Abstract
A growth medium was developed for cultivation of Clostridium thermocellum ATCC 27405 in which “background” carbon present in buffers, reducing agents, chelating agents, and growth factors was a small fraction of the carbon present in the primary growth substrate. Background carbon was 1.6% of primary substrate carbon in the low-carbon (LC) medium, whereas it accounts for at least 40% in previously reported media. Fermentation of cellulose in LC medium was quite similar to Medium for Thermophilic Clostridia (MTC), a commonly used growth medium that contains background carbon at 88% of primary substrate carbon. Of particular note, we found that the organism can readily be cultivated by eliminating some components, lowering the concentrations of others, and employing a tenfold lower concentration of reducing agent. As such, we were able to reduce the amount of background carbon 55-fold compared to MTC medium while reaching the same cell biomass concentration. The final mass ratios of the products acetate:ethanol:formate were 5:3.9:1 for MTC and 4.1:1.5:1 for LC medium. LC medium is expected to facilitate metabolic studies involving identification and quantification of extracellular metabolites. In addition, this medium is expected to be useful in studies of cellulose utilization by anaerobic enrichment cultures obtained from environmental inocula, and in particular to diminish complications arising from metabolism of carbon-containing compounds other than cellulose. Finally, LC medium provides a starting point for industrial growth media development.
References
Atlas R (2004) Handbook of microbiological media, 3rd edn. CRC press, Boca Raton, FL
Fleming RW, Quinn LY (1971) Chemically defined medium for growth of Clostridium thermocellum, a cellulolytic thermophilic anaerobe. Appl Microbiol 21:967
Garcia-Martinez DV, Shinmyo A, Madia A, Demain AL (1980) Studies on cellulase production by Clostridium-thermocellum. Eur J Appl Microbiol Biotechnol 9:189–197. doi:10.1007/BF00504485
Glusker J (1980) Citrate conformation and chelation: enzymatic implications. Acc Chem Res 13:345–352. doi:10.1021/ar50154a002
Guedon E, Payot S, Desvaux M, Petitdemange H (1999) Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium. J Bacteriol 181:3262–3269
Hogsett D (1995) Cellulose hydrolysis and fermentation by clostridium thermocellum for the production of ethanol. Dartmouth College, Hanover
Johnson EA, Madia A, Demain AL (1981) Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum. Appl Environ Microbiol 41:1060–1062
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Molec Biol Rev 66:739. doi:10.1128/mmbr.66.4.739.2002
McBee RH (1948) The culture and physiology of a thermophilic cellulose-fermenting bacterium. J Bacteriol 56:653–663
McBee RH (1950) The anaerobic thermophilic cellulolytic bacteria. Bacteriol Rev 14:51–63
Ozkan M, Desai SG, Zhang Y, Stevenson DM, Beane J, White EA, Guerinot EL, Lynd LR (2001) Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria. J Ind Microbiol Biotechnol 27:275–280. doi:10.1038/sj.jim.7000082
Payot S, Guedon E, Cailliez C, Gelhaye E, Petitdemange H (1998) Metabolism of cellobiose by Clostridium cellulolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth. Microbiol 144:375–384
Quinn LY (1949) Isolation and morphological study of a thermophilic cellulose digesting organism. Purdue University, Lafayette, Indiana
Raman B, McKeown CK, Rodriguez M, Brown SD, Mielenz JR (2011) Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation. BMC Microbiol 11:134. doi:10.1186/1471-2180-11-134
Raman B, Pan C, Hurst GB, Rodriguez M Jr, McKeown CK, Lankford PK, Samatova NF, Mielenz JR (2009) Impact of pretreated switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis. PLoS ONE 4:e5271. doi:10.1371/journal.pone.0005271
Saeman JF, Bubl JL, Harris EE (1945) Quantitative saccharification of wood and cellulose. Ind Eng Chem Anal Ed 17:35–37. doi:10.1021/i560137a008
Sluiter A, Hames B, Ruiz R, Scarlata C, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. Technical Report NREL/TP-510-42618
Weimer PJ, Zeikus JG (1977) Fermentation of cellulose and cellobiose by Clostridium-thermocellum in absence and presence of Methanobacterium-thermoautotrophicum. Appl Environ Microbiol 33:289–297
Zhang YH, Lynd LR (2003) Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation: development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures. Anal Chem 75:219–227. doi:10.1021/ac020271n
Zhang YHP, Lynd LR (2005) Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum. J Bacteriol 187:99–106. doi:10.1128/jb.187.1.99-106.2005
Acknowledgments
Primary support for this work was from the Bioenergy Science Center (BESC), a U.S. Department of Energy (DOE) Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Additional support was provided by Mascoma Corporation, in which LL has a financial interest. We acknowledge Dr. Julie Paye, Dr. Xiongjun Shao, Dr. Douwe van der Veen and Dr. Javier Izquierdo for useful discussions and advice. All authors have agreed to submit this manuscript to the “Journal of Industrial Microbiology and Biotechnology”.
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Holwerda, E.K., Hirst, K.D. & Lynd, L.R. A defined growth medium with very low background carbon for culturing Clostridium thermocellum . J Ind Microbiol Biotechnol 39, 943–947 (2012). https://doi.org/10.1007/s10295-012-1091-3
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DOI: https://doi.org/10.1007/s10295-012-1091-3