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Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities. However, genetic control of C. acetobutylicum biofilm has not been dissected so far. Here, to identify potential genes controlling C. acetobutylicum biofilm formation, over 40 gene candidates associated with extracellular matrix, cell surface, cell signaling or gene transcription, were tried to be disrupted to examine their individual impact. A total of 25 disruptants were finally obtained over years of attempts, for which biofilm and relevant phenotypes were characterized. Most of these disruptants formed robust biofilm still, or suffered both growth and biofilm defect. Only a strain with a disrupted histidine kinase gene (CA_C2730, designated bfcK in this study) abolished biofilm formation without impairing cell growth or solvent production. Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels. The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase (encoded by CA_C0404) that might negatively regulate biofilm formation. Based on these findings, possible bfcK-mediated mechanisms for biofilm formation were proposed. This is a big step toward understanding the biofilm formation in C. acetobutylicum and will help further engineering of its biofilm-based industrial processes.

Dunia kini menghadapi masalah pencemaran lingkungan karena penggunaan bahan bakar fosil yang ekstensif dalam industri atau transportasi. Oleh karena itu, sangat mendesak untuk menemukan sumber daya terbarukan alternatif untuk produksi bahan bakar cair dan bahan kimia. Energi alternatif dari senyawa alkohol yang telah dikembangkan di Indonesia adalah bioetanol. Biofuel dari senyawa alkohol lainnya yang telah dikembangkan adalah bio-butanol. Penelitian ini bertujuan untuk mengembangkan energy alternative pengganti minyak bumi berupa biobutanol. Bio-butanol adalah senyawa alkohol yang memiliki rumus molekul C4H9OH dan dihasilkan dari proses fermentasi bahan-bahan organik yang memiliki kandungan karbohidrat dengan menggunakan bakteri Clostridium Acetobutylicum. Fruktosa food grade diencerkan dengan air lalu difermentasi dengan penambahan bakteri Clostridium acetobutylicum selama 12 hari pada temperature ruang. Pelaksanaan penelitian dengan memfermentasi larutan fruktosa (6%v/v; 9%v/v; 12%v/v; 15%v/v; dan 18%v/v) dan jumlah bakteri Clostridium acetobutylicum (1; 2; 3; 4; 5 ml). Pengujian kadar biobutanol dianalisa menggunakan GC (Gas Chromatography). Kadar biobutanol yang relative baik dihasilkan dari fermentasi larutan fruktosa 15%v/v dan jumlah bakteri sebanyak 5 ml dengan hasil 0.9058%v/v.

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in the butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 A resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferase, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggest that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.

Clostridium acetobutylicum DSM 792 has been characterized as a native acetone-butanol-ethanol (ABE) fermenting bacterium. C. acetobutylicm is used as a model organism to improve solvent production by metabolic engineering strategies. In this study, the ABE pathway in C. acetobutylicum was modified by overexpressing the pyruvate decarboxylase gene from Zymomonas mobilis under the control of 2 promoters, namely Pferr and Ppta (promoters of genes encoding pyruvate ferredoxin oxidoreductase and phosphotransacetylase) and the recombinant strains were designated as DSM 792(pPDC1) and DSM 792(pPDC2). The specific activity of the alcohol dehydrogenases in the pdc-expressing strains was higher than that in the wild-type strain. In batch fermentation experiments, the total ABE production from the recombinant strains DSM 792 (pPDC1) and DSM 792 (pPDC2) was 1.57 and 7.9 g/L, respectively, which was higher when compared to the wild type ABE production (0.64 g/L) under uncontrolled pH. The alcohol to acetone ratio (BE/A) was found to be 2.28, 2.77, and 5.26 in wild type, DSM 792 (pPDC1), and DSM 792 (pPDC2), respectively. This suggests that the re-routing of pyruvate by overexpression of the pdc could play a role in the generation of more reducing equivalents towards ethanol and butanol production.

Bovine ruminal fluid (BRF) bioaugmented with Clostridium acetobutylicum (Clac) was assessed for hydrolyzing cellulose and produce biohydrogen (BioH2) simultaneously from pretreated corncob in a single step, without the use of external hydrolytic biocatalysts. The corncob was pretreated using three thermochemical methods: H2SO4 2%, 160 °C; NaOH 2%, 140 °C; NaOCl 2%, 140 °C; autohydrolysis: H2O, 190 °C. Subsequently, BioH2 production was carried out using the pretreated material with the highest digestibility applying a Taguchi experimental array to identify the optimal operating conditions. The results showed a higher glucose released from pretreated corncob with H2SO4 (134.7 g/L) compared to pretreated materials by autohydrolysis, NaOH and NaOCl (123 g/L, 89.8 g/L and 52.9 g/L, respectively). The mixed culture was able to hydrolyze the pretreated corncob and produce 575 mL of H2 (at 35 °C, pH 5.5, 1:2 ratio of BRF:Clac and 5% of solids loading) equivalent to 132 L H2/Kg of biomass.

Despite its advantages, lignocellulosic butanol cannot become a real alternative without processes for obtaining credits from non-cellulosic fraction of lignocellulose. In this study, the process of “cellulosic butanol” was integrated with “hemicellulosic methane” process, i.e., anaerobic digestion (AD), for higher energy return on investment (EROI). Aqueous pretreatment was evaluated as a connecting chain between the processes. The alkaline (1% NaOH), acidic (1% H2SO4), and neutral (autohydrolysis) pretreatments were utilized at 140, 160, and 180 °C for bioenergy recovery from triticale straw. Enzymatic hydrolysis of the straw pretreated by alkaline (180 °C), dilute acid (140 °C), and autohydrolysis (180 °C) and fermentation of the hydrolysates by Clostridium acetobutylicum led to 13.6, 8.5, and 10.2 g/L acetone-butanol-ethanol (ABE), respectively. AD of the liquors remained after pretreatment by alkaline, dilute acid, and autohydrolysis led to 83–121, 240–247, and 380–430 mL/g VSS methane leading to more than 60% increase in EROI.

Microorganisms harbor bulks of functionally similar or undefined genes, which belong to paralogous gene family. There is a necessity of exploring combinatorial or interactive functions of these genes, but conventional loss-of-function strategy with one-by-one rounds suffers extremely low efficiency for generating mutant library with all genes permutations. Here, taking histidine kinases (HKs) in Clostridium acetobutylicum as a proof-of-concept, we developed a multi-plasmid cotransformation strategy for generating all theoretical HKs combinations in one round. For five HKs with 31 theoretical combinations, the library containing 22 mutants within all the possible HKs-inactivated combinations was constructed with 11 days compared to 242 days by conventional strategy, while the other 9 combinations cannot survive. Six mutants with the enhanced butanol production and tolerance were obtained with changes of cell development during fermentation, one of which could produce 54.2% more butanol (56.4% more solvents), while the butanol production of other mutants was unchanged or decreased. The cotransformation strategy demonstrated potentials for fast exploring pleiotropic function of paralogous family genes in cell survival, cell development, and target product metabolism. This article is protected by copyright. All rights reserved.

The production of butanol, acetone and ethanol by Clostridium acetobutylicum is a biphasic fermentation process. In the first phase the carbohydrate substrate is metabolized to acetic and butyric acid, in the following second phase the product spectrum is shifted towards the economically interesting solvents. Here we present a cascade of six continuous stirred tank reactors (CCSTR), which allows performing the time dependent metabolic phases of an acetone-butanol-ethanol (ABE) batch fermentation in a spatial domain. Experimental data of steady states under four operating conditions—with variations of the pH in the first bioreactor between 4.3 and 5.6 as well as the total dilution rate between 0.042 h−1 and 0.092 h−1—were used to optimize and validate a corresponding mathematical model. Beyond a residence time distribution representation and substrate, biomass and product kinetics this model also includes the differentiation of cells between the metabolic states. Model simulations predict a final product concentration of 8.2 g butanol L−1 and a productivity of 0.75 g butanol L−1 h−1 in the CCSTR operated at pHbr1 of 4.3 and D = 0.092 h−1, while 31% of the cells are differentiated to the solventogenic state. Aiming at an enrichment of solvent-producing cells, a feedback loop was introduced into the cascade, sending cells from a later state of the process (bioreactor 4) back to an early stage of the process (bioreactor 2). In agreement with the experimental observations, the model accurately predicted an increase in butanol formation rate in bioreactor stages 2 and 3, resulting in an overall butanol productivity of 0.76 g L−1 h−1 for the feedback loop cascade. The here presented CCSTR and the validated model will serve to investigate further ABE fermentation strategies for a controlled metabolic switch.

The present study is focused on biobutanol production from potato peel waste in dilute orange peel extract by Clostridium acetobutylicum MTCC 11274 at three concentrations of 20, 40, and 60 g/L as ...

Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

A new wild type solventogenic Clostridium bacterium, which could produce a large amount of solvent from a mixture of 70% glucose and 30% xylose, was isolated. Based on 16S rRNA gene analysis, this strain was identified as Clostridium beijerinckii. Batch fermentations with this strain (C. beijerinckii GSC1) resulted in the production of 19.65 g/L total solvents (Acetone-Butanol-Ethanol), which is 31% higher than that with the typical wild type strain Clostridium acetobutylicum ATCC 824. This new strain utilized glucose and xylose simultaneously without genetic modification. The selectivity of GSC1 for butanol was 81.8%, which is much higher than that of C. acetobutylicum ATCC 824 (68.7%). Simple genetic modification was performed to obtain a more improved performance. The acid production in batch fermentation by C. beijerinckii GSC1_R1 (gene-modified strain) was reduced to 2.6 g/L from 6.02 g/L. The solvent productivity of GSC1_R1 in continuous fermentation was 3.6 g/L/h. These results indicate that the newly isolated strain is very promising and applicable for the production of biobutanol from second-generation biomass owing to the superior performance of the wild type strain.

Simultaneous saccharification and fermentation (SSF) is a widely used approach for lignocellulosic butanol production. However, the major drawbacks for SSF lie in poor cellulase activity, sugar yield and butanol productivity at temperature below 37 °C. For the first time, a genetically engineered Clostridium acetobutylicum with enhanced thermotolerance at 39–45 °C was applied to high temperature SSF of corn stover pretreated by dilute sulfuric acid and aqueous ammonia. The optimized SSF at 42 °C with 12 h pre-hydrolysis gave 10.8 g/L butanol and 18.2 g/L ABE using pretreated solids and overlimed acid-treated liquid, obtaining butanol yield and productivity of 0.18 g/g-substrate and 0.18 g/L/h. Significant improvements on butanol production and productivity were achieved compared to conventional SSF process. The demonstrated SSF made full use of the sugars from acid pretreatment and acid-treated liquid for hydrolysis, reduced total cellulase usage, and contributed to hydrolysis and fermentation efficiency, indicating its potential for improving lignocellulosic butanol production.

The food wastage problem and its associated issues have become a significant threat to sustainable development, particularly in developing countries. Cooked rice, which has been reported to be one of the most abundant regional food wastes, is a substantial feedstock for biofuel production due to its rich carbon content. In this study, both solid-state and acetone–butanol–ethanol (ABE) fermentations were conducted in series with Aspergillus oryzae and aero-tolerant Clostridium acetobutylicum YM1 for the biobutanol production. To validate the process parameters for an industrial-scale process, it is critical to examine the effects of process parameters using a process simulation tool. To this end, by adapting lab-scale process results, a techno-economic evaluation of the biobutanol production from the cooked rice by two-step fermentation was performed using SuperPro Designer®. This study aims to examine various scenarios for cooked rice utilization to produce butanol while examining process optimization and minimization of operational expenses for the aerobic ABE fermentation and microbial saccharification. According to the simulation results, butanol production cost was estimated to be 1.24 $ kg−1, incorporating a cost reduction of 0.58 $ kg−1 from heat integration, steam and power generation, while eliminating costs for the unnecessary commercial enzymes and N2 gas for purging. Finally, sensitivity analysis was applied to demonstrate the plant feasibility concerning potential fluctuations in the changing biofuel market and environmental conditions.

Clostridiumacetobutylicum ATCC 824 effectively utilizes a wide range of substrates to produce commodity chemicals. When grown on substrates of different oxidation states, the organism exhibits different recycling needs of reduced intracellular electron carrying co-factors. Ratios of substrates with different oxidation states were used to modulate the need to balance electron carriers and demonstrate fine-tuned control of metabolic output. Three different oxidized substrates were first fed singularly, then in different ratios to three different strains of Clostridium sp. Growth was most robust when fed glucose in exclusive fermentations. However, the use of the other two more oxidized substrates was strain-dependent in exclusive feeds. In glucose-galacturonate mixed fermentation, the main products (acetate and butyrate) were dependant on the ratios of the substrates. Exclusive fermentation on galacturonate was nearly homoacetic. Co-utilization of galacturonate and glucose was observed from the onset of fermentation in growth conditions using both substrates combined, with the proportion of galacturonate present dictating the amount of acetate produced. For all three strains, increasing galacturonate content (%) in a mixture of galacturonate and glucose from 0 to 50, and 100, resulted in a corresponding increase in the amount of acetate produced. For example, C.acetobutylicum increased from ~ 10 mM to ~ 17 mM, and then ~ 23 mM. No co-utilization was observed when galacturonate was replaced with gluconate in the two substrate co-feed.

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h−1), a higher butyric acid production of 6.8 g·L−1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h−1, with a constant hydrogen production of 507 mL·h−1, corresponding to a volumetric rate of 6.10 L·L−1 d−1, and a yield of 2.39 mol of H2 per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.

Under stress, Clostridium acetobutylicum sporulates and halts its metabolism, which limits its use in industrial acetone–butanol–ethanol (ABE) fermentation. It is challenging to manipulate the highly regulated sporulation program used by clostridia for survival. Four orphan histidine kinases (HKs) involved in the phosphorylation of Spo0A, a global regulator, were found to control sporulation, cell growth, and metabolism (acidogenesis and solventogenesis). Single knockout of cac3319 aborted sporulation at the initiation stage but enhanced butanol tolerance and production, whereas cac0437 knockout caused premature sporulation and inhibited the transition from acidogenesis to solventogenesis, resulting in early autolysis and low butanol production. No solvent was produced when both cac3319 and cac0903 were knocked out because the mutant lost the megaplasmid carrying solventogenesis genes. On the other hand, a mutant with double knockouts of cac3319 and cac0323 showed enhanced butanol production of >20 g L−1 by a repeated batch fermentation in a fibrous bed bioreactor, which enabled in situ product recovery via vapor stripping-vapor permeation to achieve the highest butanol production of 441.9 g L−1 ever reported. This study elucidated the regulatory roles of 4 HKs in controlling clostridial sporulation and solventogenesis with morphological, transcriptomics, and fermentation kinetics analyses, and demonstrated combinatory knockouts of HKs as a novel metabolic engineering approach to abort sporulation and enhance butanol production in ABE fermentation suitable for industrial application.

Abstract N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, β-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria. Graphic abstract

The anaerobe Clostridium acetobutylicum belongs to the most important industrially used bacteria. Whereas genome mining points to a high potential for secondary metabolism in C. acetobutylicum, the functions of most biosynthetic gene clusters are cryptic. We report that the addition of supra‐physiological concentrations of cysteine triggered the formation of a novel natural product, clostrisulfone (1). Its structure was fully elucidated by NMR, MS and the chemical synthesis of a reference compound. Clostrisulfone is the first reported natural product with a diphenylsulfone scaffold. A biomimetic synthesis suggests that pentamethylchromanol‐derived radicals capture sulfur dioxide to form 1. In a cell‐based assay using murine macrophages a biphasic and dose‐dependent regulation of the LPS‐induced release of nitric oxide was observed in the presence of 1., Induction of the industrial anaerobe Clostridium acetobutylicum with cysteine led to the discovery of an unprecedented diarylsulfone natural product named clostrisulfone that likely results from sulfur dioxide capture by chromane‐derived radicals. Its structure was elucidated by NMR and confirmed by synthesis. The tocopherol‐related molecule exerts immunomodulatory activities (see figure).

Potato peel waste (PPW) is a carbohydrate-rich waste from potato industries, which is an environmental threat worldwide. In this study, it was evaluated for biobutanol production via acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. The results showed that PPW contained a considerable amount of glycoalkaloids, severe inhibitors for the bacterium. Thus, three processes, i.e., dilute acid pretreatment (Process I), the inhibitors extraction followed by dilute acid hydrolysis (Process II) and ethanol organosolv pretreatment (Process III), were employed before hydrolysis and fermentation to produce ABE. The extraction of glycoalkaloids with ethanol, dilute acid hydrolysis at 180 °C for 60 min, and enzymatic hydrolysis led to a hydrolysate with 36 g/L glucose, which was successfully fermented to 11.6 g/L ABE. In process II, the organosolv pretreatment led to the removal of the major fraction of inhibitors, in the range of 77–88% of glycoalkaloids. The enzymatic hydrolysis of PPW pretreated with 75% ethanol at 180 °C for 60 min resulted in a fermentable hydrolysate with 38 g/L glucose. The fermentation of overall hydrolysate resulted in a high ABE concentration of 24.8 g/L, indicating that PPW is an appropriate substrate for butanol production after the removal of its bacterial inhibitors.

The pulp and paper industry produces a diverse range of side-streams from multi-stage processes, but these remain underutilized despite their high potential for use as biofuels. This study investigated acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum DSM 1731 from the side-streams of three different stages of the pulp and paper manufacturing process (PI, PII, and PIII). Biomass specimens with and without water washing were pretreated with 0.2% H2SO4 at 180 °C for 10 min, followed by enzymatic hydrolysis, to obtain fermentable sugars. The results showed that the produced ABE solvent concentrations were 12.8 g/L, 5.2 g/L, and 6.3 g/L from PI, PII, and PIII, respectively. The butanol yields of PI, PII, and PIII were 0.25, 0.18, and 0.19 g/g sugars, respectively. Among the tested side-streams, PI was shown to have potential as a feedstock for butanol production without prewashing prior to dilute acid pretreatment, enzymatic hydrolysis, and microbial fermentation.

In this study, the role of CaCO3 in n-butanol production was further investigated using corn straw hydrolysate (CSH) media by Clostridium acetobutylicum CICC 8016. CaCO3 addition stimulated sugars utilization and butanol production. Further study showed that calcium salts addition to CSH media led to the increase in Ca2+ concentration both intracellularly and extracellularly. Interestingly, without calcium salts addition, intracellular Ca2+ concentration in the synthetic P2 medium was much higher than that in the CSH medium despite the lower extracellular Ca2+ concentrations in the P2 medium. These results indicated that without additional calcium salts, Ca2+ uptake by C. acetobutylicum CICC 8016 in the CSH medium may be inhibited by non-sugar biomass degradation compounds, such as furans, phenolics and organic acids. Comparative proteomics analysis results showed that most enzymes involved in glycolysis, redox balance and amino acids metabolism were up-regulated with CaCO3 addition. This study provides further insights into the role of CaCO3 in n-butanol production using real biomass hydrolysate.

The effects of supplementation of standard media with metabolic intermediates (2 g/L butyric acid, 2 g/L butyric acid + 1.5 g/L acetic acid) on butanol and hydrogen production/yield were evaluated....

The accumulation of short-chain carboxylic acids (SCCAs), such as acetic acid (C2), propionic acid (C3), butyric acid (C4), and valeric acid (C5), produced by acetogens in the anaerobic digestion (AD) process hampers the maintenance of stable AD processes in biomethane production. The conversion of various SCCAs to the corresponding alcohols can be a good solution not only for utilizing abandoned or harmful SCCAs and producing useful alcohols but also for increasing the efficiency of the biogas production process. ACT01_02765 (acyl-CoA transferase) from Megasphaera hexanoica quickly and easily converted C2-C8 carboxylic acids into the corresponding alcohols in Escherichia coli with AdhE2 (alcohol dehydrogenase) from Clostridium acetobutylicum. E. coli (ACT01_02765 and AdhE2) converted carboxylic acids to the corresponding alcohols with a conversion yield that was approximately 40 times higher and a conversion rate that was approximately 10–50 times faster than those of E. coli (Ptb-Buk and AdhE2). The enzymatic machinery in E. coli (ACT01_02765 and AdhE2) is effective for carboxylic acids of different carbon chain lengths, resulting in the production of propanol, butanol, pentanol, hexanol, heptanol, and octanol.

Bioacetone, biobutanol, and bioethanol (BioABE) production is dependent on the fermentable sugars produced from lignocellulosic biomass and on the composition and initial pH of the medium. Understanding these process variables and their interconnectedness could enhance the BioABE product yield. Acacia mangium is available abundantly and it is a potential feedstock for BioABE production. In this study, BioABE was produced from fermentable sugars of A. mangium using Clostridium acetobutylicum YM1. Alkaline treated A. mangium (70 °C, 3 h, 5.50 %w/v NaOH) was further hydrolyzed via enzymatic hydrolysis using a multi-enzyme of white rot fungi to convert it into fermentable sugars. Approximately 15 g/L of fermentable sugars was produced from A. mangium (100 g/L) and was used for BioABE production in comparison with glucose. Initial findings showed that only 0.94 g/L of BioABE was produced in comparison with glucose (2.86 g/L) at a pH of 6.2. Decreasing the initial pH of the medium to 4.50 increased the BioABE (2.87 g/L), and after the medium was supplemented with tryptone-yeast-acetate (TYA), the BioABE yield increased by more than 100% to 6.84 g/L. This study discovered that BioABE produced from A. mangium was comparable to using commercial glucose, thus offering high potential as a low-cost feedstock.

A strictly anaerobic bacterial strain designated EA1Twas isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1Tare spore-forming rods (1–3×0.4–0.8 µm) and stain Gram-negative, albeit they possess a Gram-positive type of cell-wall ultrastructure. Growth of strain EA1Twas observed at 30 and 37 °C and within a pH range of pH 5–9. The major components recovered in the fatty acid fraction were C14:0, C16:0, C16:0DMA (dimethyl acetal) and C16:1ω7c. Strain EA1Tfermented several mono- and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO2and H2were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1Trepresents a novel taxon within the familyOscillospiraceae. The most closely related type strains of EA1T, based on 16S rRNA gene sequence identity, areCaproiciproducens galactitolivoransBS-1T(94.9 %), [Clostridium]leptumDSM 753T(93.8 %), [Clostridium]sporosphaeroidesDSM 1294T(91.7 %) andRuminococcus bromiiATCC 27255T(91.0 %). Further phenotypic characteristics of strain EA1Tdifferentiate it from related, validly described bacterial species. Strain EA1Trepresents a novel genus and novel species within the familyOscillospiraceae. The proposed name isCaproicibacter fermentansgen. nov., sp. nov. The type strain is EA1T(DSM 107079T=JCM 33110T).

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell–cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum .

Objective We previously described the structure and activity of a glycoside hydrolase family 30 subfamily 8 (GH30-8) endoxylanase, CaXyn30A, from Clostridium acetobutylicum which exhibited novel glucuronic acid (GA)-independent activity. Immediately downstream from CaXyn30A is encoded another GH30-8 enzyme, CaXyn30B. While CaXyn30A deviated substantially in the highly conserved β7-α7 and β8-α8 loop regions of the catalytic cleft which are responsible for GA-dependence, CaXyn30B maintains these conserved subfamily 8 amino acid residues thus predicting canonical GA-dependent activity. In this report, we show that CaXyn30B functions as a canonical GA-dependent GH30-8 endoxylanase in contrast to its GA-independent neighbor, CaXyn30A. Results A clone expressing the catalytic domain of CaXyn30B (CaXyn30B-CD) exhibited GA-dependent endoxylanase activity. Digestion of glucuronoxylan generated a ladder of aldouronate limit products as anticipated for canonical GA-dependent GH30-8 enzymes. Unlike the previously described CaXyn30A-CD, CaXyn30B-CD showed no activity on arabinoxylan or the generation of appreciable neutral oligosaccharides from glucuronoxylan substrates. These results are consistent with amino acid sequence comparisons of the catalytic cleft and phylogenetic analysis.

BackgroundThe industrial vinegar residue (VR) from solid-state fermentation, mainly cereals and their bran, will be a potential feedstock for future biofuels because of their low cost and easy availability. However, utilization of VR for butanol production has not been as much optimized as other sources of lignocellulose, which mainly stem from two key elements: (i) high biomass recalcitrance to enzymatic sugar release; (ii) lacking of suitable industrial biobutanol production strain. Though steam explosion has been proved effective for bio-refinery, few studies report SE for VR pretreatment. Much of the relevant knowledge remains unknown. Meanwhile, recent efforts on rational metabolic engineering approaches to increase butanol production inClostridiumstrain are quite limited. In this study, we assessed the impact of SE pretreatment, enzymatic hydrolysis kinetics, overall sugar recovery and applied atmospheric and room temperature plasma (ARTP) mutant method for theClostridiumstrain development to solve the long-standing problem.ResultsSE pretreatment was first performed. At the optimal condition, 29.47% of glucan, 71.62% of xylan and 22.21% of arabinan were depolymerized and obtained in the water extraction. In the sequential enzymatic hydrolysis process, enzymatic hydrolysis rate was increased by 13-fold compared to the VR without pretreatment and 19.60 g glucose, 15.21 g xylose and 5.63 g arabinose can be obtained after the two-step treatment from 100 g VR. Porous properties analysis indicated that steam explosion can effectively generate holes with diameter within 10–20 nm. Statistical analysis proved that enzymatic hydrolysis rate of VR followed the Pseudop-second-order kinetics equation and the relationship between SE severity and enzymatic hydrolysis rate can be well revealed by Boltzmann model. Finally, a superior inhibitor-tolerant strain,Clostridium acetobutylicumTust-001, was generated with ARTP treatment. The water extraction and enzymolysis liquid gathered were successfully fermented, resulting in butanol titer of 7.98 g/L and 12.59 g/L of ABE.ConclusionsSE proved to be quite effective for VR due to high fermentable sugar recovery and enzymatic hydrolysate fermentability. Inverse strategy employing ARTP and repetitive domestication for strain breeding is quite feasible, providing us with a new tool for solving the problem in the biofuel fields.

The biological production of H2 represents a renewable and eco-friendly energy alternative compared to fossil fuels. However, its production from lignocellulose involves the use of expensive enzymatic complexes. In the present work, the production of H2 from pretreated agave biomass was evaluated by means of a Consolidated Bioprocess (CBP). This strategy was carried through the interaction of cellulose-degrading microorganisms obtained from bovine ruminal fluid (BRF) capable of enhancing H2 production by Clostridium acetobutylicum. The results obtained show the capacity of BRF to hydrolyze the acid pretreated agave, improving the production of H2 in the experiments where the inoculum of Clostridium was greater. According to the results, production of H2 is significantly affected by the increase of the solids loading, obtaining a maximum H2 production at a 10% of solids loading, pH 5.5 and 35 °C, representing a yield of 150 L of H2 per Kg of biomass in 264 h.

Innovative processes to transform plant biomass into renewable chemicals are needed to replace fossil fuels and limit climate change. Clostridium acetobutylicum is of industrial interest because it ferments sugars into acetone, butanol and ethanol (ABE). However, this organism is unable to depolymerize cellulose, limiting its use for the direct transformation of lignocellulose. In this study, the freely secreted family 5 cellulase cphy2058 from Clostridium phytofermentans was efficiently expressed in C. acetobutylicum to enhance cellulolysis. RT-qPCR confirmed the transcription of the cellulase gene, and galactomannan and carboxymethyl cellulose plate assays showed endoglucanase and mannanase activity of cphy2058. ABE titers by the recombinant, cellulase-expressing C. acetobutylicum reached 85.83 mM in cellobiose cultures, whereas ABE titers by wild-type were 44.52 mM. ABE titers by the cellulase-expressing strain were similarly elevated relative to wild-type in cellulose and galactomannan cultures. These results demonstrate a novel strategy to enhance complex substrate utilization by heterologous cellulase expression in C. acetobutylicum.

Butanol is an attractive second-generation biofuel as well as an important chemical. Production of butanol from lignocellulosic feedstock via consolidated bioprocessing has attracted increasing int...

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni2+–NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. K M and k cat/K M of the enzyme were 24.90 µM and 25.13 s−1 µM−1, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

Process stability is a key operational issue when operating dark fermentation with mixed microbial cultures for hydrogen production. This study aimed at mitigating the instability of hydrogen production by separately adding exogenous pure strains suspected to have key roles in fermentative cultures. Among them, Clostridium acetobutylicum, Clostridium pasteurianum and Lactobacillus bulgaris which became predominant within the mixed culture strongly reduced the spectrum of produced metabolites and H2 production variability. Interestingly, Escherichia coli and Cupriavidus necator, which remained in minor abundance, maintained a high and stable H2 production while lowering the metabolic variability. 16S rRNA revealed that this could correlate to a simplification of the microbial diversity and the non-emergence of spore-forming competitors such as Sporolactobacillus sp. These results illustrate the potential beneficial role of minor OTUs as keystone species on H2-producing complex ecosystem and support the possibility of using them to engineer the ecosystem and maintain high and stable performances.

The processes of oxidative depolymerization and acidic hydrolysis have consistently been used for the pretreatment of lignocellulosic biomass (wheat straw, sawdust, and lignin), which makes it possible to obtain a high content of soluble organic compounds in the hydrolysate (44–94 g COD/L) and to enhance the concentration of reducing sugars from 1 to 36%. The introduction of Clostridium acetobutylicum bacteria to an acidogenic biocatalyst resulted in an even greater increase in the content of volatile fatty acids and ethanol in the end product. The maxima among the studied substrates yields of butyric acid and ethanol were observed when the pretreated straw was hydrolyzed (27% (0.82 g/L) and 17.4% (0.44 g/L), respectively). It was shown that the addition of glycerol as a substrate makes it possible to increase the yields of butyric and acetic acids as a result of the conversion of the pretreated biomass; the addition of glycerol to straw in a 1 : 1 ratio enhanced the maximum butyric acid and VFA yields up to 32% (0.96 g/L) and 72% (2.67 g/L), respectively.

Background Butyl acetate has shown wide applications in food, cosmetics, medicine, and biofuel sectors. These short-chain fatty acid esters can be produced by either chemical or biological synthetic process with corresponding alcohols and acids. Currently, biosynthesis of short chain fatty acid esters, such as butyl butyrate, through microbial fermentation systems has been achieved; however, few studies regarding biosynthesis of butyl acetate were reported. Results In this study, three proof-of-principle strategies for the one-pot butyl acetate production from glucose through microbial fermentation were designed and evaluated. (1) 7.3 g/L of butyl acetate was synthesized by butanol-producing Clostridium acetobutylicum NJ4 with the supplementation of exogenous acetic acid; (2) With the addition of butanol, 5.76 g/L of butyl acetate can be synthesized by acetate-producing Actinobacillus succinogenes130z (ΔpflA); (3) Microbial co-culture of C. acetobutylicum NJ4 and A. succinogenes130z (ΔpflA) can directly produce 2.2 g/L of butyl acetate from glucose by using microbial co-culture system with the elimination of precursors. Through the further immobilization of A. succinogenes130z (ΔpflA), butyl acetate production was improved to 2.86 g/L. Conclusion Different microbial mono- and co-culture systems for butyl acetate biosynthesis were successfully constructed. These strategies may be extended to the biosynthesis of a wide range of esters, especially to some longer chain ones.

ATPase, a key enzyme involved in energy metabolism, has not yet been well studied in Clostridium acetobutylicum. Here, we knocked down the atpG gene encoding the ATPase gamma subunit in C. acetobutylicum ATCC 824 using a mobile group II intron system and analyzed the physiological characteristics of the atpG gene knockdown mutant, 824-2866KD. Properties investigated included cell growth, glucose consumption, production of major metabolites, and extracellular pH. Interestingly, in 2-L batch fermentations, 824-2866KD showed no significant difference in metabolite biosynthesis or cell growth compared with the parent ATCC 824. However, the pH value in 824-2866KD cultures at the late stage of the solventogenic phase was abnormally high (pH 6.12), compared with that obtained routinely in the culture of ATCC 824 (pH 5.74). This phenomenon was also observed in batch cultures of another C. acetobutylicum, BEKW-2866KD, an atpG-knockdown and pta-buk double-knockout mutant. The findings reported in this study suggested that ATPase is relatively minor than acid-forming pathway in ATP metabolism in C. acetobutylicum.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730 and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the non-sporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (stage 0) whereas cac3319 overexpression promoted spore development (stage VII), and cac0437 knockout initiated but blocked sporulation before stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis. This article is protected by copyright. All rights reserved.

Clostridium acetobutylicum is an important industrial platform capable of producing a variety of biofuels and bulk chemicals. Biofilm of C. acetobutylicum renders many production advantages and has been long and extensively applied in fermentation. However, molecular and genetic mechanisms underlying the biofilm have been much less studied and remain largely unknown. Here, we review studies to date focusing on C. acetobutylicum biofilms, especially on its physiological and molecular aspects, summarizing the production advantages, cell physiological changes, extracellular matrix components and regulatory genes of the biofilm. This represents the first review dedicated to the biofilm of C. acetobutylicum. Hopefully, it will deepen our understanding toward C. acetobutylicum biofilm and inspire more research to learn and develop more efficient biofilm processes in this industrially important bacterium.

BACKGROUND: Butanol production through ABE (acetone, butanol, and ethanol) fermentation has attracted a great deal of attention because of its potential to replace petroleum‐based fuels. Low butanol production and productivity due to butanol inhibition are the main challenges to make ABE fermentation viable economically. In this work, polydimethylsiloxane (PDMS) sponge and thermos‐responsive poly(N‐isopropylacrylamide)‐grafted PDMS (PNIPAAm‐PDMS) sponges (with different mass fractions of PNIPAAm on the PDMS surface) were investigated as adsorbents for butanol. RESULTS: The butanol model solutions showed that the adsorption–desorption efficiency of different types of sponges for butanol is highly temperature‐dependent. Higher temperatures exhibited higher adsorption efficiency. In contrast, lower temperatures markedly increased the desorption efficiency for butanol. PNIPAAm‐PDMS with 8.0 wt% of PNIPAAm on the PDMS surface showed as the best adsorbent for butanol. It had adsorption efficiency of 14, 24 and 46% at 25, 37 and 50 °C, respectively. Moreover, 96% of butanol adsorbed was recovered at 25 °C. When it was coupled with a batch ABE fermentation by Clostridium acetobutylicum, butanol production increased by 102% compared to batch fermentation without adsorption. A semi‐continuous fermentation with the initial feeding time at the 60th hour exhibited as the optimum condition. However, it failed to enhance butanol productivity due to a high accumulation of butanol in the broth. Then, a semi‐continuous fermentation with adsorption was conducted, yielding total butanol production of 20 g/L. CONCLUSION: PNIPAAm‐PDMS sponge is proposed as an adsorbent to overcome butanol inhibition in ABE fermentation by temperature manipulation to recover butanol. © 2019 Society of Chemical Industry

C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.

Background: Fuels and chemicals from renewable feedstocks have a growing demand, and acetone, butanol and ethanol (ABE) are some relevant examples. These molecules can be produced by the bacterial fermentation process using hydrolysates generated from lignocellulosic biomass as sugarcane bagasse, one of the most abundant sources of lignocellulosic biomass in Brazil. It originates as a residue in mills and distilleries in the production of sugar and ethanol. Results: In the present work, two strategies to generate hydrolysates of sugarcane bagasse were adopted. The fermentation of the first hydrolysate by Clostridium acetobutylicum DSM 6228 resulted in final concentrations of butanol, acetone and ethanol of 6.4, 4.5 and 0.6 g/L, respectively. On the other hand, the second hydrolysate presented better results (averages of 9.1, 5.5 and 0.8 g/L, respectively), even without the need for nutrient supplementation, since key elements were already present in the medium. The productivity (QP) and yield (YP/S) of the solvents with second hydrolysate were 0.5 g/L·h-1 and 0.4 g/g, respectively. Conclusions: The results described herein open new perspectives for the production of important molecules from residual lignocellulosic biomass for the fuel and chemical industries within the context of second-generation biorefinery.How to cite: Gomes AC, Rodrigues MI, Passos DF, et al. Acetone-butanol-ethanol fermentation from sugarcane bagasse hydrolysates: utilization of C5 and C6 sugars. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.004. Keywords: Acetone, Bagasse, Biofuels, Butanol, Clostridium acetobutylicum, Ethanol, Fermentation, Hydrolysate, Saccharum, Sugarcane bagasse

The cell degeneration and the fermentation performance decline during repeated cycle fermentation with immobilized Clostridium acetobutylicum on bagasse for hydrogen and butanol production was studied. The cell degeneration has been characterized in abnormality of a long-chain morphology through 7 cycles of repeated fermentation. The fermentation performance decline has been indicated by decrease of glucose consumption rate from 0.82 g/L/h to 0.22 g/L/h, reduction of hydrogen production and productivity from 6 L/L to 2.5 L/L and 170 mL/L/h to 40 mL/L/h, as well as the decrease of butanol production and butanol productivity from 6.5 g/L to 1.0 g/L and 0.18 g/L/h to 0.02 g/L/h, respectively. The ratio of hydrogen production and butanol production was in the range of 6–10 and 17–20 during the earlier three and later four cycles, respectively. The carbon flow directed to ethanol was higher during the later period of fermentation.

The co-factor NADH plays an important role in butanol biosynthesis. In this study, we found that aspartate could effectively improve the butanol production of Clostridium acetobutylicum ATCC 824. Further study showed that aspartate could be used as the precursor of NADH de novo synthesis in C. acetobutylicum ATCC 824. When 2 g/L aspartate was added, the transcription levels of essential genes (nadA, nadB and nadC) for NADH de novo synthesis were significantly higher than that of without aspartate addition. The levels of intracellular NAD+, NADH, total NAD(H) and the ratio of NADH/NAD+ were also significantly increased, which were 63.9 ± 8.0%, 85.0 ± %, 77.7 ± 8.0% and 12.7 ± 2.9% higher than those of without aspartate addition, respectively. Furthermore, the butanol production was improved by overexpressing the NADH de novo synthesis genes, and the fermentation performance could be further enhanced by strengthening the VB1 biosynthesis and NADH de novo synthesis pathway simultaneously. As a result, the butanol titer of the engineered strain 824(thiCGE–nadC) reached 13.96 ± 0.11 g/L, 7.2 ± 0.4%, 18.1 ± 0.1%, 34.1 ± 0.1% higher than that of 824(thiCGE), 824(nadC) and the wild type strain, respectively. This study has a reference value for the NADH related researches of other microbes, and the engineering strategy used in this study provides a new idea for construction of efficient fuel-producing strains.

Studies on acetone–butanol–ethanol (ABE) fermentation of sugars from lignocellulosic biomass have been actively carried out in recent years, and recombinant strains have been developed to efficiently coutilize glucose and xylose, which are the dominant sugar types in most lignocellulosic biomass. However, there is a lack of mathematical models for describing and predicting the simultaneous utilization of glucose and xylose in ABE fermentation, particularly for the purpose of supporting optimization and control of the process. This study proposes a kinetic model for ABE fermentation with coutilization of glucose and xylose by recombinant Clostridium acetobutylicum. The model is developed based on unstructured models, that is, the Monod equation and the Luedeking–Piret model, and a modified concentration-dependent weighting factor is suggested to describe the simultaneous utilization of glucose and xylose based on the experimental analysis. A systematic identification approach is employed to obtain reliable...

The conventional acetone-butanol-ethanol fermentation process suffers from several key hurdles viz. low solvent titer, insufficient yield and productivity, and solvent intolerance which largely affect butanol commercialization. To counteract these issues, the effect of stimulator, namely, folic acid was investigated in the present study to improve butanol titer. Folic acid is involved in biosynthesis of a diverse range of cellular components, which subsequently alter the amino acid balance. Therefore, different concentrations of folic acid were screened, and 10 mg/L supplementation resulted in a maximum butanol production of 10.78 ± 0.09 g/L with total solvents of 18.91 ± 0.21 g/L. Folic acid addition at different time intervals was also optimized to get additional improvements in final butanol concentration. Overall, folic acid supplementation resulted in two-fold increase in butanol concentration and thus could be considered as a promising strategy to enhance solvent titers.

Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Electronic supplementary material The online version of this article (10.1186/s13068-019-1524-6) contains supplementary material, which is available to authorized users.

The red seaweed, Gelidium amansii, contains proteins such as R-phycoerythrin and R-phycocyanin, as well as polysaccharides. Initial protein extraction from G. amansii yielded 53 μg/g R-phycoerythrin and 56 μg/g R-phycocyanin. After protein extraction, monosaccharides were produced using hyper thermal acid hydrolysis and enzymatic saccharification. Acetone–butanol–ethanol (ABE) was produced by Clostridium acetobutylicum KCTC 1790. To overcome the different pH requirements for monosaccharide consumption and solvent production, a two-stage pH control culture strategy was developed. Fermentation at pH 6.0 supported a high cell growth rate and both glucose and galactose were consumed completely. The pH was then shifted from 6.0 to 4.5 to produce ABE after 84 h. The maximum ABE concentration reached 15.5 g/L in comparison to 3.1 g/L under fermentation without pH control. The two-stage pH control strategy is a suitable method for ABE production from G. amansii.