Sylvia Schnell

Molecular Analyses of Novel Methanotrophic Communities in Forest Soil That Oxidize Atmospheric Methane

ABSTRACT Forest and other upland soils are important sinks for atmospheric CH4, consuming 20 to 60 Tg of CH4 per year. Consumption of atmospheric CH4 by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH4 oxidation rates in a German forest soil and characterized the methanotrophic populations by PCR and denaturing gradient gel electrophoresis (DGGE) with primer sets targeting thepmoA gene, coding for the α subunit of the particulate methane monooxygenase, and the small-subunit rRNA gene (SSU rDNA) of all life. The forest soil was a sink for atmospheric CH4 in situ and in vitro at all times. In winter, atmospheric CH4was oxidized in a well-defined subsurface soil layer (6 to 14 cm deep), whereas in summer, the complete soil core was active (0 cm to 26 cm deep). The content of total extractable DNA was about 10-fold higher in summer than in winter. It decreased with soil depth (0 to 28 cm deep) from about 40 to 1 μg DNA per g (dry weight) of soil. The PCR product concentration of SSU rDNA of all life was constant both in winter and in summer. However, the PCR product concentration of pmoAchanged with depth and season. pmoA was detected only in soil layers with active CH4 oxidation, i.e., 6 to 16 cm deep in winter and throughout the soil core in summer. The same methanotrophic populations were present in winter and summer. Layers with high CH4 consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH4oxidation activity was positively correlated with the number of methanotrophic populations present. The pmoA sequences derived from excised DGGE bands were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in atmospheric CH4 consumption.

Localization of iron-reducing activity in paddy soil by profile studies.

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm (≈ 100 µmol/cm3). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm (≈ 100--200 µmol/cm3).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw−1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO2 by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations (≈ 350µmol/cm3) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO3−, 40 µM SO42−) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.

Unexpected Stability of Bacteroidetes and Firmicutes Communities in Laboratory Biogas Reactors Fed with Different Defined Substrates

ABSTRACT In the present study, bacterial communities in 200-liter biogas reactors containing liquid manure consecutively fed with casein, starch, and cream were investigated over a period of up to 33 days. A 16S rRNA gene clone library identified Bacteroidetes and Firmicutes as the most abundant bacterial groups in the starting material, at 58.9% and 30.1% of sequences, respectively. The community development of both groups was monitored by real-time PCR and single-strand conformation polymorphism (SSCP) analysis. The Firmicutes and Bacteroidetes communities were unexpectedly stable and hardly influenced by batch-feeding events. The continuous feeding of starch led to community shifts that nevertheless contributed to a stable reactor performance. A longer starving period and a change in the pH value resulted in further community shifts within the Bacteroidetes but did not influence the Firmicutes. Predominant DNA bands from SSCP gels were cloned and sequenced. Sequences related to Peptococcaceae, Cytophagales, and Petrimonas sulfuriphila were found in all samples from all experiments. Real-time PCR demonstrated the abundance of members of the phylum Bacteroidetes and also reflected changes in gene copy numbers in conjunction with a changing pH value and acetate accumulation.

Simultaneous Determination of Iron(III), Iron(II), and Manganese(II) in Environmental Samples by Ion Chromatography

A chromatographic method was developed for simultaneous determination of Fe(III), Fe(II), Mn(II), and other transition metals. A high-performance polymer-coated silica-based cation exchange column was used for the separation of metals from hydrochloric acid-extracted environmental samples. After separation, the metals were mixed with PAR [4-(2-pyridylazo)resorcinol] and passed through a supercoiled Teflon postcolumn reactor. The absorbance of the colored complexes was recorded on line at 520 nm. Detection limits for Fe(III), Fe(II), and Mn(II) were 19, 9, and 25 fmol, respectively. Linear detector response was observed up to concentrations of 5-20 nmol. At concentrations of 2 nmol, the analyses were reproducible with 0.4% for Fe(III), 0.2% for Fe(II), and 0.5% for Mn(II). The method was compared with a photometric assay using ferrozine for Fe(II) determinations and hydroxylamine as reducing agent for Fe(III). Concentrations of Fe(III) were calculated from ferrozine determinations prior to and after the reduction of samples. Good agreement of both methods was obtained for various applications. Fe(III) and Fe(II) concentrations were determined in a depth profile of a flooded rice paddy soil. The profile showed increasing Fe(II) concentrations from the upper 1 mm soil layer to a depth of 10 mm, indicating the highest iron-reducing activity in a soil depth of 3 mm. A growth experiment with the metalreducing bacterium Geobacter metallireducens showed Fe(III) reduction concomitant with the production of Fe(II) and consumption of acetate as a carbon and energy source. Production of Mn(II) from manganese oxide was followed with an enrichment culture of Mn(IV)-reducing bacteria.

Strain FAc12, a dissimilatory iron‐reducing member of the Anaeromyxobacter subgroup of Myxococcales

Dissimilatory iron reduction is of quantitative importance during anaerobic degradation of organic matter in flooded rice field soils. To isolate dissimilatory Fe(III)-reducing microorganisms from rice soil, enrichments were carried out with acetate and ferrihydrite. One of these resulted in the isolation of strain FAc12. This organism grew anaerobically in defined mineral medium with acetate as electron donor and with ferric citrate, ferrihydrite, or nitrate as electron acceptor. Strain FAc12 also grew well aerobically in defined mineral medium with acetate, citrate, glucose, or with complex medium. Comparative sequence analysis of its 16S rRNA gene revealed that strain FAc12 is most closely related to the very recently described Anaeromyxobacter dehalogenans within the order Myxococcales. The overall similarity value between the 16S rRNA gene sequences of strain FAc12 and the type strain of A. dehalogenans (2CP-1) is 99.5%. A. dehalogenans has been reported to be the first facultative anaerobic myxobacterium, while all other members of the Myxococcales were known to be strict aerobes. A. dehalogenans is able to grow by chlororespiration and to utilize nitrate as terminal electron acceptor for growth. Cultivation-independent retrieval of 16S rRNA gene sequences revealed that rice roots are also colonized by various members of this novel subgroup. This information and the metabolic capacity of strain FAc12 allows the assumption that these organisms are physiologically adapted to environments characterized by spatial and temporal fluctuations between oxic and anoxic conditions, as is typically the case for flooded rice soil.

Suppression of methane emission from rice paddies by ferric iron fertilization

Abstract Rice microcosms incubated in the greenhouse showed that methane emission was reduced after fertilization of the soil with ferric iron oxide–ferrihydrite. The total methane emission during the vegetation period of rice was reduced by 43% and 84%, with the addition of 15 and 30 g of ferrihydrite per kg of soil, respectively. Growth of the rice plants was reduced during the first few weeks in the ferrihydrite fertilized soil but was unchanged after 14 weeks. The number of rice seeds produced per plant was identical after the lower iron fertilization and was 82% of the control after the higher iron fertilization. The presence of ferric iron suppressed methanogenesis and methane emission in rice microcosms.

Evidence for methane production by saprotrophic fungi

Methane in the biosphere is mainly produced by prokaryotic methanogenic archaea, biomass burning, coal and oil extraction, and to a lesser extent by eukaryotic plants. Here we demonstrate that saprotrophic fungi produce methane without the involvement of methanogenic archaea. Fluorescence in situ hybridization, confocal laser-scanning microscopy and quantitative real-time PCR confirm no contribution from microbial contamination or endosymbionts. Our results suggest a common methane formation pathway in fungal cells under aerobic conditions and thus identify fungi as another source of methane in the environment. Stable carbon isotope labelling experiments reveal methionine as a precursor of methane in fungi. These findings of an aerobic fungus-derived methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Effects of silver nanoparticles on microbial growth dynamics

Engineered metal nanoparticles are increasingly used in consumer products, in part as additives that exhibit advantageous antimicrobial properties. Conventional nanoparticle susceptibility testing is based largely on determination of nontemporal growth profiles such as measurements of inhibition zones in common agar diffusion tests, counting of colony‐forming units, or endpoint or regular‐interval growth determination via optical density measurements. For better evaluation of the dynamic effects from exposure to nanoparticles, a cultivation‐based assay was established in a 96‐well format and adapted for time‐resolved testing of the effects of nanoparticles on micro‐organisms.

Hydrogenotrophic methanogens dominate in biogas reactors fed with defined substrates

Methanogenic communities in 200L biogas reactors containing liquid manure were investigated for 33 d. The reactors were consecutively fed with casein, starch and cream. Real-time PCR with primers targeting the gene for methyl coenzyme-M reductase (mcrA) resulted in copy numbers of up to 2.1×10(9) g dry mass(-1). Single strand conformation polymorphism (SSCP) analysis revealed a stable community consisting of few hydrogenotrophic methanogens. One of the two most abundant species was closely related to Methanospirillum hungatei, whereas the other one was only distantly related to other methanogens, with Methanopyrus kandleri being the closest cultivated relative. Most probable number (MPN) cultivations were accomplished with a sample from a 600 m(3) reactor from which all manures used in the experiments originated, and equal cell counts of ca. 10(9) g dry mass(-1) were found for cultivations with acetate, H(2) and methanol. SSCP analysis of these samples and sequencing of the DNA bands identified different hydrogenotrophic methanogens in all samples, and acetoclastic methanogens closely related to Methanosarcina mazei in the samples cultivated with acetate and methanol. As the acetoclastic species were not found in any other SSCP sample, it was supposed that the ammonia values in the manure of the laboratory biogas reactor, which ranged from 2.48 to 3.61 g NH(4)-NL(-1), inhibited the growth of the acetoclastic methanogens.

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.