Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields.

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

Interactions between Cyanobacteria and Methane Processing Microbes Mitigate Methane Emissions from Rice Soils.

Cyanobacteria play a relevant role in rice soils due to their contribution to soil fertility through nitrogen (N2) fixation and as a promising strategy to mitigate methane (CH4) emissions from these systems. However, information is still limited regarding the mechanisms of cyanobacterial modulation of CH4 cycling in rice soils. Here, we focused on the response of methane cycling microbial communities to inoculation with cyanobacteria in rice soils. We performed a microcosm study comprising rice soil inoculated with either of two cyanobacterial isolates (Calothrix sp. and Nostoc sp.) obtained from a rice paddy. Our results demonstrate that cyanobacterial inoculation reduced CH4 emissions by 20 times. Yet, the effect on CH4 cycling microbes differed for the cyanobacterial strains. Type Ia methanotrophs were stimulated by Calothrix sp. in the surface layer, while Nostoc sp. had the opposite effect. The overall pmoA transcripts of Type Ib methanotrophs were stimulated by Nostoc. Methanogens were not affected in the surface layer, while their abundance was reduced in the sub surface layer by the presence of Nostoc sp. Our results indicate that mitigation of methane emission from rice soils based on cyanobacterial inoculants depends on the proper pairing of cyanobacteria-methanotrophs and their respective traits.

Experimental erosion of microbial diversity decreases soil CH4 consumption rates.

Biodiversity-ecosystem functioning (BEF) experiments have predominantly focused on communities of higher organisms, in particular plants, with comparably little known to date about the relevance of biodiversity for microbially driven biogeochemical processes. Methanotrophic bacteria play a key role in Earth's methane (CH4 ) cycle by removing atmospheric CH4 and reducing emissions from methanogenesis in wetlands and landfills. Here, we used a dilution-to-extinction approach to simulate diversity loss in a methanotrophic landfill cover soil community. Replicate samples were diluted 101 -107 -fold, preincubated under a high CH4 atmosphere for microbial communities to recover to comparable size, and then incubated for 86 days at constant or diurnally cycling temperature. We hypothesize that (1) CH4 consumption decreases as methanotrophic diversity is lost, and (2) this effect is more pronounced under variable temperatures. Net CH4 consumption was determined by gas chromatography. Microbial community composition was determined by DNA extraction and sequencing of amplicons specific to methanotrophs and bacteria (pmoA and 16S gene fragments). The richness of operational taxonomic units (OTU) of methanotrophic and nonmethanotrophic bacteria decreased approximately linearly with log-dilution. CH4 consumption decreased with the number of OTUs lost, independent of community size. These effects were independent of temperature cycling. The diversity effects we found occured in relatively diverse communities, challenging the notion of high functional redundancy mediating high resistance to diversity erosion in natural microbial systems. The effects also resemble the ones for higher organisms, suggesting that BEF relationships are universal across taxa and spatial scales.

The intrinsic methane mitigation potential and associated microbes add product value to compost.

Conventional agricultural activity reduces the uptake of the potent greenhouse gas methane by agricultural soils. However, the recently observed improved methane uptake capacity of agricultural soils after compost application is promising but needs mechanistic understanding. In this study, the methane uptake potential and microbiomes involved in methane cycling were assessed in green compost and household-compost with and without pre-digestion. In bottle incubations of different composts with both high and near-atmospheric methane concentrations (∼10.000 & ∼10 ppmv, respectively), green compost showed the highest potential methane uptake rates (up to 305.19 ± 94.43 nmol h-1 g dw compost-1 and 25.19 ± 6.75 pmol h-1 g dw compost-1, respectively). 16S, pmoA and mcrA amplicon sequencing revealed that its methanotrophic and methanogenic communities were dominated by type Ib methanotrophs, and more specifically by Methylocaldum szegediense and other Methylocaldum species, and Methanosarcina species, respectively. Ordination analyses showed that the abundance of type Ib methanotrophic bacteria was the main steering factor of the intrinsic methane uptake rates of composts, whilst the ammonium content was the main limiting factor, being most apparent in household composts. These results emphasize the potential of compost to contribute to methane mitigation, providing added value to compost as a product for industrial, commercial, governmental and public interests relevant to waste management. Compost could serve as a vector for the introduction of active methanotrophic bacteria in agricultural soils, potentially improving the methane uptake potential of agricultural soils and contributing to global methane mitigation, which should be the focus of future research.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave.

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g-1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g-1 DW, 1-3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g-1 DW·h-1 in sediment versus 11.02 ng CH4·g-1 DW·h-1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Archaea and their interactions with bacteria in a karst ecosystem.

Karst ecosystems are widely distributed around the world, accounting for 15-20% of the global land area. However, knowledge on microbial ecology of these systems does not match with their global importance. To close this knowledge gap, we sampled three niches including weathered rock, sediment, and drip water inside the Heshang Cave and three types of soils overlying the cave (forest soil, farmland soil, and pristine karst soil). All these samples were subjected to high-throughput sequencing of V4-V5 region of 16S rRNA gene and analyzed with multivariate statistical analysis. Overall, archaeal communities were dominated by Thaumarchaeota, whereas Actinobacteria dominated bacterial communities. Thermoplasmata, Nitrosopumilaceae, Aenigmarchaeales, Crossiella, Acidothermus, and Solirubrobacter were the important predictor groups inside the Heshang Cave, which were correlated to NH4 + availability. In contrast, Candidatus Nitrososphaera, Candidatus Nitrocosmicus, Thaumarchaeota Group 1.1c, and Pseudonocardiaceae were the predictors outside the cave, whose distribution was correlated with pH, Ca2+, and NO2 -. Tighter network structures were found in archaeal communities than those of bacteria, whereas the topological properties of bacterial networks were more similar to those of total prokaryotic networks. Both chemolithoautotrophic archaea (Candidatus Methanoperedens and Nitrosopumilaceae) and bacteria (subgroup 7 of Acidobacteria and Rokubacteriales) were the dominant keystone taxa within the co-occurrence networks, potentially playing fundamental roles in obtaining energy under oligotrophic conditions and thus maintaining the stability of the cave ecosystem. To be noted, all the keystone taxa of karst ecosystems were related to nitrogen cycling, which needs further investigation, particularly the role of archaea. The predicted ecological functions in karst soils mainly related to carbohydrate metabolism, biotin metabolism, and synthesis of fatty acid. Our results offer new insights into archaeal ecology, their potential functions, and archaeal interactions with bacteria, which enhance our understanding about the microbial dark matter in the subsurface karst ecosystems.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Zwartia hollandica gen. nov., sp. nov., Jezberella montanilacus gen. nov., sp. nov. and Sheuella amnicola gen. nov., comb. nov., representing the environmental GKS98 (betIII) cluster.

We present two strains affiliated with the GKS98 cluster. This phylogenetically defined cluster is representing abundant, mainly uncultured freshwater bacteria, which were observed by many cultivation-independent studies on the diversity of bacteria in various freshwater lakes and streams. Bacteria affiliated with the GKS98 cluster were detected by cultivation-independent methods in freshwater systems located in Europe, Asia, Africa and the Americas. The two strains, LF4-65T (=CCUG 56422T=DSM 107630T) and MWH-P2sevCIIIbT (=CCUG 56420T=DSM 107629T), are aerobic chemoorganotrophs, both with genome sizes of 3.2 Mbp and G+C values of 52.4 and 51.0 mol%, respectively. Phylogenomic analyses based on concatenated amino acid sequences of 120 proteins suggest an affiliation of the two strains with the family Alcaligenaceae and revealed Orrella amnicola and Orrella marina (= Algicoccus marinus) as being the closest related, previously described species. However, the calculated phylogenomic trees clearly suggest that the current genus Orrella represents a polyphyletic taxon. Based on the branching order in the phylogenomic trees, as well as the revealed phylogenetic distances and chemotaxonomic traits, we propose to establish the new genus Zwartia gen. nov. and the new species Z. hollandica sp. nov. to harbour strain LF4-65T and the new genus Jezberella gen. nov. and the new species J. montanilacus sp. nov. to harbour strain MWH-P2sevCIIIbT. Furthermore, we propose the reclassification of the species Orrella amnicola in the new genus Sheuella gen. nov. The new genera Zwartia, Jezberella and Sheuella together represent taxonomically the GKS98 cluster.

Insights into the Genomic Potential of a Methylocystis sp. from Amazonian Floodplain Sediments.

Although floodplains are recognized as important sources of methane (CH4) in the Amazon basin, little is known about the role of methanotrophs in mitigating CH4 emissions in these ecosystems. Our previous data reported the genus Methylocystis as one of the most abundant methanotrophs in these floodplain sediments. However, information on the functional potential and life strategies of these organisms living under seasonal flooding is still missing. Here, we described the first metagenome-assembled genome (MAG) of a Methylocystis sp. recovered from Amazonian floodplains sediments, and we explored its functional potential and ecological traits through phylogenomic, functional annotation, and pan-genomic approaches. Both phylogenomics and pan-genomics identified the closest placement of the bin.170_fp as Methylocystis parvus. As expected for Type II methanotrophs, the Core cluster from the pan-genome comprised genes for CH4 oxidation and formaldehyde assimilation through the serine pathway. Furthermore, the complete set of genes related to nitrogen fixation is also present in the Core. Interestingly, the MAG singleton cluster revealed the presence of unique genes related to nitrogen metabolism and cell motility. The study sheds light on the genomic characteristics of a dominant, but as yet unexplored methanotroph from the Amazonian floodplains. By exploring the genomic potential related to resource utilization and motility capability, we expanded our knowledge on the niche breadth of these dominant methanotrophs in the Amazonian floodplains.

Molecular evidence for stimulation of methane oxidation in Amazonian floodplains by ammonia-oxidizing communities.

Ammonia oxidation is the rate-limiting first step of nitrification and a key process in the nitrogen cycle that results in the formation of nitrite (NO2 -), which can be further oxidized to nitrate (NO3 -). In the Amazonian floodplains, soils are subjected to extended seasons of flooding during the rainy season, in which they can become anoxic and produce a significant amount of methane (CH4). Various microorganisms in this anoxic environment can couple the reduction of different ions, such as NO2 - and NO3 -, with the oxidation of CH4 for energy production and effectively link the carbon and nitrogen cycle. Here, we addressed the composition of ammonium (NH4 +) and NO3 --and NO2 --dependent CH4-oxidizing microbial communities in an Amazonian floodplain. In addition, we analyzed the influence of environmental and geochemical factors on these microbial communities. Soil samples were collected from different layers of forest and agroforest land-use systems during the flood and non-flood seasons in the floodplain of the Tocantins River, and next-generation sequencing of archaeal and bacterial 16S rRNA amplicons was performed, coupled with chemical characterization of the soils. We found that ammonia-oxidizing archaea (AOA) were more abundant than ammonia-oxidizing bacteria (AOB) during both flood and non-flood seasons. Nitrogen-dependent anaerobic methane oxidizers (N-DAMO) from both the archaeal and bacterial domains were also found in both seasons, with higher abundance in the flood season. The different seasons, land uses, and depths analyzed had a significant influence on the soil chemical factors and also affected the abundance and composition of AOA, AOB, and N-DAMO. During the flood season, there was a significant correlation between ammonia oxidizers and N-DAMO, indicating the possible role of these oxidizers in providing oxidized nitrogen species for methanotrophy under anaerobic conditions, which is essential for nitrogen removal in these soils.

Niche differentiation of atmospheric methane-oxidizing bacteria and their community assembly in subsurface karst caves.

Karst caves are recently proposed as atmospheric methane sinks in terrestrial ecosystems. Despite of the detection of atmospheric methane-oxidizing bacteria (atmMOB) in caves, we still know little about their ecology and potential ability of methane oxidation in this ecosystem. To understand atmMOB ecology and their potential in methane consumption, we collected weathered rocks and sediments from three different caves in southwestern China. We determined the potential methane oxidization rates in the range of 1.25 ± 0.08 to 1.87 ± 0.41 ng CH4  g-1 DW h-1 , which are comparable to those reported in forest and grassland soils. Results showed that alkaline oligotrophic caves harbour high numbers of atmMOB, particularly upland soil cluster (USC), which significantly correlated with temperature, CH4 and CO2 concentrations. The absolute abundance of USCγ was higher than that of USCα. USCγ-OPS (open patch soil) and USCγ-SS (subsurface soil) dominated in most samples, whereas USCα-BFS (boreal forest soil) only predominated in the sediments near cave entrances, indicating niche differentiation of atmMOB in caves. Overwhelming dominance of homogenous selection in community assembly resulted in convergence of atmMOB communities. Collectively, our results demonstrated the niche differentiation of USC in subsurface alkaline caves and their non-negligible methane-oxidizing potential, providing brand-new knowledge about atmMOB ecology in subsurface biosphere.

PhyloFunDB: A Pipeline to Create and Update Functional Gene Taxonomic Databases.

The increase in sequencing capacity has amplified the number of taxonomically unclassified sequences in most databases. The classification of such sequences demands phylogenetic tree construction and comparison to currently classified sequences, a process that demands the processing of large amounts of data and use of several different software. Here, we present PhyloFunDB, a pipeline for extracting, processing, and inferring phylogenetic trees from specific functional genes. The goal of our work is to decrease processing time and facilitate the grouping of sequences that can be used for improved taxonomic classification of functional gene datasets.

Producing organic amendments: Physicochemical changes in biowaste used in anaerobic digestion, composting, and fermentation.

Organic amendments (OAs) produced via composting, anaerobic digestion, or lactic acid fermentation, can be used to replenish soil carbon. Not all OAs production technologies preserve C and nutrients in the same way. In this study, we compared the influence of these technologies (i.e., treatments) on C and nutrient preservation and OAs chemical composition after production. We produced compost, digestate, and lactic-acid fermentation product using the same biowaste-resembling model substrate using three reactors under laboratory conditions. We compared the chemical conversions and end-products using mass balances over C, N, and P. Overall results show that losses are minimal under reducing production conditions. Fermentation and digestion conserved 99% and 64% of C; and 93% and 100% of N, respectively. While compost conservation of nutrients was limited to 25% of C and 38% of N. Digestate had the highest concentrations of C, N, and P in the water-soluble phase, enabling their accessibility for soil microbes. Concentrations in the fermentation product were one order of magnitude lower but still higher than in compost. The treatments also influence the final availability of C, N, and P, which could potentially improve the fertilising and soil-improving properties of produced OAs. Our results show that under reducing conditions, losses of C, N, and P can be decreased while increasing OAs applications in terms of sources for soil-microbial development.

Methane-Derived Carbon as a Driver for Cyanobacterial Growth.

Methane, a potent greenhouse gas produced in freshwater ecosystems, can be used by methane-oxidizing bacteria (MOB) and can therefore subsidize the pelagic food web with energy and carbon. Consortia of MOB and photoautotrophs have been described in aquatic ecosystems and MOB can benefit from photoautotrophs which produce oxygen, thereby enhancing CH4 oxidation. Methane oxidation can account for accumulation of inorganic carbon (i.e., CO2) and the release of exometabolites that may both be important factors influencing the structure of phytoplankton communities. The consortium of MOB and phototroph has been mainly studied for methane-removing biotechnologies, but there is still little information on the role of these interactions in freshwater ecosystems especially in the context of cyanobacterial growth and bloom development. We hypothesized that MOB could be an alternative C source to support cyanobacterial growth in freshwater systems. We detected low δ13C values in cyanobacterial blooms (the lowest detected value -59.97‰ for Planktothrix rubescens) what could be the result of the use of methane-derived carbon by cyanobacteria and/or MOB attached to their cells. We further proved the presence of metabolically active MOB on cyanobacterial filaments using the fluorescein isothiocyanate (FITC) based activity assay. The PCR results also proved the presence of the pmoA gene in several non-axenic cultures of cyanobacteria. Finally, experiments comprising the co-culture of the cyanobacterium Aphanizomenon gracile with the methanotroph Methylosinus sporium proved that cyanobacterial growth was significantly improved in the presence of MOB, presumably through utilizing CO2 released by MOB. On the other hand, 13C-CH4 labeled incubations showed the uptake and assimilation of MOB-derived metabolites by the cyanobacterium. We also observed a higher growth of MOB in the presence of cyanobacteria under a higher irradiance regime, then when grown alone, underpinning the bidirectional influence with as of yet unknown environmental consequences.

USCγ Dominated Community Composition and Cooccurrence Network of Methanotrophs and Bacteria in Subterranean Karst Caves.

Karst caves have recently been demonstrated to act as a sink for atmospheric methane, due in part to consumption by microbes residing in caves that can oxidize methane at atmospheric levels. However, our knowledge about the responsible atmospheric methane-oxidizing bacteria (atmMOB) in this vast habitat remains limited to date. To address this issue, weathered rock samples from three karst caves were collected in Guilin City and subjected to high-throughput sequencing of pmoA and 16S rRNA genes. The results showed that members of the high-affinity upland soil cluster (USC), especially upland soil cluster gamma (USCγ), with absolute abundances of 104 to 109 copies · g-1 dry sample, dominated the atmMOB communities, while Proteobacteria and Actinobacteria dominated the overall bacterial communities. Moreover, USCγ was a keystone taxon in cooccurrence networks of both the atmMOB and the total bacterial community, whereas keystone taxa in the bacterial network also included Gaiella and Aciditerrimonas. Positive links overwhelmingly dominated the cooccurrence networks of both atmMOB and the total bacterial community, indicating a consistent response to environmental disturbances. Our study shed new insights on the diversity and abundances underlining atmMOB and total bacterial communities and on microbial interactions in subterranean karst caves, which increased our understanding about USC and supported karst caves as a methane sink. IMPORTANCE Karst caves have recently been demonstrated to be a potential atmospheric methane sink, presumably due to consumption by methane-oxidizing bacteria. However, the sparse knowledge about the diversity, distribution, and community interactions of methanotrophs requires us to seek further understanding of the ecological significance of methane oxidation in these ecosystems. Our pmoA high-throughput results from weathered rock samples from three karst caves in Guilin City confirm the wide occurrence of atmospheric methane-oxidizing bacteria in this habitat, especially those affiliated with the upland soil cluster, with a gene copy number of 104 to 109 copies per gram dry sample. Methanotrophs and the total bacterial communities had more positive than negative interactions with each other as indicated by the cooccurrence network, suggesting their consistent response to environmental disturbance. Our results solidly support caves as an atmospheric methane sink, and they contribute to a comprehensive understanding of the diversity, distribution, and interactions of microbial communities in subsurface karst caves.

Complete and Draft Genome Sequences of Aerobic Methanotrophs Isolated from a Riparian Wetland.

Wetlands are important sources of methane emissions, and the impacts of these emissions can be mitigated by methanotrophic bacteria. The genomes of methanotrophs Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A, as well as Methylocystis sp. strains H4A, H15, H62, and L43, were sequenced and are reported here.

Response of a methane-driven interaction network to stressor intensification.

Microorganisms may reciprocally select for specific interacting partners, forming a network with interdependent relationships. The methanotrophic interaction network, comprising methanotrophs and non-methanotrophs, is thought to modulate methane oxidation and give rise to emergent properties beneficial for the methanotrophs. Therefore, microbial interaction may become relevant for community functioning under stress. However, empirical validation of the role and stressor-induced response of the interaction network remains scarce. Here, we determined the response of a complex methane-driven interaction network to a stepwise increase in NH4Cl-induced stress (0.5-4.75 g L-1, in 0.25-0.5 g L-1 increments) using enrichment of a naturally occurring complex community derived from a paddy soil in laboratory-scale incubations. Although ammonium and intermediates of ammonium oxidation are known to inhibit methane oxidation, methanotrophic activity was unexpectedly detected even in incubations with high ammonium levels, albeit rates were significantly reduced. Sequencing analysis of the 16S rRNA and pmoA genes consistently revealed divergent communities in the reference and stressed incubations. The 16S rRNA-based co-occurrence network analysis revealed that NH4Cl-induced stress intensification resulted in a less complex and modular network, likely driven by less stable interaction. Interestingly, the non-methanotrophs formed the key nodes, and appear to be relevant members of the community. Overall, stressor intensification unravels the interaction network, with adverse consequences for community functioning.

Niche Differentiation of Host-Associated Pelagic Microbes and Their Potential Contribution to Biogeochemical Cycling in Artificially Warmed Lakes.

It has been proposed that zooplankton-associated microbes provide numerous beneficial services to their "host". However, there is still a lack of understanding concerning the effect of temperature on the zooplankton microbiome. Furthermore, it is unclear to what extent the zooplankton microbiome differs from free-living and particle-associated (PA) microbes. Here, we explicitly addressed these issues by investigating (1) the differences in free-living, PA, and zooplankton associated microbes and (2) the impact of temperature on these microbes in the water column of a series of lakes artificially warmed by two power plants. High-throughput amplicon sequencing of the 16S rRNA gene showed that diversity and composition of the bacterial community associated to zooplankton, PA, and bacterioplankton varied significantly from one another, grouping in different clusters indicating niche differentiation of pelagic microbes. From the abiotic parameters measured, temperature significantly affected the diversity and composition of all analyzed microbiomes. Two phyla (e.g., Proteobacteria and Bacteroidetes) dominated in zooplankton microbiomes whereas Actinobacteria was the dominant phylum in the bacterioplankton. The microbial species richness and diversity was lower in zooplankton compared to bacterioplankton and PA. Surprisingly, genera of methane-oxidizing bacteria, methylotrophs and nitrifiers (e.g., Nitrobacter) significantly associated with the microbiome of zooplankton and PA. Our study clearly demonstrates niche differentiation of pelagic microbes and their potential link to biogeochemical cycling in freshwater systems.