Journal of Bioscience and Bioengineering

VOL. 111 No. 1, 41 46, 2011

www.elsevier.com/locate/jbiosc

Methanogenic pathway and community structure in a thermophilic anaerobic

digestion process of organic solid waste

Daisuke Sasaki,1 Tomoyuki Hori,1,2 Shin Haruta,1,3, Yoshiyuki Ueno,4 Masaharu Ishii,1 and Yasuo Igarashi1

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 1
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukisamu-Higashi 2-17-2-1, Toyohira-ku,
Sapporo 062-8517, Japan 2 Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji-shi, Tokyo 192-0397,
Japan 3 and Kajima Technical Research Institute, Tobitakyu 2-19-1, Chofu-shi, Tokyo 182-0036, Japan 4

Received 29 June 2010; accepted 20 August 2010

Available online 18 September 2010

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 non-
aceticlastic oxidative pathway.
2010, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Thermophilic methanogenic bioreactor; Organic solid waste; Methanogenic pathway; Microbial community structure]

Reuse and recycling of organic wastes, including garbage and waste
from the food industry, have been attracting social attention and
concern. Anaerobic digestion is one of the effective technologies for
recovering energetic materials from organic waste and is a simple and
environmentally acceptable means of reducing and stabilizing organic
waste (1).
The thermophilic anaerobic digestion process has advantages over
the mesophilic process with respect to digestion efficiency and
disinfection of pathogenic organisms (2). Unlike the mesophilic process,
the thermophilic process is characterized by limited species of
aceticlastic methanogens and simple structure of the bacterial commu-
nities (3,4). Several researchers have provided information about
microbial composition and distribution in thermophilic reactors
treating organic solid wastes (47). Their reports have indicated that
methanogenesis from solid wastes required microbial members distinct
from those in other reactors treating liquid slurry such as industrial
Corresponding author. Department of Biotechnology, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657,
Japan. Tel.: +81 42 677 2580; fax: +81 42 677 2559.
E-mail address: sharuta@tmu.ac.jp (S. Haruta).
1389-1723/$ - see front matter 2010, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2010.08.011
wastewater. However, little is known about the methanogenetic
pathway in the reactors.
The metabolic pathway to produce methane from organic polymers
consists basically of three steps: first, complex polymeric substances
such as cellulose and protein are hydrolyzed (hydrolysis), then the
hydrolyzed products are degraded to volatile fatty acids (VFAs) and H2/
CO2 (acidogenesis), and finally methane gas is produced from acetate or
H2/CO2 (methanogenesis) (8). Acetate has been known as a key
intermediate metabolite during methanogenesis, and the decomposi-
tion of acetate is considered to be the rate-limiting step of the over-all
reaction process (9,10). So far, two methanogenic pathways from
acetate have been reported (1113). One is the direct methanogenesis
by aceticlastic methanogenic archaea, such as Methanosarcina spp.
(aceticlastic cleavage, reaction formula 1). The aceticlastic methanogens
convert the methyl and carboxyl groups of acetate to CH4 and CO2,
respectively. The other is non-aceticlastic oxidation, i.e., the co-
metabolism pathway by acetate-oxidizing bacteria (reaction formula 2)
and hydrogenotrophic methanogens (reaction formula 3). During the
latter pathway, acetate is first oxidized to CO2 and then the produced
CO2 is reduced to CH4. The decomposition pathway of acetate is known
to be affected by the operation temperature, the composition of organic
substances, the types of reactors, and the organic loading rate (14).
42 SASAKI ET AL.
Aceticlastic cleavage
CH3 COO H2 O CH4 HCO
3 1
Non-aceticlastic oxidation
CH3 COO 4H2 OH CO
3 HCO3 4H2 H
2 filter was defined as the solid fraction. The fraction passing through the net filter was
H CO
3 or HCO3 4H2 H CH4 or CH4 3H2 O 3 fraction, solid fraction, and liquid fraction by the benzyl-chloride method (16). The
(Asterisks represent the carbon of methyl group in acetate.)
In the present study, a lab-scale thermophilic continuous-flow
stirred-tank reactor (CFSTR) was operated using artificial garbage
slurry (AGS) as a model of organic solid waste. After establishing
efficient digestion performance for 18 months (organic loading rate
[OLR], 6.25 gCODcr l1 day1), we analyzed the microbial composi-
tion of the enriched microflora and the pathway of acetate
degradation in the process by using stable isotopes.
MATERIALS AND METHODS
Operation of thermophilic CFSTR Two liters of seed sludge collected from a
thermophilic anaerobic digester treating garbage slurry (15) was cultivated at 55 C in
a 3 L jar fermenter (MDL-301s; B.E. Marubishi, Tokyo, Japan) with agitation at 100 rpm.
The initial anaerobic condition in the bioreactor was established by replacing the gas
phase with argon gas. AGS was prepared from 20 g of a commercial dog food (Vita-One;
Nihon Pet Food, Tokyo, Japan) dissolved in 1 L of sterilized water. The physicochemical
characteristics of the AGS are as follows: total chemical oxygen demand (COD), 25
gCODcr l1; soluble COD concentration, 8.5 gCODcr l1; suspended solid (SS),
16.5 g l1; volatile suspended solid (VSS), 15.5 g l1. The supply of AGS to the reactor
was accompanied by the concomitant removal of an equal amount of broth from the
reactor. The hydraulic retention time (HRT) was controlled using timer-controlled
peristaltic pumps at a constant value. The reactor was initially operated at HRT of
10 days (OLR, 2.5 gCODcr l1 day1) for 150 days. Thereafter, the OLR was stepwise
increased to 3.13 gCODcr l1 day1 (for 30 days at HRT of 8.0 days), 3.75 gCODcr l1
day1 (for 30 days at HRT of 6.7 days), and 5.0 gCODcr l1 day1 (for 15 days at HRT of
5.0 days). Finally, the reactor was stably operated at OLR of 6.25 gCODcr l1 day1 (i.e.,
HRT of 4.0 days) for 600 days. The pH value in the reactor was maintained at 7.2 by
automatic titration with 5 N NaOH.
Analyses of the reactor performance The gas production rate was measured
periodically by water displacement with a graduated cylinder. The broth in the reactor
was collected for analyses of physicochemical parameters at 3-day intervals. The COD
concentration was determined by using the dichromate method with a COD analyzer
(DR-300; Hach, Loveland, CO, USA). To determine the concentration of total SS, 2 ml of
the broth was passed through the membrane (Cellulose Acetate Membrane Filter;
0.2 m47 mm, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) after which the membrane was
weighed after drying at 105 C for 3 h. The concentration of VFAs was determined using
a liquid chromatograph (L6300; Hitachi, Tokyo, Japan) equipped with a TSKgel OAPak-
A column (Tohso, Tokyo, Japan) and a UV spectrophotometric detector (SPD-7A;
Shimadzu, Kyoto, Japan). The generated gas composition was analyzed by gas
chromatography-mass spectrometry (GC-MS) (GC17A-QP5050; Shimadzu) equipped
with a CP-PoraPLOT Q (L: 25 m, ID: 0.32 mm, df: 10 m; GL Science Inc., Tokyo, Japan).
Analysis of isotope distribution in the generated gas with 13C-labeled
acetate First, 10 ml of broth (600 days, HRT of 4.0 days) was taken from the
reactor and transferred to 25 ml vials. The vials were sealed with a butyl rubber stopper
and an aluminum cap. The gas phase was replaced with nitrogen gas using a
Deoxygenized Gas Pressure Injector (IP-8; Sanshin, Yokohama, Japan). In order to
equalize the degradation activity among the vials and to reduce the unlabeled acetate
originally presented in the broth, the vials were pre-incubated for 6 days at 55 C with
shaking. After the pre-cultivation, the gas phase was replaced with nitrogen gas. Then,
4 mM of sodium acetate-2-13C, sodium acetate-1-13C, or sodium acetate-1,2-13C (99
atom %; Sigma-Aldrich, Tokyo, Japan) were added to the pre-cultured vials. After 24 h of
incubation with shaking, the gaseous products were analyzed using the selected ion
monitoring (SIM) method by a GC-MS (GC17A-QP5050; Shimadzu) equipped with a
CP-PoraPLOT Q (L: 25 m, ID: 0.32 mm, df: 10 m; GL Science Inc.,). Helium was used as a
TABLE 1. Operational parameters and reactor performance during stable operation at the shortest HRT in this study.
Operational parameters
1 1 1 1
HRT (day) OLR (gCODcr l day ) Controlled pH Gas production rate (ml l
4.0 6.25 7.2 1810 ( 118)
a
Data are average values obtained between the 60th and 600th days of operation at HRT of 4.0 days.
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
defined as the liquid fraction. The genomic DNA was extracted from the total
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 maximum-
likelihood 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.
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
Reactor performance a
day ) COD removal ratio (%) SS removal ratio (%) Acetate (mM) Propionate (mM)
65.8 ( 2.80) 61.8 ( 5.59) 1.85 ( 1.81) 0.86 ( 0.83)
VOL. 111, 2011 METHANOGENESIS FROM ORGANIC SOLID WASTE 43

TABLE 2. Distribution of 13C-labeled acetate into CH4 and CO2 production. Methane production from 2-13C acetate or 1-13C acetate showed
CH4 produced from labeled acetate that 44% or 37% of methane was derived from the carboxyl group of
Substrate Peak intensities acetate, respectively (Table 2). These results indicated that a portion of
the acetate was converted to methane through non-aceticlastic
m/z 15 (12CH4) m/z 17 (13CH4) CH4 from CH4 from
Actual methyl carboxyl oxidation. Accordingly, 38% or 42% of CO2 was derived from the
Actual Background group/ group/ methyl group of acetate when 2-13C acetate or 1-13C acetate was used
subtracted a total CH4 total CH4 (Table 2). Non-aceticlastic oxidation produces equal amounts of
13
CH12
3 COONa 425,568 48,813 63,155 0.56 0.44 methane from the carboxyl and methyl groups of acetate; hence
12
CH13
3 COONa 458,789 82,034 47,673 0.63 0.37 non-aceticlastic oxidation accounted for 7488% (multiply the above
13
CH13
3 COONa 376,755 98,850 values, 3744%, by 2) of the total degradation of acetate. Consequently,
12
CH12
3 COONa 618,274 21,971
the aceticlastic cleavage of acetate accounted for 1226%.
no addition 346,025 17,301
Diversity of bacterial and archaeal communities Table 3
shows the phylogenetic affiliation and appearance frequency of the
CO2 produced from labeled acetate
detected clones. In the domain Bacteria, 12 OTUs were detected
Substrate Peak intensities among 94 clones in the broth. The bacterial community was
m/z 44 (12CO2) m/z 45 (13CO2) CO2 from CO2 from composed mainly of OTU-B1 (53% of the total number of clones)
Actual methyl carboxyl and OTU-B12 (21%). OTU-B1 belonged to phylum Thermotogae, and
Actual Background group/ group/
its closest relative was Petrotoga mobilis (Y15479; 90% sequence
subtracted b total CO2 total CO2
similarity), which has been detected from anaerobic bioreactors and
13
CH12
3 COONa 5,673,250 246,785 148,805 0.38 0.62
12
thermophilic microbial fuel cells (2729). The second dominant
CH13
3 COONa 5,564,999 138,534 191,833 0.42 0.58
13
CH13
clone, OTU-B12, was classified into uncultured municipal solid waste
3 COONa 5,426,465 195,295
12 12
CH3 COONa 6,889,539 51,474 (MSW) cluster I in phylum Firmicutes (6) (Fig. 1). OTU-B10, -B13,
no addition 3,763,455 37,634 -B14, and -B15 were also classified in the MSW cluster I.
All values are the average of three individual experiments. The broth in the reactor was divided into a solid fraction and a
a
The peak area at an m/z value of 15 from 13CH13
3 COONa was regarded as the background. liquid fraction. Forty-five and 46 bacterial clones were analyzed for
b
The peak area at an m/z value of 44 from 13CH13 3 COONa was regarded as the these fractions, respectively. The bacterial community in the liquid
background.
fraction resembled that in the broth (i.e., the total fraction), indicating
that the liquid slurry was the major habitat of microflora in the
operation at HRT of 4.0 days, and the acetate decomposition pathway reactor. The solid fraction was characterized by the appearance of
was analyzed. After pre-incubation, the broth supplemented with 13C- OTU-B5, -B6, -B7, -B8, and -B9, which belonged to Clostridium clusters
labeled acetate was anaerobically incubated. The incubation condi- in phylum Firmicutes (Fig. 1).
tions were chosen to simulate the concentration of acetate in the In the domain Archaea, 3 OTUs were detected among 55 clones
reactor and to minimize cross feeding. from the broth. The archaeal community was dominated by OTU-A1

TABLE 3. Phylogenetic affiliation and numbers of bacterial and archaeal clones obtained from each fraction.
OTU a No. of clones b Phylogenetic group c (genus or The closest relative (accession no., similarity %)
specific cluster)
Broth Solid Liquid

Bacteria
B1 50 7 24 Thermotogae Petrotoga mobilis (Y15479, 90)
B2 2 1 1 Firmicutes Coprothermobacter proteolyticus (X69335, 97)
B3 0 1 0 Firmicutes Coprothermobacter proteolyticus (X69335, 97)
B4 1 0 1 Firmicutes Coprothermobacter proteolyticus (X69335, 99)
B5 0 6 0 Firmicutes Clostridium populeti (X71853, 91)
B6 1 5 0 Firmicutes (Clostridium) cattle fecal clone EMP_I21 (EU794242, 87)
B7 6 9 1 Firmicutes Clostridium straminisolvens (AB125279, 94)
B8 1 4 0 Firmicutes Clostridium stercorarium (AJ310082, 94)
B9 0 1 0 Firmicutes Clostridium stercorarium (AJ310082, 95)
B10 1 0 0 Firmicutes (MSW cluster I) anaerobic digester clone G55_D25_M_B_E12 (DQ887948, 100)
B11 3 0 1 Firmicutes anaerobic digester clone HTB1-B2 (AB374111, 98)
B12 20 8 11 Firmicutes (MSW cluster I) anaerobic MSW digester clone MBA01 (AB114311, 99)
B13 0 1 0 Firmicutes (MSW cluster I) landfill leachate bioreactor clone AC007 (AY330129, 96)
B14 7 1 5 Firmicutes (MSW cluster I) anaerobic MSW digester clone MBA06 (AB114316, 99)
B15 0 0 1 Firmicutes (MSW cluster I) anaerobic digester clone A55_D21_L_B_B02 (EF559037, 100)
B16 1 0 1 Synergistetes Anaerobaculum mobile (AJ243189, 99)
B17 1 0 0 Firmicutes marine sediment clone MFC-7 (EU194835, 99)
B18 0 1 0 Planctomycetes hot spring clone OPB17 (AF027057, 96)
Sum 94 45 46

Archaea
A1 28 17 17 Methanomicrobiales Methanoculleus thermophilus (EF118904, 100)
A2 25 11 26 Methanomicrobiales Methanoculleus thermophilus (EF118904, 98)
A3 2 15 2 Methanosarcinales Methanosarcina thermophila (M59140, 98)
A4 0 4 0 Methanosarcinales Methanosarcina thermophila (M59140, 95)
A5 0 1 0 Methanosarcinales Methanosarcina thermophila (M59140, 94)
A6 0 2 0 Euryarchaeota (Rice cluster III) MSW landfill leachate clone (AJ576219, 99)
Sum 55 50 45
a
The sequences with more than 99.5% homology were collected as the same OTU.
b
Clone libraries of the total fraction of broth (Broth), the solid fraction (Solid), and the liquid fraction (Liquid) are presented.
c
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 metha-
nogenic 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
example, the accumulation of VFAs increased in the population of
Methanosarcina sp. (32). It has been reported that the population of
hydrogenotrophic methanogens was larger than that of aceticlastic
methanogens in various thermophilic digesters (33,34). In particular,
the dominance of Methanoculleus spp. and the decline of Methano-
sarcina spp. were detected from stable thermophilic reactors degrad-
ing organic solid wastes (4,35). This tendency in the methanogenic
population was similarly observed in our reactor (Table 3).
Isotope tracer experiments indicated that non-aceticlastic oxida-
tion is the major pathway (approximately 80%) of acetate decompo-
sition in the reactor as a result of the limited population of aceticlastic
methanogens. A thermophilic anaerobic reactor reported by Petersen
and Ahring converted 4.1% and 14.1% of acetate via the non-aceticlastic
oxidation pathway when the acetate concentration was 12 mM
and b1 mM, respectively (36). A higher rate of acetate oxidation,
approximately 70%, was determined for practical thermophilic
anaerobic digesters where VFAs were effectively removed (37).
These studies indicated that the decrease in the acetate concentration
promoted non-aceticlastic oxidation in thermophilic reactors. A low
acetate concentration for long-term operation in this study would
efficiently enrich the non-aceticlastic oxidation pathway. The non-

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.
VOL. 111, 2011
aceticlastic oxidation of acetate could be realized by syntrophic acetate-
oxidizing bacteria and Methanoculleus sp.; their growth rate and af-
finity 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 Thermo-
toga 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 ex-
periment, although the actual metabolic functions of individual micro-
organisms in the digester are still unclear (6,7).
Clone library analysis showed the specific distribution of micro-
organisms 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 long-
chain fatty acids in chemostat cultivation, J. Biosci. Bioeng., 102, 535544 (2006).
METHANOGENESIS FROM ORGANIC SOLID WASTE 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 micro-
aeration 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. Micro-
biol., 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 methano-
genic 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 DNA-
and 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 large-
scale 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 Methano-
saetaceae, 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 Methano-
sarcina 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-compart-
ment 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).