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This study evaluated the energy recovery of citrus wastes with pure cultures (Clostridium acetobutylicum ATCC 824, Clostridium beijerinckii ATCC 10132 – Inocula 1 and 2, respectively) and an autochthonous microbial (Inoculum 3) from citrus wastewater (CW) in anaerobic batch reactors to produce biofuels (H 2 and ethanol) fed with different carbon sources (glucose, maltose, fructose, sucrose, xylose, lactose, starch, acetate, and glycerol) in addition to CW. 4 conditions are tested: 1st - Inoculum 3, composed by Bacillus (20.44%), Clostridium (13.65%) and Lactobacillus (10.61%) was able to produce H 2 with sucrose, glucose and fructose. 2nd - The highest metabolites were produced with TYA medium with Inocula 1 (374 mg L−1 of acetic acid) and 2 (611 mg L−1 of butanol). Inoculum 3 generated the highest ethanol (1571.8 mg L−1) in PYG medium. 3rd - In the same nutritional conditions (PYG), Inocula 1 and 2 produced H 2 (36.80 and 64.10 mmol L−1, respectively) and acetic acid (246 and 201 mg L−1, respectively); the H 2 was suppressed with inoculum 3 due to Lactobacillus genus. 4th H 2 production was favored by autochthonous bacteria in CW. This study valued the autochthonous microorganisms in the possibility of bioconversion of organic matter into value-added products in addition to contribute to an environment-sustainable solution for citrus processing. [Display omitted] • Autochthonous consortium from citrus wastewater was able to produce 37.8 mmol H 2 L−1. • Bacillus , Clostridium and Lactobacillus were identified in autochthonous microbial. • Clostridium beijerinckii ATCC 10132 was able to produce 611 mg L−1 of butanol. • Autochthonous microbial generated the highest ethanol (1571.8 mg L−1) in PYG medium. [ABSTRACT FROM AUTHOR]

The present study investigated, the effects of different antimicrobial compounds on Clostridium species, responsible for the late-blowing defect (LBD) in the dairy industry. The antimicrobial effect of nisin, lysozyme and water-soluble extracts (WSE) on tested Clostridium spp. strains. The minimum inhibition concentration (MIC) of nisin against vegetative cells of C. sporogenes 73, C. sporogenes 97, and C. butyricum 99 was determined to be 6.25 µg mL-1, 12.5 µg mL-1 and 6.25 µg mL-1, respectively. The MIC of nisin against spores of all Clostridium species/strains was found to be 1.56 µg mL-1. However, it was determined that lysozyme, known to be effective on gram-positive microorganisms, and did not affect Clostridium spp. vegetative cells and spores at the 10 mg mL-1 maximum concentration. In addition to nisin and lysozyme, the anticlostridial effect of WSEs from different cheeses was also investigated. The WSEs (50 mg mL-1) obtained from various ripened cheeses did not have an affect on Clostridium spores, whereas they showed a strong antimicrobial effect on vegetative cells. Furthermore, the peptide profiles of these WSEs were determined in reserve-phase high performance liquid chromatography (RP-HPLC), and these peptide profiles were found to differ. [ABSTRACT FROM AUTHOR]

Mesophilic, anaerobic, and cellulolytic Ruminiclostridium-type bacterial species can secrete an extracellular, multi-enzyme machinery cellulosome, which efficiently degrades cellulose. In this study, we first reported the complete genome of Ruminiclostridium papyrosolvens DSM2782, a single circular 5,027,861-bp chromosome with 37.1% G  +  C content, and compared it with other Ruminiclostridium-type species. Pan-genome analysis showed that Ruminiclostridium-type species share a large number of core genes to conserve basic functions, although they have a high level of intraspecific genetic diversity. Especially, KEGG mapping revealed that Ruminiclostridium-type species mainly use ABC transporters regulated by two-component systems (TCSs) to absorb extracellular sugars but not phosphotransferase systems (PTSs) that are employed by solventogenic clostridia, such as Clostridium acetobutylicum. Furthermore, we performed comparative analyses of the species-specific repertoire of CAZymes for each of the Ruminiclostridium-type species. The high similarity of their cohesins suggests a common ancestor and potential cross-species recognition. Additionally, both differences between the C-terminal cohesins and other cohesins of scaffoldins and between the dockerins linking with cellulases and other catalytic domains indicate a preference for the location of cellulosomal catalytic subunits at scaffoldins. The information gained in this study may be utilized directly or developed further by genetic engineering and optimizing enzyme systems or cell factories for enhanced biotechnological biomass deconstruction and biofuel production. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited therapeutic options. The diversity and composition of the intratumoral microbiota are associated with PDAC outcomes, and modulating the tumor microbiota has the potential to influence tumor growth and the host immune response. Here, we explore whether intervention with butyrate-producing probiotics can limit PDAC progression. Methods: Based on the TCGA (PAAD) database, we analyzed the differential communities of intratumoral microbiota in PDAC patients with long survival and short survival and explored the relevant mechanisms of Clostridium butyricum and its metabolite butyrate in the treatment of PDAC. Treatment with Clostridium butyricum or butyrate in combination with the ferroptosis inducer RSL3 in a PDAC mouse model has an inhibitory effect on PDAC progression. The potential molecular mechanisms were verified by flow cytometry, RNA-seq, Western blotting, qRT‒PCR and immunofluorescence. Results: We found that the tumoral butyrate-producing microbiota was linked to a better prognosis and less aggressive features of PDAC. Intervention with Clostridium butyricum or its metabolite butyrate triggered superoxidative stress and intracellular lipid accumulation, which enhanced ferroptosis susceptibility in PDAC. Conclusion: Our study reveals a novel antitumor mechanism of butyrate and suggests the therapeutic potential of butyrate-producing probiotics in PDAC. [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 Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

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]

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]

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain's genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol. [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 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]

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 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.

The Correction Notice in the journal "Applied Biological Chemistry" addresses errors in the article "Characterization of acidogenic phase metabolism in Clostridium acetobutylicum ATCC 824 (pCD07239) under different culture conditions." The corrections include adjusting values in the text and table, as well as providing a corrected figure. The authors have acknowledged and rectified these errors for accuracy. [Extracted from the article]

Abstract 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.

In Germany alone, more than 5·106 tons of municipal green waste is produced each year. So far, this material is not used in an economically worthwhile way. In this work, grass clippings and tree pruning as examples of municipal green waste were utilized as feedstock for the microbial production of platform chemicals. A pretreatment procedure depending on the moisture and lignin content of the biomass was developed. The suitability of grass press juice and enzymatic hydrolysate of lignocellulosic biomass pretreated with an organosolv process as fermentation medium or medium supplement for the cultivation of Saccharomyces cerevisiae, Lactobacillus delbrueckii subsp. lactis, Ustilago maydis, and Clostridium acetobutylicum was demonstrated. Product concentrations of 9.4 gethanol L−1, 16.9 glactic acid L−1, 20.0 gitaconic acid L−1, and 15.5 gsolvents L−1 were achieved in the different processes. Yields were in the same range as or higher than those of reference processes grown in established standard media. By reducing the waste arising in cities and using municipal green waste as feedstock to produce platform chemicals, this work contributes to the UN sustainability goals and supports the transition toward a circular bioeconomy. [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]

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described. [ABSTRACT FROM AUTHOR]

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PP i) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PP i sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum , i CTH669. Hundreds of changes to the former GEM, i CBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of i CTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PP i synthesis routes, newly identified and previously proposed, were assessed through the lens of the i CTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PP i -generating cycles. In all cases, the metabolic cost of PP i synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PP i could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PP i sources likely account for most PP i production in wild-type strains. Second, alternate metabolic routes as encoded by i CTH669 can collectively maintain PP i levels even when previously investigated synthesis cycles are disrupted. Model i CTH669 is available at github.com/maranasgroup/iCTH669. • Lignocellulolytic Clostridium thermocellum glycolysis is pyrophosphate (PP i)-driven. • A highly curated genome-scale model, i CTH669, of C. thermocellum is reconstructed. • i CTH669 recapitulates recent yield data of ten PP i -linked knockout strains. • Previously proposed and newly identified sources of PP i were investigated. • PP i sources are evaluated by i CTH669, genomics, thermodynamics, and transcriptomics. [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]

Clostridium thermocellum, a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (.50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum, we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum. We addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations. IMPORTANCE: The accurate measurement of intracellular metabolites (metabolomics) is an integral part of metabolic engineering for the enhanced production of industrially important compounds and a useful technique to understand microbial physiology. Previous work tended to focus on model organisms under laboratory conditions. As we try to perform metabolomic studies with a wider range of organisms under conditions that more closely represent those found in nature or industry, we have found limitations in existing techniques. For example, fast filtration is an important step in quenching metabolism in preparation for metabolite extraction; however, it does not work for cultures of C. thermocellum at high substrate concentrations. In this work, we characterize the extent of the problem and develop techniques to overcome it. [ABSTRACT FROM AUTHOR]

The experiment was conducted to study the effect of Clostridium butyricum on production performance, serum biochemical indexes and economic benefits of laying hens. Four thousand 38-week-old Hyline brown laying hens were randomly divided into four groups with five replicates in each group and 200 laying hens in each replicate. The laying hens in control group were fed with basal diet, and the laying hens in experimental groups were fed with basal diet supplemented with 250, 500 and 1 000 mg/kg Clostrium butyric, respectively. The pre-test period lasted for 7 d and the formal test period lasted for 60 d. The results showed that the qualified egg rate of 1 000 mg/kg group was significantly higher than that of control group ( P<0.05), and the feed to egg ratio was significantly lower than that of control group ( P<0.05). The Haugh unit of eggs in experimental groups was significantly higher than that in control group ( P<0.05). The intestinal Lactobacillus in 500 mg/kg group and 1 000 mg/kg group was significantly higher than that in control group ( P<0.05), and the number of Escherichia coli in 500 mg/kg group was significantly lower than that in control group ( P<0.05). The T-AOC, GSH-Px and SOD activity in experimental groups were significantly higher than those in control group ( P<0.05), and the MDA content was significantly lower than that in control group ( P<0.05). The content of IgG in serum of laying hens in 1 000 mg/kg group was significantly higher than that in control group ( P<0.05), and the content of IgM in serum of laying hens in experimental groups was significantly higher than that in control group ( P<0.05). The profit per layer of the experimental groups was 8.36%, 17.72% and 19.40% higher than that of control group, respectively. The experiment indicates that dietary supplementation of Clostridium butyricum can improve the production performance and egg quality, and effectively improve the body health level. The dietary supplementation level of Clostridium butyricum is recommended to be 1 000 mg/kg. [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]

Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines 2H and 13C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.

Abstract 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.

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.

Riboswitches are RNA elements involved in regulating genes that participate in the biosynthesis or transport of essential metabolites. They are characterized by their ability to recognize their target molecules with high affinity and specificity. Riboswitches are commonly cotranscribed with their target genes and are located at the 5' end of their transcriptional units. To date, only two exceptional cases of riboswitches being situated at the 3' end and transcribing in the antisense direction of their regulated genes have been described. The first case involves a SAM riboswitch located at the 3' end of the ubiG-mccB-mccA operon in Clostridium acetobutylicum involved in converting methionine to cysteine. The second case concerns a Cobalamin riboswitch in Listeria monocytogenes that regulates the transcription factor PocR related to this organism's pathogenic process. In almost a decade since the first descriptions of antisense-acting riboswitches, no new examples have been described. In this work, we performed a computational analysis to identify new examples of antisense-acting riboswitches. We found 292 cases in which, according to the available information, we infer that the expected regulation of the riboswitch is consistent with the signaling molecule it senses and the metabolic function of the regulated gene. The metabolic implications of this novel type of regulation are thoroughly discussed. [ABSTRACT FROM AUTHOR]

Background: Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO2, and H2, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands. Results: This work reports the development and optimization of an electrotransformation protocol for A. wieringae strain JM, which can also be used in A. wieringae DSM 1911, and A. woodii DSM 1030. We also show the functionality of the thiamphenicol resistance marker, catP, and the functionality of the origins of replication pBP1, pCB102, pCD6, and pIM13 in all tested Acetobacterium strains, with transformation efficiencies of up to 2.0 × 103 CFU/μgDNA. Key factors affecting electrotransformation efficiency include OD600 of cell harvesting, pH of resuspension buffer, the field strength of the electric pulse, and plasmid amount. Using this method, the acetone production operon from Clostridium acetobutylicum was efficiently introduced in all tested Acetobacterium spp., leading to non-native biochemical acetone production via plasmid-based expression. Conclusions: A. wieringae can be electrotransformed at high efficiency using different plasmids with different replication origins. The electrotransformation procedure and tools reported here unlock the genetic and metabolic manipulation of the biotechnologically relevant A.wieringae strains. For the first time, non-native acetone production is shown in A. wieringae. [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]

Coffee waste is an abundant biomass that can be converted into high value chemical products, and is used in various renewable biological processes. In this study, oil was extracted from spent coffee grounds (SCGs) and used for polyhydroxyalkanoate (PHA) production through Pseudomonas resinovorans. The oil–extracted SCGs (OESCGs) were hydrolyzed and used for biohydrogen production through Clostridium butyricum DSM10702. The oil extraction yield through n–hexane was 14.4%, which accounted for 97% of the oil present in the SCGs. OESCG hydrolysate (OESCGH) had a sugar concentration of 32.26 g/L, which was 15.4% higher than that of the SCG hydrolysate (SCGH) (27.96 g/L). Hydrogen production using these substrates was 181.19 mL and 136.58 mL in OESCGH and SCGH media, respectively. The consumed sugar concentration was 6.77 g/L in OESCGH and 5.09 g/L in SCGH media. VFA production with OESCGH (3.58 g/L) increased by 40.9% compared with SCGH (2.54 g/L). In addition, in a fed–batch culture using the extracted oil, cell dry weight was 5.4 g/L, PHA was 1.6 g/L, and PHA contents were 29.5% at 24 h. [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]

The current study was conducted to explore the effects of dietary Clostridium butyricum (C. butyricum) and fermented calcium (Ca) butyrate produced by C. butyricum on the performance and egg quality of post‐peak laying. A total of 384 50‐week‐old hens were fed a basal diet, the basal diet with 300 mg/kg of fermented Ca butyrate or 1 × 109 CFU/kg C. butyricum for 8 weeks. Hens received a C. butyricum exhibited higher yolk properties, albumen height, and Haugh unit. A diet with fermented Ca butyrate or C. butyricum increased the egg mass and the pre‐grade yellow follicle number. RNA‐sequencing (RNA‐seq) data showed that these observations were associated with cytokine–cytokine receptor interaction and intestinal immune status. Accordingly, when compared with the basal diet group, Ca butyrate and C. butyricum addition decreased serum pro‐inflammatory cytokine levels and increased the concentration of immunoglobulin A, along with improved intestinal barrier. In addition, dietary C. butyricum inclusion induced a higher abundance of Ruminococcaceae and Lachnospiraceae at the family level. In summary, dietary fermented Ca butyrate or C. butyricum supplementation improved egg quality and ovarian function, which might be related to the enhanced intestinal barrier and immunity in post‐peak laying hens. [ABSTRACT FROM AUTHOR]

The solvent-producing bacterium Clostridium acetobutylicum is able to grow on a variety of carbohydrates. The main hexose transport system is the phosphoenolpyruvate-dependent phosphotransferase system (PTS). When the gene glcG that encodes the glucose transporter was inactivated, the resulting mutant glcG::int(1224) grew as well as the wild type, yet its glucose consumption was reduced by 17% in a batch fermentation. Transcriptomics analysis of the phosphate-limited continuous cultures showed that the cellobiose transporter GlcCE was highly up-regulated in the mutant glcG::int(1224). The glcCE mutation did not affect growth and even consumed slightly more glucose during solventogenesis growth compared to wild type, indicating that GlcG is the primary glucose-specific PTS. Poor growth of the double mutant glcG::int(1224)-glcCE::int(193) further revealed that GlcCE was the secondary glucose PTS and that there must be other PTSs capable of glucose uptake. The observations obtained in this study provided a promising foundation to understand glucose transport in C. acetobutylicum. [ABSTRACT FROM AUTHOR]

Biobutanol produced in acetone–butanol–ethanol (ABE) fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs, but are rarely used in industrial fermentation because of increased risks of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single‐pass fibrous‐bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At a dilution rate of 1.88 h−1, butanol was produced at 9.55 g/L, with a yield of 0.24 g/g and productivity of 16.8 g/L/h, which was the highest productivity ever achieved for biobutanol fermentation and an 80‐fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over 1 month. [ABSTRACT FROM AUTHOR]

Purpose: The microbial community in the pit mud correlated closely with the quality of the final product of Chinese strong-flavored Baijiu (CSFB). However, environmental conditions and brewing processes can vary by region and distilleries. This may lead to differences in microbial composition and function in pit mud. Therefore, revealing the features of the pit mud microbial community structure and functions of different distilleries will provide key information for understanding the diversity and difference of microbes in the brewing of CSFB, which will be beneficial for the improvement of the quality of pit mud and CSFB in the future. Methods and results: Illumina MiSeq sequencing of 16S rRNA gene amplicons was used to analyze the similarities and differences in microbial community structure and function in pit muds of different distilleries located in Shihezi (Xinjiang), Xiangyang (Hubei), and Yibin (Sichuan). At the genus level, Clostridium, Lactobacillus, Aminobacterium, Petrimonas, Syntrophomonas, Methanoculleus, Syntrophaceticus, Sedimentibacter, Caloramator, Ruminococcus, Bacillus, Methanosarcina, and Garciella were the dominated genera of pit muds. There were great differences in the composition of microorganisms in pit muds used by different distilleries. The significantly enriched prokaryotic microbiotas of pit muds collected in the distilleries of Xiangyang were mainly affiliated with Bacillus, Lactobacillus, and Croceifilum, and the relative abundance of methanogens, such as Methanomicrobia and Methanobacteria, were only significantly enriched in the pit mud collected from the distilleries of Yibin (P < 0.05). Functional analysis indicated that the difference of microbial composition in pit mud will further lead to significant differences in various metabolic functions. Conclusion: The compositions and functions of dominant microorganisms in pit mud used for the production of CSFB by different enterprises across regions in China were greatly different, and there was a close relationship between the compositions and functions of microorganisms in pit mud. Therefore, it may be an effective method to improve CSFB fermentation processes by directionally regulating the microbial community functions of pit mud using specific strains. [ABSTRACT FROM AUTHOR]

Efficient production of second-generation biofuels (bioethanol and biobutanol) from renewable lignocellulosic biomass holds strategic importance and aligns with sustainability objectives. However, it poses a persistent long-term challenge because of the biomass recalcitrance. Therefore, development of mild, environmentally friendly biomass pretreatment methods is a critical concern. To this end, in this study, a ternary deep eutectic solvent (DES) was synthesized with choline chloride, succinic acid, and glycerol at a molar ratio of 1:0.5:0.5. The ternary DES pretreatment largely enhanced glucan digestibility of corn stover (CS) to 94.7 % which was 4.60-fold to untreated CS. Mechanism of improved enzymatic efficiency of DES-pretreated CS was revealed by characterization methods such as SEM, XRD, and FTIR technologies. In addition, adding 1.0 % H 2 SO 4 to the ternary DES further increased enzymatic hydrolysis yield to nearly 100 %. Subsequently, the H 2 SO 4 -assisted DES-pretreated CS was used as substrate to produce C2-C4 bioalcohols (ethanol and butanol) by microbial fermentations. The results indicate that 46.6–49.2 g/L ethanol could be obtained by S. cerevisiae under separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF) strategies. Meanwhile, up to 11.9 g/L butanol was achieved from CS hydrolysate by Clostridium acetobutylicum with fed-batch mode. Finally, an artificial microbial consortium by co-culturing C. acetobutylicum and S. cerevisiae was developed which efficiently co-produced 12.4 g/L biobutanol and 5.7 g/L bioethanol from CS hydrolysate. Overall, this study provides a sustainable and green pretreatment technology that deconstruct lignocellulose and possess universality to produce various low-carbon bioalcohols by microbial fermentations. [Display omitted] ● A ternary deep eutectic solvent (DES) was synthesized for biomass pretreatment. ● Glucose yield of DES-pretreated corn stover (CS) reached 94.7 % after enzymolysis. ● Combined H 2 SO 4 with DES pretreatment facilitated sugars yield to nearly 100 %. ● Up to 46.6–49.2 g/L ethanol and 11.9 g/L butanol were produced by monocultures. ● A co-culture system co-produced 18.1 g/L bioalcohols and 26.2 g/L ABE from CS. [ABSTRACT FROM AUTHOR]

Advances in strain breeding for butanol biosynthesis were quite limited because of physiological complexity of solventogenic Clostridia. Using AI, this study developed a high-throughput screening method for Clostridium acetobutylicum to find strains with inhibitor tolerance and high butanol production. A mutant library was generated from C. acetobutylicum ATCC 824 through ARTP mutagenesis and physiological traits were digitized using color indicators. The classification performance of Machine learning algorithms (PCA, PLS, SVM, ANN) were compared for different butanol-producing strains. Among 2000 strains screened, C. acetobutylicum Tust-f3 was identified, which could tolerate 4.5 g/L furfural and yield 10.5 g/L butanol from undetoxified lignocellulosic hydrolysate. Proteome analysis reveals that 38 proteins may play a crucial role. Subsequently, seven universal detoxification components for furfural were identified via heterologous expression in E. coli Genes CA_RS19590 and CA_RS08810 showed significant growth improvement (14.44 and 14.28-fold, respectively, compared to control). This study highlights the potential of machine learning in strain selection and breeding. [Display omitted] • An AI-based screening method was developed to identify clostridia strains with high butanol production and furfural tolerance. • Strain Tust-f3 tolerated 4.5 g/L furfural, yielding 10.5 g/L butanol from undetoxified lignocellulosic hydrolysate. • Discovery of seven functional proteins associated with furfural tolerance. • Exogenous expression of protein CA_RS19590 enhanced furfural tolerance of Escherichia coli by 14.44-fold. [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]

[Display omitted] • H 2 fermentation process of C. tyrobutyricum was optimized systematically. • The effect of endogenous hydrogenases overexpression on H 2 production was studied. • Introducing sucrose metabolic pathway realized H 2 production from sugarcane molasses. • The highest H 2 production achieved 1098.4 mL with a yield of 248.2 mL/g sugar. Clostridia is the dominant microorganism in dark-fermentative biohydrogen production, but there have been relatively few studies on hydrogen production by Clostridium tyrobutyricum using cheap biomass. In this study, the important process parameters for hydrogen fermentation, such as substrate concentration, pH and temperature, were optimized systematically. Then, the effect of endogenous hydrogenases overexpression on hydrogen production was studied. To achieve economical hydrogen production from cheap substrate sugarcane molasses, the sucrose metabolic pathway was further introduced into C. tyrobutyricum. As a result, the engineered C. tyrobutyricum produced 1098.4 mL hydrogen with a yield of 248.2 mL/g sugar and productivity of 199.6 mL/L/h under the optimum fermentation conditions, which is the highest hydrogen production (910.37 mmol/L) reported so far using molasses. This study demonstrates that C. tyrobutyricum is a promising cell factory for hydrogen production from cheap and renewable biomass resources. [ABSTRACT FROM AUTHOR]

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain’s genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol.

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.

Clostridium butyricum has good tolerance and stress resistance to bile salt, high temperature and gastric acid and plays a significant role in protecting stability of intestinal microbiota, reducing intestinal pathogenic bacteria, enhancing animal immunity, reducing diarrhea rate, promoting digestion and absorption and improving growth performance. The paper reviews biological characteristics of Clostridium butyricum and its impact on livestock and poultry production, in order to provide reference for further application of Clostridium butyricum in animal husbandry. [ABSTRACT FROM AUTHOR]

L‐5‐Methyltetrahydrofolate (L‐5‐MTHF) is the only biologically active form of folate in the human body. Production of L‐5‐MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L‐5‐MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L‐5‐MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene‐THF) reductase, introducing the genes encoding formate‐THF ligase, formyl‐THF cyclohydrolase and methylene‐THF dehydrogenase from the one‐carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L‐5‐MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT‐PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 μg g−1 of dry cell weight for L‐5‐MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L‐5‐MTHF in E. coli. [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]