Bianca Pommerenke

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

ABSTRACT Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H2-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [13C]propionate (<10 mM) and the oxidation of ∼30 μmol of 13C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” 13C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of 13C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [13C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.

DNA-, rRNA- and mRNA-based stable isotope probing of aerobic methanotrophs in lake sediment

A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms.

Structure and Topology of Microbial Communities in the Major Gut Compartments of Melolontha melolontha Larvae (Coleoptera: Scarabaeidae)

ABSTRACT Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and γ-Proteobacteria were restricted to the lumen, whereas clones related to the β- and δ-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5′-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.

Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment

In this study, we demonstrate the possibility of obtaining a targeted metatranscriptome from a functional group of microorganisms using a stable isotope probing (SIP) approach. Methanotrophs in lake sediment were labelled using (13)CH4, and both labelled and unlabelled-RNA were isolated and sequenced by 454 pyrosequencing. The unlabelled metatranscriptome had a large diversity of bacterial, archaeal, eukaryotic and viral sequences as expected from a diverse sediment community. In contrast, the labelled-RNA metatranscriptome was dominated by methanotroph sequences, particularly from Methylococcaceae. Transcripts of the methane monooxygenase genes pmoCAB were the most abundant in this metatranscriptome, and the pathway of methane oxidation to CO2 could be traced, as well as many steps in the ribulose monophosphate pathway for carbon assimilation. A high abundance of mRNA transcripts for proteins related to motility was detected, suggesting an importance for methanotrophs in lake sediments. This combination of SIP and metatranscriptomics should be broadly applicable, and will enhance the detection and identification of mRNA from target organisms.

Geobacter, Anaeromyxobacter and Anaerolineae populations are enriched on anodes of root exudate-driven microbial fuel cells in rice field soil

Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC.

Diversity and Activity of Denitrifiers of Chilean Arid Soil Ecosystems

The Chilean sclerophyllous matorral is a Mediterranean semiarid ecosystem affected by erosion, with low soil fertility, and limited by nitrogen. However, limitation of resources is even more severe for desert soils such as from the Atacama Desert, one of the most extreme arid deserts on Earth. Topsoil organic matter, nitrogen and moisture content were significantly higher in the semiarid soil compared to the desert soil. Although the most significant loss of biologically preferred nitrogen from terrestrial ecosystems occurs via denitrification, virtually nothing is known on the activity and composition of denitrifier communities thriving in arid soils. In this study we explored denitrifier communities from two soils with profoundly distinct edaphic factors. While denitrification activity in the desert soil was below detection limit, the semiarid soil sustained denitrification activity. To elucidate the genetic potential of the soils to sustain denitrification processes we performed community analysis of denitrifiers based on nitrite reductase (nirK and nirS) genes as functional marker genes for this physiological group. Presence of nirK-type denitrifiers in both soils was demonstrated but failure to amplify nirS from the desert soil suggests very low abundance of nirS-type denitrifiers shedding light on the lack of denitrification activity. Phylogenetic analysis showed a very low diversity of nirK with only three distinct genotypes in the desert soil which conditions presumably exert a high selection pressure. While nirK diversity was also limited to only few, albeit distinct genotypes, the semiarid matorral soil showed a surprisingly broad genetic variability of the nirS gene. The Chilean matorral is a shrub land plant community which form vegetational patches stabilizing the soil and increasing its nitrogen and carbon content. These islands of fertility may sustain the development and activity of the overall microbial community and of denitrifiers in particular.

Chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of actinobacterial subdivision lineage TM3

Anaerobic nitrate-dependent Fe(II) oxidation is widespread in various environments and is known to be performed by both heterotrophic and autotrophic microorganisms. Although Fe(II) oxidation is predominantly biological under acidic conditions, to date most of the studies on nitrate-dependent Fe(II) oxidation were from environments of circumneutral pH. The present study was conducted in Lake Grosse Fuchskuhle, a moderately acidic ecosystem receiving humic acids from an adjacent bog, with the objective of identifying, characterizing and enumerating the microorganisms responsible for this process. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions have shown the enrichment of TM3 group of uncultured Actinobacteria. A time-course experiment done on these Actinobacteria showed a consumption of Fe(II) and nitrate in accordance with the expected stoichiometry (1:0.2) required for nitrate-dependent Fe(II) oxidation. Quantifications done by most probable number showed the presence of 1 × 104 autotrophic and 1 × 107 heterotrophic nitrate-dependent Fe(II) oxidizers per gram fresh weight of sediment. The analysis of microbial community by 16S rRNA gene amplicon pyrosequencing showed that these actinobacterial sequences correspond to ∼0.6% of bacterial 16S rRNA gene sequences. Stable isotope probing using 13CO2 was performed with the lake sediment and showed labeling of these Actinobacteria. This indicated that they might be important autotrophs in this environment. Although these Actinobacteria are not dominant members of the sediment microbial community, they could be of functional significance due to their contribution to the regeneration of Fe(III), which has a critical role as an electron acceptor for anaerobic microorganisms mineralizing sediment organic matter. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteria.

Drying effects on archaeal community composition and methanogenesis in bromeliad tanks

Tank bromeliads are highly abundant epiphytes in neotropical forests and form a unique canopy wetland ecosystem which is involved in the global methane cycle. Although the tropical climate is characterized by high annual precipitation, the plants can face periods of restricted water. Thus, we hypothesized that water is an important controller of the archaeal community composition and the pathway of methane formation in tank bromeliads. Greenhouse experiments were established to investigate the resident and active archaeal community targeting the 16S rDNA and 16S rRNA in the tank slurry of bromeliads at three different moisture levels. Archaeal community composition and abundance were determined using terminal restriction fragment length polymorphism and quantitative PCR. Release of methane and its stable carbon isotopic signature were determined in a further incubation experiment under two moisture levels. The relative abundance of aceticlastic Methanosaetaceae increased up to 34% and that of hydrogenotrophic Methanobacteriales decreased by more than half with decreasing moisture. Furthermore, at low moisture levels, methane production was up to 100-fold lower (≤0.1-1.1 nmol gdw(-1) d(-1)) than under high moisture levels (10-15 nmol gdw(-1) d(-1)). The rapid response of the archaeal community indicates that the pathway of methane formation in bromeliad tanks may indeed be strongly susceptible to periods of drought in neotropical forest canopies.

Methane emission from feather moss stands

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH4 . However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH4 turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH4 cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH4 emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH4 h-1 gdw-1 . Temperature and water content positively influenced CH4 emission. Methanogenic potentials determined under N2 atmosphere in darkness ranged between 22 and 157 pmol CH4 h-1 gdw-1 . Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH4 was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 104 and 105 copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH4 was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH4 produced was depleted in 13 C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3-62 mM). The δ13 C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH4 by hydrogenotrophic methanogenesis and causing net emission of CH4 under ambient conditions, albeit at low rates.

Hydrogenotrophic methanogenesis is the dominant methanogenic pathway in Neotropical tank bromeliad wetlands

Several thousands of tank bromeliads per hectare of neotropical forest create a unique wetland ecosystem that emits substantial amounts of CH4 . Tank bromeliads growing in the forest canopy (functional type-II tank bromeliads) were found to emit more CH4 than tank bromeliads growing on the forest floor (functional type-I tank bromeliads) but the reasons for this difference and the underlying microbial CH4 -cycling processes have not been studied. Therefore, we characterized archaeal communities in bromeliad tanks of the two different functional types in a neotropical montane forest of southern Ecuador using terminal-restriction fragment length polymorphism (T-RFLP) and performed tank-slurry incubations to measure CH4 production potential, stable carbon isotope fractionation and pathway of CH4 formation. The archaeal community composition was dominated by methanogens and differed between bromeliad functional types. Hydrogenotrophic Methanomicrobiales were the dominant methanogens and hydrogenotrophic methanogenesis was the dominant methanogenic pathway among all bromeliads. The relative abundance of aceticlastic Methanosaetaceae and the relative contribution of aceticlastic methanogenesis increased in type-I tank bromeliads probably due to more oxic conditions in type-I than in type-II bromeliads leading to the previously observed lower in situ CH4 emissions from type-I tank bromeliads but to higher CH4 production potentials in type-I tank bromeliad slurries.