Methanogenic Degradation and Microbial Metabolism of Trace Gases
We want to learn which groups of soil microorganisms are responsible for particular
biogeochemical processes and to understand the reason why. For this purpose we investigate
biogeochemical processes involved in the exchange of climatically relevant trace gases (CH4,
N2O, H2) between soil and atmosphere. A particular focus is on processes in flooded rice
fields, which we have used during the last thirty years as a model system for studying
biogeochemistry and ecology of soil microbes.
Our experimental approach includes analytical chemical techniques and isotopic tracer studies
(14C, 13C) for investigating the biogeochemical cycling of microbial substrates and effectors in
soil. Similar techniques are used to investigate metabolic processes in microbial cultures, in
particular studying stable isotope fractionation. Culture studies are the basis for the use of
natural
13
C abundance measurements to elucidate paths of C-flux. Another important
experimental approach is the molecular characterization of rRNA genes and different proteinencoding genes for elucidating the composition of microbial communities, including
transcript analysis and fluorescent in situ hybridization (FISH). Furthermore, stable isotope
probing (SIP) of DNA and RNA allows the identification of metabolically active populations
that incorporate 13C-labelled substrates.
The research group of Ralf Conrad also encompasses the project groups of Dr. Martin Blaser
and Dr. Marc G. Dumont The present report describes the progress of these groups in the
years 2012 and 2013.
1. Research group Ralf Conrad
(1) Microbial control of CH4 production in various anoxic environments, such as rice field
ecosystems, lake sediments, ephemeral flooded soils, canopy wetlands, and arid land. In
previous study we found that drainage of flooded rice fields results in dramatic decrease of
CH4 production, albeit the community structure of methanogens was little affected [33]. We
therefore systematically investigated the effect drying and rewetting in a wide diversity of
terrestrial systems differing in the extent of inundation. These systems covered arid lands as
the one extreme and permanently inundated lake sediments as the other. In all cases we
determined rates and pathways of methanogenesis and characterized the bacterial and archaeal
communities involved. Arid lands had only very low population densities of methanogens,
which all belonged to the genera Methanocella and Methanosarcina. However, after wetting,
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these methanogenic populations proliferated and produced methane [2-5]. The cascade of
microbial resuscitation in biological desert soil crusts was studied using stable isotope probing
with H218O (Fig. 1) and showed an initial dominance by Bacillales and Clostridiales, which
apparently served as primary fermenting bacteria providing substrate to the methanogens [1].
Lake sediments are well known as methanogenic environments. Community composition and
activity in sediments on the Tibetan plateau differed primarily with respect to salinity [31],
those in Danish grasslands on drained peat mainly in soil pH [19]. In Brazilian lake sediments
we found differences between sediments from white water, black water and clear water lakes.
Intermittent drying of such sediments resulted in drastic change in the composition of both
archaeal and bacterial populations. Like in desert soils, populations of Clostridiales and of the
methanogenic Methanocellales and Methanosarcinaceae became dominant after drying. The
diversity of Bacteria decreased in particular, however, without impeding the activity of CH4
production [8]. Rice cultivation in Uruguay is a crop rotation of four years pasture and two
years flooded rice. The soil thus changes from four years aerated upland condition to two
years anoxic flooded conditions. Interestingly, the community of methanogenic archaea was
dominated by Methanocellales and Methanosarcinaceae and that of the Bacteria by
Firmicutes. However, population densities of methanogens were generally quite high, despite
aerated soil conditions for four years [17]. Adaptation of methanogenic communities to
temporary and permanent dryness seems to involve a stepwise change of abundance and/or
composition of the fermenting bacteria and methanogenic archaeal communities.
(2) Partitioning of carbon flow from root, straw and soil organic matter to methane in rice
fields. Methane production in rice fields originates from three possible sources of carbon,
which is soil organic matter, plant residues (straw) and root exudates (plant photosynthesis).
Quantitative partitioning of C flow from these three sources can be achieved by labeling
studies. We used
13
C-labeled rice straw and straw derived from maize (C4) and rice (C3)
having different isotopic composition. Maize straw comes from crop rotation between rice
and maize, which is increasingly common in Asia. The studies showed that application of
maize and rice straw resulted in similar abundance of the major methanogens, and similar
rates and pathways of CH4 production [9, 10]. Root exudation (plant photosynthesis) was
shown to be the main source of methane production [40]. Addition of straw resulted in
increased methane production, which however, was not only due to the decomposition of
straw organic matter, but also due to stimulation of methane production from soil organic
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matter. The mechanism of this stimulation is unclear, but seems to involve microbial activity
that is increased by the addition of straw [10, 41].
(3) Niche differentiation of bacterial and archaeal ammonia oxidation in different soil
environments. The first step in nitrification is caused by ammonia-oxidizing bacteria (AOB)
and the newly discovered ammonia-oxidizing archaea (AOA), which belong to the phylum of
Thaumarchaeota. They both occur in soils and sediments, but their relative role in ammonia
oxidation is unclear. We addressed this question using different environments, i.e. different
compartments (bulk soil, surface soil, rhizosphere, roots) of rice fields [28, 29], freshwater
sediments at different temperature [39], and acidic volcanic soils of different age [21]. We
found niche differentiation in rice fields with AOB being mainly active in soil and AOA in
root compartments. We also found that AOA were the main pioneers in colonization of young
volcanic soil, but that AOB were the predominant ammonia oxidizers in lake sediment, except
at increased temperature.
2. Project group Martin Blaser
(1) Isotopic enrichment in methanogenic and acetogenic microbial cultures. Most biological
systems discriminate against the heavier 13C isotope characterized by the isotope enrichment
factor (ε). We recently showed that cultures of methylotrophic methanogens exhibited very
strong fractionation with ε = -83‰ [35]. Likewise, eleven different chemolithotrophic
homoacetogenic bacterial cultures grown on H2/CO2 also exhibited very strong fractionation,
on average about -57‰ [6]. However, fractionation was affected by the availability of total
CO2 and by the buffer used for the growth medium [14]. Temperature had no effect on
fractionation, but hydrogenotrophic and methylotrophic methanogenesis could be
distinguished by their overall fractionation [36]. For homoacetogenic bacterial cultures we
have found that fractionation was strongly affected by the used substrate. C1 compounds
(methanol, formate, CO2) are directly incorporated via the acetyl-CoA pathway and were
associated with strong isotopic enrichment. The same organisms can also ferment hexose
producing two mole acetate via glycolysis / pyruvate decarboxylation and one mole acetate
via the acetyl-CoA pathway. By inhibiting the acetyl-CoA pathway with nitrate we could
show that the released acetate is an isotopic mixture of the two underlying pathways
(Bachelor Thesis, C. Freude).
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(2) Isotopic enrichment in methanogenic and acetogenic environmental samples. The
importance of homoacetogenesis in the environment was addressed by measuring isotopic
fractionation in anaerobically incubated rice field soil under N2/CO2 or H2/CO2, at different
temperatures (15°C, 37°C, 50°C) and in the presence and absence of bromoethanesulfonate
(inhibits methanogenesis). Strong isotopic enrichment into acetate was only detectable under
elevated hydrogen. Low temperature clearly favored homoacetogenesis, while at higher
temperatures both methanogenesis and homoacetogenesis were operating. Under natural
conditions the contribution of the acetyl-CoA pathway to the overall isotopic acetate signal
was rather small. Further studies will include molecular analysis of the methanogenic and the
homoacetogenic microbial communities. To characterize the diversity and phylogeny of
methanogens in the environment, we investigated the stability of the mcrA transcripts (qPCR)
upon drying Philippine rice field soil in the laboratory (Bachelor Thesis, P. Pledl).
3. Project group Marc G. Dumont
(1) Methanotrophic diversity. Methane oxidation by methanotrophic bacteria is an important
process that mitigates methane release to the atmosphere. We are interested in determining the
factors that affect the diversity and activity of different genera of methanotrophs in various
environments. In the past two years we have moved to using a next-generation sequencing
(NGS) approach to study methanotroph diversity, for example by pyrosequencing pmoA gene
amplicons. We developed two approaches for assigning pmoA sequences to methanotroph
taxa using either a naïve Bayesian classifier or a lowest common ancestor classifier based on
BLAST alignments [15]. We are now using this technology to investigate factors controlling
methanotroph diversity in wetlands [13], lake sediments (Dumont, in prep.) and terra preta
soil in Amazonia (Lima & Dumont, in prep.). We find that environmental factors, such as
water content and pH, affect the relative abundance of methanotroph genera and species. We
are now trying to get better insights into the mechanisms by which methanotrophs respond to
the environment using metatranscriptomics.
(2) Metatranscriptomics targeting methanotrophs. We have developed a method to obtain a
targeted metatranscriptome of methane oxidizers in environmental samples by using stable
isotope probing (SIP) with
13
CH4 to recover
13
C-enriched RNA. We used this approach to
target methanotroph RNA in aerobic lake sediment, which is a zone of high methane
oxidation activity [16]. Shotgun sequencing of the
13
C-RNA indicated that we enriched the
metatranscriptome to >70% methanotroph-associated sequences, compared to 5% in the
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unenriched RNA. We could identify pathways of carbon and nitrogen metabolism in the
methanotrophs as well as identify highly expressed genes, for example encoding proteins
involved in motility, that give clues to their behaviour [16]. We are now using this method to
answer longstanding questions such as how nitrogen stimulates methane oxidation activity
(Hu & Dumont, in prep.). We are also complementing these studies with experiments using
methanotroph pure cultures, for example to investigate how methanotrophs respond to
starvation [7].
(3) Microbial ecology of methane-producing environments. We are also interested in
obtaining a broader understanding of microbial communities and processes occurring in
environments with high methane production and consumption potentials. Rice paddy systems
are one of the most important natural sources of atmospheric methane and we are studying the
effect of rice plants on the composition of the community and soil processes. For example, we
have used deep sequencing of 16S rRNA genes and the GeoChip to investigate the effect of
plant growth stage on the microbial community structure and function. We find that the rice
plant has a significant effect on the microbial community and potential carbon and nitrogen
transforming processes (Breidenbach & Dumont, in prep.). Wetlands are another important
source of methane and again we have used 16S rRNA amplicon pyrosequencing to
characterize the microbial communities in wetlands on the Qinghai-Tibet plateau [12]. Lastly,
in lake sediments we have shown using a combination of SIP and cultivation approaches that
previously uncultivated actinobacteria could be anaerobically oxidizing Fe(II) to Fe(III),
thereby indirectly mitigating methanogenesis by favouring iron reduction [27].
Publications
1. Angel, R. and Conrad, R. (2013) Elucidating the microbial resuscitation cascade in
biological soil crusts following a simulated rain event. Environ. Microbiol. 15, 2799-2815.
2. Angel, R., Claus, P., and Conrad, R. (2012) Methanogenic archaea are globally ubiquitous
in aerated soils and become active under wet anoxic conditions. ISME J. 6, 847-862.
3. Angel, R., Kammann, C., Claus, P., and Conrad, R. (2012) Effect of long-term free-air CO2
enrichment on the diversity and activity of soil methanogens in a periodically waterlogged
grassland. Soil Biol. Biochem. 51, 96-103.
4. Angel, R., Paternak, Z., Soares, M. I. M., Conrad, R., and Gillor, O. (2013) Active and total
prokaryotic communities in dryland soils. FEMS Microbiol. Ecol. 86, 130-138.
5. Aschenbach, K., Conrad, R., Rehakova, K., Dolezal, J., Janatkova, K., and Angel, R.
(2013) Methanogens at the top of the world: occurrence and potential activity of methanogens
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in newly deglaciated soils in high-altitude cold deserts in the Western Himalayas. Frontiers
Microbiol. 4, 359, doi:10.3389/fmicb.2013.00359.
6. Blaser, M. B., Dreisbach, L. K., and Conrad, R. (2013) Carbon isotope fractionation of 11
acetogenic strains grown on H2 and CO2. Appl. Environ. Microbiol. 79, 1787-1794.
7. Brandt, F., Pommerenke, B., and Dumont, M.G. (2013) Continued presence of pmoA
mRNA and 16S rRNA in aerobic methanotrophs under anoxic conditions without methane;
submitted.
8. Conrad, R., Ji, Y., Noll, M., Klose, M., Claus, P., and Enrich-Prast, A. (2013) Response of
the methanogenic microbial communityies in Amazonian oxbow lake sediments to
desiccation stress. Environ. Microbiol., in press, doi:10.1111/1462-2920.12267.
9. Conrad, R., Klose, M., Lu, Y., and Chidthaisong, A. (2012) Methanogenic pathway and
archaeal communities in three different anoxic soils amended with rice straw and maize straw.
Front. Microbiol. 3, 4, doi:10.3389/fmicb.2012.00004.
10. Conrad, R., Klose, M., Yuan, Q., Lu, Y., and Chidthaisong, A. (2012) Stable carbon
isotope fractionation, carbon flux partitioning and priming effects in anoxic soils during
methanogenic degradation of straw and soil organic matter. Soil Biol. Biochem. 49, 193-199.
11. Daebeler, A., Metje, M., and Frenzel, P. (2013) Methyl fluoride affects methanogenesis
rather than community composition of methanogenic archaea in a rice field soil. PLoS One 8,
e53656.
12. Deng, Y., Cui, X., Hernández, M., and Dumont, M.G. (2013) Bacterial diversity in
hummock and hollow soils of three wetlands on the Qinghai-Tibetan Plateau revealed by 16S
rRNA pyrosequencing; submitted.
13. Deng, Y., Cui, X., Lüke, C., and Dumont, M.G. (2013) Aerobic methanotroph diversity in
Riganqiao peatlands on the Qinghai-Tibetan Plateau. Environ. Microbiol. Reports 5, 566-574.
14. Dreisbach, L.K., Blaser, M., and Conrad, R. (2013) Carbon isotope fractionation of
Thermoanaerobacter kivui in different growth media and with different total inorganic carbon
concentrations, submitted.
15. Dumont, M.G., Lüke, C., Deng, Y., and Frenzel, P. (2013) Classification of pmoA
amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN;
under revision.
16. Dumont, M.G., Pommerenke, B., and Casper, P. (2013) Using stable isotope probing to
obtain a targeted metatranscriptome of methanotrophs in lake sediment.
Environ.Microbiol.Reports 5, 757-764.
17. Fernandez Scavino, A., Ji, Y., Pump, J., Klose, M., Claus, P., and Conrad, R. (2013)
Structure and function of the methanogenic microbial communities in Uruguayan soils shifted
between pasture and irrigated rice fields. Environ. Microbiol. 15, 2588-2602.
18. Fröhlich-Nowoisky, J., Burrows, S. M., Xie, Z. Q., Engling, G., Solomon, P. A., Fraser,
M. P., Mayol-Bracero,O.L., Artaxo,P., Begerow,D., Conrad,R., Andreae,M.O., Despres,V.,
and Pöschl,U. (2012) Biogeography in the air: fungal diversity over land and oceans.
Biogeosciences 9, 1125-1136.
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19. Görres, C. M., Conrad, R., and Petersen, S. (2013) Effect of soil properties and hydrology
on Archaeal community composition in three temperate grasslands on peat. FEMS Microbiol.
Ecol. 85, 227-240.
20. Henneberger, R., Lüke, C., Mosberger, L., and Schroth, M.H. (2012) Structure and
function of methanotrophic communities in a landfill-cover soil. FEMS Microbiol. Ecol. 81,
52-65.
21. Hernandez, M., Dumont, M. G., Calabi, M., Basualto, D., and Conrad, R. (2013)
Ammonia-oxidizers are pioneer microorganisms in the colonization of new acidic volcanic
soils from South of Chile. Environ. Microbiol. Reports, in press, doi:10.1111/17582229.12109.
22. Ho, A. and Frenzel, P. (2012) Heat stress and methane-oxidizing bacteria: effects on
activity and population dynamics. Soil Biol. Biochem. 50, 22–25.
23. Ho, A., Erens, H., Mujinya. B.B., Boeckx, P., Baert, G., Schneider, B., Frenzel, P., Boon,
N., and Van Ranst, E. (2013) Termites facilitate methane oxidation and shape the
methanotrophic community. Appl. Environ. Microbiol. 79,7234-7240.
24. Ho, A., Kerckhof, F.M., Lüke, C., Reim, A., Krause, S., Boon, N., and Bodelier, P.L.E.
(2013) Conceptualizing functional traits and ecological characteristics of methane-oxidizing
bacteria as life strategies. Environ. Microbiol. Rep. 5, 335-345.
25. Ho, A., Lüke, C., Reim, A., and Frenzel, P. (2013) Selective stimulation in a natural
community of methane oxidizing bacteria: effects of copper on pmoA transcription and
activity. Soil Biol. Biochem. 65, 211-216.
26. Ho, A., Vlaeminck, S.E., Ettwig, K.F., Schneider, B., Frenzel, P., and Boon, N. (2013)
Revisiting methanotrophic communities in sewage treatment plants. Appl. Environ.
Microbiol. 79, 2841-2846.
27. Kanaparthi, D., Pommerenke, B., Casper, P., and Dumont, M.G. (2013)
Chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of actinobacterial subdivision
lineage TM3. ISME J. 7, 1582-1594.
28. Ke, X., Angel, R., Lu, Y., and Conrad, R. (2013) Niche differentiation of ammonia
oxidizers and nitrite oxidizers in rice paddy soil. Environ. Microbiol. 15, 2275-2292.
29. Ke, X., Lu, Y., and Conrad, R. (2013) Different behavior of methanogenic archaea and
Thaumarchaeota in rice field microcosms. FEMS Microbiol. Ecol., in press,
doi:10.1111/1574-6941.12188.
30. Krause, S., Lüke, C., and Frenzel, P. (2012) Methane source strength and energy flow
shape methanotrophic communities in oxygen-methane counter-gradients. Environ.
Microbiol. Rep. 4, 203-208.
31. Liu, Y., Yao, T., Gleixner, G., Claus, P., and Conrad, R. (2013) Methanogenic pathways,
C isotope fractionation, and archaeal community compoistion in lake sediments and wetland
soils on the Tibetan Plateau. J. Geophys. Res. Biogeosci. 118, 650-664.
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32. Lüke, C., Frenzel, P., Ho, A., Fiantes, D., Schad, P., Schneider, B., Schwark, L., and
Utami, S.R. (2013) Macroecology of methane oxidizing bacteria: The β-diversity of pmoA
genotypes in tropical and subtropical rice paddies. Environ. Microbiol., in press.
33. Ma, K., Conrad, R., and Lu, Y. (2012) Responses of methanogen mcrA genes and their
transcripts to alternate dry wet cycle of paddy field soil. Appl. Environ. Microbiol. 78, 445454.
34. Ma, K., Conrad, R., and Lu, Y. (2013) Dry/wet cycles change the activity and population
dynamics of methanotrophs in rice field soil. Appl. Environ. Microbiol. 79, 4932-4.
35. Penger, J., Conrad, R., and Blaser, M. (2012) Stable carbon isotope fractionation by
methylotrophic methanogenic archaea. Appl. Environ. Microbiol. 78, 7596-7602.
36. Penger, J., Conrad, R., and Blaser, M. (2013) The stable carbon isotope fractionation of
strongly fractionating microorganisms is not affected by growth temperature, submitted.
37. Reim, A., Lüke, C., Krause, S., Pratscher, J., and Frenzel, P. (2012) One millimetre makes
the difference: high-resolution analysis of methane-oxidizing bacteria and their specific
activity at the oxic-anoxic interface in a flooded paddy soil. ISME J. 6, 2128-2139.
38. Roth, P.J., Lehndorff, E., Hahn, A., Frenzel, P., and Amelung, W. (2013) Cycling of rice
rhizodeposits through peptide-bound amino acid enantiomers in soils under 50 and 2000 years
of paddy management. Soil. Biol. Biochem. 65, 227-235.
39. Wu, Y., Ke, X., Hernandez, M., Wang, B., Dumont, M. G., Jia, Z., and Conrad, R. (2013)
Autotrophic growth of bacterial and archaeal ammonia oxidizers in freshwater sediment
microcosms incubated at different temperatures. Appl. Environ. Microbiol. 79, 3076-3084.
40. Yuan, Q., Pump, J., and Conrad, R. (2012) Partitioning of CH4 and CO2 production
originating from rice straw, soil and root organic carbon in rice microcosms. PLoS One 7,
e49073, doi:10.1371/journal.pone.0049073.
41. Yuan, Q., Pump, J., and Conrad, R. (2013) Straw application in paddy soil enhances
methane production also from other carbon sources. Biogeosciences Discuss. 10, 1416914193.
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Figure 1: Network depicting the mutual association of operational taxonomic units (OUT)
of bacterial (16S rRNA genes) communities in dry soil crusts and crusts incubated
under different conditions. Only OTUs from the ‘heavy’ fraction of samples labeled
with H218O were used. 1. arid/dry crust. 2. arid/light–oxic. 3. arid/dark-anoxic. 4.
hyperarid/dry crust. 5. hyperarid/light–oxic. 6. hyperarid/dark-anoxic. 7. unaffiliated
(taken from Angel & Conrad 2013, doi:10.1111/1462-2920.12140).
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