Comprehensive mutation profiling from wastewater in southern Germany extends evidence of circulating SARS-CoV-2 diversity beyond mutations characteristic for Omicron.

Tracking SARS-CoV-2 variants in wastewater is primarily performed by detecting characteristic mutations of the variants. Unlike the Delta variant, the emergence of the Omicron variant and its sublineages as variants of concern has posed a challenge in using characteristic mutations for wastewater surveillance. In this study, we monitored the temporal and spatial variation of SARS-CoV-2 variants by including all the detected mutations and compared whether limiting the analyses to characteristic mutations for variants like Omicron impact the outcomes. We collected 24-hour composite samples from 15 wastewater treatment plants (WWTP) in Hesse and sequenced 164 wastewater samples with a targeted sequencing approach from September 2021 to March 2022. Our results show that comparing the number of all the mutations against the number of the characteristic mutations reveals a different outcome. A different temporal variation was observed for the ORF1a and S gene. As Omicron became dominant, we observed an increase in the overall number of mutations. Based on the characteristic mutations of the SARS-CoV-2 variants, a decreasing trend for the number of ORF1a and S gene mutations was noticed, though the number of known characteristic mutations in both genes is higher in Omicron than Delta.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Wastewater surveillance allows early detection of SARS-CoV-2 omicron in North Rhine-Westphalia, Germany.

Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany). Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021. In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.

Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities.

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Genome Sequencing of Wastewater Confirms the Arrival of the SARS-CoV-2 Omicron Variant at Frankfurt Airport but Limited Spread in the City of Frankfurt, Germany, in November 2021.

Wastewater-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance of Frankfurt Airport by genome sequencing was used to detect SARS-CoV-2 variants entering the region. In November 2021, we found all characteristic mutations of Omicron in wastewater originating from Frankfurt Airport before the first confirmed clinical report from an arriving passenger on 26 November 2021.

Elevated levels of antibiotic resistance in groundwater during treated wastewater irrigation associated with infiltration and accumulation of antibiotic residues.

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Monitoring SARS-CoV-2 in sewage: Toward sentinels with analytical accuracy.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

Metagenomic Insights Into Functional and Taxonomic Compositions of an Activated Sludge Microbial Community Treating Leachate of a Completed Landfill: A Pathway-Based Analysis.

Upcycling wastes into valuable products by mixed microbial communities has recently received considerable attention. Sustainable production of high-value substances from one-carbon (C1) compounds, e.g., methanol supplemented as an external electron donor in bioreactors for wastewater treatment, is a promising application of upcycling. This study undertook a gene-centric approach to screen valuable production potentials from mixed culture biomass, removing organic carbon and nitrogen from landfill leachate. To this end, the microbial community of the activated sludge from a landfill leachate treatment plant and its metabolic potential for the production of seven valuable products were investigated. The DNA extracted from the activated sludge was subjected to shotgun metagenome sequencing to analyze the microbial taxonomy and functions associated with producing the seven products. The functional analysis confirmed that the activated sludge could produce six of the valuable products, ectoine, polyhydroxybutyrate (PHB), zeaxanthin, astaxanthin, acetoin, and 2,3-butanediol. Quantification of the detected functional gene hit numbers for these valuable products as a primary trial identified a potential rate-limiting metabolic pathway, e.g., conversion of L-2,4-diaminobutyrate into N-γ-acetyl-L2,4,-diaminobutyrate during the ectoine biosynthesis. Overall, this study demonstrated that primary screening by the proposed gene-centric approach can be used to evaluate the potential for the production of valuable products using mixed culture or single microbe in engineered systems. The proposed approach can be expanded to sites where water purification is highly required, but resource recovery, or upcycling has not been implemented.

Metatranscriptomic Analysis Reveals SARS-CoV-2 Mutations in Wastewater of the Frankfurt Metropolitan Area in Southern Germany.

We report a sequencing analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples collected in the Frankfurt metropolitan area of Germany. The majority of the detected mutations have been identified only in clinical genomes outside Frankfurt, indicating that the sequencing of SARS-CoV-2 RNA in wastewater can provide insights into emerging variants in a city.

Long-term monitoring of SARS-CoV-2 RNA in wastewater of the Frankfurt metropolitan area in Southern Germany.

Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 RNA concentration in sewage, we continuously collected 24 h composite samples twice a week from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 × 1013 copies/day, which represented similar levels compared to April with approx. 2 × 1014 copies/day. This corresponds to a continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 RNA load to the WWTP Niederrad ranged from approx. 4 × 1011 to 1 × 1015 copies/day, the load to the WWTP Sindlingen from approx. 1 × 1011 to 2 × 1014 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as an early warning system for SARS-CoV-2 infections and a monitoring system to identify global hotspots of COVID-19.

Antibiotic resistance gene load and irrigation intensity determine the impact of wastewater irrigation on antimicrobial resistance in the soil microbiome.

Treated wastewater (TWW) irrigation is a useful counter-measure against the depletion of freshwater (FW) resources. However, TWW contains several contaminants of emerging concern, such as antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs). Thus, TWW irrigation might promote the spread of antimicrobial resistance in soil environments. In the present work, we hypothesized that the ARG load and irrigation intensity define the effect of TWW irrigation on ARG spread dynamics in soil. This hypothesis was tested using a multiphase approach: a) comparing soil from a full-scale, commercially operated, TWW irrigated field with non-irrigated soil, b) long-term sampling of the TWW irrigated field over one year with different irrigation intensities and intercepted by irrigation breaks and c) laboratory-scale soil microcosms irrigated with TWW compared to FW. Six ARGs, the integrase gene intI1 and the 16S rRNA were quantified using qPCR. In addition, effects of TWW irrigation on bacterial community composition of microcosm-samples were analysed with 16S rRNA amplicon sequencing. The genes sul1, qnrS, blaOXA-58, tet(M) and intI1 were significantly more abundant in the TWW irrigated field soil, whereas blaCTX--M-32 and blaTEM, the least abundant genes in the TWW irrigation, showed higher abundance in the non-irrigated soil. The relative abundance of sul1, qnrS, blaOXA-58, tet(M) and intI1 correlated with TWW irrigation intensity and decreased during irrigation breaks. Despite the decrease, the levels of these genes remained consistently higher than the non-irrigated soil indicating persistence upon their introduction into the soil. Microcosm experiments verified observations from the field study: TWW irrigation promoted the spread of ARGs and intI1 into soil at far elevated levels compared to FW irrigation. However, the impact of TWW irrigation on 16S rRNA absolute abundance and the soil microbial community composition was negligible. In conclusion, the impact of TWW irrigation depends mainly on the introduced ARG load and the irrigation intensity.

Targeted metagenomics reveals extensive diversity of the denitrifying community in partial nitritation anammox and activated sludge systems.

The substantial presence of denitrifiers has already been reported in partial nitritation anammox (PNA) systems using the 16S ribosomal RNA (rRNA) gene, but little is known about the phylogenetic diversity based on denitrification pathway functional genes. Therefore, we performed a metagenomic analysis to determine the distribution of denitrification genes and the associated phylogeny in PNA systems and whether a niche separation between PNA and conventional activated sludge (AS) systems exists. The results revealed a distinct abundance pattern of denitrification pathway genes and their association to the microbial species between PNA and AS systems. In contrast, the taxonomic analysis, based on the 16S rRNA gene, did not detect notable variability in denitrifying community composition across samples. In general, narG and nosZa2 genes were dominant in all samples. While the potential for different stages of denitrification was redundant, variation in species composition and lack of the complete denitrification gene pool in each species appears to confer niche separation between PNA and AS systems. This study suggests that targeted metagenomics can help to determine the denitrifying microbial composition at a fine-scale resolution while overcoming current biases in quantitative polymerase chain reaction approaches due to a lack of appropriate primers.

Treated wastewater irrigation promotes the spread of antibiotic resistance into subsoil pore-water.

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory microcosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the microcosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled microcosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab microcosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.

Time to act-assessing variations in qPCR analyses in biological nitrogen removal with examples from partial nitritation/anammox systems.

Quantitative PCR (qPCR) is broadly used as the gold standard to quantify microbial community fractions in environmental microbiology and biotechnology. Benchmarking efforts to ensure the comparability of qPCR data for environmental bioprocesses are still scarce. Also, for partial nitritation/anammox (PN/A) systems systematic investigations are still missing, rendering meta-analysis of reported trends and generic insights potentially precarious. We report a baseline investigation of the variability of qPCR-based analyses for microbial communities applied to PN/A systems. Round-robin testing was performed for three PN/A biomass samples in six laboratories, using the respective in-house DNA extraction and qPCR protocols. The concentration of extracted DNA was significantly different between labs, ranged between 2.7 and 328 ng mg-1 wet biomass. The variability among the qPCR abundance data of different labs was very high (1-7 log fold) but differed for different target microbial guilds. DNA extraction caused maximum variation (3-7 log fold), followed by the primers (1-3 log fold). These insights will guide environmental scientists and engineers as well as treatment plant operators in the interpretation of qPCR data.

High-throughput profiling of antibiotic resistance genes in wastewater: comparison between a pond system in Namibia and an activated sludge treatment in Germany.

There are increasing concerns about wastewater treatment plants (WWTPs) acting as hotspots for antibiotic resistance genes (ARG). However, their role largely depends upon the treatment methods and antibiotics in the wastewater. To better understand these influences, we compared the occurrence and fate of ARG between a pond system in a developing country (Namibia) and an advanced WWTP (activated sludge system) in a developed country (Germany). A targeted metagenomic approach was used to investigate the wide-spectrum profiles of ARGs and their co-occurrence patterns at both locations. In total, 93 ARG subtypes were found in the German influent wastewater, 277 in the Namibian influent wastewater. The abundant ARG types found in Namibia and Germany differed, especially for multidrug resistance genes. The differences in occurrence and reduction can help to understand the performance of simple WWTP such as pond systems common in Namibia, where direct contact with wastewater is a potential risk for contamination.

Membrane aerated biofilm reactors for mainstream partial nitritation/anammox: Experiences using real municipal wastewater.

This study investigated the potential of Membrane-Aerated Biofilm Reactors (MABRs) for mainstream nitrogen removal via partial nitration/anaerobic ammonium oxidation (anammox). Four laboratory-scale MABRs were operated with real municipal wastewater characterized by low concentrations of nitrogen (varying between 31 and 120 mg-NH4-N L-1) and the presence of biodegradable organic carbon (soluble COD (sCOD) between 7 and 230 mg-O2 L-1). Two reactors were operated with different aeration strategies (intermittent vs. continuous), the other two with differences in biomass retention (recirculation or removal of detached biomass). Keeping a constant HRT caused instabilities due to difficulties with setting the optimal oxygen flux for the respective surface loadings (1.6-6 g-NH4-N m-2 d-1). Operating the MABRs with a constant surface loading (2 g-NH4-N m-2 d-1) resulted in higher and more stable total nitrogen (TN) removal independent of the aeration strategy. The intermittently aerated MABR improved from an average TN removal of 23%-69%, the continuously aerated MABR from 20% to 50% TN removal. Independent of the feeding strategy, the continuously aerated reactor removed slightly more ammonium (80-95%) compared to the intermittently aerated reactor (74-93%). Limiting the oxygen supply by intermittent aeration proofed successful to favor partial nitritation and anammox. Continuous aeration did not achieve stable suppression of nitrite oxidizing bacteria (NOB). Of the removed ammonium, approx. 26% were left in the effluent as nitrate (only 10% with intermittent aeration). Recirculation of the detached biomass resulted in reattachment onto the biofilm or membrane surface. This recirculation led to significantly higher biomass retention times and thus to better performance. Removing detached biofilm from the reactor caused a slightly lower TN removal of 33% compared to 45% with reattachment, while average ammonium removal was 58% compared to 63%, respectively. Scouring events had a significant impact on the overall operation, resulting in short term losses of TN removal capacities of 50-100%. The microbial community composition was different depending on the aeration strategy and biomass retention. The continuously aerated reactor contained significantly more AOB than the intermittently aerated MABR. The reactor with biomass retention contained less ammonium oxidizing bacteria (AOB), compared to the reactor with low biomass retention. In all MABRs, anammox bacteria established in the biofilm after an initial drop in abundance.

WITHDRAWN: Membrane Aerated Biofilm Reactors for mainstream partial nitritation / anammox: Experiences using real municipal wastewater.

The Publisher regrets that this article is an accidental duplication of a published article,  http://dx.doi.org/10.1016/j.wroa.2020.100066. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Identification of a Metagenome-Assembled Genome of an Uncultured Methyloceanibacter sp. Strain Acquired from an Activated Sludge System Used for Landfill Leachate Treatment.

Using metagenome sequencing, a nearly complete genome sequence was retrieved for the uncultured Methyloceanibacter sp. strain A49, recovered from an activated sludge system used for landfill leachate treatment at a closed landfill site. The total size and encoded sequences are 3,407,434 bp and 3,280 genes, respectively.

Lost in translation: the quest for Nitrosomonas cluster 7-specific amoA primers and TaqMan probes.

The choice of primer and TaqMan probes to quantify ammonia-oxidizing bacteria (AOB) in environmental samples is of crucial importance. The re-evaluation of primer pairs based on current genomic sequences used for quantification of the amoA gene revealed (1) significant misrepresentations of the AOB population in environmental samples, (2) and a lack of perfect match primer pairs for Nitrosomonas europaea and Nitrosomonas eutropha. We designed two new amoA cluster 7-specific primer pairs and TaqMan probes to quantify N. europaea (nerF/nerR/nerTaq) and N. eutropha (netF/netR/netTaq). Specificity and quantification biases of the newly designed primer sets were compared with the most popular primer pair (amoA1f/amoA2r) using DNA from various AOB cultures as individual templates as well as DNA mixtures and environmental samples. Based on the qPCR results, we found that the newly designed primer pairs and the most popular one performed similarly for individual templates but differed for the DNA mixtures and environmental samples. Using the popular primer pair introduced a high underestimation of AOB in environmental samples, especially for N. eutropha. Thus, there is a strong need for more specific primers and probes to understand the occurrence and competition between N. europaea and N. eutropha in different environments.

Exploration and enrichment of methane-oxidizing bacteria derived from a rice paddy field emitting highly concentrated methane.

Methane-oxidizing bacteria (MOB) possess the metabolic potential to assimilate the highly potent greenhouse gas, CH4, and can also synthesize valuable products. Depending on their distinct and fastidious metabolic pathways, MOB are mainly divided into Type I and Type II; the latter are known as producers of polyhydroxyalkanoate (PHA). Despite the metabolic potential of MOB to synthesize PHA, the ecophysiology of MOB, especially under high CH4 flux conditions, is yet to be understood. Therefore, in this study, a rice paddy soil receiving a high CH4 flux from underground was used as an inoculum to enrich MOB using fed-batch operation, then the enriched Type II MOB were characterized. The transitions in the microbial community composition and CH4 oxidation rates were monitored by 16S rRNA gene amplicon sequencing and degree of CH4 consumption. With increasing incubation time, the initially dominant Methylomonas sp., affiliated with Type I MOB, was gradually replaced with Methylocystis sp., Type II MOB, resulting in a maximum CH4 oxidation rate of 1.40 g-CH4/g-biomass/day. The quantification of functional genes encoding methane monooxygenase, pmoA and PHA synthase, phaC, by quantitative PCR revealed concomitant increases in accordance with the Type II MOB enrichment. These increases in the functional genes underscore the significance of Type II MOB to mitigate greenhouse gas emission and produce PHA.