The methanogenic pathway and microbial community in a thermophilic anaerobic digestion process of organic solid waste
were investigated in a continuous-flow stirred-tank reactor using artificial garbage slurry as a feedstock. The decomposition
pathway of acetate, a significant precursor of CH4 and a key intermediate metabolite in the anaerobic digestion process, was
analyzed by using stable isotopes. A tracer experiment using 13C-labeled acetate revealed that approximately 80% of the
acetate was decomposed via a non-aceticlastic oxidative pathway, whereas the remainder was converted to methane via an
aceticlastic pathway. Archaeal 16S rRNA analyses demonstrated that the hydrogenotrophic methanogens Methanoculleus spp.
accounted for N 90% of detected methanogens, and the aceticlastic methanogens Methanosarcina spp. were the minor
constituents. The clone library targeting bacterial 16S rRNA indicated the predominance of the novel Thermotogales
bacterium (relative abundance: ~ 53%), which is related to anaerobic acetate oxidizer Thermotoga lettingae TMO, although the
sequence similarity was low. Uncultured bacteria that phylogenetically belong to municipal solid waste cluster I were also
predominant in the microflora (~ 30%). These results imply that the microbial community in the thermophilic degrading
process of organic solid waste consists exclusively of unidentified bacteria, which efficiently remove acetate through a nonaceticlastic oxidative pathway.
2010, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Thermophilic methanogenic bioreactor; Organic solid waste; Methanogenic pathway; Microbial community structure]
1389-1723/$ - see front matter 2010, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2010.08.011
42
SASAKI ET AL.
J. BIOSCI. BIOENG.,
carrier gas at a flow rate of 1.5 ml min1, and the column temperature was 35 C. The
peaks at m/z 15 and 17 were regarded as the fragment ion for 12CH4 and the molecular
ion for 13CH4, respectively. The peaks at m/z 44 and 45 were regarded as the molecular
ion for 12CO2 and 13CO2, respectively.
Extraction and purification of DNA
The broth (300 days, HRT of 4.0 days)
from the reactor was divided into two fractions by filtration using a nylon net filter
(pore size: 41 m) (NY41; Millipore, Tokyo, Japan). The fraction remaining on the net
filter was defined as the solid fraction. The fraction passing through the net filter was
defined as the liquid fraction. The genomic DNA was extracted from the total
fraction, solid fraction, and liquid fraction by the benzyl-chloride method (16). The
concentration of genomic DNA was measured by a UV spectrophotometer (DU 7400;
Beckman-Coulter Co., Fullerton, CA, USA) at 260 and 280 nm, and checked by 0.8%
agarose gel electrophoresis. For clone analyses, 10 ng of the genomic DNA was used as
the template of PCR amplification.
Cloning and phylogenetic analysis
Clone libraries of bacterial and archaeal
16S rRNA gene sequences were constructed from genomic DNA. PCR amplification of
the partial 16S rRNA genes was carried out by using AmpliTaqGold with GeneAmp
(Applied Biosystems, Tokyo, Japan) according to the manufacturer's instructions. PCR
was performed with a thermal cycler (PTC-200 DNA Engine; MJ Japan, Tokyo, Japan).
The 16S rRNA genes were amplified using the bacterial primers Ba27f/Ba907r (17,18)
or the archaeal primers Ar109f/Ar912rt (19,20). The thermal cycle condition was
started with an initial denaturation at 94 C for 10 min, followed by 25 cycles of
denaturation at 94 C for 30 s, annealing at 52 C for 45 s, extension at 72 C for 90 s,
and the final extension step at 72 C for 5 min. The PCR products purified using the
QIAquick Gel Extraction kit (Qiagen, Tokyo, Japan) were ligated to the pGEM-T Easy
Vector (Promega, Tokyo, Japan) according to the manufacturer's instructions. The
ligation products were transformed into Escherichia coli JM109 (Toyobo, Tokyo, Japan).
Plasmids were extracted using a GenElute plasmid Miniprep Kit (Sigma-Aldrich).
Sequencing was performed by using a 3130xl Genetic Analyzer (Applied Biosystems)
with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The
sequences were checked for chimeric artifacts by using the CHIMERA_CHECK program
from the Ribosomal Database Project (21). Homology searches were performed with
the BLAST program at the web site of the National Center for Biotechnology
Information. Sequences with more than 99.5% identity were treated as the same
operational taxonomy unit (OTU) (22,23). Phylogenetic analyses were performed
using the ARB software package (24). A phylogenetic tree was calculated and
represented by using the neighbor-joining, maximum-parsimony, and maximumlikelihood methods. Bootstrap values were obtained from 1000 replications.
Nucleotide sequence accession numbers
The nucleotide sequence data
obtained in this study have been deposited in the DDBJ/EMBL/GenBank nucleotide
sequence databases under the following accession numbers: bacterial sequences,
AB428524, AB428526 to AB428539, and AB428540 to AB428543; archaeal sequences,
AB428544 to AB428549.
Aceticlastic cleavage
Non-aceticlastic oxidation
H CO
3 or HCO3 4H2 H CH4 or CH4 3H2 O
RESULTS
Reactor operation and performance Table 1 summarizes the
average values of reactor performance from 60 to 600 days when the
reactor was operated at HRT of 4.0 days (OLR, 6.25 gCODcr l1 day1).
Gas production was stable during this period, and the accumulation of
acetate and propionate was negligible at concentrations of 1.85
(1.81) mM and 0.86 (0.83) mM, respectively. More than 60% of
SS supplied to the reactor was solubilized, and COD removal efficiency
was over 65%. The gas produced contained approximately 80% CH4,
and the remainder was CO2. Low content of CO2 could be due to
increase of alkalinity caused by NaOH titration. It was calculated that
more than 80% of decomposed COD equivalent was recovered as
methane gas.
These reactor performance values approximated those of previous
thermophilic CFSTRs treating organic solid wastes at similar OLR
(6,25,26). Our reactor's operation and performance also stably
digested AGS at the short HRT, whereas the previous reactors were
operated at HRT of 10 days or more.
Isotope distribution in the generated gas from 13C-labeled
acetate
Broth was taken from the reactor after 600 days of
TABLE 1. Operational parameters and reactor performance during stable operation at the shortest HRT in this study.
Reactor performance a
Operational parameters
HRT (day)
4.0
a
OLR (gCODcr l
6.25
day
Controlled pH
7.2
day
Acetate (mM)
Propionate (mM)
65.8 ( 2.80)
61.8 ( 5.59)
1.85 ( 1.81)
0.86 ( 0.83)
Data are average values obtained between the 60th and 600th days of operation at HRT of 4.0 days.
Peak intensities
m/z 15 (12CH4)
Actual
CH12
3 COONa
CH13
3 COONa
13
CH13
3 COONa
12
CH12
3 COONa
no addition
13
12
Background
subtracted a
425,568
458,789
376,755
618,274
346,025
48,813
82,034
0.56
0.63
0.44
0.37
m/z 45 (13CO2)
Actual
CO2 from
methyl
group/
total CO2
CO2 from
carboxyl
group/
total CO2
148,805
191,833
195,295
51,474
37,634
0.38
0.42
0.62
0.58
Peak intensities
m/z 44 (12CO2)
CH12
3 COONa
CH13
3 COONa
13
CH13
3 COONa
12
12
CH3 COONa
no addition
13
12
Actual
Background
subtracted b
5,673,250
5,564,999
5,426,465
6,889,539
3,763,455
246,785
138,534
43
TABLE 3. Phylogenetic affiliation and numbers of bacterial and archaeal clones obtained from each fraction.
OTU a
No. of clones b
Broth
Solid
Liquid
Bacteria
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
B16
B17
B18
Sum
50
2
0
1
0
1
6
1
0
1
3
20
0
7
0
1
1
0
94
7
1
1
0
6
5
9
4
1
0
0
8
1
1
0
0
0
1
45
24
1
0
1
0
0
1
0
0
0
1
11
0
5
1
1
0
0
46
Thermotogae
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes (Clostridium)
Firmicutes
Firmicutes
Firmicutes
Firmicutes (MSW cluster I)
Firmicutes
Firmicutes (MSW cluster I)
Firmicutes (MSW cluster I)
Firmicutes (MSW cluster I)
Firmicutes (MSW cluster I)
Synergistetes
Firmicutes
Planctomycetes
Archaea
A1
A2
A3
A4
A5
A6
Sum
28
25
2
0
0
0
55
17
11
15
4
1
2
50
17
26
2
0
0
0
45
Methanomicrobiales
Methanomicrobiales
Methanosarcinales
Methanosarcinales
Methanosarcinales
Euryarchaeota (Rice cluster III)
a
b
c
The sequences with more than 99.5% homology were collected as the same OTU.
Clone libraries of the total fraction of broth (Broth), the solid fraction (Solid), and the liquid fraction (Liquid) are presented.
Results of phylogenetic grouping were derived from the phylogenetic tree by ARB software.
44
SASAKI ET AL.
J. BIOSCI. BIOENG.,
(51% of the total number of clones) and OTU-A2 (45%), which were
closely related to Methanoculleus thermophilus (EF118904; 100% and
98% sequence similarity, respectively). The remaining clone, OTU-A3,
was related to Methanosarcina thermophila (M59140; 98% sequence
similarity), implying that the archaeal population consists mainly of
hydrogenotrophic methanogens. The population of the OTUs related
to M. thermophila was increased in the solid fraction. The OTU-A6
detected in the solid fraction was an uncultured archaeon (AJ576219;
99% sequence similarity), which was classified into rice cluster III
found in a MSW landfill leachate (30).
DISCUSSION
In this study, we analyzed the microbial composition in a
thermophilic anaerobic reactor stably treating organic solid waste
during long-time operation at OLR of 6.25 gCODcr l1 day1. Acetate
degradation pathway by the microflora was also analyzed. Relatives to
Methanoculleus sp. and Methanosarcina sp. were detected as methanogenic archaea. These methanogens had been widely distributed in
thermophilic anaerobic reactors (31), and their population ratio
seems to be affected by HRT, OLR, or the concentration of VFAs; for
FIG. 1. Phylogenetic tree showing the relationship between bacterial OTUs detected in this study and reference sequences based on a comparison of 16S rRNA gene sequences by
using ARB software. Hydrogenobacter thermophilus (Z30214) was used as an outgroup. The bar corresponds to a 10% difference in nucleotide sequences, as determined by measuring
the lengths of the horizontal lines connecting any two organisms. The symbols (closed circles, open circles, closed square, open squares) at nodes show bootstrap values (N 95%, N85%,
N75%, N 65%) obtained after 1000 resamplings.
aceticlastic oxidation of acetate could be realized by syntrophic acetateoxidizing bacteria and Methanoculleus sp.; their growth rate and affinity to acetate may have been appropriate to the reactor conditions.
The dominant bacterial clones, i.e., OTUB1, which accounted for
more than half of the bacterial clones, were found in a cluster of
Thermotogales in the phylogenetic tree (Fig. 1). P. mobilis and Thermotoga lettingae within this cluster have both been known as fermentative
and sulfate/thiosulfate reducing bacteria (38,39). T. lettingae strain
TMO was reported to have syntrophically oxidized acetate without
sulfate ions under co-culture conditions with a hydrogenotrophic
methanogen (39). OTU-B1 was expected to be a probable candidate of
acetate-oxidizer. The second dominant bacterial group, consisting of
OTU-B10, -B12, and -B14, was found in MSW cluster I (Table 3, Fig. 1).
The clones in this group accounted for approximately 30% of all clones
(i.e., 28 of 94 clones). Microorganisms in uncultured MSW cluster I
probably play a role in the decomposition of the complex organic
matter and/or soluble matter, such as proteins and lipids, in the
feedstock during the initial step of the methanogenesis in this experiment, although the actual metabolic functions of individual microorganisms in the digester are still unclear (6,7).
Clone library analysis showed the specific distribution of microorganisms in the solid fraction. Bacterial clones OTU-B5, OTU-B6, and
OTUs-B7B9, which were frequently found in the solid fraction, were
affiliated with the Clostridium clusters XIVa, IV, and III, respectively.
These clusters were characterized by cellulolytic, proteolytic, and/or
fermentative bacteria (4043). These bacteria possibly digest the
insoluble polymeric substances in the AGS, since more than 60% of SS
decomposition occurred in the experimental period. In addition,
aceticlastic methanogens related to M. thermophila (OTUs-A3A5)
were highly distributed in the solid fraction (Table 3). It has been
widely realized that Methanosarcina spp. form aggregate (44) and
easily adhere to solid substances such as cellulosic materials. They
could directly produce methane from acetate that derived from the
fermentation of solid organic matter. Sasaki et al. also reported the
predominance of M. thermophila on supporting materials in a
thermophilic packed-bed reactor (45).
The present study is the first to relate the methanogenic pathway
from acetate to the microbial structure in a thermophilic anaerobic
digester degrading organic solid waste. Our results suggest the strong
contribution of the non-aceticlastic oxidative pathway to acetate
degradation, which was widely recognized as a rate-limiting step in
methanogenic bioreactors. Recently, several studies have found the
syntrophic acetate oxidizing reaction coupled with hydrogenotrophic
methanogens (14,46,47). The OTU-B1 showed low sequence similarity
(b90%) with the reference sequences in databases, implying that this
bacterium may be a novel species of the acetate oxidizer. The isolation
and physiological characterization of this bacterium will be of interest
for further examinations and will contribute to the understanding of
acetate decomposition in thermophilic methanogenesis while also
leading to deeper insight into anaerobic digestion of organic solid waste.
ACKNOWLEDGMENTS
This work was supported by New Energy and Industrial Technology
Development Organization (NEDO).
References
1. Speece, R. E.: Anaerobic biotechnology for industrial wastewater, Archae Press,
Nashville, Tennessee, (1996).
2. Kim, J. K., Oh, B. R., Chun, Y. N., and Kim, S. W.: Effects of temperature and
hydraulic retention time on anaerobic digestion of food waste, J. Biosci. Bioeng.,
102, 328332 (2006).
3. Shigematsu, T., Tang, Y., Mizuno, Y., Kawaguchi, H., Morimura, S., and Kida, K.:
Microbial diversity of mesophilic methanogenic consortium that can degrade longchain fatty acids in chemostat cultivation, J. Biosci. Bioeng., 102, 535544 (2006).
45
4. Liu, K., Tang, Y. Q., Matsui, T., Morimura, S., Wu, X. L., and Kida, K.: Thermophilic
anaerobic co-digestion of garbage, screened swine and dairy cattle manure,
J. Biosci. Bioeng., 107, 5460 (2009).
5. Tang, Y. Q., Matsui, T., Morimura, S., Wu, X. L., and Kida, K.: Effect of
temperature on microbial community of a glucose-degrading methanogenic
consortium under hyperthermophilic chemostat cultivation, J. Biosci. Bioeng.,
106, 180187 (2008).
6. Tang, Y., Shigematsu, T., Ikbal, Morimura, S., and Kida, K.: The effects of microaeration on the phylogenetic diversity of microorganisms in a thermophilic
anaerobic municipal solid-waste digester, Water Res., 38, 25372550 (2004).
7. Sasaki, K., Haruta, S., Ueno, Y., Ishii, M., and Igarashi, Y.: Microbial population in
the biomass adhering to supporting material in a packed-bed reactor degrading
organic solid waste, Appl. Microbiol. Biotechnol., 75, 941952 (2007).
8. McCarty, P. L.: One hundred years of anaerobic digestion, Anaerobic Digestion
1981, Elsevier Biochemical Press B.V, Amsterdam, 1982, pp. 322 (1982).
9. Ueno, Y., Sasaki, D., Fukui, H., Haruta, S., Ishii, M., and Igarashi, Y.: Changes in
bacterial community during fermentative hydrogen and acid production from
organic waste by thermophilic anaerobic microflora, J. Appl. Microbiol., 101,
331343 (2006).
10. Mountfort, D. O. and Asher, R. A.: Changes in proportions of acetate and carbon
dioxide used as methane precursors during the anaerobic digestion of bovine
waste, Appl. Environ. Microbiol., 35, 648654 (1978).
11. Shigematsu, T., Tang, Y., Kobayashi, T., Kawaguchi, H., Morimura, S., and Kida,
K.: Effect of dilution rate on metabolic pathway shift between aceticlastic and
nonaceticlastic methanogenesis in chemostat cultivation, Appl. Environ. Microbiol., 70, 40484052 (2004).
12. Schnrer, A., Houwen, F. P., and Svensson, B. H.: Mesophilic syntrophic acetate
oxidation during methane formation by a triculture at high ammonium
concentration, Arch. Microbiol., 162, 7074 (1994).
13. Petersen, S. P. and Ahring, B. K.: Acetate oxidation in a thermophilic anaerobic
sewage-sludge digestor: the importance of non-aceticlastic methanogenesis from
acetate, FEMS Microbiol. Ecol., 86, 149158 (1991).
14. Hattori, S.: Syntrophic acetate-oxidizing microbes in methanogenic environments,
Microb. Environ., 23, 118127 (2008).
15. Ueno, Y., Haruta, S., Ishii, M., and Igarashi, Y.: Changes in product formation and
bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor, Appl. Microbiol.
Biotechnol., 57, 6573 (2001).
16. Zhu, H., Qu, F., and Zhu, L. H.: Isolation of genomic DNAs from plants, fungi and
bacteria using benzyl chloride, Nucleic Acids Res., 21, 52795280 (1993).
17. Lane, D. J.: 16S/23S rRNA sequencing, in: E. Stackebrandt, M. Goodfellow (Eds.),
Nucleic acid techniques in bacterial systematics, Wiley, New York, 1991, pp. 115147
(1991).
18. Lueders, T. and Friedrich, M. W.: Effects of amendment with ferrihydrite and
gypsum on the structure and activity of methanogenic populations in rice field soil,
Appl. Environ. Microbiol., 68, 24842494 (2002).
19. Grokopf, R., Janssen, P. H., and Liesack, W.: Diversity and structure of the
methanogenic community in anoxic rice paddy soil microcosms as examined by
cultivation and direct 16S rRNA gene sequence retrieval, Appl. Environ. Microbiol.,
64, 960969 (1998).
20. Lueders, T., Manefield, M., and Friedrich, M. W.: Enhanced sensitivity of DNAand rRNA-based stable isotope probing by fractionation and quantitative analysis
of isopycnic centrifugation gradients, Environ. Microbiol., 6, 7378 (2004).
21. Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker Jr., C. T., Jr., Saxman, P. R., Farris, R.
J., Garrity, G. M., Olsen, G. J., Schmidt, T. M., and Tiedje, J. M.: The RDP-II
(Ribosomal Database Project), Nucleic Acids Res., 29, 173174 (2001).
22. Stackebrandt, E. and Goebel, B. M.: Taxonomic note: a place for DNA-DNA
reassociation and 16S rRNA sequence analysis in the present species definition in
bacteriology, Int. J. Syst. Bacteriol., 44, 846849 (1994).
23. Chouari, R., Paslier, D. Le., Daegelen, P., Ginestet, P., Weissenbach, J. H., and
Sghir, A.: Molecular evidence for novel planctomycete diversity in a municipal
wastewater treatment plant, Appl. Environ. Microbiol., 69, 73547363 (2003).
24. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner,
A., Lai, T., Steppi, S., Jobb, G., and other 22 authors: ARB: a software environment for
sequence data, Nucleic Acids Res., 32, 13631371 (2004).
25. Karakashev, D., Batstone, D. J., and Angelidaki, I.: Influence of environmental
conditions on methanogenic compositions in anaerobic biogas reactors, Appl.
Environ. Microbiol., 71, 331338 (2005).
26. Griffin, M. E., McMahon, K. D., Mackie, R. I., and Raskin, L.: Methanogenic
population dynamics during start-up of anaerobic digesters treating municipal
solid waste and biosolids, Biotechnol. Bioeng., 57, 342355 (1998).
27. Rivire, D., Desvignes, V., Pelletier, E., Chaussonnerie, S., Guermazi, S.,
Weissenbach, J., Li, T., Camacho, P., and Sghir, A.: Towards the definition of a
core of microorganisms involved in anaerobic digestion of sludge, ISME J., 3,
700714 (2009).
28. Weiss, A., Jrme, V., Burghardt, D., Likke, L., Peiffer, S., Hofstetter, E. M., Gabler,
R., and Freitag, R.: Investigation of factors influencing biogas production in a largescale thermophilic municipal biogas plant, Appl. Microbiol. Biotechnol., 84,
9871001 (2008).
46
SASAKI ET AL.
29. Wrighton, K. C., Agbo, P., Warnecke, F., Weber, K. A., Brodie, E. L., DeSantis, T. Z.,
Hugenholtz, P., Andersen, G. L., and Coates, J. D.: A novel ecological role of the
Firmicutes identified in thermophilic microbial fuel cells, ISME J., 2, 11461156 (2008).
30. Luton, P. E., Wayne, J. M., Sharp, R. J., and Riley, P. W.: The mcrA gene as an
alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in
landfill, Microbiology, 148, 35213530 (2002).
31. Demirel, B. and Scherer, P.: The roles of acetotrophic and hydrogenotrophic
methanogens during anaerobic conversion of biomass to methane: a review, Rev.
Environ. Sci. Biotechnol. Bioeng., 7, 173190 (2008).
32. Hori, T., Haruta, S., Ueno, Y., Ishii, M., and Igarashi, Y.: Dynamic transition of a
methanogenic population in response to the concentration of volatile fatty acids in a
thermophilic anaerobic digester, Appl. Environ. Microbiol., 72, 16231630 (2006).
33. Syutsubo, K., Sinthurat, N., Ohashi, A., and Harada, H.: Population dynamics of
anaerobic microbial consortia in thermophilic granular sludge in response to feed
composition change, Water Sci. Technol., 43, 5966 (2001).
34. Bourque, J. S., Guiot, S. R., and Tartakovsky, B.: Methane production in an UASB
reactor operated under periodic mesophilicthermophilic conditions, Biotechnol.
Bioeng., 100, 11151121 (2008).
35. Krakat, N., Westphal, A., Schmidt, S., and Scherer, P.: Anaerobic digestion of
renewable biomass: thermophilic temperature governs methanogen population
dynamics, Appl. Environ. Microbiol., 76, 18421850 (2010).
36. Petersen, S. P. and Ahring, B. K.: Acetate oxidation in a thermophilic anaerobic
sewage-sludge digestor: the importance of non-aceticlastic methanogenesis from
acetate, FEMS Microbiol. Ecol., 86, 149158 (1991).
37. Karakashev, D., Batstone, D. J., Trably, E., and Angelidaki, I.: Acetate oxidation is
the dominant methanogenic pathway from acetate in the absence of Methanosaetaceae, Appl. Environ. Microbiol., 72, 51385141 (2006).
38. Lien, T., Madsen, M., Rainey, F. A., and Birkeland, N. K.: Petrotoga mobilis sp. nov.,
from a North Sea oil-production well, Int. J. Syst. Bacteriol., 48, 10071013 (1998).
J. BIOSCI. BIOENG.,
39. Balk, M., Weijma, J., and Stams, A. J.: Thermotoga lettingae sp. nov., a novel
thermophilic, methanol-degrading bacterium isolated from a thermophilic
anaerobic reactor, Int. J. Syst. Evol. Microbiol., 52, 13611368 (2002).
40. Varel, V. H., Tanner, R. S., and Woese, C. R.: Clostridium herbivorans sp. nov., a
cellulolytic anaerobe from the pig intestine, Int. J. Syst. Bacteriol., 45, 490494
(1995).
41. Zellner, G., Stackebrandt, E., Nagel, D., Messner, P., Weiss, N., and Winter, J.:
Anaerofilum pentosovorans gen. nov., sp. nov., and Anaerofilum agile sp. nov., two
new, strictly anaerobic, mesophilic, acidogenic bacteria from anaerobic bioreactors,
Int. J. Syst. Bacteriol., 46, 871875 (1996).
42. Kato, S., Haruta, S., Cui, Z. J., Ishii, M., Yokota, A., and Igarashi, Y.: Clostridium
straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic
bacterium isolated from a cellulose-degrading bacterial community, Int. J. Syst.
Evol. Microbiol., 54, 20432047 (2004).
43. Madden, R. H.: Isolation and characterization of Clostridium stercorarium sp. nov.,
cellulolytic thermophile, Int. J. Syst. Bacteriol., 33, 837840 (1983).
44. Ferry, J. G.: Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila, Biofactors, 6, 2535 (1997).
45. Sasaki, K., Haruta, S., Ueno, Y., Ishii, M., and Igarashi, Y.: Archaeal population on
supporting material in methanogenic packed-bed reactor, J. Biosci. Bioeng., 102,
244246 (2006).
46. Briones, A. M., Daugherty, B. J., Angenent, L. T., Rausch, K. D., Tumbleson, M. E.,
and Raskin, L.: Microbial diversity and dynamics in multi- and single-compartment anaerobic bioreactors processing sulfate-rich waste streams, Environ.
Microbiol., 9, 93106 (2007).
47. Nsslein, B., Chin, K. J., Eckert, W., and Conrad, R.: Evidence for anaerobic
syntrophic acetate oxidation during methane production in the profundal
sediment of subtropical Lake Kinneret (Israel), Environ. Microbiol., 3, 460470
(2001).