Effect of dilution rate on metabolic pathway shift between aceticlastic and nonaceticlastic methanogenesis in chemostat cultivation.

Abstract

Acetate conversion pathways of methanogenic consortia in acetate-fed chemostats at dilution rates of 0.025 and 0.6 day(-1) were investigated by using (13)C-labeled acetates, followed by gas chromatography-mass spectrometry (GC-MS) analysis of the CH(4) and CO(2) produced. Nonaceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens was suggested to occupy a primary pathway (approximately 62 to 90%) in total methanogenesis at the low dilution rate. In contrast, aceticlastic cleavage of acetate by aceticlastic methanogens was suggested to occupy a primary pathway (approximately 95 to 99%) in total methanogenesis at the high dilution rate. Phylogenetic analyses of transcripts of the methyl coenzyme M reductase gene (mcrA) confirmed that a significant number of transcripts of the genera Methanoculleus (hydrogenotrophic methanogens) and Methanosarcina (aceticlastic methanogens) were present in the chemostats at the low and high dilution rates, respectively. The mcrA transcripts of the genus Methanosaeta (aceticlastic methanogens), which dominated the population in a previous study (T. Shigematsu, Y. Tang, H. Kawaguchi, K. Ninomiya, J. Kijima, T. Kobayashi, S. Morimura, and K. Kida, J. Biosci. Bioeng. 96:547-558, 2003), were poorly detected at both dilution rates due to the limited coverage of the primers used. These results demonstrated that the dilution rate could cause a shift in the primary pathway of acetate conversion to methane in acetate-fed chemostats.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2004, p. 4048–4052
0099-2240/04/$08.00?0DOI: 10.1128/AEM.70.7.4048–4052.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 7
Effect of Dilution Rate on Metabolic Pathway Shift between
Aceticlastic and Nonaceticlastic Methanogenesis
in Chemostat Cultivation
Toru Shigematsu,1* Yueqin Tang,1Tsutomu Kobayashi,1Hiromi Kawaguchi,1
Shigeru Morimura,1and Kenji Kida1,2
Graduate School of Science and Technology1and Department of Applied Chemistry and Biochemistry,
Faculty of Engineering,2Kumamoto University, Kumamoto City,
Kumamoto 860-8555, Japan
Received 23 January 2004/Accepted 17 March 2004
Acetate conversion pathways of methanogenic consortia in acetate-fed chemostats at dilution rates of 0.025
and 0.6 day?1were investigated by using13C-labeled acetates, followed by gas chromatography-mass spectrom-
etry (GC-MS) analysis of the CH4and CO2produced. Nonaceticlastic syntrophic oxidation by acetate-oxidizing
syntrophs and hydrogenotrophic methanogens was suggested to occupy a primary pathway (approximately 62
to 90%) in total methanogenesis at the low dilution rate. In contrast, aceticlastic cleavage of acetate by aceti-
clastic methanogens was suggested to occupy a primary pathway (approximately 95 to 99%) in total metha-
nogenesis at the high dilution rate. Phylogenetic analyses of transcripts of the methyl coenzyme M reductase
gene (mcrA) confirmed that a significant number of transcripts of the genera Methanoculleus (hydrogenotrophic
methanogens) and Methanosarcina (aceticlastic methanogens) were present in the chemostats at the low and
high dilution rates, respectively. The mcrA transcripts of the genus Methanosaeta (aceticlastic methanogens),
which dominated the population in a previous study (T. Shigematsu, Y. Tang, H. Kawaguchi, K. Ninomiya, J.
Kijima, T. Kobayashi, S. Morimura, and K. Kida, J. Biosci. Bioeng. 96:547-558, 2003), were poorly detected at
both dilution rates due to the limited coverage of the primers used. These results demonstrated that the dilu-
tion rate could cause a shift in the primary pathway of acetate conversion to methane in acetate-fed chemostats.
Under methanogenic conditions, acetate is quantitatively
the most dominant intermediate of anaerobic degradation of
organic matter. It is estimated that approximately 70 to 80% of
methane is derived from acetate in anoxic environments (11,
13, 14). Two processes by which acetate is converted to meth-
ane have been described. The acetate-utilizing methanogens,
the genera Methanosaeta and Methanosarcina, use the aceti-
clastic cleavage pathway in which the methyl group of acetate
is converted to methane, while the carboxyl group is converted
to CO2(4). The second process includes the syntrophic oxi-
dation of acetate to CO2and hydrogen by one organism and
the subsequent reduction of carbon dioxide to methane by a
hydrogenotrophic methanogen. Two thermophilic bacteria,
strain AOR and Thermacetogenium phaeum, and one meso-
philic bacterium, Clostridium ultunense, were demonstrated to
be capable of acetate oxidation in cocultures with hydro-
genotrophic methanogens (7, 10, 19). The net reaction is the
same as the reaction for aceticlastic cleavage of acetate in the
syntrophic acetate oxidation process, but
strates have been used to differentiate between the two pro-
cesses (16, 18, 24). The quantitative information for these two
acetate conversion pathways in total methanogenic microbial
communities has been limited to date.
In a previous study, chemostat cultures of mesophilic ace-
14C-labeled sub-
tate-degrading methanogenic consortia were constructed (8).
The relative concentration of coenzyme F420, which is involved
in hydrogenotrophic methanogenesis, was much higher at low
dilution rates than that at high dilution rates. Microbial com-
munity structure analysis of the chemostat cultures at dilution
rates of 0.025 and 0.6 day?1revealed that a significant number
of cells of the genus Methanoculleus, which is a hydrogenotro-
phic methanogen, were detected only at the low dilution rate,
although larger populations of aceticlastic methanogens affil-
iated with the genera Methanosaeta and Methanosarcina were
detected at both dilution rates (21). The detection of hydro-
genotrophic methanogens and higher F420concentrations in
the chemostat cultures at the low dilution rate suggests that a
significant proportion of methanogenesis occurs by syntrophic
acetate oxidation rather than by aceticlastic cleavage of ace-
tate. In this study, we analyzed the acetate conversion path-
ways of methanogenic consortia using13C-labeled substrates
and gas chromatography-mass spectrometry (GC-MS) analysis
of the CH4and CO2produced. We also performed a phyloge-
netic analysis of transcripts of the mcrA gene, which encodes
the ?-subunit of methyl coenzyme M reductase I (MCR I), at
both dilution rates.
MATERIALS AND METHODS
Operation of acetate-fed chemostats. Two anaerobic chemostats were oper-
ated for more than 2 years at 37°C with acetate as the only added carbon source
and electron donor at dilution rates of 0.025 and 0.6 day?1. Completely stirred
tank reactors, each with a working volume of 1.7 liters, were used as the che-
mostats. The inoculum for these reactors was digested sludge acclimatized in our
laboratory for 6 months. Detailed operation methods have been described pre-
viously (8). Under steady-state conditions, the acetate concentrations in the
* Corresponding author. Mailing address: Department of Materials
and Life Science, Graduate School of Science and Technology, Kum-
amoto University, 2-39-1 Kurokami, Kumamoto City, Kumamoto 860-
8555, Japan. Phone: 81 96 342 3668. Fax: 81 96 342 3679. E-mail: shige
@kumamoto-u.ac.jp.
4048

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chemostats were approximately 10 and 250 mg/liter at dilution rates of 0.025 and
0.6 day?1, respectively.
Batchwise gas evolution test with13C-labeled substrate. A 10-ml sample of
culture broth was taken from a chemostat and centrifuged at 25,000 ? g for 15
min. The precipitate was washed with 30 ml of synthetic wastewater (8) contain-
ing no carbon source and was resuspended in 10 ml of synthetic wastewater
containing no carbon source. The precipitate was transferred to vials and sup-
plemented with [1-13C]sodium acetate, [2-13C]sodium acetate, or [1,2-13C]so-
dium acetate to give a final concentration of 100 mM. Washing and suspension
of the pellet were performed by using an anaerobic glove box (model 1025;
Forma Scientific, Marietta, Ohio) supplemented with helium gas. The vials were
immersed in a thermostat-controlled water bath at 37°C. After 12 h of incubation
with mixing, the CH4and CO2in the headspace were analyzed by using a
GCMS-QP5000 GC-MS (Shimadzu, Kyoto, Japan) equipped with a GS-GasPro
column (30 m by 0.32 mm; J & W Scientific, Folsom, Calif.). Helium was used as
carrier gas at a flow rate of 1.7 ml/min. The column temperature was 30°C. The
peaks at m/z 15 and 17 in the mass spectrum, which were derived from a
retention time of 1.2 min in the gas chromatogram, were regarded as the frag-
ment ion for12CH4and the molecular ion for13CH4, respectively. The peaks at
m/z 44 and 45, which were derived from a retention time of 1.5 min, were
regarded as the molecular ions for12CO2and13CO2, respectively.
RT-PCR amplification and cloning. Total nucleic acids from the culture broth
in a chemostat were extracted by a method described previously (21). RNA was
then extracted by the method of Griffiths et al. (5) and purified with an RNeasy
kit (QIAGEN, Hilden, Germany). Reverse transcription (RT) reactions were
performed with a Gene Amp Gold RNA PCR reagent kit (Applied Biosystems,
Foster City, Calif.) by using 500 and 100 ng of the template RNA extracted from
the chemostats at dilution rates of 0.025 and 0.6 day?1, respectively, and the
reverse primer ME2 (5?-TCAT(G/T)GC(A/G)TAGTT(A/G/T)GG(A/G)TAG
T-3?) (6). The resulting cDNA was purified with a MicroSpin S-400 HR column
(Amersham Biosciences, Piscataway, N.J.) and was used as the template for
amplification of mcrA with AmpliTaq (Applied Biosystems, Foster City, Calif.)
used according to the manufacturer’s instructions (1? PCR buffer, 2.5 U of
AmpliTaq DNA polymerase, each deoxynucleoside triphosphate at a concentra-
tion of 250 ?M, and 40 pmol of each primer in a 100-?l reaction mixture). The
PCR primers used in the amplification were the forward primer ME1 (5?-GC(
A/C)ATGCA(A/G)AT(A/C/T)GG(A/T)ATGTC-3?) (6) and the reverse primer
ME2b (5?-TCCTG(G/C)AGGTCG(A/T)A(A/G)CCGAAGAA-3?). Reactions
were performed with a GeneAmp PCR System 2400 (Applied Biosystems) with
the following cycle conditions: preincubation at 95°C for 2 min; 25 cycles of 95°C
for 1 min, 50°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for
7 min. The amplified mcrA fragments were cloned into a plasmid pT7Blue T
vector (Novagen Inc., Madison, Wis.) by using a DNA ligation kit (version 2;
Takara, Kyoto, Japan).
Sequencing and phylogenetic analysis. Cloned RT-PCR products were pre-
pared from randomly selected recombinants and used as templates for sequenc-
ing. Sequencing was performed by using a DNA sequencer (CEQ8000; Beckman
Coulter, Fullerton, Calif.) with a CEQ Quick Start Master Mix kit (Beckman
Coulter). DNA and deduced amino acid sequences were analyzed with the
GENETYX-WIN software package (version 5.1; Software Development, Tokyo,
Japan). The search for homologous proteins was conducted with the BLAST
program (1). Multiple alignments were run by using the Clustal X program,
version 1.8 (22). Phylogenetic analyses were conducted with MEGA, version 2.1
(9). Identical sequences (100% similarity) were recognized by matrix analysis and
manual comparison and were used in subsequent analyses as an operational
taxonomic unit (OTU). The OTUs were designated ALM01 to ALM08 for
clones from the culture broth at a dilution rate of 0.025 day?1and AHM01 to
AHM08 for clones at a dilution rate of 0.6 day?1.
Quantitative RT-PCR of mcrA transcripts. Real-time quantitative RT-PCR
experiments were conducted to quantify mcrA transcripts of different taxonomic
groups by using the TaqMan fluorogenic PCR system. The RT reaction was
performed with a Gene Amp Gold RNA PCR reagent kit (Applied Biosystems)
by using 10 ?g of the extracted RNA as a template and the reverse primer ME2
in a 100-?l reaction mixture. The resulting cDNA was precipitated by ethanol
precipitation and then vacuum dried and resuspended in 20 ?l of Tris-EDTA
buffer (pH 7.4). The cDNA was purified by using a MicroSpin S-400 HR column
(Amersham Biosciences) and was then used as a template for a quantitative
PCR. The quantitative PCR was carried out by using primers ME1 and ME2b
and a genus-specific TaqMan probe. Three TaqMan probes, SAE716TAQ (5?-
AGGCCTTCCCCACTCTGCTTGAGGAT-3?), SAR716TAQ (5?-AGAAATT
CCCAACAGCCCTTGAAGAC-3?), and MCU716TAQ (5?-AGCAGTACCC
GACCATGATGGAGGAC-3?), were used for detection of the mcrA gene prod-
ucts of the genera Methanosaeta, Methanosarcina, and Methanoculleus, respec-
tively. The specificities of these probes for the target mcrA genes were confirmed
by manual comparison of the nucleotide sequences of mcrA genes in the DDBJ/
EMBL/GenBank database. All TaqMan probes were 5? end labeled with 6-car-
boxyfluorescein and 3? end labeled with 6-carboxytetramethyl rhodamine, ob-
tained from Applied Biosystems. In order to evaluate the selectivity of the
primer-probe sets, three clones, ALM07, AHM01, and ALM01, were used as
controls. By using the three sets of primers and TaqMan probes, fluorescence
signal monitoring was performed with the GeneAmp 5700 sequence detection
system (Applied Biosystems). Reaction mixtures for fluorogenic PCR in which
the concentrations of both the primer and the TaqMan probe were optimized
(300 and 200 nM, respectively) were prepared. The concentration of control
DNA varied between 15.63 and 625 pg per 50 ?l of reaction mixture. The
TaqMan Universal PCR Master Mix (Applied Biosystems) was used with the
following cycle conditions: an initial step of 50°C for 2 min and then 95°C for 10
min; and two-step cycles of 95°C for 15 s and 60°C for 1 min. All assays were
performed at least in duplicate. Post-PCR analysis was performed by using
GeneAmp 5700 SDS software.
Nucleotide sequence accession numbers. The DDBJ/EMBL/GenBank acces-
sion numbers for the sequences of clones ALM01 to ALM08 and AHM01 to
AHM08 are AB158524 to AB158539.
RESULTS
Batchwise gas evolution test with
Two acetate-fed mesophilic anaerobic chemostats were oper-
ated for more than 2 years at dilution rates of 0.025 and 0.6
day?1. Under steady-state conditions, the acetate concentra-
tions in the chemostats were approximately 0.2 and 4 mM at
dilution rates of 0.025 and 0.6 day?1, respectively. To evaluate
the acetate conversion pathway in the two chemostats, batch-
wise cultivation of culture broth extracted from the chemostats
was carried out by using forms of13C-labeled sodium acetate
as the substrates. If the culture broth used aceticlastic cleavage
of acetate, the methyl and carboxyl groups of acetate would
have been converted to methane and CO2, respectively (4), but
if the culture broth used syntrophic acetate oxidation, 2 mol of
CO2would have been produced from 1 mol of acetate, while 1
mol of the CO2produced would have been concurrently re-
duced to methane (24). In the latter case, the methyl and
carboxyl groups of acetate would have been converted to equal
amounts of methane and CO2.
When the culture broth of the chemostat at a dilution rate of
0.025 day?1was used for batch cultivation, 33 and 45% of the
methane were considered to be derived from the carboxyl
group of acetate when [2-13C]sodium acetate and [1-13C]so-
dium acetate, respectively, were used as the substrates (Table
1). For CO2, 31 and 32% were considered to be derived from
the methyl group when [2-13C]sodium acetate and [1-13C]so-
dium acetate, respectively, were used as the substrates (Table
2). On the other hand, only about 2% of the methane and 6%
of the CO2were considered to be derived from the carboxyl
and methyl groups of acetate, respectively, when the culture
broth of the chemostat at a dilution rate of 0.6 day?1was used.
These results suggested that the syntrophic oxidation pathway
accounted for approximately 62 to 90% of the total methano-
genesis in the chemostat at the low dilution rate. In contrast, at
the high dilution rate, the aceticlastic cleavage of acetate was
suggested to account for 95 to 99% of total methanogenesis.
Because we used batchwise cultivation for the13C-labeled sub-
strate assay, the results might not precisely reflect the in situ
activities of the microorganisms in the chemostats. But the
culture broth at the low dilution rate had an obviously larger
potential for syntrophic acetate oxidation than for aceticlastic
13C-labeled substrates.
VOL. 70, 2004METABOLIC PATHWAY SHIFT IN METHANOGENESIS 4049

Page 3

cleavage of acetate, whereas the culture broth at the high
dilution rate had a larger potential for aceticlastic cleavage
than for syntrophic acetate oxidation.
Analysis of mcrA gene transcripts from the chemostats.
MCR I appears to be unique to methanogens and to be present
in all methanogens. The mcrA gene encoding the ?-subunit of
MCR I has been used a marker gene for the specific detection
of methanogens in various environments (6, 12). The mcrA
transcripts in community RNA extracted from the acetate-fed
chemostats were amplified by RT-PCR and were used to con-
struct two clone libraries, designated ALM (mcrA transcripts
from the chemostat at a dilution rate of 0.025 day?1) and
AHM (mcrA transcripts from the chemostat at a dilution rate
of 0.6 day?1). Twenty-one clones from each library were ran-
domly selected and sequenced. All nucleotide sequences and
deduced amino acid sequences showed significant similarities
with sequences of known mcrA genes and McrAs, respectively.
In the ALM library, eight different sequences (OTUs) were
obtained. Six OTUs (ALM01 to ALM06, 17 clones) were
closely related to the mcrA gene of Methanoculleus thermophi-
licus (Fig. 1) and were regarded as mcrA genes of the genus
Methanoculleus (Table 3). The other two OTUs (ALM07 and
ALM08, four clones) were closely related to the mcrA gene of
Methanosaeta concilii and were regarded as mcrA genes of the
genus Methanosaeta. In the AHM library, eight OTUs were
obtained. Seven OTUs (AHM01 to AHM07, 20 clones) were
closely related to the mcrA gene of Methanosarcina mazei and
were regarded as mcrA genes of the genus Methanosarcina.
Another OTU (AHM08, one clone) was closely related to
mcrA of M. concilii and was regarded as an mcrA gene of the
genus Methanosaeta.
Real-time quantitative RT-PCR experiments were con-
ducted to quantify mcrA transcripts of different taxonomic
groups. mcrA transcripts of the genus Methanosaeta could not
be detected in the RNA from the chemostat at either dilution
rate (Table 3). mcrA transcripts of the genus Methanosarcina
could be detected only in the RNA from the chemostat at a
dilution rate of 0.6 day?1. In contrast, mcrA transcripts of the
genus Methanoculleus could be detected only in the RNA from
the chemostat at a dilution rate of 0.025 day?1. These results
agreed with our clonal sequence analysis which showed that
the mcrA transcripts of the genera Methanoculleus and Meth-
anosarcina were dominant in RNA from the chemostats at the
low dilution rate and the high dilution rate, respectively. Be-
cause the ME1 and ME2 primers were reported not to be
suitable for amplification of the mcrA genes of the genus Meth-
anosaeta (12), the selectivity of the primer sets should be con-
sidered. However, our results still indicated that the levels of
mcrA transcription activity of the genera Methanoculleus and
Methanosarcina were significant in the chemostats at the low
and high dilution rates, respectively.
DISCUSSION
We used a stable-isotope technique to analyze the primary
pathway of acetate conversion to methane in acetate-fed che-
TABLE 1. GC-MS analysis of CH4produced from13C-labeled acetate
Dilution rate
(day?1)
Substrate
Peak intensities
CH4from carboxyl
base/total CH4
CH4from methyl
base/total CH4
m/z 15 (12CH4)
m/z 17(13CH4)
(actual)
Actual
Background
subtracteda
0.025
13CH3
12CH3
13CH3
12COONa
13COONa
13COONa
2,695b
1,139
2,695
1,139
5,474
928
1,361
0.33
0.45
0.67
0.55
0
0.6
13CH3
12CH3
13CH3
12COONa
13COONa
13COONa
81,973
636,401
74,312
14,066
562,089
614,114
13,860
588,433
0.022
0.024
0.98
0.98
aThe peak intensities at an m/z value of 15 from13CH313COONa were regarded as the background.
bAll values are averages for duplicate experiments.
TABLE 2. GC-MS analysis of CO2produced from13C-labeled acetate
Dilution rate
(day?1)
Substrate
Peak intensities
CO2from methyl
base/total CO2
CO2from carboxyl
base/total CO2
m/z 44 (12CO2)
m/z 45(13CO2)
(actual)
Actual
Background
subtracteda
0.025
13CH3
12CH3
13CH3
12COONa
13COONa
13COONa
4,041b
3,424
1,406
2,635
2,018
1,196
4,296
3,380
0.31
0.32
0.69
0.68
0.6
13CH3
12CH3
13CH3
12COONa
13COONa
13COONa
87,919
12,951
8,004
79,915
4,947
5,302
80,702
73,096
0.062
0.058
0.94
0.94
aThe peak intensities at an m/z value of 44 from13CH313COONa were regarded as the background.
bAll values are averages for duplicate experiments.
4050 SHIGEMATSU ET AL.APPL. ENVIRON. MICROBIOL.

Page 4

mostats. To our knowledge, this was the first application of
13C-labeled substrates combined with GC-MS analysis of the
methane and CO2produced to analyze acetate conversion
pathways to methane. This technique is more useful and con-
venient for determining which groups of acetate are converted
to methane and CO2than the conventional technique using
14C-labeled substrates. The results obtained in the RT-PCR
experiment targeting mcrA transcripts supported the findings
obtained by using13C-labeled substrates, although the limited
coverage of the primers used requires further consideration.
The specific growth rate of the mesophilic acetate-oxidizing
syntroph C. ultunense cocultured with a hydrogenotrophic
methanogen by using acetate as a substrate was reported to be
0.027 to 0.035 day?1(20). The specific growth rates of the
mesophilic aceticlastic methanogens M. concilii and M. mazei
were 0.24 to 0.28 and 0.98 day?1, respectively (2). At the high
dilution rate (0.6 day?1), the cells affiliated with the genus
Methanosarcina, which were able to grow rapidly, would have
FIG. 1. Phylogenetic relationships of deduced amino acid sequences of ?-subunits of MCR I (McrA). The tree was constructed from
phylogenetic distances obtained by the neighbor-joining method (17). ALM and AHM indicate clones from cultivation at the low (0.025 day?1)
and high (0.6 day?1) dilution rates, respectively. The numbers of clones that had identical sequences are shown in parentheses. Bar ? 5 amino acid
substitutions per 100 amino acids. Bootstrap probabilities (3) are indicated at branch nodes. The DDBJ/EMBL/GenBank accession numbers for
reference strains are shown in parentheses. The tree was rooted by using McrA of Methanobacterium bryantii as the outgroup.
TABLE 3. Composition of and quantification of mcrA
transcripts of three taxonomic groups
Phylogenetic
affiliationa
Dilution rate of
0.025 day?1
Dilution rate of
0.6 day?1
No. of
clones
Quantitative
RT-PCR
(copies/?g of
total RNA)b
No. of
clones
Quantitative
RT-PCR
(copies/?g of
total RNA)b
Methanosaeta
spp. mcrA
Methanosarcina
spp. mcrA
Methanoculleus
spp. mcrA
4NDc
1ND
0 ND 204.15 ? 107
179.06 ? 106
0 ND
aPrimers ME1 and ME2 used in this study were reported not to be suitable for
amplification of the mcrA genes of the genus Methanosaeta (12).
bThe values for quantitative RT-PCR are averages for duplicate experiments.
cND, not detected.
VOL. 70, 2004METABOLIC PATHWAY SHIFT IN METHANOGENESIS 4051

Page 5

been predominant in the chemostat and engaged in aceticlastic
cleavage of acetate to methane as the primary pathway. On the
other hand, the low dilution rate (0.025 day?1) was sufficiently
low for growth of the three acetate-utilizing members, acetate-
oxidizing syntrophs, Methanosaeta, and Methanosarcina. In this
case, competition among the substrate affinities of the three
acetate-utilizing members would have been decisive for dom-
inance. The apparent Kmfor acetate of a thermophilic acetate-
oxidizing syntroph was reported to be 0.65 mM (16), although
no Kmvalue of a mesophilic acetate-oxidizing syntroph is cur-
rently available. The apparent Kmvalues for acetate of the
genera Methanosaeta and Methanosarcina were approximately
0.8 to 0.9 and 3 to 5 mM, respectively (23). The acetate-
oxidizing syntroph associated with Methanoculleus, according
to the high substrate affinity, was better adapted to convert
acetate primarily in the chemostat at a low dilution rate. The
genus Methanosaeta, whose population was previously shown
to be the largest among the three members by previous ribo-
somal DNA analyses (21), played a secondary role for acetate
conversion by aceticlastic cleavage in the chemostat at the low
dilution rate. It is possible that even cells of a dominant pop-
ulation could not show a dominant metabolic function in a
consortium because of their lower metabolic activity. The cor-
relation between the population dominance of the genus Meth-
anosaeta and its lower metabolic activity for acetate conversion
at the low dilution rate still requires further analysis in terms of
quantification and comparison of mcrA transcripts of the gen-
era Methanosaeta and Methanoculleus with more universal
primers. We have no direct evidence of which bacteria are
responsible for acetate oxidation in our chemostats. However,
the dominance of bacteria belonging to the phylum Firmicutes,
with which C. ultunense and T. phaeum are affiliated, was
shown by 16S ribosomal DNA clonal sequence analysis (21).
Some members of this phylum, which is related to the known
acetate-oxidizing syntrophs, may contribute to the syntrophic
acetate oxidation in the chemostat at the low dilution rate.
The results described above, combined with previous find-
ings (8, 21) demonstrate that the dilution rate could cause a
shift in the primary pathway of acetate conversion to methane
in acetate-fed chemostats. At the low dilution rate, the acetate-
oxidizing syntrophs, associated with hydrogenotrphic methano-
gens, could metabolically overcome the aceticlastic methano-
gens and play a primary role in the conversion of acetate to
methane. Recently, Nu ¨sslein et al. reported that a large pro-
portion of methanogenesis in lake sediment occurs by syntro-
phic acetate oxidation rather than by aceticlastic cleavage of
acetate (15). Most natural environments fulfill the conditions
of low dilution rate and low acetate concentration that were
present in our chemostat. Syntrophic acetate oxidation might
be a common mechanism in natural methanogenic environ-
ments.
ACKNOWLEDGMENT
This study was financially supported by a grant-in-aid for scientific
research (project 14580593) from the Japan Society for the Promotion
of Science.
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