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.

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.

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.

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.

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.

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.