Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii

  • Joel Farkas
  • Daehwan Chung
  • Minseok Cha
  • Jennifer Copeland
  • Philip Grayeski
  • Janet Westpheling
Bioenergy/Biofuels/Biochemicals

Abstract

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD680) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.

Keywords

Caldicellulosiruptor Biomass utilization Defined growth media 

Notes

Acknowledgments

We thank Irina Kataeva for helpful discussions during the course of this work. This work was supported by The BioEnergy Science Center supported by a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. JF was supported by a predoctoral Graduate Training In Genetics grant (NIH 5T32GM007103-30) to the Genetics Department of the University of Georgia.

Supplementary material

10295_2012_1202_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 13 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2012

Authors and Affiliations

  • Joel Farkas
    • 1
    • 2
  • Daehwan Chung
    • 1
    • 2
  • Minseok Cha
    • 1
    • 2
  • Jennifer Copeland
    • 1
    • 2
  • Philip Grayeski
    • 1
    • 2
  • Janet Westpheling
    • 1
    • 2
  1. 1.Department of GeneticsUniversity of GeorgiaAthensUSA
  2. 2.The BioEnergy Science Center, Department of EnergyOak Ridge National LaboratoryOak RidgeUSA

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