Ralf Conrad

Publications (107)437.57 Total impact

• Article: Elucidating the microbial resuscitation cascade in biological soil crusts following a simulated rain event.

  Roey Angel, Ralf Conrad
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  ABSTRACT: Biological soil crusts (biocrusts) are photosynthetic mats formed through an association of prokaryotic and eukaryotic microorganisms with soil particles. Biocrusts are found in virtually any terrestrial ecosystem where vascular plant coverage is abiotically limited, with drylands comprising the primary habitat for them. We studied the dynamics of the active bacterial community in two biocrusts from an arid and a hyperarid region in the Negev Desert, Israel, under light-oxic and dark-anoxic incubation conditions after simulated rainfall. We used H2 (18) O for hydrating the crusts and analysed the bacterial community in the upper and lower parts of the biocrust using an RNA-stable isotope probing approach coupled with 454-pyrosequencing. In both biocrusts, two distinct bacterial communities developed under each incubation condition. The active anaerobic communities were initially dominated by members of the order Bacillales which were later replaced by Clostridiales. The aerobic communities on the other hand were dominated by Sphingobacteriales and several Alphaproteobacteria (Rhizobiales, Rhodobacterales, Rhodospirillales and Rubrobacteriales). Actinomycetales were the dominant bacterial order in the dry crusts but quickly collapsed and accounted for < 1% of the community by the end of the incubation. Our study shows that biocrusts host a diverse community whose members display complex interactions as they resuscitate from dormancy.
  Environmental Microbiology 04/2013; · 5.84 Impact Factor
• Source

  Available from: Zhongjun Jia

  Article: Autotrophic growth of bacterial and archaeal ammonia oxidizers in freshwater sediment microcosms incubated at different temperatures.

  Yucheng Wu, Xiubin Ke, Marcela Hernández, Baozhan Wang, Marc G Dumont, Zhongjun Jia, Ralf Conrad
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  ABSTRACT: Both bacteria and archaea potentially contribute to ammonia oxidation, but their roles in freshwater sediment are still poorly understood. Seasonal differences in the relative activity of these groups might exist since cultivated archaeal ammonia oxidizers have higher temperature optima than their bacterial counterparts. In this study, sediment collected from the eutrophic freshwater Lake Taihu (China) was incubated at different temperatures (4°C, 15°C, 25°C and 37°C) for up to 8 weeks. We examined the active bacterial and archaeal ammonia oxidizers in these sediment microcosms by combined stable isotope probing (SIP) and molecular community analysis. The results showed that accumulation of nitrate in microcosms correlated negatively with temperature although ammonium depletion was the same, which might be related to an enhanced activity of other nitrogen transformation processes. Incubation at different temperatures significantly changed the microbial community composition as revealed by 454 pyrosequencing targeting bacterial 16S rRNA genes. After 8-weeks incubation, 13C-bicarbonate labeling of bacterial amoA genes, which encode the ammonia monooxygenase subunit A, and an observed increase in copy numbers indicated the activity of ammonia-oxidizing bacteria in all microcosms. Nitrosomonas sp. Is79A3 and Nitrosomonas communis lineages dominated the heavy fraction of CsCl gradients at low and high temperatures, respectively, indicating a niche differentiation of active bacterial ammonia oxidizers along the temperature gradient. The 13C-labelling of ammonia-oxidizing archaea in microcosms incubated at 4-25°C was minor. In contrast, significant 13C-labelling of Nitrososphaera-like archaea and changes in the abundance and composition of archaeal amoA genes were observed at 37°C, implicating autotrophic growth of ammonia oxidizing archaea in warmer conditions.
  Applied and environmental microbiology 03/2013; · 3.69 Impact Factor
• Article: Niche differentiation of ammonia oxidizers and nitrite oxidizers in rice paddy soil.

  Xiubin Ke, Roey Angel, Yahai Lu, Ralf Conrad
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  ABSTRACT: The dynamics of populations and activities of ammonia-oxidizing and nitrite-oxidizing microorganisms were investigated in rice microcosms treated with two levels of nitrogen. Different soil compartments (surface, bulk, rhizospheric soil) and roots (young and old roots) were collected at three time points (the panicle initiation, heading and maturity periods) of the season. The population dynamics of bacterial (AOB) and archaeal (AOA) ammonia oxidizers was assayed by determining the abundance (using qPCR) and composition (using T-RFLP and cloning/sequencing) of their amoA genes (coding for a subunit of ammonia monooxygenase), that of nitrite oxidizers (NOB) by quantifying the nxrA gene (coding for a subunit of nitrite oxidase of Nitrobacter spp.) and the 16S rRNA gene of Nitrospira spp. The activity of the nitrifiers was determined by measuring the rates of potential ammonia oxidation and nitrite oxidation and by quantifying the copy numbers of amoA and nxrA transcripts. Potential nitrite oxidation activity was much higher than potential ammonia oxidation activity and was not directly affected by nitrogen amendment demonstrating the importance of ammonia oxidizers as pace makers for nitrite oxidizer populations. Marked differences in the distribution of bacterial and archaeal ammonia oxidizers, and of Nitrobacter-like and Nitrospira-like nitrite oxidizers were found in the different compartments of planted paddy soil indicating niche differentiation. In bulk soil, ammonia-oxidizing bacteria (Nitrosospira and Nitrosomonas) were at low abundance and displayed no activity, but in surface soil their activity and abundance was high. Nitrite oxidation in surface soil was dominated by Nitrospira spp. By contrast, ammonia-oxidizing Thaumarchaeota and Nitrobacter spp. seemed to dominate nitrification in rhizospheric soil and on rice roots. In contrast to soil compartment, the level of N fertilization and the time point of sampling had only little effect on the abundance, composition and activity of the nitrifying communities. The results of our study show that in rice fields population dynamics and activity of nitrifiers is mainly differentiated by the soil compartments rather than by nitrogen amendment or season.
  Environmental Microbiology 01/2013; · 5.84 Impact Factor
• Article: Sulfide and methane evolution in the hypolimnion of a subtropical lake: a three-year study

  Werner Eckert, Ralf Conrad
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  ABSTRACT: The differential impact of microbial sulfate reduction and methanogenesis on the mineralization of particulate organic carbon (POC) in warm monomictic Lake Kinneret (LK), Israel was studied during three consecutive lake cycles. The hypolimnetic accumulation of total sulfide and dissolved methane was examined in relation to the physical forcing of the water column and the settling flux of particulate matter. With the on-set of thermal stratification in spring, both solutes increased concomitantly with the depletion of oxygen, first in the benthic boundary layer, followed by the upper hypolimnion. Methane production was restricted to the sediments as emphasized by the persistently linear concentration gradient in the hypolimnion. Sulfate reduction occurred both in the sediments and the water column as revealed by the hypolimnetic distribution of sulfide and recurring metalimnetic sulfide peaks. Annual differences in the accumulation pattern of both solutes appeared to be primarily linked to the settling flux of POC and the length of the stratified season. Relatively lower hypolimnetic concentrations of dissolved methane during the stratified season of 2000 coincided with increased ebullition of gaseous methane, likely as the result of a nearly a 2m drop in the lake level. Overall, sulfate reduction accounted for more than 60% of the POC settling flux, a finding that differs from similar studies made in temperate lakes where methanogenesis was shown to be the primary mode of terminal carbon mineralization. Intensive organic carbon turnover at the sediment water interface and comparatively high sulfate concentrations in LK are the most likely reason.
  Biogeochemistry 04/2012; 82(1):67-76. · 3.07 Impact Factor
• Article: Effect of long-term free-air CO2 enrichment on the diversity and activity of soil methanogens in a periodically waterlogged grassland

  Roey Angel, Claudia Kammann, Peter Claus, Ralf Conrad
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  ABSTRACT: As atmospheric CO2 levels continue to rise researchers now identify concomitant changes in plant biomass and diversity, which are postulated to alter the quality and quantity of the organic carbon entering the soil. In anoxic soils, CH4 is the end product of the degradation of organic carbon and the system's terminal electron sink. Some soils (such as wetlands) are usually waterlogged and therefore constitute permanent CH4 sources, while others (hydromorphic soils) are only occasionally saturated with water and alternate between acting as net CH4 sources or sinks. Since methanogenesis is ultimately dependent on soil organic carbon, we hypothesized that a long term alteration of the latter will cause changes in type and magnitude of the former. To test this, we studied the effect of 10 years of atmospheric CO2 enrichment on the methanogenic potential and community in a hydromorphic temperate grassland soil at the experimental Free Air Carbon dioxide Enrichment (FACE) site in Giessen, Germany. While all soils demonstrated methanogenic potential, we detected no significant changes in CH4 production rates, lag times, methanogenic pathways, diversity, or population sizes in soils that were exposed to either 20 or 30% elevated ambient CO2. Our findings suggest that the methanogenic potential of the soil and the methanogenic community might be insensitive to changes in atmospheric CO2 concentrations, at least not on a decadal timescale. Thus, if our results can be extrapolated to other temporarily flooded or even wetland ecosystems, the often-observed increase in CH4 emissions under elevated CO2 may simply be due to an increase in labile-C input via living root and increasing fresh litter deposition, but not due to shifts in the microbial population characteristics. This could make it easier to model and extrapolate the global effect of elevated CO2 on terrestrial CH4 emissions.
  Soil Biology and Biochemistry 01/2012; 51:96-103. · 3.50 Impact Factor
• Article: Methanogenic pathway and archaeal communities in three different anoxic soils amended with rice straw and maize straw.

  Ralf Conrad, Melanie Klose, Yahai Lu, Amnat Chidthaisong
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  ABSTRACT: Addition of straw is common practice in rice agriculture, but its effect on the path of microbial CH(4) production and the microbial community involved is not well known. Since straw from rice (C3 plant) and maize plants (C4 plant) exhibit different δ(13)C values, we compared the effect of these straw types using anoxic rice field soils from Italy and China, and also a soil from Thailand that had previously not been flooded. The temporal patterns of production of CH(4) and its major substrates H(2) and acetate, were slightly different between rice straw and maize straw. Addition of methyl fluoride, an inhibitor of acetoclastic methanogenesis, resulted in partial inhibition of acetate consumption and CH(4) production. The δ(13)C of the accumulated CH(4) and acetate reflected the different δ(13)C values of rice straw versus maize straw. However, the relative contribution of hydrogenotrophic methanogenesis to total CH(4) production exhibited a similar temporal change when scaled to CH(4) production irrespectively of whether rice straw or maize straw was applied. The composition of the methanogenic archaeal communities was characterized by terminal restriction fragment length polymorphism (T-RFLP) analysis and was quantified by quantitative PCR targeting archaeal 16S rRNA genes or methanogenic mcrA genes. The size of the methanogenic communities generally increased during incubation with straw, but the straw type had little effect. Instead, differences were found between the soils, with Methanosarcinaceae and Methanobacteriales dominating straw decomposition in Italian soil, Methanosarcinaceae, Methanocellales, and Methanobacteriale in China soil, and Methanosarcinaceae and Methanocellales in Thailand soil. The experiments showed that methanogenic degradation in different soils involved different methanogenic population dynamics. However, the path of CH(4) production was hardly different between degradation of rice straw versus maize straw and was also similar for the different soil types.
  Frontiers in microbiology. 01/2012; 3:4.
• Article: Responses of methanogen mcrA genes and their transcripts to an alternate dry/wet cycle of paddy field soil.

  Ke Ma, Ralf Conrad, Yahai Lu
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  ABSTRACT: Intermittent drainage can substantially reduce methane emission from rice fields, but the microbial mechanisms remain poorly understood. In the present study, we determined the rates of methane production and emission, the dynamics of ferric iron and sulfate, and the abundance of methanogen mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase) and their transcripts in response to alternate dry/wet cycles in paddy field soil. We found that intermittent drainage did not affect the growth of rice plants but significantly reduced the rates of both methane production and emission. The dry/wet cycles also resulted in shifts of soil redox conditions, increasing the concentrations of ferric iron and sulfate in the soil. Quantitative PCR analysis revealed that both mcrA gene copies and mcrA transcripts significantly decreased after dry/wet alternation compared to continuous flooding. Correlation and regression analyses showed that the abundance of mcrA genes and transcripts positively correlated with methane production potential and soil water content and negatively correlated with the concentrations of ferric iron and sulfate in the soil. However, the transcription of mcrA genes was reduced to a greater extent than the abundance of mcrA genes, resulting in very low mcrA transcript/gene ratios after intermittent drainage. Furthermore, terminal restriction fragment length polymorphism analysis revealed that the composition of methanogenic community remained stable under dry/wet cycles, whereas that of metabolically active methanogens strongly changed. Collectively, our study demonstrated a stronger effect of intermittent drainage on the abundance of mcrA transcripts than of mcrA genes in rice field soil.
  Applied and environmental microbiology 11/2011; 78(2):445-54. · 3.69 Impact Factor
• Article: Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions.

  Roey Angel, Peter Claus, Ralf Conrad
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  ABSTRACT: The prototypical representatives of the Euryarchaeota--the methanogens--are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina, whose gene copy numbers also correlated with methane production rates. Analysis of the δ(13)C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.
  The ISME Journal 11/2011; 6(4):847-62. · 7.38 Impact Factor
• Article: Fluxes and production pathways of nitrous oxide in different types of tropical forest soils in Thailand

  Supika Vanitchung, Ralf Conrad, Narumon W. Harvey, Amnat Chidthaisong
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  ABSTRACT: Nitrous oxide (N2O) emissions from the soil surface of five different forest types in Thailand were measured using the closed chamber method. Soil samples were also taken to study the N2O production pathways. The monthly average emissions (±SD, n = 12) of N2O from dry evergreen forest (DEF), hill evergreen forest (HEF), moist evergreen forest (MEF), mixed deciduous forest (MDF) and acacia reforestation (ARF) were 13.0 ± 8.2, 5.7 ± 7.1, 1.2 ± 12.1, 7.3 ± 8.5 and 16.7 ± 9.2 µg N m−2 h−1, respectively. Large seasonal variations in fluxes were observed. Emission was relatively higher during the wet season than during the dry season, indicating that soil moisture and denitrification were probably the main controlling factors. Net N2O uptake was also observed occasionally. Laboratory studies were conducted to further investigate the influence of moisture and the N2O production pathways. Production rates at 30% water holding capacity (WHC) were 3.9 ± 0.2, 0.5 ± 0.06 and 0.87 ± 0.01 ng N2O-nitrogen (N) g-dw−1day−1 in DEF, HEF and MEF respectively. At 60% WHC, N2O production rates in DEF, HEF and MEF soils increased by factors of 68, 9 and 502, respectively. Denitrification was found to be the main N2O production pathway in these soils except in MEF.
  Soil Science and Plant Nutrition. 10/2011; 57(5):650-658.
• Source

  Available from: Zhongjun Jia

  Article: Active autotrophic ammonia-oxidizing bacteria in biofilm enrichments from simulated creek ecosystems at two ammonium concentrations respond to temperature manipulation.

  Sharon Avrahami, Zhongjun Jia, Josh D Neufeld, J Colin Murrell, Ralf Conrad, Kirsten Küsel
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  ABSTRACT: The first step of nitrification, the oxidation of ammonia to nitrite, is important for reducing eutrophication in freshwater environments when coupled with anammox (anaerobic ammonium oxidation) or denitrification. We analyzed active formerly biofilm-associated aerobic ammonia-oxidizing communities originating from Ammerbach (AS) and Leutra South (LS) stream water (683 ± 550 [mean ± standard deviation] and 16 ± 7 μM NH(4)(+), respectively) that were developed in a flow-channel experiment and incubated under three temperature regimens. By stable-isotope probing using (13)CO(2), we found that members of the Bacteria and not Archaea were the functionally dominant autotrophic ammonia oxidizers at all temperatures under relatively high ammonium loads. The copy numbers of bacterial amoA genes in (13)C-labeled DNA were lower at 30°C than at 13°C in both stream enrichment cultures. However, the community composition of the ammonia-oxidizing bacteria (AOB) in the (13)C-labeled DNA responded differently to temperature manipulation at two ammonium concentrations. In LS enrichments incubated at the in situ temperature (13°C), Nitrosomonas oligotropha-like sequences were retrieved with sequences from Nitrosospira AmoA cluster 4, while the proportion of Nitrosospira sequences increased at higher temperatures. In AS enrichments incubated at 13°C and 20°C, AmoA cluster 4 sequences were dominant; Nitrosomonas nitrosa-like sequences dominated at 30°C. Biofilm-associated AOB communities were affected differentially by temperature at two relatively high ammonium concentrations, implicating them in a potential role in governing contaminated freshwater AOB distributions.
  Applied and environmental microbiology 09/2011; 77(20):7329-38. · 3.69 Impact Factor
• Article: Assimilation of acetate by the putative atmospheric methane oxidizers belonging to the USCα clade.

  Jennifer Pratscher, Marc G Dumont, Ralf Conrad
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  ABSTRACT: Forest soils are a major biological sink for atmospheric methane, yet the identity and physiology of the microorganisms responsible for this process remain unclear. Although members of the upland soil cluster α (USCα) are assumed to represent methanotrophic bacteria adapted to the oxidation of the trace level of methane in the atmosphere and to be an important sink of this greenhouse gas, so far they have resisted isolation. In particular, the question of whether the atmospheric methane oxidizers are able to obtain all their energy and carbon solely from atmospheric methane still waits to be answered. In this study, we performed stable-isotope probing (SIP) of RNA and DNA to investigate the assimilation of (13) C-methane and (13) C-acetate by USCα in an acidic forest soil. RNA-SIP showed that pmoA mRNA of USCα was not labelled by (13) C of supplemented (13) C methane, although catalysed reporter deposition - fluorescence in situ hybridization (CARD-FISH) targeting pmoA mRNA of USCα detected its expression in the incubated soil. In contrast, incorporation of (13) C-acetate into USCαpmoA mRNA was observed. USCαpmoA genes were not labelled, indicating that they had not grown during the incubation. Our results indicate that the contribution of alternative carbon sources, such as acetate, to the metabolism of the putative atmospheric methane oxidizers in upland forest soils might be substantial.
  Environmental Microbiology 08/2011; 13(10):2692-701. · 5.84 Impact Factor
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  Available from: Zhongjun Jia

  Article: Simazine application inhibits nitrification and changes the ammonia-oxidizing bacterial communities in a fertilized agricultural soil.

  Marcela Hernández, Zhongjun Jia, Ralf Conrad, Michael Seeger
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  ABSTRACT: s-Triazine herbicides are widely used for weed control, and are persistent in soils. Nitrification is an essential process in the global nitrogen cycle in soil, and involves ammonia-oxidizing Bacteria (AOB) and ammonia-oxidizing Archaea (AOA). In this study, we evaluated the effect of the s-triazine herbicide simazine on the nitrification and on the structure of ammonia-oxidizing microbial communities in a fertilized agricultural soil. The effect of simazine on AOB and AOA were studied by PCR-amplification of amoA genes of nitrifying Bacteria and Archaea in soil microcosms and denaturing gradient gel electrophoresis (DGGE) analyses. Simazine [50 μg g(-1) dry weight soil (d.w.s)] completely inhibited the nitrification processes in the fertilized agricultural soil. The inhibition by simazine of ammonia oxidation observed was similar to the reduction of ammonia oxidation by the nitrification inhibitor acetylene. The application of simazine-affected AOB community DGGE patterns in the agricultural soil amended with ammonium, whereas no significant changes in the AOA community were observed. The DGGE analyses strongly suggest that simazine inhibited Nitrosobacteria and specifically Nitrosospira species. In conclusion, our results suggest that the s-triazine herbicide not only inhibits the target susceptible plants but also inhibits the ammonia oxidation and the AOB in fertilized soils.
  FEMS Microbiology Ecology 08/2011; 78(3):511-9. · 3.41 Impact Factor
• Source

  Available from: Soumitra Paul Chowdhury

  Article: Genome data mining and soil survey for the novel group 5 [NiFe]-hydrogenase to explore the diversity and ecological importance of presumptive high-affinity H(2)-oxidizing bacteria.

  Philippe Constant, Soumitra Paul Chowdhury, Laura Hesse, Jennifer Pratscher, Ralf Conrad
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  ABSTRACT: Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.
  Applied and environmental microbiology 07/2011; 77(17):6027-35. · 3.69 Impact Factor
• Article: Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil.

  Sindy Hunger, Oliver Schmidt, Maik Hilgarth, Marcus A Horn, Steffen Kolb, Ralf Conrad, Harold L Drake
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  ABSTRACT: Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO(2) as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO(2)-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [(13)C]formate or (13)CO(2) (i.e., [(13)C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were (13)C enriched (i.e., labeled) in [(13)C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in (13)CO(2) treatments. [(13)C]acetate was enriched in [(13)C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.
  Applied and Environmental Microbiology 06/2011; 77(11):3773-85. · 3.83 Impact Factor
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  Available from: Soumitra Paul Chowdhury

  Article: Co-localization of atmospheric H 2 oxidation activity and high affinity H 2 -oxidizing bacteria in non-axenic soil and sterile soil amended with Streptomyces sp. PCB7

  Philippe Constant, Soumitra Paul Chowdhury, Laura Hesse, Ralf Conrad
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  ABSTRACT: a b s t r a c t Two complementary experimental approaches were utilized to examine the extent to which free soil hydrogenases and H 2 -oxidizing bacteria contribute to the soil uptake of atmospheric H 2 . First, high affinity hydrogenase activity and H 2 -oxidizing bacteria were fractionated in non-axenic soil and axenic soil colonized with the high affinity H 2 -oxidizing bacterium Streptomyces sp. PCB7. Non-axenic soil was frac-tionated by buoyant density centrifugation. High affinity H 2 oxidation activity measured in individual fractions was proportional to the copy number of hhyL gene, specifying the large subunit of putative high affinity [NiFe]-hydrogenases. 2.5% of the hydrogenase activity was recovered in bacteria-free soil extract. Similarly, sequential centrifugation and wet filtrations of strain PCB7-colonized soil dispersed in solubi-lization buffer caused a loss of the activity, at a ratio proportional to the number of living cells removed. No abiontic hydrogenase activity was detected in bacteria-free fractions. The second experimental approach was designed to verify whether or not the [NiFe]-hydrogenase of strain PCB7 retains high affinity H 2 oxidation activity in soil, under the abiontic state. H 2 oxidation rates of crude enzyme extract of strain PCB7 measured under aerobic and anaerobic conditions were indistinguishable, indicating that the high affinity hydrogenase of strain PCB7 is oxygen-tolerant. The hydrogenase activity of sterile soil spiked with as much as 0.14 mg (protein) g (soil-dw) À1 was equivalent to the H 2 -oxidation activity of only 10 6 e10 7 CFU of strain PCB7 g (soil-dw) À1 . Taken together, our results indicate that high affinity hydrogenase activity is proportional to the abundance of H 2 -oxidizing bacteria in soil and, that abiontic hydrogenases contribute only a few percent of the total high affinity H 2 oxidation activity detected in soil.
  Soil Biology and Biochemistry 05/2011; 43:1888. · 3.50 Impact Factor
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  Available from: Fanghua Liu

  Article: Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil.

  Fanghua Liu, Ralf Conrad
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  ABSTRACT: Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H(2) (80%) and (13)CO(2) (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from (13)CO(2). At 50 °C, acetate was first produced but later on consumed with formation of CH(4). Acetate was also produced in the absence of exogenous H(2) albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, (13)C-labeled bacterial rRNA was detected after 20 days of incubation with (13)CO(2). In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated (13)CO(2) in the presence and absence of exogenous H(2), respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the (13)C treatment at 50 °C. Assimilation of (13)CO(2) into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H(2) at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.
  The ISME Journal 03/2011; 5(9):1526-39. · 7.38 Impact Factor
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  Available from: pnas.org

  Article: Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil.

  Jennifer Pratscher, Marc G Dumont, Ralf Conrad
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  ABSTRACT: Ammonia oxidation is an essential part of the global nitrogen cycling and was long thought to be driven only by bacteria. Recent findings expanded this pathway also to the archaea. However, most questions concerning the metabolism of ammonia-oxidizing archaea, such as ammonia oxidation and potential CO(2) fixation, remain open, especially for terrestrial environments. Here, we investigated the activity of ammonia-oxidizing archaea and bacteria in an agricultural soil by comparison of RNA- and DNA-stable isotope probing (SIP). RNA-SIP demonstrated a highly dynamic and diverse community involved in CO(2) fixation and carbon assimilation coupled to ammonia oxidation. DNA-SIP showed growth of the ammonia-oxidizing bacteria but not of archaea. Furthermore, the analysis of labeled RNA found transcripts of the archaeal acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed and labeled. These findings strongly suggest that ammonia-oxidizing archaeal groups in soil autotrophically fix CO(2) using the 3-hydroxypropionate-4-hydroxybutyrate cycle, one of the two pathways recently identified for CO(2) fixation in Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH targeting the gene encoding subunit A of ammonia monooxygenase (amoA) mRNA and 16S rRNA of archaea also revealed ammonia-oxidizing archaea to be numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxidizing archaea in soil to nitrification and CO(2) assimilation and that their importance to the overall archaeal community might be larger than previously thought.
  Proceedings of the National Academy of Sciences 02/2011; 108(10):4170-5. · 9.68 Impact Factor
• Article: Transcriptional response of methanogen mcrA genes to oxygen exposure of rice field soil

  Yanli Yuan, Ralf Conrad, Yahai Lu
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  ABSTRACT: Methane production in paddy soil is substantially suppressed after even a brief exposure of soil to oxygen. We hypothesized that the strong response of methanogen activity is reflected in the transcription of functional genes rather than in the composition of the community of methanogens. Therefore, we determined the community composition and the transcriptional response of methanogens in a rice field soil by targeting the mcrA gene (encoding the α subunit of methyl-coenzyme M reductase). Transcription of mcrA genes measured by quantitative PCR decreased by an order of magnitude after brief exposure to O2. Terminal restriction fragment length polymorphism of mcrA genes and gene transcripts showed that although the community structure of methanogens did not change, the composition of transcripts dramatically responded to O2 exposure. In the beginning, transcripts of Methanocellales were the relatively most abundant, indicating resistance of these hydrogenotrophic methanogens against O2 stress. Later on, mcrA transcripts of acetoclastic methanogens became relatively more abundant coinciding with the turnover of acetate. The transcription of Methanosarcinaceae was relatively greater when acetate accumulated while Methanosaetaceae became more active when acetate concentrations decreased. In the presence of methyl fluoride, a specific inhibitor of acetoclastic methanogenesis, mcrA transcription by Methanosaetaceae was greatly suppressed while that of Methanosarcinaceae was less affected. Our study showed that in contrast to constant community structure as revealed by DNA-based fingerprinting the transcription of functional mcrA genes strongly responded to O2 stress and the presence of inhibitor CH3F. The response patterns reflected the genomic and physiological traits of individual methanogens.
  Environmental Microbiology Reports 01/2011; 3(3):320 - 328. · 3.23 Impact Factor
• Article: Diversity, structure, and size of n(2)o-producing microbial communities in soils-what matters for their functioning?

  Gesche Braker, Ralf Conrad
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  ABSTRACT: Nitrous oxide (N(2)O) is mainly generated via nitrification and denitrification processes in soils and subsequently emitted into the atmosphere where it causes well-known radiative effects. How nitrification and denitrification are affected by proximal and distal controls has been studied extensively in the past. The importance of the underlying microbial communities, however, has been acknowledged only recently. Particularly, the application of molecular methods to study nitrifiers and denitrifiers directly in their habitats enabled addressing how environmental factors influence the diversity, community composition, and size of these functional groups in soils and whether this is of relevance for their functioning and N(2)O production. In this review, we summarize the current knowledge on community-function interrelationships. Aerobic nitrification (ammonia oxidation) and anaerobic denitrification are clearly under different controls. While N(2)O is an obligatory intermediate in denitrification, its production during ammonia oxidation depends on whether nitrite, the end product, is further reduced. Moreover, individual strains vary strongly in their responses to environmental cues, and so does N(2)O production. We therefore conclude that size and structure of both functional groups are relevant with regard to production and emission of N(2)O from soils. Diversity affects on function, however, are much more difficult to assess, as it is not resolved as yet how individual nitrification or denitrification genotypes are related to N(2)O production. More research is needed for further insights into the relation of microbial communities to ecosystem functions, for instance, how the actively nitrifying or denitrifying part of the community may be related to N(2)O emission.
  Advances in applied microbiology 01/2011; 75:33-70. · 5.23 Impact Factor
• Article: Bacterial and archaeal communities involved in the in situ degradation of 13C-labelled straw in the rice rhizosphere

  Minita Shrestha†, Pravin Malla Shrestha†, Ralf Conrad
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  ABSTRACT: Rice straw is a major substrate for the production of methane in flooded rice fields and results in increase of CH4 emission into the atmosphere. We investigated the bacteria and archaea involved in straw degradation by adding 13C-labelled straw to the rhizosphere of planted rice microcosms in the greenhouse. The degradation of added straw resulted in the production of 13C-labelled CH4 as end-product, which was detected in the pore water. The incorporation of 13C into ribosomal RNA of Bacteria and Archaea present in the rhizospheric soil and on the roots was assessed by stable isotope probing (SIP) followed by terminal restriction fragment polymorphism (T-RFLP) fingerprinting and cloning/sequencing of RNA fractions with different buoyant densities. Members of the Clostridium cluster I, III and XIVa were actively involved in straw degradation both in rhizospheric soil and on roots. However, on roots, Proteobacteria, Bacilli, Actinobacteria, Bacteroidetes and Chlorobi were also involved in the straw degradation process. Mostly Methanosarcina and to a less degree also Methanobacteriaceae were the dominant Archaea that assimilated straw-derived carbon in the rhizospheric soil. Both Bacteria and Archaea together were most likely responsible for the conversion of rice straw to CH4. In conclusion, this study tackled the important and interesting issue of linking active microorganisms responsible for the straw degradation process to CH4 emission into the atmosphere.
  Environmental Microbiology Reports 01/2011; · 3.23 Impact Factor