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JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2006, The Society for Biotechnology, Japan

Vol. 101, No. 3, 271–273. 2006

DOI: 10.1263/jbb.101.271

Microbial Community in Methanogenic Packed-Bed ReactorSuccessfully Operating at Short Hydraulic Retention Time

Kengo Sasaki,1 Shin Haruta,1* Masahiro Tatara,2 Akira Yamazawa,2

Yoshiyuki Ueno,2 Masaharu Ishii,1 and Yasuo Igarashi1

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan1 and Kajima Technical Research Institute,

2-19-1 Tobitakyu, Chofu-shi, Tokyo 182-0036, Japan2

Received 24 October 2005/Accepted 12 December 2005

The microbial community in a thermophilic anaerobic packed-bed reactor, which had been suc-cessfully operated to convert acetic and butyric acids to methane at a short hydraulic retentiontime (from 24 h to 1.9 h), was investigated. Archaea closely related to known methanogens weredetected by 16S rRNA gene analyses of the effluents, together with diverse types of unidentifiedbacteria.

[Key words: packed-bed reactor, short-chain fatty acid, methane fermentation]

Tatara et al. operated a thermophilic (55°C) down-flowanaerobic packed-bed reactor (TDAPR) (TDAPR1 in Ref. 1)on an artificial medium containing acetic and butyric acidsas major carbon sources (1). Briefly, the anaerobic reactorpacked with unwoven carbon fiber textiles was suppliedwith medium (pH 5.0) containing KH

2PO

4, 0.91; Na

2HPO

4⋅

12H2O, 2.39; NH

4Cl, 0.50; MgCl

2⋅6H

2O, 0.18; yeast ex-

tract, 2.00; acetic acid, 1.40; butyric acid, 3.90 (g/l) and sup-plementary minerals and vitamins. The hydraulic retentiontime (HRT) was gradually shortened from 20 d by increas-ing the dilution rate and, finally, stable methane productionat 1.9-h HRT was achieved, where the substrates were sup-plied at an organic loading rate (OLR) of 126 kg-COD/m3/d.To date, this operation at a short HRT, that is, high dilutionrate (>0.5 h–1) and high OLR, has been reported to show thehighest performance among continuous reactors (1). In thisstudy, we carried out clone library analysis with 16S rRNAgene sequences to characterize the microbial composition ineffluents from the TDAPR1 (1). Reactor conditions on daysinvestigated are summarized in Table 1. The sample taken at24-h HRT was representative of the efficient operation atlow acetic and butyric acid concentrations and neutral pH.The second sample taken at 1.9-h HRT was characterized bythe maximum allowable OLR. An additional sample takenat 1.4-h HRT, where no gas was produced due to overload-ing, was also examined.

Effluents at each HRT were collected after the operationhad been carried out for a period at least 4-fold longer timethan the HRT. Total DNA was extracted and purified as re-ported previously (2). PCR was performed to amplify 16SrRNA gene as described previously (2) with primer sets 27f(Escherichia coli positions 8–27) plus 1492r (E. coli posi-tions 1492–1510) for Bacteria (3), or Arch21F (E. coli posi-

tions 8–25) (4) plus Arch915 (E. coli positions 915–934) (5)for Archaea. For the archaeal sample at 24-h HRT, addi-tional PCR with primers Arch21F and Arch958R (4) wasconducted prior to PCR with Arch21F and Arch915. Ampli-fied fragments were cloned into E. coli JM109 using thepGEM-T Easy vector system (Promega, Tokyo). To defineoperational taxonomic units (OTUs), each clone was ana-lyzed by PCR-denaturing gradient gel electrophoresis withthe following primers: B342If (E. coli positions 342–358)plus U806Ir-GC (E. coli positions 788–806) for Bacteria,or A348If (E. coli positions 348–365) plus U806Ir-GC forArchaea (2, 6). A GC clamp (7) was attached to U806Ir atthe 5′-terminus. Sequencing was conducted using an ABI 377DNA sequencer after reaction using a BigDye kit (AppliedBiosystems). The sequence similarity of two to three cloneswith the same mobility was also checked. Nucleotide se-quences reported here were deposited in the GSDB, DDBJ,EMBL and NCBI under accession nos. AB233994–AB234022.

At 24-h HRT, OTU A-4 related to the aceticlastic metha-nogen Methanosarcina thermophila (M59140, 98.9% simi-larity) was frequently detected (Table 2). In addition, threearchaeal OTUs (A-1, A-2, and A-3) affiliated with the ge-nus Methanoculleus were detected: A-1, Methanoculleuschicugoensis (AB038795, 97.8% similarity); A-2, Methano-culleus bourgensis (AB065298, 99.8% similarity); and A-3,

* Corresponding author. e-mail: aharuta@mail.ecc.u-tokyo.ac.jpphone: +81-(0)3-5841-5144 fax: +81-(0)3-5841-5272

TABLE 1. Reactor conditions at sampling points (1)

Operation(d)

HRT(h)

OLRa

(kg/m3/d)

Biogasproduction

(l/d)pH

Acetate(mM)

Butyrate(mM)

139 24.0 10 8 7.0 0.6 0.0197 1.9 126 80 6.3 8.0 10.8202 1.4 169 0 4.8 28.5 44.3

a Organic loading rate is represented as COD determined by thedichromate method.

SASAKI ET AL. J. BIOSCI. BIOENG.,272

Methanoculleus thermophilicus (AB065297, 100% similar-ity). These hydrogenotrophic methanogens were consideredto take hydrogen from syntrophic degraders (8).

At 1.9-h HRT, OTU A-4 was still detected (Table 2). M.thermophila has been shown to have a generation time, withacetate as the substrate at 50 °C, close to 12 h (9). The abilityof Methanosarcina cells to aggregate (10) might explaintheir existence in the reactor. Sawayama et al. detectedMethanosarcina sp. on carbon felt in a mesophilic packed-bed system (11). On the other hand, OTUs A-1, A-2 and A-3were replaced by OTU A-5, which is closely related toMethanothermobacter thermautotrophicus (AE000930,99.8% similarity). This change in population of hydrogeno-trophic methanogens may be caused by the increase in ace-tate and/or butyrate concentrations (Table 1) (12, 13). Allclones detected at 1.4-h HRT were related to M. therm-autotrophicus, implying that the packed-bed system influ-enced the existence of OTU A-5.

The bacterial species detected at 24-h HRT were differentfrom those detected at 1.9-h HRT (Table 2). The bacterialcommunity at 24-h HRT was characterized by the predomi-nance of OTU B-3 (Table 2). B-3 showed low sequencesimilarities to known bacteria (<93%) and was one of thecharacteristic bacteria in this reactor. At 1.9-h HRT, the fre-quently observed OTUs, namely, B-7, B-9, B-11, and B-13were not closely related to reference organisms (Fig. 1).OTU B-7 seemed to be related to sulfate-reducing bacteria(SRB); however, this OTU was placed in a different clusterfrom the one for known SRB. OTU B-9 was not related toany known phyla and was classified in the cluster composedof uncultured bacteria, such as clone R5p16 (AF482444) ob-tained from granular sludge. OTU B-13 possibly belongedto the genus Clostridium, but the relative bacteria have notyet been isolated. No sequence related to OTU B-11 (>90%similarity) was found in the database indicating the existenceof a unique bacterium in this reactor. In addition, we de-tected OTUs B-14 and B-15, which were related to the ace-tate- or butyrate-oxidizing syntroph, Clostridium ultunense(14) or Syntrophomonas sp. TB6 (15), respectively (Fig. 1).However, their sequences also exhibited low similarities(<94%) to the 16S rRNA of these known species. Thus,many specific and unidentified organisms existed in the re-actor, which was supplied with selected substrates.

We compared the HRT with the generation time of each

bacterium in the literature. Rapid growth with a generationtime of <1 h under anaerobic conditions has been reportedfor Thermosyntropha lipolytica and Marinitoga piezophila(16, 17). However, if the bacteria detected at 1.9-h HRTshow sufficiently fast growth, they should also predominateat 24-h HRT. Because this was not the case, it is possible

TABLE 2. Distribution of 16S rRNA gene clones among samples tested

OTUHRT (h)

OTUHRT (h)

OTUHRT (h)

24 1.9 1.4 24 1.9 1.4 24 1.9 1.4

Archaeal clones B-4 2 – – B-15 – 1 –A-1 4 – – B-5 1 – 4 B-16 – 2 –A-2 2 – – B-6 1 – – B-17 – 1 –A-3 1 – – B-7 3 7 – B-18 – 1 –A-4 7 11 – B-8 2 5 1 B-19 – 1 –A-5 – 34 30 B-9 – 5 – B-20 – 1 –

B-10 – 3 – B-21 – 1 –Bacterial clones B-11 – 7 1 B-22 – 1 –

B-1 1 – – B-12 – 1 – B-23 – – 41B-2 1 – – B-13 – 7 – B-24 – – 1B-3 7 2 – B-14 – 2 –

–, Not detected.

FIG. 1. Phylogenetic tree of bacterial clones. Sequences werealigned using the CLUSTAL X program V1.8. A phylogenetic treewas constructed by the neighbor-joining method. Numbers at nodesare bootstrap values (1000 replicates). DDBJ/EMBL/GenBank acces-sion numbers for reference strains are shown in parentheses. Bootstrapvalues less than 60% are not shown.

NOTESVOL. 101, 2006 273

that microorganisms grew on the packed-bed and were re-leased to the effluent. For example, OTU B-8, which ishighly related to Anaerobaculum mobile (AJ243189, 99.4%similarity) which has a generation time of 3 h (18), wasfound in all samples (Table 2). Overloading at 1.4-h HRTresulted in the reduction in pH and the disappearance ofmost of the OTUs detected at 1.9-h HRT (Table 2). The pre-dominance of OTU B-23, which is related to Thermoan-aerobacterium thermosaccharolyticum (AF247003, 98.2%similarity), might have been influenced by the packed-bedsystem.

In this study, the archaea detected were closely related tomethanogens which have been well characterized. Many bac-teria found were hitherto unknown species. Because aceticand butyric acids were applied, the metabolism of these mi-crobes attracted much attention.

This project was supported by the New Energy and IndustrialTechnology Development Organization of Japan.

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