Bioresource Technology 123 (2012) 599–607

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Bioresource Technology

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Identification and quantification of key microbial trophic groupsof methanogenic glucose degradation in an anaerobic digester sludge

Tsukasa Ito a, Kazumi Yoshiguchi b, Herto Dwi Ariesyady b,c, Satoshi Okabe b,⇑a Department of Civil and Environmental Engineering, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japanb Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japanc Faculty of Civil and Environmental Engineering, Institute of Technology Bandung, Bandung 40132, Indonesia

h i g h l i g h t s

" Key trophic groups in anaerobic glucose degradation were identified and quantified." Population size of propionate degraders was the smallest among the trophic groups." Population size of acetate degraders was comparable to the glucose degraders." The specific degradation rate of acetate was low (1/28 of that of glucose)." We explained why the degradation of propionate and acetate was rate-limiting.

a r t i c l e i n f o

Article history:Received 10 May 2012Received in revised form 23 July 2012Accepted 28 July 2012Available online 7 August 2012

Keywords:Glucose degradersPropionate degradersAcetate degradersRNA-SIPMAR-FISH

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.07.108

⇑ Corresponding author. Address: Division of Enviroof Engineering, Hokkaido University, North 13 West 8,Japan. Tel./fax: +81 (0)11 706 6266.

E-mail address: sokabe@eng.hokudai.ac.jp (S. Okab

a b s t r a c t

We investigated the major phylogenetic groups and population size of glucose-, propionate-, and acetate-degrading bacteria in the glucose-degrading anaerobic digester sludge by stable-isotope probing analysisof 16S rRNA (RNA-SIP) with [13C6]glucose followed by time course analysis of microautoradiographycombined with fluorescent in situ hybridization (MAR-FISH) with [U-14C]glucose. The results indicatedthat glucose was predominately degraded to CH4 and CO2 by glucose-degrading Propionibacterium andOlsenella that are belonging to the phylum Actinobacteria, propionate-degrading Smithella and Syntrop-hobacter, and acetate-degrading Methanosaeta and Synergistes group 4 in this anaerobic sludge. The pop-ulation size of propionate degraders was the smallest among three trophic groups and the specificdegradation rate of propionate was also low. The specific degradation rate of acetate was low eventhough their population size was comparable to the glucose degraders. These results could explainwhy the degradation of propionate and acetate was the rate-limiting step in methanogenic glucosedegradation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion is a widely used process for the productionof methane (CH4) from complex organic matter and requires atleast four physiologically different microbial groups (trophicgroups), hydrolyzing bacteria, fermenting bacteria, acetogenic bac-teria and two types (i.e., acetoclastic and hydrogenotrophic) ofmethanogenic archaea. The major intermediate metabolites inanaerobic digestion were volatile fatty acids (VFAs) such as propi-onate and acetate. In addition to the hydrolysis step, the conver-sion of VFAs is rate limiting, and propionate and acetate aremore or less present or accumulated in the anaerobic digester,

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nmental Engineering, FacultySapporo Hokkaido 060-8628,

e).

even if the hydrogen partial pressure is low enough to thermody-namically allow syntrophic oxidation of VFAs. The rate of the VFAsconversion relies on the metabolic interaction among those fer-menting bacteria, acetogenic bacteria and methanogenic archaea(Schink, 1997). Therefore, more detailed information of diversity,population size, and in situ activity of these microbial trophicgroups is required to improve the performance of anaerobic diges-ter. Although plenty of phylogenic analyses of individual microbialpopulations of methanogens, propionate degraders, and acetatedegraders have been reported to date (Ariesyady et al., 2007b;Ito et al., 2011; Shin et al., 2010), comprehensive and quantitativestudy on a series of active trophic groups involved in anaerobicdigestion is scarce. Therefore, we attempted to identify and quan-tify active populations responsible for degradation of the impor-tant intermediates for methanogenesis; glucose, propionate, andacetate in anaerobic digester sludge.

600 T. Ito et al. / Bioresource Technology 123 (2012) 599–607

In fact, it is difficult to quantify each trophic group by 16S rRNAbased techniques such as fluorescent in situ hybridization (FISH)because FISH is based on phylogenetic sequences and is not alwaysrelated to physiological properties (Okabe et al., 2010). In addition,substrate utilization capability of microorganisms in pure culturestudy does not necessarily reflect real substrate utilization in com-plex microbial community (Nielsen et al., 2000; Okabe et al., 2004),indicating that microbial metabolic functions should be directlymeasured in situ. Microautoradiography combined with fluores-cent in situ hybridization (MAR-FISH) and stable-isotope probingof 16S rRNA (RNA-SIP) are powerful techniques to simultaneouslydetermine the phylogenetic identity and in situ specific metabolicfunction at a single cell level and have been applied to variouscomplex microbial communities (Ariesyady et al., 2007a,b; Itoet al., 2002, 2011; Kindaichi et al., 2004; Kong et al., 2004; Luederset al., 2004; Manefield et al., 2002; Okabe et al., 2005). MAR-FISHdetermines active population quantitatively but is limited by thelack of taxonomic resolution of FISH probes, while RNA-SIP deter-mines phylogenetic affiliation of active populations but is limitedby the lack of quantitative resolution of relative clone distribution.Therefore, combining approach of MAR-FISH and RNA-SIP wasused to address the objective of this study. First, RNA-SIP with[13C6]glucose followed by MAR-FISH with [U-14C]glucose was per-formed for anaerobic digester sludge to identify dominant glucose-, propionate-, and acetate-degrading bacteria. Second, time courseanalysis of MAR-FISH during [U-14C]glucose degradation to meth-ane and CO2 was performed to visualize and quantify thepopulation size of each trophic group by tracking the fate of radio-activity originated from [U-14C]glucose. Finally, the populationsizes of three trophic groups and their degradation rates weredetermined and compared to explain why degradation of propio-nate and acetate was the rate-liming step in methanogenic glucosedegradation in this anaerobic digester sludge.

2. Methods

2.1. Anaerobic sludge samples

Anaerobic sludge samples were taken from the mesophilicanaerobic fed-batch reactors that were operated stably for morethan 2 years in our laboratory. The seed sludge for the anaerobicdigesters was obtained from an anaerobic mesophilic digester atthe Ebetsu municipal wastewater treatment plant located at Ebe-tsu city, Japan. The typical characteristics (total solids, TS; total vol-atile solids, TVS) of the Ebetsu anaerobic digester sludge were26 g TS/L, and 15 g TVS/L, and the digester sludge was flocculent.The operation conditions of the Ebetsu anaerobic digester plantwere as follows: gas production rate, 0.44 m3-biogas/m3/d; volatilesolid destruction, 65%; average running temperature, 40 �C; organ-ic load, 2.5 kg VS/m3/day. Powdered whole-milk (Meiji Dairies Cor-poration, Tokyo, Japan) composed of carbohydrate (57%), lipid(25%) and protein (13%) was fed every 2 days at a loading rate of1.5 g COD L–1 day–1. Mineral solution was supplemented with thepowdered whole-milk. Other details of the reactor operation andthe composition of the mineral solution were described elsewhere(Ariesyady et al., 2007b).

2.2. Analytical measurements

Methane, carbon dioxide, and hydrogen gas were analyzed bygas chromatography (Shimazu, Kyoto, Japan) equipped with a ther-mal conductivity detector (TCD) and a 6 m, 2 mm i.d. SHINCARBONT column (Shinwa Chemical, Kyoto, Japan). Volatile fatty acids(VFAs) were determined with an ion chromatograph equippedwith an ICE-AS1 column (model DX-100, Dionex, Sunnyvale, CA).

Glucose was determined with an ion chromatograph equippedwith an CarboPac PA1 column (Dionex).

2.3. RNA-SIP

2.3.1. Incubation with [13C6]glucoseTwenty-five ml slurry samples were taken from the anaerobic

reactor, transferred to a 50 ml polycarbonate tube, and centrifugedat 2,500g for 5 min. The centrifugation resulted in approximately5 ml of sludge and 20 ml of supernatant. After replacing the gasphase of the sample tube with N2 and CO2 (80:20) gas, the sampletube was transferred to an anaerobic chamber containing N2 andCO2 (80:20). The following preparation processes and incubationwere conducted in the anaerobic chamber. The supernatant wasfirst replaced with 20 ml of the mineral solution, and then the mix-ture of the mineral solution and sludge was transferred to a 30 mlglass vial. The mineral solution was flushed and evacuated in ad-vance with oxygen-free N2 gas to remove the oxygen. For incubationwith 13C-labeled glucose (13C6, >99 atom% 13C, Isotec, OH), the vialwas incubated with 2.5 mM of glucose for 48 h with gently shakingat 30 rpm at 37 �C. The temperature of 37 �C was to mimic the incu-bation condition of the mesophilic anaerobic fed-batch reactors. The2.5 mM glucose was degraded to CH4 and CO2 via acetate and propi-onate as detectable intermediates during the 48 h incubation.

2.3.2. RNA extraction and fractionationThe sludge sample (25 ml) was centrifuged at 15,000g for 10 min

immediately after the incubation with 13C6-glucose. Total RNA wasextracted from the entire harvested pellet with the FastRNA ProSoil-Direct Kit (Qbiogene Inc., CA). The extracted RNA was purifiedprior to DNase I digestion (Lueders et al., 2004). The purified RNA(12 lg) was subsequently loaded with cesium trifluoroacetate(CsTFA) equilibrium density gradient in 2.0 ml Beckman Quick-Sealpolyallomer Bell Top tubes, and subjected to density gradient centri-fugation with Optima TLX (Beckmann Coulter) at 64,000 rpm and20 �C for 36 h (Manefield et al., 2002). Centrifuged gradients werefractionated into 20 gradient fractions with the fraction recoverysystem (Bechmann Coulter) at the flow rate of 3.3 lL s�1 by displace-ment with ddH2O using a syringe pump (Manefield et al., 2002). Infractions 1 to 20, fraction 1 was the 1st fraction collected from thebottom of the gradient. The amount of RNA of each gradient fractionwas quantified fluorometrically by RiboGreen assay (Invitrogen,Carlsbad, CA) (Lueders et al., 2004). In control experiment with unla-beled RNA from the digester sludge, RiboGreen measurementsshowed that unlabeled RNA enriched between fractions 10 and 15(with peak fraction 12). After the incubation with 13C6-glucose,RNA also appeared between fractions 5 and 7 (with peak fraction 6).

2.3.3. RT-PCR, cloning, sequencing, and phylogenetic analysisFor cloning and sequencing analysis, the fraction six that con-

tained heavy 13C-labeled RNA was amplified with the SuperscriptIII one-step RT-PCR kit (Invitrogen) using bacterial primer pair 8fand 1492r (Lane, 1991), and archaeal primer pair A109f and1492r (Lane, 1991; Grosskopf et al., 1998). The RT-PCR was carriedout with the following amplification program: one cycle consistingof 55 �C for 30 min (reverse transcription) and 94 �C for 2 min, then40 cycles consisting of 94 �C for 15 s; 54 �C for 30 s and 68 �C for2 min, followed by final extension at 68 �C for 7 min. The RT-PCRproduct was gel-purified and cloned by using a TOPO� XL PCR clon-ing kit (Invitrogen).

Randomly selected clones were sequenced on an ABI model3100-Avant genetic analyzer with a BigDye terminator ReadyReaction kit (Applied Biosystems, Foster City, CA). The sequencesobtained were compared with reference 16S rRNA gene sequencesavailable in the GenBank/EMBL/DDBJ databases using the BLASTsearch (Altschul et al., 1997). The closest relatives of the clones

T. Ito et al. / Bioresource Technology 123 (2012) 599–607 601

and the taxonomic classification were confirmed by the followingphylogenetic analysis. Phylogenetic analysis was performed usingthe MEGA3 software package (Kumar et al., 2004) after multiplealignments of data by CLUSTAL W (Thompson et al., 1994). Thephylogenetic trees were constructed using neighbour-joining andmaximum-parsimony methods. The confidence level for nodeswas ascertained by performing a bootstrap analysis (1,000replications).

2.4. 16S rRNA gene-cloning and phylogenetic analysis

One milliliter of slurry was sampled and then immediately sub-jected to three cycles of freezing in liquid N2 and thawing in a 65 �Cwater bath (3 min at each temperature). After the freeze–thaw cy-cles, 0.2 ml of the slurry samples were subjected to DNA extraction.DNA was extracted using a FastDNA Spin kit for soil (BIO 101, Qbi-ogene, Carlsbad, CA). The nearly full-length 16S rRNA genes frommixed bacterial and archaeal DNA were amplified by PCR usingthe two universal primer set for Bacteria, 8f and 1492r, and for Ar-chaea, A109f and 1492r (Lane, 1991; Grosskopf et al., 1998). Tominimize nonspecific annealing of the primers to nontarget DNA,a hot-start PCR program was used for all amplifications. The PCRwas carried out with the following amplification program: afterinitial denaturation at 94 �C for 5 min, 21 cycles consisting of94 �C for 1 min; 55 �C for 1 min and 72 �C for 2 min, followed by fi-nal extension at 72 �C for 8 min. To reduce possible bias associatedwith PCR amplification, the 16S rRNA gene was amplified in tripli-cate tubes and then combined for the next cloning step. The PCRproduct was gel-purified and cloned by using a TOPO� XL PCR clon-ing kit (Invitrogen). Subsequent sequencing of randomly selectedclones and phylogenetic analysis of the sequences obtained werethe same as described in the Section 2.3.3 RT-PCR, cloning,sequencing, and phylogenetic analysis.

2.5. MAR-FISH

2.5.1. Incubation with [U-14C]glucoseTen-ml slurry samples were taken from the anaerobic digester

and centrifuged at 2,500g for 5 min. Eight milliliter of the superna-tant was replaced with the mineral solution (without powderedmilk). The sludge and mineral solution were gently mixed, and then3 ml of the mixtures were transferred to 5 ml serum bottles. Theserum bottles were sealed with gas-tight rubber stoppers andanaerobically incubated with gently shaking at 30 rpm at 37 �Cwith [U-14C]glucose for 1, 3, 12, 36, and 48 h to investigate time-course shift of active community during glucose conversion tomethane and CO2. Three serum bottles were prepared for each incu-bation time. [U-14C]Glucose was added with unlabeled glucose(14C/(12C + 14C); 1%) to give a final concentration of 2.5 mM. Con-trols were prepared by pasteurizing the sludge at 70 �C for 30 minand run in parallel for all analyses. The radiolabeled [U-14C]glucosewas purchased from the Amersham pharmacia biotech (Bucking-hamshire, UK), and the specific activity was 11.7 GBq mmol–1.

The uptakes of radiolabeled substrates were measured for allcultures by liquid scintillation counting prior to FISH and microau-toradiographic procedures as described by Ariesyady et al. (2007b)and Ito et al. (2002).

In parallel to the incubation with [U-14C]glucose, cold runexperiment, that is without [U-14C]glucose, was conducted to mea-sure the time-course concentration changes of glucose, volatilefatty acids, CH4, H2, and CO2.

2.5.2. Sample fixation, washing, and fluorescent in situ hybridization(FISH)

The incubation was terminated at 1, 3, 12, 36, and 48 h by add-ing 4% paraformaldehyde. The fixation of the samples, washing,

and fluorescent in situ hybridization (FISH) were conductedaccording to Okabe et al. (1999) and Ito et al. (2002).

2.5.3. FISH probesFollowing oligonucleotide probes were used: EUB338, EUB338-

II, EUB338-III, CFB719, CFB286, GNSB-941, CFX1223, SmiSR354,Synbac824, SmiLR150, Syner195, MSMX860, MX825 and ARC915(Table 1). The probes were labeled with fluorescein isothiocyanate(FITC) or tetramethylrhodamine 5-isothiocyanate (TRITC) at the 50

end.

2.5.4. Autoradiographic developing procedureFollowing the FISH, the autoradiographic procedure was per-

formed directly on the cover glasses by using liquid film emulsion(LM-1; Amersham Pharmacia Biotech, Piscataway, NJ) (Lee et al.,1999; Kindaichi et al., 2004). The optimum exposure time wasdetermined to be 4 days for the samples incubated with[U-14C]glucose in preliminary experiments.

2.5.5. Microscopy and enumeration by MAR-FISHA model LSM510 confocal laser scanning microscope (CLSM)

(Carl Zeiss, Oberkochen, Germany) equipped with a UV laser (351and 364 nm), an Ar ion laser (450 to 514 nm), and two HeNe lasers(543 and 633 nm) were used. The formation of silver grains in theautoradiographic film was observed by using the transmissionmode of the CLSM system. A MAR-positive cell was defined as a cellcovered with at least five silver grains in this study. The numbers ofMAR-positive cells and total probe-hybridized cells (or total MAR-positive cells) were determined in triplicate by directly counting atleast 1,000 silver grain-covered cells in randomly chosen micro-scopic fields of a few slides prepared for each sample. More than3,000 DAPI (40,6-diamidino-2-phenylindole) -stained cells werecounted for each sample and FISH probe. For filamentous archaealcells such as Methanosaeta, probe-hybridized filaments werecounted.

2.6. Determination of substrate degradation rates for glucose,propionate and acetate

Substrate degradation rates for glucose, propionate, and acetatewere determined from batch experiments. The incubation condi-tion was the same as that for MAR-FISH but without radiolabeledsubstrates. Twenty-five milliliter slurry samples were taken fromthe anaerobic digester and centrifuged at 2,500g for 5 min. Twentymilliliter of the supernatant was replaced with the mineral solu-tion (Ariesyady et al., 2007b). The sludge and mineral solutionwere gently mixed and then transferred to 35 ml serum bottles.The serum bottles were sealed with gas-tight rubber stoppersand anaerobically incubated with gently shaking at 30 rpm at37 �C with glucose, propionate, or acetate. The initial glucose con-centration was adjusted at 2.5 mM. Triplicate bottles were pre-pared. Every thirty minutes subsamples were withdrawn forglucose measurement. The glucose concentrations during the ini-tial 1 h incubation were used to calculate the glucose degradationrate. The propionate concentrations were adjusted at 0.2, 0.5, 2.5,5.0, 10, 15, and 20 mM. Duplicate bottles were prepared for eachconcentration. Every two hours subsamples were withdrawn forpropionate measurement. Propionate degradation rates at differentpropionate concentrations were calculated from the slopes of linerfitting for time-dependent changes in propionate concentrationsduring the 6 h incubation. The maximum propionate degradationrate was determined by the Lineweaver–Burke plot of the ratesof propionate degradation versus propionate concentrations. Themaximum acetate degradation rate was determined with[2-14C]acetate by liquid scintillation counting as described by Itoet al. (2011). Acetate concentrations were adjusted at 0.5, 1.0,

Table 1FISH oligonucleotide probes used in this study

Probe Sequence (5’-3’) Specificity % FAa Reference

EUB338 GCT GCC TCC CGT AGG AGT Most but not all Bacteria –b Amann et al., 1990EUB338-II GCA GCC ACC CGT AGG TGT Bacterial groups not covered by EUB338 and EUB338-III –b Daims et al., 1999EUB338-III GCT GCC ACC CGT AGG TGT Bacterial groups not covered by EUB338 and EUB338-II –b Daims et al., 1999ARC915 GTG CTC CCC CGC CAA TTC CT Archaea 35 Raskin et al., 1994MSMX860 GGC TCG CTT CAC GGC TTC CCT Methanosarcinales (all Methanosarcina and Methanosaeta) 45 Raskin et al., 1994MX825 TCG CAC CGT GGC CGA CAC CTA

GCSome Methanosaetaceae 50 Raskin et al., 1994

GNSB-941 AAA CCA CAC GCT CCG CT Chloroflexi 35 Gich et al., 2001CFX1223 CCA TTG TAG CGT GTG TGT MG Chloroflexi 35 Björnsson et al., 2002CFB719 AGC TGC CTT CGC AAT CGG most members of the class Bacteriodetes, some Flavobacteria and Sphingobacteria 30 Weller et al., 2000CFB286 TCC TCT CAG AAC CCC TAC most members of the genus Tannerella and the genus Prevotella of the class

Bacteriodetes50 Weller et al., 2000

HGC69A TAT AGT TAC CAC CGC CGT Actinobcteria 25 Roller et al., 1994Synbac824 GTA CCC GCT ACA CCT AGT Syntrophobacter spp. 10 Ariesyady et al.,

2007aSmiSR354 CGC AAT ATT CCT CAC TGC Smithella propionica, Smithella sp. short rod (SR) 10 Ariesyady et al.,

2007aSmiLR150 CCT TTC GGC ACG TTA TTC Smithella sp. long rod (LR) 10 Ariesyady et al.,

2007bSyner195 GCA GTA CTC GCG TAC CTT Synergistes group 4 (Synergistetes PD-UASB-13) 10-

20Ito et al., 2011

a FA, formamide concentration in the hybridization buffer.b The probe can be used at any formamide concentrations.

602 T. Ito et al. / Bioresource Technology 123 (2012) 599–607

2.5, 5, 10, and 20 mM. The acetate concentrations during the initial10 h incubation were used to calculate the acetate degradationrate. Each rate was estimated during the initial incubation time(1–10 h) before entering the log phase. The pH was within therange of 7.0–7.5 during the incubation periods.

2.7. Nucleotide sequence accession numbers

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNAgene sequences of the clones used for the phylogenetic analysisare AB669227–AB669271.

3. Results

3.1. Phylogenetic analysis of heavy 13C-labeled bacterial 16S rRNAoriginated from [13C6]glucose

The phylogenetic affiliation of 16S rRNA sequences was ana-lyzed for total 48 clones of heavy 13C-labeled bacterial rRNA de-rived from the anaerobic digester sludge incubated with 2.5 mM[13C6]glucose for 48 h (Fig. 1 and Table 2). Fifty percent of total16S rRNA clones belonged to Actinobacteria in which Olsenella

Spirochaeta2%

Others3%Firmicutes 2%

Betaproteobacteria 4%

2% 3%

Deltaproteobacteria 6%

Betaproteobacteria 4%

Actinobacteria

Deltaproteobacteria 6%

Actinobacteria50%

Chloroflexi8%8%

BacteroidetesBacteroidetes10%

S i t tSynergistetes15%

Fig. 1. Community composition of whole bacterial 16S rRNA clones retrieved from‘heavy’ fraction of the RNA-SIP experiment of the anaerobic digester sludgeincubated with 2.5 mM [13C6]glucose for 48 h.

(23%) and Propionibacterium (17%) were predominant. Another fiftypercent of total 16S rRNA clones represented Synergistetes (15%),Bacteroidetes (10%), Chloroflexi (8%), Deltaproteobacteria (6%), Beta-proteobacteria (4%), Firmicutes (2%), Spirochaeta (2%) and others(3%). Among the clones belonging to Synergistetes, thirteen percentbelonged to acetate-utilizing Synergistes group 4 (Table 2). Acetateutilization by Synergistes group 4 was demonstrated in our previ-ous study (Ito et al., 2011). Among the clones belonging to Delta-proteobacteria, two percent were affiliated with syntrophicpropionate-degrading Syntrophobacter, and another two percentwere affiliated with syntrophic propionate-degrading Smithella(Table 2). Thus, propionate- and acetate-degrading bacteria weredetected in addition to glucose degraders (such as Actinobacteria,Bacteroidetes, and Chloroflexi). This is probably because 13C wascross-fed into these acetate- and propionate-degrading bacteriaduring 48 h incubation. For archaea, only two clones of heavy13C-labeled archaeal rRNA were retrieved from the anaerobic di-gester sludge, which are affiliated with Methanosaeta (acetoclasticmethanogen).

3.2. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes

Phylogenetic analysis of bacterial and archaeal 16S rRNA geneswas performed in order to analyze total microbial communitycomposition of the anaerobic digester sludge and compare withthe 13C-labeled bacterial community composition obtained byRNA-SIP analysis with [13C6]glucose. In total, 100 clones were ana-lyzed for bacteria (Fig. 2a and Table 3). Thirty-three percent of thetotal bacterial clones belonged to the Bacteroidetes. Spirochaeta(17%), Deltaproteobacteria (14%), Chloroflexi (11%), and Firmicutes(9%) followed. Only one percent was affiliated with Propionibacte-rium (Table 3) which was predominant in the RNA-SIP analysiswith [13C6]glucose. One percent belonged to Synergistes group 4(Table 3). Fourteen percent was affiliated with syntrophic propio-nate-degrading Smithella and Syntrophobacter of Deltaproteobacte-ria. For archaea, thirty-five clones were also analyzed (Fig. 2b).Eighty percent of the total clones belonged to Methanosaetaceae,in which acetate-degrading Methanosaeta concilii and Methanosaetasoehngenii were the closest relatives. Fourteen percent was affili-ated with the genus Methanosarcina, and six percent was affiliatedwith the hydrogenotrophic Methanoculleus.

Table 2Distribution of 16S rRNA clones retrieved from ‘heavy’ fraction of the RNA-SIP experiment of the anaerobic digester sludge incubated with 2.5 mM [13C6]glucose for 48 h. Closestrelative of the clones related to phylum Actinobacteria, Synergistetes, and Deltaproteobacteria.

Clone Closest relative Nucleotide similarity (%) Relative abundance (%)

Actinobacteria13-1 Olsenella uli DSM 7084 (CP002106) 1352/1390 (97%) 2313-3 Propionibacteriaceae bacterium FH044 (AB298766) 1387/1392 (99%) 1713-31 Propionibacteriaceae bacterium SH081 (AB298752) 1340/1392 (96%) 413-2 uncultured Actinobacteria bacterium (CU917992) 1354/1362 (99%) 413-9a uncultured methanogenic UASB reactor bacterium clone (EF063623) 1401/1404 (99%) 2

Georgenia thermotolerans (AB436534) 1269/1347 (94%)

Synergistetes13-29 uncultured mesophilic biogas digester bacterium clone (FN563164) of Synergistes group 4/PD-UASB-13 1397/1404 (99%) 1313-6 uncultured mesophilic anaerobic digester clone (CU918827) 1118/1130 (98%) 2

Deltaproteobacteria13-8a Syntrophus sp. 16S rRNA gene, partial, Clone B1 (AJ133794) 1396/1405 (99%) 2

Smithella propionica (AF126282) 1286/1346 (95%)13-37a uncultured granular sludge bacterium clone R1p32 (AF482435) 1429/1445 (98%) 2

Syntrophobacter sulfatireducens (AY651787) 1339/1433 (93%)13-35a uncultured phenol-degrading sludge bacterium clone (EU399668) 1345/1432 (93%) 2

Syntrophus gentianae (X85132) 1323/1431 (92%)

a The closest relative was uncultured clone. Therefore, the closest cultivated relative was also listed.

(a)Synergistetes 1%

Gammaproteobacteria1% 9%

Bacteroidetes33%

Actinobacteria 1%y g 1%

2%OP5 33%2%Verrucomicrobia2%OP5

Firmicutes9%9%

Chloroflexi11%

Spirochaetes17%

Deltaproteobacteria14% 17%14%

Methanoculleus 6%

Methanosarcina(b)

Methanosaeta14%

80%

Others

Fig. 2. Community composition of whole bacterial (a) and archaeal (b) 16S rRNAgene clones retrieved from anaerobic digester sludge.

T. Ito et al. / Bioresource Technology 123 (2012) 599–607 603

3.3. Identification of anaerobic [14C]glucose-degrading bacteria usingMAR-FISH

As the results of phylogenetic analyses of the heavy 13C-labeled16S rRNA from 13C-glucose, Actinobacteria was thought to be adominant glucose degrader since Actinobacteria dominated a halfof the clone libraries. Therefore, FISH probe specific for Actinobac-teria, HGC69A, was used for MAR-FISH with [U-14C]glucose to con-firm that the Actinobacteria was glucose-degrading bacteria in theanaerobic digester sludge. FISH and MAR-FISH analyses revealedthat the HGC69A probe-hybridized bacterial cells were mostly

clustered and abundantly present in the digester sludge. TheHGC69A probe-hybridized cells were covered densely with silvergrains (Fig. S1), indicating a significant utilization of 14C-glucose.This clearly indicated that bacteria belonging to the Actinobacteria,in which the genera Olsenella and Propionibacterium dominated,were glucose degraders in this sludge. The HGC69A probe-hybridized Actinobacteria cells in the digester sludge were MAR-negative when 14C-acetate and 14C-propionate were used (datanot shown).

Bacteroidetes and Chloroflexi were also thought to be dominantglucose degraders because they were subdominant in the clonelibraries of RNA-SIP. However, cells hybridized with Bacteroide-tes-specific CFB719 and CFB286 probes and Chloroflexi-specificGNSB-941 and CFX1223 probes were MAR-negative after incuba-tion with [U-14C]glucose for 1 h (data not shown).

3.4. Time course analysis of MAR-FISH with [14C]glucose

Time course analysis of MAR-FISH with different incubationperiods of 1, 3, 12 and 36 h was carried out to investigate the fateof 14C derived from [U-14C]glucose (Fig. 3). In parallel with theexperiment with [U-14C]glucose (hot run), the same incubationexperiment with 12C-glucose (2.5 mM) (cold run) was performedto monitor the fate of volatile fatty acids for 48 h. Acetate and pro-pionate significantly accumulated within the first 12 h and thengradually decreased to undetectable levels after 24 h. Methaneand carbon dioxide was constantly produced. The gas compositionat 48 h was 68 mol% of CH4 and 32 mol% of CO2. Organic carbonadded as glucose (2.5 mM, equivalent to 15 mM carbon) at thebeginning of the incubation was almost recovered as CH4

(10 mM) and CO2 (5 mM). Headspace hydrogen gas was always be-low the detection limit (detection limit, 1 � 10�4 atm) during theentire experiment. Lactate, normal-butyrate, iso-butyrate, and for-mate were always below the detection limit. Thus, glucose wasmainly converted to methane via propionate pathway in thisstudy.

Time course analysis of MAR-FISH revealed that the HGC69Aprobe-hybridized Actinobacteria was most abundant MAR positivemicrobial group (8.3 ± 1.6% of total cells, n = 3) at 1 h and rapidlyincreased to 15.2 ± 0.6% (n = 3) at 3 h. This population increasecoincided with decreasing glucose concentration. After 3 h, theMAR-positive Actinobacteria population decreased from15.2 ± 0.6% (n = 3) to 6.2 ± 1.7% (n = 3) at 36 h.

(a)

(b)

(c)

Fig. 3. Time-course concentration changes of glucose and fermentation products (a), probe-identified active microbial cells (b) and active bacterial and archaeal cells (c)during the glucose degradation by the anaerobic digester sludge. Glucose (2.5 mM) was added at 0 h. The right y-axis of (a) is CH4 and CO2 concentrations in liquid phase,which were converted from the gas composition (%) in the gas phase. Active cells in (b) and (c) were identified by MAR-FISH with general and specific probes listed in Table 1.Total bacterial and archaeal populations of the digester sludge were relatively constant during the 48 h incubation.

Table 3Distribution of bacterial 16S rRNA gene clones retrieved from anaerobic digester sludge. Closest relative of the clones related to phylum Actinobacteria, Synergistetes, andDeltaproteobacteria.

Clone Closest relative Nucleotide similarity (%) Relative abundance (%)

Actinobacteriaa88 Propionibacteriaceae bacterium FH044 (AB298766) 1404/1420 (98%) 1Synergistetesa42a Synergistetes UASB granular sludge bacterium (AB558582) 915/929 (98%) 1

uncultured bacterium clone PD-UASB-13 (AY261810) 834/862 (96%)Deltaproteobacteriaa13a uncultured industrial anaerobic digestor bacterium clone 50c (FJ462108) 1484/1519 (97%) 10

Smithella propionica (AF126282) 1308/1372 (95%)a14a uncultured household biogas digester bacterium clone (EU407214) 1468/1501 (97%) 2

Smithella propionica (AF126282) 1268/1354 (93%)a74a uncultured anaerobic wastewater treatment sludge bacterium clone (EF688231) 1516/1521 (99%) 1

Smithella propionica (AF126282) 1315/1372 (95%)a11a uncultured mesophilic anaerobic digester bacterium clone (CU920818) 1369/1374 (99%) 1

Syntrophobacter wolinii DSM 2805 (X70906) 1341/1367 (98%)

a The closest relative was uncultured clone. Therefore, the closest cultivated relative was also listed.

604 T. Ito et al. / Bioresource Technology 123 (2012) 599–607

Smithella- and Syntrophobacter-specific probes-hybridized MARpositive cells slightly increased during 36 h incubation but only

less than 2% of total cells. Methanosaeta- and Synergistes group 4specific probes-hybridized MAR positive cells gradually increased

T. Ito et al. / Bioresource Technology 123 (2012) 599–607 605

during 36 h incubation and reached 7.9 ± 1.1% (n = 3) and4.5 ± 1.4% (n = 3) of total cells, respectively. These population in-creases corresponded with methane production and decrease inacetate concentration. However, 30–40% of MAR-positive bacterialcells were unidentified at 12 and 36 h. Based on the results of MAR-FISH analysis during the 36 h incubation period, the maximumvalues of active MAR-positive glucose-, propionate-, and acetate-degrading microbial populations were estimated to be 15.2% (at3 h), 2.0% (at 12 h) and 12.4% (at 36 h) of total cells, respectively.

3.5. Glucose-, propionate-, and acetate-degrading populations andtheir specific degradation rate

Total DAPI count of the anaerobic digester sludge was4 � 1010 cells mg VSS–1 (standard deviation of triplicate measure-ments (SD); <5%). Based on the total DAPI count and the relativeabundance of each trophic group, the population size of eachtrophic group in the anaerobic digester sludge was calculated asfollows: 6.1 � 109 cells mg VSS–1; glucose-degrading population,0.8 � 109 cells mg VSS–1; propionate-degrading population, and5.0 � 109 cells mg VSS–1; acetate-degrading population. The deg-radation rate for glucose, propionate, and acetate of the anaerobicdigester sludge was determined to be 83� 10–2 lmol mg VSS–1 h–1

for glucose, 4.1 � 10–2 lmol mg VSS–1 h–1 for propionate, and2.4 � 10–2 lmol mg VSS–1 h–1 for acetate, respectively. Specificdegradation rate (i.e., degradation rate per cell) for glucose, propi-onate and acetate of each trophic group was determined by divid-ing each substrate-degradation rate of the anaerobic digestersludge by the population size of each trophic group. The specificdegradation rate for glucose, propionate, and acetate was13.6 � 10–11, 5.1 � 10–11, and 0.5 � 10–11 lmol cell–1 h–1, respec-tively. Fig. 4 represented a graphic view of these degradationrates, in which the specific degradation rate was on x-axis

Fig. 4. Comparison among glucose-, propionate-, and acetate-degradation of the anaeroboverall degradation rate of anaerobic digester sludge for each substrate, and the specific sof graphical bar represent the specific substrate degradation rate, the population sizesubstrate.

(numbers in parentheses); the population size was on y-axis(numbers in parentheses); and the potential overall degradationrate of anaerobic digester sludge for each substrate was displayedas enclosed area and numbers in boxes.

4. Discussion

4.1. Glucose-degrading methanogenic microbial community

Time course analysis of MAR-FISH with [U-14C]glucose andRNA-SIP analysis with [13C6]glucose successfully identified glu-cose-degrading methanogenic microbial community, whichmainly composed of glucose-degrading Olsenella and Propionibac-terium, propionate-degrading Smithella and Syntrophobacter, andacetate-degrading Methanosaeta and Synergistes group 4. It is notedthat this study directly demonstrated their involvement in glucosedegradation to methane in complex anaerobic digester sludgein situ.

The genera Olsenella and Propionibacterium in the phylumActinobacteria were frequently detected from the [13C6]glucoseRNA-SIP clone library in this study and other anaerobic digestersludge in the literature (Ariesyady et al., 2007a; Chouari et al.,2005; Godon et al., 1997; Rivière et al., 2009; Shin et al., 2010).Both the genera Olsenella and Propionibacterium have ability to de-grade glucose anaerobically (Dewhirst et al., 2001; Nakamura et al.,2003). Most strains of Propionibacterium could utilize glucose andproduce propionic and acetic acids as the fermentation end prod-ucts (Nakamura et al., 2003). This study clearly showed thatglucose was mainly converted to propionate and acetate byglucose-degrading Propionibacterium and Olsenella in the anaerobicdigester sludge, resulting in a significant accumulation of propio-nate and acetate. The RNA-SIP analysis revealed that other possibleglucose-degrading bacteria such as Chloroflexi and Bacteroidetes

ic digester sludge in terms of the population size of each trophic group, the potentialubstrate degradation rate (degradation rate per cell). The x-axis, y-axis, and the area, and the potential overall degradation rate of anaerobic digester sludge for each

606 T. Ito et al. / Bioresource Technology 123 (2012) 599–607

were present in the sludge. However, the MAR-FISH analysis con-firmed they were minor glucose degraders in the anaerobic diges-ter sludge.

Glucose was immediately converted to propionate and acetatewithin 3 h (Fig. 3a). There were only two major propionate-degrad-ing groups in the anaerobic digester sludge. Propionate-degradingSmithella was first more activated than Syntrophobacter (Fig. 3b andFig. S2) (60% of Smithella was MAR-positive, whereas 13% of Syn-trophobacter was MAR-positive at 1 h). Thereafter, Syntrophobacterbecame MAR positive as propionate concentration increased. Thisis probably because Smithella has higher affinity to propionate thanSyntrophobacter, and thus Smithella was more active than Syntrop-hobacter at lower propionate concentration (<0.5 mM), namely atthe beginning of the incubation (Ariesyady et al., 2007b).

On the other hand, acetate was accumulated only at first 12 h,and thereafter acetate concentration decreased to less than0.1 mM while propionate was still accumulated (Fig. 3). MAR-posi-tive Methanosaeta and Synergistes group 4 steadily increased withtime and became major MAR-positive populations at 36 h. Thus,acetate-degrading Methanosaeta and Synergistes group 4 must bewell associated with propionate-degrading Smithella and Syntrop-hobacter. In our previous study, propionate-degrading bacteriaand acetate-degrading microorganisms were phylogeneticallyand functionally diverse in the same culture, though acetate-degrading microbial community had less diversity than propio-nate-degrading community (Ariesyady et al., 2007b; Ito et al.,2011). Both populations were dynamically responding to the peri-odical changes in the concentrations of propionate and acetate inthe anaerobic digester operated in a fill and draw mode.

4.2. Propionate and acetate degradation as the rate-limiting steps

During 36 h incubation, only propionate and acetate were accu-mulated, indicating anaerobic degradations of propionate and ace-tate are slow and rate-limiting. It has been recognized thatpropionate is degraded by propionate-degrading bacteria associ-ated syntrophically with hydrogenotrophic methanogens if sulfateis absent. Methanogenic acetate degradation is carried out byeither the methanogens or some acetate-oxidizing bacteriacoupled to hydrogenotrophic methanogens (Hattori, 2008).Hydrogenotrophic methanogen, Methanoculleus, was detectedfrom the anaerobic digester sludge. Methanosarcina was also de-tected. Methanosarcina has been generally recognized as an aceto-trophic methanogen but many Methanosarcina spp. can also growby using hydrogen to reduce CO2 to CH4 (Boone et al., 1993). Inaddition, sulfate concentration and headspace H2 partial pressurewas under detection limit in this study. Thus, syntrophic propio-nate and acetate degradations are thermodynamically favorablein this experimental setup, but yield limited amounts of energy(Hattori, 2008). Syntrophs and methanogens must share this en-ergy for their growth.

In this study, the propionate-degradation rate of the digestersludge was approximately one-twentieth of the glucose-degrada-tion rate (Fig. 4). This slow rate is probably due to the small popu-lation size of propionate-degrading bacteria, which correspondedto one eighth of glucose-degrading bacteria and to one sixth of ace-tate degrader. This is probably because the energy yield and propi-onate load to propionate-degraders were limited. Propionate wasthe sole substrate for propionate-degraders in the digester sludge,while the degradation of whole milk (the original anaerobic sludgewas cultured with whole milk) would provide a variety of organiccompounds, which can be utilized by the glucose-degrading bacte-ria. Therefore, the smallest population size of propionate-degrad-ers among three trophic groups is thought to be a necessaryconsequence.

The acetate-degradation rate of the digester sludge was approx-imately one-thirty-fifth of the glucose-degradation rate, whereasthe acetate-degrading population size was close to the glucose-degrading population (Fig. 4). The slow rate of acetate degradationwas probably because specific acetate degradation rate (acetatedegradation rate per cell) was significantly low, which was one-twenty-eighth of that for glucose and one-eleventh of that for pro-pionate, indicating that the acetate-turnover rate of the cells wasslow. Judging from the studies on propionate-degrading bacteriaand Methanosaeta in pure culture and defined co-culture (Booneet al., 1993; Liu et al., 1999), the doubling time of acetate degradersand propionate degraders was in the similar range of 2–4 days.However, in situ assimilation rate of acetate in the anaerobic diges-ter sludge seems to be significantly slower than that of propionate,which could be expected from the MAR-FISH analysis. In thisstudy, at least three times larger amount of radiolabeled substratewas required to visualize acetate degraders than propionatedegraders. The ratio of 14C-substrate to 12C-substrate (i.e., hot/cold)was 25% for acetate degraders and 8% for propionate degraders, inwhich the specific radioactivity of 14C-acetate and 14C-propionatewas 2.1 GBq mmol–1 and 2.3 GBq mmol–1, respectively. The incu-bation time for acetate degraders was also 2.5 times longer thanpropionate degraders to obtain similar number of silver grains pro-duced by MAR (i.e., 5 h for acetate and 2 h for propionate, respec-tively). Thus, the assimilation rate of acetate was about 8 timesslower than that of propionate (25/8 (%) � 5/2 (h) = 8). Althoughthe specific acetate degradation rate was very slow, it was ex-pected that acetate load was relatively large during the reactoroperation because acetate is the major and final intermediate ofsecondary fermentation processes. In addition, the SRT of the reac-tor (ca. 35 days) was long enough for retaining the population ofacetate degraders including Methanosaeta. Therefore, the popula-tion size of acetate degraders was comparable to glucosedegraders.

5. Conclusions

RNA-SIP analysis with [13C6]glucose followed by MAR-FISHwith [U-14C]glucose were conducted to directly identify and quan-tify three key microbial trophic populations: glucose-, propionate-,and acetate-degrading populations, in anaerobic digester sludge.The digester sludge was mainly composed of glucose-degradingPropionibacterium and Olsenella, propionate-degrading Smithellaand Syntrophobacter, and acetate-degrading Methanosaeta and Syn-ergistes group 4. Furthermore, the population size of propionatedegraders was the smallest among three trophic groups, and thespecific degradation rate of propionate was also low. On the otherhand, the specific degradation rate of acetate-degrading populationwas low even though their population size was comparable to glu-cose degraders.

Acknowledgements

We gratefully thank the Central Institute of Isotope Science,Hokkaido University, for providing the facilities for the isotopeexperiments. This study was supported by NEDO and JSPS.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2012.07.108.

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