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 (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.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

“…To determine the concentration at which growth of C. bescii is inhibited by ethanol, wild-type C. bescii was grown in low osmolarity-defined (LOD) medium (27) with 1% cellobiose as the sole carbon source, and subjected to different levels of added ethanol at both 65°C and 75°C (Fig. S1).…”

“…C. bescii strains and plasmids used in this study are listed in Table 1. All C. bescii strains were grown anaerobically in liquid or on solid surface in LOD medium (27), final pH 7.0, with maltose [0.5% (wt/vol); catalog no. M5895, Sigma) as the carbon source unless otherwise noted.…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…Strains of C. bescii were grown on the glucose-containing modified DSM 516 (CG516) medium containing 20 M uracil, as previously described (5). For sequencing analysis, cultures were grown in 500 ml of cellobiosecontaining low-osmolarity complex (LOC) medium, as described previously (28). The 500-ml cultures for sequencing analysis were grown overnight statically at 70°C under anaerobic conditions.…”

“…S1 and was sequence verified. Competent cells were prepared by growing 500-ml cultures in LOD medium with amino acids (28) and washed with 10% (wt/vol) sucrose, as described previously (5). Cells were mixed with 0.5 to 1 g of plasmid DNA and transferred to a 1-mm-gap electroporation cuvette, and electroporation was carried out as described previously (5).…”

“…Recovery was in 20 ml of LOC medium at various intervals, 1-ml samples of recovery cultures were centrifuged for 1 min at 14,000 rpm, and the supernatant was removed to minimize uracil carryover to selective medium. Cell pellets were resuspended in 0.2 ml of selective LOD medium without uracil supplementation and transferred to the selective medium (28). Transformation isolates were purified once on solid LOD medium without uracil and screened for complete plasmid insertion at the ΔcbeI locus.…”