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Wastewater treatment plants (WWTPs) are among the major anthropogenic sources of N 2 O, 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 proofof-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 N 2 O-containing gas streams of varied flow rates (200−2000 mL•min −1 ) and O 2 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 N 2 O at an elimination capacity of 0.402 ± 0.009 g N 2 O•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 N 2 O•m −3 •h −1 . These data were used to validate the mathematical model developed to estimate the performance of the N 2 O 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 N 2 O removal and the performance enhancement achieved with the serial configuration.

SummaryAs 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.

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.

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, and the dilution rates were increased with an increment of 0.05 h 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; 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.

14 Despite the recent interest in nosZ-possessing organisms as the sole N 2 O sink in soil and aquatic 15 environments, quantification of nosZ has relied on undependable qPCR techniques prone to unspecific 16 amplification and compromised accuracy. Here, we have combined culture-based methods with 17 computational methods to develop TaqMan-based qPCR for quantification of microorganisms actively 18 involved in N 2 O consumption in activated sludge reactors. A sewage sample was enriched in a batch 19 reactor fed continuous stream of N 2 containing 20-10,000 ppmv N 2 O, where 14 genera of potential N 2 O-20 reducers were identified. All available amino acid sequences of NosZ affiliated to these taxa were grouped 21 into five subgroups (two clade I and three clade II groups), and primer/probe sets exclusively and 22 comprehensively targeting each subgroups were designed and validated with in silico PCR. The nosZ 23 profiles of four activated sludge samples determined with the qPCR assays were compared with those 24 analyzed from shotgun metagenome. The results of the group-specific qPCR assays were generally in 25 agreement with the results of the metagenomic analyses, with the two subgroups (Flavobacterium-like and 26 Dechloromonas-like) of clade II nosZ dominating the nosZ-possessing population. These quantitative tools 27 will be immensely useful for identification of the key N 2 O-reducing populations in environments with 28 rapid nitrogen turnover. 29 30

Wastewater treatment plants (WWTPs) are a major source of N 2 O, a potent greenhouse gas with 300 times higher global warming potential than CO 2 . Several approaches have been proposed for mitigation of N 2 O 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 N 2 O 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 N 2 O, exhibiting elimination capacities as high as 5.25 g N 2 O m −3 •h −1 . Additionally, the bench-scale experiments performed abreast corroborated the resilience of the system to short-term N 2 O starvations. Our results corroborate the feasibility of biotrickling filtration for mitigating N 2 O emitted from WWTPs and demonstrate its robustness toward suboptimal field operating conditions and N 2 O starvation, as also supported by analyses of the microbial compositions and nosZ gene profiles.