Abstract
BackgroundCurrently, 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.ResultsTo 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0776-6) contains supplementary material, which is available to authorized users.
Highlights
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
These remarkable features indicate that different mutant strains generated from the wild-type strain following chemical mutagenesis STTY may present different physiological characteristics when grown in STTY medium
In order to verify whether the changes in butanol of the mutant strains were resulted from the altered α-amylase activities, we further investigated the metabolic pathways that clostridium utilizes for decomposing starch
Summary
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. Targeting the acetone biosynthetic pathway and reducing or eliminating the unwanted byproducts; through this method, the butanol titer was reduced compared to the wild-type strain [8,9,10] Besides these efforts with C. acetobutylicum, there have no reports that high butanol yield was obtained through breeding with the C. beijerinckii strain to directly ferment starch crops in industrial applications. This strategy reduced shunt of metabolic precursors from competing metabolic pathways. The transformation of the Clostridium metabolic pathway via metabolic engineering approaches to obtain mutant strains that produce high yields of butanol has been very difficult due to the fact that solventogenic clostridia has complicated physiological and metabolic networks. Traditional methods, such as classic chemical mutagenesis, are still an option for an effective approach to obtain desired strains capable of producing high yields of butanol
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