Response to nitrogen addition reveals metabolic and ecological strategies of soil bacteria.

The nitrogen (N) cycle represents one of the most well-studied systems, yet the taxonomic diversity of the organisms that contribute to it is mostly unknown, or linked to poorly characterized microbial groups. While new information has allowed functional groups to be refined, they still rely on a priori knowledge of enzymes involved and the assumption of functional conservation, with little connection to the role the transformations, plays for specific organisms. Here, we use soil microcosms to test the impact of N deposition on prokaryotic communities. By combining chemical, genomic and transcriptomic analysis, we are able to identify and link changes in community structure to specific organisms catalysing given chemical reactions. Urea deposition led to a decrease in prokaryotic richness, and a shift in community composition. This was driven by replacement of stable native populations, which utilize energy from N-linked redox reactions for physiological maintenance, with fast responding populations that use this energy for growth. This model can be used to predict response to N disturbances and allows us to identify putative life strategies of different functional and taxonomic groups, thus providing insights into how they persist in ecosystems by niche differentiation.

One-step PCR amplicon sequencing libraries perform better than two-step when assessing soil microbial diversity and community profiles.

Despite adoption of high-throughput sequencing of PCR-amplified microbial taxonomic markers for ecological analyses, distinct approaches for preparing amplicon libraries exist. One approach utilises long fusion primers and a single PCR (one-step) while another utilises shorter primers in a first reaction, before transferring diluted amplicons to a second reaction for barcode index incorporation (two-step). We investigated whether transferring diluted amplicons risked creating artificially simplified, poorly diverse communities. In soils from three sites with paired cropland and forest, one-step yielded higher alpha-diversity indices, including detection of two-four times more unique taxa. Modelling expected taxa per sequence observation predicted that one-step reaches full coverage by 104 sequences per sample while two-step needs 105-109. Comparisons of rank abundance demonstrated that two-step covered only 38%-69% of distributions. Beta-diversity showed better separation of communities in response to land use change under one-step, although both approaches showed a significant effect. Driving differences was underestimation of relatively minor taxa with the two-step procedure. These taxa were low in abundance, yet play important roles in carbon cycling, secondary metabolite production, anaerobic metabolism, and bacterial predation. We conclude that one-step amplicon libraries are advisable for studies focussed on diversity or relatively minor yet functionally important taxa.

Competition and community succession link N transformation and greenhouse gas emissions in urine patches.

Nitrous oxide (N2O) is a strong greenhouse gas produced by biotic/abiotic processes directly linked to both fungal and prokaryotic communities that produce, consume or create conditions leading to its emission. In soils exposed to nitrogen (N) in the form of urea, an ecological succession is triggered resulting in a dynamic turnover of microbial populations. However, knowledge of the mechanisms controlling this succession and the repercussions for N2O emissions remain incomplete. Here, we monitored N2O production and fungal/prokaryotic community changes (via 16S and 18S amplicon sequencing) in soil microcosms exposed to urea. Contributions of microbes to emissions were determined using biological inhibitors. Results confirmed that urea leads to shifts in microbial community assemblages by selecting for certain microbial groups (fast growers) as dictated through life history strategies. Urea reduced overall community diversity by conferring dominance to specific groups at different stages in the succession. The diversity lost under urea was recovered with inhibitor addition through the removal of groups that were actively growing under urea indicating that species replacement is mediated in part by competition. Results also identified fungi as significant contributors to N2O emissions, and demonstrate that dominant fungal populations are consistently replaced at different stages of the succession. These successions were affected by addition of inhibitors which resulted in strong decreases in N2O emissions, suggesting that fungal contributions to N2O emissions are larger than that of prokaryotes.

Composition and Diversity of Natural Bacterial Communities in Mabisi, a Traditionally Fermented Milk.

Many traditionally fermented milk products such as mabisi involve spontaneous fermentation, which can result in bacterial community composition variation due to selection pressure. The aim of this study was to determine the composition of bacterial communities in the different types of mabisi produced across Zambia and identify the factors that influence their composition. Samples of mabisi were collected across the country, and analyzed for pH and bacterial communities using 16S rRNA amplicon sequencing. We found that the bacterial community composition was dominated by members of two phyla, i.e., Firmicutes and Proteobacteria, from which the top 10 most abundant genera were Lactococcus, Lactobacillus, Streptococcus, Enterobacter, Citrobacter, Klebsiella, Kluyvera, Buttiauxella, Aeromonas, and Acinetobacter. The most dominant genus was Lactococcus, which was present in all types of mabisi produced from all regions. The mabisi products from traditional mabisi production regions (TMPRs) were dominated by lactic acid bacteria (LAB) whereas products from non-TMPRs were dominated by non-LAB species. Tonga mabisi, the most popular type of mabisi produced in non-TMPRs, had the most complex and diverse bacterial community composition compared to the other types, which included barotse, backslopping, creamy, and thick-tonga mabisi. Other factors that influenced bacterial community composition were geographical location, fermentation duration and pH while the type of fermentation container and producer did not. This study provides new insights that can be applied in starter culture development as well as microbial functionality studies.

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.

Author Correction: Influence of soil moisture on codenitrification fluxes from a urea-affected pasture soil.

A correction has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Seasonal Variation in Abundance and Diversity of Bacterial Methanotrophs in Five Temperate Lakes.

Lakes are significant sources of methane (CH4) to the atmosphere. Within these systems, methanotrophs consume CH4 and act as a potential biofilter mitigating the emission of this potent greenhouse gas. However, it is still not well understood how spatial and temporal variation in environmental parameters influence the abundance, diversity, and community structure of methanotrophs in lakes. To address this gap in knowledge, we collected water samples from three depths (surface, middle, and bottom) representing oxic to suboxic or anoxic zones of five different Swedish lakes in winter (ice-covered) and summer. Methanotroph abundance was determined by quantitative real time polymerase chain reaction and a comparison to environmental variables showed that temperature, season as well as depth, phosphate concentration, dissolved oxygen, and CH4 explained the observed variation in methanotroph abundance. Due to minimal differences in methane concentrations (0.19 and 0.29 µM for summer and winter, respectively), only a weak and even negative correlation was observed between CH4 and methanotrophs, which was possibly due to usage of CH4. Methanotrophs were present at concentrations ranging from 105 to 106 copies/l throughout the oxic (surface) and suboxic/anoxic (bottom) water mass of the lakes, but always contributed less than 1.3% to the total microbial community. Relative methanotroph abundance was significantly higher in winter than in summer and consistently increased with depth in the lakes. Phylogenetic analysis of pmoA genes in two clone libraries from two of the ice-covered lakes (Ekoln and Ramsen) separated the methanotrophs into five distinct clusters of Methylobacter sp. (Type I). Terminal restriction fragment length polymorphism analysis of the pmoA gene further revealed significant differences in methanotrophic communities between lakes as well as between winter and summer while there were no significant differences between water layers. The study provides new insights into diversity, abundance, community composition and spatial as well as temporal distribution of freshwater methanotrophs in low-methane dimictic lakes.

Influence of soil moisture on codenitrification fluxes from a urea-affected pasture soil.

Intensively managed agricultural pastures contribute to N2O and N2 fluxes resulting in detrimental environmental outcomes and poor N use efficiency, respectively. Besides nitrification, nitrifier-denitrification and heterotrophic denitrification, alternative pathways such as codenitrification also contribute to emissions under ruminant urine-affected soil. However, information on codenitrification is sparse. The objectives of this experiment were to assess the effects of soil moisture and soil inorganic-N dynamics on the relative contributions of codenitrification and denitrification (heterotrophic denitrification) to the N2O and N2 fluxes under a simulated ruminant urine event. Repacked soil cores were treated with 15N enriched urea and maintained at near saturation (-1 kPa) or field capacity (-10 kPa). Soil inorganic-N, pH, dissolved organic carbon, N2O and N2 fluxes were measured over 63 days. Fluxes of N2, attributable to codenitrification, were at a maximum when soil nitrite (NO2-) concentrations were elevated. Cumulative codenitrification was higher (P = 0.043) at -1 kPa. However, the ratio of codenitrification to denitrification did not differ significantly with soil moisture, 25.5 ± 15.8 and 12.9 ± 4.8% (stdev) at -1 and -10 kPa, respectively. Elevated soil NO2- concentrations are shown to contribute to codenitrification, particularly at -1 kPa.

Remnants of marine bacterial communities can be retrieved from deep sediments in lakes of marine origin.

Some bacteria can be preserved over time in deep sediments where they persist either in dormant or slow-growing vegetative stages. Here, we hypothesized that such cells can be revived when exposed to environmental conditions similar to those before they were buried in the sediments. To test this hypothesis, we collected bacteria from sediment samples of different ages (140-8500 calibrated years before present, cal BP) from three lakes that differed in the timing of their physical isolation from the Baltic Sea following postglacial uplift. After these bacterial communities were grown in sterile water from the Baltic Sea, we determined the proportion of 16S rRNA sequence reads associated with marine habitats by extracting the environment descriptive terms of homologous sequences retrieved from public databases. We found that the proportion of reads associated with marine descriptive term was significantly higher in cultures inoculated with sediment layers formed under Baltic conditions and where salinities were expected to be similar to current levels. Moreover, a similar pattern was found in the original sediment layers. Our study, therefore, suggests that remnants of marine bacterial communities can be preserved in sediments over thousands of years and can be revived from deep sediments in lakes of marine origin.

High-Resolution Denitrification Kinetics in Pasture Soils Link N2O Emissions to pH, and Denitrification to C Mineralization.

Denitrification in pasture soils is mediated by microbial and physicochemical processes leading to nitrogen loss through the emission of N2O and N2. It is known that N2O reduction to N2 is impaired by low soil pH yet controversy remains as inconsistent use of soil pH measurement methods by researchers, and differences in analytical methods between studies, undermine direct comparison of results. In addition, the link between denitrification and N2O emissions in response to carbon (C) mineralization and pH in different pasture soils is still not well described. We hypothesized that potential denitrification rate and aerobic respiration rate would be positively associated with soils. This relationship was predicted to be more robust when a high resolution analysis is performed as opposed to a single time point comparison. We tested this by characterizing 13 different temperate pasture soils from northern and southern hemispheres sites (Ireland and New Zealand) using a fully automated-high-resolution GC detection system that allowed us to detect a wide range of gas emissions simultaneously. We also compared the impact of using different extractants for determining pH on our conclusions. In all pH measurements, soil pH was strongly and negatively associated with both N2O production index (IN2O) and N2O/(N2O+N2) product ratio. Furthermore, emission kinetics across all soils revealed that the denitrification rates under anoxic conditions (NO+N2O+N2 µmol N/h/vial) were significantly associated with C mineralization (CO2 µmol/h/vial) measured both under oxic (r2 = 0.62, p = 0.0015) and anoxic (r2 = 0.89, p<0.0001) conditions.

Phylogenetic and functional potential links pH and N2O emissions in pasture soils.

Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir &nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.