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Received 29 June 2010; accepted 20 August 2010Available online 18 September 2010

Reuse anfrom the foconcern. Anrecovering eenvironmentally acceptable means of reducing and stabilizing organic

microbial composition and distribution in thermophilic reactorstreating organic solid wastes (47). Their reports have indicated that

anogenetic

ic polymerssubstances

such as cellulose and protein are hydrolyzed (hydrolysis), then the

by aceticlastic methanogenic archaea, such as Methanosarcina spp.(aceticlastic cleavage, reaction formula 1). The aceticlasticmethanogens

Journal of Bioscience anVOL. 111 No. 1, 41

www.elsevier.com/locate/jbioscmethanogenesis from solidwastes requiredmicrobialmembers distinctfrom those in other reactors treating liquid slurry such as industrial

convert the methyl and carboxyl groups of acetate to CH4 and CO2,respectively. The other is non-aceticlastic oxidation, i.e., the co-waste (1).The thermophilic anaerobic digestion process has advantages over

the mesophilic process with respect to digestion efficiency anddisinfection of pathogenic organisms (2). Unlike themesophilic process,the thermophilic process is characterized by limited species ofaceticlastic methanogens and simple structure of the bacterial commu-nities (3,4). Several researchers have provided information about

hydrolyzed products are degraded to volatile fatty acids (VFAs) and H2/CO2 (acidogenesis), and finallymethane gas is produced fromacetate orH2/CO2 (methanogenesis) (8). Acetate has been known as a keyintermediate metabolite during methanogenesis, and the decomposi-tion of acetate is considered to be the rate-limiting step of the over-allreaction process (9,10). So far, two methanogenic pathways fromacetate have been reported (1113). One is the direct methanogenesis Correspondand Life SciencJapan. Tel.: +8

E-mail add

1389-1723/$doi:10.1016/jd recycling of organic wastes, including garbage and wasteod industry, have been attracting social attention andaerobic digestion is one of the effective technologies fornergetic materials from organic waste and is a simple and

wastewater. However, little is known about the methpathway in the reactors.

The metabolic pathway to producemethane from organconsists basically of three steps: first, complex polymericThe methanogenic pathway and microbial community in a thermophilic anaerobic digestion process of organic solid wastewere investigated in a continuous-flow stirred-tank reactor using artificial garbage slurry as a feedstock. The decompositionpathway of acetate, a significant precursor of CH4 and a key intermediate metabolite in the anaerobic digestion process, wasanalyzed by using stable isotopes. A tracer experiment using 13C-labeled acetate revealed that approximately 80% of theacetate was decomposed via a non-aceticlastic oxidative pathway, whereas the remainder was converted to methane via anaceticlastic pathway. Archaeal 16S rRNA analyses demonstrated that the hydrogenotrophic methanogens Methanoculleus spp.accounted for N90% of detected methanogens, and the aceticlastic methanogens Methanosarcina spp. were the minorconstituents. The clone library targeting bacterial 16S rRNA indicated the predominance of the novel Thermotogalesbacterium (relative abundance: ~53%), which is related to anaerobic acetate oxidizer Thermotoga lettingae TMO, although thesequence similarity was low. Uncultured bacteria that phylogenetically belong to municipal solid waste cluster I were alsopredominant in the microflora (~30%). These results imply that the microbial community in the thermophilic degradingprocess 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]Methanogenic pathway and communidigestion process o

Daisuke Sasaki,1 Tomoyuki Hori,1,2 Shin Haruta,1,3,

Department of Biotechnology, Graduate School of Agricultural and Life ScieBioproduction Research Institute, National Institute of Advanced Indu

Sapporo 062-8517, Japan2 Graduate School of Science and Engineering, TokJapan3 and Kajima Technical Research Institute, Ting author. Department of Biotechnology, Graduate School of Agriculturales, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657,1 42 677 2580; fax: +81 42 677 2559.ress: sharuta@tmu.ac.jp (S. Haruta).

- see front matter 2010, The Society for Biotechnology, Japan. All.jbiosc.2010.08.011structure in a thermophilic anaerobicrganic solid waste

shiyuki Ueno,4 Masaharu Ishii,1 and Yasuo Igarashi1

The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan1

l Science and Technology, Tsukisamu-Higashi 2-17-2-1, Toyohira-ku,etropolitan University, Minami-Osawa 1-1, Hachioji-shi, Tokyo 192-0397,akyu 2-19-1, Chofu-shi, Tokyo 182-0036, Japan4

d Bioengineering46, 2011metabolismpathway by acetate-oxidizing bacteria (reaction formula 2)and hydrogenotrophic methanogens (reaction formula 3). During thelatter pathway, acetate is first oxidized to CO2 and then the producedCO2 is reduced to CH4. The decomposition pathway of acetate is knownto be affected by the operation temperature, the composition of organicsubstances, the types of reactors, and the organic loading rate (14).

rights reserved.

Aceticlastic cleavage

CH3COO H2OCH4 HCO3 1Non-aceticlastic oxidation

carrier gas at a flow rate of 1.5 ml min1, and the column temperature was 35 C. Thepeaks at m/z 15 and 17 were regarded as the fragment ion for 12CH4 and the molecularion for 13CH4, respectively. The peaks at m/z 44 and 45 were regarded as the molecularion for 12CO2 and 13CO2, respectively.

Extraction and purification of DNA The broth (300 days, HRT of 4.0 days)

ce d

ay1

42 SASAKI ET AL. J. BIOSCI. BIOENG.,CH3COO 4H2OHCO3 HCO3 4H2 H 2

HCO3 or HCO3 4H2 HCH4or CH4 3H2O 3

(Asterisks represent the carbon of methyl group in acetate.)

In the present study, a lab-scale thermophilic continuous-flowstirred-tank reactor (CFSTR) was operated using artificial garbageslurry (AGS) as a model of organic solid waste. After establishingefficient 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 acetatedegradation in the process by using stable isotopes.

MATERIALS AND METHODS

Operation of thermophilic CFSTR Two liters of seed sludge collected from athermophilic anaerobic digester treating garbage slurry (15) was cultivated at 55 C ina 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 gasphase 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 physicochemicalcharacteristics of the AGS are as follows: total chemical oxygen demand (COD), 25gCODcr 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 reactorwas accompanied by the concomitant removal of an equal amount of broth from thereactor. The hydraulic retention time (HRT) was controlled using timer-controlledperistaltic pumps at a constant value. The reactor was initially operated at HRT of10 days (OLR, 2.5 gCODcr l1 day1) for 150 days. Thereafter, the OLR was stepwiseincreased 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 of5.0 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 byautomatic titration with 5 N NaOH.

Analyses of the reactor performance The gas production rate was measuredperiodically by water displacement with a graduated cylinder. The broth in the reactorwas collected for analyses of physicochemical parameters at 3-day intervals. The CODconcentration 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 ofthe broth was passed through the membrane (Cellulose Acetate Membrane Filter;0.2 m47 mm, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) after which the membrane wasweighed after drying at 105 C for 3 h. The concentration of VFAs was determined usinga 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 gaschromatography-mass spectrometry (GC-MS) (GC17A-QP5050; Shimadzu) equippedwith 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-labeledacetate First, 10 ml of broth (600 days, HRT of 4.0 days) was taken from thereactor and transferred to 25 ml vials. The vials were sealed with a butyl rubber stopperand an aluminum cap. The gas phase was replaced with nitrogen gas using aDeoxygenized Gas Pressure Injector (IP-8; Sanshin, Yokohama, Japan). In order toequalize the degradation activity among the vials and to reduce the unlabeled acetateoriginally presented in the broth, the vials were pre-incubated for 6 days at 55 C withshaking. 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 (99atom %; Sigma-Aldrich, Tokyo, Japan)were added to the pre-cultured vials. After 24 h ofincubation with shaking, the gaseous products were analyzed using the selected ionmonitoring (SIM) method by a GC-MS (GC17A-QP5050; Shimadzu) equipped with aCP-PoraPLOT Q (L: 25 m, ID: 0.32 mm, df: 10 m; GL Science Inc.,). Heliumwas used as a

TABLE 1. Operational parameters and reactor performan

Operational parameters

HRT (day) OLR (gCODcr l1 day1 ) Controlled pH Gas production rate (ml l1 d4.0 6.25 7.2 1810 (118)

a Data are average values obtained between the 60th and 600th days of operation at HRTfrom 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 netfilter was defined as the solid fraction. The fraction passing through the net filter wasdefined as the liquid fraction. The genomic DNA was extracted from the totalfraction, solid fraction, and liquid fraction by the benzyl-chloride method (16). Theconcentration 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 asthe template of PCR amplification.

Cloning and phylogenetic analysis Clone libraries of bacterial and archaeal16S rRNA gene sequences were constructed from genomic DNA. PCR amplification ofthe partial 16S rRNA genes was carried out by using AmpliTaqGold with GeneAmp

(Applied Biosystems, Tokyo, Japan) according to the manufacturer's instructions. PCRwas 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 wasstarted with an initial denaturation at 94 C for 10 min, followed by 25 cycles ofdenaturation 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 theQIAquick Gel Extraction kit (Qiagen, Tokyo, Japan) were ligated to the pGEM-T EasyVector (Promega, Tokyo, Japan) according to the manufacturer's instructions. Theligation 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). Thesequences were checked for chimeric artifacts by using the CHIMERA_CHECK programfrom the Ribosomal Database Project (21). Homology searches were performed withthe BLAST program at the web site of the National Center for BiotechnologyInformation. Sequences with more than 99.5% identity were treated as the sameoperational taxonomy unit (OTU) (22,23). Phylogenetic analyses were performedusing the ARB software package (24). A phylogenetic tree was calculated andrepresented 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 dataobtained in this study have been deposited in the DDBJ/EMBL/GenBank nucleotidesequence 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 theaverage values of reactor performance from 60 to 600 days when thereactor was operated at HRT of 4.0 days (OLR, 6.25 gCODcr l1 day1).Gas production was stable during this period, and the accumulation ofacetate and propionate was negligible at concentrations of 1.85(1.81) mM and 0.86 (0.83) mM, respectively. More than 60% ofSS supplied to the reactor was solubilized, and COD removal efficiencywas over 65%. The gas produced contained approximately 80% CH4,and the remainder was CO2. Low content of CO2 could be due toincrease of alkalinity caused by NaOH titration. It was calculated thatmore than 80% of decomposed COD equivalent was recovered asmethane gas.

These reactor performance values approximated those of previousthermophilic CFSTRs treating organic solid wastes at similar OLR(6,25,26). Our reactor's operation and performance also stablydigested AGS at the short HRT, whereas the previous reactors wereoperated at HRT of 10 days or more.

Isotope distribution in the generated gas from 13C-labeledacetate Broth was taken from the reactor after 600 days of

uring stable operation at the shortest HRT in this study.

Reactor performancea

) 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)

of 4.0 days.

Methane production from 2-13C acetate or 1-13C acetate showedthat 44% or 37% of methane was derived from the carboxyl group ofacetate, respectively (Table 2). These results indicated that a portion ofthe acetate was converted to methane through non-aceticlasticoxidation. Accordingly, 38% or 42% of CO2 was derived from themethyl group of acetate when 2-13C acetate or 1-13C acetate was used(Table 2). Non-aceticlastic oxidation produces equal amounts ofmethane from the carboxyl and methyl groups of acetate; hencenon-aceticlastic oxidation accounted for 7488% (multiply the abovevalues, 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 3shows the phylogenetic affiliation and appearance frequency of thedetected clones. In the domain Bacteria, 12 OTUs were detectedamong 94 clones in the broth. The bacterial community wascomposed mainly of OTU-B1 (53% of the total number of clones)and OTU-B12 (21%). OTU-B1 belonged to phylum Thermotogae, andits closest relative was Petrotoga mobilis (Y15479; 90% sequencesimilarity), which has been detected from anaerobic bioreactors andthermophilic microbial fuel cells (2729). The second dominantclone, 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 aliquid fraction. Forty-five and 46 bacterial clones were analyzed forthese fractions, respectively. The bacterial community in the liquidfraction resembled that in the broth (i.e., the total fraction), indicatingthat the liquid slurry was the major habitat of microflora in thereactor. The solid fraction was characterized by the appearance of

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

CH4 produced from labeled acetate

Substrate Peak intensities

m/z 15 (12CH4) m/z 17 (13CH4)Actual

CH4 frommethyl

CH4 fromcarboxyl

Actual Backgroundsubtracteda

group/total CH4

group/total CH4

13CH312COONa 425,568 48,813 63,155 0.56 0.4412CH313COONa 458,789 82,034 47,673 0.63 0.3713CH313COONa 376,755 98,85012CH312COONa 618,274 21,971no addition 346,025 17,301

CO2 produced from labeled acetate

Substrate Peak intensities

m/z 44 (12CO2) m/z 45 (13CO2)Actual

CO2 frommethyl

CO2 fromcarboxyl

Actual Backgroundsubtractedb

group/total CO2

group/total CO2

13CH312COONa 5,673,250 246,785 148,805 0.38 0.6212CH313COONa 5,564,999 138,534 191,833 0.42 0.5813CH313COONa 5,426,465 195,29512CH312COONa 6,889,539 51,474no addition 3,763,455 37,634

All values are the average of three individual experiments.a The peak area at anm/z value of 15 from13CH313COONawas regarded as the background.b The peak area at an m/z value of 44 from 13CH313COONa was regarded as the

background.

METHANOGENESIS FROM ORGANIC SOLID WASTE 43VOL. 111, 2011operation at HRT of 4.0 days, and the acetate decomposition pathwaywas analyzed. After pre-incubation, the broth supplementedwith 13C-labeled acetate was anaerobically incubated. The incubation condi-tions were chosen to simulate the concentration of acetate in thereactor and to minimize cross feeding.TABLE 3. Phylogenetic affiliation and numbers of bacteri

OTUa No. of clonesb Phylogenetic groupspecific clus

Broth Solid Liquid

BacteriaB1 50 7 24 ThermotogB2 2 1 1 FirmicuteB3 0 1 0 FirmicuteB4 1 0 1 FirmicuteB5 0 6 0 FirmicuteB6 1 5 0 Firmicutes (ClosB7 6 9 1 FirmicuteB8 1 4 0 FirmicuteB9 0 1 0 FirmicuteB10 1 0 0 Firmicutes (MSWB11 3 0 1 FirmicuteB12 20 8 11 Firmicutes (MSWB13 0 1 0 Firmicutes (MSWB14 7 1 5 Firmicutes (MSWB15 0 0 1 Firmicutes (MSWB16 1 0 1 SynergistetB17 1 0 0 FirmicuteB18 0 1 0 PlanctomyceSum 94 45 46

ArchaeaA1 28 17 17 MethanomicroA2 25 11 26 MethanomicroA3 2 15 2 MethanosarciA4 0 4 0 MethanosarciA5 0 1 0 MethanosarciA6 0 2 0 Euryarchaeota (RiceSum 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 lc Results of phylogenetic grouping were derived from the phylogenetic tree by ARB softwOTU-B5, -B6, -B7, -B8, and -B9, which belonged to Clostridium clustersin phylum Firmicutes (Fig. 1).

In the domain Archaea, 3 OTUs were detected among 55 clonesfrom the broth. The archaeal community was dominated by OTU-A1

al and archaeal clones obtained from each fraction.c (genus orter)

The closest relative (accession no., similarity %)

ae Petrotoga mobilis (Y15479, 90)s Coprothermobacter proteolyticus (X69335, 97)s Coprothermobacter proteolyticus (X69335, 97)s Coprothermobacter proteolyticus (X69335, 99)s Clostridium populeti (X71853, 91)tridium) cattle fecal clone EMP_I21 (EU794242, 87)s Clostridium straminisolvens (AB125279, 94)s Clostridium stercorarium (AJ310082, 94)s Clostridium stercorarium (AJ310082, 95)cluster I) anaerobic digester clone G55_D25_M_B_E12 (DQ887948, 100)s anaerobic digester clone HTB1-B2 (AB374111, 98)cluster I) anaerobic MSW digester clone MBA01 (AB114311, 99)cluster I) landfill leachate bioreactor clone AC007 (AY330129, 96)cluster I) anaerobic MSW digester clone MBA06 (AB114316, 99)cluster I) anaerobic digester clone A55_D21_L_B_B02 (EF559037, 100)es Anaerobaculum mobile (AJ243189, 99)s marine sediment clone MFC-7 (EU194835, 99)tes hot spring clone OPB17 (AF027057, 96)

biales Methanoculleus thermophilus (EF118904, 100)biales Methanoculleus thermophilus (EF118904, 98)nales Methanosarcina thermophila (M59140, 98)nales Methanosarcina thermophila (M59140, 95)nales Methanosarcina thermophila (M59140, 94)cluster III) MSW landfill leachate clone (AJ576219, 99)iquid fraction (Liquid) are presented.are.

(51% of the total number of clones) and OTU-A2 (45%), which wereclosely related to Methanoculleus thermophilus (EF118904; 100% and98% sequence similarity, respectively). The remaining clone, OTU-A3,was related to Methanosarcina thermophila (M59140; 98% sequencesimilarity), implying that the archaeal population consists mainly ofhydrogenotrophic methanogens. The population of the OTUs relatedto M. thermophila was increased in the solid fraction. The OTU-A6detected in the solid fraction was an uncultured archaeon (AJ576219;99% sequence similarity), which was classified into rice cluster IIIfound in a MSW landfill leachate (30).

DISCUSSION

In this study, we analyzed the microbial composition in athermophilic anaerobic reactor stably treating organic solid wasteduring long-time operation at OLR of 6.25 gCODcr l1 day1. Acetatedegradation pathway by themicroflorawas also analyzed. Relatives toMethanoculleus sp. and Methanosarcina sp. were detected as metha-nogenic archaea. These methanogens had been widely distributed inthermophilic anaerobic reactors (31), and their population ratioseems to be affected by HRT, OLR, or the concentration of VFAs; for

example, the accumulation of VFAs increased in the population ofMethanosarcina sp. (32). It has been reported that the population ofhydrogenotrophic methanogens was larger than that of aceticlasticmethanogens 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 methanogenicpopulation 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 aceticlasticmethanogens. A thermophilic anaerobic reactor reported by PetersenandAhring converted 4.1% and14.1% of acetate via the non-aceticlasticoxidation pathway when the acetate concentration was 12 mMand b1 mM, respectively (36). A higher rate of acetate oxidation,approximately 70%, was determined for practical thermophilicanaerobic digesters where VFAs were effectively removed (37).These studies indicated that the decrease in the acetate concentrationpromoted non-aceticlastic oxidation in thermophilic reactors. A lowacetate concentration for long-term operation in this study wouldefficiently enrich the non-aceticlastic oxidation pathway. The non-

his

44 SASAKI ET AL. J. BIOSCI. BIOENG.,FIG. 1. Phylogenetic tree showing the relationship between bacterial OTUs detected in t

using ARB software. Hydrogenobacter thermophilus (Z30214) was used as an outgroup. The bathe lengths of the horizontal lines connecting any two organisms. The symbols (closed circlesN75%, N65%) obtained after 1000 resamplings.study and reference sequences based on a comparison of 16S rRNA gene sequences by

r corresponds to a 10% difference in nucleotide sequences, as determined by measuring, open circles, closed square, open squares) at nodes show bootstrap values (N95%, N85%,

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 formore than half of the bacterial clones, were found in a cluster ofThermotogales in the phylogenetic tree (Fig. 1). P. mobilis and Thermo-toga lettingaewithin this cluster have both been known as fermentativeand sulfate/thiosulfate reducing bacteria (38,39). T. lettingae strainTMO was reported to have syntrophically oxidized acetate withoutsulfate ions under co-culture conditions with a hydrogenotrophicmethanogen (39). OTU-B1 was expected to be a probable candidate ofacetate-oxidizer. The second dominant bacterial group, consisting ofOTU-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 Iprobably play a role in the decomposition of the complex organicmatter and/or soluble matter, such as proteins and lipids, in thefeedstock 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, andOTUs-B7B9, which were frequently found in the solid fraction, wereaffiliated with the Clostridium clusters XIVa, IV, and III, respectively.These clusters were characterized by cellulolytic, proteolytic, and/orfermentative bacteria (4043). These bacteria possibly digest theinsoluble polymeric substances in the AGS, since more than 60% of SSdecomposition 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 beenwidely realized that Methanosarcina spp. form aggregate (44) andeasily adhere to solid substances such as cellulosic materials. Theycould directly produce methane from acetate that derived from thefermentation of solid organic matter. Sasaki et al. also reported thepredominance of M. thermophila on supporting materials in athermophilic packed-bed reactor (45).

The present study is the first to relate the methanogenic pathwayfrom acetate to the microbial structure in a thermophilic anaerobicdigester degrading organic solid waste. Our results suggest the strongcontribution of the non-aceticlastic oxidative pathway to acetatedegradation, which was widely recognized as a rate-limiting step inmethanogenic bioreactors. Recently, several studies have found thesyntrophic acetate oxidizing reaction coupled with hydrogenotrophicmethanogens (14,46,47). The OTU-B1 showed low sequence similarity(b90%) with the reference sequences in databases, implying that thisbacterium may be a novel species of the acetate oxidizer. The isolationand physiological characterization of this bacterium will be of interestfor further examinations and will contribute to the understanding ofacetate decomposition in thermophilic methanogenesis while alsoleading to deeper insight into anaerobic digestion of organic solidwaste.

ACKNOWLEDGMENTS

This work was supported by New Energy and Industrial TechnologyDevelopment Organization (NEDO).

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46 SASAKI ET AL. J. BIOSCI. BIOENG.,

Methanogenic pathway and community structure in a thermophilic anaerobic digestion process of organic solid wasteMaterials and &INS id=Outline placeholderOperation of thermophilic CFSTRAnalyses of the reactor performanceAnalysis of isotope distribution in the generated gas with 13C-labeled acetateExtraction and purification of DNACloning and phylogenetic analysisNucleotide sequence accession numbers

ResultsReactor operation and performanceIsotope distribution in the generated gas from 13C-labeled acetateDiversity of bacterial and archaeal communities

DiscussionAcknowledgmentsReferences