Matthias Noll

Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing

Methane-oxidizing bacteria (MOB) in soil are not only controlled by their main substrates, methane and oxygen, but also by nitrogen availability. We compared an unfertilized control with a urea-fertilized treatment and applied RNA-stable-isotope-probing to follow activity changes upon fertilization as closely as possible. Nitrogen fertilization of an Italian rice field soil increased the CH4 oxidation rates sevenfold. In the fertilized treatment, isopycnic separation of 13C-enriched RNA became possible after 7 days when 300 micromol 13CH4 g(dry soil)(-1) had been consumed. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprints and clone libraries documented that the type I methanotrophic genera Methylomicrobium and Methylocaldum assimilated 13CH4 nearly exclusively. Although previous studies had shown that the same soil contains a much larger diversity of MOB, including both type I and type II, nitrogen fertilization apparently activated only a small subset of the overall diversity of MOB, type I MOB in particular.

Applying stable isotope probing of phospholipid fatty acids and rRNA in a Chinese rice field to study activity and composition of the methanotrophic bacterial communities in situ

Methanotrophs in the rhizosphere play an important role in global climate change since they attenuate methane emission from rice field ecosystems into the atmosphere. Most of the CH4 is emitted via transport through the plant gas vascular system. We used this transport for stable isotope probing (SIP) of the methanotrophs in the rhizosphere under field conditions and pulse-labelled rice plants in a Chinese rice field with CH4 (99% 13C) for 7 days. The rate of 13CH4 loss rate during 13C application was comparable to the CH4 oxidation rate measured by the difluoromethane inhibition technique. The methanotrophic communities on the roots and in the rhizospheric soil were analyzed by terminal-restriction fragment length polymorphism (T-RFLP), cloning and sequencing of the particulate methane monooxygenase (pmoA) gene. Populations of type I methanotrophs were larger than those of type II. Both methane oxidation rates and composition of methanotrophic communities suggested that there was little difference between urea-fertilized and unfertilized fields. SIP of phospholipid fatty acids (PLFA-SIP) and rRNA (RNA-SIP) were used to analyze the metabolically active methanotrophic community in rhizospheric soil. PLFA of type I compared with type II methanotrophs was labelled more strongly with 13C, reaching a maximum of 6.8 atom-%. T-RFLP analysis and cloning/sequencing of 16S rRNA genes showed that methanotrophs, especially of type I, were slightly enriched in the ‘heavy’ fractions. Our results indicate that CH4 oxidation in the rice rhizosphere under in situ conditions is mainly due to type I methanotrophs.

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH(4) into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH(4) flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using (13)C-labelled CH(4) directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to (13)CH(4) for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

ABSTRACT Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a γ-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G+C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH4+ was lower and the level of extractable SO42− was higher without protists, indicating that nitrogen mineralization and SO42− reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O2 was a major control, and O2 availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.

Identification of Acetate-Assimilating Microorganisms under Methanogenic Conditions in Anoxic Rice Field Soil by Comparative Stable Isotope Probing of RNA

ABSTRACT Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

Functional and structural response of the methanogenic microbial community in rice field soil to temperature change.

The microbial community in anoxic rice field soil produces CH(4) over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH(4) production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC-1) changes to almost complete dominance of RC-1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH(4) production measuring δ(13)C in CH(4) and CO(2) and calculating the apparent fractionation factor (α(app)) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T-RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T-RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre-incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well-defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42-46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.

Effect of temperature change on the composition of the bacterial and archaeal community potentially involved in the turnover of acetate and propionate in methanogenic rice field soil

The microbial community structure was investigated together with the path of methane production in Italian rice field soil incubated at moderate (35 degrees C) and high (45 degrees C) temperature using terminal restriction fragment length polymorphism and stable isotope fractionation. The structure of both the archaeal and bacterial communities differed at 35 degrees C compared with 45 degrees C, and acetoclastic and hydrogenotrophic methanogenesis dominated, respectively. Changing the incubation of the 45 degrees C soil to different temperatures (25, 30, 35, 40, 45, 50 degrees C) resulted in a dynamic change of both microbial community structure and stable isotope fractionation. In all treatments, acetate first accumulated and then decreased. Propionate was also transiently produced and consumed. It is noteworthy that acetate was also consumed at thermophilic conditions, although archaeal community composition and stable isotope fractionation indicated that acetoclastic methanogenesis did not operate. Instead, acetate must have been consumed by syntrophic acetate oxidizers. The transient accumulation and subsequent consumption of acetate at thermophilic conditions was specifically paralleled by terminal restriction fragments characteristic for clostridial cluster I, whereas those of clostridial clusters I and III, Acidaminococcaceae and Heliobacteraceae, paralleled the thermophilic turnover of both acetate and propionate.

Response of the methanogenic microbial communities in Amazonian oxbow lake sediments to desiccation stress

Methanogenic microbial communities in soil and sediment function only when the environment is inundated and anoxic. In contrast to submerged soils, desiccation of lake sediments happens only rarely. However, some predictions suggest that extreme events of drying will become more common in the Amazon region, and this will promote an increase in sediments drying and exposure. We asked whether and how such methanogenic communities can withstand desiccation stress. Therefore, we determined the rates and pathways of CH(4) production (analysis of CH(4) and δ(13) C of CH(4), CO(2) and acetate), the copy numbers of bacterial and archaeal 16S rRNA genes and mcrA genes (quantitative PCR), and the community composition of Archaea and Bacteria (T-RFLP and pyrosequencing) in oxbow lake sediments of rivers in the Brazilian Amazon region. The rivers were of white water, black water and clear water type. The measurements were done with sediment in fresh state and after drying and rewetting. After desiccation and rewetting the composition of both, the archaeal and bacterial community changed. Since lake sediments from white water rivers exhibited only negligible methanogenic activity, probably because of relatively high iron and low organic matter content, they were not further analysed. The other sediments produced CH(4), with hydrogenotrophic methanogenesis usually accounting for > 50% of total activity. After desiccation and rewetting, archaeal and bacterial gene copy numbers decreased. The bacterial community showed a remarkable increase of Clostridiales from about 10% to > 30% of all Bacteria, partially caused by proliferation of specific taxa as the numbers of OTU shared with fresh sediment decreased from about 9% to 3%. Among the Archaea, desiccation specifically enhanced the relative abundance of either Methanocellales (black water) and/or Methanosarcinaceae (clear water). Despite the changes in gene copy numbers and composition of the microbial community, rates of CH(4) production even increased after desiccation-rewetting, demonstrating that the function of the methanogenic microbial community had not been impaired. This result indicates that the increase in extreme events of drying may increase methane production in flooded sediments.

Biodegradation of a Biocide (Cu-N-Cyclohexyldiazenium Dioxide) Component of a Wood Preservative by a Defined Soil Bacterial Community

ABSTRACT The wood protection industry has refined their products from chrome-, copper-, and arsenate-based wood preservatives toward solely copper-based preservatives in combination with organic biocides. One of these is Cu-HDO, containing the chelation product of copper and N-cyclohexyldiazenium dioxide (HDO). In this study, the fate of isotope-labeled (13C) and nonlabeled (12C) Cu-HDO incorporated in wood sawdust mixed with soil was investigated. HDO concentration was monitored by high-pressure liquid chromatography. The total carbon and the δ13C content of respired CO2, as well as of the soil-wood-sawdust mixture, were determined with an elemental analyzer-isotopic ratio mass spectrometer. The concentration of HDO decreased significantly after 105 days of incubation, and after 24 days the 13CO2 concentration respired from soil increased steadily to a maximum after 64 days of incubation. Phospholipid fatty acid-stable isotope probing (PFA-SIP) analysis revealed that the dominant PFAs C19:0d8,9, C18:0, C18:1ω7, C18:2ω6,9, C17:1d7,8, C16:0, and C16:1ω7 were highly enriched in their δ13C content. Moreover, RNA-SIP identified members of the phylum Acidobacteria and the genera Phenylobacterium and Comamonas that were assimilating carbon from HDO exclusively. Cu-HDO as part of a wood preservative effectively decreased fungal wood decay and overall microbial respiration from soil. In turn, a defined bacterial community was stimulated that was able to metabolize HDO completely.

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 106–108 gsoilDW−1. Methanol dissimilation was measured based on conversion of supplemented 14C-methanol, and occurred at concentrations down to 0.002 μmol methanol gsoilDW−1. Tested soils exhibited specific affinities to methanol (a0s=0.01 d−1) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (⩽0.6 μmol methanol gsoilDW−1) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant–methylotroph interactions were determinative for soil methylotrophs.