Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria

Biotechnology for Biofuels

Vitali A Svetlitchnyi¹  Oliver Kensch¹  Doris A Falkenhan¹  Svenja G Korseska¹  Nadine Lippert¹  Melanie Prinz¹  Jamaleddine Sassi¹  Anke Schickor¹  Simon Curvers¹ 

[1] Direvo Industrial Biotechnology GmbH, Nattermannallee 1, D-50259, Köln, Germany

关键词: Thermoanaerobacter;    Lignocellulose;    Lactate;    High temperature;    Extremely thermophilic bacteria;    Ethanol;    Consolidated bioprocessing;    Caldicellulosiruptor;    Anaerobic;   

Others  :  798138
DOI  :  10.1186/1754-6834-6-31

 received in 2012-07-17, accepted in 2013-02-04,  发布年份 2013

【 摘 要 】

Background

Consolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.

Results

We have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.

Conclusions

The newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

【 授权许可】

   
2013 Svetlitchnyi et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Mussatto SI, Dragone G, Guimarães PMR, Silva JPA, Carneiro LM, Roberto IC, Vicente A, Domingues L, Teixeira JA: Technological trends, global market, and challenges of bio-ethanol production. Biotechnol Adv 2010, 28:817-830.
• [2]Farrell AE, Plevin RJ, Turner BT, Jones AD, O'Hare M, Kammen DM: Ethanol can contribute to energy and environmental goals. Science 2006, 311:506-508.
• [3]Olson DG, McBride JE, Joe Shaw A, Lynd LR: Recent progress in consolidated bioprocessing. Curr Opin Biotechnol 2011, 23:1-10.
• [4]Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW: The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 2012, 109:1083-1087.
• [5]Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 2002, 66:506-577.
• [6]La Grange DC, den Haan R, van Zyl WH: Engineering cellulolytic ability into bioprocessing organisms. Appl Microbiol Biotechnol 2010, 87:1195-1208.
• [7]Demain AL, Newcomb M, Wu JH: Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev 2005, 69:124-154.
• [8]Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC: High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microbiol 2011, 77:8288-8294.
• [9]Taylor MP, Eley KL, Martin S, Tuffin MI, Burton SG, Cowan DA: Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol 2009, 27:398-405.
• [10]Venkateswaran S, Demain AL: The Clostridium thermocellum-Clostridium thermosaccharolyticum ethanol production process: nutritional studies and scale-down. Chem Eng Commun 1986, 45:53-60.
• [11]Wang DIC, Avgerinos GC, Biocic I, Wang S-D, Fang H-Y: Ethanol from cellulosic biomass. Phil Trans R Soc Lond 1983, B300:323-333.
• [12]He Q, Hemme CL, Jiang H, He Z, Zhou J: Mechanisms of enhanced cellulosic bioethanol fermentation by co-cultivation of Clostridium and Thermoanaerobacter spp. Bioresour Technol 2011, 102:9586-9592.
• [13]Ng TK, Ben-Bassat A, Zeikus JG: Ethanol production by thermophilic bacteria: fermentation of cellulosic substrates by cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum. Appl Environ Microbiol 1981, 41:1337-1343.
• [14]Wiegel J: Formation of ethanol by bacteria. A pledge for the use of extreme thermophilic anaerobic bacteria in industrial ethanol fermentation processes. Experientia 1980, 36:1434-1446.
• [15]Willquist K, Zeidan AA, van Niel EW: Physiological characteristics of the extreme thermophile Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell factory. Microb Cell Fact 2010, 9:89. BioMed Central Full Text
• [16]Hamilton-Brehm SD, Mosher JJ, Vishnivetskaya T, Podar M, Carroll S, Allman S, Phelps TJ, Keller M, Elkins JG: Caldicellulosiruptor obsidiansis sp. nov., an anaerobic, extremely thermophilic, cellulolytic bacterium isolated from Obsidian Pool, Yellowstone National Park. Appl Environ Microbiol 2010, 76:1014-1020.
• [17]Yang S-J, Kataeva I, Hamilton-Brehm SD, Engle NL, Tschaplinski TJ, Doeppke C, Davis M, Westpheling J, Adams MWW: Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe "Anaerocellum thermophilum" DSM 6725. Appl Environ Microbiol 2009, 75:4762-4769.
• [18]Donnison AM, Brockelsby CM, Morgan HW, Daniel RM: The degradation of lignocellulosics by extremely thermophilic microorganisms. Biotechnol Bioeng 1989, 33:1495-1499.
• [19]Shaw AJ, Podkaminer KK, Desai SG, Bardsley JS, Rogers SR, Thorne PG, Hogsett DA, Lynd LR: Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc Natl Acad Sci USA 2008, 105:13769-13774.
• [20]Larsen L, Nielsen P, Ahring BK: Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland. Arch Microbiol 1997, 168:114-119.
• [21]Lee Y-E, Jain MK, Lee C, Lowe SE, Zeikus JG: Taxonomic distinction of saccharolytic thermophilic anaerobes: description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp.nov.; reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfiricum E100–69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Int J Syst Bacteriol 1993, 43:41-51.
• [22]Klinke HB, Thomsen AB, Ahring BK: Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 2004, 66:10-26.
• [23]Rainey FA, Donnison AM, Janssen PH, Saul D, Rodrigo A, Bergquisr PL, Daniel RM, Stackebrandt E, Morgan HW: Description of Caldicellulosiruptor saccharolyticusgen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiol Lett 1994, 120:263-266.
• [24]NREL: Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure (LAP). Golden CO: National Renewable Energy Laboratory; 2011.
• [25]Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY, Mitchinson C, Saddler JN: Comparative sugar recovery and fermentation data following pretreatment of poplar wood by leading technologies. Biotechnol Prog 2009, 25:333-339.
• [26]Sato K, Goto S, Yonemura S, Sekine K, Okuma E, Takagi Y, Hon-Nami K, Saiki T: Effect of yeast extract and vitamin B12 on ethanol production from cellulose by Clostridium thermocellum I-1-B. Appl Environ Microbiol 1992, 58:734-736.
• [27]Sato K, Tomita M, Yonemura S, Goto S, Sekine K, Okuma E, Takagi Y, Hon-Nami K, Saiki T: Characterization of and ethanol hyper-production by Clostridium thermocellum I-1-B. Biosci Biotech Biochem 1993, 57:2116-2121.
• [28]Wiegel J, Ljungdahl LG: Thermoanaerobacter ethanolicus gen nov., spec. nov., a new, extremely thermophilic, anaerobic bacterium. Arch Microbiol 1981, 128:343-348.
• [29]Wiegel J, Ljungdahl LG, Rawson JR: Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum. J Bacteriol 1979, 139:800-810.
• [30]Van de Werken HJ, Verhaart MR, VanFossen AL, Willquist K, Lewis DL, Nichols JD, Goorissen HP, Mongodin EF, Nelson KE, van Niel EW, Stams AJ, Ward DE, de Vos WM, van der Oost J, Kelly RM, Kengen SW: Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Appl Environ Microbiol 2008, 74:6720-6729.
• [31]VanFossen AL, Verhaart MR, Kengen SM, Kelly RM: Carbohydrate utilization patterns for the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus reveal broad growth substrate preferences. Appl Environ Microbiol 2009, 75:7718-7724.
• [32]Dam P, Kataeva I, Yang SJ, Zhou F, Yin Y, Chou W, Poole FL 2nd, Westpheling J, Hettich R, Giannone R, Lewis DL, Kelly R, Gilbert HJ, Henrissat B, Xu Y, Adams MW: Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725. Nucleic Acids Res 2011, 39:3240-3254.
• [33]Lynd LR, Baskaran S, Casten S: Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacterium thermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture. Biotechnol Prog 2001, 17:118-125.
• [34]Willquist K, Claassen PAM, van Niel EWJ: Evaluation of the influence of CO2 as stripping gas on the performance of the hydrogen producer Caldicellulosiruptor saccharolyticus. Int J Hydrogen Energy 2009, 34:4718-4726.
• [35]Chung D, Farkas J, Huddleston JR, Olivar E, Westpheling J: Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725. PLoS One 2012, 7:e43844.
• [36]Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J: Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 2013, 40:41-49.
• [37]Hungate RE: A roll tube method for cultivation of strict anaerobes. Methods Microbiol 1969, 3B:117-132.
• [38]Lane DJ: 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Edited by Stackebrandt E, Goodfellow M. Chichester: Wiley; 1991:115-175.
• [39]Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol 1987, 4:406-425.
• [40]Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24:1596-1599.
• [41]Shaw AJ, Hogsett DA, Lynd LR: Identification of the [Fe-Fe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout. J Bacteriol 2009, 191:6457-6464.