ng C 27 (2007) 767–772www.elsevier.com/locate/msec

Materials Science and Engineeri

Performance and methanogenic community of rotating disk reactor packedwith polyurethane during thermophilic anaerobic digestion

Yingnan Yang a, Kenichiro Tsukahara a,⁎, Shigeki Sawayama b

a Ethanol and Bioconversion Team, Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology,16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan

b Ethanol and Bioconversion Team, Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology,2-2-2 Hirosuehiro, Kure, Hiroshima 737-0197, Japan

Received 19 May 2006; received in revised form 9 August 2006; accepted 9 August 2006Available online 18 September 2006

Abstract

A newly developed anaerobic rotating disk reactor (ARDR) packed with polyurethane was used in continuous mode for organic waste removalunder thermophilic (55 °C) anaerobic conditions. This paper reports the effects of the rotational speed on the methanogenic performance andcommunity in an ARDR supplied with acetic acid synthetic wastewater as the organic substrate. The best performance was obtained from theARDR with the rotational speed (ω) of 30 rpm. The average removal of dissolved organic carbon was 98.5%, and the methane production rate was393 ml/l-reactor/day at an organic loading rate of 2.69 g/l-reactor/day. Under these operational conditions, the reactor had a greater biomassretention capacity and better reactor performance than those at other rotational speeds (0, 5 and 60 rpm). The results of 16S rRNA phylogeneticanalysis indicated that the major methanogens in the reactor belonged to the genus Methanosarcina spp. The results of real-time polymerase chainreaction (PCR) analysis suggested that the cell density of methanogenic archaea immobilized on the polyurethane foam disk could be concentratedmore than 2000 times relative to those in the original thermophilic sludge. Scanning electron microphotographs showed that there were moreimmobilized microbes at ω of 30 rpm than 60 rpm. A rotational speed on the outer layer of the disk of 6.6 m/min could be appropriate foranaerobic digestion using the polyurethane ARDR.© 2006 Elsevier B.V. All rights reserved.

Keywords: Anaerobic rotating disk reactor (ARDR); Immobilization; Methanogens; SEM; 16S rRNA; Real-time PCR

1. Introduction

Several new processes have been developed to provide moreefficient and economical anaerobic treatment for a broad rangeof organic wastes [1–3]. Depending on the treatment needs,different processes are designed to optimize methane produc-tion, and to minimize operational costs.

The development of the fixed-bed system has provedeffective in reducing reactor volume and improving the organicloading ratio because of microbes immobilized on the fixed-bedand reactor walls [2,4]. However, the clogging, hydraulic short-circuiting and decrease in the reactor's active volume after along period of operation are the main disadvantages of this

⁎ Corresponding author. Tel.: +81 29 861 8471; fax: +81 29 861 8187.E-mail address: k-tsukahara@aist.go.jp (K. Tsukahara).

0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2006.08.007

process. The active reactor volume was found to be reduced byabout 60% after 3.5 years of operation [5]. A rotating biologicalcontactor is widely applied in treating municipal and industrialwastewaters aerobically [6,7]. The rotating biological contactorwith combination of fixed-film and suspended growth systemshas been identified as an alternative bioreactor in studies on thetreatment of high-strength industrial wastewaters originatedfrom pulp and paper mills [8]. Generally, the discs are fixed on ahorizontal shaft and partially immersed in the wastewater undermesophilic aerobic conditions [9].

The anaerobic rotating biological contactor process, firstintroduced by Tait and Freidman [10], used a simple reactor withseveral baffle plates fixed on a horizontal shaft to treat syntheticcarbonaceous wastewater under mesophilic conditions. Theyemployed two identical four-stage reactors which were operatedwith 70% of the disk surface area submerged. They concluded

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that the anaerobic rotating biological contactor process is bothfeasible and practical for the removal of high-strength organiccompounds in wastewater. However, using a similar system, thestage chemical oxygen demand (COD) removal efficiency de-creases with an increase of stage number and about 90% oforganic compounds were removed in the first two stages, indi-cating that a two-stage reactor may be sufficient in practicalapplications and 100% submergence of the disk achieved thebest COD removal efficiency in the anaerobic rotating biologicalcontactor process [11]. Furthermore, there is little information inthe literature on process design with respect to disk rotationalspeed. Therefore, an adequate understanding of the effects ofchanging the rotational speed on the performance of the rotatingdisk reactor is essential before practical application.

The thermophilic digestion process will intrinsically havehigher loading rates, but the process is considered to be moresensitive to environmental changes than the mesophilic process[12,13]. The effectiveness of thermophilic anaerobic digestionof the organic waste using a rotating disk reactor, with the disksfixed on a vertical shaft and 100% disk submergence with dif-ferent rotational speeds, has not yet been actively discussed.

Reticular polyurethane foam has a high specific surface areaand porosity. It appeared to be an excellent colonization matrixand have good endurance for an anaerobic filter reactor [3]. Inthe present study, a newly developed anaerobic rotating diskreactor (ARDR) packed with polyurethane was used in con-tinuous mode for organic waste removal under thermophilicanaerobic conditions. The effects of the rotational speed on themethanogenic performance and community in the ARDR wereinvestigated.

2. Materials and methods

2.1. Reactor design and operation

In this study, reticular polyurethane foam (Type CFH)(INOAC, Nagoya, Japan) was used for microbial immobilization.

Fig. 1. Schematic diagram of anaerobic rotating di

A schematic diagram of the ARDR is shown in Fig. 1. The reactorwas a glass vessel with an inner diameter of 10 cm and a height of15 cm. Five pieces of 7-cm-diameter and 1-cm-thick polyurethanefoam disks were fixed on a vertical shaft and completely sub-merged in the liquid content of the reactor. Thewhole surface areafor biofilm was 495 cm2. The shaft and attached disks wererotated by an electric motor. The shaft was rotated at a speed of 0,5, 30 or 60 rpm.

The reactor was operated in continuous mode at a hydraulicretention time of 2 days and the temperature was maintained at55 °C. To the reactor was firstly added 900 ml of a mixture ofthermophilic methanogenic sludge (16% w/w) from the waste-water treatment plant (Kyoto, Japan) and a synthetic medium(84% w/w) containing CH3COONa (5 g/l), yeast extract(300 mg/l), NH4Cl (200 mg/l), KH2PO4 (16 mg/l) and tracemineral solution (200 ml/l). The chemical composition of thetrace metal solution was according to the paper of Yang et al. [3].After incubation for 20 days, the feeding with the syntheticmedium at an organic load rate (OLR) of 2.69 g/l-reactor/daywas continued until day 211. The rotational speed (ω) wascontrolled at 5 rpm for 24 days, 0 rpm for 35 days, 30 rpm for72 days and 60 rpm for 60 days.

2.2. Analyses

A portion of the reactor contents was sampled twice a week.The effluent was centrifuged at 10,000 rpm for 10 min to allowprecipitation of the microbes. The supernatant was used tomeasure the dissolved organic carbon (DOC) with a TOC ana-lyzer (TOC-5000A, Shimadzu, Kyoto, Japan). Biogas produc-tion and pH in the reactor were measured daily. The compositionof the biogas was determined using a gas chromatograph (GC-8A, Shimadzu) with a thermal conductivity detector equippedwith a steel Porapak Q column (Shinwakakou, Kyoto, Japan) at90 °C.

The microbes present in the biofilms were observed through ascanning electron microscope (SEM) (DS-720, Topcon, Tokyo,

sk reactor (ARDR) packed with polyurethane.

Fig. 2. Methane concentration and production rate in the ARDR continuouslysupplied with synthetic acetic acid after incubation for 20 days.

769Y. Yang et al. / Materials Science and Engineering C 27 (2007) 767–772

Japan). Under ω of 30 and 60 rpm, respectively, part of theattached bed materials was taken out and the cells were washedwith buffer solution (pH 7.0). Samples were prepared for SEMaccording to Yang et al. [3].

2.3. Phylogenetic analysis of 16S rRNA gene

Samples from five different places in the disk bed materialswere collected and the Phylogenetic of 16S rRNA gene ana-lyzed with the procedures described as Sawayama et al. [14].

2.4. Quantification of microbes by real-time PCR

DNAwas extracted from the original thermophilic anaerobicdigested sludge (0.5 ml, volatile solid 2.1%) and from themicrobes in the free-living liquid, and immobilized on thepolyurethane disks (0.1 cm3) at a rotational speed of 30 and60 rpm, respectively. Real-time PCR was conducted with anABI7000 sequence detection system (Applied Biosystems) andTaqMan Universal PCR Master Mix (Applied Biosystems).Quantitative measurement by real-time PCR was conducted inquadruplicate. The analysis methods were according toSawayama et al. [14]. The real-time PCR amplificationfollowed a three-step PCR (40 cycles) with 20 s denaturation(95 °C), 20 s annealing (55 °C), and 120 s elongation (72 °C).The annealing temperature for the bacteria only was 50 °C. TheR2 range of the standard curves obtained by the real-time PCRmeasurements was 0.956–0.996.

The standard DNA for real-time PCR of bacteria wasprepared by PCR with a 2-bp-longer PCR primer set of S-D-Bact-0346-S-a-19 (5′–GGAGGCAGCAGTDRGGAAT–3′)and S-D-Bact-0786-A-a-22 (5′–GTGGACTACYVGGGTATCTAAT–3′). The standard DNAs for the methanogenic archaeaMethanosarcina spp. and Methanobacterium spp. were pre-pared by PCR with Methanosarcina barkeri (DSM 800) andMethanobacterium formicicum (DSM 1535), respectively.

2.5. Accession numbers

The sequences determined in the present study have beendeposited in the DDBJ/EMBL/GenBank databanks under theaccession numbers from AB244634–AB244636. The organ-isms, together with their GenBank and EMBL accession num-bers, whose 16S rRNA sequences were used for the phylogenicanalysis were as follows: Methanobacterium bryantii,AF028688; Methanobacterium congolense, AF233586;Methanobacterium curvum, AF276958; Methanobacteriumformicicum, AF028689; Methanobacterium palustre,AF093061; Methanobacterium subterraneum, X99044;Methanobrevibacter arboriphilus, AB065294; Methano-sphaera stadtmanae, M59139; Methanococcus voltae,M59290; Methanomicrobium mobile, M59142; Methanofollistationis, AF095272; Methanospirillum hungatei, M60880;Methanosarcina acetivorans, M59137; Methanosarcina bar-keri, AB065295; Methanosarcina frisius, M59138; Methano-sarcina mazei, AB065295; Methanosarcina siciliae, U20153;Methanosarcina thermophila, M59140; Methanococcoides

burtonii, X65537; Methanolobus tindarius, M59135; Metha-nomethylovorans hollandica, AF120163; Methanosaeta con-cilii, M59146; Methanosaeta thermoacetophila, M59141;Sulfolobus acidocaldarius, D14053.

3. Results and discussion

3.1. Reactor performance

The methane concentration in the continuous ARDRpromptly increased with feeding. It reached 92.7% at day 7and remained at that level until the end of digestion (Fig. 2).This high methane concentration was caused by the relativelyhigh pH. This short start-up period could be due to the rotationalspeed of 5 rpm improving the contact ratio between substrateand inoculum. Higher biomass concentration could be immo-bilized on the high porosity of the rotating polyurethane diskbeds.

Fig. 2 shows the methane concentration and production rateas a function of ω. After incubation for 20 days, the feedingwith the synthetic medium at an organic load rate (OLR) of2.69 g/l-reactor/day was continued until day 211. The rotationalspeed (ω) was controlled at 5 rpm for 24 days, 0 rpm for35 days, 30 rpm for 72 days and 60 rpm for 60 days until the testfinished. During this period, the average methane productionrates of 295 ml/l-reactor/day, 290 ml/l-reactor/day, 393 ml/l-reactor/day and 297 ml/l-reactor/day were achieved from day 21through 44, 45 through 79, 80 through 151, and 152 through211 of operation, respectively. As shown in Fig. 2, the averagemethane production rates increased with an increase of ω in therange of 0–30 rpm. However, the opposite trend was observedat ω of 60 rpm. The optimal ω value was around 30 rpm, atwhich the rotational speed on the outer layer of the disk was6.6 m/min in the present operation. A steady period is generallyconsidered to be when stable methane formation lasts for morethan twice the hydraulic retention time. The methane yield canprovide information on the dynamic steps of biofilm develop-ment [1]. The yields in the present experiments indicate that theARDR achieved a stable and well-performing anaerobic diges-tion especially at ω of 30 rpm.

The DOC average removal efficiencies at different ω areshown in Fig. 3. The DOC removal efficiency increased with an

Fig. 3. Average DOC removal efficiency (%) under different rotational speeds inthe ARDR continuously supplied with synthetic acetic acid.

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increase of ω in the range of 0–30 rpm. However, the oppositetrend was observed at ω values above 30 rpm. The optimal ωvalue is around 30 rpm with the average DOC removal effi-ciency of 98.5%. The increase of ARDR treatment efficiencywith increasing ω in the range of 0–30 rpm could be explainedby the fact that the flow is more mixed and the contact ratiobetween substrate and microorganism improved at a higher ω.The decrease of ARDR treatment efficiency with increasingω atω values above 30 rpm may be explained by the dominance ofthe fluid shear stress over the mass transfer. The average DOCremoval efficiencies were 91.7% at ω of 60 rpm. The rotationalspeed of the outer layer was 6.6 and 13.2 m/min at ω of 30 and60 rpm, respectively. A rotational speed of the outer layer of

Fig. 4. 16S rRNA-based phylogenetic relationship between the 30 clones (RDC01, RDthe methanogen-specific primer set and recorded methanogens. Values in parent(100 replicates). The phylogenetic tree was constructed by using the neighbor-joinin

6.6 m/min could be appropriate for anaerobic digestion using thepolyurethane ARDR.

DOC of more than 98% could be removed at an OLR of2.69 g/l-reactor/day, which indicates that this ARDR systemwas highly efficient and capable of removing more substrate athigher loading rates. The better performance by the ARDRcould be explained by the improved efficiency of contact bet-ween substrate and microorganism under an optimum rotationalspeed such as 30 rpm, thus increasing the quantity of metha-nogens immobilized on the polyurethane foam; as a conse-quence, enough methanogens were supplied to the wholereactor, contributing to the high system efficiency.

3.2. Phylogenetic clone analyses

The results of the 16S rRNA phylogenetic analysis of theoriginal thermophilically and anaerobically digested sludge formethanogens suggested that themajor methanogens in the originalsludge consisted of Methanobrevibacter spp. (9/20 clones) andother members of the family Methanobacteriaceae (9/20 clones).Two clones of Methanosaeta spp. were also detected in the orig-inal sludge [14].

Fig. 4 presents the phylogenetic tree of the methanogens,showing the distribution of the major clones among methano-genic biodiversity in the rotating disks of the ARDR system.The major methanogenic clones immobilized on the disks werethe Methanosarcina spp. (28/30 clones) and members of theorder Methanobacterium spp. (2/30 clones) (Fig. 4). The

C03 and RDC21) amplified from the rotating disks at ω of 30 rpm by PCR withheses indicate clone numbers. Numbers at nodes represent bootstrap valuesg method.

Table 116S rRNA gene copy numbers of original anaerobically digested sludge, immobilized microbes on rotating disk, and free-living cells in the reactor at ω 30 and 60 rpm,respectively

DNA source Bacterial copy number(copies/cm3)

Methanogenic archaea copynumber (copies/cm3)

Methanosarcina spp. copynumber (copies/cm3)

Methanobacterium spp. copynumber (copies/cm3)

Original anaerobicallydigested sludge

(3.9±0.2a)× l08 (1.4±0.3)×107 (4.6±0.3)×106 (1.8±0.2)×105

Free-living cells in the reactorat ω 30 rpm

(2.2±1.2)×107 (6.4±0.4)×107 (2.9±0.9)×107 (2.7±0.8)×104

Immobilized microbes on rotatingdisk at ω 30 rpm

(6.1±0.7)×109 (3.9±0.7)×1010 (9.8±0.6)×109 (2.6±0.4)×107

Free-living cells in the reactorat ω 60 rpm

(1.8±0.7)×107 (5.5±0.6)×106 (4.9±2.0)×106 NDb

Immobilized microbes on rotatingdisk at ω 60 rpm

(1.4±0.1)×107 (9.2±1.1)×109 (8.8±0.9)×109 (1.4±0.1)×107

The R2 range of the standard curves obtained by the real-time PCR measurements was 0.956–0.996.a S.D.b ND: not detected.

Fig. 5. SEM photographs of microbes immobilized on the polyurethane foamdisk: (A) ω=30 rpm; (B) ω=60 rpm; scale bars are indicated on thephotographs. Magnification: ×10000.

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dominant methanogenic clones immobilized on the bed weredifferent from those in the original sludge. The change inmicrobial environment from free-living to immobilization andchange in organic substrate could be the reason.

Most of the methanogenic sequence clones of immobilizedcells were phylogenetically associated with the aceticlasticMethanosarcina spp. and the hydrogenotrophic Methanobac-terium spp. The reactors were supplied only with acetic acid asan organic substrate. Methanosarcina was the most abundantaceticlastic methanogens in the ARDR reactor (Fig. 4). Onlytwo methanogenic genera, Methanosarcina and Methanosaeta,are known to be able to produce methane from acetate, andMethanosaeta spp. generally has lower competitiveness thanMethanosarcina spp. at high acetate concentrations [15].Much further quantitative investigation is necessary in orderto understand the microbial community in the ARDR.

3.3. Quantitative description of microbial community

The results of real-time PCR analysis indicated that themethanogenic and Methanosarcina spp. rRNA gene copydensity increased more than 2000 times from the originalthermophilic sludge to the immobilized microbes, and that thebacterial rRNA gene copy density increased 10 times at ω of30 rpm but decreased at ω of 60 rpm (Table 1). The increase inthe quantity of methanogens contributed to the effectiveanaerobic digestion. The rRNA gene copy density of methano-genic archaea immobilized on rotating disks was more than 600and 1600 times that of the free-living cells at ω of 30 and60 rpm, respectively. A similar course was observed onMethanosarcina spp. Under the optimal ω of 30 rpm, enoughmethanogens were immobilized on the rotating disks, and anoptimum quantity of methanogens was supplied simultaneouslyto the free-living liquid, thus improving the system efficiency.Under the higher ω of 60 rpm, although many methanogenswere immobilized on the rotating disks, the fluid shear stresshindered the transfer of the methanogens to the free-livingliquid and thus lowered the efficiency of the system.

16S rRNA gene densities of Methanosarcina spp. andMethanobacterium spp. immobilized on the polyurethane disk

were determined as 9.8×109 and 2.6×107 copies/cm3 at ω of30 rpm, 8.8×109 and 1.4×107 copies/cm3 at ω of 60 rpm,respectively, by real-time PCR analyses. Those free-living cellsin the reactor were 2.9×107 and 2.7×104 copies/cm3 at ω of30 rpm, and 4.9×106 and no detected copies/cm3 at ω of60 rpm, respectively.

The results of real-time analysis showed that Methanosar-cina spp. form a major methanogenic group not only in themethanogenic community immobilized on the rotating disks but

772 Y. Yang et al. / Materials Science and Engineering C 27 (2007) 767–772

also in the free-living broth. The results coincide with those ofclone analyses (Fig. 4).

Molecular techniques are now an invaluable tool for thecharacterization of complex microbial communities and, whenused in conjunction with process engineering and physiologicalmeasurements, they will allow a more extensive knowledge andunderstanding of the physiology and biochemistry of themicrobial populations involved in anaerobic digestion.

3.4. Microscopic observation

Fig. 5 shows SEM photographs of the microbes immobilizedon the disk materials at ω of 30 and 60 rpm. More biomass wasimmobilized on the polyurethane disk at ω of 30 rpm than60 rpm (Fig. 5 A, B). These results indicate that the rotationalspeed plays an important role in the adhesion capacity. Thehigher quantity of immobilized biomass was due to the bettercontact ratio between substrate and microorganism under anoptimum rotational speed such as 30 rpm.

The morphologies of the major immobilized cells were alsocharacterized (Fig. 5 A, B). These microphotographs revealedthat the biofilm was primarily composed of long rods ofMethanobacterium-like cells and coccal, diplococcal-shapedMethanosarcina-like cells [16,17]. The observation reflectedthe phylogenetic clone analyses.

4. Conclusions

From the performance of the reactor under different rotationalspeeds, we can conclude that the rotational speed influencedmethane production rate and DOC removal efficiency. Theoptimal rotational speed of disks for microbial treatment ofacetic acid synthetic wastewater in an ARDR was found to be30 rpm in the present investigation. The results of 16S rRNAphylogenetic analysis indicated that the major methanogens inthe reactor belonged to the genus Methanosarcina spp. Theresults of real-time PCR analysis indicated that the cell densitiesof the immobilized methanogens increased relative to those ofthe free-living methanogens in the original anaerobically di-gested sludge. The increase in biogas production was explainedby the increase in the quantity of methanogens immobilized onthe polyurethane disks and in the free-living broth. The mor-phologies observed by microscopic analyses indicated that theimmobilized microbes were primarily composed of long rods ofMethanobacterium-like cells and coccal, diplococcal-shaped

Methanosarcina-like cells. These results indicated that it isimportant to appropriately adjust the rotational speed forefficient digestion. The better biogas production by the ARDRcould be explained by the improved contact ratio betweensubstrate and microorganism under an optimum rotational speedsuch as 30 rpm, thus increasing the quantity of methanogensimmobilized on the polyurethane foam; as a consequence,enough methanogens were supplied to the whole reactor. Arotational speed of 6.6 m/min on the outer layer of the disk couldbe appropriate for anaerobic digestion using the polyurethaneARDR. Although the system employed in this work was rathersmall, the results positively suggested that the upscale in-vestigation for this particular system was highly attractive.Further scaled-up investigation with different kinds of organicsubstrate is necessary in order to understand the effectiveness forpractical application.

Acknowledgements

This work was supported by the New Energy and IndustrialTechnology Development Organization (NEDO), Japan.

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