Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes.

Real-time quantitative PCR (qPCR) has been widely used to quantify gene copy numbers in microbial ecology. Despite its simplicity and straightforwardness, establishing qPCR assays is often impeded by the tedious process of producing qPCR standards by cloning the target DNA into plasmids. Here, we designed double-stranded synthetic DNA fragments from consensus sequences as qPCR standards by aligning microbial gene sequences (10-20 sequences per gene). Efficiency of standards from synthetic DNA was compared with plasmid standards by qPCR assays for different phylogenetic marker and functional genes involved in carbon (C) and nitrogen (N) cycling, tested with DNA extracted from a broad range of soils. Results showed that qPCR standard curves using synthetic DNA performed equally well to those from plasmids for all the genes tested. Furthermore, gene copy numbers from DNA extracted from soils obtained by using synthetic standards or plasmid standards were comparable. Our approach therefore demonstrates that a synthetic DNA fragment as qPCR standard provides comparable sensitivity and reliability to a traditional plasmid standard, while being more time- and cost-efficient.

Hydrodynamic regimes modulate nitrogen fixation and the mode of diazotrophy in Lake Tanganyika.

The factors that govern the geographical distribution of nitrogen fixation are fundamental to providing accurate nitrogen budgets in aquatic environments. Model-based insights have demonstrated that regional hydrodynamics strongly impact nitrogen fixation. However, the mechanisms establishing this physical-biological coupling have yet to be constrained in field surveys. Here, we examine the distribution of nitrogen fixation in Lake Tanganyika - a model system with well-defined hydrodynamic regimes. We report that nitrogen fixation is five times higher under stratified than under upwelling conditions. Under stratified conditions, the limited resupply of inorganic nitrogen to surface waters, combined with greater light penetration, promotes the activity of bloom-forming photoautotrophic diazotrophs. In contrast, upwelling conditions support predominantly heterotrophic diazotrophs, which are uniquely suited to chemotactic foraging in a more dynamic nutrient landscape. We suggest that these hydrodynamic regimes (stratification versus mixing) play an important role in governing both the rates and the mode of nitrogen fixation.

Differential effects of wastewater treatment plant effluents on the antibiotic resistomes of diverse river habitats.

Wastewater treatment plants (WWTPs) are key sources of antimicrobial resistance genes (ARGs) that could influence the resistomes of microbial communities in various habitats of the receiving river ecosystem. However, it is currently unknown which habitats are most impacted and whether ARGs, like certain chemical contaminants, could be accumulated or enriched in the river ecosystem. We conducted a systematic metagenomic survey on the antibiotic resistomes of WWTP effluent, four riverine habitats (water, suspended particles, sediment, epilithic biofilm), and freshwater amphipod gut microbiomes. The impact of WWTP effluent on the downstream habitats was assessed in nine Swiss rivers. While there were significant differences in resistomes across habitats, the wastewater resistome was more similar to the resistome of receiving river water than to the resistomes of other habitats, and river water was the habitat most strongly impacted by the WWTPs effluent. The sulfonamide, beta-lactam, and aminoglycoside resistance genes were among the most abundant ARGs in the WWTP effluents, and especially aadA, sul1, and class A beta-lactamase genes showed significantly increased abundance in the river water of downstream compared to upstream locations (p < 0.05). However, this was not the case for the sediment, biofilm, and amphipod gut habitats. Accordingly, evidence for accumulation or enrichment of ARGs through the riverine food web was not identified. Our study suggests that monitoring riverine antimicrobial resistance determinants could be conducted using "co-occurrence" of aadA, sul1, and class A beta-lactamase genes as an indicator of wastewater-related pollution and should focus on the water as the most affected habitat.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes.

Wastewater-based surveillance (WBS) for disease monitoring is highly promising but requires consistent methodologies that incorporate predetermined objectives, targets, and metrics. Herein, we describe a comprehensive metagenomics-based approach for global surveillance of antibiotic resistance in sewage that enables assessment of 1) which antibiotic resistance genes (ARGs) are shared across regions/communities; 2) which ARGs are discriminatory; and 3) factors associated with overall trends in ARGs, such as antibiotic concentrations. Across an internationally sourced transect of sewage samples collected using a centralized, standardized protocol, ARG relative abundances (16S rRNA gene-normalized) were highest in Hong Kong and India and lowest in Sweden and Switzerland, reflecting national policy, measured antibiotic concentrations, and metal resistance genes. Asian versus European/US resistomes were distinct, with macrolide-lincosamide-streptogramin, phenicol, quinolone, and tetracycline versus multidrug resistance ARGs being discriminatory, respectively. Regional trends in measured antibiotic concentrations differed from trends expected from public sales data. This could reflect unaccounted uses, captured only by the WBS approach. If properly benchmarked, antibiotic WBS might complement public sales and consumption statistics in the future. The WBS approach defined herein demonstrates multisite comparability and sensitivity to local/regional factors.

Microbial Nitrogen Transformation Potential in Sediments of Two Contrasting Lakes Is Spatially Structured but Seasonally Stable.

The nitrogen (N) cycle is of global importance, as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbially mediated processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates from pore water concentration gradients in sediments. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in sediments were associated with distinct microbial community compositions and significant differences in abundances of various N transformation genes. For both characteristics, we observed a more pronounced spatial than seasonal variability within each lake. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundances and higher nirS:nirK (nitrite reductase) ratios, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for nitrification. Specifically, it harbored a high abundance of Nitrospira, including some with the potential for comammox. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding how the lacustrine sedimentary N cycle responds to variations in trophic conditions. IMPORTANCE Anthropogenic nitrogen (N) inputs can lead to eutrophication in surface waters, especially in N-limited coastal ecosystems. Lakes effectively remove reactive N by transforming it to N2 through microbial denitrification or anammox. The rates and distributions of these microbial processes are affected by factors such as the amount and quality of settling organic material and nitrate concentrations. However, the microbial communities mediating these N transformation processes in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in sediments of two lakes with different trophic states. We show that lakes with different trophic states select for distinct communities of N-cycling microorganisms with contrasting functional potentials for N transformation.

Microeukaryotic gut parasites in wastewater treatment plants: diversity, activity, and removal.

BACKGROUND: During wastewater treatment, the wastewater microbiome facilitates the degradation of organic matter, reduction of nutrients, and removal of gut parasites. While the latter function is essential to minimize public health risks, the range of parasites involved and how they are removed is still poorly understood. RESULTS: Using shotgun metagenomic (DNA) and metatranscriptomic (RNA) sequencing data from ten wastewater treatment plants in Switzerland, we were able to assess the entire wastewater microbiome, including the often neglected microeukaryotes (protists). In the latter group, we found a surprising richness and relative abundance of active parasites, particularly in the inflow. Using network analysis, we tracked these taxa across the various treatment compartments and linked their removal to trophic interactions. CONCLUSIONS: Our results indicate that the combination of DNA and RNA data is essential for assessing the full spectrum of taxa present in wastewater. In particular, we shed light on an important but poorly understood function of wastewater treatment - parasite removal. Video Abstract.

Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge.

BACKGROUND: There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. RESULTS: The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75-90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40-73 to 31-68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35-13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4-2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. CONCLUSIONS: Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention. Video abstract.

Wastewater bypass is a major temporary point-source of antibiotic resistance genes and multi-resistance risk factors in a Swiss river.

Untreated combined sewage (bypass) is often discharged by wastewater treatment plants to receiving rivers during stormwater events, where it may contribute to increased levels of antibiotic resistance genes (ARGs) and multi-resistance risk factors (multi-resistant bacteria and multi-resistance genomic determinants (MGDs)) in the receiving water. Other contamination sources, such as soil runoff and resuspended river sediment could also play a role during stormwater events. Here we report on stormwater event-based sampling campaigns to determine temporal dynamics of ARGs and multi-resistance risk factors in bypass, treated effluent, and the receiving river, as well as complimentary data on catchment soils and surface sediments. Both indicator ARGs (qPCR) and resistome (ARG profiles revealed by metagenomics) indicated bypass as the main contributor to the increased levels of ARGs in the river during stormwater events. Furthermore, we showed for the first time that the risk of exposure to bypass-borne multi-resistance risk factors increase under stormwater events and that many of these MGDs were plasmid associated and thus potentially mobile. In addition, elevated resistance risk factors persisted for some time (up to 22 h) in the receiving water after stormwater events, likely due to inputs from distributed overflows in the catchment. This indicates temporal dynamics should be considered when interpreting the risks of exposure to resistance from event-based contamination. We propose that reducing bypass from wastewater treatment plants may be an important intervention option for reducing dissemination of antibiotic resistance.

Pathogenic and Indigenous Denitrifying Bacteria are Transcriptionally Active and Key Multi-Antibiotic-Resistant Players in Wastewater Treatment Plants.

The global rise and spread of antibiotic resistance greatly challenge the treatment of bacterial infections. Wastewater treatment plants (WWTPs) harbor and discharge antibiotic resistance genes (ARGs) as environmental contaminants. However, the knowledge gap on the host identity, activity, and functionality of ARGs limits transmission and health risk assessment of the WWTP resistome. Hereby, a genome-centric quantitative metatranscriptomic approach was exploited to realize high-resolution qualitative and quantitative analyses of bacterial hosts of ARGs (i.e., multiresistance, pathogenicity, activity, and niches) in the 12 urban WWTPs. We found that ∼45% of 248 recovered genomes expressed ARGs against multiple classes of antibiotics, among which bacitracin and aminoglycoside resistance genes in Proteobacteria were the most prevalent scenario. Both potential pathogens and indigenous denitrifying bacteria were transcriptionally active hosts of ARGs. The almost unchanged relative expression levels of ARGs in the most resistant populations (66.9%) and the surviving ARG hosts including globally emerging pathogens (e.g., Aliarcobacter cryaerophilus) in treated WWTP effluent prioritize future examination on the health risks related to resistance propagation and human exposure in the receiving environment.

Distinct growth stages controlled by the interplay of deterministic and stochastic processes in functional anammox biofilms.

Mainstream anaerobic ammonium oxidation (anammox) represents one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from wastewaters. However, little is known about the exact processes and drivers of microbial community assembly within the complex microbial biofilms that support anammox in engineered ecosystems. Here, we followed anammox biofilm development on fresh carriers in an established 8m3 mainstream anammox reactor that is exposed to seasonal temperature changes (~25-12°C) and varying NH4+ concentrations (5-25 mg/L). We use fluorescence in situ hybridization and 16S rRNA gene sequencing to show that three distinct stages of biofilm development emerge naturally from microbial community composition and biofilm structure. Neutral modelling and network analysis are employed to elucidate the relative importance of stochastic versus deterministic processes and synergistic and antagonistic interactions in the biofilms during their development. We find that the different phases are characterized by a dynamic succession and an interplay of both stochastic and deterministic processes. The observed growth stages (Colonization, Succession and Maturation) appear to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure that supports the desired metabolic and functional capacities observed for biofilm carriers already present in the system (~100gNH4-N m3 d-1). We discuss the relevance of this improved understanding of anammox-community ecology and biofilm development in the context of its practical application in the start-up, configuration, and optimization of anammox biofilm reactors.

Linking seasonal N2O emissions and nitrification failures to microbial dynamics in a SBR wastewater treatment plant.

Nitrous oxide (N2O) is a strong greenhouse gas and causal for stratospheric ozone depletion. During biological nitrogen removal in wastewater treatment plants (WWTP), high N2O fluxes to the atmosphere can occur, typically exhibiting a seasonal emission pattern. Attempts to explain the peak emission phases in winter and spring using physico-chemical process data from WWTP were so far unsuccessful and new approaches are required. The complex and diverse microbial community of activated sludge used in biological treatment systems also exhibit substantial seasonal patterns. However, a potentially causal link between the seasonal patterns of microbial diversity and N2O emissions has not yet been investigated. Here we show that in a full-scale WWTP nitrification failure and N2O peak emissions, bad settleability of the activated sludge and a turbid effluent strongly correlate with a significant reduction in the microbial community diversity and shifts in community composition. During episodes of impaired performance, we observed a significant reduction in abundance for filamentous and nitrite oxidizing bacteria in all affected reactors. In some reactors that did not exhibit nitrification and settling failures, we observed a stable microbial community and no drastic loss of species. Standard engineering approaches to stabilize nitrification, such as increasing the aerobic sludge age and oxygen availability failed to improve the plant performance on this particular WWTP and replacing the activated sludge was the only measure applied by the operators to recover treatment performance in affected reactors. Our results demonstrate that disturbances of the sludge microbiome affect key structural and functional microbial groups, which lead to seasonal N2O emission patterns. To reduce N2O emissions from WWTP, it is therefore crucial to understand the drivers that lead to the microbial population dynamics in the activated sludge.

Nitrogen isotope effects can be used to diagnose N transformations in wastewater anammox systems.

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, - 16 to - 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19-32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems.

Unraveling the riverine antibiotic resistome: The downstream fate of anthropogenic inputs.

River networks are one of the main routes by which the public could be exposed to environmental sources of antibiotic resistance, that may be introduced e.g. via treated wastewater. In this study, we applied a comprehensive integrated analysis encompassing mass-flow concepts, chemistry, bacterial plate counts, resistance gene quantification and shotgun metagenomics to track the fate of the resistome (collective antibiotic resistance genes (ARGs) in a microbial community) of treated wastewater in two Swiss rivers at the kilometer scale. The levels of certain ARGs and the class 1 integron integrase gene (intI1) commonly associated with anthropogenic sources of ARGs decreased quickly over short distances (2-2.5 km) downstream of wastewater discharge points. Mass-flow analysis based on conservative tracers suggested this decrease was attributable mainly to dilution but ARG loadings frequently also decreased (e.g., 55.0-98.5 % for ermB and tetW) over the longest studied distances (6.8 and 13.7 km downstream). Metagenomic analysis confirmed that ARG of wastewater-origin did not persist in rivers after 5 ~ 6.8 km downstream distance. sul1 and intI1 levels and loadings were more variable and even increased sharply at 5 ~ 6.8 km downstream distance on one occasion. While input from agriculture and in-situ positive selection pressure for organisms carrying ARGs cannot be excluded, in-system growth of biomass is a more probable explanation. The potential for direct human exposure to the resistome of wastewater-origin thus appeared to typically abate rapidly in the studied rivers. However, the riverine aquatic resistome was also dynamic, as evidenced by the increase of certain gene markers downstream, without obvious sources of anthropogenic contamination. This study provides new insight into drivers of riverine resistomes and pinpoints key monitoring targets indicative of where human sources and exposures are likely to be most acute.

Temperature modulates stress response in mainstream anammox reactors.

Autotrophic nitrogen removal by anaerobic ammonium oxidizing (anammox) bacteria is an energy-efficient nitrogen removal process in wastewater treatment. However, full-scale deployment under mainstream conditions remains challenging for practitioners due to the high stress susceptibility of anammox bacteria towards fluctuations in dissolved oxygen (DO) and temperature. Here, we investigated the response of microbial biofilms with verified anammox activity to DO shocks under 20 °C and 14 °C. While pulse disturbances of 0.3 mg L-1 DO prompted only moderate declines in the NH4+ removal rates, 1.0 mg L-1 DO led to complete but reversible inhibition of the NH4+ removal activity in all reactors. Genome-centric metagenomics and metatranscriptomics were used to investigate the stress response on various biological levels. We show that temperature regime and strength of DO perturbations induced divergent responses from the process level down to the transcriptional profile of individual taxa. Community-wide gene expression differed significantly depending on the temperature regime in all reactors, and we found a noticeable impact of DO disturbances on genes involved in transcription, translation, replication and posttranslational modification at 20 °C but not 14 °C. Genome-centric analysis revealed that different anammox species and other key biofilm taxa differed in their transcriptional responses to distinct temperature regimes and DO disturbances.

Environmental and Microbial Interactions Shape Methane-Oxidizing Bacterial Communities in a Stratified Lake.

In stratified lakes, methane-oxidizing bacteria (MOB) are strongly mitigating methane fluxes to the atmosphere by consuming methane entering the water column from the sediments. MOB communities in lakes are diverse and vertically structured, but their spatio-temporal dynamics along the water column as well as physico-chemical parameters and interactions with other bacterial species that drive the community assembly have so far not been explored in depth. Here, we present a detailed investigation of the MOB and bacterial community composition and a large set of physico-chemical parameters in a shallow, seasonally stratified, and sub-alpine lake. Four highly resolved vertical profiles were sampled in three different years and during various stages of development of the stratified water column. Non-randomly assembled MOB communities were detected in all compartments. We could identify methane and oxygen gradients and physico-chemical parameters like pH, light, available copper and iron, and total dissolved nitrogen as important drivers of the MOB community structure. In addition, MOB were well-integrated into a bacterial-environmental network. Partial redundancy analysis of the relevance network of physico-chemical variables and bacteria explained up to 84% of the MOB abundances. Spatio-temporal MOB community changes were 51% congruent with shifts in the total bacterial community and 22% of variance in MOB abundances could be explained exclusively by the bacterial community composition. Our results show that microbial interactions may play an important role in structuring the MOB community along the depth gradient of stratified lakes.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Every fifth published metagenome is not available to science.

Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? Here, we reveal that metagenomes from no fewer than 20% of the papers found in our literature search, published between 2016 and 2019, were not deposited in a repository or were simply inaccessible. The proportion of inaccessible data within the literature has been increasing year-on-year. Noncompliance with Open Data is best predicted by the scientific discipline of the journal. The number of citations, journal type (e.g., Open Access or subscription journals), and publisher are not good predictors of data accessibility. However, many publications in high-impact factor journals do display a higher likelihood of accessible metagenomic data sets. Twenty-first century science demands compliance with the ethical standard of data sharing of metagenomes and DNA sequence data more broadly. Data accessibility must become one of the routine and mandatory components of manuscript submissions-a requirement that should be applicable across the increasing number of disciplines using metagenomics. Compliance must be ensured and reinforced by funders, publishers, editors, reviewers, and, ultimately, the authors.

Growth and rapid succession of methanotrophs effectively limit methane release during lake overturn.

Lakes and reservoirs contribute substantially to atmospheric concentrations of the potent greenhouse gas methane. Lake sediments produce large amounts of methane, which accumulate in the oxygen-depleted bottom waters of stratified lakes. Climate change and eutrophication may increase the number of lakes with methane storage in the future. Whether stored methane escapes to the atmosphere during annual lake overturn is a matter of controversy and depends critically on the response of the methanotroph assemblage. Here we show, by combining 16S rRNA gene and pmoA mRNA amplicon sequencing, qPCR, CARD-FISH and potential methane-oxidation rate measurements, that the methanotroph assemblage in a mixing lake underwent both a substantial bloom and ecological succession. As a result, methane oxidation kept pace with the methane supplied from methane-rich bottom water and most methane was oxidized. This aspect of freshwater methanotroph ecology represents an effective mechanism limiting methane transfer from lakes to the atmosphere.

Niche partitioning of methane-oxidizing bacteria along the oxygen-methane counter gradient of stratified lakes.

Lakes are a significant source of atmospheric methane, although methane-oxidizing bacteria consume most methane diffusing upward from anoxic sediments. Diverse methane-oxidizing bacteria form an effective methane filter in the water column of stratified lakes, yet, niche partitioning of different methane-oxidizing bacteria along the oxygen-methane counter gradient remains poorly understood. In our study, we reveal vertical distribution patterns of active methane-oxidizing bacteria along the oxygen-methane counter gradient of four lakes, based on amplicon sequencing analysis of 16S rRNA and pmoA genes, and 16S rRNA and pmoA transcripts, and potential methane oxidation rates. Differential distribution patterns indicated that ecologically different methane-oxidizing bacteria occupied the methane-deficient and oxygen-deficient part above and below the oxygen-methane interface. The interface sometimes harbored additional taxa. Within the dominant Methylococcales, an uncultivated taxon (CABC2E06) occurred mainly under methane-deficient conditions, whereas Crenothrix-related taxa preferred oxygen-deficient conditions. Candidatus Methylomirabilis limnetica (NC10 phylum) abundantly populated the oxygen-deficient part in two of four lakes. We reason that the methane filter in lakes is structured and that methane-oxidizing bacteria may rely on niche-specific adaptations for methane oxidation along the oxygen-methane counter gradient. Niche partitioning of methane-oxidizing bacteria might support greater overall resource consumption, contributing to the high effectivity of the lacustrine methane filter.