Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms.

Concentrations of biosynthate (microbial biomass plus extracellular proteins) and residual substrate were inferred using elemental analysis for batch cultures of Clostridium thermocellum. Inferring residual substrate based on elemental analysis for a cellulose (Avicel)-grown culture shows similar results to residual substrate determined by quantitative saccharification using acid hydrolysis. Inference based on elemental analysis is also compared to different on-line measurements: base addition, CO2 production, and Near Infra Red optical density (OD850 ). Of these three on-line techniques, NIR OD850 has the best correlation with residual substrate concentration and is the most practical to use. Both biosynthate and residual substrate concentration demonstrate typical sigmoidal trends that can easily be fitted with a five-parameter Richards curve. The sigmoidal character of the inferred concentrations and on-line data, especially the CO2 production rate, suggest that there is a maximum in cell-specific rates of growth and substrate utilization during batch fermentations of crystalline cellulose, which is not observed during grown on cellobiose. Using a sigmoidal fit curve, the instantaneous specific growth rate was determined. While soluble substrate grown cultures show a constant growth rate, cultures grown on solid substrate do not. Features of various approaches are compared, with some more appropriate for rapid general indication of metabolic activity and some more appropriate for quantitative physiological studies.