Ruth Henneberger

Ecology and microbial structures of archaeal/bacterial strings‐of‐pearls communities and archaeal relatives thriving in cold sulfidic springs

Recently, a unique microbial community, growing in a whitish, macroscopically visible strings-of-pearls-like structure was discovered in the cold, sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. The pearls interior is predominated by microcolonies of the non-methanogenic SM1 euryarchaeon; the outer part of the pearls is mainly composed of Thiothrix. To screen sulfidic ecosystems for the distribution of such unique microbial communities, comparative microbial and geochemical analyses of cold, sulfidic springs of three geographically distinct locations in Bavaria, Germany, and Dalyan, Turkey, were performed. Here, we report on the discovery and study of another type of strings-of-pearls revealing a new microbial community structure. While the SM1 euryarchaeon is again the predominant archaeal constituent, the bacterial partner is the so-called IMB1 eta-proteobacterium. Due to the predominance of the IMB1 eta-proteobacterium, the strings-of-pearls reveal a fluffy and greyish macroscopical appearance. The phylogenetic survey revealed SM1 euryarchaeal relatives, designated as SM1 group, in all sites studied, indicating a widespread distribution of these archaea in terrestrial ecosystems.

New Insights into the Lifestyle of the Cold-Loving SM1 Euryarchaeon: Natural Growth as a Monospecies Biofilm in the Subsurface

ABSTRACT In the surface waters of sulfidic springs near Regensburg, Bavaria, Germany, the SM1 euryarchaeon, together with filamentous bacteria, forms the recently described unique string-of-pearls community. In addition to naturally occurring string-of-pearls communities, the growth of these communities was also observed on polyethylene nets provided as an artificial attachment material in the streamlets of springs. In order to learn more about the distribution and origin of the SM1 euryarchaeon and its possible occurrence in the subsurface, polyethylene nets were incubated as deeply as possible in different spring holes. After a short residence time, slime-like, milky drops, almost completely composed of SM1 euryarchaeon, were attached to the nets, indicating that this organism grows independent of a partner in deeper earth layers. A newly designed in situ biofilm trapping system allowed the quantitative harvesting of organisms exhibiting this newly discovered lifestyle of the SM1 euryarchaeon for detailed biological studies. The discovery of naturally occurring archaeal biofilms extends our knowledge of the biology and ecological significance of archaea in their environments.

Structure and function of methanotrophic communities in a landfill-cover soil.

In landfill-cover soils, aerobic methane-oxidizing bacteria (MOB) convert CH(4) to CO(2), mitigating emissions of the greenhouse gas CH(4) to the atmosphere. We investigated overall MOB community structure and assessed spatial differences in MOB diversity, abundance and activity in a Swiss landfill-cover soil. Molecular cloning, terminal restriction-fragment length polymorphism (T-RFLP) and quantitative PCR of pmoA genes were applied to soil collected from 16 locations at three different depths to study MOB community structure, diversity and abundance; MOB activity was measured in the field using gas push-pull tests. The MOB community was highly diverse but dominated by Type Ia MOB, with novel pmoA sequences present. Type II MOB were detected mainly in deeper soil with lower nutrient and higher CH(4) concentrations. Substantial differences in MOB community structure were observed between one high- and one low-activity location. MOB abundance was highly variable across the site [4.0 × 10(4) to 1.1 × 10(7) (g soil dry weight)(-1)]. Potential CH(4) oxidation rates were high [1.8-58.2 mmol CH(4) (L soil air)(-1) day(-1) ] but showed significant lateral variation and were positively correlated with mean CH(4) concentrations (P < 0.01), MOB abundance (P < 0.05) and MOB diversity (weak correlation, P < 0.17). Our findings indicate that Methylosarcina and closely related MOB are key players and that MOB abundance and community structure are driving factors in CH(4) oxidation at this landfill.

Field‐scale labelling and activity quantification of methane‐oxidizing bacteria in a landfill‐cover soil

Aerobic methane-oxidizing bacteria (MOB) play an important role in soils, mitigating emissions of the greenhouse gas methane (CH(4)) to the atmosphere. Here, we combined stable isotope probing on MOB-specific phospholipid fatty acids (PLFA-SIP) with field-based gas push-pull tests (GPPTs). This novel approach (SIP-GPPT) was tested in a landfill-cover soil at four locations with different MOB activity. Potential oxidation rates derived from regular- and SIP-GPPTs agreed well and ranged from 0.2 to 52.8 mmol CH(4) (L soil air)(-1) day(-1). PLFA profiles of soil extracts mainly contained C(14) to C(18) fatty acids (FAs), with a dominance of C(16) FAs. Uptake of (13) C into MOB biomass during SIP-GPPTs was clearly indicated by increased δ(13)C values (up to c. 1500‰) of MOB-characteristic FAs. In addition, (13)C incorporation increased with CH(4) oxidation rates. In general, FAs C(14:0) , C(16:1ω8), C(16:1ω7) and C(16:1ω6) (type I MOB) showed highest (13)C incorporation, while substantial (13)C incorporation into FAs C(18:1ω8) and C(18:1ω7) (type II MOB) was only observed at high-activity locations. Our findings demonstrate the applicability of the SIP-GPPT approach for in situ quantification of potential CH(4) oxidation rates and simultaneous labelling of active MOB, suggesting a dominance of type I MOB over type II MOB in the CH(4)-oxidizing community in this landfill-cover soil.

Field-scale tracking of active methane-oxidizing communities in a landfill-cover soil reveals spatial and seasonal variability

Aerobic methane-oxidizing bacteria (MOB) in soils mitigate methane (CH4 ) emissions. We assessed spatial and seasonal differences in active MOB communities in a landfill cover soil characterized by highly variable environmental conditions. Field-based measurements of CH4 oxidation activity and stable-isotope probing of polar lipid-derived fatty acids (PLFA-SIP) were complemented by microarray analysis of pmoA genes and transcripts, linking diversity and function at the field scale. In situ CH4 oxidation rates varied between sites and were generally one order of magnitude lower in winter compared with summer. Results from PLFA-SIP and pmoA transcripts were largely congruent, revealing distinct spatial and seasonal clustering. Overall, active MOB communities were highly diverse. Type Ia MOB, specifically Methylomonas and Methylobacter, were key drivers for CH4 oxidation, particularly at a high-activity site. Type II MOB were mainly active at a site showing substantial fluctuations in CH4 loading and soil moisture content. Notably, Upland Soil Cluster-gamma-related pmoA transcripts were also detected, indicating concurrent oxidation of atmospheric CH4 . Spatial separation was less distinct in winter, with Methylobacter and uncultured MOB mediating CH4 oxidation. We propose that high diversity of active MOB communities in this soil is promoted by high variability in environmental conditions, facilitating substantial removal of CH4 generated in the waste body.

Methane dynamics in an alpine fen: a field-based study on methanogenic and methanotrophic microbial communities

Wetlands are important sources of the greenhouse gas methane (CH4). We provide an in situ study of CH4 dynamics in the permanently submerged soil of a Swiss alpine fen. Physico-chemical pore water analyses were combined with structural and microbiological analyses of soil cores at high vertical resolution down to 50 cm depth. Methanotrophs and methanogens were active throughout the depth profile, and highest abundance of active methanotrophs and methanogens [6.1 × 10(5) and 1.1 × 10(7) pmoA and mcrA transcripts (g soil)(-1), respectively] was detected in the uppermost 2 cm of the soil. Active methanotrophic communities in the near-surface zone, dominated by viable mosses, varied from the communities in the deeper zones, but further changes with depth were not pronounced. Apart from a distinct active methanogenic community in the uppermost sample, a decrease of acetoclastic Methanosaetaceae with depth was observed in concomitance with decreasing root surface area. Overall, root surface area correlated with mcrA transcript abundance and CH4 pore water concentrations, which peaked (137.1 μM) at 10 to 15 cm depth. Our results suggest that stimulation of methanogenesis by root exudates of vascular plants had a stronger influence on CH4 dynamics than stimulation of CH4 oxidation by O2 input.

Methanotrophic and Methanogenic Communities in Swiss Alpine Fens Dominated by Carex rostrata and Eriophorum angustifolium.

ABSTRACT Vascular plants play a key role in controlling CH4 emissions from natural wetlands, because they influence CH4 production, oxidation, and transport to the atmosphere. Here we investigated differences in the abundance and composition of methanotrophic and methanogenic communities in three Swiss alpine fens dominated by different vascular plant species under natural conditions. The sampling locations either were situated at geographically distinct sites with different physicochemical properties but the same dominant plant species (Carex rostrata) or were located within the same site, showing comparable physicochemical pore water properties, but had different plant species (C. rostrata or Eriophorum angustifolium). All three locations were permanently submerged and showed high levels of CH4 emissions (80.3 to 184.4 mg CH4 m−2 day−1). Soil samples were collected from three different depths with different pore water CH4 and O2 concentrations and were analyzed for pmoA and mcrA gene and transcript abundance and community composition, as well as soil structure. The dominant plant species appeared to have a significant influence on the composition of the active methanotrophic communities (transcript level), while the methanogenic communities differed significantly only at the gene level. Yet no plant species-specific microbial taxa were discerned. Moreover, for all communities, differences in composition were more pronounced with the site (i.e., with different physicochemical properties) than with the plant species. Moreover, depth significantly influenced the composition of the active methanotrophic communities. Differences in abundance were generally low, and active methanotrophs and methanogens coexisted at all three locations and depths independently of CH4 and O2 concentrations or plant species.

In-Situ Sonication for Enhanced Recovery of Aquifer Microbial Communities

Sampling methods for characterization of microbial communities in aquifers should target both suspended and attached microorganisms (biofilms). We investigated the effectiveness and reproducibility of low-frequency (200 Hz) sonication pulses on improving extraction efficiency and quality of microorganisms from a petroleum-contaminated aquifer in Studen (Switzerland). Sonication pulses at different power levels (0.65, 0.9, and 1.1 kW) were applied to three different groundwater monitoring wells. Groundwater samples extracted after each pulse were compared with background groundwater samples for cell and adenosine tri-phosphate concentration. Turbidity values were obtained to assess the release of sediment fines and associated microorganisms. The bacterial community in extracted groundwater samples was analyzed by terminal-restriction-fragment-length polymorphism and compared with communities obtained from background groundwater samples and from sediment cores. Sonication enhanced the extraction efficiency up to 13-fold, with most of the biomass being associated with the sediment fines extracted with groundwater. Consecutive pulses at constant power were decreasingly effective, while pulses with higher power yielded the best results both in terms of extraction efficiency and quality. Our results indicate that low-frequency sonication may be a viable and cost-effective tool to improve the extraction of microorganisms from aquifers, taking advantage of existing groundwater monitoring wells.

Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome.

A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative β-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any β-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies.

Positive diversity-functioning relationships in model communities of methanotrophic bacteria

Biodiversity enhances ecosystem functions such as biomass production and nutrient cycling. Although the majority of the terrestrial biodiversity is hidden in soils, very little is known about the importance of the diversity of microbial communities for soil functioning. Here, we tested effects of biodiversity on the functioning of methanotrophs, a specialized group of soil bacteria that plays a key role in mediating greenhouse gas emissions from soils. Using pure strains of methanotrophic bacteria, we assembled artificial communities of different diversity levels, with which we inoculated sterile soil microcosms. To assess the functioning of these communities, we measured methane oxidation by gas chromatography throughout the experiment and determined changes in community composition and community size at several time points by quantitative PCR and sequencing. We demonstrate that microbial diversity had a positive overyielding effect on methane oxidation, in particular at the beginning of the experiment. This higher assimilation of CH4 at high diversity translated into increased growth and significantly larger communities towards the end of the study. The overyielding of mixtures with respect to CH4 consumption and community size were positively correlated. The temporal CH4 consumption profiles of strain monocultures differed, raising the possibility that temporal complementarity of component strains drove the observed community-level strain richness effects; however, the community niche metric we derived from the temporal activity profiles did not explain the observed strain richness effect. The strain richness effect also was unrelated to both the phylogenetic and functional trait diversity of mixed communities. Overall, our results suggest that positive biodiversity-ecosystem-function relationships show similar patterns across different scales and may be widespread in nature. Additionally, biodiversity is probably also important in natural methanotrophic communities for the ecosystem function methane oxidation. Therefore, maintaining soil conditions that support a high diversity of methanotrophs may help to reduce the emission of the greenhouse gas methane.