Photoenhancement of biogas production from thermophilic anaerobic digestion

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<ul><li><p>387</p><p>JOURNAL OF BIOSCIENCE AND BIOENGINEERING</p><p>Vol. 98, No. 5, 387390. 2004</p><p>Photoenhancement of Biogas Productionfrom Thermophilic Anaerobic Digestion</p><p>CHIKA TADA1 AND SHIGEKI SAWAYAMA1*</p><p>Biomass Group, Energy Technology Research Institute, National Institute of AdvancedIndustrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan1</p><p>Received 6 April 2004/Accepted 24 August 2004</p><p>After 46 d of anaerobic digestion at 55C, the volume of methane produced from an illuminatedreactor was 2.2 times as high as that from a dark reactor. Light at a wavelength of 390540 nm en-hanced the methane production.</p><p>[Key words: light, anaerobic digestion, thermophilic, methane, Methanothermobacter]</p><p>The anaerobic digestion of organic waste and treatmentof wastewater has the advantages of relatively little sludgeproduction, low energy consumption, and methane produc-tion, which have led to this process being widely studiedand used (1). Olson et al. reported that the growth of metha-nogens was inhibited by the blue end (370430 nm) of thevisible spectrum (2). Most conventional systems of anaero-bic digestion are operated under dark conditions.</p><p>On the other hand, photoactivation of 2-(methylthio)ethanesulfonic acid reductase (CH</p><p>3-S-CoM reductase) from</p><p>Methanobacterium (now Methanothermobacter) has beenreported (3; List of Bacterial Names Standing in Nomen-clature; http://www.bacterio.cict.fr/). This enzyme catalyzesCH</p><p>3-S-CoM to form CH</p><p>4 at the terminal step of methano-</p><p>genesis only in the presence of light. Thus, the photoen-hancement of biogas production might be possible throughhydrogenotrophic methanogens; however, this has not yetbeen reported.</p><p>The use of the lighted upflow anaerobic sludge blanket(LUASB) method under mesophilic conditions has beenstudied (4). Phototrophic bacteria grew in the LUASB re-actor and removed ammonium and phosphate in the light;however, the methane production was lower than that in thedark. Phototrophic bacteria competed with methanogens forelectron donors. Lighted anaerobic digestion under thermo-philic conditions has not yet been studied.</p><p>We studied the effect of light on thermophilic anaerobicdigestion.</p><p>Thermophilically and anaerobically digested sludge wascollected from a cattle waste treatment plant in Kyoto. Ther-mophilic methanogenic sludge (80 ml) was mixed with 320ml of organic medium, containing the following chemicalsper liter: CH</p><p>3COONa, 2 g; glucose, 2 g; KH</p><p>2PO</p><p>4, 16 mg;</p><p>MgCl26H</p><p>2O, 25 mg; CaCl</p><p>22H</p><p>2O, 25 mg; FeEDTA, 5 mg;</p><p>CoCl2 6H</p><p>2O, 5 mg; and MnCl</p><p>2 4H</p><p>2O, 5 mg. The volatile</p><p>solids content of the organic medium was 1.2% (w/w). Apiece of polyurethane foam (7 181 cm; no. 20; Keiyo,Chiba) was placed in each of two reactors made from 500-ml</p><p>glass bottles. A gas bag was connected to the reactor forbiogas yield measurement. The reactors were then placed ina thermostated incubator at 55C. The dark reactor was en-tirely wrapped in aluminum foil to exclude light. The lowerpart of the illuminated reactor was covered with foil, and theupper part was illuminated by 60-W incandescent lamps(LW110V60W; Mitsubishi Osram, Tokyo). The illuminatedreactor was set 7 cm apart from the incandescent light, thelight intensity of which was 520 mol/s/m2 which was mea-sured with a light meter LI-250 (LI-COR; Lincoln, NE,USA). Yeast extract (0.8 g) was fed to the reactors once aweek. The experiments were repeated three times, and stan-dard deviations were calculated.</p><p>The biogas composition was determined with a gas chro-matograph (GC-8A; Shimadzu, Kyoto). Ammonium andphosphate concentrations in the reactor effluent were deter-mined with an ion chromatograph (DX120; Dionex, Sunny-vale, CA, USA). Dissolved organic carbon (DOC) concen-tration was determined with a TOC meter (TOC-5000A;Shimadzu). The concentration of organic acids was deter-mined with an ion chromatograph (DX120; Dionex). Thephototrophic bacterial cell density was measured as bac-teriochlorophyll a (BChl-a) (5).</p><p>After 46 d of digestion, 0.5 ml of the liquid fraction ineach reactor was collected. DNA was extracted with a Fast-Prep kit (Bio 101; Carlsbad, CA, USA) according to themanufacturers instructions. The specific DNA fragments ofthe 16S rRNA gene were amplified by polymerase chain re-action (PCR) with the methanogen-specific primer set S-P-March-0348-S-a-17 (5-GYGCAGCAGGCGCGAAA-3) (6)and S-D-Arch-0786-A-a-20 (5-GGACTACVSGGGTATCTAAT-3) (7). PCR program consisted of 15 cycles each of1 min at 95C, 1 min at 55C, and 2 min at 72C. PCR am-plification and cloning were performed as described bySekiguchi et al. (8).</p><p>The sequences (418 bp) of 20 clones were determinedwith a dRhodamine Dye Terminator Cycle Sequencing FSReady Reaction kit (Applied Biosystems, Foster City, CA,USA) and an automated sequence analyzer (model 377;Applied Biosystems). The sequences were compared withknown 16S rRNA gene sequences by a nucleotidenucleo-</p><p>* Corresponding author. e-mail: s.sawayama@aist.go.jpphone/fax: +81-(0)29-861-8184</p></li><li><p>TADA AND SAWAYAMA J. BIOSCI. BIOENG.,388</p><p>tide BLAST search of the NCBI DNA database (http://www.ncbi.nlm.nih.gov/). Sequence data were aligned witha CLUSTAL W package for phylogenetic analysis (9). Aphylogenetic tree was constructed by the neighbor-joiningmethod with the MEGA V2.1 package (10).</p><p>The sequences determined in the present study have beendeposited in the DDBJ/EMBL/GenBank databanks (IC1AB126040; IC6 AB126041; IC14 AB126042; IC20AB126043; DC1 AB126044). The organisms whose 16SrRNA sequences were used for the phylogenic analysis wereas follows: Methanobacterium bryantii (AF028688), M.congolense (AF233586), M. curvum (AF276958), M. palus-tre (AF093061), M. subterraneum (X99044), Methano-thermobacter defluvii (X99046), M. thermoautotrophicus(AB020530), M. thermoformicicum (X68716), M. wolfeii(AB104858), Methanobrevibacter smithii (AF054208), M.arboriphilus (AB065294), Methanosphaera stadtmanae(M59139), Methanococcus voltae (U38461), Methanomicro-bium mobile (M59142), Methanofollis tationis (AF095272),Methanospirillum hungatei (M60880), Methanosarcinaacetivorans (M59137), M. barkeri (AB065295), M. mazei(AB065295), Methanococcoides burtonii (X65537), Metha-nolobus tindarius (M59135), Methanomethylovorans hol-landica (AF120163), Methanosaeta concilii (M59146), M.thermoacetophila (M59141), and Sulfolobus acidocaldarius(D14053).</p><p>To investigate the effect of light wavelength on methaneproduction, we controlled the wavelength with two filters, a&lt; 390 nm and a &lt; 540 nm sharp cut filter (each 5 5 cm0.5 mm; JASCO, Tokyo). The filters were set in front offluorescent bulbs. The light intensity at the reactor surfacewas adjusted to 520 mol/s/m2 with a light meter LI-250(LI-COR). The experimental method of thermophilic anaer-obic digestion was the same as described previously in thispaper except for the light source. The experiments were re-peated three times, and standard deviations were calculated.</p><p>Figure 1 shows the methane production from the reactors.In the first 6 d of operation, methane yields were not sig-</p><p>nificantly different between dark and light conditions. After13 d, the methane yield from the light reactor became higherthan that from the dark reactor. The maximum methane pro-duction rate from the light reactor (28.1 ml/d reactor) wasapproximately three times as high as that from the dark re-actor (9.7 ml/d reactor) from day 13 to day 21. The conver-sion ratios of added organic carbon (1.31 g DOC) to meth-ane and carbon dioxide were 68.4% in the light reactorthroughout the 46 d of operation and 22.5% in the dark re-actor. After 46 d of incubation, the volume of methane pro-duced from the light reactor was 2.2 times as high as thatfrom the dark reactor.</p><p>BChl-a was not detected in the illuminated or dark re-actor effluent. Phototrophic bacteria did not grow in thelight thermophilic reactor, unlike in the LUASB reactor (4).The concentrations of ammonium and phosphate ions werenot significantly different between the illuminated and darkreactors (data not shown). The DOC concentration in theilluminated reactors was approximately two times higherthan that in the dark reactors.</p><p>The concentration of lactate in the dark reactor was morethan two times higher than that in the illuminated reactor.The concentration of formate, acetate, propionate and bu-tyrate in the illuminated reactor was higher than that in thedark reactor. Acetate in the dark reactor was not detectedfrom day 28 to day 42. Butyrate in the dark reactor was notdetected after day 13. The enhancement of fatty acid pro-duction in the illuminated reactor suggested that hydro-genotrophic methanogenesis activated acid fermentation inthe illuminated reactor. The pH of both reactors was 7.57.7.</p><p>The 16S rRNA-based phylogenetic relationship betweenclones amplified by PCR from the illuminated and dark re-actors is shown in Fig. 2. In the illuminated reactor, the 20clones were divided into four groups (designated as IC1,IC6, IC14, and IC20) belonging to Methanothermobacter.In the dark reactor, all 20 clones had the same sequence(designated DC1), also belonging to Methanothermobacter.A nucleotidenucleotide BLAST search of the DNA data-base matched the 16S rRNA gene sequences of clones fromboth reactors with M. thermoautotrophicus delta H (similar-ity 100%) or M. wolfeii (similarity 100%). Either speciescould have been the major methanogenic population in bothreactors. Ahring et al. reported that M. thermoautotrophicusdelta H increased in granules in a thermophilic UASB reac-tor (11). In the present study, the thermophilic fixed-bed re-actors loaded with yeast extract could accumulate Methano-thermobacter spp.</p><p>Inactive CH3-S-CoM reductase extracted from M. ther-</p><p>moautotrophicus delta H was activated by exposure to light(3). This enzyme catalyzes CH</p><p>3-S-CoM to form CH</p><p>4 at the</p><p>terminal step of methanogenesis. We consider that its pho-toactivation in methanogens by illumination is the reasonfor the enhancement of methane production. CH</p><p>3-S-CoM is</p><p>reductively demethylated with reducing equivalents fromN-(7-mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP)according to the following equation (12):</p><p>CH3-S-CoMHS-HTP CH</p><p>4CoM-S-S-HTP</p><p>The enzyme catalyzes CH3-S-CoM to form CH</p><p>4 only in the</p><p>FIG. 1. Methane production from illuminated and dark reactors dur-ing thermophilic (55C) anaerobic digestion. Symbols: open squares,methane production from the illuminated reactor without any filters;closed squares, methane production from the dark reactor. Bars indi-cate SD. SDs were evaluated by error propagation from the data of ex-periments repeated three times.</p></li><li><p>NOTESVOL. 98, 2004 389</p><p>presence of light, and CH3-S-CoM reductase from M. ther-</p><p>moautotrophicus was activated by light between 400 and515 nm (3). Bonacker et al. reported that M. wolfeii also hasthe same CH</p><p>3-S-CoM reductase as M. thermoautotrophicus</p><p>(13).We considered that if the CH</p><p>3-S-CoM reductase is acti-</p><p>vated by light, the methane production from the illuminatedreactor would change according to wavelength. Figure 3shows the ratio of methane yield from the illuminated reac-tors based on that from the dark reactors during 14 or 21 dof operation. Methane production at an illumination wave-length of more than 390 nm was higher than that at a wave-length of more than 540 nm and that in the dark. By day 21of operation, the methane yield from the reactor with anillumination wavelength of more than 390 nm was 843 ml/g</p><p>DOC added. The methane yield from the reactor with anillumination wavelength of more than 540 nm was 690 ml/gDOC added, and that from the reactor without illuminationwas 668 ml/g DOC added. Comparing the methane yields atmore than 390 nm and more than 540 nm, the value was sta-tistically different (p</p></li><li><p>TADA AND SAWAYAMA J. BIOSCI. BIOENG.,390</p><p>the development of an efficient methane production tech-nology.</p><p>This work was supported by the New Energy and IndustrialTechnology Development Organization (NEDO), Japan.</p><p>REFERENCES</p><p>1. Fannin, K. F., Conrad, J. R., Srivastava, V. J., Jerger, D. E.,and Chynoweth, D. P.: Anaerobic processes. J. Water Pollut.Control Fed., 55, 623632 (1983).</p><p>2. Olson, K. D., Mcmathon, C. W., and Wolfe, R. S.: Lightsensitivity of methanogenic archaebacteria. Appl. Environ.Microbiol., 57, 26832686 (1991).</p><p>3. Olson, K. D., Mcmathon, C. W., and Wolfe, R. S.: Photo-activation of the 2-(methylthio) ethanesulfonic acid reduc-tase from Methanobacterium. Proc. Natl. Acad. Sci. USA, 88,</p><p>40994103 (1991).4. Sawayama, S., Tsukahara, K., and Yagishita, T.: Waste-</p><p>water treatment and poly--hydroxybutyrate production usinglighted upflow anaerobic sludge blanket method. J. Biosci.Bioeng., 87, 683689 (1999).</p><p>5. Cohen-Bazire, G., Sistrom, W. R., and Stanier, R. Y.: Ki-netic studies of pigment synthesis by non-sulfur purple bac-teria. J. Cell Comp. Physiol., 49, 2568 (1957).</p><p>6. Sawayama, S., Tada, C., Tsukahara, K., and Yagishita, T.:Effect of ammonium addition on methanogenic communityin a fluidized bed anaerobic digestion. J. Biosci. Bioeng., 97,6570 (2004).</p><p>7. Takai, K. and Horikoshi, K.: Rapid detection and quantifi-cation of members of the Archaeal community by quantitativePCR using fluorogenic probes. Appl. Environ. Microbiol., 66,50665072 (2000).</p><p>8. Sekiguchi, Y., Kamagata, Y., Syutsubo, K., Ohashi, A.,Harada, H., and Nakamura, K.: Phylogenetic diversity ofmesophilic and thermophilic granular sludge determined by16S rRNA gene analysis. Microbiology, 144, 26552665(1998).</p><p>9. Thompson, J. D., Higgins, D. G., and Gibson, T. J.:CLUSTAL W: improving the sensitivity of progressive mul-tiple sequence alignment through sequence weighting, posi-tions-specific gap penalties and weight matrix choice. Nu-cleic Acids Res., 22, 46734680 (1994).</p><p>10. Kumar, S., Tamura, K., and Nei, M.: MEGA-molecularevolutionary genetics analysis software for microcomputers.Comput. Appl. Biosci., 10, 189191 (1994).</p><p>11. Ahring, B. K., Schmidt, J. E., Nielsen, M. W., Macario,A. J. L., and Macario, E. C.: Effect of medium compositionand sludge removal on the production. Composition, and ar-chitecture of thermophilic (55C) acetate-utilizing granulesfrom an upflow anaerobic sludge blanket reactor. Appl. Envi-ron. Microbiol., 59, 25382545 (1993).</p><p>12. Ellermann, J., Hedderich, R., Bocher, R., and Thauer,R. K.: The final step in methane formation. Investigationswith highly purified methyl-CoM reductase (component C)from Methanobacterium thermoautotrophicum (strain Mar-burg). Eur. J. Biochem., 172, 669677 (1988).</p><p>13. Bonacker, L. G., Baudner, S., and Thauer, R. K.: Differen-tial expression of the two methyl-coenzyme M reductases inMethanobacterium thermoautotrophicum as determined im-munochemically via isoenzyme-specific antisera. Eur. J. Bio-chem., 206, 8792 (1992).</p><p>FIG. 3. Ratios of methane yield at different wavelengths of lightbased on that in the dark during 14 or 21 d of operation. Symbols: dot-ted patterns, over 14 d; gray patterns, over 21 d. Bars indicate SD. SDswere evaluated by error propagation from the data of experiments re-peated three times. Asterisk shows that t...</p></li></ul>