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Methanogenic community and performance of fixed- and fluidized-bed reactors with reticular polyurethane foam with different pore sizes
Materials Science and Engineering C 24 (2004) 803 – 813 www.elsevier.com/locate/msec
Methanogenic community and performance of fixed- and fluidized-bed reactors with reticular polyurethane foam with different pore sizes Yingnan Yang, Chika Tada, Kenichiro Tsukahara, Shigeki Sawayama* Biomass Group, Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8569, Japan Received 10 June 2004; accepted 11 August 2004 Available online 30 September 2004
Abstract This paper reports the effects of the pore size of bed material and bed type on the methanogenic performance and community in fixed and fluidized bed reactors supplied with acetic acid as the sole organic substrate. Fixed-bed reactors with polyurethane foam had a greater biomass retention capacity and better reactor performance than fluidized-bed reactors. A polyurethane pore size of 20 cells/25 mm was better for both beds than a pore size of 13 cells/25 mm. These results indicated that the adjusted pore size is also important for the efficient digestion. The best performance was obtained from the fixed-bed reactor with the 20 cells/25 mm polyurethane foam. Scanning electron microphotographs indicated that the immobilized microbes were primarily composed of coccal, diplococcal-shaped Methanosarcina-like cells, short-rods of Methanosaeta-like cells and long rods of Methanobacterium. Methanosarcina-like cells prevailed in the fixed-bed reactor. The results of 16S rRNA phylogenetic analysis indicated that the major immobilized methanogens belonged to the genus Methanosarcina. The results of real-time polymerase chain reaction (PCR) analysis indicated that the cell density of immobilized methanogens in the fixed-bed reactor was higher than that in the fluidized-bed reactor. The better biogas production by the fixed-bed reactor could be explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. D 2004 Elsevier B.V. All rights reserved. Keywords: Anaerobic digestion; Pore size; Bed type; Immobilization; Methanogens; SEM; 16S rRNA; Real-time PCR
1. Introduction Anaerobic digestion is a biological treatment process that has many advantages over more conventional aerobic processes, including low levels of excess sludge production, low space requirements and production of biogas [1]. The social requirement for low-cost and high-efficiency treatment of organic waste and wastewater has prompted studies of advanced technologies for anaerobic digestion, including the development of novel reactor designs and operating conditions. One of the successful methods in anaerobic digestion is immobilization on inert support media. Specific surface
* Corresponding author. Tel.: +81 29 861 8184; fax: +81 29 861 8184. E-mail address: [email protected] (S. Sawayama). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.022
area, porosity, surface roughness, pore size, and orientation of the packing material were found to play an important role in anaerobic filter reactor performance [2,3]. Picanco et al. [4] reported that the efficiency of removing organic matter in fixed-bed reactors is directly related to the characteristics of the support material used for immobilization of anaerobes. It is widely accepted that organic support material has a higher affinity than inorganic material [5]. Reticular polyurethane foam has a high specific surface area which can reach 2400 m2/m3, and a porosity of 97%. It appeared to be an excellent colonization matrix for an anaerobic filter reactor [6]. Pore size was one of the most important parameter for microbiological and engineering requirements in high-efficiency beds [7]. Many kinds of bedding model have been considered for degrading a variety of organic wastes in anaerobic digestion reactors. A simple, low-cost and high-efficiency bedding method is necessary for
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2. Materials and methods 2.1. Bed materials In this study, two kinds of reticular polyurethane foam (Type CFH) (INOAC, Nagoya, Japan) with the similar shape and same characteristics but different pore size were used for microbial immobilization. Fig. 1 shows the porous structure of the bed materials. Fig. 1A shows the bigger pore size (PS-13, 13F3 cells/25 mm) and Fig. 1B shows the smaller pore size (PS-20, 20F4 cells/25 mm) of reticular polyurethane foam structure. 2.2. Reactor operation
Fig. 1. SEM photos of the bed materials. (A) PS-13 reticular polyurethane foam; (B) PS-20 reticular polyurethane form. Scale bars are indicated on photographs. Magnification: 25.
practical use. Two kinds of bedding type (fixed and fluidized) were tested in the present study. Aldrich reported that the electron microscope can be used to obtain detailed knowledge of events in digesters containing mixed populations of anaerobes [8]. Recently, several studies using 16S rRNA gene sequence analyses of microbial communities of anaerobic digestion have been reported [9–11]. They have unprecedentedly provided detailed insights into the microbial communities in these poorly characterized ecosystems and have recovered a number of unknown 16S rRNA gene sequences. Nevertheless, most molecular studies of anaerobic digestion system have focused on free-living microbes, except upflow anaerobic sludge blanket (UASB) granules. The determination of bacterial and archaeal loads by real-time PCR using a broad-range TaqMan probe and primer set was reported [12–14]. For an immobilized anaerobic digestion system, it is worthwhile studying immobilized microbes for a better understanding of the immobilization systems. In the present study, we used an integrated approach combining chemical analysis to monitor reactor performance, scanning electron microscopy (SEM) to observe the main immobilized cellular morphologies, 16S rRNA gene sequence analysis and real-time PCR analysis to compare communities of attached microbes. An aim of this study was to investigate the influence of the bed material’s pore size and the bedding method on methanogenic characteristics and immobilized methanogens.
Four reactors (300 ml) were set up. Each contained reticular polyurethane foam with one of two pore sizes but with the same working volume (about 20%, v/v) as either a fluidized-bed reactor or a fixed-bed reactor (Fig. 2). Another reactor without bed material was used as a control. The reactors were fed with synthetic medium composed of CH3COONa (5 g/l), NH4Cl (200 mg/l), KH2PO4 (16 mg/l), and a mineral solution (200 ml/l). This solution contained FeSO 4 d 7H 2 O (1.11 g/l), MgSO 4 d 7H 2 O (24.65 g/l), CaCl2d 2H2O (2.94 g/l), NaCl (23.4 g/l), MnSO4d 4H2O (111 mg/l), ZnSO4d 7H2O (28.8 mg/l), Co(NO3)2d 6H2O (29.2 mg/l), CuSO4d 5H2O (25.2 mg/l), Na2MoO4d 2H2O (24.2 mg/l), and H3BO3 (31.0 mg/l). NaOH (5 N) was added to adjust the initial pH of the synthetic medium to 7.2. The reactors were inoculated with 15% (v/v) methanogenic sludge from a communal wastewater treatment plant (Ibaraki Prefecture, Japan), and were acclimatized at 35 8C by feeding with synthetic medium containing 5.0 g/l total organic carbon (TOC) for 1 month. The reactors were placed in an incubator at 35 8C with shaking at 120 rpm to keep the bed materials in an uniform condition. The bed reactors were operated in semi-continuous mode at a hydraulic retention time of 14 days. The active volume in the reactor was 150 ml. The produced biogas was collected in a syringe.
Fig. 2. Schematic diagram of reactors with (A) fluidized-bed reactor; (B) fixed-bed reactor.
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2.3. Chemical analysis A portion of the reactor contents was sampled once a week. The equivalent volume of medium was anaerobically added to the reactor every time after the sample collection. The liquid sample was centrifuged at 10,000 rpm for 10 min to allow precipitation of the microbes. The supernatant was used to measure the dissolved organic carbon (DOC) with a TOC analyzer (TOC-5000A, Shimadzu, Kyoto, Japan). Biogas production and pH in the reactor were measured daily. The composition of the biogas was determined by using a gas chromatograph (GC-8A, Shimadzu) with a thermal conductivity detector equipped with a steel column packed with Porapak Q (Shinwakakou, Kyoto, Japan) at 90 8C. The immobilized biomass was indirectly quantified through determination of total volatile solids expressed as unit of support, since the supports presented the same dimensions [4]. 2.4. Scanning electron microscope The bed materials and the main cellular morphologies present in the biofilms were observed through a scanning electron microscope (DS-720, Topcon, Tokyo, Japan). At the end of the experiment, the attached bed materials were taken out and the cells were washed with buffer solution (pH 7.0). Then the samples were fixed with 10% (v/v) glutaraldehyde solution overnight. Fixed samples were desalted with ultrapure water, and refrigerated under 20 8C for 3 h. They were then desiccated in a freeze dryer (FD5, RIKAKIKAI, Tokyo, Japan) for 1 day. Before microscopic examination, samples were coated with gold powder. Samples were prepared for SEM according to Yase et al. [15].
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Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) and an automated sequence analyzer (model 377; Perkin-Elmer Applied Biosystems). The sequences of 20 DNA clones measuring 417 bp were determined, and the sequence data were aligned with the CLUSTALW package for phylogenetic analysis [19]. A phylogenetic tree was constructed by the neighbor-joining method using the MEGA V2.1 package [20]. 2.6. Quantification of immobilized microbes by real-time PCR DNA was extracted from the original sludge (0.5 ml), and microbes immobilized on the polyurethane foam (PS-20, 20F4 cells/25 mm). Real-time PCR was conducted with an ABI7000 sequence detection system (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems). Quantitative measurement by real-time PCR was conducted in quadruplicate. The amplifying primer set of SD-Bact-0348-S-a-17 (5V-AGGCAGCAGTDRGGAAT-3V) and S-D-Bact-0786-A-a-20 (5V-GGACTACYVGGGTATCTAAT-3V), and the double dye probe of S-D-Bact-0515-S-a25 (5V-TGCCAGCAGCCGCGGTAATACRDAG-3V) were used for measuring the 16S rRNA copy number of bacteria [18]. The amplifying primer set of S-P-MArch-0348-S-a-17 and S-D-Arch-0786-A-a-20 and the double dye probe of S-PMArch-0515-S-a-25 (5-TGCCAGCMGCCGCGGTAAYACCGGC-3) were used for the methanogens [14]. The amplifying primer set of S-F-Msaet-0387-S-a-21 (5VGATAAGGGRAYCTCGAGTGCY-3V) and S-F-Msaet0573-A-a-17 (5V-GGCCGRCTACAGACCCT-3V) and the double dye probe of S-F-Msaet-0540-A-a-31 (5V-AGACC-
2.5. Phylogenetic analysis of 16S rRNA gene Samples from two different places in the bed materials were collected from the PS-20 fluidized and fixed-bed reactors after 42 days. All the samples were collected as duplicates or triplicates. The DNA was extracted by using a Fast DNA SPIN kit for Soil (Qbiogene, Carlsbad, CA, USA) and a Fast Prep FP120 instrument (Qbiogene) according to the manufacturer’s instructions. The archaeal 16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) with the primer set S-P-MArch-0348S-a-17 (5V-GYGCAGCAGGCGCGAAA-3V) and S-DArch-0786-A-a-20 (5V-GGACTACVSGGGTATCTAAT-3V) [14,16–18]. The PCR reaction was performed with a program which cycling consisted of 15 cycles each of 1 min at 95 8C, 1 min at 50 8C and 2 min at 72 8C. The PCR product was purified in a Microspin S-400 HR column (Amersham Biosciences, Piscataway, NJ, USA) and cloned with a TA Cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cloned DNA was sequenced using a dRhodamin Dye
Fig. 3. Change in cumulative methane production from the reactors. Bed materials with different pore sizes but the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed or fluidized beds. The reactors were operated in semi-continuous mode at a hydraulic retention time of 4 days. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
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Fig. 4. Methane yield from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed types.
CAATAAHARCGGTTACCACTCGRGCC-3V) were used for the Methanosaeta sp. [14]. The real-time PCR amplification followed a three-step PCR (40 cycles) with 20 s denaturation (95 8C), 20 s annealing (55 8C), and 120 s elongation (95 8C). The annealing temperature for the bacteria only was 50 8C. The R2 range of the standard curves obtained by the real-time PCR measurements was 0.962– 0.989. The standard DNA for real-time PCR of bacteria was prepared by PCR with a 2-bp-longer PCR primer set of S-DBact-0346-S-a-19 (5V-GGAGGCAGCAGTDRGGAAT-3V)
Fig. 6. Change in DOC removal efficiency (%) from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed type. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
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and S-D-Bact-0786-A-a-22 (5V-GTGGACTACYVGGGTATCTAAT-3V). The standard DNAs for the methanogens and Methanosaeta sp. were prepared from Methanosarcina barkeri (DSM 800) and Methanosaeta thermophila strain PT (DSM 6194), respectively, by PCR. The average 16S ribosomal RNA gene copy numbers of the bacteria (4 copies/cell) and methanogens (2.5 copies/cell) were referred to the Ribosomal RNA Operon Copy Number Database for the conversion of copy number to cell number [21].
3. Results and discussion 3.1. Reactor performances Fig. 3 shows the change of cumulative methane yield in the reactors containing fixed and fluidized beds with PS-20 and PS-13, and without bed during the operational days. The cumulative methane yield decreased in the following
Fig. 5. Change in methane concentration from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed type. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
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Fig. 7. Quantity of the immobilized biomass in each bed reactor at the end of the experiment.
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Fig. 8. SEM photos of microbes immobilized on the polyurethane foam. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed types. (A) PS-13 fluidized-bed reactor; (B) PS-13 fixed-bed reactor; (C) PS-20 fluidized-bed reactor; (D) PS-20 fixed-bed reactor. Scale bars are indicated on photographs. Magnification: 1000.
order: PS-20 fixed-bed reactorNPS-13 fixed-bed reactorNPS-20 fluidized-bed reactorNPS-13 fluidized-bed reactorNno-bed reactor. Fig. 4 shows the average methane yield in five different anaerobic digesters. The PS-20 fixed reactor presented the highest methane yield about 0.59 l CH4/gDOCadded. The methane yields showed the same order as above. Methane yield during the anaerobic fixed-bed system seems to be a representative parameter for process monitor-
ing [22]. These results indicated that the PS-20 fixed-bed reactor gives better digestion performance than the others. The methane concentration in each reactor was shown in Fig. 5. A similar course was observed in all reactors. The lowest methane concentration was measured in the no-bed reactor. A change in DOC removal efficiency was shown in Fig. 6. The DOC removal ratio in all four bed reactors was 92%.
Fig. 9. SEM photos of the major cellular morphologies immobilized on the polyurethane form. (A) PS-13 fluidized-bed reactor; (B) PS-13 fixed-bed reactor; (C) PS-20 fluidized-bed reactor; (D) PS-20 fixed-bed reactor. Scale bars are indicated on the photographs. Magnification: (A, B, C, 10 000; D, 5000).
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Removal was fastest in the PS-20 fixed-bed reactor, where 90% of DOC was removed within 14 days; it took 27–35 days in the PS-13 fixed-bed, PS-20 fluidized-bed and PS-13 fluidized-bed reactors. In the no-bed reactor, the DOC
removal ratio (55%) was relatively low. These results indicate that the fixed-bed reactor with the pore size of 20 cells/25 mm was the most efficient for the anaerobic digestion.
Fig. 10. 16S rRNA-based phylogenetic relationship between the clones (MSC01-MSC15) amplified from the original anaerobic digested sludge by PCR with the methanogen-specific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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3.2. Quantification and microscopic observation After 42 days of reactor operation, the bed materials were removed to measure the quantification of the attached biomass and for microscopic observation. The immobilized biomass quantification from each reactor was shown in Fig. 7. The fixed-bed reactors retained more biomass than the fluidized-bed reactors. The biomass retained in the fluidized-bed support corresponded to only 39% of that retained in the fixed-bed support in PS-13, and 54% in PS-20. Fig. 8 was the SEM photos of the microbes immobilized on the bed materials in each reactor. More biomass was immobi-
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lized in the fixed-bed reactors than in the fluidized-bed reactors. These results indicate that the bedding type plays an important role in the adhesion capacity. The higher quantity of immobilized biomass was due to the smaller pore size of the polyurethane foam. The morphologies of the major immobilized cells were also characterized (Fig. 9). These microphotographs revealed that the biofilm was primarily composed of coccal, diplococcal-shaped Methanosarcina-like cells, short rods of Methanosaeta-like cells and long rods of Methanobacterium-like cells [23,24]. Similar numbers of each were immobilized on the PS-13 and PS-20 fluidized
Fig. 11. 16S rRNA-based phylogenetic relationship between the clones (FL20C02-FL20C03) amplified from the PS-20 fluidized-bed by PCR with the methanogen-specific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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bed reactors (Fig. 9A,C). Although short-rods of Methanosaeta-like cells and long rods of Methanobacteriumlike cells were immobilized (Fig. 9B,D), a large quantity of coccal methanogens, closely resembling to Methanosarcina [25] prevailed in the better performing fixed-bed reactors, especially in PS-20. 3.3. Phylogenetic clone analyses The results of the 16S rRNA phylogenetic analysis of the original anaerobically digested sludge for methanogens
suggested that the major methanogens in the original sludge were Methanosaeta spp. (17/20 clones) and members of the order Methanomicrobiales (3/20 clones) (Fig. 10). This dominance of the Methanosaeta sp. in the anaerobically digested sludge of the large scale digester agrees with other reports [26,1]. Figs. 11 and 12 present the phylogenetic trees of the methanogens, showing the distribution of the major clones among methanogenic biodiversity in the PS-20 fluidized and fixed-bed reactors, respectively. The major methanogenic clones immobilized in the PS-20 fluidized-bed reactor
Fig. 12. 16S rRNA-based phylogenetic relationship between the clones (FI20C01-FI20C07) amplified from the PS-20 fixed-bed by PCR with the methanogenspecific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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were the Methanosarcina spp. (20/20 clones) (Fig. 11). The major methanogenic clones immobilized on the PS-20 fixed-bed reactor were the Methanosarcina spp. (19/20 clones) and members of the order Methanomicrobiales (1/ 20 clones) (Fig. 12). The SEM analyses also suggested that the coccal, diplococcal-shaped of Methanosarcina-like cells were immobilized on the bed materials (Figs. 9C,D). The dominant methanogenic clones immobilized on the bed were different from those in the original sludge. This could be caused by the change in microbial environment from free-living to immobilization and change in organic substrate. Most of the methanogenic sequence clones of immobilized cells were phylogenetically associated with the hydrogenotrophic Methanobacterium and the aceticlastic Methanosarcina and Methanosaeta. The reactors were supplied only with acetic acid as an organic substrate. Methanosarcina was the most abundant aceticlastic methanogens in both of the bed reactors (Figs. 11 and 12). To date, only two methanogenic genera, Methanosarcina and Methanosaeta, are known to be able to produce methane from acetate, and Methanosaeta spp. generally have lower competitiveness than Methanosarcina spp. at high acetate concentrations [27]. Further quantitative investigation of especially Methanosarcina spp. could be necessary in order to understand the immobilized methanogenic community. This work gave a preliminary description of the methanogenic community immobilized on the fluidized and fixed-bed materials in the anaerobic digesters by the 16S rRNA gene phylogenetic analyses. These results did not show a clear difference in diversity between the two bedding types. The major immobilized methanogens of both bedding types were highly similar to the methanogenic genus Methanosarcina. However, the reactor performance from the present study shows that the fixed-bed reactor had a greater biomass retention capacity and better reactor performance than the fluidized-bed reactor. 3.4. Quantitative change in methanogens The results of real-time PCR analysis indicated that the cell densities of the immobilized methanogens and bacteria increased relative to those of the free-living methanogens and bacteria, respectively, in the original anaerobically digested sludge (Table 1). The cell density of immobilized methanogens in the fixed-bed reactor was higher than that in the fluidized-bed reactor. The increase in biogas production was explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. The real-time PCR analysis enabled the quantification of the immobilized microbes on the bed materials in the anaerobic digester, allowing us to evaluate the effectiveness of the bed-type bioreactors. The results of the clone analysis of the original sludge suggest that the major methanogens were Methanosaeta
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Table 1 16S rRNA gene copy numbers of microbes immobilized on PS-20 polyurethane foam in fluidized- and fixed-bed reactors DNA source
Bacterial copy number (copies/cm3)
Methanogenic copy number (copies/cm3)
Methanosaeta copy number (copies/cm3)
Anaerobically digested sludge Immobilized microbes in the fluidizedbed reactor Immobilized microbes in the fixedbed reactor
(5.1F3.7a)108
(1.3F0.2)109
(6.8F0.8)108
(2.4F0.8)109
(2.2F0.2)109
Not detected
(6.1F0.5)108
(7.1F0.5)109
(5.6F2.5)105
The R 2 range of the standard curves obtained by the real-time PCR measurements was 0.962–0.989. a SD.
spp. and members of the order Methanomicrobiales, and that after immobilization, these were changed to Methanobacterium and Methanosarcina. The results of real-time PCR analysis also indicated that the dominant methanogen in the original anaerobically digested sludge was Methanosaeta and that the immobilized cell density of the Methanosaeta decreased from the free-living cell density of the original sludge, especially in the fluidized-bed reactor (Table 1). The results of real-time PCR analysis coincide with those of clone analyses (Figs. 11 and 12).
4. Conclusions From the performance of all reactors under the same operational conditions, we can conclude that the bedding method and pore sizes of the bed material influenced methane yield. The smaller pore size of PS-20 performed better than the larger pore size of PS-13 under the present experimental conditions. The fixed-bed system seems to be more efficient for immobilized methanogens, organic removal and methane yield than the fluidized-bed system. The fixed-bed reactor with a pore size of 20 cells/25 mm was the most efficient. Microscopic observations of immobilized microbes showed that the fixed-bed reactors immobilized more microorganisms than the fluidized-bed reactors. Distinct methanogens colonized the bed material. The morphologies observed by microscopic analyses indicated that the immobilized microbes were primarily composed of coccaland diplococcal-shaped Methanosarcina-like cells, shortrods of Methanosaeta and long-rods of Methanobacterium. Methanosarcina prevailed in the fixed-bed reactors. The results of 16S rRNA gene phylogenetic analyses indicated that the major immobilized methanogens were belonged to the genus Methanosarcina. The results of real-time PCR analysis indicated that the cell densities of
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the immobilized methanogens increased relative to those of the free-living methanogens in the original anaerobically digested sludge. The increase in biogas production was explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. Real-time PCR analysis enabled the quantification of the immobilized microbes on the bed material in the anaerobic digester, allowing us to evaluate the effectiveness of the bed-type bioreactors.
[5]
[6]
[7]
[8]
5. Accession numbers The sequences determined in the present study have been deposited in the DDBJ/EMBL/GenBank databanks under the accession numbers from AB112676 to AB112700. The organisms, together with their GenBank and EMBL accession numbers, whose 16S rRNA sequences were used for the phylogenic analysis were as follows: Methanobacterium bryantii, AF028688; M. congolense, AF233586; M. curvum, AF276958; M. formicicum, AF028689; M. palustre, AF093061; M. subterraneum, X99044; Methanobrevibacter arboriphilus , AB065294; Methanosphaera stadtmanae, M59139; Methanococcus voltae, M59290; Methanomicrobium mobile, M59142; Methanofollis tationis, AF095272; Methanospirillum hungatei, M60880; Methanosarcina acetivorans, M59137; M. barkeri, AB065295; M. frisius, M59138; M. mazei, AB065295; M. siciliae, U20153; M. thermophila, M59140; Methanococcoides burtonii, X65537; Methanolobus tindarius, M59135; Methanomethylovorans hollandica, AF120163; Methanosaeta concilii, M59146; Methanosaeta thermoacetophila, M59141; Sulfolobus acidocaldarius, D14053.
Acknowledgements
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This work was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
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Y. Yang et al. / Materials Science and Engineering C 24 (2004) 803–813 [25] E. Koorneef, A.J.L. Macario, J.T.C. Grotenhuis, E.C. Demacario, Methanogens revealed immunologically in granules from five upflow anaerobic sludge blanket (UASB) bioreactors grown on different substrates, FEMS Microbiol. Ecol. 73 (1990) 225 – 230. [26] L. Raskin, L.K. Poulsen, D.R. Noguera, B.E. Rittmann, D.A. Stahl, Quantification of methanogenic groups in anaerobic biological
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Methanogenic community and performance of fixed- and fluidized-bed reactors with reticular polyurethane foam with different pore sizes Yingnan Yang, Chika Tada, Kenichiro Tsukahara, Shigeki Sawayama* Biomass Group, Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8569, Japan Received 10 June 2004; accepted 11 August 2004 Available online 30 September 2004
Abstract This paper reports the effects of the pore size of bed material and bed type on the methanogenic performance and community in fixed and fluidized bed reactors supplied with acetic acid as the sole organic substrate. Fixed-bed reactors with polyurethane foam had a greater biomass retention capacity and better reactor performance than fluidized-bed reactors. A polyurethane pore size of 20 cells/25 mm was better for both beds than a pore size of 13 cells/25 mm. These results indicated that the adjusted pore size is also important for the efficient digestion. The best performance was obtained from the fixed-bed reactor with the 20 cells/25 mm polyurethane foam. Scanning electron microphotographs indicated that the immobilized microbes were primarily composed of coccal, diplococcal-shaped Methanosarcina-like cells, short-rods of Methanosaeta-like cells and long rods of Methanobacterium. Methanosarcina-like cells prevailed in the fixed-bed reactor. The results of 16S rRNA phylogenetic analysis indicated that the major immobilized methanogens belonged to the genus Methanosarcina. The results of real-time polymerase chain reaction (PCR) analysis indicated that the cell density of immobilized methanogens in the fixed-bed reactor was higher than that in the fluidized-bed reactor. The better biogas production by the fixed-bed reactor could be explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. D 2004 Elsevier B.V. All rights reserved. Keywords: Anaerobic digestion; Pore size; Bed type; Immobilization; Methanogens; SEM; 16S rRNA; Real-time PCR
1. Introduction Anaerobic digestion is a biological treatment process that has many advantages over more conventional aerobic processes, including low levels of excess sludge production, low space requirements and production of biogas [1]. The social requirement for low-cost and high-efficiency treatment of organic waste and wastewater has prompted studies of advanced technologies for anaerobic digestion, including the development of novel reactor designs and operating conditions. One of the successful methods in anaerobic digestion is immobilization on inert support media. Specific surface
* Corresponding author. Tel.: +81 29 861 8184; fax: +81 29 861 8184. E-mail address: [email protected] (S. Sawayama). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.022
area, porosity, surface roughness, pore size, and orientation of the packing material were found to play an important role in anaerobic filter reactor performance [2,3]. Picanco et al. [4] reported that the efficiency of removing organic matter in fixed-bed reactors is directly related to the characteristics of the support material used for immobilization of anaerobes. It is widely accepted that organic support material has a higher affinity than inorganic material [5]. Reticular polyurethane foam has a high specific surface area which can reach 2400 m2/m3, and a porosity of 97%. It appeared to be an excellent colonization matrix for an anaerobic filter reactor [6]. Pore size was one of the most important parameter for microbiological and engineering requirements in high-efficiency beds [7]. Many kinds of bedding model have been considered for degrading a variety of organic wastes in anaerobic digestion reactors. A simple, low-cost and high-efficiency bedding method is necessary for
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2. Materials and methods 2.1. Bed materials In this study, two kinds of reticular polyurethane foam (Type CFH) (INOAC, Nagoya, Japan) with the similar shape and same characteristics but different pore size were used for microbial immobilization. Fig. 1 shows the porous structure of the bed materials. Fig. 1A shows the bigger pore size (PS-13, 13F3 cells/25 mm) and Fig. 1B shows the smaller pore size (PS-20, 20F4 cells/25 mm) of reticular polyurethane foam structure. 2.2. Reactor operation
Fig. 1. SEM photos of the bed materials. (A) PS-13 reticular polyurethane foam; (B) PS-20 reticular polyurethane form. Scale bars are indicated on photographs. Magnification: 25.
practical use. Two kinds of bedding type (fixed and fluidized) were tested in the present study. Aldrich reported that the electron microscope can be used to obtain detailed knowledge of events in digesters containing mixed populations of anaerobes [8]. Recently, several studies using 16S rRNA gene sequence analyses of microbial communities of anaerobic digestion have been reported [9–11]. They have unprecedentedly provided detailed insights into the microbial communities in these poorly characterized ecosystems and have recovered a number of unknown 16S rRNA gene sequences. Nevertheless, most molecular studies of anaerobic digestion system have focused on free-living microbes, except upflow anaerobic sludge blanket (UASB) granules. The determination of bacterial and archaeal loads by real-time PCR using a broad-range TaqMan probe and primer set was reported [12–14]. For an immobilized anaerobic digestion system, it is worthwhile studying immobilized microbes for a better understanding of the immobilization systems. In the present study, we used an integrated approach combining chemical analysis to monitor reactor performance, scanning electron microscopy (SEM) to observe the main immobilized cellular morphologies, 16S rRNA gene sequence analysis and real-time PCR analysis to compare communities of attached microbes. An aim of this study was to investigate the influence of the bed material’s pore size and the bedding method on methanogenic characteristics and immobilized methanogens.
Four reactors (300 ml) were set up. Each contained reticular polyurethane foam with one of two pore sizes but with the same working volume (about 20%, v/v) as either a fluidized-bed reactor or a fixed-bed reactor (Fig. 2). Another reactor without bed material was used as a control. The reactors were fed with synthetic medium composed of CH3COONa (5 g/l), NH4Cl (200 mg/l), KH2PO4 (16 mg/l), and a mineral solution (200 ml/l). This solution contained FeSO 4 d 7H 2 O (1.11 g/l), MgSO 4 d 7H 2 O (24.65 g/l), CaCl2d 2H2O (2.94 g/l), NaCl (23.4 g/l), MnSO4d 4H2O (111 mg/l), ZnSO4d 7H2O (28.8 mg/l), Co(NO3)2d 6H2O (29.2 mg/l), CuSO4d 5H2O (25.2 mg/l), Na2MoO4d 2H2O (24.2 mg/l), and H3BO3 (31.0 mg/l). NaOH (5 N) was added to adjust the initial pH of the synthetic medium to 7.2. The reactors were inoculated with 15% (v/v) methanogenic sludge from a communal wastewater treatment plant (Ibaraki Prefecture, Japan), and were acclimatized at 35 8C by feeding with synthetic medium containing 5.0 g/l total organic carbon (TOC) for 1 month. The reactors were placed in an incubator at 35 8C with shaking at 120 rpm to keep the bed materials in an uniform condition. The bed reactors were operated in semi-continuous mode at a hydraulic retention time of 14 days. The active volume in the reactor was 150 ml. The produced biogas was collected in a syringe.
Fig. 2. Schematic diagram of reactors with (A) fluidized-bed reactor; (B) fixed-bed reactor.
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2.3. Chemical analysis A portion of the reactor contents was sampled once a week. The equivalent volume of medium was anaerobically added to the reactor every time after the sample collection. The liquid sample was centrifuged at 10,000 rpm for 10 min to allow precipitation of the microbes. The supernatant was used to measure the dissolved organic carbon (DOC) with a TOC analyzer (TOC-5000A, Shimadzu, Kyoto, Japan). Biogas production and pH in the reactor were measured daily. The composition of the biogas was determined by using a gas chromatograph (GC-8A, Shimadzu) with a thermal conductivity detector equipped with a steel column packed with Porapak Q (Shinwakakou, Kyoto, Japan) at 90 8C. The immobilized biomass was indirectly quantified through determination of total volatile solids expressed as unit of support, since the supports presented the same dimensions [4]. 2.4. Scanning electron microscope The bed materials and the main cellular morphologies present in the biofilms were observed through a scanning electron microscope (DS-720, Topcon, Tokyo, Japan). At the end of the experiment, the attached bed materials were taken out and the cells were washed with buffer solution (pH 7.0). Then the samples were fixed with 10% (v/v) glutaraldehyde solution overnight. Fixed samples were desalted with ultrapure water, and refrigerated under 20 8C for 3 h. They were then desiccated in a freeze dryer (FD5, RIKAKIKAI, Tokyo, Japan) for 1 day. Before microscopic examination, samples were coated with gold powder. Samples were prepared for SEM according to Yase et al. [15].
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Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) and an automated sequence analyzer (model 377; Perkin-Elmer Applied Biosystems). The sequences of 20 DNA clones measuring 417 bp were determined, and the sequence data were aligned with the CLUSTALW package for phylogenetic analysis [19]. A phylogenetic tree was constructed by the neighbor-joining method using the MEGA V2.1 package [20]. 2.6. Quantification of immobilized microbes by real-time PCR DNA was extracted from the original sludge (0.5 ml), and microbes immobilized on the polyurethane foam (PS-20, 20F4 cells/25 mm). Real-time PCR was conducted with an ABI7000 sequence detection system (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems). Quantitative measurement by real-time PCR was conducted in quadruplicate. The amplifying primer set of SD-Bact-0348-S-a-17 (5V-AGGCAGCAGTDRGGAAT-3V) and S-D-Bact-0786-A-a-20 (5V-GGACTACYVGGGTATCTAAT-3V), and the double dye probe of S-D-Bact-0515-S-a25 (5V-TGCCAGCAGCCGCGGTAATACRDAG-3V) were used for measuring the 16S rRNA copy number of bacteria [18]. The amplifying primer set of S-P-MArch-0348-S-a-17 and S-D-Arch-0786-A-a-20 and the double dye probe of S-PMArch-0515-S-a-25 (5-TGCCAGCMGCCGCGGTAAYACCGGC-3) were used for the methanogens [14]. The amplifying primer set of S-F-Msaet-0387-S-a-21 (5VGATAAGGGRAYCTCGAGTGCY-3V) and S-F-Msaet0573-A-a-17 (5V-GGCCGRCTACAGACCCT-3V) and the double dye probe of S-F-Msaet-0540-A-a-31 (5V-AGACC-
2.5. Phylogenetic analysis of 16S rRNA gene Samples from two different places in the bed materials were collected from the PS-20 fluidized and fixed-bed reactors after 42 days. All the samples were collected as duplicates or triplicates. The DNA was extracted by using a Fast DNA SPIN kit for Soil (Qbiogene, Carlsbad, CA, USA) and a Fast Prep FP120 instrument (Qbiogene) according to the manufacturer’s instructions. The archaeal 16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) with the primer set S-P-MArch-0348S-a-17 (5V-GYGCAGCAGGCGCGAAA-3V) and S-DArch-0786-A-a-20 (5V-GGACTACVSGGGTATCTAAT-3V) [14,16–18]. The PCR reaction was performed with a program which cycling consisted of 15 cycles each of 1 min at 95 8C, 1 min at 50 8C and 2 min at 72 8C. The PCR product was purified in a Microspin S-400 HR column (Amersham Biosciences, Piscataway, NJ, USA) and cloned with a TA Cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cloned DNA was sequenced using a dRhodamin Dye
Fig. 3. Change in cumulative methane production from the reactors. Bed materials with different pore sizes but the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed or fluidized beds. The reactors were operated in semi-continuous mode at a hydraulic retention time of 4 days. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
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Fig. 4. Methane yield from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed types.
CAATAAHARCGGTTACCACTCGRGCC-3V) were used for the Methanosaeta sp. [14]. The real-time PCR amplification followed a three-step PCR (40 cycles) with 20 s denaturation (95 8C), 20 s annealing (55 8C), and 120 s elongation (95 8C). The annealing temperature for the bacteria only was 50 8C. The R2 range of the standard curves obtained by the real-time PCR measurements was 0.962– 0.989. The standard DNA for real-time PCR of bacteria was prepared by PCR with a 2-bp-longer PCR primer set of S-DBact-0346-S-a-19 (5V-GGAGGCAGCAGTDRGGAAT-3V)
Fig. 6. Change in DOC removal efficiency (%) from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed type. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
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and S-D-Bact-0786-A-a-22 (5V-GTGGACTACYVGGGTATCTAAT-3V). The standard DNAs for the methanogens and Methanosaeta sp. were prepared from Methanosarcina barkeri (DSM 800) and Methanosaeta thermophila strain PT (DSM 6194), respectively, by PCR. The average 16S ribosomal RNA gene copy numbers of the bacteria (4 copies/cell) and methanogens (2.5 copies/cell) were referred to the Ribosomal RNA Operon Copy Number Database for the conversion of copy number to cell number [21].
3. Results and discussion 3.1. Reactor performances Fig. 3 shows the change of cumulative methane yield in the reactors containing fixed and fluidized beds with PS-20 and PS-13, and without bed during the operational days. The cumulative methane yield decreased in the following
Fig. 5. Change in methane concentration from the reactors. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed type. Symbols: ( ) PS-20 fixed-bed reactor; (o) PS-20 fluidized-bed reactor; (x) PS-13 fixed-bed reactor; ( w ) PS-13 fluidized-bed reactor; (D) no-bed reactor; (A) feed timing.
.
Fig. 7. Quantity of the immobilized biomass in each bed reactor at the end of the experiment.
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Fig. 8. SEM photos of microbes immobilized on the polyurethane foam. Two pore sizes of the bed material with the same working volume (about 20% v/v) were put into each reactor (300 ml) with fixed and fluidized bed types. (A) PS-13 fluidized-bed reactor; (B) PS-13 fixed-bed reactor; (C) PS-20 fluidized-bed reactor; (D) PS-20 fixed-bed reactor. Scale bars are indicated on photographs. Magnification: 1000.
order: PS-20 fixed-bed reactorNPS-13 fixed-bed reactorNPS-20 fluidized-bed reactorNPS-13 fluidized-bed reactorNno-bed reactor. Fig. 4 shows the average methane yield in five different anaerobic digesters. The PS-20 fixed reactor presented the highest methane yield about 0.59 l CH4/gDOCadded. The methane yields showed the same order as above. Methane yield during the anaerobic fixed-bed system seems to be a representative parameter for process monitor-
ing [22]. These results indicated that the PS-20 fixed-bed reactor gives better digestion performance than the others. The methane concentration in each reactor was shown in Fig. 5. A similar course was observed in all reactors. The lowest methane concentration was measured in the no-bed reactor. A change in DOC removal efficiency was shown in Fig. 6. The DOC removal ratio in all four bed reactors was 92%.
Fig. 9. SEM photos of the major cellular morphologies immobilized on the polyurethane form. (A) PS-13 fluidized-bed reactor; (B) PS-13 fixed-bed reactor; (C) PS-20 fluidized-bed reactor; (D) PS-20 fixed-bed reactor. Scale bars are indicated on the photographs. Magnification: (A, B, C, 10 000; D, 5000).
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Removal was fastest in the PS-20 fixed-bed reactor, where 90% of DOC was removed within 14 days; it took 27–35 days in the PS-13 fixed-bed, PS-20 fluidized-bed and PS-13 fluidized-bed reactors. In the no-bed reactor, the DOC
removal ratio (55%) was relatively low. These results indicate that the fixed-bed reactor with the pore size of 20 cells/25 mm was the most efficient for the anaerobic digestion.
Fig. 10. 16S rRNA-based phylogenetic relationship between the clones (MSC01-MSC15) amplified from the original anaerobic digested sludge by PCR with the methanogen-specific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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3.2. Quantification and microscopic observation After 42 days of reactor operation, the bed materials were removed to measure the quantification of the attached biomass and for microscopic observation. The immobilized biomass quantification from each reactor was shown in Fig. 7. The fixed-bed reactors retained more biomass than the fluidized-bed reactors. The biomass retained in the fluidized-bed support corresponded to only 39% of that retained in the fixed-bed support in PS-13, and 54% in PS-20. Fig. 8 was the SEM photos of the microbes immobilized on the bed materials in each reactor. More biomass was immobi-
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lized in the fixed-bed reactors than in the fluidized-bed reactors. These results indicate that the bedding type plays an important role in the adhesion capacity. The higher quantity of immobilized biomass was due to the smaller pore size of the polyurethane foam. The morphologies of the major immobilized cells were also characterized (Fig. 9). These microphotographs revealed that the biofilm was primarily composed of coccal, diplococcal-shaped Methanosarcina-like cells, short rods of Methanosaeta-like cells and long rods of Methanobacterium-like cells [23,24]. Similar numbers of each were immobilized on the PS-13 and PS-20 fluidized
Fig. 11. 16S rRNA-based phylogenetic relationship between the clones (FL20C02-FL20C03) amplified from the PS-20 fluidized-bed by PCR with the methanogen-specific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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bed reactors (Fig. 9A,C). Although short-rods of Methanosaeta-like cells and long rods of Methanobacteriumlike cells were immobilized (Fig. 9B,D), a large quantity of coccal methanogens, closely resembling to Methanosarcina [25] prevailed in the better performing fixed-bed reactors, especially in PS-20. 3.3. Phylogenetic clone analyses The results of the 16S rRNA phylogenetic analysis of the original anaerobically digested sludge for methanogens
suggested that the major methanogens in the original sludge were Methanosaeta spp. (17/20 clones) and members of the order Methanomicrobiales (3/20 clones) (Fig. 10). This dominance of the Methanosaeta sp. in the anaerobically digested sludge of the large scale digester agrees with other reports [26,1]. Figs. 11 and 12 present the phylogenetic trees of the methanogens, showing the distribution of the major clones among methanogenic biodiversity in the PS-20 fluidized and fixed-bed reactors, respectively. The major methanogenic clones immobilized in the PS-20 fluidized-bed reactor
Fig. 12. 16S rRNA-based phylogenetic relationship between the clones (FI20C01-FI20C07) amplified from the PS-20 fixed-bed by PCR with the methanogenspecific primer set and recorded methanogens. Values in parentheses indicate clone numbers. Numbers at nodes represent bootstrap values (100 replicates). The phylogenetic tree was constructed by using the neighbor-joining method.
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were the Methanosarcina spp. (20/20 clones) (Fig. 11). The major methanogenic clones immobilized on the PS-20 fixed-bed reactor were the Methanosarcina spp. (19/20 clones) and members of the order Methanomicrobiales (1/ 20 clones) (Fig. 12). The SEM analyses also suggested that the coccal, diplococcal-shaped of Methanosarcina-like cells were immobilized on the bed materials (Figs. 9C,D). The dominant methanogenic clones immobilized on the bed were different from those in the original sludge. This could be caused by the change in microbial environment from free-living to immobilization and change in organic substrate. Most of the methanogenic sequence clones of immobilized cells were phylogenetically associated with the hydrogenotrophic Methanobacterium and the aceticlastic Methanosarcina and Methanosaeta. The reactors were supplied only with acetic acid as an organic substrate. Methanosarcina was the most abundant aceticlastic methanogens in both of the bed reactors (Figs. 11 and 12). To date, only two methanogenic genera, Methanosarcina and Methanosaeta, are known to be able to produce methane from acetate, and Methanosaeta spp. generally have lower competitiveness than Methanosarcina spp. at high acetate concentrations [27]. Further quantitative investigation of especially Methanosarcina spp. could be necessary in order to understand the immobilized methanogenic community. This work gave a preliminary description of the methanogenic community immobilized on the fluidized and fixed-bed materials in the anaerobic digesters by the 16S rRNA gene phylogenetic analyses. These results did not show a clear difference in diversity between the two bedding types. The major immobilized methanogens of both bedding types were highly similar to the methanogenic genus Methanosarcina. However, the reactor performance from the present study shows that the fixed-bed reactor had a greater biomass retention capacity and better reactor performance than the fluidized-bed reactor. 3.4. Quantitative change in methanogens The results of real-time PCR analysis indicated that the cell densities of the immobilized methanogens and bacteria increased relative to those of the free-living methanogens and bacteria, respectively, in the original anaerobically digested sludge (Table 1). The cell density of immobilized methanogens in the fixed-bed reactor was higher than that in the fluidized-bed reactor. The increase in biogas production was explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. The real-time PCR analysis enabled the quantification of the immobilized microbes on the bed materials in the anaerobic digester, allowing us to evaluate the effectiveness of the bed-type bioreactors. The results of the clone analysis of the original sludge suggest that the major methanogens were Methanosaeta
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Table 1 16S rRNA gene copy numbers of microbes immobilized on PS-20 polyurethane foam in fluidized- and fixed-bed reactors DNA source
Bacterial copy number (copies/cm3)
Methanogenic copy number (copies/cm3)
Methanosaeta copy number (copies/cm3)
Anaerobically digested sludge Immobilized microbes in the fluidizedbed reactor Immobilized microbes in the fixedbed reactor
(5.1F3.7a)108
(1.3F0.2)109
(6.8F0.8)108
(2.4F0.8)109
(2.2F0.2)109
Not detected
(6.1F0.5)108
(7.1F0.5)109
(5.6F2.5)105
The R 2 range of the standard curves obtained by the real-time PCR measurements was 0.962–0.989. a SD.
spp. and members of the order Methanomicrobiales, and that after immobilization, these were changed to Methanobacterium and Methanosarcina. The results of real-time PCR analysis also indicated that the dominant methanogen in the original anaerobically digested sludge was Methanosaeta and that the immobilized cell density of the Methanosaeta decreased from the free-living cell density of the original sludge, especially in the fluidized-bed reactor (Table 1). The results of real-time PCR analysis coincide with those of clone analyses (Figs. 11 and 12).
4. Conclusions From the performance of all reactors under the same operational conditions, we can conclude that the bedding method and pore sizes of the bed material influenced methane yield. The smaller pore size of PS-20 performed better than the larger pore size of PS-13 under the present experimental conditions. The fixed-bed system seems to be more efficient for immobilized methanogens, organic removal and methane yield than the fluidized-bed system. The fixed-bed reactor with a pore size of 20 cells/25 mm was the most efficient. Microscopic observations of immobilized microbes showed that the fixed-bed reactors immobilized more microorganisms than the fluidized-bed reactors. Distinct methanogens colonized the bed material. The morphologies observed by microscopic analyses indicated that the immobilized microbes were primarily composed of coccaland diplococcal-shaped Methanosarcina-like cells, shortrods of Methanosaeta and long-rods of Methanobacterium. Methanosarcina prevailed in the fixed-bed reactors. The results of 16S rRNA gene phylogenetic analyses indicated that the major immobilized methanogens were belonged to the genus Methanosarcina. The results of real-time PCR analysis indicated that the cell densities of
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the immobilized methanogens increased relative to those of the free-living methanogens in the original anaerobically digested sludge. The increase in biogas production was explained by the increase in the quantity of methanogens immobilized on the polyurethane foam. Real-time PCR analysis enabled the quantification of the immobilized microbes on the bed material in the anaerobic digester, allowing us to evaluate the effectiveness of the bed-type bioreactors.
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[7]
[8]
5. Accession numbers The sequences determined in the present study have been deposited in the DDBJ/EMBL/GenBank databanks under the accession numbers from AB112676 to AB112700. The organisms, together with their GenBank and EMBL accession numbers, whose 16S rRNA sequences were used for the phylogenic analysis were as follows: Methanobacterium bryantii, AF028688; M. congolense, AF233586; M. curvum, AF276958; M. formicicum, AF028689; M. palustre, AF093061; M. subterraneum, X99044; Methanobrevibacter arboriphilus , AB065294; Methanosphaera stadtmanae, M59139; Methanococcus voltae, M59290; Methanomicrobium mobile, M59142; Methanofollis tationis, AF095272; Methanospirillum hungatei, M60880; Methanosarcina acetivorans, M59137; M. barkeri, AB065295; M. frisius, M59138; M. mazei, AB065295; M. siciliae, U20153; M. thermophila, M59140; Methanococcoides burtonii, X65537; Methanolobus tindarius, M59135; Methanomethylovorans hollandica, AF120163; Methanosaeta concilii, M59146; Methanosaeta thermoacetophila, M59141; Sulfolobus acidocaldarius, D14053.
Acknowledgements
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This work was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
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