Abstract
Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T opt = 55 °C), Caldicellulosiruptor obsidiansis (T opt = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T opt = 100 °C) and Thermotoga maritima (T opt = 80 °C). Multi-well plates were incubated at various temperatures for approximately 72–120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD600. Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.
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Acknowledgments
We thank Sue Carroll and Marilyn Kerley who provided excellent technical assistance for many of the procedures described in this chapter. Jennifer J. Moser provided additional metabolite analysis. Martin Keller and Anthony V. Palumbo participated in helpful discussions. We also thank the National Park Service for coordinating and allowing sampling under permit YELL-2008-SCI-5714.
This work was supported by the BioEnergy Science Center (BESC), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science, Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US Department of Energy under contract DE-AC05-00OR22725.
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Hamilton-Brehm, S.D., Vishnivetskaya, T.A., Allman, S.L., Mielenz, J.R., Elkins, J.G. (2012). Anaerobic High-Throughput Cultivation Method for Isolation of Thermophiles Using Biomass-Derived Substrates. In: Himmel, M. (eds) Biomass Conversion. Methods in Molecular Biology, vol 908. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-956-3_15
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DOI: https://doi.org/10.1007/978-1-61779-956-3_15
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