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

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 (OD(680)) 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.

Fig. 1

Growth of C. bescii in various media measured by optical density at 680 nM. a Modified defined DSMZ 640 medium with 0 g/l (blue), 0.5 g/l (red), or 1 g/l (green) sodium sulfide. b Sulfide-free modified defined DSMZ 640 with casein and amino acids (blue), casein only (red), amino acids only (green), or neither (purple). c Defined modified DSMZ 640 without sulfide, casein, or amino acids with 0 μM (blue), 1 μm (red), 10 μM (green), 100 μM (purple), 1 mM (aqua), or 2.4 mM (orange) phosphate. d Modified defined DSMZ 640 medium without sulfide, casein, or amino acids, 10 μM phosphate and 11.9 mM sodium bicarbonate, 5.7 mM cysteine (blue) or 23.8 mM sodium bicarbonate, 17.1 mM cysteine (red). Error bars represent standard deviation and each point is the average of three replicates

Fig. 2

Relationship between cell number and OD₆₈₀. Cell concentration is linearly related to OD₆₈₀. OD = 1 corresponds to a cell density of 10⁹ cells/ml. R² = 0.977

Fig. 3

NaCl tolerance in defined modified DSMZ 640 [7] and LOD media. Growth measured by optical density at 680 nM. a Defined modified DSMZ 640 (~150 mOsm), without added NaCl (blue), and with NaCl added to ~170 mOsm (red), ~190 mOsm (green), ~210 mOsm (purple), ~230 mOsm (aqua), and ~250 mOsm (orange). b LOD medium with NaCl added to ~170 mOsm (red), ~190 mOsm (green), ~210 mOsm (purple), ~230 mOsm (aqua), and ~250 mOsm (orange). Error bars represent standard deviation and each point is the average of three replicates

Fig. 4

Growth of C. bescii in low osmolarity defined (LOD) media (blue) and low osmolarity complex (LOC) media (red). Error bars represent standard deviation and each point is the average of three replicates