Abstract
BackgroundThe formation of by-products, mainly acetone in acetone–butanol–ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process. In this study, we genetically engineered Clostridium acetobutylicum XY16 isolated by our lab to eliminate acetone production and altered ABE to isopropanol–butanol–ethanol (IBE). Meanwhile, process optimization under pH control strategies and supplementation of calcium carbonate were adopted to investigate the interaction between the reducing force of the metabolic networks and IBE production.ResultsAfter successful introduction of secondary alcohol dehydrogenase into C. acetobutylicum XY16, the recombinant XY16 harboring pSADH could completely eliminate acetone production and convert it into isopropanol, indicating great potential for large-scale production of IBE mixtures. Especially, pH could significantly improve final solvent titer through regulation of NADH and NADPH levels in vivo. Under the optimal pH level of 4.8, the total IBE production was significantly increased from 3.88 to 16.09 g/L with final 9.97, 4.98 and 1.14 g/L of butanol, isopropanol, and ethanol. Meanwhile, NADH and NADPH levels were maintained at optimal levels for IBE formation compared to the control one without pH adjustment. Furthermore, calcium carbonate could play dual roles as both buffering agency and activator for NAD kinase (NADK), and supplementation of 10 g/L calcium carbonate could finally improve the IBE production to 17.77 g/L with 10.51, 6.02, and 1.24 g/L of butanol, isopropanol, and ethanol.ConclusionThe complete conversion of acetone into isopropanol in the recombinant C. acetobutylicum XY16 harboring pSADH could alter ABE to IBE. pH control strategies and supplementation of calcium carbonate were effective in obtaining high IBE titer with high isopropanol production. The analysis of redox cofactor perturbation indicates that the availability of NAD(P)H is the main driving force for the improvement of IBE production.
Highlights
The formation of by-products, mainly acetone in acetone–butanol–ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process
isopropanol– butanol–ethanol (IBE) fermentation profile by the recombinant C. acetobutylicum XY16 harboring pSADH After successful introduction of plasmid harboring pSADH into C. acetobutylicum XY16, batch fermentation without pH control was carried out to investigate the effects of sadh gene expression on solvent production and cell growth (Fig. 1)
The recombinant strain XY16 did not undergo a typical acid re-assimilation phase with the residual acetate and butyrate concentration reaching approximately 2.59 and 2.39 g/L, respectively. These results indicated that the metabolic profile of XY16 was dramatically affected by the expression of sadh gene
Summary
The formation of by-products, mainly acetone in acetone–butanol–ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process. Several solventogenic Clostridium sp., such as C. beijerinckii NRRL B-593, have shown indigenous conversion of acetone into isopropanol, which possesses higher energy density than acetone (23.9 MJ/L vs 22.6 MJ/L) and shows broader usage as fuel, solvents, and chemical intermediates. In this biological process, the secondary alcohol dehydrogenase (s-ADH) encoding by sadh gene could efficiently catalyze acetone into isopropanol, resulting in an alcohol fuel mixture of isopropanol, butanol, and ethanol (IBE) [6,7,8]. The fuel mixture in the fermentation broth has a direct end-usage as fuel additive, which would further eliminate the need for expensive recovery process and greatly improves the economic feasibility of IBE production
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