RESEARCH ARTICLE
Organic acids and ethanol inhibit the oxidation of methane by
mire methanotrophs
Adam S. Wieczorek, Harold L. Drake & Steffen Kolb
Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany
Correspondence: Steffen Kolb, Department
of Ecological Microbiology, University of
Bayreuth, 95440 Bayreuth, Germany. Tel.:
149 921 55 5620; fax: 149 921 55 5799;
e-mail: [email protected]
Received 20 December 2010; revised 28
February 2011; accepted 28 February 2011.
Final version published online 13 April 2011.
DOI:10.1111/j.1574-6941.2011.01080.x
Editor: Gary King
MICROBIOLOGY ECOLOGY
Keywords
atmospheric methane oxidation; acidic fen;
Methylocystis ; pmoA; mmoX; terrestrial
microbiology.
Abstract
Aerobic methane (CH4) oxidation reduces the emission of CH4 from mires and is
regulated by various environmental factors. Organic acids and alcohols are
intermediates of the anaerobic degradation of organic matter or are released by
plant roots. Methanotrophs isolated from mires utilize these compounds preferentially to CH4. Thus, the effect of organic acids and ethanol on CH4 oxidation by
methanotrophs of a mire was evaluated. Slurries of mire soil oxidized supplemental CH4 down to subatmospheric concentrations. The dominant pmoA and
mmoX genotypes were affiliated with sequences from Methylocystis species capable
of utilization of acetate and atmospheric CH4. Soil slurries supplemented with
acetate, propionate or ethanol had reduced CH4 oxidation rates compared with
unsupplemented or glucose-supplemented controls. Expression of Methylocystisaffiliated pmoA decreased when CH4 consumption decreased in response to acetate
and was enhanced after acetate was consumed, at which time the consumption of
CH4 reached control levels. The inhibition of methanotroph activity might have
been due to either toxicity of organic compounds or their preferred utilization.
CH4 oxidation was reduced at 5 and 0.5 mM of supplemental organic compounds.
Acetate concentrations may exceed 3 mM in the investigated mire. Thus, the
oxidation of CH4 might decrease in microzones where organic acids occur.
Introduction
Mires are the largest single source of atmospheric methane
(CH4) on Earth (100–237 Gt yr1) (Denman et al., 2007)
and are represented by various wetland types, such as peat
bogs and fens (Gore, 1983). Mire-inhabiting aerobic methanotrophic bacteria (hereafter methanotrophs) consume up
to 90% of endogenously produced CH4 (Shannon et al.,
1996; Hornibrook et al., 2009). Thus, methanotrophs are
important to CH4 fluxes in such wetland ecosystems. Mire
methanotrophs are active at anoxic–oxic interfaces (Sundh
et al., 1994, 1995; Kettunen et al., 1999). The upper 0.3 m of
peat bog soils has the spontaneous capacity to oxidize CH4
in the presence of oxygen (O2) (Macdonald et al., 1996;
Chen et al., 2008a; Tuomivirta et al., 2009). O2 is a key factor
in mires that alters both the oxidation and the production of
CH4, and hence net emission rates of CH4 (Kettunen et al.,
1999). O2 concentrations in mire soils vary due to fluctuations in the water table and vascular plants (e.g. sedges) that
leak O2 (Moog & Bruggemann, 1998; Kettunen et al., 1999;
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Mainiero & Kazda, 2005; Paul et al., 2006). Mires may not
only be net sources of CH4 but may also be temporary net
sinks of CH4 (Dedysh & Panikov, 1997; Denman et al., 2007;
Dalal & Allen, 2008). Thus, the factors that create varying
zones of CH4 consumption and CH4 production make it
difficult to predict emission rates of CH4 (Dalal & Allen,
2008; Knorr et al., 2008, 2009; Hornibrook et al., 2009).
Methanotrophic communities in mires are dominated by
species of Alphaproteobacteria (Dedysh, 2009). Methanotroph pure cultures have primarily been isolated from moss
(Sphagnum)-covered mires (i.e. peat bogs) and are acidotolerant species of the genera Methylocystis, Methylocella and
Methylocapsa (Alphaproteobacteria) (Dedysh et al., 2001,
2002, 2003; Chen et al., 2008a, b; Dedysh, 2009). Uncultivated alphaproteobacterial methanotrophs are associated
with Sphagnum cuspidatum (Raghoebarsing et al., 2005;
Kip et al., 2010). An uncultivated taxon containing pmoA
(gene encoding the hydroxylase subunit of the particulate
CH4, monooxygenase pMMO) genotype MHP was detected
in Calluna-covered wetlands (Chen et al., 2008a). MHP is
FEMS Microbiol Ecol 77 (2011) 28–39
29
Organic compounds alter methanotroph activity in fen soil
closely affiliated with Methylocapsa acidiphila (Chen et al.,
2008a; Degelmann et al., 2010). Gammaproteobacterial
methanotrophs may also be present in mire communities
(Morris et al., 2002; Jaatinen et al., 2005; Chen et al., 2008a).
Methanotroph abundances in fen soils are not known, but
their abundances have been measured in various peat bog
soils and range from 106 to 108 cells g1 soil wet weight
(Sundh et al., 1995; Dedysh et al., 2001, 2002, 2003; Dedysh,
2009; Belova et al., 2011).
Most cultured methanotrophs are restricted to the utilization of CH4 and other single carbon compounds (Trotsenko & Murrell, 2008). However, species of the genera
Beijerinckiaceae and Methylocystaceae may utilize alternative
compounds such as acetate (Dedysh et al., 2005; Dunfield
et al., 2010; Belova et al., 2011). Mire-derived isolates belong
to the genera Methylocystis and Methylocella. Methylocystis
strain H2s was isolated from a bog and grows slowly on
acetate in the absence of CH4 (Belova et al., 2011). Strain
H2s-related phylotypes are abundant in northern mires,
comprising 18–58% (i.e. 0.4 107–3.4 107 cells g1 soil
wet weight) of detected alphaproteobacterial methanotrophs (Belova et al., 2011). Methylocella species are facultative methanotrophs that appear to utilize acetate, malate,
succinate or ethanol in preference to CH4 (Dedysh et al.,
2005). Acetate inhibits the ability of Methylocella silvestris
BL2 to utilize CH4 by repressing the synthesis of soluble CH4
monooxygenase (sMMO; the gene for the hydroxylase
subunit is mmoX) (Theisen et al., 2005).
Acetate and ethanol are typical fermentation products
that are intermediates of the anaerobic degradation of
organic matter in mires (Drake et al., 2009; Wüst et al.,
2009). These compounds are also root exudates of mire
plants (Strom et al., 2003; Crow & Wieder, 2005). Acetate
concentrations in soil pore waters of mires vary both
temporally and spatially and can range as high 3.2 mM
(Shannon et al., 1996; Hines et al., 2001; Duddleston et al.,
2002; Rooney-Varga et al., 2007; Knorr et al., 2008; Küsel
et al., 2008). Thus, organic compounds may be determinants of CH4 oxidation in habitats that are inhabited by
facultative methanotrophs (Dedysh et al., 2005; Theisen &
Murrell, 2005). The objective of the current study was to
determine the effect of organic compounds on methanotrophic communities and CH4 oxidation of Sphagnum
fallax-covered patches of a temperate fen.
Materials and methods
Study site and sampling
Four soil cores (diameter 8 cm, depth 25 cm) were sampled
in August 2008 and three in May 2009 from different
S. fallax-covered patches of the acidic (pH 4.5) fen
Schlöppnenbrunnen (Fichtelgebirge, Bavaria, Germany;
FEMS Microbiol Ecol 77 (2011) 28–39
50107 0 5300 N, 11152 0 5100 E, 700 m a.s.l.). The moss patches
were dominated by S. fallax. Sphagnum palustre was occasionally present, but such patches were not sampled. Areas
lacking mosses were dominated by grasses and sedges, i.e.
Molinia caerulea, Eriophorum vaginatum, Juncus effusus and
Carex canescens (Gerstenberger, 2001; Knorr et al., 2009;
Küsel et al., 2008). The mean annual air temperature was
6.3 1C and the mean annual precipitation was 1020 mm
(Knorr et al., 2009). The soil type is a fibric histosol on
granite bedrock (Paul et al., 2006; Wüst et al., 2009). Intact
soil cores were placed in water-tight plastic bags and
incubated at 20 1C under day–night light conditions to
maintain photosynthesis of mosses. Evaporated water was
regularly refilled.
Sphagnum fallax-associated consumption of CH4
Living mosses were removed from soil cores. Dead plant
material was removed. The plants were pooled and washed
three times with distilled water. The washed plants were
placed in sterile flasks (three replicates) that were closed
with gas-tight stoppers. The atmosphere was ambient air
supplemented with 1000 mL L1. CH4 (approximately
1.4 mM in the aqueous phase; Riessner Gase GmbH, Germany). Plants were incubated for 50 days under a continuous day–night (12 h light, 12 h darkness) cycle at 20 1C in
closed bottles. Light was supplied with a standard light bulb.
Fresh sterile air was regularly added with syringes.
Effect of organic compounds on methanotrophs
and oxidation of CH4
Maximal concentrations of organic acids in the Schlöppnerbrunnen fen soil may exceed 3.5 mM (Küsel et al., 2008).
Thus, supplemental substrate concentrations of 0.5–5 mM
were chosen to investigate the effect of different organic
compounds on methanotrophs and CH4 oxidation. Mossfree fen soil samples from the upper 20 cm of three different
soil cores were homogenized and pooled. Forty grams of the
homogenate was placed into 500 mL flasks, diluted with
80 mL sterile water, sealed with gas-tight rubber stoppers.
Treatments were performed in triplicate. Anoxic controls
had a gas phase of sterile argon (100%; Riessner Gase
GmbH). Experiments were initiated by adding CH4
(1000 mL L1). Slurries were incubated in the dark at 20 1C
in a rotary shaker (60 r.p.m.). Liquid samples (2 mL) were
taken with sterile syringes (10 mL; BD, Germany), frozen
immediately on liquid nitrogen and stored at 80 1C until
analysed.
Analysis
CH4 was determined with a gas chromatograph equipped
with a flame ionization detector (Hewlett Packard)
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30
(Degelmann et al., 2010). Organic acids and alcohols were
measured by HPLC (Hewlett Packard) (Küsel & Drake,
1995). pH and soil dry weight were determined according
to previously published protocols (Matthies et al., 1993;
Maurer et al., 2008). Dry weight was determined by drying
plants at 60 1C until plant weight was constant.
CH4 oxidation rates were calculated from the linear part
of the first-order kinetics plotted through at least six data
points in each experimental replicate (Maurer et al., 2008).
Concentrations of CH4 are combined from both the gas and
the liquid phases and were calculated from the ideal gas law
taking into consideration the actual pressure, temperature,
pH, volume of gas and liquid phases in incubation flasks,
and the Bunsen coefficient (Matthies et al., 1993). A pairwise
t-test was applied to test if CH4 oxidation rates of incubations that were supplemented with CH4 plus acetate or
another organic compound (three experimental replicates)
were different from rates measured in the respective control
incubations (three experimental replicates) that were only
supplied with CH4.
DNA and RNA extraction
DNA was extracted from 0.3 g of fen soil with a modification
of a published protocol (Stralis-Pavese et al., 2004). Soil was
frozen and thawed, lysozyme was added to lysis buffer 1 and
proteinase K was omitted. DNA and RNA for enumeration
of the pmoA gene and transcript numbers by quantitative
PCR (qPCR) were coextracted according to another protocol optimized for simultaneous recovery of DNA and RNA
from soil samples (Griffiths et al., 2000), followed by
separation of DNA and RNA using the Qiagen RNA/DNA
Mini Kit (Qiagen GmbH, Germany). DNA and RNA
extracts were quantified using the Quant-iTTM PicoGreens
dsDNA kit and the Quant-iTTM RiboGreens RNA assay kit
(Invitrogen, Germany), respectively.
Construction of pmoA and mmoX gene libraries
DNA extracts from soil cores were pooled. One microlitre
DNA extract was used in PCRs. The reaction mix contained
Premix F (Epicentre) combined with a Taq DNA polymerase
recombinant (Invitrogen). The pmoA gene was amplified
with primers A189F (5 0 -GGNGACTGGGACTTCTGG-3 0 )
and A682R (5 0 -GAASGCNGAGAAGAASGC-3 0 ) (500 mM
final concentration) (Holmes et al., 1995). The PCR protocol was: initial denaturation at 94 1C for 8 min, 11-cycle
touchdown (denaturation at 94 1C for 1 min, elongation at
72 1C for 1 min) including a stepwise decrease of the
annealing temperature from 62 to 52 1C (1 min), 24 repeated cycles with denaturation, annealing (52 1C) and
elongation, followed by a final elongation at 72 1C (7 min).
The pmoA2 gene was amplified with primers pmoA206f (5 0 GGNGACTGGGACTTCTGGATCGACTTCAAGGATCG-3 0 )
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A.S. Wieczorek et al.
and pmoA703b (5 0 -GAASGCNGAGAAGAASGCGGC
GACCGGAACGACGT-3 0 ) (125 mM final concentration)
(Yimga et al., 2003). The mmoX gene was amplified with
primers mmoX206f (5 0 -ATCGCBAARGAATAYGCSCG-3 0 )
and mmoX886r (5 0 -ACCCANGGCTCGACYTTGAA-3 0 )
(500 mM) (Hutchens et al., 2004). Temperature profiles
and annealing conditions (including primer and Mg21
concentrations) were as published elsewhere (Yimga et al.,
2003; Hutchens et al., 2004). Genomic DNA of Methylococcus capsulatus Bath (pmoA and mmoX) and Methylocystis
parvus (pmoA and pmoA2) were used as positive controls.
PCR products were purified using a MinElute gel extraction
kit (Qiagen GmbH), ligated into vector pGEM-T (Promega,
Germany) and transformed into competent Escherichia coli
JM109 cells (Promega) according to the manufacturer’s
protocol. Clones were screened for insert-positive vectors
by amplification with primers M13uni and M13rev
(Messing, 1983) and subsequent fragment length analysis
with agarose gel electrophoresis. M13-PCR products were
purified before sequencing with a Millipore Multiscreen
96-well filtration system (Millipore Corp.) and sequenced
commercially (Macrogen Inc., Seoul, South Korea).
pmoA and mmoX analyses
pmoA and mmoX gene sequences were edited, translated
into amino acid sequences and aligned (CLUSTALW algorithm
in MEGA, version 4.0) (Tamura et al., 2007). The alignments
were refined manually. Similarity-based distance matrices
were calculated from alignments of amino acid sequences.
PmoA and MmoX sequences were grouped into specieslevel operational taxonomic units (OTUs) with the software
DOTUR (Schloss & Handelsman, 2005) and a species-level
OTU threshold value of 7% dissimilarity (Degelmann et al.,
2010). Phylogenetic trees were constructed from sequences
representative of each OTU, their closest related genotypes
(BLAST analysis using the latest version of the GenBank
nucleotide database; Altschul et al., 1990) and distantly
related genotypes using ARB (Ludwig et al., 2004). pmoA
and mmoX gene sequences were deposited at the European
Molecular Biology Laboratory (EMBL) database (http://
www.ebi.ac.uk/embl/) (FR726166–FR726182).
Quantification of Methylocystis -related pmoA
qPCR was performed on an iCycler thermocycler with the
iQ5 multicolor PCR detection system (Bio-Rad, Germany),
and data were analysed with IQ5 OPTICAL SYSTEM software
(version 2.0; Bio-Rad). pmoA of Methylocystis-related genes
was quantified using the qPCR assay TYPEII with modified
primers [II223f (5 0 -CGTCGTATGTGGCCGAC-3 0 ; Kolb
et al., 2003) and II646mR (5 0 -CGTGCCGCGCTCGAC
CATGCG-3 0 ; current study)]. Reactions were performed in
duplicate in Thermosprint 96 PCR plates sealed with
FEMS Microbiol Ecol 77 (2011) 28–39
31
Organic compounds alter methanotroph activity in fen soil
Thermosprint transparent sealing tapes (Bilatec, Germany).
The reaction volume was 20 mL. Five microlitres of DNA
solution containing 1–6 ng DNA was added to 15 mL of
reaction mix containing primers and twofold SensiMix Plus
SYBR and Fluorescein Supermix (Quantace, Germany). The
final primer concentration of all assays was 500 nM. The
initial denaturation was at 95 1C for 8 min, followed by 45
PCR cycles (denaturation at 95 1C for 25 s, annealing at
66 1C for 20 s and elongation at 72 1C for 45 s). Fluorescence
data of TYPEII assay were collected at an additional step
after elongation (82 1C, 10 s), as primer dimer formation
was evident by melting curve analysis. Quantitative DNA
standards were dilution series of quantified (Quant-iT PicoGreen dsDNA kit; Invitrogen) pmoA gene inserts flanked by
partial pGEM-T vector sequences (Degelmann et al., 2010)
that had identical primer binding sites as those of pmoA
genes of M. parvus.
Specificity of pmoA measurements
Target group specificity of all qPCR measurements was
evaluated. qPCR products obtained by the TYPEII assay
from each treatment (including all time points, products of
DNA- and cDNA-based measurements) were pooled. Specificity was evaluated by cloning of qPCR products and BLAST
analysis (Altschul et al., 1990) of at least 46 vector insert
sequences per cloned amplicon. All sequences (n = 140) were
affiliated with the target sequence group of the TYPEII assay
and were Z93% similar to pmoA genes of Methylocystis
species (data not shown). pmoA gene sequences were
deposited at the EMBL database (http://www.ebi.ac.uk/
embl/) (FR726183–FR726200).
Results
Oxidation of CH4 by S. fallax and fen soil
Quantification of pmoA transcripts
RNA extracts were reverse transcribed using random hexamer primers (SuperScript Vilo cDNA Synthesis Kit; Invitrogen) and pmoA cDNA quantities were then analysed by
qPCR as described above. cDNA numbers were corrected for
the amount of extracted total RNA and related to gene
numbers and variable in vitro transcription efficiencies.
Bias correction of qPCR measurements
Coextracted inhibitory compounds, such as humic substances, may inhibit qPCR measurements leading to incorrect and underestimated gene numbers (Degelmann et al.,
2010). Thus, every DNA extract was spiked with 2 106
inhibition-control DNA molecules mL1. DNA extracts were
quantified with the pmoA-specific assay, and additionally
with assay INHIB-CORR to calculate a factor that allows for
correction of pmoA gene numbers (Degelmann et al., 2010).
Transcript quantification may also be biased due to variable
reverse transcription efficiencies. Thus, RNA extracts were
spiked with 2 108 inhibition-control RNA molecules mL1.
The single-stranded inhibition-control RNA was synthesized
by in vitro transcription of pGEM-T vector DNA using the
T7 transcription kit (Fermentas, Germany). The product
was purified according the manufacturer’s instructions and
quantified (Quant-iTTM RiboGreens RNA assay kit; Invitrogen). The RNA extracts were then spiked with inhibitioncontrol RNA and quantified with the newly established assay
RNA-INHIB-CORR [IHCf (5 0 -ATTGGGCCCGACGTC-3 0 ),
IHCr (5 0 -ATTTAGGTGACACTATAGAATA-3 0 )]. The thermo profile was similar as for the TYPEII assay, but annealing
was optimal at 60 1C (20 s) and fluorescence data were
acquired during elongation. The correction factor was
calculated as described for DNA-based measurements (Degelmann et al., 2010)
FEMS Microbiol Ecol 77 (2011) 28–39
Initial experiments with fen material (i.e. a combination of
S. fallax and soil) demonstrated that supplemental acetate
and ethanol decreased the consumption (presumably due to
oxidation) of 1000 mL L1. CH4 under oxic conditions (see
Supporting Information, Fig. S1). In subsequent, more
detailed analyses, all tested samples (i.e. S. fallax and
different depths of fen soil) consumed CH4 (Table 1).
Consumption of CH4 followed first-order reaction kinetics
(Fig. 1). Production of CH4 under anoxic conditions was
negligible (data not shown). Fen soil displayed a greater
capacity to consume CH4 than did S. fallax (Table 2).
Nonetheless, washed moss oxidized CH4 for 4 100 days
(data not shown). CH4 was consumed to below atmospheric
concentrations (i.e. o 1.8 p.p.m.; Fig. 1), indicating the
presence of methanotrophs that were capable of utilizing
atmospheric CH4.
Methanotrophic community composition
The number of sequences of pmoA (n = 260) and mmoX
(n = 170) covered 4 90% of the estimated genotypes based
on rarefaction analyses (Fig. S2). A total of 10 and seven
OTUs were estimated for pmoA and mmoX, respectively
(Fig. S2). Dominant pmoA and mmoX genotypes (82% and
64% for pmoA and mmoX, respectively) belonged to Methylocystis (Methylocystaceae; Figs 2 and 3). The closest cultured
relatives of the dominant pmoA genotype OTU 1 were
Methylocystis strains SC2, DWT and LR1 (Fig. 2), strains
capable of atmospheric CH4 consumption (Kolb, 2009).
OTU 1 was also similar (o 10% difference based on amino
acid sequence) to the bog-derived Methylocystis strain H2s
(Belova et al., 2011). OTU 7 was closely related to pmoA2,
and its detection suggested the presence of methanotrophs
with an alternative CH4 monooxygenase that utilizes atmospheric CH4 (Yimga et al., 2003; Baani & Liesack, 2008).
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32
A.S. Wieczorek et al.
Table 1. Apparent CH4 oxidation rates (Vapp) of Sphagnum fallax and
fen soil
Treatment
S. fallaxw
Control
Ethanol (5 mM)
Ethanol (0.5 mM)
Acetate (5 mM)
Acetate (0.5 mM)
Control
Ethanol (5 mM)
Ethanol (0.5 mM)
Acetate (5 mM)
Acetate (0.5 mM)
Control
Ethanol (5 mM)
Ethanol (0.5 mM)
Acetate (5 mM)
Acetate (0.5 mM)
Control
Glucose (5 mM)
Acetate (5 mM)
Propionate (2.5 mM)
Ethanol (2.5 mM)
1.0 0.3
0.5 0.0z
ND
ND
ND
4.7 0.9
1.6 0.5z
4.1 0.8
0.8 0.1z
4.0 0.9z
5.1 0.8
2.2 0.3z
3.4 0.6z
1.0 0.3z
4.5 0.1
6.9 0.5
6.6 0.2
1.6 0.2z
2.7 0.3z
5.3 0.3z
0–6 cm soilw
6–15 cm soilw
0–20 cm soil‰
Supplemental CH was 1000 mL L1. CH (7 mmol g1 dry weight), and
4
4
rates were measured for 19 h after supplementation with organic
compounds. Values are mean SD.
w
The sampling date was August 2008.
z
Respective rates were statistically different (p 0.05) from the unsupplemented control (pairwise t-test).
‰
The sampling date was May 2009.
ND, not determined.
0.03
6
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c
0.01
40
60
80
100
4
2
0
0
20
40
60
Time (h)
80
100
Fig. 1. Utilization of supplemental CH4 by slurries of fen soil. Soil was
obtained in August 2008. Inset: a magnified view of the latter time
period. Filled circles, CH4 in treatment with ambient air and supplemental CH4; empty circles, CH4 in anoxic treatment without supplemental
CH4. The dotted line in the inset is the atmospheric mixing ratio
(1.8 p.p.m. CH4). Error bars are the SD of mean values of three replicates.
Table 2. Apparent CH4 oxidation rates of Sphagnum fallax and fen soil
Material
w
The mmoX gene library was dominated by OTUs 1 and 2,
genotypes that were closely related to isolates H2s and LR1.
These OTUs displayed o 10% dissimilar mmoX-encoded
amino acid sequences (Fig. 3). Approximately 13% of
the detected MmoX OTUs affiliated with facultative methanotrophic Beijerinckiaceae (Methylocella spp.; Fig. 3).
The MmoX OTU 6 represented a novel deep branching
genotype with a nucleotide sequence that was Z23%
dissimilar to those of published mmoX genes. However, the
low relative abundance of OTU 6 (0.6%; Fig. 3) indicates
that it was probably unimportant to the in situ oxidation
of CH4.
Methylococcaceae were also detected with the two gene
markers. However, Methylococcaceae-affiliated OTUs contributed only 4.7% of all pmoA and mmoX sequences
(Figs 2 and 3). Gammaproteobacterial PmoA proteins were
related ( o 10% based on amino acid sequence) to uncultured species previously detected in a peat bog (Fig. 2; Chen
et al., 2008a), and the respective MmoX OTUs were highly
similar ( o 3% based on amino acid sequence) to sequences
of M. capsulatus and Methylomonas sp. LW13 (OTUs 5 and
7, respectively; Fig. 3).
0.05
8
CH4 (µmol g–1soil DW)
Material
Vapp CH4 (mmol g1 dry
weight soil day1)
S. fallax
0–6 cm soilz
6–15 cm soilz
CH4 (mmol g1 dry weight soil day1)
1.0 0.3
4.7 0.9
5.1 0.8
The sampling date was August 2008. Values are means with SDs.
w
Sphagnum fallax plants were incubated for 4 100 days under an oxic
atmosphere with 1000 mL L1. CH4 (7 mmol g1 dry weight).
z
Soil slurries were incubated for 24 h.
Effect of organic compounds on the oxidation of
CH4 and expression of Methylocystis pmoA
CH4 oxidation was impaired when CH4-supplemented fen
soil slurries were supplemented with acetate, ethanol or
propionate (Figs 4c and 5) as compared with unsupplemented soil slurries (Fig. 4a). In contrast, glucose did not affect
CH4 oxidation (Fig. 4b). CH4 oxidation rates decreased
significantly with 5 mM organic compounds. Although
CH4 oxidation rates were decreased less with 0.5 mM
organic compounds than with 5 mM organic compounds,
the differences between the rates obtained with these two
concentrations were statistically significant (Table 1).
The pH at the start of incubations varied between 4.4 and
5.0, and stayed constant or increased up to a maximum of
FEMS Microbiol Ecol 77 (2011) 28–39
0.10
54%
53%
86%
69%
OTU 8 | 0.8%
OTU 9 | 0.8%
OTU 6 | 1.9%
Environmental sequence (EF644624)
OTU 1 | 81.9%
Environmental sequence (FJ930092)
OTU 2 | 1.9%
Methylocystis sp. SC2 (AJ431386)
Methylocystis sp. DWT (AJ868404)
M th l
ti sp. LR1 (Y18443)
55% Methylocystis
67% OTU 4 | 6.9%
Environmental sequence (AY781164)
52% 85% Methylocystis sp. H2s (FN422005)
81% OTU 5 | 2.3%
97%
Environmental sequence (AJ868287)
64%
‘Methylosinus acidophilus’ (DQ076755)
Methylosinus sporium (DQ119048)
70% Methylosinus trichosporium BF1 (AJ868409)
69%
56%
Cluster 5 (AJ868265)
RA14 (AF148521)
54%Methylocapsa acidiphila B2 (AJ278727)
77%
AC (AY550736)
Cl t 4 (AJ868259)
Cluster
97% OTU 7 | 1.9%
Environmental sequence (AY781169)
Methyloc
cystaceae
33
Organic compounds alter methanotroph activity in fen soil
100%
Methylocystis sp. SC2 (AJ431387)
Fig. 2. Phylogenetic tree of PmoA OTUs and
reference sequences. Gene libraries were
prepared from the August 2008 sampling.
Percentage values after OTUs indicate the relative
frequency of sequences assigned to a given OTU.
The total number of sequences was 260.
Accession numbers are given in parentheses. The
tree was calculated using translated amino acid
sequences by applying the TREEPUZZLE algorithm
(126 amino acids, JTTsubstitution model; Schmidt
et al., 2002). Percentage values at nodes are tree
puzzle estimates (based on 25 000 replicates).
Open circles at nodes indicate that these nodes
were confirmed by the ARB-implemented
neighbour-joining algorithm using the same
dataset (Ludwig et al., 2004). Scale bar, 10%
sequence divergence.
59%
96% OTU 10 | 0.4%
Environmental sequence (AY781167)
83%
96%
OTU 3 | 1.2%
Environmental sequence (AY781161)
Cluster 3 (AJ868281)
68%
64% Methylococcus capsulatus BL4 (AB484601)
Methylomonas
y
methanica ((EU722434))
90%
52%
JR3 (FJ970601)
USC γ (AJ579669)
Cluster 2 (AJ868278)
89%
Cluster 1 (AJ868245)
Crenothrix polyspora (DQ295899)
80%
Nitrosomonas europaea (AF037107)
MR1 (AF200729)
RA21 (AF148522)
Methylacidiphilum fumariolicum SolV (EF591085)
6.0 when supplemental organic compounds were consumed
(Figs 4 and 5). Glucose, acetate and ethanol were utilized
without apparent delay (Fig. 5). Propionate consumption
started after 20 h (Fig. 5b). CH4 was consumed without
apparent delay in all treatments, but rates were lowered
by 75%, 22% and 60% in the presence of supplemental
acetate, propionate or ethanol, respectively (Figs 4 and 5;
Table 1).
CH4 oxidation rates and pmoA gene expression increased
when supplemental acetate was consumed to 0.5 mM or less
(Fig. 4c). The decreased oxidation of CH4 was maximal
above 1 mM acetate (Fig. 4c). pmoA expression in the
FEMS Microbiol Ecol 77 (2011) 28–39
Methylo
ococcaceae
77%
pmoA2
88%
presence of glucose was similar to expression patterns
observed in the unsupplemented control (Fig. 4a and b).
The number of pmoA transcripts increased concomitantly to
the consumption of supplemental CH4 and decreased subsequent to the consumption of CH4 (Fig. 4). In contrast,
pmoA gene numbers remained relatively constant over time
(Table S1) and approximated 7.9 106 g1 soil dry weight.
The in silico translated amino acid sequences of the pmoA
genes amplified by qPCR from soils obtained in May 2009
were at least 86–92% similar to PmoA of known Methylocystis strains and 88–99% similar to the dominant OTU 1
that was detected in soil collected in August 2008 and used
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Published by Blackwell Publishing Ltd. All rights reserved
c
34
A.S. Wieczorek et al.
Methylocystis sp. LR1 (AJ458522)
65%
‘Methylosinus acidophilus’ (DQ076756)
93%
92%
‘Methylocystis bryophila’ strain H2s (FN422004)
OTU 1 | 63.5%
OTU 2 | 18.2%
Methylocystaceae
0.10
Environmental sequence (AY781159)
100% Environmental sequence (EF644584)
OTU 6 | 0.6%
Methylocella palustris (AJ458535)
OTU 3 | 8.8%
Environmental sequence (EF633470)
70%
%
Methylocella silvestris (AJ491848)
Methylocella tundrae (AJ555245)
93%
OTU 4| 4.1%
92% Environmental sequence (EF644587)
100%
OTU 5 | 4.1%
Methylococcus capsulatus (AE017282)
OTU 7 | 0.6%
97%
Environmental sequence (EF644603)
Methylomonas sp. LW13 (AY007290)
Methylomicrobium buryatense (DQ295843)
for gene library construction. These results indicated that
the Methylocystis species that responded to CH4 and organic
compounds in the May 2009 sampling were the same as the
dominant Methylocystis species detected in the August 2008
sampling (i.e. Figs 4 and 5).
Discussion
The methanotrophic community of Schlöppnerbrunnen
S. fallax-covered patches comprised facultative and obligate
methanotrophs. Uncultured Methylocystis species were the
main constituents of the methanotrophic community, and
their abundances approximated 4 106 cells g1 soil dry
weight (based on the number of detected pmoA sequences
and the assumption that a methanotrophic cell contains two
copies of pmoA) (Kolb et al., 2005). This is within the range
of values from previous studies on mire methanotrophs
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Methylococcaceae
77%
c
Beijerinckiaceae
67%
Fig. 3. Phylogenetic tree of MmoX OTUs and
reference sequences. Gene libraries were
prepared from the August 2008 sampling.
Percentage values after OTUs indicate the relative
frequency of sequences assigned to a given OTU.
The total number of sequences was 260.
Accession numbers are given in parentheses. The
tree was calculated using translated amino acid
sequences by applying the TREEPUZZLE algorithm
(225 amino acids, JTT substitution model;
Schmidt et al., 2002). Percentage values at nodes
are tree puzzle estimates (based on 25 000
replicates). Open circles at nodes indicate that
these nodes were confirmed by the
ARB-implemented neighbour-joining algorithm
using the same dataset (Ludwig et al., 2004).
Scale bar, 10% sequence divergence.
(Dedysh et al., 2001, 2003; Dedysh, 2009; Belova et al.,
2011). Pore water concentrations of CH4 may vary between
0 and 460 mM in the investigated fen (Knorr et al., 2008,
2009). Concentrations of 6.4 nM CH4 would be sufficient to
maintain the number of detected methanotroph cells (Kolb
et al., 2005). The accumulation of storage polymers and
their subsequent utilization (Dunfield, 2007), the utilization
of alternative substrates (Dedysh et al., 2005), or the utilization of high-affinity pMMO (Baani & Liesack, 2008) might
constitute strategies for coping with highly variable CH4
availability in mires.
Atmospheric CH4 consumption and dominant
genotypes
Methanotrophs capable of utilizing atmospheric CH4 may
be favoured in zones with low concentrations of CH4 that
FEMS Microbiol Ecol 77 (2011) 28–39
35
Organic compounds alter methanotroph activity in fen soil
8
CH4 (µmol g–1soil DW)
8
8
4
4
0
(c)
CH4 (µmol g–1soil DW)
8
Fig. 4. Effect of supplemental glucose and
acetate on the consumption of supplemental
CH4 and the expression of Methylocystisaffiliated pmoA in slurries of fen material (i.e. a
mixture of fen soil and Sphagnum fallax). Fen
material was obtained in May 2009. (a) Control
slurries without supplemental organic compounds. (b) Slurries supplemented with 5 mM
glucose. (c) Slurries supplemented with 5 mM
acetate. Small filled black circles, CH4; cross, pH;
open polygons, glucose; open triangles, acetate;
large filled black circles (connected with thick
line), pmoA transcripts. Error bars are the SD of
mean values of three replicates.
Acetate
4
occur in the investigated fen soil (Knorr et al., 2009). Indeed,
structural gene analyses indicated that dominant methanotrophs in the fen (i.e. pmoA OTU 1 and mmoX OTU 1) were
closely related to Methylocystis strains that are capable of
atmospheric CH4 oxidation (Dunfield et al., 1999, 2002;
Dunfield & Conrad, 2000; Knief & Dunfield, 2005; Baani &
Liesack, 2008). It is likely that pmoA OTU 1 and mmoX OTU
1 belong to the same novel uncultured Methylocystis species
as they are both closely related (o 5% difference in amino
acid sequence) to Methylocystis strain LR1 (Figs 2 and 3).
The detected genotypes probably represented a novel species
most closely related to the mire-associated Methylocystis
FEMS Microbiol Ecol 77 (2011) 28–39
20
40
pmoA transcript–gene ratio
pH
10–2
5
10–3
10–4
4
8
6
0
0
6
0
4
0
10–4
4
0
Glucose
10–3
10–2
5
10–3
10–4
pmoA transcript–gene ratio
(b)
5
pmoA transcript–gene ratio
0
10–2
pH
4
Organic compounds (mM)
4
6
pH
Control
Organic compounds (mM)
CH4 (µmol g–1 soil DW)
8
Organic compounds (mM)
(a)
4
60
Time (h)
heyeri and ‘Methylocystis bryophila’ H2s. The high relative
abundance of OTU 1 in pmoA and mmoX libraries indicated
that this putative species was the main driver of CH4
oxidation in the investigated fen soil (Figs 2 and 3).
Distribution of CH4 oxidation in S. fallax patches
Endophytic methanotrophs in the moss S. cuspidatum
belong to the Alphaproteobacteria and may oxidize
up to 80 mmol CH4 g1 dry weight moss day1 (Raghoebarsing et al., 2005; Kip et al., 2010). CH4 oxidation rates by
washed S. fallax in the current study were approximately
2011 Federation of European Microbiological Societies
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c
36
A.S. Wieczorek et al.
5
4
0
0
4
8
6
8
Propionate
4
4
0
0
0
20
40
Time (h)
5
pH
6
pH
8
4
(b)
CH4 (µmol g–1soil DW)
Ethanol
Organic compounds (mM)
CH4 (µmol g–1soil DW)
8
Organic compounds (mM)
(a)
4
60
Fig. 5. Effect of supplemental ethanol and propionate on the consumption of supplemental CH4 by slurries of fen material (i.e. a mixture of fen
soil and Sphagnum fallax). Fen material was obtained in May 2009. (a)
Slurries supplemented with 2.5 mM ethanol. (b) Slurries supplemented
with 2.5 mM propionate. Filled black circles, CH4; cross, pH; empty
diamonds, ethanol; empty rectangles, propionate. Error bars are the SD
of mean values of three replicates.
160-fold lower than previously reported rates. Thus, it
seems likely that such an endophytic association of methanotrophic Alphaproteobacteria in the S. fallax of the
Schlöppnerbrunnen fen was not significant. CH4 oxidation
occurred without apparent delay in the upper 15 cm of the
fen, and CH4 oxidation rates of fen soil were higher than
those of mosses. The experimental CH4 concentrations
(2.5 mM) were in situ relevant (Knorr et al., 2009),
suggesting that moss patches constituted a highly active
zone of methanotrophy. The spontaneous activity of
6–15 cm fen soil suggested that O2 is at least temporarily
available down to 15 cm, which is in accordance with
previous studies on the availability of O2 in peat bogs and
the Schlöppnerbrunnen fen (Sundh et al., 1995; Knorr et al.,
2008).
Methanotroph community composition
Members of Methylocystaceae (e.g. Methylocystis and Methylosinus) and Beijerinckiaceae (e.g. Methylocella) are dominant in
various northern peat lands (Dedysh et al., 2001, 2002, 2003;
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
Chen et al., 2008a, b; Dedysh, 2009). mmoX sequences
indicative of facultative methanotrophic Beijerinckiaceae
were detected in the current study, but their relative
abundance was low (o 13%). Likewise, uncultured, peat
land-specific Methylococcaceae-affiliated genotypes occur in
mires (e.g. Jaatinen et al., 2005) and were also detected in the
present study, although their abundance was low (o 4.7%).
It has been speculated that methanotrophic Verrucomicrobia (Methylacidiphilaceae; Op den Camp et al., 2009) may
go undetected, as these recently discovered methanotrophs
harbour pmoA genes that cannot be amplified with certain
primers (Dunfield et al., 2007; Degelmann et al., 2010).
Nonetheless, the acidophilic nature of methanotrophic
Verrucomicrobia suggests that they might be common inhabitants of acidic peat lands (Dedysh, 2009). Primers
targeting pmoA of known Verrucomicrobia (Degelmann
et al., 2010) did not yield PCR products from fen soil DNA
extracts (data not shown), indicating that methanotrophic
Verrucomicrobia were either not present in detectable abundances or were distantly related to known methanotrophic
Verrucomicrobia.
Response of fen methanotrophs to organic
compounds
Although supplemental acetate decreased CH4 oxidation
rates and pmoA expression, detected pmoA numbers were
not appreciably affected by this treatment (Table S1),
suggesting that these compounds primarily altered the
activity rather than growth (i.e. replication) of methanotrophs. That glucose did not affect CH4 oxidation indicated
that there was an organic compound-specific effect on the
activity of methanotrophs.
The apparent inhibition of methanotrophic activity of fen
soil by certain fatty acids and ethanol might have been due
to the preferred utilization of the organic supplements. This
hypothesis is supported by the presence of (1) facultative
methanotrophic species (Beijerinckiaceae; Dedysh et al.,
2005) and (2) a Methylocystis-affiliated species that is closely
related to ‘M. bryophila’ H2s, an organism capable of growth
on 3.5 mM acetate (Belova et al., 2011). Thus, facultative
methanotrophs may have altered their substrate utilization
and reduced their biosynthesis of pMMO in response to
alternative carbon sources, and obligate methanotrophs
(e.g. Methylococcaceae) may have consumed CH4 in the
presence of organic compounds. These possibilities are
consistent with the spontaneous but reduced oxidation of
CH4 at the beginning of incubations with supplemental
acetate and ethanol (Figs 4 and 5).
Alternatively, the inhibitory effects of supplemental organic compounds may have been caused by toxic effects.
High concentrations of acetate (pKa = 4.8) and propionate
(pKa = 4.9) might be toxic at pH values o 4.8, as their
FEMS Microbiol Ecol 77 (2011) 28–39
37
Organic compounds alter methanotroph activity in fen soil
protonated forms may perturb membrane potentials and
uncouple ATP synthesis (Russel, 1992). Ethanol had less
effect on the oxidation of CH4 than did propionate. Supplemental acetate, glucose and ethanol were consumed without
apparent delay (Figs 4 and 5), suggesting that aerobes were
poised to do so. Although the taxa involved in the consumption of supplemental organic compounds remain unresolved, it is highly likely that some of these aerobes were
not methanotrophs. Propionate is not known to be utilized
by methanotrophs (Dedysh et al., 2005; Trotsenko & Murrell, 2008), suggesting that propionate affected methanotrophs mainly by toxicity.
Millimolar concentrations of organic acids (up to
3.5 mM) temporally occur in pore water of the investigated
fen (Küsel et al., 2008). Similar concentrations of acetate,
propionate and ethanol decreased the oxidation of CH4
(Table 1). However, the decrease in the oxidation of CH4 was
minimal at lower concentrations (i.e. 0.5 mM) of organic
compounds, i.e. concentrations that are typical of bulk soil
pore water of the investigated fen (Knorr et al., 2008) (Table
1). Although CH4 might under most in situ conditions be a
more readily available methanotrophic substrate than an
alternative utilizable organic compound, the occurrence of
O2 and acetate in deeper fen soil (Knorr et al., 2008; Küsel
et al., 2008) indicates that methanotrophs may be periodically confronted with alternative utilizable organic compounds that could support their aerobic growth. Thus, it is
likely that the oxidation of CH4 in the Schlöppnerbrunnen
fen is affected locally and temporally by organic compounds
derived from root exudation and fermentation, and resolving the quantitative in situ importance of these organic
compounds on ecosystem CH4 fluxes is a topic that warrants
additional study.
Acknowledgements
We thank K.-H. Knorr, coordinator for the field site at the
Schlöppnerbrunnen fen, for permission to obtain soil samples and M.A. Horn for constructive discussion. Support for
this study was provided by the University of Bayreuth and
the Deutsche Forschungsgemeinschaft (DFG; Ko2912/2-1,
Dr310/5-1).
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Supporting Information
Additional supporting information can be found in the
online version of this article:
Table S1. Numbers of pmoA genes and pmoA transcripts
that were the basis for the transcript-gene ratios presented in
Fig. 4.
Fig. S1. Effect of supplemental acetate and ethanol on the
consumption of supplemental CH4 by slurries of fen soil.
Fig. S2. Rarefaction analyses of PmoA (black line) MmoX
(grey line) amino acid sequences obtained from in silico
translation of gene sequences.
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