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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 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]

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 communities (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).

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 decomposition 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 cometabolism 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).

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

CH3 COO H2 O CH4 HCO


3

Non-aceticlastic oxidation

CH3 COO 4H2 OH CO


3 HCO3 4H2 H

H CO
3 or HCO3 4H2 H CH4 or CH4 3H2 O

(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 composition 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 OAPakA 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

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

Gas production rate (ml l


1810 ( 118)

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)

Data are average values obtained between the 60th and 600th days of operation at HRT of 4.0 days.

VOL. 111, 2011

METHANOGENESIS FROM ORGANIC SOLID WASTE

TABLE 2. Distribution of 13C-labeled acetate into CH4 and CO2 production.


CH4 produced from labeled acetate
Substrate

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

m/z 17 (13CH4) CH4 from CH4 from


Actual
methyl
carboxyl
group/
group/
total CH4 total CH4
63,155
47,673
98,850
21,971
17,301

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

CO2 produced from labeled acetate


Substrate

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

All values are the average of three individual experiments.


a
The peak area at an m/z value of 15 from 13CH13
3 COONa was regarded as the background.
b
The peak area at an m/z value of 44 from 13CH13
3 COONa was regarded as the
background.

operation at HRT of 4.0 days, and the acetate decomposition pathway


was analyzed. After pre-incubation, the broth supplemented with 13Clabeled acetate was anaerobically incubated. The incubation conditions were chosen to simulate the concentration of acetate in the
reactor and to minimize cross feeding.

43

Methane production from 2-13C acetate or 1-13C acetate showed


that 44% or 37% of methane was derived from the carboxyl group of
acetate, respectively (Table 2). These results indicated that a portion of
the acetate was converted to methane through non-aceticlastic
oxidation. Accordingly, 38% or 42% of CO2 was derived from the
methyl group of acetate when 2-13C acetate or 1-13C acetate was used
(Table 2). Non-aceticlastic oxidation produces equal amounts of
methane from the carboxyl and methyl groups of acetate; hence
non-aceticlastic oxidation accounted for 7488% (multiply the above
values, 3744%, by 2) of the total degradation of acetate. Consequently,
the aceticlastic cleavage of acetate accounted for 1226%.
Diversity of bacterial and archaeal communities
Table 3
shows the phylogenetic affiliation and appearance frequency of the
detected clones. In the domain Bacteria, 12 OTUs were detected
among 94 clones in the broth. The bacterial community was
composed mainly of OTU-B1 (53% of the total number of clones)
and OTU-B12 (21%). OTU-B1 belonged to phylum Thermotogae, and
its closest relative was Petrotoga mobilis (Y15479; 90% sequence
similarity), which has been detected from anaerobic bioreactors and
thermophilic microbial fuel cells (2729). The second dominant
clone, OTU-B12, was classified into uncultured municipal solid waste
(MSW) cluster I in phylum Firmicutes (6) (Fig. 1). OTU-B10, -B13,
-B14, and -B15 were also classified in the MSW cluster I.
The broth in the reactor was divided into a solid fraction and a
liquid fraction. Forty-five and 46 bacterial clones were analyzed for
these fractions, respectively. The bacterial community in the liquid
fraction resembled that in the broth (i.e., the total fraction), indicating
that the liquid slurry was the major habitat of microflora in the
reactor. The solid fraction was characterized by the appearance of
OTU-B5, -B6, -B7, -B8, and -B9, which belonged to Clostridium clusters
in phylum Firmicutes (Fig. 1).
In the domain Archaea, 3 OTUs were detected among 55 clones
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


specific cluster)

The closest relative (accession no., similarity %)

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

Petrotoga mobilis (Y15479, 90)


Coprothermobacter proteolyticus (X69335, 97)
Coprothermobacter proteolyticus (X69335, 97)
Coprothermobacter proteolyticus (X69335, 99)
Clostridium populeti (X71853, 91)
cattle fecal clone EMP_I21 (EU794242, 87)
Clostridium straminisolvens (AB125279, 94)
Clostridium stercorarium (AJ310082, 94)
Clostridium stercorarium (AJ310082, 95)
anaerobic digester clone G55_D25_M_B_E12 (DQ887948, 100)
anaerobic digester clone HTB1-B2 (AB374111, 98)
anaerobic MSW digester clone MBA01 (AB114311, 99)
landfill leachate bioreactor clone AC007 (AY330129, 96)
anaerobic MSW digester clone MBA06 (AB114316, 99)
anaerobic digester clone A55_D21_L_B_B02 (EF559037, 100)
Anaerobaculum mobile (AJ243189, 99)
marine sediment clone MFC-7 (EU194835, 99)
hot spring clone OPB17 (AF027057, 96)

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)

Methanoculleus thermophilus (EF118904, 100)


Methanoculleus thermophilus (EF118904, 98)
Methanosarcina thermophila (M59140, 98)
Methanosarcina thermophila (M59140, 95)
Methanosarcina thermophila (M59140, 94)
MSW landfill leachate clone (AJ576219, 99)

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

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 Methanosarcina spp. were detected from stable thermophilic reactors degrading 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 oxidation is the major pathway (approximately 80%) of acetate decomposition 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

METHANOGENESIS FROM ORGANIC SOLID WASTE

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).
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