Investigation of availability of a high throughput screening method for predicting butanol solvent -producing ability of Clostridium beijerinckii

HaiFeng Su, Jun Zhu, Gang Liu, Furong Tan

Currently, efficient screening methods for selection of desired bacterial phenotypes from large populations are not easy feasible or readily available due to the complicated physiological and metabolic networks of solventogenic clostridia. In this study, to contribute to the improvement of methods for predicting the butanol-producing ability of Clostridium beijerinckii based on starch substrate, we further investigate a simple, visualization screening method for selecting target strains from mutant library of Clostridium beijerinckii NCIMB 8052 by using trypan blue dye as an indicator in solid starch via statistical survey and validation of fermentation experiment with controlling pH. To verify an effective, efficient phenotypic screening method for isolating high butanol-producing mutants, the revalidation process was conducted based on Trypan Blue was used for visualization, and starch was used as the bacterial metabolic substrate. The availability of the screening system was further evaluated based on the relationship between characteristics of mutant strains and their α-amylase activities. Mutant clones were analyzed in detail based on their distinctive growth patterns and rate of fermentation of soluble starch to form butanol and were compared by statistical method. Significant correlations were identified between colony morphology and changes in butanol concentrations. The screening method was validated via statistical analysis for characterizing phenotypic parameters. The fermentation experiment of mutant strains with controlling pH value also demonstrated a positive correlation between increased α-amylase activity and increased solvent production by Clostridium beijerinckii was observed, and therefore indicated that the trypan blue dyeing method can be used as a fast method to screen target mutant strain for better solvent producers from, for instance, a mutant library. The suitability of the novel screening procedure was validated, opening up a new indicator of approach to select mutant solventogenic clostridia with improved fermentation of starch to increase butanol concentrations. The applicability can easily be broadened to a wide range of interesting microbes such as cellulolytic or acetogenic microorganisms, which produce biofuels from feedstock rich in starch.