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[Display omitted] • The effects of phenolic acids/aldehydes on butyrate fermentation were investigated. • Phenolic inhibitors were reduced to low toxicity alcohols/acids by C. tyrobutyricum. • Some reductase, HSP, and membrane transporter genes were induced by phenolic stress. • Accelerating the reduction of PCs could enhance the strain's tolerance to PC stress. • Valuable insights for enhancing utilization of lignocellulosic biomass were provided. Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum , enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain's tolerance to PCs. Under 1.0 g/L p -coumaric acid stress, compared with the wild-type strain, ATCC 25755/ sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia , which holds promise for improving biofuel and chemical production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

[Display omitted] • Heterologous α-amylases were screened and expressed in C. tyrobutyricum. • α-glucosidase was co-expressed with α-amylases for better starch hydrolyzation. • Engineered C. tyrobutyricum produced up to 27.0 g/L butyrate from cassava starch. • 26.8 g/L butyl butyrate was produced from cassava starch by the engineered strain. Clostridium tyrobutyricum has been successfully engineered to produce butyrate, butanol, butyl butyrate, and γ-aminobutyric acid. It would be interesting to produce bio-chemicals and bio-fuels directly using starch from non-food crop, e.g., cassava, by engineered C. tyrobutyricum. In this study, heterologous α-amylases were screened and expressed in C. tyrobutyricum , resulting in successfully starch hydrolyzation. Furthermore, α-glucosidase (AgluI) was co-expressed with α-amylases, resulting in enhancement in the capacity of starch hydrolyzation and butyrate production. When increasing the cassava starch concentration to 100 g/L, the engineered strain CTAA05 produced 27.0 g/L butyrate. In addition, when introducing butyl butyrate synthetic pathway, strain MU3-AAV produced 26.8 g/L butyl butyrate with 100 g/L cassava starch as substrate. This study showed a generalizable framework to engineered anaerobes for anaerobic production of bio-chemicals and bio-fuels from starchy biomass. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Butyric acid production using mannitol in a brown alga S. japonica was investigated. • Addition of acetate was critical for the efficient consumption of mannitol. • Mixture of S. japonica /rice straw was used to supply mannitol, glucose & acetate. • Mixed biomass enabled high butyric acid production, yield, and selectivity. • Using mixed biomass is a noteworthy method overcoming limits of single biomass. Abstract A brown alga Saccharina japonica and rice straw are attractive feedstock for microbial butyric acid production. However, inefficient fermentation of mannitol (a dominant component in S. japonica) and toxicity of inhibitors in lignocellulosic hydrolysate are limitations. This study demonstrated that mixed biomass with S. japonica and rice straw was effective in butyric acid production over those restrictions. Mannitol was consumed only when acetic acid was present. Notably, acetic acid was not produced but consumed along with mannitol. By mixing S. japonica and rice straw to take advantage of glucose and acetic acid in rice straw, Clostridium tyrobutyricum effectively consumed mannitol by utilizing acetic acid in hydrolysate and acetic acid derived from glucose with the enhanced butyric acid production. Furthermore, cell growth was restored owing to the decreased inhibitor concentration. The results demonstrate the potential of butyric acid production from mixed biomass of macroalgae/lignocellulose overcoming the drawbacks of single biomass. [ABSTRACT FROM AUTHOR]

Highlights • Furfural severely inhibited the butyrate productivity of C. tyrobutyricum. • Heterologous expression of sdr increased furfural tolerance of C. tyrobutyricum. • Coexpression of sdr and groESL enhanced the utilization of corncob acid hydrolysate. • 32.8 g/L butyrate at a productivity of 0.29 g/L·h and yield of 0.40 g/g was obtained. Abstract Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/ sdr + groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/ sdr + groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The first attempt to produce caproic acid by M. hexanoica with s-HF/MBRs. • Caproic acid of 10.08 g L−1 was produced with the fastest productivity of 0.69 g L−1 h−1. • The s-HF/MBRs showing potentials on caproic acid production by co-culture process. Abstract In this study, a coculture bioprocess was developed with Clostridium strains producing butyric acid and Megasphaera hexanoica producing caproic acid from the butyric acid. The two bacterial strains were each cultivated in two submerged hollow-fiber membrane bioreactors (s-HF/MBRs), separately. Each fermentation broth was filtered through the membrane modules, and the filtered broth was either interchanged on another reactor or obtained sequentially through. Using s-HF/MBRs, the caproic acid concentration increased to 10.08 g L−1, with the fastest productivity of 0.69 g L−1 h−1, which higher than that previously reported. [ABSTRACT FROM AUTHOR]

One-pot bioconversion is an economically attractive biorefinery strategy to reduce enzyme consumption. Direct conversion of lignocellulosic biomass for butyric acid production is still challenging because of competition among microorganisms. In a consolidated hydrolysis/fermentation bioprocessing (CBP) the microbial structure may eventually prefer the production of caproic acid rather than butyric acid production. This paper presents a new bioaugmentation approach for high butyric acid production from rice straw. By dosing 0.03 g/L of Clostridium tyrobutyricum ATCC 25755 in the CBP, an increase of 226% higher butyric acid was yielded. The selectivity and concentration also increased to 60.7% and 18.05 g/L, respectively. DNA-sequencing confirmed the shift of bacterial community in the augmented CBP. Butyric acid producer was enriched in the bioaugmented bacterial community and the bacteria related to long chain acids production was degenerated. The findings may be useful in future research and process design to enhance productivity of desired bio-products. [ABSTRACT FROM AUTHOR]

Butyric acid is an important platform chemical, which is widely used in the fields of food, pharmaceutical, energy, etc. Microbial fermentation as an alternative approach for butyric acid production is attracting great attention as it is an environmentally friendly bioprocessing. However, traditional fermentative butyric acid production is still not economically competitive compared to chemical synthesis route, due to the low titer, low productivity, and high production cost. Therefore, reduction of butyric acid production cost by utilization of alternative inexpensive feedstock, and improvement of butyric acid production and productivity has become an important target. Recently, several advanced strategies have been developed for enhanced butyric acid production, including bioprocess techniques and metabolic engineering methods. This review provides an overview of advances and strategies in process and strain engineering for butyric acid production by microbial fermentation. Additionally, future perspectives on improvement of butyric acid production are also proposed. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/ groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum , were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20–0.21 g/L·h for the wild-type strain) due to the shortened lag phase. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum can utilize glucose and xylose as carbon source for butyric acid production. However, xylose catabolism is inhibited by glucose, hampering butyric acid production from lignocellulosic biomass hydrolysates containing both glucose and xylose. In this study, an engineered strain of C. tyrobutyricum Ct-pTBA overexpressing heterologous xylose catabolism genes (xylT, xylA, and xylB) was investigated for co-utilizing glucose and xylose present in hydrolysates of plant biomass, including soybean hull, corn fiber, wheat straw, rice straw, and sugarcane bagasse. Compared to the wild-type strain, Ct-pTBA showed higher xylose utilization without significant glucose catabolite repression, achieving near 100% utilization of glucose and xylose present in lignocellulosic biomass hydrolysates in bioreactor at pH 6. About 42.6 g/L butyrate at a productivity of 0.56 g/L'h and yield of 0.36 g/g was obtained in batch fermentation, demonstrating the potential of C. tyrobutyricum Ct-pTBA for butyric acid production from lignocellulosic biomass hydrolysates. [ABSTRACT FROM AUTHOR]

The production of n -butanol from sugarcane juice by metabolically engineered Clostridium tyrobutyricum Ct(Δack)-pscrBAK overexpressing scr operon genes ( scrB , scrA , and scrK ) for sucrose catabolism and an aldehyde/alcohol dehydrogenase gene ( adhE2 ) for butanol biosynthesis was studied with corn steep liquor (CSL) as a low-cost nitrogen source. In free cell fermentation, butanol production of ∼16 g/L at a yield of 0.31 ± 0.02 g/g and productivity of 0.33 ± 0.02 g/L·h was obtained from sucrose and yield of 0.24 ± 0.02 g/g and productivity of 0.30 ± 0.01 g/L·h from sugarcane juice containing sucrose, glucose and fructose. The fermentation was also studied in a fibrous bed bioreactor (FBB) operated in a repeated batch mode for 10 consecutive cycles in 10 days, achieving an average butanol yield of 0.21 ± 0.02 g/g and productivity of 0.53 ± 0.05 g/L·h from sugarcane juice, demonstrating its long-term stability without applying the antibiotic selection pressure. [ABSTRACT FROM AUTHOR]

[Display omitted] • Xylose pathway genes in C. tyrobutyricum were identified and verified. • The potential mechanisms of CCR in C. tyrobutyricum were proposed and confirmed. • Double knockout of hprK and xylR eliminated CCR in C. tyrobutyricum. • Undetoxified lignocellulosic hydrolysates were used as the substrates. Clostridium tyrobutyricum , a gram-positive anaerobic bacterium, is recognized as the promising butyric acid producer. But, the existence of carbon catabolite repression (CCR) is the major drawback for C. tyrobutyricum to efficiently use the lignocellulosic biomass. In this study, the xylose pathway genes were first identified and verified. Then, the potential regulatory mechanisms of CCR in C. tyrobutyricum were proposed and the predicted engineering targets were experimental validated. Inactivation of hprK blocked the CcpA-mediated CCR and resulted in simultaneous conversion of glucose and xylose, although xylose consumption was severe lagging behind. Deletion of xylR further shortened the lag phase of xylose utilization. When hprK and xylR were inactivated together, the CCR in C. tyrobutyricum was completely eliminated. Consequently, ATCC 25755/Δ hprK Δ xylR showed significant increase in butyrate productivity (1.8 times faster than the control) and excellent butyric acid fermentation performance using both mixed sugars (11.0–11.9 g/L) and undetoxified lignocellulosic hydrolysates (12.4–13.4 g/L). [ABSTRACT FROM AUTHOR]

Highlights: [•] MTT assay is fast, high efficiency and feasible for different irradiation levels. [•] High-energy carbon ion enhances biosynthesis of butyric acid for C. tyrobutyricum. [•] Butyrate yields by Eucommia ulmoides Oliv. as a carbon source was carried out. [Copyright &y& Elsevier]

Highlights: [•] A novel anaerobic strain Clostridium tyrobutyricum RPT-4213 was discovered and identified. [•] RPT-4213 produced butyric acid from MRS with a yield of 0.48g/g glucose. [•] RPT-4213 converted diluted wheat straw hydrolysate to butyrate at 0.44g/g glucose. [•] RPT-4213 converted diluted switchgrass hydrolysate to butyrate at 0.42g/g glucose. [Copyright &y& Elsevier]

Abstract: A fermentation process using Clostridium tyrobutyricum immobilized in a fibrous-bed bioreactor (FBB) was developed for butyric acid production from sugarcane bagasse (SCB) hydrolysate. SCB was first treated with dilute acid and then hydrolyzed with cellulases. The hydrolysate containing glucose and xylose was used as carbon source for the fermentation without detoxification. The bacterium was able to grow at a specific growth rate of ∼0.06h−1 in media containing 15–20% (w/v) SCB in serum bottles. In batch cultures in the FBB, both glucose and xylose in the SCB hydrolysate were simultaneously converted to butyrate with a high yield (0.45–0.54g/gsugar) and productivity (0.48–0.60g/Lh). A final butyrate concentration of 20.9g/L was obtained in a fed-batch culture, with an overall productivity of 0.51g/Lh and butyrate yield of 0.48g/g sugar consumed. This work demonstrated the feasibility of using SCB as a low-cost feedstock to produce butyric acid. [Copyright &y& Elsevier]

Abstract: In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to enrich hydrogen-producing bacteria. By varying the influent substrate concentrations and hydraulic retention times (HRT), the volumetric loading rate (VLR) of 55.64kg-COD/m3/day seemed to be optimum for this enriched culture for fermentative hydrogen production from lactate and acetate. The results of batch experiments confirmed that the enriched culture tended to fulfill the e − equiv requirement for cell growth at a lower VLR condition (21.77kg-COD/m3/day), while it could largely distribute the e − equiv for hydrogen production at a higher VLR condition. However, a maximum lactate/acetate concentration allowed for enriching this culture existed, especially at a lower HRT condition in which wash-out can be an issue for this enriched culture. Finally, the results of cloning and sequencing indicated that Clostridium tyrobutyricum was considered the major hydrogen-producing bacteria in the CSTR fed with lactate and acetate. [Copyright &y& Elsevier]

Abstract: Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44g/g), high productivity (2.75g/L/h) and a butyrate concentration of 27.5g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4g/L, with the yield of 0.38g/g and the selectivity of ∼85.1 (85.1g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work. [ABSTRACT FROM AUTHOR]

Abstract: The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate. [Copyright &y& Elsevier]

Abstract: The effects of the hydraulic retention time (HRT=8, 10, 12 or 16.7h) and glucose concentration (30, 40 or 50g/L) on the production of hydrogen and butyrate by an immobilized Clostridium tyrobutyricum culture, grown under continuous culturing conditions, were evaluated. With 30g/L glucose, the higher HRTs tested led to greater butyrate concentrations in the culture, i.e., 9.3g/L versus 12.9g/L with HRTs of 8h and 16.7h, respectively. In contrast, higher biogas and hydrogen production rates were generally seen when the HRT was lower. Experiments with different glucose concentrations saw a significant amount of glucose washed out when 50g/L was used, the highest being 22.7g/L when the HRT was 16.7h. This study found the best conditions for the continuous production of hydrogen and butyric acid by C. tyrobutyricum to be with an HRT of 12h and a glucose concentration of 50g/L, respectively. [Copyright &y& Elsevier]

Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2gl−1 with yield of 0.47gg−1 and reactor productivity up to 4.13gl−1 h−1 at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6±0.8gl−1), yield (0.58±0.01gg−1), and sugar utilization (90.8±0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2gl−1. [Copyright &y& Elsevier]

The effects of pH on hydrogen fermentation of glucose by newly isolated H2-producing bacterium Clostridium tyrobutyricum JM1 were investigated in batch cultivations. The changes of carbon material and energy balances by pH conditions provided useful information for understanding and interpreting the regulatory system of the microorganism, and for optimization of a desired product, in this case, molecular hydrogen. The most probable metabolic pathways of C. tyrobutyricum JM1 were determined through an accurate analysis of stoichiometry and the consistency of the experimental data, checked by high carbon recovery. The carbon material and energy balances were adequately applied to estimate the carbon-flow distribution. They suggested that pH 6.3 was appropriate to maximize hydrogen production with a high concentration of butyrate and balanced activities of NADH. [Copyright &y& Elsevier]

A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2l H2 l−1 d−1 at 2h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223ml (g-hexose)−1 with an influent glucose concentration of 5gl−1. Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production. [Copyright &y& Elsevier]

[Display omitted] • Butanol production from S. japonica hydrolysate by C. tyrobutyricum was studied. • The effect of pretreatment method on butanol production was investigated. • Ultrasonic-assisted acid hydrolysis was proposed for efficient butanol production. • Heat shock protein overexpression was employed for enhanced butanol production. • The highest butanol production from macroalgal biomass was obtained by Ct-pMA12G. Macroalgal biomass is currently considered as a potential candidate for biofuel production. In this study, the effects of pretreatment method and heat shock protein overexpression were investigated for efficient butanol production from Saccharina japonica using engineered Clostridium tyrobutyricum. First, various pretreatment methods including acid hydrolysis, acid hydrolysis and enzymatic saccharification, and ultrasonic-assisted acid hydrolysis were employed to obtain the fermentable sugars, and the resulted hydrolysates were evaluated for butanol fermentation. The results showed that ultrasonic-assisted acid hydrolysate obtained the highest butanol yield (0.26 g/g) and productivity (0.19 g/L⋅h). Then, the effects of homologous or heterologous heat shock protein overexpression on butanol production and tolerance were examined. Among all the engineered strains, Ct-pMA12G exhibited improved butanol tolerance and enhanced butanol production (12.15 g/L butanol with a yield of 0.34 g/g and productivity of 0.15 g/L⋅h) from 1.8-fold concentrated S. japonica hydrolysate, which was the highest level ever reported for macroalgal biomass. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new process Ms-SSF to enhanced butyric acid production was first proposed. • Cell viability of coated CtΔack::cat1 was remarkably increased. • The thermostability and pH stability of immobilized cellulase was notably enhanced. • Rice straw hydrolysis and glucose conversion was considerable in Ms-SSF. • 26.25 g/L butyrate with a productivity of 0.41 g/L·h was achieved in Ms-SSF. Here, Metal-Organic Frameworks (MOFs) coupling simultaneous saccharification and fermentation for butyric acid production using rice straw was constructed. Clostridium tyrobutyricum Δ ack :: cat1 , with deleted ack gene and overexpressed cat1 gene, was used as the butyric-acid-fermentation strain. MOFs was employed as a photocatalyst to improve butyric acid production, as well as a cytoprotective exoskeleton with immobilized cellulase for the hydrolysis of rice straw. Thus, the survival of MOFs-coated strain, the thermostability and pH stability of cellulase both remarkably increased. As a result, 55% of rice straw was hydrolyzed in 24 h, and the final concentration of butyric acid in visible light was increased by 14.23% and 29.16% compared to uncoated and coated strain without visible light, respectively. Finally, 26.25 g/L of butyric acid with a productivity of 0.41 g/L·h in fed-batch fermentation was obtained. This novel process inspires green approach of abundant low-cost feedstocks utilization for chemical production. [ABSTRACT FROM AUTHOR]

• C. tyrobutyricum was engineered to overexpress galactose metabolism-related genes. • Removing lipids could significantly enhance the hydrolysis efficiency of SCGs. • The engineered strain utilized galactose and glucose simultaneously. • 34.3 g/L butyrate at a productivity of 0.36 g/L·h and yield of 0.37 g/g was obtained. • This provides an economically viable approach for butyric acid production. Clostridium tyrobutyricum cannot utilize galactose, which is abundant in lignocellulose and red algae, as a carbon source for butyric acid production. Hence, when using galactose-rich coffee ground hydrolysate as the substrate, the fermentation performance of C. tyrobutyricum is poor. In this work, a recombinant strain, C. tyrobutyricum ATCC 25755/ ketp , overexpressing galactose catabolism genes (galK , galE , galT , and galP) from Clostridium acetobutylicum ATCC 824 was constructed for the co-utilization of glucose and galactose. Batch fermentation in the bioreactor showed that ATCC 25755/ ketp could efficiently utilize galactose without glucose-induced carbon catabolite repression and consume nearly 100% of the galactose present in the spent coffee ground hydrolysate. Correspondingly, the butyric acid concentration and productivity of ATCC 25755/ ketp reached 34.3 g/L and 0.36 g/L·h, respectively, an increase of 78.6% and 56.5% compared with the wild-type strain, indicating its potential for butyric acid production from hydrolysates of inexpensive and galactose-rich biomass. [ABSTRACT FROM AUTHOR]

• scrA , scrB and scrK were successfully overexpressed in C. tyrobutyricum ATCC 25755. • The cane molasses could be utilized efficiently by C. tyrobutyricum ATCC 25755/ scrBAK. • The medium composition and initial sugar concentration were optimized. • 45.71 g/L butyrate at a yield of 0.39 g/g was obtained in fed-batch fermentation. The sucrose metabolic genes (scrA, scrB and scrK) from C. acetobutylicum ATCC 824 were successfully overexpressed in C. tyrobutyricum ATCC 25755, endowing it with the ability to co-utilize sucrose, fructose and glucose in the cane molasses. As a result, the engineering strain C. tyrobutyricum ATCC 25755/ scrBAK produced 18.07 g/L and 18.98 g/L butyric acid when sucrose and cane molasses were used as the carbon source, respectively. Furthermore, the medium composition and initial cane molasses concentration were optimized to make full use of the untreated cane molasses. Based on these results, 45.71 g/L butyric acid with a yield of 0.39 g/g was obtained in fed-batch fermentation, and the feedstock cost of using untreated cane molasses was decreased by ~47% when compared with the conventional glucose fermentation. This study demonstrated the potential application of C. tyrobutyricum ATCC 25755/ scrBAK for economic butyric acid production from untreated cane molasses. [ABSTRACT FROM AUTHOR]

• C. tyrobutyricum can assimilate lactic acid for enhanced butanol production. • CSL is a nitrogen source and provides lactic acid to enhance butanol production. • Efficient butanol production was obtained from PMS (and CSL) via either SHF or SRHF. In this study, hyper-butanol producing Clostridium tyrobutyricum Δ cat1::adhE2 was used for butanol production from paper mill sludge (PMS) and corn steep liquor (CSL). Our results demonstrated that CSL can not only serve as a cheap nitrogen source, but also provide lactic acid that can be assimilated by C. tyrobutyricum for enhanced butanol production. Through a separate hydrolysis and fermentation, 16.5 g/L butanol with a yield of 0.26 g/g was obtained from PMS hydrolysates supplemented with 5% CSL. Further, a separate repeated hydrolysis was conducted to improve PMS hydrolysis rate and enhance sugar yield. Fermentation using hydrolysates from such process also generated high-level butanol with high yield. Our results suggested an innovative bioprocess for efficient biobutanol production from low-value waste streams. [ABSTRACT FROM AUTHOR]

• n -Butanol production from low-cost cassava bagasse hydrolysate was studied. • Engineered Clostridium tyrobutyricum was immobilized in a fibrous-bed bioreactor. • Methyl viologen or benzyl viologen was added to enhance n -butanol production. • High-level n -butanol (>15 g/L) with a >0.3 g/g yield was obtained in fermentation. • Techno-economic analysis shows the potential for industrial application. The production of biofuels such as butanol is usually limited by the availability of inexpensive raw materials and high substrate cost. Using food crops as feedstock in the biorefinery industry has been criticized for its competition with food supply, causing food shortage and increased food prices. In this study, cassava bagasse as an abundant, renewable, and inexpensive byproduct from the cassava starch industry was used for n -butanol production. Cassava bagasse hydrolysate containing mainly glucose was obtained after treatments with dilute acid and enzymes (glucoamylases and cellulases) and then supplemented with corn steep liquor for use as substrate in repeated-batch fermentation with engineered Clostridium tyrobutyricum CtΔ ack - adhE2 in a fibrous-bed bioreactor. Stable butanol production with high titer (>15.0 g/L), yield (>0.30 g/g), and productivity (~0.3 g/L∙h) was achieved, demonstrating the feasibility of an economically competitive process for n -butanol production from cassava bagasse for industrial application. [ABSTRACT FROM AUTHOR]

• n -Butanol production from lignocellulosic biomass hydrolysates was studied. • Engineered Clostridium tyrobutyricum was immobilized in a fibrous-bed bioreactor. • Co-utilization of glucose and xylose in the hydrolysates was achieved. • High-level butanol production was obtained from hydrolysates without detoxification. • Techno-economic analysis shows the potential for industrial application. Acetone-butanol-ethanol fermentation suffers from high substrate cost and low butanol titer and yield. In this study, engineered Clostridium tyrobutyricum CtΔ ack - adhE2 immobilized in a fibrous-bed bioreactor was used for butanol production from glucose and xylose present in the hydrolysates of low-cost lignocellulosic biomass including corn fiber, cotton stalk, soybean hull, and sugarcane bagasse. The biomass hydrolysates obtained after acid pretreatment and enzymatic hydrolysis were supplemented with corn steep liquor and used in repeated-batch fermentations. Butanol production with high titer (∼15 g/L), yield (∼0.3 g/g), and productivity (∼0.3 g/L∙h) was obtained from cotton stalk, soybean hull, and sugarcane bagasse hydrolysates, while corn fiber hydrolysate with higher inhibitor contents gave somewhat inferior results. The fermentation process was stable for long-term operation without any noticeable degeneration, demonstrating its potential for industrial application. A techno-economic analysis showed that n -butanol could be produced from lignocellulosic biomass using this novel fermentation process at ∼$2.5/gal for biofuel application. [ABSTRACT FROM AUTHOR]