and indirect impacts on the viability 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). 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-. The transcription analyses observed decreased nosZ/(nirS1+nirS2) ratios coinciding with N2O accumulation. The N2O consumption rates of resting D. aromatica cells subjected to the salt and alkaline stress conditions were significantly lower than the rates of N2O production from NO2- reduction at N2O / NO2- concentration of 0.1 mM, but not at a higher concentration (1.0 mM). These results indicate that alteration in N2O consumption kinetics was another cause of enhanced N2O production observed under the stress conditions. The findings in this study suggest that canonical denitrifiers may become a significant N2O source when faced with abrupt environmental changes.

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.

Aerobic methanotrophs have long been known to play a critical role in the global carbon cycle, being capable of converting methane to biomass and carbon dioxide. Interestingly, these microbes exhibit great sensitivity to copper and rare-earth elements, with the expression of key genes involved in the central pathway of methane oxidation controlled by the availability of these metals. That is, these microbes have a “copper switch” that controls the expression of alternative methane monooxygenases and a “rare-earth element switch” that controls the expression of alternative methanol dehydrogenases. Further, it has been recently shown that some methanotrophs can detoxify inorganic mercury and demethylate methylmercury; this finding is remarkable, as the canonical organomercurial lyase does not exist in these methanotrophs, indicating that a novel mechanism is involved in methylmercury demethylation. Here, we review recent findings on methanotrophic interactions with metals, with a particular focus on these metal switches and the mechanisms used by methanotrophs to bind and sequester metals.

In this study, oxidized dye wastewaters were tested for their potential to be used as a cheap coagulant for microalgae harvesting. Two dyes (methylene blue (MB) and methyl orange (MO)) were selected as model dyes, and the Fenton-like reaction under high temperature (90 °C, 1 min) employed as an oxidative treatment option. A maximum harvesting efficiency over 90% was obtained with both MB and MO at a dilution ratio of 5:1 (dye wastewater: cell culture), when the optimal oxidation condition was 20 mg/L of dye, 1 mM of FeCl3, and 0.5% of H2O2 concentration. This phenomenon could be explained by the possibility that amine groups are formed and exposed in oxidized dyes, which act as a kind of amine-based coagulant just like chitosan. This study clearly showed that dye wastewater, when properly oxidized, could serve as a potent coagulant for microalgae harvesting, potentially rendering the harvesting cost reduced to a substantial degree.

Shewanella loihica strain PV-4 harbors both a functional denitrification (NO 3 − →N 2 ) and a respiratory ammonification (NO 3 − →NH 4 + ) pathway. Batch and chemostat experiments revealed that NO 2 − affects pathway selection and the formation of reduced products. Strain PV-4 cells grown with NO 2 − as the sole electron acceptor produced exclusively NH 4 + . With NO 3 − as the electron acceptor, denitrification predominated and N 2 O accounted for ∼90% of reduced products in the presence of acetylene. Chemostat experiments demonstrated that the NO 2 − :NO 3 − ratio affected the distribution of reduced products, and respiratory ammonification dominated at high NO 2 − :NO 3 − ratios, whereas low NO 2 − :NO 3 − ratios favored denitrification. The NO 2 − :NO 3 − ratios affected nirK transcript abundance, a measure of denitrification activity, in the chemostat experiments, and cells grown at a NO 2 − :NO 3 − ratio of 3 had ∼37-fold fewer nirK transcripts per cell than cells grown with NO 3 − as the sole electron acceptor. In contrast, the transcription of nrfA , implicated in NO 2 − -to-NH 4 + reduction, remained statistically unchanged under continuous cultivation conditions at NO 2 − :NO 3 − ratios below 3. At NO 2 − :NO 3 − ratios above 3, both nirK and nrfA transcript numbers decreased and the chemostat culture washed out, presumably due to NO 2 − toxicity. These findings implicate NO 2 − as a relevant modulator of NO 3 − fate in S. loihica strain PV-4, and, by extension, suggest that NO 2 − is a relevant determinant for N retention (i.e., ammonification) versus N loss and greenhouse gas emission (i.e., denitrification).

N

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a “copper monopoly” that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N(2)O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N(2)O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N(2)O production was observed from NO(3)(−) reduction, whereas no significant N(2)O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N(2)O emissions, with the potential for increasing net greenhouse gas emissions.

Injecting and storing of carbon dioxide (CO2) in deep geologic formations is considered as one of the promising approaches for geologic carbon storage (GCS). Microbial wettability alteration of injected CO2 is expected to occur naturally by microorganisms indigenous to the geologic formation or microorganisms intentionally introduced to increase CO2 storage capacity in the target reservoirs. The question as to the extent of microbial CO2 wettability alteration under reservoir conditions still warrants further investigation. This study investigated the effect of a lipopeptide biosurfactant—surfactin, on interfacial tension (IFT) reduction and contact angle alteration in CO2/water/quartz systems under a laboratory setup simulating in situ reservoir conditions. The temporal shifts in the IFT and the contact angle among CO2, brine, and quartz were monitored for different CO2 phases (3 MPa, 30°C for gaseous CO2; 10 MPa, 28°C for liquid CO2; 10 MPa, 37°C for supercritical CO2) upon cultivation of Bacillus subtilis strain ATCC6633 with induced surfactin secretion activity. Due to the secreted surfactin, the IFT between CO2 and brine decreased: from 49.5 to 30 mN/m, by ~39% for gaseous CO2; from 28.5 to 13 mN/m, by 54% for liquid CO2; and from 32.5 to 18.5 mN/m, by ~43% for supercritical CO2, respectively. The contact angle of a CO2 droplet on a quartz disc in brine increased: from 20.5° to 23.2°, by 1.16 times for gaseous CO2; from 18.4° to 61.8°, by 3.36 times for liquid CO2; and from 35.5° to 47.7°, by 1.34 times for supercritical CO2, respectively. With the microbially altered CO2 wettability, improvement in sweep efficiency of injected and displaced CO2 was evaluated using 2-D pore network model simulations; again the increment in sweep efficiency was the greatest in liquid CO2 phase due to the largest reduction in capillary factor. This result provides novel insights as to the role of naturally occurring biosurfactants in CO2 storage and suggests that biostimulation of biosurfactant production may be a feasible technique for enhancement of CO2 storage capacity.

N 2 O-reducing organisms with nitrous oxide reductases (NosZ) are known as the only biological sink of N 2 O in the environment. Among the most abundant nosZ genes found in the environment are nosZ genes affiliated with the understudied Gemmatimonadetes phylum. In this study, a unique regulatory mechanism of N 2 O reduction in Gemmatimonas aurantiaca strain T-27, an isolate affiliated with the Gemmatimonadetes phylum, was examined. Strain T-27 was incubated with N 2 O and/or O 2 as the electron acceptor. Significant N 2 O reduction was observed only when O 2 was initially present. When batch cultures of strain T-27 were amended with O 2 and N 2 O, N 2 O reduction commenced after O 2 was depleted. In a long-term incubation with the addition of N 2 O upon depletion, the N 2 O reduction rate decreased over time and came to an eventual stop. Spiking of the culture with O 2 resulted in the resuscitation of N 2 O reduction activity, supporting the hypothesis that N 2 O reduction by strain T-27 required the transient presence of O 2 . The highest level of nosZ transcription (8.97 nosZ transcripts/ recA transcript) was observed immediately after O 2 depletion, and transcription decreased ∼25-fold within 85 h, supporting the observed phenotype. The observed difference between responses of strain T-27 cultures amended with and without N 2 O to O 2 starvation suggested that N 2 O helped sustain the viability of strain T-27 during temporary anoxia, although N 2 O reduction was not coupled to growth. The findings in this study suggest that obligate aerobic microorganisms with nosZ genes may utilize N 2 O as a temporary surrogate for O 2 to survive periodic anoxia. IMPORTANCE Emission of N 2 O, a potent greenhouse gas and ozone depletion agent, from the soil environment is largely determined by microbial sources and sinks. N 2 O reduction by organisms with N 2 O reductases (NosZ) is the only known biological sink of N 2 O at environmentally relevant concentrations (up to ∼1,000 parts per million by volume [ppmv]). Although a large fraction of nosZ genes recovered from soil is affiliated with nosZ found in the genomes of the obligate aerobic phylum Gemmatimonadetes , N 2 O reduction has not yet been confirmed in any of these organisms. This study demonstrates that N 2 O is reduced by an obligate aerobic bacterium, Gemmatimonas aurantiaca strain T-27, and suggests a novel regulation mechanism for N 2 O reduction in this organism, which may also be applicable to other obligate aerobic organisms possessing nosZ genes. We expect that these findings will significantly advance the understanding of N 2 O dynamics in environments with frequent transitions between oxic and anoxic conditions.

hglee@kari.re.krABSTRACT A blisk with rotor shroud is usually adopted in LRE turbine to maximize its performance. However it experiences the severe thermal load and resulting damage dur ing engine stating and stop. Shroud splitting is devised to relieve the thermal stress on the turbine rotor. Structural analysis confirmed the reduction of plastic strain at the blade hub and tip. However, split gap at the rotor shroud entails additional tip leakage and results performance degradation. In order to assess the effect of shroud split on the turbine performance, tests have been performed for various settings of shroud split. For the maximum number of shroud splitting, measured efficiency red uction ratio was 2.65% to the value of original shape rotor.초 록 액체로켓 엔진의 시동과 종료 시, 터빈에 가해지는 열충격을 완화시키기 위해 초음속 충동형 터빈로터의 일체형 슈라우드를 여러 조각으로 분할하는 방법을 고안하였다. 구조해석 수행결과 슈라우드 분할은 동익의 허브와 팁에 나타나는 소성변형량을 크게 줄일 수 있는 것으로 나타났다. 그러나 슈라우드 분할은 의도하지 않은 누설손실로 인해 성능손실이 불가피하며 이에 대한 정량적인 성능감소를 측정하기 위해 다양한 슈라우드 분할 형상에 대해 시험을 수행하였다. 연구대상 터빈의 경우 슈라우드 분할 수를 최대로 할 경우 설계점 효율은 2.65% 비율로 감소하는 것으로 나타났다.Key Words: Supersonic Impulse Turbine(초음속 충동형 터빈), Split(스플릿), Shroud(슈라우드), Blisk(블리스크), Leakage(누설)1. 서 론 한국항공우주연구원(이하 항우연)에서는 한국

Lactate but not acetate oxidation was reported to support electron acceptor reduction by Shewanella spp. under anoxic conditions. We demonstrate that the denitrifiers Shewanella loihica strain PV-4 and Shewanella denitrificans OS217 utilize acetate as an electron donor for denitrification but not for fumarate or ferric iron reduction.

Summary A non-motile strain of Methylocystis, strain SB2, isolated from a spring bog in southeast Michigan, had a curved rod morphology with a typical type II intracytoplasmic membrane system. This organism expressed the membrane-bound or particulate methane monooxygenase (pMMO) as well as a chalkophore with high affinity for copper and did not express the cytoplasmic or soluble methane monooxygenase (sMMO). Strain SB2 was found to grow within the pH range of 6–9, with optimal growth at 6.8. Growth was observed at temperatures ranging between 10°C and 30°C, with no growth at 37°C. The DNA G+C content was 62.9 mol%. Predominant fatty acids were 18:1ω7c (72.7%) and 18:1ω9c (24%) when grown on methane. Phylogenetic comparisons based on both pmoA and 16S rRNA sequences indicated that this organism belonged to the Methylocystis genus, and was closely related to Methylocystis rosea SV97T and Methylocystis echinoides IMET10491T (98% 16S rRNA gene sequence similarity to both strains). DNA : DNA hybridizations indicated that strain SB2 had 70% similarity with M. rosea SV97T. Unlike M. rosea SV97T, strain SB2 was able to utilize not only methane for growth, but also ethanol and acetate. Furthermore, the predominant fatty acids in strain SB2 were different from those found in M. rosea SV97T, i.e. 54.2% and 39.7% of fatty acids are 18:1ω8 and 18:1ω7 in M. rosea SV97T, while 18:1ω8 is completely absent in strain SB2.

Summary The particulate methane monooxygenase (pMMO) in Methylocystis strain SB2 was found to be constitu- tively expressed in the absence of methane when the strain was grown on either acetate or ethanol. Real- timequantitativepolymerasechainreaction(PCR)and reversetranscription-PCRshowedthattheexpression of pmoA decreased by one to two orders of magnitude when grown on acetate as compared with growth of strain SB2 on methane. The capability of strain SB2 to degrade a mixture of chlorinated ethenes in the absence of methane was examined to verify the pres- ence and activity of pMMO under acetate-growth con- ditions as well determine the effectiveness of such conditions for bioremediation. It was found that when strain SB2 was grown on acetate and exposed to 40 mM each of trichloroethylene (TCE), trans- dichloroethylene (t-DCE) and vinyl chloride (VC), approximately 30% of VC and t-DCE was degraded but no appreciable TCE removal was measured after 216 h of incubation. The ability to degrade VC andt-DCE was lostwhenacetylenewasadded,confirmingthatpMMO was responsible for the degradation of these chlori- nated ethenes by Methylocystis strain SB2 when the strain was grown on acetate.

Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron-CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs (Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe-CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis.

Methane is a potent greenhouse gas with a global warming potential ~23 times that of carbon dioxide. Here, we describe the modeling of a biotrickling filtration system composed of methane-consuming bacteria, i.e., methanotrophs, to assess the utility of these systems in removing methane from the atmosphere. Model results indicate that assuming the global average atmospheric concentration of methane, 1.7 ppmv, methane removal is ineffective using these methanotrophic biofilters as the methane concentration is too low to enable cell survival. If the concentration is increased to 500-6,000 ppmv, however, similar to that found above landfills and in concentrated animal feeding operations (factory farms), 4.98-35.7 tons of methane can be removed per biofilter per year assuming biotrickling filters of typical size (3.66 m in diameter and 11.5 m in height). Using reported ranges of capital, operational, and maintenance costs, the cost of the equivalent ton of CO(2) removal using these systems is $90-$910 ($2,070-$20,900 per ton of methane), depending on the influent concentration of methane and if heating is required. The use of methanotrophic biofilters for controlling methane emissions is technically feasible and, provided that either the costs of biofilter construction and operation are reduced or the value of CO(2) credits is increased, can also be economically attractive.

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 degrees C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 degrees C to examine the effect of mixtures of trichloroethylene, trans-dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Delta model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 degrees C. Finally, it was also discovered that at 20 degrees C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 degrees C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.