Stefan Ratering

Biochar and hydrochar effects on greenhouse gas (carbon dioxide, nitrous oxide, and methane) fluxes from soils.

With a growing world population and global warming, we are challenged to increase food production while reducing greenhouse gas (GHG) emissions. We studied the effects of biochar (BC) and hydrochar (HC) produced via pyrolysis or hydrothermal carbonization, respectively, on GHG fluxes in three laboratory incubation studies. In the first experiment, ryegrass was grown in sandy loam mixed with equal amounts of a nitrogen-rich peanut hull BC, compost, BC+compost, double compost, or no addition (control); wetting-drying cycles and N fertilization were applied. Biochar with or without compost significantly reduced NO emissions and did not change the CH uptake, whereas ryegrass yield was significantly increased. In the second experiment, 0% (control) or 8% (w/w) of BC (peanut hull, maize, wood chip, or charcoal) or 8% HC (beet chips or bark) was mixed into a soil and incubated at 65% water-holding capacity (WHC) for 140 d. Treatments included simulated plowing and N fertilization. All BCs reduced NO emissions by ∼60%. Hydrochars reduced NO emissions only initially but significantly increased them after N fertilization to 302% (HC-beet) and 155% (HC-bark) of the control emissions, respectively. Large HC-associated CO emissions suggested that microbial activity was stimulated and that HC was less stable than BC. In the third experiment, nutrient-rich peanut hull BC addition and incubation over 1.5 yr at high WHCs did not promote NO emissions. However, NO emissions were significantly increased with BC after NHNO addition. In conclusion, BC reduced NO emissions and improved the GHG-to-yield ratio under field-relevant conditions. However, the risk of increased NO emissions with HC addition must be carefully evaluated.

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.

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.

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.

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.

Paradox of plant growth promotion potential of rhizobacteria and their actual promotion effect on growth of barley (Hordeum vulgare L.) under salt stress

From the rhizosphere of two salt tolerant plant species, Hordeum secalinum and Plantago winteri growing in a naturally salt meadow, 100 strains were isolation on enrichment media for various plant growth-promoting (PGP) functions (ACC deaminase activity, auxin synthesis, calcium phosphate mobilization and nitrogen fixation). Based on the taxonomic affiliation of the isolated bacteria and their enrichment medium 22 isolates were selected to test their growth promotion effect on the crop barley (Hordeum vulgare) under salt stress in pot experiment. In parallel the isolates were characterized in pure culture for their plant growth-promoting activities. Surprisingly the best promotors did not display a promising set of PGP activities. Isolates with multiple PGP-activities in pure culture like Microbacterium natoriense strain E38 and Pseudomonas brassicacearum strain E8 did not promote plant growth. The most effective isolate was strain E108 identified as Curtobacterium flaccumfaciens, which increased barley growth up to 300%. In pure culture strain E108 showed only two out of six plant growth promoting activities and would have been neglected. Our results highlight that screening based on pure culture assays may not be suitable for recognition of best plant growth promotion candidates and could preclude the detection of both new PGPR and new plant promotion mechanisms.

Bacterial microbiota associated with flower pollen is influenced by pollination type, and shows a high degree of diversity and species-specificity

Diverse microorganisms colonise the different plant-microhabitats, such as rhizosphere and phyllosphere, and play key roles for the host. However, bacteria associated with pollen are poorly investigated, despite its ecological, commercial and medical relevance. Due to structure and nutritive composition, pollen provides a unique microhabitat. Here the bacterial abundance, community structure, diversity and colonization pattern of birch, rye, rapes and autumn crocus pollens were examined, by using cultivation, high-throughput sequencing and microscopy. Cultivated bacteria belonged to Proteobacteria, Actinobacteria and Firmicutes, with remarkable differences at species level between pollen species. High-throughput sequencing of 16S rRNA gene amplicon libraries showed Proteobacteria as the dominant phylum in all pollen species, followed by Actinobacteria, Acidobacteria and Firmicutes. Both plant species and pollination type significant influenced structure and diversity of the pollen microbiota. The insect-pollinated species possessed a more similar microbiota in comparison to the wind-pollinated ones, suggesting a levelling effect by insect vectors. Scanning electron microscopy as well as fluorescent in situ hybridisation coupled with confocal laser scanning microscopy (FISH-CLSM) indicated the tectum surface as the preferred niche of bacterial colonisation. This work is the most comprehensive study of pollen microbiology, and strongly increases our knowledge on one of the less investigated plant-microhabitats.

Cellvibrio diazotrophicus sp. nov., a nitrogen-fixing bacteria isolated from the rhizosphere of salt meadow plants and emended description of the genus Cellvibrio.

Two Gram-reaction-negative, aerobic, nitrogen-fixing, rod-shaped bacteria, designated strains E20 and E50(T), were isolated from the rhizosphere of salt meadow plants Plantago winteri and Hordeum secalinum, respectively, near Münzenberg, Germany. Based on the 16S rRNA gene sequence analysis both strains E20 and E50(T) are affiliated with the genus Cellvibrio, sharing the highest similarity with Cellvibrio gandavensis LMG 18551(T) (96.4%) and (97.1%), respectively. Strains E20 and E50(T) were oxidase and catalase-positive, grew at a temperature range between 16 and 37 °C and in the presence of 0-5% NaCl (w/v). The DNA G+C contents were 52.1 mol% (E20) and 51.6 mol% (E50(T)). Major fatty acids of strains E20 and E50(T) were summed feature 3 (C16 : 1ω7c and/or iso-C(15 : 0) 2-OH), C(16 : 0), C(18 : 1)ω7c, C(12 : 0), C(18 : 0) and C(12 : 0) 3-OH. The DNA-DNA relatedness of the strains to Cellvibrio gandavensis LMG 18551(T) was 39% for strain E20 and 58% for strain E50(T). The nitrogen fixation capability of strains E20 and E50(T) was confirmed by the acetylene reduction assay. On the basis of our polyphasic taxonomic study, strains E20 and E50(T) represent a novel species of the genus Cellvibrio, for which the name Cellvibrio diazotrophicus is proposed. The type strain of Cellvibrio diazotrophicus is E50(T) ( = LMG 27267(T) = KACC 17069(T)). An emended description of the genus Cellvibrio is proposed based on the capability of fixing nitrogen and growth in presence of up to 5% NaCl (w/v).

Rheinheimera hassiensis sp. nov. and Rheinheimera muenzenbergensis sp. nov., two species from the rhizosphere of Hordeum secalinum.

Two motile, Gram-staining-negative, aerobic, rod-shaped bacteria designated strains E48(T) and E49(T) were isolated from the rhizosphere of Hordeum secalinum from a natural salt meadow near Münzenberg, Germany. 16S rRNA gene sequence similarity analysis revealed that strains E48(T) and E49(T) shared similarities of 97.6 % with Rheinheimera pacifica KMM 1406(T) and 98.5 % with Rheinheimera nanhaiensis E407-8(T), respectively. Major fatty acids of strain E48(T) were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C17 : 1ω8c, and of strain E49(T) were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The DNA G+C contents were 50.5 mol% (E48(T)) and 50.0 mol% (E49(T)). Strains E48(T) and E49(T) grew at 4-37 °C (optimum 28 °C) and with 0-6 % NaCl (optimum 0-3 %) and 0-5 % NaCl (optimum 0-3 %), respectively. The potential for nitrogen fixation by strains E48(T) and E49(T) was evaluated by molecular techniques and the acetylene reduction assay. The DNA-DNA hybridization, physiological and molecular data demonstrated that strains E48(T) and E49(T) represent two novel species of the genus Rheinheimera, and therefore the names Rheinheimera hassiensis sp. nov. (type strain E48(T) = LMG 27268(T) = KACC 17070(T)) and Rheinheimera muenzenbergensis sp. nov. (type strain E49(T) = LMG 27269(T) = KACC 17071(T)) are proposed.

Changes of the microbial population structure in an overloaded fed-batch biogas reactor digesting maize silage.

Two parallel, stable operating biogas reactors were fed with increasing amounts of maize silage to monitor microbial community changes caused by overloading. Changes of microorganisms diversity revealed by SSCP (single strand conformation polymorphism) indicating an acidification before and during the pH-value decrease. The earliest indicator was the appearance of a Methanosarcina thermophila-related species. Diversity of dominant fermenting bacteria within Bacteroidetes, Firmicutes and other Bacteria decreased upon overloading. Some species became dominant directly before and during acidification and thus could be suitable as possible indicator organisms for detection of futurity acidification. Those bacteria were related to Prolixibacter bellariivorans and Streptococcus infantarius subsp. infantarius. An early detection of community shifts will allow better feeding management for optimal biogas production.