Enhanced direct fermentation of cassava to butanol by Clostridium species strain BOH3 in cofactor-mediated medium

Abstract

BackgroundThe main challenge of cassava-based biobutanol production is to enhance the simultaneous saccharification and fermentation with high hyperamylolytic activity and butanol yield. Manipulation of cofactor [e.g., Ca2+ and NAD/(P)H] levels as a potential tool to modulate carbon flux plays a key role in the cassava hydrolysis capacity and butanol productivity. Here, we aimed to develop a technology for enhancing butanol production with simultaneous hydrolysis of cassava (a typical model as a non-cereal starchy material) using a cofactor-dependent modulation method to maximize the production efficacy of biobutanol by Clostridium sp. stain BOH3.ResultsSupplementing CaCO3 to the medium containing cassava significantly promotes activities of α-amylase responsible for cassava hydrolysis and butanol production due to the role of Ca2+ cofactor-dependent pathway in conversion of cassava starch to reducing sugar and its buffering capacity. Also, after applying redox modulation with l-tryptophan (a precursor as de novo synthesis of NADH and NADPH), the levels of cofactor NADH and NADPH increased significantly by 67 % in the native cofactor-dependent system of the wild-type Clostridium sp. stain BOH3. Increasing availability of NADH and NADPH improved activities of NADH- and NADPH-dependent butanol dehydrogenases, and thus could selectively open the valve of carbon flux toward the more reduced product, butanol, against the more oxidized acid or acetone products. By combining CaCO3 and l-tryptophan, 17.8 g/L butanol with a yield of 30 % and a productivity of 0.25 g/L h was obtained with a hydrolytic capacity of 88 % towards cassava in a defined medium. The metabolic patterns were shifted towards more reduced metabolites as reflected by higher butanol–acetone ratio (76 %) and butanol–bioacid ratio (500 %).ConclusionsThe strategy of altering enzyme cofactor supply may provide an alternative tool to enhance the stimulation of saccharification and fermentation in a cofactor-dependent production system. While genetic engineering focuses on strain improvement to enhance butanol production, cofactor technology can fully exploit the productivity of a strain and maximize the production efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0351-7) contains supplementary material, which is available to authorized users.

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol

“…l -Tryptophan, a precursor in de novo synthesis of NADH and NADPH, induces redox modulation by the highly transcribed tryptophan biosynthesis genes and triggers the availabilities of NADH and NADPH. Therefore, the NADH-dependent Bdh activities may be enhanced by triggering de novo synthesis of cofactors via supplementation of the precursor l -tryptophan, demonstrating that salvaging cofactors (for example, NADH and NADPH) is a major approach to elevating overexpression of the bdh gene and, thus, to redistributing the metabolic flux from the bioacid synthesis pathway to the central NAD/(P)H-dependent butanol pathway ( 45 ).…”

Simultaneous saccharification and fermentation of hemicellulose to butanol by a non-sporulating Clostridium species

“…In vitro activity tests were performed inside an anaerobic chamber. Cell extraction, solventogenic enzyme activity assays, and determination of protein concentration in supernatant and cell extracts were conducted as described previously (Li et al, 2015). Xylanase activity was assayed by measuring the amount of reducing sugar released from beechwood xylan.…”

“…Extraction of genomic DNA or RNA from active cultures was performed as described previously (Li et al, 2015). Genomic DNA was used for enumeration of cells in the culture by quantitative PCR (qPCR) (ABI 7500 Fast Real-Time PCR system, Foster, CA, USA).…”

Enhanced isopropanol–butanol–ethanol mixture production through manipulation of intracellular NAD(P)H level in the recombinant Clostridium acetobutylicum XY16

“…Intracellular concentrations of NAD(P)H and NAD(P) + were assayed using the cycling method [ 22 , 40 ]. Briefly, cells were harvested by centrifugation at 12,000 rpm and 4 °C.…”