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In this study, we investigated the metabolic behavior of the engineered Clostridium acetobutylicum ATCC 824 (pCD07239) strain during the acidogenic phase under varying glucose concentrations and pH conditions. Unlike the wild-type C. acetobutylicum ATCC 824, the engineered strain exhibited negligible butyrate production and simultaneous butanol production during the acidogenic phase under limited glucose condition of 25 g/L. Specifically, batch fermentations of the engineered strain with 25 g/L glucose at a pH of around 5.0 (initially uncontrolled) demonstrated butanol production of 2.99 g/L, while butyrate remained below 0.30 g/L. Separately, in batch fermentations at pH 6.0 with 90 g/L glucose, acetate production nearly doubled compared to fermentations at pH 5.0 with the same glucose concentrations, reaching a maximum concentration of 11.43 g/L, while butyrate production remained relatively low at 4.04 g/L. Under these pH 6.0 and 90 g/L glucose conditions, butanol production reached 9.86 g/L. These findings indicate that C. acetobutylicum ATCC 824 (pCD07239) maintained low butyrate production, even under conditions favoring acidogenesis, and consistently produced butanol. Additionally, the negligible production of acetone at pH 6.0 further indicates that the traditional phase transition was not prominent, suggesting altered regulation mechanisms in the engineered strain. These findings highlight C. acetobutylicum ATCC 824 (pCD07239) strain's unique metabolic profile and its potential for efficient biobutanol production under diverse conditions. [ABSTRACT FROM AUTHOR]

Abstract In this study, we investigated the metabolic behavior of the engineered Clostridium acetobutylicum ATCC 824 (pCD07239) strain during the acidogenic phase under varying glucose concentrations and pH conditions. Unlike the wild-type C. acetobutylicum ATCC 824, the engineered strain exhibited negligible butyrate production and simultaneous butanol production during the acidogenic phase under limited glucose condition of 25 g/L. Specifically, batch fermentations of the engineered strain with 25 g/L glucose at a pH of around 5.0 (initially uncontrolled) demonstrated butanol production of 2.99 g/L, while butyrate remained below 0.30 g/L. Separately, in batch fermentations at pH 6.0 with 90 g/L glucose, acetate production nearly doubled compared to fermentations at pH 5.0 with the same glucose concentrations, reaching a maximum concentration of 11.43 g/L, while butyrate production remained relatively low at 4.04 g/L. Under these pH 6.0 and 90 g/L glucose conditions, butanol production reached 9.86 g/L. These findings indicate that C. acetobutylicum ATCC 824 (pCD07239) maintained low butyrate production, even under conditions favoring acidogenesis, and consistently produced butanol. Additionally, the negligible production of acetone at pH 6.0 further indicates that the traditional phase transition was not prominent, suggesting altered regulation mechanisms in the engineered strain. These findings highlight C. acetobutylicum ATCC 824 (pCD07239) strain’s unique metabolic profile and its potential for efficient biobutanol production under diverse conditions.

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.

The study aims to optimize the medium composition for biobutanol production using a Palm Kernel Cake (PKC) hydrolysate by Clostridium saccharoperbutylacetonicum N1-4. Various nutrient factors affecting biobutanol production were screened using the Plackett-Burman design. These factors included: NH4NO3, KH2PO4, K2HPO4, MgSO4.7H2O, MnSO4.7H2O, FeSO4.7H2O, yeast extract, cysteine, PABA, biotin, and thiamin. The results were analyzed by an analysis of variance (ANOVA), which showed that cysteine (P=0.008), NH4NO3 (P=0.011) dan yeast extract (P=0.036) had significant effects on biobutanol production. The established model from the ANOVA analysis had a significant value of Pmodel>F = 0.0299 with an F-value of 32.82 which explains that the factors can explain in detail the variation in the data about the average and the interpretation is true with an R2 value of 0.993. The estimated maximum biobutanol production was 10.56 g/L, whereas the optimized medium produced 15.49 g/L of biobutanol. Process optimizations with optimum concentration of cysteine, NH4NO3, and yeast extract have produced 21.33 g/L biobutanol which is a 37.7% improvement from the non-optimized medium. The findings show that PKC hydrolysate with the addition of optimal concentrations of the three types of medium namely, cysteine (0.15 g/L), NH4NO3 (0.50 g/L), and yeast extract (1.5 g/L) during ABE fermentation, yielded a maximum biobutanol concentration of 21.33 g/L. Therefore, the results of this study provide good indications for promoting PKC hydrolysate as a new source of novel substrates with great potential in producing high biobutanol through ABE fermentation by C. saccharoperbutylacetonicum N1-4. [ABSTRACT FROM AUTHOR]

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured. Key points: • Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum • Cultivation conditions of the pre-culture are a reason for the acid crash • Inoculum from cells in stationary growth phase ensures shift to solventogenesis [ABSTRACT FROM AUTHOR]

Jackfruit (Artocarpus heterophyllus Lam) waste (JFW), a lignocellulosic biomass, is a potential raw material that can be tapped to produce energy and valuable products for human use. In the present work, biobutanol production was studied using jackfruit waste. Initially, the jackfruit waste was pretreated using acid and alkaline agents and hydrolyzed to produce fermentable sugars. The hydrolyzed jackfruit waste was fermented using Clostridium acetobutylicum NCIM 2877 to produce biobutanol. Among the studied pretreatments, sulfuric acid pretreatment produced a maximum sugar yield of 84.21 ± 2.6 mggds−1 in the acid liquor. Enzyme hydrolysis of pretreated jackfruit waste produced a total glucose yield of 215.12 ± 13.44 mggds−1 after optimization. After detoxification using activated charcoal, the JFW hydrolysate produced a maximum biobutanol titer and yield of 1.60 ± 0.5 g L−1, and 106.59 ± 5.1 mg/gglucose, respectively. Central composite design optimization of acetone‐butanol‐ethanol fermentation produced a maximum biobutanol titer of 5.6 ± 0.2 g L−1 (250.00 ± 11.4 mg/gglucose) and total solvent titer of 7.28 ± 0.3 g L−1 (325.00 ± 13.1 mg/gglucose). A 31 ton annual biobutanol production from JFW, simulated using SuperPro Designer, revealed the unit production cost and payback time to be $10.49/kg and 8.94 years. [ABSTRACT FROM AUTHOR]

The concept of energy conversion into platform chemicals using bioelectrochemical systems (BES) has gained increasing attention in recent years, as the technology simultaneously provides an opportunity for sustainable chemical production and tackles the challenge of Power‐to‐X technologies. There are many approaches to realize the industrial scale of BES. One concept is to equip standard bioreactors with static electrodes. However, large installations resulted in a negative influence on various reactor parameters. In this study, we present a new single‐chamber BES based on a stirred tank reactor in which the stirrer was replaced by a carbon fiber brush, performing the functions of the working electrode and the stirrer. The reactor is characterized in abiotic studies and electro‐fermentations with Clostridium acetobutylicum. Compared to standard reactors an increase in butanol production of 20.14±3.66 % shows that the new BES can be efficiently used for bioelectrochemical processes. [ABSTRACT FROM AUTHOR]

Background: Sucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium. Results: The sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses. Conclusions: Our work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks. [ABSTRACT FROM AUTHOR]

Solvent-producing Clostridium acetobutylicum was purified and used in an acetone-butanol-ethanol (ABE) fermentation process. The objective of this study is to design a fermentation medium for the synthesis of butanol and determine the ideal glucose concentration for appropriate microbe ingestion. The fermentation medium was incubated at 37 °C for up to 90 h before inoculation while being sparged with nitrogen gas under anaerobic conditions. Based on the optical density of fermentation media, the growth rate was also monitored. At 60 g/L of glucose, which was the optimum condition for fermentation, the process followed a log phase pattern until the death phase, with the largest growth taking place between 10 h and 50 h after incubation. The C. acetobutylicum steadily consumed the glucose content, reaching its maximal consumption with only around 12 g/L remaining. In contrast to acetone and ethanol, which produced the highest concentrations at 6.4 g/L and 5.2 g/L, respectively, butanol productions were seen appropriately, with the greatest concentration yielding 11.2 g/L of butanol. This shows that C. acetobutylicum expressed its active metabolism for up to 60 g/L and further increase of glucose content will deteriorate the performance of butanol production. [ABSTRACT FROM AUTHOR]

The preparation of bio-butanol from corn starch requires saccharification and fermentation processes. In view of the fact that the pH value at the later stage of fermentation is applicable to the enzymatic hydrolysis of glucoamylase, the effects of simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were compared in this paper. 11.2 g/L butanol and 21.5 g/L total solvent could be obtained by the SSF process, while the yield was 9.74 g/L butanol and 17.2 g/L total solvent in the SHF process. The SSF process required a shorter overall process time (120 h) than the SHF process (144 h) and resulted in a large increase of 38.9% in butanol productivity (2.25 g/Ld for SSF compared to 1.62 g/Ld for SHF). These results show that the application of SSF can reduce the fermentation overall time, simplify the fermentation process, and reduce equipment investment and operating costs.

Different strategies have been assessed for the revalorization of guishe to obtain biomolecules. The juice obtained after the mechanical extraction of guishe is rich in phytochemicals and sugars which can be converted to other products. The objective of the present study was to evaluate the production of hydrogen and butanol at different guishe juice concentrations (and therefore, different sugar concentrations) via fermentation in batch mode using Clostridium acetobutylicum ATCC 824. Fermentation assays were performed in triplicate under anaerobic conditions at 35 °C for 142 h. Guishe juice was supplemented with all components of synthetic medium (salts, vitamins and reducing agents), except glucose, and diluted at different concentrations: 20%, 40%, 60%, 80% and 100%. For comparison purposes, a control was carried out in a synthetic medium using glucose as carbon source. Results showed a maximum butanol concentration of 5.39 g/L using 80% guishe juice, corresponding to a productivity and yield of 0.04 g/L h−1 and 0.24 g/g, respectively. Meanwhile, the highest productivity (1.16 L H2/L d−1; 1.99 mmol H2/L h−1) and yield (18.4 L/kg) of hydrogen were obtained with 40% guishe juice. This study demonstrates the potential of guishe juice to be used as a low-cost substrate for hydrogen and butanol production. [ABSTRACT FROM AUTHOR]

The review provides an overview of the current status of the solvent-producing clostridia. The origin and development of industrial clostridial species, as well as the history of the industrial Acetone Butanol Ethanol fermentation process, is reexamined, and the recent resurgence of interest in the production of biobutanol is reviewed. Over 300 fully sequenced genomes for solvent-producing and closely related clostridial species are currently available in public databases. These include 270 genomes sourced from the David Jones culture collection. These genomes were allocated arbitrary DJ codes, and a conversion table to identify the species and strains has now been provided. The expanded genomic database facilitated new comparative genomic and phylogenetic analysis. A synopsis of the common features, molecular taxonomy, and phylogeny of solvent-producing clostridia and the application of comparative phylogenomics are evaluated. A survey and analysis of resident prophages in solvent-producing clostridia are discussed, and the discovery, occurrence, and role of novel R-type tailocins are reported. Prophage genomes with R-type tailocin-like features were detected in all 12 species investigated. The widespread occurrence of tailocins in Gram-negative species is well documented; this survey has indicated that they may also be widespread in clostridia. [ABSTRACT FROM AUTHOR]

Generating hydrogen gas (H 2) using the dark fermentation method has attracted much attention due to its lower energy requirement and environmental friendliness. However, producing a high yield of bio-H 2 is as challenging as ever due to low energy conversion by microorganisms. In this respect, the advancement of genome editing tools including the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology could overcome the established maximum ceiling of product yield. To date, CRISPR-Cas systems, particularly those based on Type II CRISPR-Cas9 and Type V CRISPR-Cas12, are widely used in manipulating novel bacteria to improve the yield of specific biofuel. However, studies using the CRISPR-Cas technology for improving bio-H 2 production remain scarce. Understanding the metabolic pathways of Clostridium spp. is essential for using the CRISPR-Cas technology Thus, this review highlighted the state-of-the-art in CRISPR-Cas systems for bacterial genome editing while paying attention to bioprocess optimization strategies for modulating the biohydrogen production. • Cas9, Cas12 and endogenous CRISPR-Cas can be used for genome editing in Clostridium. • Template vectors for easy deliveries of CRISPR-Cas components in Clostridium spp. • Overexpress butyrate and knockout acetate production pathway improve H 2 production. • Simultaneous repression of solvent and lactate pathway for efficient H 2 production. • Modulation of lignocellulose and sugar utilization genes to improve H 2 production. [ABSTRACT FROM AUTHOR]

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile , aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum , we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD 600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD 600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase. • THF cycle to yield CH 3 -THF was evaluated in C. acetobutylicum. • Methyl branch of Wood-Ljungdahl pathway was validated by 13C-tracing. • Heterologous Wood-Ljungdahl pathway was installed in C. acetobutylicum. • Butanol production at early growth phase was achieved in engineered strain. [ABSTRACT FROM AUTHOR]

Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of xylR (CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of araR (CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both araR and xylR genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both araR and xylR in C. acetobutylicum. As suggested by these data, overexpression of xylA and xylB led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars. IMPORTANCE C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them. [ABSTRACT FROM AUTHOR]

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_Ppta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach. [ABSTRACT FROM AUTHOR]

As an essential lignocellulosic source, corn stover (CS) arouses an extensive interest in transforming into bio-butanol and high-value-added products. Optimization experiments of medium and kinetic fermentation experiments of Clostridium acetobutylicum CICC 8008 were conducted. The CS was pretreated by a combination of steam explosion and NaOH, followed by saccharification by cellulase. The saccharification liquor contained 65.91 g/L of total reducing sugar. The optimum fermentation medium was 1,000 mL saccharification liquor supplemented with 4.89 g/L yeast extract, 6.9 g/L CaCO 3 , and 10.56 mg/L biotin. When the optimum medium was used in the butanol fermentation process, the maximum acetone-butanol-ethanol (ABE) content was 41.78 g/L, and the contents of acetone, butanol, and ethanol were 9.73, 23.82, and 8.23 g/L, respectively. The maximum specific growth rate was 0.066 h−1, the coefficient of growth was 2.29 g/(gL), and no substrate and production inhibition were found. ABE was the growth-associated metabolite. Collectively, our current findings provided valuable insights into the efficient application of lignocellulose biomass into the biorefinery of butanol. [Display omitted] [ABSTRACT FROM AUTHOR]

A new alkaline tolerant Clostridium felsineum strain (CUEA03) was isolated from a mangrove sediment in Thailand. Genomics revealed that this strain contained a 5,081,113 bp genome sequence with 4797 predicted protein coding sequences, comprised of a large number of genes that are linked to several processes of carbon utilization, hydrogen (H 2), and butanol production. Chemical tests revealed that the bacteria have a high potential for utilization of a wide range of carbon sources. After optimization by batch fermentation, a maximum cumulative H 2 production (CHP) of 5425 mL/L in 72 h was obtained at pH 9, 30 °C, 0.9 g/L NaCl, and 35 g/L glucose. Moreover, this strain was compatible with agro-industrial waste, giving a CHP of 5187 mL/L (893 mL/g COD consumed). This demonstrates that environmentally isolated organisms have the potential to be used as robust high H 2 producers from various complex organic substrates. [Display omitted] • New alkaline tolerant Clostridium felsineum strain CUEA03. • Draft genome sequencing and genomics information of CUEA03. • CUEA03 was compatible with xylose, MCC, mannose, lactose, starch, etc. • Several important fermentation factors were optimized for H 2 production by CUEA03. [ABSTRACT FROM AUTHOR]

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7–11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4–9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system. [ABSTRACT FROM AUTHOR]

In this study, two types of fermentation methods: SSF and consolidation SHF/SSF were used for production of acetone-butanol-ethanol (ABE) from corn straw as a feedstock. Clostridium acetobutylicum DSM1731 was used as the fermenting organism. Corn straw was thermochemically pretreated and then hydrolyzed using three types of enzymes. The impact has been investigated on the effect of mineral compounds supplementation ((NH4)2SO4, MgSO4, (NH4)3PO4) on ABE productivity and butanol content. From the SSF, where mineral salts were supplemented into the fermentation medium, it was found that the maximum ABE and butanol concentrations were 28.35 g/L and 24.03 g/L, respectively, corresponding to a productivities of 0.295 g/L/h (ABE) and 0.250 g/L/h (butanol). In the consolidation SHF/SSF method with mineral compounds supplementation, the maximum ABE and butanol concentrations were 31.35 g/L and 28.64 g/L, respectively, corresponding to productivities of 0.327 g/L/h (ABE) and 0.298 g/L/h (butanol). Compared to control samples, mineral salts supplementation had a positive effect on cellular metabolic activities, leading to an earlier initiation of the solventogenesis stage. In supplemented samples, an increase in the rate of ABE fermentation by Clostridium was observed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Economic sustainability of biofuels relies on efficient C5 and C6 sugar use. • Optimized DA pretreatment: C6-rich solid, C5-rich liquid, and minimal inhibitors. • Fermentative routes evaluated: ABE fermentation for 2G butanol, SIcF for 2G. • ethanol. • Cellulosic and hemicellulosic hydrolysates were suitably fermentable. • Pretreatment severity to predict sugars and by-products in other conditions. A systematic study of dilute acid pretreatment of sugarcane bagasse was performed to optimize the recovery of hexose-rich solid and pentose-rich liquid while minimizing fermentation inhibitors. Pretreatment experiments evaluated the effects of pretreatment time (10–90 min) and acid concentration (0.5–3.0 % w/w). The optimized pretreatment (1.4 % w/w sulfuric acid, 56.4 min at 121 °C) led to a solid fraction with 60.4 % cellulose and a liquid stream rich in pentose, in which 92.7 % of sugars was recovered as xylose (17.8 g/L). Besides that, low concentrations of by-products (3 g/L – the sum of acetic and formic acid, furfural, and HMF) were identified in the liquid fraction. Both fractions were integrated into enzymatic processes for further co-fermentation of C5- and C6- sugars. Two fermentative routes were evaluated: simultaneous isomerization of xylose and co-fermentation of glucose and xylulose (SIcF) for 2G ethanol production and acetone-butanol-ethanol (ABE) fermentation of glucose and xylose for biobutanol, utilizing Saccharomyces cerevisiae and Clostridium acetobutylicum , respectively. SIcF yielded 0.48 g ethanol /g sugars (24 g/L ethanol) with complete glucose consumption and 58.2 % xylose conversion. ABE fermentation produced 0.36 g ABE /g sugars (15.7 g/L ABE and 9.5 g/L butanol), with 96 % glucose consumption and 29.6 % xylose conversion. The proof-of-concept demonstrated that the hydrolysates were satisfactorily fermentable by microorganisms, thus validating the pretreatment optimization for fermentation applications. [ABSTRACT FROM AUTHOR]

Biobutanol, a promising green alternative fuel, fermented from Clostridium acetobutylicum , while its high-cost and limited yield constraining its development. ABE (acetone-butanol-ethanol) and IBE (isopropanol-butanol-ethanol) are mixed fermentation products from non-edible biomass raw materials, using them together with water as alternative fuels will reduce industrial production costs and save fossil fuels. Therefore, this study conducted a multifaceted experimental evaluation on ABE/IBE mixed fuels with different water content, demonstrating that it has good water holding capacity when mixed with traditional fossil fuels. Taking ABE (3: 6: 1) as an example, its water holding capacity after mixing with diesel at 10–90 % is 0.37–7.83 % at 20 °C. Meanwhile, the particle size of ABE/IBE mixed fuels is about 2–30 nm, exhibiting a microemulsion with thermodynamic stability. The anhydrous or water-containing mixed fuel with the ratio of ABE (IBE) of 10%–50 % meets the range of the density and kinematic viscosity of diesel engine fuel. The mixed fuel is non-corrosive to copper without water, and a water content of about 3 % or higher will increase the risk of engine corrosion at 20 °C. Despite the addition of biofuel and water, studies on energy combustion performance and pollutant emission performance have found that appropriate addition of biofuel and water can produce higher power output and lower pollutant emissions than traditional fossil fuels, with ABE20W0.5 being the optimal. This study demonstrates the great potential of ABE and IBE as biofuels to achieve carbon neutrality goals, providing novel research direction for green alternative fuels in the future. [Display omitted] • The Clostridium acetobutylicum fermentation products can be used as biofuel. • The mixed fuel with water forms a microemulsion with particle size 2–30 nm. • The microstructure of the mixed fuel changes as the mixed ratio changes. • Only the mixed fuel with higher water content is corrosive to copper flakes. • The fermentation mixed fuel can improve energy performance and save production cost. [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain. Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes. [ABSTRACT FROM AUTHOR]

An integrated bioprocessing approach was applied to enhance energy recovery from the biodegradable fraction of municipal solid waste (BFMSW). Clostridium acetobutylicum and Enterobacter aerogenes with complementary characteristics were cocultivated for coproduction of hydrogen, 2,3-butanediol, and acetone-butanol-ethanol (ABE). Following the fermentation process, the remaining solids were used for biomethane production. The BFMSW was first subjected to ethanolic organosolv pretreatment and then enzymatic cellulose and hemicellulose hydrolysis at 5 and 8% solid loadings. The glucose (328 g/kg raw BFMSW), xylose (37.8 g/kg raw BFMSW), and starch (152.1 g/kg raw BFMSW) portions of the pretreated BFMSW hydrolysate with 5% solid loading were cofermented to hydrogen (162.8 L/kg raw BFMSW), 2,3-butanediol (42.5 g/kg raw BFMSW), butanol (121.9 g/kg raw BFMSW), ethanol (38.9 g/kg raw BFMSW), and acetone (45.2 g/kg raw BFMSW) by the coculture, and subsequently, the residual unfermented solids were further digested to biomethane (31.7 L/kg raw BFMSW). From the bioenergy perspective, 311.8 mL gasoline equivalent (10 MJ energy) was obtained from each kg of raw BFMSW, indicating the potential of the integrated fermentation-anaerobic digestion bioprocess for biofuels production from the zero-cost substrate. [Display omitted] [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.

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

BACKGROUND Butanol is a versatile chemical that can be used as fuel, solvent or intermediate in chemical synthesis. Several strategies have been used to increase the availability of reducing power in Clostridium sp. with the aim of maximizing the production of butanol, the use of electron carriers being one of the most effective. Moreover, electrons from an external power source can be transferred to cells through electron carriers such neutral red, or directly reducing NAD+. From a previous metabolic analysis, we hypothesized that adding butyrate to the culture medium and using an external electron source as a co‐reducing agent, along with glucose‐derived reducing power (electro‐fermentation), would result in an increase in the specific butanol productivity. RESULTS: Butyrate caused a decrease in the maximum concentration of butanol reached at the concentration used (6 g L−1). The supplementation of neutral red to the culture medium by itself and along with electro‐fermentation were effective in stimulating the production of butanol. The mean specific rate of butanol production obtained for electro‐fermentation in medium supplemented with butyrate and neutral red was 307% and 156% higher than that recorded using medium supplemented with butyrate, or butyrate and neutral red, respectively, this value being even 113% higher than that recorded for the non‐supplemented medium. CONCLUSIONS: The presence of butyrate and absence of neutral red in the electro‐fermentation were detrimental for the microorganism. The electro‐fermentation in medium supplemented with neutral red led to an increase in both volumetric and specific productivities of butanol. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Arthrobacter sp. BCC 72131, an obligate aerobic bacterium, was used to create anaerobic conditions, and Clostridium beijerinckii TISTR 1461 was used as a butanol producer in an acetone-butanol–ethanol (ABE) fermentation. Sweet sorghum juice (SSJ) medium containing 60 g/L of total sugar supplemented with 1.27 g/L of (NH4)2SO4 was used as a butanol production (BP) medium. Arthrobacter sp. was inoculated into the BP medium in 1-L screw-capped bottles. After 2, 4, 6 and 12 h of Arthrobacter sp. cultivation at 30 °C, C. beijerinckii was transferred into the BP medium to start butanol production at 37 °C. The results showed that C. beijerinckii inoculation after 6 h of Arthrobacter sp. cultivation gave the highest butanol titer (PB) at 12.56 g/L, with a butanol yield (YB/S) and volumetric butanol productivity (QB) of 0.34 g/g and 0.23 g/L·h, respectively. These values are approximately 10–27% higher than those of the control experiment using a single culture of C. beijerinckii TISTR 1461 and oxygen-free nitrogen (OFN) gas flushing to create anaerobic conditions. Field emission scanning electron microscopic (FE-SEM) images of Clostridium cells, as well as protein and free amino nitrogen concentrations in the broth during butanol fermentation were also studied to confirm the results. The butanol fermentation was then carried out in a 5.6-L stirred-tank and a 1.2-L low-cost gas-lift bioreactor by the mixed cultures using the optimal time of Clostridium inoculation. The PB, YB/S and QB values obtained were not significantly different from those in the 1-L screw-capped bottles. Hence, Arthrobacter sp. can be used as a novel method to create anaerobic conditions instead of a traditional method employing OFN gas flushing. Using mixed cultures of Arthrobacter sp. BCC 72131 and C. beijerinckii TISTR 1461 is a practical method to produce butanol on a large-scale, both in complex and low-cost bioreactors. [ABSTRACT FROM AUTHOR]

[Display omitted] • EF aids early onset of solventogenesis and suppresses acid forming pathway. • Higher butanol production (68.86%) in EF as compared to conventional fermentation. • Production of butanol is governed by crt , hbd and bcd gene. • EF system downregulated buk gene (butyric acid) and upregulated bcd gene (butanol) • Bioenergetics analysis suggested that butanol production via EF is more spontaneous. This investigation scrutinized the quantitative aspects of biobutanol production in conventional and bioelectrogenic induced ABE fermentation using Clostridium acetobutylicum ATCC-824. The primary objective of the study was to investigate how alterations in the extracellular redox potential (-0.4 V vs. Ag/AgCl (3.5 M KCl)) affect the hetero-fermentation towards the production of acetone, butanol, and ethanol during the bi-phasic fermentation, utilizing different glucose concentrations from 20 g/L − 60 g/L. The study showed significant results with 68.86 % increased butanol titer (6.67 g/L) compared to conventional fermentation (3.95 g/L butanol). Additionally, the acetone production also increased to 2.34 g/L as compared to conventional fermentation (1.96 g/L). Furthermore, gene expression and network analysis showed that various ABE pathway genes were significantly up or down regulated during the bi-phasic fermentation, indicating a difference between the conventional and EF reactors. The external potential influenced the overexpression of genes like pta, crt, bcd, ask, adh (CA_C1574), and hbd (CA_C3375), while simultaneously downregulation of buk gene , suggesting the acidogenic decoupling and transition from cold channel (butyric acid) towards hot channel pathway (directly from acetyl CoA through butyryl-CoA) for improved butanol production. Moreover, the study also revealed that entropy and enthalpy changes played a significant role in butanol production and ABE fermentation. The EF showed more negative free energy (-3.33 × 104 kJ/mol) when compared with conventional fermentation (-2.8 × 104 kJ/mol) suggesting the mechanism to be more spontaneous and viable. Overall, this study provided evidence of metabolic rerouting with non-genetic approach by regulating electron flow, culminating in an impact on the bi-phasic fermentation towards specific metabolites. [ABSTRACT FROM AUTHOR]

[Display omitted] • A robust strain with enhanced butanol production was metabolically engineered. • The butanol/ABE ratio increased from 63.2 %–87.6 % by knocking out adc gene. • The knockout of adc gene limited butanol production to ∼11 g/L. • Maximum butanol titer of 19.7 g/L was obtained by overexpressing adhE2 gene. Biobutanol is naturally produced by solventogenic clostridia during acetone-butanol-ethanol (ABE) fermentation, which is hampered by low butanol titer, yield and productivity. Here, Clostridium acetobutylicum ATCC 55025 and its robust mutant HKKO with histidine kinase genes cac3319/cac0323 knockout were metabolically engineered to eliminate acetone formation and improve butanol production. By knocking out acetoacetate decarboxylase gene adc in ATCC 55025 and HKKO, the acetone titer decreased to 0.1 g/L and the butanol/ABE ratio increased by 39.0 % and 40.9 %, respectively. However, the butanol production was limited to ∼11 g/L. To improve butanol production, an alternative strategy was used by overexpressing acetaldehyde/alcohol dehydrogenase gene adhE2 in HKKO, and the mutant produced butanol of 19.7 g/L than that of 18.2 g/L from HKKO with high butanol productivity of 0.41 g/L/h and yield of 0.26 g/g. This study provides novel insights into the roles of adc and adhE2 on butanol biosynthesis and generates a hyper-butanol-producing strain. [ABSTRACT FROM AUTHOR]

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, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating 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. [ABSTRACT FROM AUTHOR]

Two bacterial strains (CL11A and CL11D) that are capable of ABE fermentation, identified as Leuconostoc mesenteroides and Weissella cibari, were isolated from the soil surrounding the roots of bean plants. Another strain (ZM 3A), identified as Lactobacillus plantarum, which is capable of purely ethanolic fermentation was isolated from sugarcane. Glucose was used as a standard substrate to investigate the performance of these strains in mono--and co-culture fermentation for ABE production. The performance parameters employed in this study were substrate degradation rates, product and metabolite yields, pH changes and microbial growth rates. Both ABE isolates were capable of producing the three solvents but Leuconostoc mesenteroides had a higher specificity for ethanol than Weissella cibari. The co-culturing of Leuconostoc mesenteroides and Lactobacillus plantarum enhanced ethanol production at the expense of both acetone and butanol, and also influenced the final substrate consumption rate and product yield. The experiments indicated the potential of these niche environments for the isolation of ABE-producing microorganisms. This study contributes to the formulation of ideal microbial co-culture and consortia fermentation, which seeks to maximize the yield and production rates of favored products. [ABSTRACT FROM AUTHOR]

Clostridium fermentations have been developed for producing butanol and other value-added chemicals, but their development is constrained by some limitations, such as relatively high substrate cost and the need to maintain an anaerobic condition. Recently, co-culture is emerging as a popular way to address these limitations by introducing a partner strain with Clostridium. Generally speaking, the co-culture strategy enables the use of a cheaper substrate, maintains the growth of Clostridium without any anaerobic treatment, improves product yields, and/or widens the product spectrum. Herein, we review recent developments of co-culture strategies involving Clostridium species according to their partner stains' functions with representative examples. We also discuss research challenges that need to be addressed for the future development of Clostridium co-cultures. Clostridium co-culture has emerged as a solution to some problems faced by Clostridium monoculture. Co-culturing Clostridium with cellulolytic strains or acetogens enables the production of butanol from cheaper substrates such as lignocellulosic biomass and syngas. Aerotolerance is possible in anaerobic Clostridium fermentation in co-cultures with oxygen-consuming microbes. Co-culturing solventogenic Clostridium with acetogens can improve the production of butanol and its derivatives by increasing carbon recovery and relieving solvent toxicity. Other products, such as H 2 or fatty acids, can be efficiently produced by the co-culture of Clostridium and other H 2 -producing strains (such as PNS bacteria), or chain-elongating strains respectively. [ABSTRACT FROM AUTHOR]

Background: Isobutanol is an attractive biofuel with many advantages. Third-generation biorefineries that convert CO2 into bio-based fuels have drawn considerable attention due to their lower feedstock cost and more ecofriendly refining process. Although autotrophic cyanobacteria have been genetically modified for isobutanol biosynthesis, there is a lack of stable and convenient strategies to improve their production. Results: In this study, we first engineered Synechococcus elongatus for isobutanol biosynthesis by introducing five exogenous enzymes, reaching a production titer of 0.126 g/L at day 20. It was then discovered that high salinity stress could result in a whopping fivefold increase in isobutanol production, with a maximal in-flask titer of 0.637 g/L at day 20. Metabolomics analysis revealed that high salinity stress substantially altered the metabolic profiles of the engineered S. elongatus. A major reason for the enhanced isobutanol production is the acceleration of lipid degradation under high salinity stress, which increases NADH. The NADH then participates in the engineered isobutanol-producing pathway. In addition, increased membrane permeability also contributed to the isobutanol production titer. A cultivation system was subsequently developed by mixing synthetic wastewater with seawater to grow the engineered cyanobacteria, reaching a similar isobutanol production titer as cultivation in the medium. Conclusions: High salinity stress on engineered cyanobacteria is a practical and feasible biotechnology to optimize isobutanol production. This biotechnology provides a cost-effective approach to biofuel production, and simultaneously recycles chemical nutrients from wastewater and seawater. [ABSTRACT FROM AUTHOR]

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% H 2 SO 4), 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. [Display omitted] • Aqueous pretreatments were assessed for co-production of butanol and biogas. • Dilute alkaline led to the highest yield of butanol and biogas. • The process led to a 64% improvement in the energy return on investment (EROI). • In the process 23–150 g ABE and 18–46 L methane was obtained from each kg straw. [ABSTRACT FROM AUTHOR]

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 sole nutrient sources. The biomass and solvent production from potato peel were enhanced in orange peel extract with the concentration and improved the sugar recovery. The effect on solvent production from 60 g/L potato peel in dilute orange peel extract was relatively higher than 20 and 40 g/L. The butanol yield was improved from 0.04 to 0.35 g/g from 60 g/L potato peel waste in orange peel extract. The butyric acid concentration was highly correlated with butanol production. The Clostridium acetobutylicum MTCC 11274 showed no inhibitory effect from D-limonene in orange peel extract. The potato peel waste hydrolyzate prepared in orange peel extract enhanced the growth of Clostridium acetobutylicum MTCC 11274 and fermentation of acetone, butanol, and ethanol. [ABSTRACT FROM AUTHOR]

The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed. Key Points • The regulatory network governing sporulation initiation varies in solventogenic clostridia. • Media composition and cell density are the main triggers of sporulation. • Spores can be used to improve the fermentation process. [ABSTRACT FROM AUTHOR]

[Display omitted] • CSTR with GAC is an effective strategy for Clostridium spp. immobilization. • High butanol productivity of 2.07 g L−1 h−1 from RS hydrolysate. • GS plus two PV cycles at mild conditions concentrated butanol up to 291 g L−1. • Discontinuous downstream plus distillation reduced the energy use to 20.6 MJ kg−1. This study assessed the performance of two continuous stirred tank reactors (CSTRs) to immobilize Clostridium acetobutylicum DSM 792 and Clostridium beijerinckii DSM 6422 on granular activated carbon. Dilution rates (D) from 0.05 to 0.60 h−1 were tested using a sugar mixture to mimic rice straw hydrolysate. Immobilization was shown to be a highly effective technique for processing high flows without cell washout. Both CSTRs demonstrated significantly higher butanol productivity than their free-cell counterparts, achieving the remarkable value of 2.29 g L−1 h−1 with C. beijerinckii at D = 0.60 h−1. Rice straw hydrolysate was fed to the high-rate CSTR, reaching a butanol productivity of 2.07 g L−1 h−1 at D = 0.30 h−1. A novel downstream configuration of ex situ gas stripping followed by two pervaporation cycles operated under rough vacuum was explored in discontinuous mode, that reduced the energy demand by 63 % of conventional distillation methods. This configuration holds great promise for the advance of the lignocellulosic biorefinery process. [ABSTRACT FROM AUTHOR]

[Display omitted] • Spent Substrate of Pleurotus ostreatus can act as potential biomass for biobutanol production. • Clostridium acetobutylicum MTCC 11274 produced ABE concentration of 10.80 ± 0.02 g/L. • Hybrid pre-treatment showed maximum biobutanol yield of 0.38 g/g. • SEM, FT-IR, XRD data confirmed the breakdown of lignin and hemicellulose. Addressing global energy demand, researchers sought eco-friendly biobutanol production from lignocellulosic waste biomass. In the present research work, five different pre-treatment methods viz., Microwave, Ultrasound, Alkali, Acid, and Hybrid, were investigated to explore its biobutanol production potential by utilizing Pleurotus ostreatus spent as substrate. The compositional and physico-chemical changes of the pre-treated Spent Mushroom Substrate (SMS) were assessed using SEM, FTIR, and XRD. Hybrid pre-treatment (Microwave, Alkali, Ultrasound) showed higher delignification when compared to conventional pre-treatment method. Hybrid pre-treated SMS resulted in higher total reducing sugars (521.53 ± 1.84 mg/g) than indigenous SMS (267.89 ± 1.53 mg/g). Fermentation of hybrid pre-treated SMS with Clostridium acetobutylicum MTCC 11274 produced the highest biobutanol concentration (9.84 ± 0.03 g/L) and yielded 0.38 ± 0.02 g/g of biobutanol. This study revealed that hybrid pre-treatment could be a promising solution for enhanced biobutanol production using SMS biomass. [ABSTRACT FROM AUTHOR]

Abstract 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.

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Strain thermotolerance of an engineered Clostridium acetobutylicum was enhanced at high temperature of 39–45 °C. • High temperature SSF of pretreated corn stover was developed with or without detoxification. • Overlimed acid-treated liquid contributed to efficient SSF at a matching temperature up to 42 °C. • Butanol productivity was improved to 0.18 g/L/h during SSF with 12 h pre-hydrolysis at 42 °C. 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. [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum (C. acetobutylicum) has considerable potential for use in bioenergy development. Owing to the repeated use of traditional mutagenesis methods, the strains have developed a certain tolerance. The rheology of the bioprocess and the downstream processing of the product heavily depend on the ability of C. acetobutylicum mutants to produce butanol. Carbon ion beam irradiation has advantages over traditional mutation methods for fermentative production because of its dose conformity and superb biological effectiveness. However, its effects on the specific productivity of the strains have not been clearly understood. In this study, we screened five mutants through carbon ion beam irradiation; mutant Y217 achieved a butanol-production level of 13.67 g/L, exceeding that of wild-type strain ATCC 824 (i.e., 9.77 g/L). In addition, we found that the mutant maintained normal cell membrane integrity under the stimulation of 15 g/L butanol, whereas the intracellular macromolecules of wild-type strain ATCC 824 leaked significantly. Subsequently, we used the response surface methodology (RSM) to determine if the mutant cell membrane integrity improved the butanol tolerance. We verified that with the addition of butanol, the mutant could be fermented to produce 8.35 g/L butanol, and the final butanol concentration in the fermentation broth could reach 16.15 g/L. In this study, we proved that under butanol stress, mutant Y217 features excellent butanol production and tolerance and cell membrane integrity and permeability; no prior studies have attempted to do so. This will serve as an interesting and important illustration of the complexity of genetic control of the irradiation mutation of C. acetobutylicum strains. It may also prove to be useful in the bioengineering of strains of the mutant for use in the predevelopment stage. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The present work aimed at establishing an efficient degradation and energy recovery system form sugarcane bagasse (SCB) through hydrogen peroxide-acetic acid (HPAC) pretreatment, thermophilic hydrogen production and mesophilic methane production. The degradation ratio of HPAC pretreated SCB (HPAC-SCB, 2%, w/v) exceeded 90% under the biological hydrolysis of C. thermocellum without enzyme addition. The hydrogen yield in the co-culture fermentation of T. thermosaccharolyticum and C. thermocellum from HPAC-SCB (2%, w/v) reached 226 mL/g substrate. The long-term hydrogen fermentation was successfully established with 1.59 L/(L·d), 0.159 L/g substrate for average hydrogen productivity and yield, respectively. Methane production of 0.341 L/g COD (chemical oxygen demand) added was recovered by semi-continuous methane fermentation from hydrogen-producing effluent at 12 days of hydraulic retention time (HRT). Average energy recovery of 8.79 MJ/kg SCB was obtained under the optimal conditions. The present work indicated the promising application of the established process in valorization of lignocellulosic bio-waste. Image 1 • 70 mM reducing sugar was accumulated in the saccharification of C. thermocellum. • 90% of HPAC-SCB was degraded by C. thermocellum. • 0.226 L H 2 /g HPAC-SCB was achieved by co-culture. • Energy recovery of two-stage process was higher than that of single-stage process. [ABSTRACT FROM AUTHOR]

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. Image 1 • PPW is a carbohydrate-rich but toxic waste for butanol production. • The presence of glycoalkaloids in PPW significantly affected ABE production. • Three process alternatives were compared for ABE production from PPW. • Simultaneous inhibitors removal and pretreatment led to a high butanol yield. • PPW is suitable for butanol production after removal of its bacterial inhibitors. [ABSTRACT FROM AUTHOR]

In traditional microbial biobutanol production, the solvent must be recovered during fermentation process for a sufficient space‐time yield. Thermal separation is not feasible due to the boiling point of n‐butanol. As an integrated and selective solid‐liquid separation alternative, solvent impregnated resins (SIRs) were applied. Two polymeric resins were evaluated and an extractant screening was conducted. Vacuum application with vapor collection in fixed‐bed column as bioreactor bypass was successfully implemented as butanol desorption step. In course of further increasing process economics, fermentation with renewable lignocellulosic substrates was conducted using Clostridium acetobutylicum. Utilization of SIR was shown to be a potential strategy for solvent removal from fermentation broth, while application of a bypass column allows for product removal and recovery at once. [ABSTRACT FROM AUTHOR]

Clostridium butyricum is a bacterium of rod shape, strictly anaerobic, Gram‐positive and form spores. Short‐chain fatty acids, biofuel compounds and precursors of biomaterials (H2, butanol and 1,3‐propanediol) are produced by C. butyricum during the fermentation of carbohydrates and sugars. In humans and animals, although C. butyricum has been found to be associated with botulism in infant and necrotising enterocolitis in preterm neonates, some strains have been demonstrated to be beneficial as probiotics. In aquatic animals, the application of C. butyricum as a probiotic has been commonly considered. In this review, we described and documented what we knew about the isolation, identification, phenotypic and biochemical characteristics, safety, pathogenicity and the mechanisms of action of C. butyricum in the gastrointestinal tract of aquatic animals. The evidence on the roles that C. butyricum plays in improving growth performance, digestibility, survival rate, immune response, disease resistance and the structure of intestinal tract, as well as the modulation of gut commensal microbiota and metabolic disorders in aquatic animals, is summarised. Albeit the probiotic C. butyricum has been applied in aquaculture, and further research, such as the inter‐relationships between the probiotic and the immune system of the host and the possible application of C. butyricum as an alternative to antibiotic substitution for the prevention and treatment of diseases, is required for future detailed studies. [ABSTRACT FROM AUTHOR]