Organic matter lability modifies the vertical structure of methane-related microbial communities in lake sediments.

Eutrophication increases the input of labile, algae-derived, organic matter (OM) into lake sediments. This potentially increases methane (CH4) emissions from sediment to water through increased methane production rates and decreased methane oxidation efficiency in sediments. However, the effect of OM lability on the structure of methane oxidizing (methanotrophic) and methane producing (methanogenic) microbial communities in lake sediments is still understudied. We studied the vertical profiles of the sediment and porewater geochemistry and the microbial communities (16S rRNA gene amplicon sequencing) at five profundal stations of an oligo-mesotrophic, boreal lake (Lake Pääjärvi, Finland), varying in surface sediment OM sources (assessed via sediment C:N ratio). Porewater profiles of methane, dissolved inorganic carbon (DIC), acetate, iron, and sulfur suggested that sites with more autochthonous OM showed higher overall OM lability, which increased remineralization rates, leading to increased electron acceptor (EA) consumption and methane emissions from sediment to water. When OM lability increased, the abundance of anaerobic nitrite-reducing methanotrophs (Candidatus Methylomirabilis) relative to aerobic methanotrophs (Methylococcales) in the methane oxidation layer of sediment surface decreased, suggesting that Methylococcales were more competitive than Ca. Methylomirabilis under decreasing redox conditions and increasing methane availability due to their more diverse metabolism (fermentation and anaerobic respiration) and lower affinity for methane. Furthermore, when OM lability increased, the abundance of methanotrophic community in the sediment surface layer, especially Ca. Methylomirabilis, relative to the methanogenic community decreased. We conclude that increasing input of labile OM, subsequently affecting the redox zonation of sediments, significantly modifies the methane producing and consuming microbial community of lake sediments. IMPORTANCE Lakes are important natural emitters of the greenhouse gas methane (CH4). It has been shown that eutrophication, via increasing the input of labile organic matter (OM) into lake sediments and subsequently affecting the redox conditions, increases methane emissions from lake sediments through increased sediment methane production rates and decreased methane oxidation efficiency. However, the effect of organic matter lability on the structure of the methane-related microbial communities of lake sediments is not known. In this study, we show that, besides the activity, also the structure of lake sediment methane producing and consuming microbial community is significantly affected by changes in the sediment organic matter lability.

Soil GHG dynamics after water level rise - Impacts of selection harvesting in peatland forests.

Managed boreal peatlands are widespread and economically important, but they are a large source of greenhouse gases (GHGs). Peatland GHG emissions are related to soil water-table level (WT), which controls the vertical distribution of aerobic and anaerobic processes and, consequently, sinks and sources of GHGs in soils. On forested peatlands, selection harvesting reduces stand evapotranspiration and it has been suggested that the resulting WT rise decreases soil net emissions, while the tree growth is maintained. We monitored soil concentrations of CO2, CH4, N2O and O2 by depth down to 80 cm, and CO2 and CH4 fluxes from soil in two nutrient-rich Norway spruce dominated peatlands in Southern Finland to examine the responses of soil GHG dynamics to WT rise. Selection harvesting raised WT by 14 cm on both sites, on average, mean WTs of the monitoring period being 73 cm for unharvested control and 59 cm for selection harvest. All soil gas concentrations were associated with proximity to WT. Both CH4 and CO2 showed remarkable vertical concentration gradients, with high values in the deepest layer, likely due to slow gas transfer in wet peat. CH4 was efficiently consumed in peat layers near and above WT where it reached sub-atmospheric concentrations, indicating sustained oxidation of CH4 from both atmospheric and deeper soil origins also after harvesting. Based on soil gas concentration data, surface peat (top 25/30 cm layer) contributed most to the soil-atmosphere CO2 fluxes and harvesting slightly increased the CO2 source in deeper soil (below 45/50 cm), which could explain the small CO2 flux differences between treatments. N2O production occurred above WT, and it was unaffected by harvesting. Overall, the WT rise obtained with selection harvesting was not sufficient to reduce soil GHG emissions, but additional hydrological regulation would have been needed.

Characterization, genome analysis and genetic tractability studies of a new nanocellulose producing Komagataeibacter intermedius isolate.

Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host chassis in synthetic biology have been extensively studied. However, such studies have been largely focused on the model Komagataeibacter spp. Here, we present a novel K. intermedius strain capable of utilizing glucose, and glycerol sources for biomass and BC synthesis. Genome assembly identified one bacterial cellulose synthetase (bcs) operon containing the complete gene set encoding the BC biogenesis machinery (bcsI) and three additional copies (bcsII-IV). Investigations on the genetic tractability confirmed plasmid transformation, propagation of vectors with pBBR1 and p15A origin of replications and constitutive and inducible induction of recombinant protein in K. intermedius ENS15. This study provides the first report on the genetic tractability of K. intermedius, serving as starting point towards future genetic engineering of this strain.

Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for 1-Alkene Production From Methane.

Methane (CH4) is a sustainable carbon feedstock for value-added chemical production in aerobic CH4-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed carbon from CH4 (C-CH4), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed C-CH4. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 'tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 µmol mol Csubstrate -1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Characterization of Komagataeibacter Isolate Reveals New Prospects in Waste Stream Valorization for Bacterial Cellulose Production.

Komagataeibacter spp. has been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently, studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose-producing bacterium capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from the biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in a minimal medium containing glucose (3.3 ± 0.3 g/L), pure glycerol (2.2 ± 0.1 g/L) and crude glycerol (2.1 ± 0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from the central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in a minimal medium supplemented with acetate, BC production was not observed. However, in the presence of readily utilizable substrates, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Draft genome sequence data of methanotrophic Methylovulum psychrotolerans strain S1L and Methylomonas paludis strain S2AM isolated from hypoxic water column layers of boreal lakes.

Methanotrophic bacteria inhabit a wide range of natural (e.g. wetlands, lakes and soils) and anthropogenic (e.g. wastewater treatment plants and landfills) environments. They play a crucial role in mitigating atmospheric emissions of the greenhouse gas methane. There is also a growing interest in applying methanotrophs in the bioconversion of biogas - and natural gas - methane into value-added products (e.g. chemicals and single-cell protein). Hence, isolation and genome sequencing of methanotrophic bacteria is needed to provide important data on their functional capabilities. Here, we describe the de novo assembled draft genome sequences of Methylovulum psychrotolerans strain S1L isolated from hypoxic water column layer of boreal Lake Lovojärvi (Southern Finland), comprising total of 5090628 bp in 11 contigs with G+C - content of 50.9% and containing 4554 coding sequences. The draft genome of strain S1L represents the first published genome of M. psychrotolerans strain isolated from lake ecosystems. In addition, we present the genome sequence of Methylomonas paludis strain S2AM, isolated from water column of boreal Lake Alinen Mustajärvi (Southern Finland), comprising 3673651 bp in 1 contig with G+C - content of 48.2% and 3294 coding sequences. The draft genome of strain S2AM represents the first published genome of M. paludis. The preliminary genome annotation analysis of both S1L and S2AM identified genes encoding oxidation of methane, methanol, formaldehyde and formate, assimilation of carbon, ammonium and nitrate, N2 fixation, as well as various enzymes enabling the survival in hypoxic conditions, i.e. high-affinity oxidase, hemerythrins, fermentation enzymes (for production of acetate, succinate and H2) and respiratory nitrite reductases. The draft genomes have been deposited at GenBank under the accession JAGVVN000000000 for S1L and CP073754 for S2AM.

Candidatus Methylumidiphilus Drives Peaks in Methanotrophic Relative Abundance in Stratified Lakes and Ponds Across Northern Landscapes.

Boreal lakes and ponds produce two-thirds of the total natural methane emissions above the latitude of 50° North. These lake emissions are regulated by methanotrophs which can oxidize up to 99% of the methane produced in the sediments and the water column. Despite their importance, the diversity and distribution of the methanotrophs in lakes are still poorly understood. Here, we used shotgun metagenomic data to explore the diversity and distribution of methanotrophs in 40 oxygen-stratified water bodies in boreal and subarctic areas in Europe and North America. In our data, gammaproteobacterial methanotrophs (order Methylococcales) generally dominated the methanotrophic communities throughout the water columns. A recently discovered lineage of Methylococcales, Candidatus Methylumidiphilus, was present in all the studied water bodies and dominated the methanotrophic community in lakes with a high relative abundance of methanotrophs. Alphaproteobacterial methanotrophs were the second most abundant group of methanotrophs. In the top layer of the lakes, characterized by low CH4 concentration, their abundance could surpass that of the gammaproteobacterial methanotrophs. These results support the theory that the alphaproteobacterial methanotrophs have a high affinity for CH4 and can be considered stress-tolerant strategists. In contrast, the gammaproteobacterial methanotrophs are competitive strategists. In addition, relative abundances of anaerobic methanotrophs, Candidatus Methanoperedenaceae and Candidatus Methylomirabilis, were strongly correlated, suggesting possible co-metabolism. Our data also suggest that these anaerobic methanotrophs could be active even in the oxic layers. In non-metric multidimensional scaling, alpha- and gammaproteobacterial methanotrophs formed separate clusters based on their abundances in the samples, except for the gammaproteobacterial Candidatus Methylumidiphilus, which was separated from these two clusters. This may reflect similarities in the niche and environmental requirements of the different genera within alpha- and gammaproteobacterial methanotrophs. Our study confirms the importance of O2 and CH4 in shaping the methanotrophic communities and suggests that one variable cannot explain the diversity and distribution of the methanotrophs across lakes. Instead, we suggest that the diversity and distribution of freshwater methanotrophs are regulated by lake-specific factors.

Vertical stratification patterns of methanotrophs and their genetic controllers in water columns of oxygen-stratified boreal lakes.

The vertical structuring of methanotrophic communities and its genetic controllers remain understudied in the water columns of oxygen-stratified lakes. Therefore, we used 16S rRNA gene sequencing to study the vertical stratification patterns of methanotrophs in two boreal lakes, Lake Kuivajärvi and Lake Lovojärvi. Furthermore, metagenomic analyses were performed to assess the genomic characteristics of methanotrophs in Lovojärvi and the previously studied Lake Alinen Mustajärvi. The methanotroph communities were vertically structured along the oxygen gradient. Alphaproteobacterial methanotrophs preferred oxic water layers, while Methylococcales methanotrophs, consisting of putative novel genera and species, thrived, especially at and below the oxic-anoxic interface and showed distinct depth variation patterns, which were not completely predictable by their taxonomic classification. Instead, genomic differences among Methylococcales methanotrophs explained their variable vertical depth patterns. Genes in clusters of orthologous groups (COG) categories L (replication, recombination and repair) and S (function unknown) were relatively high in metagenome-assembled genomes representing Methylococcales clearly thriving below the oxic-anoxic interface, suggesting genetic adaptations for increased stress tolerance enabling living in the hypoxic/anoxic conditions. By contrast, genes in COG category N (cell motility) were relatively high in metagenome-assembled genomes of Methylococcales thriving at the oxic-anoxic interface, which suggests genetic adaptations for increased motility at the vertically fluctuating oxic-anoxic interface.

CH4 oxidation in a boreal lake during the development of hypolimnetic hypoxia.

Freshwater ecosystems represent a significant natural source of methane (CH4). CH4 produced through anaerobic decomposition of organic matter (OM) in lake sediment and water column can be either oxidized to carbon dioxide (CO2) by methanotrophic microbes or emitted to the atmosphere. While the role of CH4 oxidation as a CH4 sink is widely accepted, neither the magnitude nor the drivers behind CH4 oxidation are well constrained. In this study, we aimed to gain more specific insight into CH4 oxidation in the water column of a seasonally stratified, typical boreal lake, particularly under hypoxic conditions. We used 13CH4 incubations to determine the active CH4 oxidation sites and the potential CH4 oxidation rates in the water column, and we measured environmental variables that could explain CH4 oxidation in the water column. During hypolimnetic hypoxia, 91% of available CH4 was oxidized in the active CH4 oxidation zone, where the potential CH4 oxidation rates gradually increased from the oxycline to the hypolimnion. Our results showed that in warm springs, which become more frequent, early thermal stratification with cold well-oxygenated hypolimnion delays the period of hypolimnetic hypoxia and limits CH4 production. Thus, the delayed development of hypolimnetic hypoxia may partially counteract the expected increase in the lacustrine CH4 emissions caused by the increasing organic carbon load from forested catchments.

Vertical stratification of bacteria and archaea in sediments of a small boreal humic lake.

Although sediments of small boreal humic lakes are important carbon stores and greenhouse gas sources, the composition and structuring mechanisms of their microbial communities have remained understudied. We analyzed the vertical profiles of microbial biomass indicators (PLFAs, DNA and RNA) and the bacterial and archaeal community composition (sequencing of 16S rRNA gene amplicons and qPCR of mcrA) in sediment cores collected from a typical small boreal lake. While microbial biomass decreased with sediment depth, viable microbes (RNA and PLFA) were present all through the profiles. The vertical stratification patterns of the bacterial and archaeal communities resembled those in marine sediments with well-characterized groups (e.g. Methanomicrobia, Proteobacteria, Cyanobacteria, Bacteroidetes) dominating in the surface sediment and being replaced by poorly-known groups (e.g. Bathyarchaeota, Aminicenantes and Caldiserica) in the deeper layers. The results also suggested that, similar to marine systems, the deep bacterial and archaeal communities were predominantly assembled by selective survival of taxa able to persist in the low energy conditions. Methanotrophs were rare, further corroborating the role of these methanogen-rich sediments as important methane emitters. Based on their taxonomy, the deep-dwelling groups were putatively organo-heterotrophic, organo-autotrophic and/or acetogenic and thus may contribute to changes in the lake sediment carbon storage.

High Nitrogen Removal in a Constructed Wetland Receiving Treated Wastewater in a Cold Climate.

Constructed wetlands provide cost-efficient nutrient removal, with minimal input of human labor and energy, and their number is globally increasing. However, in northern latitudes, wetlands are rarely utilized, because their nutrient removal efficiency has been questioned due to the cold climate. Here, we studied nutrient retention and nitrogen removal in a boreal constructed wetland (4-ha) receiving treated nitrogen-rich wastewater. On a yearly basis, most of the inorganic nutrients were retained by the wetland. The highest retention efficiency was found during the ice-free period, being 79% for ammonium-nitrogen (NH4+-N), 71% for nitrate-nitrogen (NO3--N), and 88% for phosphate-phosphorus (PO43--P). Wetland also acted as a buffer zone during the disturbed nitrification process of the wastewater treatment plant. Denitrification varied between 106 and 252 mg N m-2 d-1 during the ice-free period. During the ice-cover period, total gaseous nitrogen removal was 147 mg N m-2 d-1, from which 66% was removed as N2, 28.5% as N2O through denitrification, and 5.5% as N2 through anammox. Nearly 2600 kg N y-1 was estimated to be removed through microbial gaseous N-production which equaled 72% of NO3--N and 60% of TN yearly retention in the wetland. The wetland retained nutrients even in winter, when good oxygen conditions prevailed under ice. The results suggest that constructed wetlands are an efficient option for wastewater nitrogen removal and nutrient retention also in cold climates.

Stable carbon isotopic composition of peat columns, subsoil and vegetation on natural and forestry-drained boreal peatlands.

We studied natural and forestry-drained peatlands to examine the effect of over 34 years lowered water table on the δ13C values of vegetation, bulk peat and subsoil. In the seven studied sites, δ13C in the basal peat layer was 1.1 and 1.2 ‰ lower than that of the middle-layer and surface layer, respectively. Furthermore, there was a positive correlation between the δ13C values of the basal and surface peat layers, possibly due to carbon (C) recycling within the peat column. In the same mire complex, natural fen peat δ13C values were lower than those of the nearby bog, possibly due to the dominance of vascular plants on fen and the generally larger share of recycled C in the fens than in the bogs. Furthermore, natural and 51 years previously drained fen and bog, on the opposite sides of a ditch on the same mire complex, showed no significant differences in δ13C values. Plant δ13C values were lower, while δ13C values of subsoil were higher in the drained than in the natural site of the fen.

Resistant ammonia-oxidizing archaea endure, but adapting ammonia-oxidizing bacteria thrive in boreal lake sediments receiving nutrient-rich effluents.

Climate change along with anthropogenic activities changes biogeochemical conditions in lake ecosystems, modifying the sediment microbial communities. Wastewater effluents introduce nutrients and organic material but also novel microbes to lake ecosystems, simulating forthcoming increases in catchment loadings. In this work, we first used 16s rRNA gene sequencing to study how the overall sediment microbial community responds to wastewater in six boreal lakes. To examine forthcoming changes in the lake biogeochemistry, we focused on the ammonia-oxidizing archaea (AOA) and bacteria (AOB), and examined their functional and compositional community response to wastewater. Although we found the least diverse and least resistant prokaryotic communities from the most wastewater-influenced sediments, the community changed fast toward the natural composition with the diminishing influence of wastewater. Each lake hosted a unique resistant AOA community, while AOB communities were adapting, responding to environmental conditions as well as receiving new members from WWTPs. In general, AOB dominated in numbers in wastewater-influenced sediments, while the ratio between AOA and AOB increased when moving toward pristine conditions. Our results suggest that although future climate-change-driven increases in nutrient loading and microbial migration might significantly disrupt lake sediment microbiomes, they can promote nitrification through adapting and abundant AOB communities.

Sediment diffusion method improves wastewater nitrogen removal in the receiving lake sediments.

Sediment microbes have a great potential to transform reactive N to harmless N2, thus decreasing wastewater nitrogen load into aquatic ecosystems. Here, we examined if spatial allocation of the wastewater discharge by a specially constructed sediment diffuser pipe system enhanced the microbial nitrate reduction processes. Full-scale experiments were set on two Finnish lake sites, Keuruu and Petäjävesi, and effects on the nitrate removal processes were studied using the stable isotope pairing technique. All nitrate reduction rates followed nitrate concentrations, being highest at the wastewater-influenced sampling points. Complete denitrification with N2 as an end-product was the main nitrate reduction process, indicating that the high nitrate and organic matter concentrations of wastewater did not promote nitrous oxide (N2O) production (truncated denitrification) or ammonification (dissimilatory nitrate reduction to ammonium; DNRA). Using 3D simulation, we demonstrated that the sediment diffusion method enhanced the contact time and amount of wastewater near the sediment surface especially in spring and in autumn, altering organic matter concentration and oxygen levels, and increasing the denitrification capacity of the sediment. We estimated that natural denitrification potentially removed 3-10% of discharged wastewater nitrate in the 33 ha study area of Keuruu, and the sediment diffusion method increased this areal denitrification capacity on average 45%. Overall, our results indicate that sediment diffusion method can supplement wastewater treatment plant (WWTP) nitrate removal without enhancing alternative harmful processes.

Effects of alternative electron acceptors on the activity and community structure of methane-producing and consuming microbes in the sediments of two shallow boreal lakes.

The role of anaerobic CH4 oxidation in controlling lake sediment CH4 emissions remains unclear. Therefore, we tested how relevant EAs (SO42-, NO3-, Fe3+, Mn4+, O2) affect CH4 production and oxidation in the sediments of two shallow boreal lakes. The changes induced to microbial communities by the addition of Fe3+ and Mn4+ were studied using next-generation sequencing targeting the 16S rRNA and methyl-coenzyme M reductase (mcrA) genes and mcrA transcripts. Putative anaerobic CH4-oxidizing archaea (ANME-2D) and bacteria (NC 10) were scarce (up to 3.4% and 0.5% of archaeal and bacterial 16S rRNA genes, respectively), likely due to the low environmental stability associated with shallow depths. Consequently, the potential anaerobic CH4 oxidation (0-2.1 nmol g-1dry weight (DW)d-1) was not enhanced by the addition of EAs, nor important in consuming the produced CH4 (0.6-82.5 nmol g-1DWd-1). Instead, the increased EA availability suppressed CH4 production via the outcompetition of methanogens by anaerobically respiring bacteria and via the increased protection of organic matter from microbial degradation induced by Fe3+ and Mn4+. Future studies could particularly assess whether anaerobic CH4 oxidation has any ecological relevance in reducing CH4 emissions from the numerous CH4-emitting shallow lakes in boreal and tundra landscapes.

Microbial Community Response on Wastewater Discharge in Boreal Lake Sediments.

Despite high performance, municipal wastewater treatment plants (WWTPs) still discharge significant amounts of organic material and nitrogen and even microbes into the receiving water bodies, altering physico-chemical conditions and microbial functions. In this study, we examined how nitrified wastewater affects the microbiology of boreal lake sediments. Microbial community compositions were assessed with next generation sequencing of the 16S rRNA gene, and a more detailed view on nitrogen transformation processes was gained with qPCR targeting on functional genes (nirS, nirK, nosZI, nosZII, amoAarchaea, and amoAbacteria). In both of the two studied lake sites, the microbial community composition differed significantly between control point and wastewater discharge point, and a gradual shift toward natural community composition was seen downstream following the wastewater gradient. SourceTracker analysis predicted that ∼2% of sediment microbes were of WWTP-origin on the study site where wastewater was freely mixed with the lake water, while when wastewater was specially discharged to the sediment surface, ∼6% of microbes originated from WWTP, but the wastewater-influenced area was more limited. In nitrogen transformation processes, the ratio between nitrifying archaea (AOA) and bacteria (AOB) was affected by wastewater effluent, as the AOA abundance decreased from the control point (AOA:AOB 28:1 in Keuruu, 11:1 in Petäjävesi) to the wastewater-influenced sampling points, where AOB dominated (AOA:AOB 1:2-1:15 in Keuruu, 1:3-1:19 in Petäjävesi). The study showed that wastewater can affect sediment microbial community through importing nutrients and organic material and altering habitat characteristics, but also through bringing wastewater-originated microbes to the sediment, and may thus have significant impact on the freshwater biogeochemistry, especially in the nutrient-poor boreal ecosystems.

Methylophilaceae and Hyphomicrobium as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants.

Molecular monitoring of bacterial communities can explain and predict the stability of bioprocesses in varying physicochemical conditions. To study methanol-fed denitrification biofilters of municipal wastewater treatment plants, bacterial communities of two full-scale biofilters were compared through fingerprinting and sequencing of the 16S rRNA genes. Additionally, 16S rRNA gene fingerprinting was used for 10-week temporal monitoring of the bacterial community in one of the biofilters. Combining the data with previous study results, the family Methylophilaceae and genus Hyphomicrobium were determined as suitable target groups for monitoring. An increase in the relative abundance of Hyphomicrobium-related biomarkers occurred simultaneously with increases in water flow, NO x- load, and methanol addition, as well as a higher denitrification rate, although the dominating biomarkers linked to Methylophilaceae showed an opposite pattern. The results indicate that during increased loading, stability of the bioprocess is maintained by selection of more efficient denitrifier populations, and this progress can be analyzed using simple molecular fingerprinting.

Methylophaga and Hyphomicrobium can be used as target genera in monitoring saline water methanol-utilizing denitrification.

Which bacterial taxonomic groups can be used in monitoring saline water methanol-utilizing denitrification and whether nitrate is transformed into N2 in the process are unclear. Therefore, methylotrophic bacterial communities of two efficiently functioning (nitrate/nitrite reduction was 63-96 %) tropical and cool seawater reactors at a public aquarium were investigated with clone library analysis and 454 pyrosequencing of the 16S rRNA genes. Transformation of nitrate into N2 was confirmed using 15N labeling in incubation of carrier material from the tropical reactor. Combining the data with previous study results, Methylophaga and Hyphomicrobium were determined to be suitable target genera for monitoring the function of saline water methanol-fed denitrification systems. However, monitoring was not possible at the single species level. Interestingly, potential nitrate-reducing methylotrophs within Filomicrobium and closely related Fil I and Fil II clusters were detected in the reactors suggesting that they also contributed to methylotrophic denitrification in the saline environment.

A practical method for barcoding and size-trimming PCR templates for amplicon sequencing.

Sample barcoding facilitates the analysis of tens or even hundreds of samples in a single next-generation sequencing (NGS) run, but more efficient methods are needed for high-throughput barcoding and size-trimming of long PCR products. Here we present a two-step PCR approach for barcoding followed by pool shearing, adapter ligation, and 5' end selection for trimming sets of DNA templates of any size. Our new trimming method offers clear benefits for phylogenetic studies, since targeting exactly the same region maximizes the alignment and enables the use of operational taxonomic unit (OTU)-based algorithms.

Functional gene pyrosequencing reveals core proteobacterial denitrifiers in boreal lakes.

Denitrification is an important microbial process in aquatic ecosystems that can reduce the effects of eutrophication. Here, quantification and pyrosequencing of nirS, nirK, and nosZ genes encoding for nitrite and nitrous oxide reductases was performed in sediment samples from four boreal lakes to determine the structure and seasonal stability of denitrifying microbial populations. Sediment quality and nitrate concentrations were linked to the quantity and diversity of denitrification genes, the abundance of denitrifying populations (nirS and nosZ genes) correlated with coupled nitrification-denitrification (Dn), and the denitrification of the overlying water NO3 (-) (Dw) correlated with the nirS/nirK ratio. The number of core nirS, nirK, and nosZ operational taxonomical units (OTUs) was low (6, 7, and 3, respectively), and most of these core OTUs were shared among the lakes. Dominant nirK sequences matched best with those of the order Rhizobiales, which was one of the main bacterial orders present in the sediment microbiomes, whereas the dominant nirS sequences were affiliated with the order Burkholderiales. Over half of the nosZ sequences belonged to a single OTU of the order Burkholderiales, but coupled nitrification-denitrification rate correlated with another dominant nosZ OTU assigned to the order Rhodospirillales. The study indicates that a few core proteobacterial clusters may drive denitrification in boreal lake sediments, as the same Alpha- and Betaproteobacteria denitrifier clusters were present in different lakes and seasons.