Acetone-butanol-ethanol Production Research Articles

Pilot-scale acetone-butanol-ethanol fermentation from corn stover

Biobutanol is an advanced biofuel that can be produced from excess lignocellulose via acetone-butanol-ethanol (ABE) fermentation. Although significant technological progress has been made in this field, attempts at large-scale lignocellulosic ABE production remain scarce. In this study, 1 m3 scale ABE fermentation was investigated using high inhibitor tolerance Clostridium acetobutylicum ABE-P1201 and steam-exploded corn stover hydrolysate (SECSH). Before expanding the fermentation scale, the detoxification process for SECSH was simplified by process engineering. Results revealed that appropriate pH management during the fed-batch cultivation could largely decrease the inhibition of the toxic components in undetoxified SECSH to the solventogenesis phase of the ABE-P1201 strains, avoiding ‘acid crash’. Therefore, after naturalizing the pH by Ca(OH)2, the undetoxified SECSH, without removal of the solid components, reached 17.68±1.30g/L of ABE production with 0.34±0.01g/g of yield in 1L scale bioreactor. Based on this strategy, the fermentation scale gradually expanded from laboratory-scale apparatus to pilot-scale bioreactors. Finally, 17.05±1.20g/L of ABE titer and 0.32±0.01g/g of ABE yield were realized in 1 m3 bioreactor, corresponding to approximately 145kg of ABE production from 1 t of dry corn stover. The pilot-scale ABE fermentation demonstrated excellent stability during repeated operations. This study provided a simplified ABE fermentation strategy and verified the feasibility of the pilot process, providing tremendous significance and a solid foundation for the future industrialization of second-generation ABE plants.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone–butanol–ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Formate as a supplementary substrate facilitates sugar metabolism and solvent production by Clostridium beijerinckii NCIMB 8052

Microbial utilization and conversion of organic one-carbon compounds, such as formate and methanol that can be easily produced from CO2, has emerged as an attractive approach for biorefinery. In this study, we discovered Clostridium beijerinckii NCIMB 8052, a typical solventogenic Clostridium strain, to be a native formate-utilizing bacterium. 13C isotope analysis showed that formate could be metabolized via both assimilation and dissimilation pathways in C. beijerinckii NCIMB 8052. Notably, the use of formate as the supplementary substrate by this strain could significantly enhance its glucose consumption and ABE (acetone-butanol-ethanol) production, largely due to the up-regulation of genes responsible for glycolysis and glucose transport under formate stress. Based on these findings, we further improved formate tolerance of C. beijerinckii NCIMB 8052 by adaptive laboratory evolution, generating an evolved strain Cbei-FA01. The Cbei-FA01 strain could produce 23.0 g/L of ABE solvents using glucose and formate as dual substrates, ∼50% higher than that of the wild-type strain under the same condition. Moreover, such a promotion effect of formate on ABE production by Cbei-FA01 was also observed in fermenting a glucose-xylose mixture. This work reveals a previously unreported role of formate in biological ABE production, providing a new approach to utilize this one-carbon source.

Biobutanol production from cocoa pod husk through a sequential green method: Depectination, delignification, enzymatic hydrolysis, and extractive fermentation

The effect of various treatments on the production of reducing sugar and biobutanol from cocoa pod husk (CPH) through depectination, delignification, enzymatic hydrolysis, and fermentation was studied to obtain the best strategy for sequential processes to valorize CPH to biobutanol. Surfactants of Tween 80, Polyethylene Glycol (PEG) 6000, and Sodium Dodecyl Sulfate (SDS) was added to enhance the performance of enzymatic hydrolysis, and L-cysteine addition to support fermentation by Clostridium saccharoperbutylacetonicum N1–4. The optimum activity of the cellulase was at 0.25 FPU/g. Depectination, microwave-assisted delignification, and 2.5 g/L PEG 6000 addition sequence produced the highest sugar concentration of 9.24 g/L. The highest butanol and acetone-butanol-ethanol (ABE) concentrations of 20.4 g/L and 54.4 g/L, respectively, were obtained using immobilized cells and L-cysteine supplementation. ABE production by extractive fermentation was enhanced using immobilized cells and L-cysteine supplementation.

Waste-to-fuel technologies for the bioconversion of carrot discards into biobutanol

Carrot discard was evaluated as a raw material for acetone-butanol-ethanol (ABE) fermentation. Different strategies based on hydrothermal pretreatment and/or enzymatic hydrolysis were compared for biobutanol production from carrot discard pulp. In addition, the use of different types of enzymes and diverse enzyme mixtures were evaluated. In this way, total sugar recoveries of up to 76%, and butanol and ABE concentrations of 7.4 and 11.5 g/L, respectively (74 g butanol and 115 g ABE/kg carrot pulp), were achieved when the carrot discard pulp was enzymatically hydrolyzed, without pretreatment, using a mixture of enzymes of Cellic CTec2 and Viscozyme L at a dosage of 0.1 and 0.2 g/g, respectively. When a hydrothermal pretreatment was applied, a total sugar recovery of 88%, 6.9 g/L butanol and 10.1 g/L ABE (69 g butanol and 101 g ABE/kg carrot pulp) were attained using the same mixture of enzymes. In this way, no hydrothermal pretreatment would be necessary to produce ABE from carrot discard, which is very interesting for the profitability of the process. Furthermore, the carrot discard juice yielded 6.4 and 9 g/L butanol and ABE, respectively, showing that all the carrot discards could be used for ABE production.

Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2g/L vs. 16.8g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.

Combination of pH adjusting and intermittent feeding can improve fermentative acetone-butanol-ethanol (ABE) production from steam exploded corn stover

Steam explosion (SE) is an effective and industrially scalable lignocellulose pretreatment method that can be used for second-generation acetone-butanol-ethanol (ABE) fermentation. Nevertheless, a series technical barrier including the high inhibitors content in hydrolysate, and low concentration and low yield of ABE production always hindered the availability of the SE technique. In this study, aiming to solve the above obstacles and boosting cellulosic ABE production, the ABE fermentation process was intensified by a novel fed-batch strategy that combined the pH adjusting with intermittent feeding. Results revealed that maintaining the pH at 6.5 for initial 12 h of cultivation ensures the smoothly shifting of the acidogenesis phase to solventogenesis phase of the C. acetobutylicum cells, so that avoided the acid crash. While the lag period of fermentation was obvious shorted by intermittent feeding the concentrated SE hydrolysate into the dilute broth. Consequentially, 17.75 g/L of ABE (containing 11.75 g/L of butanol) can be obtained after 48 h of inoculation, with the yield and productivity of ABE were 0.36 g/g (of sugars) and 0.37 g/L‧h, respectively. Correspondingly, 194.9 g of ABE (including 129.0 g of butanol) can be produced from 1 kg of dried corn stover. Therefore, the novel process intensifying method could obviously improve the biobutanol production, which showed high potential and applicability in large-scale ABE productions.

Reusing the acetone-butanol-ethanol separated broth as the lignocellulose pretreatment liquor for fresh corn stalk biorefinery

In this study, fresh corn stalk was used as the feedstock for the production of bio-butanol via acetone-butanol-ethanol (ABE) fermentation, in which process the wastewater from the first stage sugar containing juice fermentation was recycled by ex situ pervaporation and reused as the pulping liquor of the residual corn stalk bagasse (CSB), affording to effective ABE production in the subsequential second stage fermentation process. Results indicated that the juice fermentation wastewater could effectively depolymerize the CSB. Under optimized conditions of the pulping process (180 °C and 10% (w/v) of biomass loading), 32.45 ± 0.85 g/L of total monomer sugars was obtained in CSB enzymatic hydrolysate, which can be directly used as the substrate for the second-stage cellulosic ABE production. Semi-simultaneous saccharification and fermentation (S-SSF) was carried out using the CSB pulps, so that improving the cellulosic ABE production. 10.94 ± 0.65 g/L of ABE concentration with 0.18 ± 0.01 g/L h of productivity were realized. After hybrid the first-stage juice fermentation and the second-stage CSB biotransformation, total 54.07 ± 1.25 g (ABE) can be produced from 1 kg of fresh corn stalk. The alternative process showed great significance in reduction of the wastewater discharge and ensuring high pulping performances.

Post-hydrolysis of cellulose oligomers by cellulase immobilized on chitosan-grafted magnetic nanoparticles: A key stage of butanol production from waste textile

The recently developed technologies for immobilization of cellulase may address the challenges in costly hydrolysis of cellulose for cellulosic butanol production. In this study, a “hybrid” hydrolysis was developed based on chemical hydrolysis of cellulose to its oligomers followed by enzymatic post-hydrolysis of the resulting “soluble oligomers” by cellulase immobilized on chitosan-coated Fe3O4 nanoparticles. This hybrid hydrolysis stage was utilized in the process of biobutanol production from a waste textile, jeans waste, leading to selective formation of glucose and high yield of butanol production by Clostridium acetobutylicum. After validating the immobilization process, the optimum immobilization parameters including enzyme concentration and time were achieved on 8 h and 15.0 mg/mL, respectively. The reusability of immobilized enzyme showed that immobilized cellulase could retain 51.5% of its initial activity after three times reuses. Dilute acid hydrolysis of regenerated cellulose at 120–180 °C for 60 min 0.5–1.0% phosphoric acid led to less than 10 g/L glucose production, and enzymatic post-hydrolysis of the oligomers resulted in up to 51.5 g/L glucose. Fermentation of the hydrolysate was accompanied by 5.3 g/L acetone-butanol-ethanol (ABE) production. The simultaneous co-saccharification and fermentation (SCSF) of soluble and insoluble oligomers of cellulose led to 17.4 g/L ABE production.

Efficient coproduction of butanol, ethanol, and biohydrogen from municipal solid waste through a cocultivated biorefinery

The cocultivation of Clostridium acetobutylicum and Mucor indicus was evaluated for the first time to take the advantages of both microorganisms for fully exploiting the energy potential of the organic fraction of municipal solid waste (OFMSW), generating high yields of acetone-butanol-ethanol (ABE) and hydrogen through a biorefinery scheme. Unlike M. indicus, C. acetobutylicum can ferment unhydrolyzed starch and pentose sugars. However, the low biofuel titer through ABE fermentation makes the bioprocess uneconomical. The fungal-bacterial symbiosis led to high ethanol titer along with high butanol and hydrogen yields without the need for starch hydrolysis. The effects of most influencing factors, i.e., initial pH, bacterium to fungus biomass ratio, and solid loading, on ABE and hydrogen production were investigated. The fermentation of organosolv pretreated OFMSW hydrolysate at initial pH of 5.5 and solid loading of 5% with 1:1 bacterium to fungus biomass ratio produced 80.2 g butanol, 30.2 g acetone, 108.1 g ethanol, and 96.3 L hydrogen from each kg raw OFMSW. From the bioenergy point of view, 7.2 MJ energy was generated, which was significantly higher than those obtained via the mono cultivation of C. acetobutylicum (6.2 MJ) and M. indicus (4.4 MJ), indicating the great potential of the fungal-bacterial cocultivation for bioenergy production from OFMSW. The economic potential of the cocultivated biorefinery was 0.16 $/kg OFMSW.

Reduction wastewater discharge in second-generation acetone-butanol-ethanol (ABE) fermentation process by adsorptive removal of organic acids toward the broth recycling system

The treatment of high chemical oxygen demand (COD) organic wastewater is one of the major technical bottlenecks for the industrialization of second-generation biobutanol production. An effective strategy to decrease the wastewater discharge in biobutanol fermentation process is broth recycling (BR). Nevertheless, the conventional BR process suffered from server organic-acid by-products inhibition, causing low butanol yield. In this study, to solve the barrier, active carbon (AC) adsorption system was hybrid with the acetone-butanol-ethanol (ABE) fermentation and ex-situ product separation (ESPR) units. After fermentation and pervaporation, organic acids that remaining in the ABE-separated broth were partially adsorbed onto AC so that improve the continuity and efficiency of the BR process. The physicochemical characterizations of different types of commercial ACs and the organic acids adsorption efficiency was evaluated by analyzing the static and dynamic adsorptions kinetics. When adopting the DD-type AC (code C299105), the better adsorbent for organic acids removal, 87.50% of fermentative wastewater can be saved whilst the ABE concentrations were also kept constantly at 15.22 ± 0.45 g L−1 in the following cycles of fermentation, and the ABE productivity was almost no changed (0.21 ± 0.01 g L−1 h−1) after 3 cycles of operation. Therefore, the AC assisted ESPR process is an attractive method for ABE production with low wastewater discharge.

Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone-butanol-ethanol production.

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n-butanol production. The combined expression of StSUT1, encoding a sucrose transporter from potato (Solanum tuberosum), and SUC2, encoding a sucrose invertase from Saccharomyces cerevisiae, remarkably enhanced n-butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium. The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

Combining acetone-butanol-ethanol production and methyl orange decolorization in wastewater by fermentation with solid food waste as substrate

Simultaneously producing acetone-butanol-ethanol (ABE) and decolorizing Methyl orange (MO) by fermentation with solid food waste (SFW) was studied. During the batch fermentation with MO concentration from 100 mgL−1 to 1000 mgL−1, carbon source utilization efficiency was above 93% with yields of acetone, butanol and ethanol being 0.13 gg-1, 0.23 gg-1 and 0.04 gg-1, respectively. During the repeated fermentation with the bagasse as the cell immobilization material, carbon source utilization efficiencies were all above 90% in the first 5 cycles fermentation. The maximum butanol and ABE concentration were 12.8 gL-1 and 22.1 gL-1, respectively. The solvent yield of acetone, butanol and ethanol were around 0.13 gg-1, 0.25 gg-1 and 0.06 gg-1, respectively. The MO decolorization had no negative effect on the ABE production and the decolorization efficiencies were all above 99% during batch fermentation or repeated fermentation with electrons transferred from intracellular to extracellular by mediator with the help of azo reductase to decolorize MO.

Efficient production of acetone butanol ethanol from sole fresh and rotten potatoes by various Clostridium strains

Efficient production of acetone butanol ethanol from sole fresh and rotten potatoes by various Clostridium strains

Acetone, Butanol, Ethanol and, Xylitol Production Through a Biorefinery Platform: An Experimental & Simulation Approach

Butanol is an interesting biofuel and a product precursor, that could be obtained with acetone and ethanol via fermentation. The biofuels production has been identified as not economically competitive, thus, the parallel production of high value-added products, such as xylitol, could be an alternative to improve the profit. Xylitol can be produced from xylose, which might be considered as a coproduct in a second generation biorefinery. This study presents a systematic biorefinery process design for the simultaneous acetone, butanol, ethanol (ABE) and xylitol production, based on experimental and simulation approaches. Experiments were performed for the pretreatment of sugarcane bagasse and ABE fermentation. The simulation part used the experimental results and experimental data from literature, to perform rigorous calculations of the ABE and xylitol production process. The economic analysis (EA) was performed relying on some indicators such as, the net present value (NPV) and payback period (PBP); EA includes several scenarios for producing only ABE and some scenarios for simultaneous ABE and xylitol production. The results showed that the combined butanol and xylitol production could reduce by 17% the selling price of butanol, compared with only producing butanol. The study also included the combustion of residual solids and carbon dioxide depletion analyses. This approach illustrates the opportunity to perform a rigorous techno-economic analysis, to identify the feasibility of the process at industrial scale, based on realistic data. This approach was implemented for ABE and xylitol production, but it can be used to any other bioproduct.

Recent innovations for reviving the ABE fermentation for production of butanol as a drop-in liquid biofuel

Butanol is a key microbial product that provides a route from renewable carbohydrate resources to a "drop-in" liquid biofuel, broadening its market in the near future. The acceptable performance of butanol as a neat or a blended fuel in different engines both from the technical and environmental points of view has attracted a wide range of research for reviving the old acetone-butanol-ethanol (ABE) fermentation. In this review, recent findings on fuel characteristics of butanol, different generations of substrate for large scale butanol production, and alternative process designs for upstream, mainstream, and downstream operations have been critically reviewed and discussed. In the upstream, studies devoted to designing and optimization of pretreatments based on prerequisites of butanol production, e.g., maximizing cellulose and hemicellulose recovery and minimizing lignin degradation, are presented. In the mainstream, different microbial systems and process integrations developed for facilitating ABE production (e.g., in-situ butanol removal) are scrutinized. Finally, innovations in ABE recovery and purification as "Achilles Heel" of butanol production processes which directly controls the energy return on investment (EROI), are reviewed and discussed.

Co-generation of acetone-butanol-ethanol and lipids by a sequential fermentation using Clostridia acetobutylicum and Rhodotorula glutinis, spaced-out by an ex-situ pervaporation step

Large amounts of effluent discharge and a costly wastewater treatment hinder the scale-up of the lignocellulosic acetone-butanol-ethanol (ABE) fermentation process. By recycling the broth after solvents removal and using it in the subsequent cycles of biomass hydrolysis and fermentation, the overall amount of effluent can be reduced. However, the ABE production is severely inhibited by the accumulated acids and other toxic by-products when reusing the cycled streams. In this study, a pervaporation assisted sequential fermentation was performed. The ABE liquor recovered after batch fermentation and ex situ pervaporation was used as the buffer for corn stover pulp hydrolysis and the following microbial lipids fermentation. In microbial lipids fermentation stage, toxic acid by-products remained in the liquor can be co-utilized as a substrate. The acids eliminated post-harvested broth was further cycled as the buffer for pulp hydrolysis and ABE fermentation. As a result, ABE yield of 0.349 g/g and concentration of 14.40 g/L were achieved after 3 cycles of operation, which show only 3.86% and 14.98% reduction compared to those of the initial cycle. Remarkably, the effluent discharge was reduced by 92.20% after applying the hybrid two-stage sequential fermentation. Meanwhile, 8.20 g/L of microbial lipids can be co-generated. The novel process offers an environmentally friendly strategy for the co-production of ABE and microbial lipids from lignocelluloses under the concept of biorefinery.

Biological production of hydrogen and acetone- butanol-ethanol from sugarcane bagasse and rice straw using co-culture of Enterobacter aerogenes and Clostridium acetobutylicum

Production of hydrogen and acetone-butanol-ethanol (ABE) from hydrolysates of sugarcane bagasse (SCB) and rice-straw (RS) was carried out via dark fermentation using co-culture of Enterobacter aerogenes and Clostridium acetobutylicum. The best improvement in the yield of hydrogen and total ABE production were observed for alkaline pretreated RS at 100 °C for 60 min obtained from the highest total reducing sugars concentration of 40.7 g L−1 after the enzymatic hydrolysis. The hydrolysates of alkaline pretreated samples at 100 °C were efficiently converted to H2 by co-culture of Enterobacter aerogenes and Clostridium acetobutylicum with the highest hydrogen yield of 136.3 and 149.9 mL H2 g−1-VS of SCB and RS, representing 3.4 and 1.5-fold enhancement in the hydrogen production yield over that obtained from the pure cultures of Enterobacter aerogenes and Clostridium acetobutylicum, respectively. Mass balance and energy generation analysis indicate that the highest specific ABE and total energy production yields per gram untreated feedstock reached 0.12 g ABE and 5.5 kJ for SCB and 0.08 g ABE and 6.6 kJ for RS.

Gene coexpression network analysis reveals a novel metabolic mechanism of Clostridium acetobutylicum responding to phenolic inhibitors from lignocellulosic hydrolysates

BackgroundLignocellulosic biomass is a promising resource of renewable biochemicals and biofuels. However, the presence of inhibitors existing in lignocellulosic hydrolysates (LCH) is a great challenge to acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. In particular, phenolic compounds (PCs) from LCH severely block ABE production even at low concentrations. Thus, it is urgent to gain insight into the intracellular metabolic disturbances caused by phenolic inhibitors and elucidate the underlying mechanisms to identify key industrial bottlenecks that undermine efficient ABE production.ResultsIn this study, a time-course of ABE fermentation by C. acetobutylicum in the presence of four typical PCs (syringaldehyde, vanillin, ferulic acid, and p-coumaric acid) was characterized, respectively. Addition of PCs caused different irreversible effects on ABE production. Specifically, syringaldehyde showed the greatest inhibition to butanol production, followed by vanillin, ferulic acid, and p-coumaric acid. Subsequently, a weighted gene co-expression network analysis (WGCNA) based on RNA-sequencing data was applied to identify metabolic perturbations caused by four LCH-derived PCs, and extract the gene modules associated with extracellular fermentation traits. The hub genes in each module were subjected to protein–protein interaction analysis and enrichment analysis. The results showed that functional modules were PC-dependent and shared some unique features. Specifically, p-coumaric acid caused the most extensive transcriptomic disturbances, particularly affecting the gene expressions of ribosome proteins and the assembly of flagella, DNA replication, repair, and recombination; the addition of syringaldehyde caused significant metabolic disturbances on the gene expressions of ribosome proteins, starch and sucrose metabolism; vanillin mainly disturbed purine metabolism, sporulation and signal transduction; and ferulic acid caused a metabolic disturbance on glycosyl transferase-related gene expressions.ConclusionThis study uncovers novel insights into the inhibitory mechanisms of PCs for the first time and provides guidance for future metabolic engineering efforts, which establishes a powerful foundation for the development of phenol-tolerant strains of C. acetobutylicum for economically sustainable ABE production with high productivity from lignocellulosic biomass.

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable.Graphical abstract