[Show abstract][Hide abstract] ABSTRACT: Crop rotation of flooded rice with upland crops is a common management scheme allowing the reduction of water consumption along with the reduction of methane emission. The introduction of an upland crop into the paddy rice ecosystem leads to dramatic changes in field conditions (oxygen availability, redox conditions). However, the impact of this practice on the archaeal and bacterial communities has scarcely been studied. Here, we provide a comprehensive study focusing on the crop rotation between flooded rice in the wet season and upland maize (RM) in the dry season in comparison to flooded rice (RR) in both seasons. The composition of the resident and active microbial communities was assessed by 454 pyrosequencing targeting the archaeal and bacterial 16S rRNA gene and 16S rRNA. The archaeal community composition changed dramatically in the rotational fields indicated by a decrease of anaerobic methanogenic lineages and an increase of aerobic Thaumarchaeota. Members of Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae, Methanocellaceae were equally suppressed in the rotational fields indicating influence on both acetoclastic and hydrogenotrophic methanogens. Contrary, members of Soil Crenarchaeotic Group, mainly Candidatus Nitrososphaera, were higher in the rotational fields possibly indicating increasing importance of ammonia-oxidation during drainage. In contrast minor effects on the bacterial community were observed. Acidobacteria and Anaeromyxobacter spp. were enriched in the rotational fields while members of anaerobic Chloroflexi and sulfate reducing members of Deltaproteobacteria were found in higher abundance in the rice fields. Combining quantitative PCR and pyrosequencing data revealed increased ribosomal numbers per cell for methanogenic species during crop rotation. This stress response, however, did not allow the methanogenic community to recover in the rotational fields during re-flooding and rice cultivation. In summary, the analyses showed that crop rotation with upland maize led to dramatic changes in the archaeal community composition whereas the bacterial community was only little affected.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Oxygen is considered as a limiting factor for nitrification in rice paddy soil. However, little is known about how the nitrifying microbial community responds to different oxygen concentrations at community and transcript level. In this study, soil and roots were harvested from 50-day-old rice microcosms and were incubated for up to 45 days under two oxygen concentrations: 2 % O2 and 20 % O2 (ambient air). Nitrification rates were measured from the accumulation of nitrite plus nitrate. The population dynamics of bacterial (AOB) and archaeal (AOA) ammonia oxidizers was determined from the abundance (using quantitative PCR (qPCR)) and composition (using terminal restriction fragment length polymorphism and cloning/sequencing) of their amoA genes, that of nitrite oxidizers (NOB) by quantifying the nxrA gene of Nitrobacter spp. and the 16S rRNA gene of Nitrospira spp. The activity of the nitrifiers was determined by quantifying the copy numbers of amoA and nxrA transcripts (using RT-qPCR). Different oxygen concentrations did not affect the community compositions of AOB, AOA, and NOB, which however were different between surface soil, bottom soil, and rice roots. However, nitrification rates were higher under ambient air than 2 % O2, and abundance and transcript activities of AOB, but not of AOA, were also higher. Abundance and transcript copy numbers of Nitrobacter were also higher at ambient air. These results indicate that AOB and NOB, but not AOA, were sensitive to oxygen availability.
[Show abstract][Hide abstract] ABSTRACT: Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet a limited number of isolates makes it difficult to understand physiology and ecological impact of the microorganisms involved. Here, two-staged cultivation was implemented to selectively enrich and isolate crystalline iron(III) reducers in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by two-year successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. The Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) media in order to stimulate proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. These isolates had 94.8-98.1% sequence similarities of 16S rRNA genes to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. The results demonstrate the successful enrichment and isolation of novel iron(III) reducers that were able to thrive by reducing highly crystallized ferric irons.
Frontiers in Microbiology 04/2015; 6. DOI:10.3389/fmicb.2015.00386 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemolithotrophic homoacetogenic bacteria apparently express a characteristic stable carbon isotope fractionation and may contribute significantly to acetate production in anoxic environments. However, fractionation factors (ε) in bacterial cultures have rarely been determined and the effect of substrate availability has not been assessed. We therefore studied the kinetic carbon isotope effect in cultures of Thermoanaerobacter kivui grown at 55 °C. The fractionation factor in HCO3- buffered medium was ca. 15‰ more negative than that in PO43- buffered medium. To test whether the difference was caused by the initial substrate ratio of H2 and total inorganic carbon (TIC; 0.5 in HCO3- vs. 4.0 in PO43- buffered medium), T. kivui was grown in either [3-(N-morpholino) propanesulfonic acid, MOPS] buffered or PO43- buffered media with different HCO3- concentration. Indeed, the fractionation factor became more negative with increasing HCO3- concentration and decreasing H2/TIC ratio. While pH had only a small effect, the fractionation was generally more negative in MOPS buffered than in phosphate buffered media, indicating that the buffer system also affected fractionation. Collectively, the results show that substrate availability and other environmental factors affect the magnitude of isotope fractionation during acetate production by chemolithotrophic homoacetogenesis.
[Show abstract][Hide abstract] ABSTRACT: The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH4) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [(13)C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H2), carbon dioxide (CO2), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and (13)C-labeling of CH4 verified that supplemental [(13)C]-glucose derived carbon was dissimilated to CH4. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H2 rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H2 to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.The ISME Journal advance online publication, 23 January 2015; doi:10.1038/ismej.2014.262.
The ISME Journal 01/2015; 9(8). DOI:10.1038/ismej.2014.262 · 9.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sampling strategy is important for unbiased analysis of the characteristics of microbial communities in the environment. During field work it is not always possible to analyze fresh samples immediately or store them frozen. Therefore, the effect of short-term storage temperature was investigated on the abundance and composition of bacterial, archaeal and denitrifying communities in environmental samples from two different sampling sites. Oxic forest soil and anoxic pond sediment were investigated by measuring microbial abundance (DNA) and transcriptional activity (RNA). Prior to investigating the effect of storage temperature, samples were immediately analyzed, in order to represent the original situation in the habitat. The effect of storage temperature was then determined after 11 days at different low temperatures (room temperature, 4 °C, −22 °C and −80 °C). Community profiling using terminal restriction fragment length polymorphism (T-RFLP) showed no significant differences between the immediately analyzed reference sample and the samples stored at different incubation temperatures, both for DNA and RNA extracts. The abundance of microbial communities was determined using quantitative PCR and it also revealed a stable community size at all temperatures tested. By contrast, incubation at an elevated temperature (37 °C) resulted in changed bacterial community composition. In conclusion, short-term storage, even at room temperature, did not affect microbial community composition, abundance and transcriptional activity in aerated forest soil and anoxic pond sediment.
[Show abstract][Hide abstract] ABSTRACT: Aims
Rice fields are an important source for the greenhouse gas methane. Plants play an essential role in carbon supply for soil microbiota, but the influence of the microbial community on carbon cycling is not well understood.
Microcosms were prepared using sand-vermiculite amended with different soils and sediments, and planted with rice. The microcosms at different growth stages were pulse-labeled with 13CO2 followed by tracing 13C in plant, soil and atmospheric carbon pools and quantifying the abundance of methanogenic archaea in rhizosphere soil.
Overall, >85 % of the freshly assimilated carbon was allocated in aboveground plant biomass, approximately 10 % was translocated into the roots and
Plant and Soil 11/2014; 384(1-2):213-229. DOI:10.1007/s11104-014-2201-y · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Archaea are widespread and abundant in many terrestrial and aquatic environments, and are thus outside extreme environments, accounting for up to ~10% of the prokaryotes. Compared to bacteria and other microorganisms, however, very little is known about the abundance, diversity, and dispersal of archaea in the atmosphere. By means of DNA analysis and Sanger sequencing targeting the 16S rRNA (435 sequences) and amoA genes in samples of air particulate matter collected over 1 year at a continental sampling site in Germany, we obtained first insights into the seasonal dynamics of airborne archaea. The detected archaea were identified as Thaumarchaeota or Euryarchaeota, with soil Thaumarchaeota (group I.1b) being present in all samples. The normalized species richness of Thaumarchaeota correlated positively with relative humidity and negatively with temperature. This together with an increase in bare agricultural soil surfaces may explain the diversity peaks observed in fall and winter. The detected Euryarchaeota were mainly predicted methanogens with a low relative frequency of occurrence. A slight increase in their frequency during spring may be linked to fertilization processes in the surrounding agricultural fields. Comparison with samples from the Cape Verde islands (72 sequences) and from other coastal and continental sites indicates that the proportions of Euryarchaeota are enhanced in coastal air, which is consistent with their suggested abundance in marine surface waters. We conclude that air transport may play an important role in the dispersal of archaea, including assumed ammonia-oxidizing Thaumarchaeota and methanogens.
[Show abstract][Hide abstract] ABSTRACT: The study of of the distribution of microorganisms through space (and time) allows evaluation of biogeographic patterns, like the species-area index (z). Due to their high dispersal ability, high reproduction rates and low rates of extinction microorganisms tend to be widely distributed, and they are thought to be virtually cosmopolitan and selected primarily by environmental factors. Recent studies have shown that, despite these characteristics, microorganisms may behave like larger organisms and exhibit geographical distribution. In this study, we searched patterns of spatial diversity distribution of bacteria and archaea in a contiguous environment. We collected 26 samples of a lake sediment, distributed in a nested grid, with distances between samples ranging from 0.01 m to 1000 m. The samples were analyzed using T-RFLP (Terminal restriction fragment length polymorphism) targeting mcrA (coding for a subunit of methyl-coenzyme M reductase) and the genes of Archaeal and Bacterial 16S rRNA. From the qualitative and quantitative results (relative abundance of operational taxonomic units) we calculated the similarity index for each pair to evaluate the taxa-area and distance decay relationship slopes by linear regression. All results were significant, with mcrA genes showing the highest slope, followed by Archaeal and Bacterial 16S rRNA genes. We showed that the microorganisms of a methanogenic community, that is active in a contiguous environment, display spatial distribution and a taxa-area relationship.
PLoS ONE 10/2014; 9(10):e110128. DOI:10.1371/journal.pone.0110128 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls CH4 emission, we pulse-labeled rice microcosms with 13CO2 to determine the rates of 13CH4 emission exclusively derived from photosynthates. We also measured emission of total CH4 (12+13CH4), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analyzed by quantitative PCR of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase, respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T-RFLP). During the vegetative growth stages, emission rates of 13CH4 linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of 13CH4 emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz-vermiculite with only 10% rice soil. Rates of total CH4 emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis-driven CH4 emission are limited by the abundance of methanogens on the roots.
[Show abstract][Hide abstract] ABSTRACT: Temperature is the major driving force for many biological as well as chemical reactions and may impact the fractionation of stable carbon isotopes. Thus, a good correlation between temperature and fractionation is observed in many chemical systems that are controlled by an equilibrium isotope effect. In contrast, biological systems that are usually controlled by a kinetic isotope effect are less well studied with respect to temperature effects and have shown contrasting results. We studied three different biological pathways (methylotrophic methanogenesis, hydrogenotrophic methanogenesis, acetogenesis by the acetyl-CoA pathway) which are characterized by very strong carbon isotope enrichment factors (−50‰ to −83‰). The microorganisms (Methanosarcina barkeri, Methanosarcina acetivorans, Methanolobus zinderi, Methanothermobacter marburgensis, Methanothermobacter thermoautotrophicus, Thermoanaerobacter kivui) exhibiting these pathways were grown at different temperatures ranging between 25 and 68 °C, and the fractionation factors were determined from 13C/12C isotope discrimination during substrate depletion and product formation.
Our experiments showed that the fractionation factors were different for the different metabolic pathways but were not much affected by the different growth temperatures. Slight variations were well within the standard errors of replication and regression analysis. Our results showed that temperature had no significant effect on the fractionation of stable carbon isotopes during anaerobic microbial metabolism with relatively strong isotope fractionation.
[Show abstract][Hide abstract] ABSTRACT: In anoxic environments, degradation of organic matter (OM) results in strong fractionation of carbon isotopes, with formation of 13C-depleted CH4. Propionate and acetate are important products of OM fermentation. Propionate is further fermented to acetate. Acetate is a direct precursor of CH4, the remainder usually being produced from H2-mediated CO2 reduction. There is a paucity of data for the turnover of acetate and, even more so, propionate. We therefore analyzed the δ13C values of organic carbon, propionate, acetate and the methyl (Me) group of acetate (acetate-Me) during the production of CH4 in anoxic incubations of various flooded and non-flooded soils and various lake sediments. Incubation in the presence of methylfluoride (CH3F), which inhibits CH4 production from acetate, allowed exclusion of isotope effects during aceticlastic methanogenesis. Despite the variation inherent in the wide diversity of sample type and origin, the data collectively showed that the δ13C value of acetate was only marginally different (-2 ± 5‰) from that of OM, while propionate was depleted in 13C relative to total acetate (-6 ± 5‰). Acetate-Me was generally depleted in 13C relative to total acetate (-8 ± 5‰). Thus, isotopic enrichment factors during the degradation of OM to total propionate and acetate were much smaller than those during hydrogenotrophic and aceticlastic methanogenesis or the intramolecular difference in δ13C between the carboxyl (CO2H) and Me of acetate, so that the δ13C value of OM may be used as a proxy when data for acetate are not available.
[Show abstract][Hide abstract] ABSTRACT: Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H2 when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD+/NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentatively-produced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments.
Proceedings of the National Academy of Sciences 07/2014; 111(31). DOI:10.1073/pnas.1407034111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence suggests that Thaumarchaeota may control nitrification in acidic soils. However, the composition of the thaumarchaeotal communities and their functioning is not well known. Therefore, we studied nitrification activity in relation to abundance and composition of Thaumarchaeota in an acidic red soil from China, using microcosms incubated with and without cellulose amendment. Cellulose was selected to simulate the input of crop residues used to increase soil fertility by local farming. Accumulation of
-N was correlated with the growth of Thaumarchaeota as determined by qPCR of 16S rRNA and ammonia monooxygenase (amoA) genes. Both nitrification activity and thaumarchaeotal growth were inhibited by acetylene. They were also inhibited by cellulose amendment, possibly due to the depletion of ammonium by enhanced heterotrophic assimilation. These results indicated that growth of Thaumarchaeota was dependent on ammonia oxidation. The thaumarchaeotal 16S rRNA gene sequences in the red soil were dominated by a clade related to soil fosmid clone 29i4 within the group I.1b, which is widely distributed but so far uncultured. The archaeal amoA sequences were mainly related to the Nitrososphaera sister cluster. These observations suggest that fosmid clone 29i4 and Nitrososphaera sister cluster represent the same group of Thaumarchaeota and dominate ammonia oxidation in acidic red soil.
[Show abstract][Hide abstract] ABSTRACT: In the Earth's lower atmosphere, H2 is maintained at trace concentrations (0.53 ppmv/0.40 nM) and rapidly turned over (lifetime ≤ 2.1 y(-1)). It is thought that soil microbes, likely actinomycetes, serve as the main global sink for tropospheric H2. However, no study has ever unambiguously proven that a hydrogenase can oxidize this trace gas. In this work, we demonstrate, by using genetic dissection and sensitive GC measurements, that the soil actinomycete Mycobacterium smegmatis mc(2)155 constitutively oxidizes subtropospheric concentrations of H2. We show that two membrane-associated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H2 at a range of concentrations [Vmax(app) = 12 nmol⋅g⋅dw(-1)⋅min(-1); Km(app) = 180 nM; threshold = 130 pM in the Δhyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [Vmax(app) = 2.5 nmol⋅g⋅dw(-1)⋅min(-1); Km(app) = 50 nM; threshold = 50 pM in the Δhyd13 (Hyd2 only) strain]. These observations strongly support previous studies that have linked group 5 [NiFe] hydrogenases (e.g., Hyd2) to the oxidation of tropospheric H2 in soil ecosystems. We further reveal that group 2a [NiFe] hydrogenases (e.g., Hyd1) can contribute to this process. Hydrogenase expression and activity increases in carbon-limited cells, suggesting that scavenging of trace H2 helps to sustain dormancy. Distinct physiological roles for Hyd1 and Hyd2 during the adaptation to this condition are proposed. Soil organisms harboring high-affinity hydrogenases may be especially competitive, given that they harness a highly dependable fuel source in otherwise unstable environments.
Proceedings of the National Academy of Sciences 03/2014; 111(11). DOI:10.1073/pnas.1320586111 · 9.67 Impact Factor