Glycerol Utilization as a Sole Carbon Source Disrupts the Membrane Architecture and Solventogenesis in Clostridium beijerinckii NCIMB 8052

Efficient bioconversion of abundant waste glycerol to value-added chemicals calls for a wider range of fermentative workhorses that can catabolize glycerol. In this study, we used quantitative gene expression and solvent profiling, qualitative metabolite analysis, and enzyme activity assays to investigate the factors that limit glycerol utilization as a sole carbon source by Clostridium beijerinckii NCIMB 8052. C. beijerinckii NCIMB 8052 did not produce acetate, acetone and butanol on glycerol. Congruently, the genes encoding the coenzyme A transferase subunits (ctfAB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE) were down-regulated up to 135- and 21-fold, respectively, at 12 h in glycerol-grown cells compared to glucose-grown cells. Conversely, NADH-dependent butanol dehydrogenase A (bdhA) was upregulated 2-fold. Glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (subunit dhaK) were upregulated up to 5- and 881-fold, respectively. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) showed mostly similar expression profiles at 12 h on glucose and glycerol. At 24 h, gapdh was downregulated 1.5-fold, while NADP⁺-dependent gapdh was upregulated up to 1.9-fold. Glycerol-grown cells showed higher or similar activity profiles for all solventogenic enzymes studied, compared to glucose-grown cells. Butyraldehyde (3 g/L) supplementation led to the production of ~0.1 g/L butanol, whilst butyrate (3.5 g/L) supplementation produced 0.7 and 0.5 g/L acetone and butanol, respectively, with glycerol. Further, the long chain saturated fatty acids cyclopentaneundecanoic acid, methyl ester and hexadecanoic acid, butyl ester were detected in glucose- but not in glycerol-grown cells. Collectively, growth on glycerol appears to disrupt synthesis of saturated long chain fatty acids, as well as solventogenesis in C. beijerinckii NCIMB 8052.