Butanol Dehydrogenase Research Articles

Enhanced butanol production from dough and okara waste through co-fermentation

Biobutanol production from waste via fermentation emerges as a promising solution for a sustainable waste-energy nexus. However, maximizing the resource potential of waste-derived feedstock for fermentation remains a hindrance to biobutanol production in the industry. To address this issue, co-fermentation of food processing waste, dough and okara, by Clostridium sp. strain BOH3 was explored to enhance biobutanol production. Results indicated that strain BOH3 generated 15.9 ± 0.90 g/L of butanol and 25.4 ± 1.43 g/L of acetone-butanol-ethanol solvents from 80 g/L co-substrate containing 85 % dough and 15 % okara, which represented a remarkable enhancement of butanol and solvent production with a notable increase of 91.6 % and 108.2 %, respectively, compared to sole fermentation of 80 g/L dough waste. In contrast, the sole fermentation of okara produced negligible amounts of solvent with acid as the dominant product due to the unsuitable carbon-to-nitrogen ratio intrinsic to okara for fermentation. Meanwhile, co-fermentation accelerated the acidogenesis-to-solventogenesis phase transition by balancing the carbon-to-nitrogen ratios and elevating the activity of amylase and butanol dehydrogenase by 1.7 folds when compared with the sole fermentation of dough waste. Findings in this study provided an effective co-fermentation strategy as well as the associated mechanistic insights regarding enhanced biobutanol production from dough and okara waste, promisingly contributing to sustainable waste-to-energy management.

Structural and Biochemical Analysis of Butanol Dehydrogenase From Thermotoga maritima.

Butanol dehydrogenase (BDH) plays a crucial role in butanol biosynthesis by catalyzing the conversion of butanal to butanol using the coenzyme NAD(P)H. In this study, we observed that BDH from Thermotoga maritima (TmBDH) exhibits dual coenzyme specificity and catalytic activity with NADPH as the coenzyme under highly alkaline conditions. Additionally, a thermal stability analysis on TmBDH demonstrated its excellent activity retention even at elevated temperatures of 80°C. These findings demonstrate the superior thermal stability of TmBDH and suggest that it is a promising candidate for large-scale industrial butanol production. Furthermore, we discovered that TmBDH effectively catalyzes the conversion of aldehydes to alcohols and exhibits a wide range of substrate specificities toward aldehydes, while excluding alcohols. The dimeric state of TmBDH was observed using rapid online buffer exchange native mass spectrometry. Additionally, we analyzed the coenzyme-binding sites and inferred the possible locations of the substrate-binding sites. These results provide insights that improve our understanding of BDHs.

Simultaneous expression of an endogenous spermidine synthase and a butanol dehydrogenase from Thermoanaerobacter pseudethanolicus in Clostridium thermocellum results in increased resistance to acetic acid and furans, increased ethanol production and an increase in thermotolerance

BackgroundSensitivity to inhibitors derived from the pretreatment of plant biomass is a barrier to the consolidated bioprocessing of these complex substrates to fuels and chemicals by microbes. Spermidine is a low molecular weight aliphatic nitrogen compound ubiquitous in microorganisms, plants, and animals and is often associated with tolerance to stress. We recently showed that overexpression of the endogenous spermidine synthase enhanced tolerance of the Gram-positive bacterium, Clostridium thermocellum to the furan derivatives furfural and HMF.ResultsHere we show that co-expression with an NADPH-dependent heat-stable butanol dehydrogenase from Thermoanaerobacter pseudethanolicus further enhanced tolerance to furans and acetic acid and most strikingly resulted in an increase in thermotolerance at 65 °C.ConclusionsTolerance to fermentation inhibitors will facilitate the use of plant biomass substrates by thermophiles in general and this organism in particular. The ability to grow C. thermocellum at 65 °C has profound implications for metabolic engineering.

Structural and Biochemical Analyses of the Butanol Dehydrogenase from Fusobacterium nucleatum.

Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that Fusobacterium nucleatum YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co2+. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co2+), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH.

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.

Oxidoreduction potential controlling for increasing the fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production

BackgroundLignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem.ResultsIn this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at − 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP+, and NADH/NAD+ were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity.ConclusionORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid “acid crash”. Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.

The mechanism of high butanol acetone ratio in ABE fermentation with fern root as substrate

The butanol acetone ratio (B/A ratio) is a critical index to evaluate the process of ABE fermentation. In this article, fern root starch with yeast extract added (FY) was used as substrate for ABE fermentation. The final B/A ratio in a 5 L anaerobic fermentor reached the highest levels of 3.42, which signified an increment of 41% compared with corn. The mechanisms for the high B/A ratio of FY substrate were as follows: 1) the weakened organic acid circuit resulted in decreased acetone synthesis; 2) the NADH level of butanol synthesis was high; 3) coA transferase activity was low and butanol dehydrogenase activity was high. The final butanol concentration and substrate utilization were examined in a 5 L anaerobic fermentor, and the results validated the feasibility of ABE fermentation with FY as substrate.

Reducing agents assisted fed-batch fermentation to enhance ABE yields

Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled cost competitive reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production, while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. The effects of ascorbic acid, L-cysteine, and dithiothreitol (DTT) on ABE fed-batch production using Clostridium acetobutylicum was investigated in current study. NADH, ATP, extracellular amino acid secretion, and NADH-dependent butanol dehydrogenase (BDH) assays were performed to study the metabolic modifications triggered by reducing agents. Incidentally, L-cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. Elevated NADH, BDH, and ATP levels in fermentation broth reflected in enhanced ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress tolerance in this study. The results demonstrate that addition of reducing agents in fed-batch ABE fermentation operation enables efficient utilization of glucose with significant improvement in solvent production.

Combination of Trace Metal to Improve Solventogenesis of Clostridium carboxidivorans P7 in Syngas Fermentation.

Higher alcohols such as butanol (C4 alcohol) and hexanol (C6 alcohol) are superior biofuels compared to ethanol. Clostridium carboxidivorans P7 is a typical acetogen capable of producing C4 and C6 alcohols natively. In this study, the composition of trace metals in culture medium was adjusted, and the effects of these adjustments on artificial syngas fermentation by C. carboxidivorans P7 were investigated. Nickel and ferrous ions were essential for growth and metabolite synthesis during syngas fermentation by P7. However, a decreased dose of molybdate improved alcohol fermentation performance by stimulating carbon fixation and solventogenesis. In response to the modified trace metal composition, cells grew to a maximum OD600nm of 1.6 and accumulated ethanol and butanol to maximum concentrations of 2.0 and 1.0 g/L, respectively, in serum bottles. These yields were ten-fold higher than the yields generated using the original composition of trace metals. Furthermore, 0.5 g/L of hexanol was detected at the end of fermentation. The results from gene expression experiments examining genes related to carbon fixation and organic acid and solvent synthesis pathways revealed a dramatic up-regulation of the Wood–Ljungdahl pathway (WLP) gene cluster, the bcs gene cluster, and a putative CoA transferase and butanol dehydrogenase, thereby indicating that both de novo synthesis and acid re-assimilation contributed to the significantly elevated accumulation of higher alcohols. The bdh35 gene was speculated to be the key target for butanol synthesis during solventogenesis.

A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

Isolation of Different Clostridium Isolates for Acetone-Butanol-Ethanol (ABE) Production from Sargassum sp.

Macroalgae with several species are an abundant, carbon-neutral renewable resource and rich in carbohydrates which make it suitable for biobutanol production. Recently, due to advantages of biobutanol as a liquid biofuel, it can be used as a substitute for gasoline and diesel. Many researches mentioned that, different Clostridium species are able to use fermentable sugars for production of biobutanol from different biomasses through ABE (Acetone- Butanol- Ethanol) fermentation process. In this study the (Sargassum sp.) was used as carbon source for biobutanol production. Thirty-three anaerobic mesophilic isolates were isolated on RCM medium from five soil cultivated with different crops. Only thirteen spore forming, mesophilic, anaerobic Clostridium isolates used for the fermentation process. 100 g/L from Sargassum sp were hydrolyzed by sulfuric acid (6 % (v/v)) followed by thermal pretreatment at 121°C for 20 minutes to produce total reducing sugars (24.151 ± 0.273 g/L), which were fermented by Clostridium isolates. The most promising isolate (HG1) which produce the highest butanol level, acetone and ethanol (3.743, 1.401 and 1.031 g/l) respectively was identified according to Butanol dehydrogenases (bdhA) gene analysis as Clostridium acetobutylicum. This paper emphasizes the importance of (Sargassum sp.) as a renewable feedstock for the biobutanol production.

Regulation of butanol biosynthesis in Clostridium acetobutylicum ATCC 824 under the influence of zinc supplementation and magnesium starvation

Present study reports modulation in butanol biosynthesis in Clostridium acetobutylicum ATCC 824 under the influence of zinc supplementation or magnesium starvation either individually or in combination. An improvement in butanol titer from 11.83 g L−1 in control to 13.72 g L−1, 15.79 g L−1, and 19.18 g L−1 was achieved when organism was grown on magnesium starved, zinc supplemented and combined zinc supplemented-magnesium starved fermentation medium, respectively. The elevation in butanol biosynthesis was associated with raised glucose utilization, reduced ethanol production and early induction of solventogenesis. Change in these phenotypic traits of the organism may be attributed to multi-level modulation in central carbon metabolism e.g., upregulation of glycolytic pathway; upregulation in thiolase activity; key intermediate enzyme for biosynthesis of acids and solvent; upregulation in the activity of butyrylaldehyde dehydrogenase & butanol dehydrogenase, the enzymes responsible for butanol biosynthesis and downregulation in alcohol dehydrogenase, redirecting carbon flux from ethanol to butanol.

Improvement of butanol production by the development and co-culture of C. acetobutylicum TSH1 and B. cereus TSH2.

Butanol fermentation comprises two successive and distinct stages, namely acidogenesis and solventogenesis. The current lack of clarity regarding the underlying metabolic regulation of fermentation impedes improvements in biobutanol production. Here, a proteomics study was performed in the acidogenesis phase, the lowest pH point (transition point), and the solventogenesis phase in the butanol-producing symbiotic system TSH06. Forty-two Clostridium acetobutylicum proteins demonstrated differential expression levels at different stages. The protein level of butanol dehydrogenase increased in the solventogenesis phase, which was in accordance with the trend of butanol concentration. Stress proteins were upregulated either at the transition point or in the solventogenesis phase. The cell division-related protein Maf was upregulated at the transition point. We disrupted the maf gene in C. acetobutylicum TSH1, and Bacillus cereus TSH2 was added to form a new symbiotic system. TSH06△maf produced 13.9 ± 1.0g/L butanol, which was higher than that of TSH06 (12.3 ± 0.9g/L). Butanol was furtherly improved in fermentation at variable temperature with neutral red addition for both TSH06 and TSH06△maf. The butanol titer of the maf deletion strain was higher than that of the wild type, although the exact mechanism remains to be determined.

Role of Trace Elements as Cofactor: An Efficient Strategy toward Enhanced Biobutanol Production

Metabolic engineering has the potential to steadily enhance product titers by inducing changes in metabolism. Especially, availability of cofactors plays a crucial role in improving efficacy of product conversion. Hence, the effect of certain trace elements was studied individually or in combinations, to enhance butanol flux during its biological production. Interestingly, nickel chloride (100 mg L–1) and sodium selenite (1 mg L–1) showed a nearly 2-fold increase in solvent titer, achieving 16.13 ± 0.24 and 12.88 ± 0.36 g L–1 total solvents with yields of 0.30 and 0.33 g g–1, respectively. Subsequently, the addition time (screened entities) was optimized (8 h) to further increase solvent production up to 18.17 ± 0.19 and 15.5 ± 0.13 g L–1 by using nickel and selenite, respectively. A significant upsurge in butanol dehydrogenase (BDH) levels was observed, which reflected in improved solvent productions. Additionally, a three-dimensional structure of BDH was also constructed using homology modeling and subsequently docked with substrate, cofactor, and metal ion to investigate proper orientation and molecular interactions.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.

High biobutanol production integrated with in situ extraction in the presence of Tween 80 by Clostridium acetobutylicum

Abstract This study investigates the improvement of butanol production in the presence – Tween 80 by Clostridium acetobutylicum NJ4. With supplementation of 15% Tween 80 (v/v), the final butanol titer could be significantly enhanced to 18.65 g/L from 70 g/L glucose, which is 38% higher than that in the control batch (13.47 g/L). With further in situ extraction using biodiesel, the butanol titer was finally improved to 33.90 g/L in the fed batch mode. The supplementation of Tween 80 could improve butanol tolerance of strain NJ4 up to 18 g/L compared to 10 g/L of control. Meanwhile, butanol dehydrogenase activity was improved to 3.89 U/mg, which is 80.93% higher than that of the control. These factors may attribute to the high butanol production. The finding of this study thus offers fundamental knowledge for the future development of alternative ABE production strategies.

Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C.saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1.

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (YP/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.

High acetone–butanol–ethanol production in pH-stat co-feeding of acetate and glucose

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao etal., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20g/L butanol and 23.27g/L ABE with a maximum specific butanol production rate of 0.26g/(gh). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13g/L butanol and 24.37g/L ABE at the high specific butanol production rate of 0.34g/(gh) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C.saccharoperbutylacetonicum N1-4.

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

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.