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Cyanobacteria are oxygen-evolving prokaryotes that can be engineered for biofuel production from solar energy, CO 2, and water. Isobutanol (IB) has the potential to serve as an alternative fuel and important chemical feedstock. The research involves engineering Synechocystis sp. PCC 6803, for photosynthetic isobutanol production via the 2-keto-acid pathway and their cultivation in lab-scale photobioreactors. This synthetic pathway involves the heterologous expression of two enzymes, α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd), under a strong light-inducible promotor, psbA2, known to show increased gene expression under high light. The use of psbA2 could be a valuable strategy for isobutanol production as economic scaling up demands the utilization of natural sunlight, which also provides very high light intensity at midday, facilitating increased production. The study reports isobutanol production from engineered strains containing both pathway genes and with only kivd. In shake flask studies, the highest isobutanol titre of 75 mg L−1 (12th day) was achieved from an engineered strain DM12 under optimized light intensity. DM12 was cultivated in a 2 L flat panel photobioreactor, resulting in a maximum isobutanol titre of 371.8 mg L−1 (10th day) with 2 % CO 2 and 200 μmol photons m−2 s−1. Cultivation of DM12 in a photobioreactor under mimic diurnal sunlight demonstrated the highest productivity of 39 mg L−1 day−1 with the maximum titre of 308.5 mg L−1 (9th day). This work lays the foundation for sustainable, large-scale biobutanol production using solar energy. • Isobutanol production was achieved from engineered Synechocystis sp. PCC 6803. • Light-inducible psbA2 promoter showed light intensity dependent butanol production. • Isobutanol titer was 371.8 mg L−1 under 200 μmol photons m−2 s−1 light in PBR. • Under simulated sunlight isobutanol productivity was 39 mg L−1 day−1 in PBR. [ABSTRACT FROM AUTHOR]

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]

In the current era there are huge quantities of waste organic matter available, creating a big burden to the environment. To address these issues, researchers started to apply effective and microbial induced biotechnological processes that can mitigate these waste matters. In this context, different nature of microbial systems are involved in hydrolysing the waste organic material into fermentable sugar. These can be easily consumed by specific microbial systems like Saccharomyces cerevisiae MTCC 3821 and Clostridium acetobutylicum that produced bioethanol and biobutanol, respectively. Saccharomyces cerevisiae was cultured in specific media and incubated at rotary shaker with 150 rpm at 30°C for 72 to 96 hours. Ethanol concentrations from different waste matters were found in the range of 1.2-1.5 g.L-1. Ethanol synthesis was done by shake flask experiment with addition of glucose (50 g.L-1) to waste organic hydrolyzed solution. Non-glucose media produced less than 3 g.L-1 ethanol but glucose media produced 4.5 g.L-1. Next, Clostridium acetobutylicum was grown in culture media containing waste organics as sole carbon substrate with pH 7 and then was incubated in anaerobic conditions at 35°C for 72 hours, produced butanol (0.7 to 1.25 g.L-1). This research work promoted biofuels synthesis by keeping a waste mitigation strategy. [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.

Methane, a potent greenhouse gas, represents both a challenge and an opportunity in the quest for sustainable energy. This work investigates the biotechnology for converting methane into clean, renewable hydrogen. The co-culture of Chlorella sacchrarophila FACHB 4 and Methylomonas sp. HYX-M1 was demonstrated to completely convert 1 mmol of methane to biomass within 96 h. After acid digestion of such biomass, up to 45.05 μmol of glucose, 4.07 μmol of xylose, and 26.5 μmol of lactic acid were obtained. Both Clostridium pasteurianum DSM525 and Clostridium sp. BZ-1 can utilize those sugars to produce hydrogen without any additional organic carbon sources. The higher light intensity in methane oxidation co-culture systems resulted in higher hydrogen production, with the BZ-1 strain producing up to 14.00 μmol of hydrogen, 8.19 μmol of lactate, and 6.09 μmol of butyrate from the co-culture biomass obtained at 12,000 lux. The results demonstrate that the co-culture biomass of microalgae and methanotroph has the potential to serve as a feedstock for dark fermentative hydrogen production. Our study highlights the complexities inherent in achieving efficient and complete methane-to-hydrogen conversion, positioning this biological approach as a pivotal yet demanding area of research for combating climate change and propelling the global energy transition. [ABSTRACT FROM AUTHOR]

Background: Acetogens, a diverse group of anaerobic autotrophic bacteria, are promising whole-cell biocatalysts that fix CO2 during their growth. However, because of energetic constraints, acetogens exhibit slow growth and the product spectrum is often limited to acetate. Enabling acetogens to form more valuable products such as volatile fatty acids during autotrophic growth is imperative for cementing their place in the future carbon neutral industry. Co-cultivation of strains with different capabilities has the potential to ease the limiting energetic constraints. The lactate-mediated co-culture of an Acetobacterium woodii mutant strain, capable of lactate production, with the Clostridium drakei SL1 type strain can produce butyrate and hexanoate. In this study, the preceding co-culture is characterized by comparison of monocultures and different co-culture approaches. Results: C. drakei grew with H2 + CO2 as main carbon and energy source and thrived when further supplemented with D-lactate. Gas phase components and lactate were consumed in a mixotrophic manner with acetate and butyrate as main products and slight accumulation of hexanoate. Formate was periodically produced and eventually consumed by C. drakei. A lactate-mediated co-culture of the A. woodii [PbgaL_ldhD_NFP] strain, engineered for autotrophic lactate production, and C. drakei produced up to 4 ± 1.7 mM hexanoate and 18.5 ± 5.8 mM butyrate, quadrupling and doubling the respective titers compared to a non-lactate-mediated co-culture. Further co-cultivation experiments revealed the possible advantage of sequential co-culture over concurrent approaches, where both strains are inoculated simultaneously. Scanning electron microscopy of the strains revealed cell-to-cell contact between the co-culture partners. Finally, a combined pathway of A. woodii [PbgaL_ldhD_NFP] and C. drakei for chain-elongation with positive ATP yield is proposed. Conclusion: Lactate was proven to be a well-suited intermediate to combine the high gas uptake capabilities of A. woodii with the chain-elongation potential of C. drakei. The cell-to-cell contact observed here remains to be further characterized in its nature but hints towards diffusive processes being involved in the co-culture. Furthermore, the metabolic pathways involved are still speculatory for C. drakei and do not fully explain the consumption of formate while H2 + CO2 is available. This study exemplifies the potential of combining metabolically engineered and native bacterial strains in a synthetic co-culture. [ABSTRACT FROM AUTHOR]

Background: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that has been found to be associated with dysregulation of gastrointestinal functions and gut microbial homeostasis (the so-called "gut-brain axis"). ASD is often accompanied by poor performances in social interaction and repetitive behaviors. Studies on the gut-brain axis provide novel insights and candidate targets for ASD therapeutics and diagnosis. Based on the ASD mice model, this work aims to reveal the mechanisms behind the interaction of intestinal barrier function and probiotics in ASD mouse models. Results: We found an altered intestinal barrier in both BTBR T+ Itpr3tf/J (BTBR) and valproic acid (VPA) mice, including increased intestinal permeability, decreased expression of intestinal tight junction proteins (claudin1, claudin3, and occludin), and increased levels of IL-6, TNF-α, and IFN-γ. Based on intestinal microbial alternation, C. butyricum can drive reduced expression of histone deacetylases 1 (HDAC1) and enhanced intestinal barrier function, significantly promoting behavioral abnormalities of ASD in BTBR mice. In parallel, we confirmed that C. butyricum was involved in the regulation of intestinal function by the Trek1 channel, indicating that it is a target of C. butyricum/butyric acid to improve intestinal barrier function in ASD mice. Conclusions: Our finding provides solid evidence for the gut microbiota involved in ASD through the brain-gut axis. In addition, the probiotics C. butyricum hold promise to improve gut health and ameliorate behavioral abnormalities associated with ASD. [ABSTRACT FROM AUTHOR]

Introduction: The prevalence of major depressive disorder (MDD) has gradually increased and has attracted widespread attention. The aim of this study was to investigate the effect of a probiotic compound consisting of Bacillus coagulans and Clostridium butyricum, on a mouse depression model. Methods: Mice were subjected to chronic unpredictable mild stress (CUMS) and then treated with the probiotics at different concentrations. And mice received behavior test such as forced swimming test and tail suspension test. After that, all mice were sacrificed and the samples were collected for analysis. Moreover, prefrontal cortex (PFC) gene expression and the gut microbiota among different groups were also analyzed. Results: Probiotics improved depressive-like behavior in CUMS mice, as indicated by decreased immobility time (p < 0.05) in the forced swimming test and tail suspension test. probiotics intervention also increased the level of 5- hydroxytryptamine (5-HT) in the prefrontal cortex and decreased the adrenocorticotropic hormone (ACTH) level in serum. In addition, by comparing the PFC gene expression among different groups, we found that the genes upregulated by probiotics were enriched in the PI3K-Akt signaling pathway in the prefrontal cortex. Moreover, we found that downregulated genes in prefrontal cortex of CUMS group such as Sfrp5 and Angpt2, which were correlated with depression, were reversed by the probiotics. Furthermore, the probiotics altered the structure of the gut microbiota, and reversed the reduction of cob(II)yrinate a,c-diamide biosynthesis I pathway in CUMS group. Several species like Bacteroides caecimuris and Parabacteroides distasoni, whose abundance was significantly decreased in the CUMS group but reversed after the probiotics intervention, showed significantly positive correlation with depression associated genes such as Tbxas1 and Cldn2. Discussion: These findings suggested that CUMS-induced depression-like behavior can be alleviated by the probiotics, possibly through alterations in the PFC gene expression and gut microbiota. [ABSTRACT FROM AUTHOR]

The discovery of immune checkpoints (CTLA-4, PD-1, and PD-L1) and their impact on the prognosis of oncological diseases have paved the way for the development of revolutionary oncological treatments. These treatments do not combat tumors with drugs "against" cancer cells but rather support and enhance the ability of the immune system to respond directly to tumor growth by attacking the cancer cells with lymphocytes. It has now been widely demonstrated that the presence of an adequate immune response, essentially represented by the number of TILs (tumor-infiltrating lymphocytes) present in the tumor mass decisively influences the response to treatments and the prognosis of the disease. Therefore, immunotherapy is based on and cannot be carried out without the ability to increase the presence of lymphocytic cells at the tumor site, thereby limiting and nullifying certain tumor evasion mechanisms, particularly those expressed by the activity (under positive physiological conditions) of checkpoints that restrain the response against transformed cells. Immunotherapy has been in the experimental phase for decades, and its excellent results have made it a cornerstone of treatments for many oncological pathologies, especially when combined with chemotherapy and radiotherapy. Despite these successes, a significant number of patients (approximately 50%) do not respond to treatment or develop resistance early on. The microbiota, its composition, and our ability to modulate it can have a positive impact on oncological treatments, reducing side effects and increasing sensitivity and effectiveness. Numerous studies published in high-ranking journals confirm that a certain microbial balance, particularly the presence of bacteria capable of producing short-chain fatty acids (SCFAs), especially butyrate, is essential not only for reducing the side effects of chemoradiotherapy treatments but also for a better response to immune treatments and, therefore, a better prognosis. This opens up the possibility that favorable modulation of the microbiota could become an essential complementary treatment to standard oncological therapies. This brief review aims to highlight the key aspects of using precision probiotics, such as Clostridium butyricum, that produce butyrate to improve the response to immune checkpoint treatments and, thus, the prognosis of oncological diseases. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria can synthesize extracellular exopolysaccharides (EPSs) that have versatile physicochemical and biological properties. In this paper, the EPSs synthesized by Lacticaseibacillus rhamnosus ŁOCK 0943 were characterized. Their structure, biological, and technological activity, as well as application potential, were analyzed. Chemical analysis showed that this strain produces mannan and β-1,6-glucan. Their emulsifying, antagonistic, and antioxidant properties, along with their prebiotic potential, were assessed. The analysis of the tested polymers' ability to create a stable emulsion showed that their emulsifying activity depends mainly on the type of oily substance used. The analysis of the antagonistic activity revealed that these EPSs can inhibit the growth of yeasts (e.g., Candida albicans ATCC 10231) and potentially pathogenic bacteria (e.g., Clostridium acetobutylicum ŁOCK 0831, Enterococcus faecalis ATCC 29212). Moreover, EPSs positively influenced the growth of all tested probiotic bacteria. Furthermore, EPSs can be successfully used as a preservative in cosmetic products. The most effective results were obtained with the use of a 0.05% solution of a chemical preservative (bronopol) and 0.25 mg/mL of the EPSs. [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.

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri. In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used.IMPORTANCEThough dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and Clostridium kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

1,4-butanediol (1,4-BDO) is an important building block in the chemical industry that has been mainly produced from fossil fuels, but now biosynthesis of 1,4-BDO has received more and more attention due to environmental issues. The Clostridia C4 pathway produces an intermediate crotonyl-CoA which could be diverted to 1,4-BDO by 4-hydroxybutyryl-CoA dehydratase (4HBD). Here, we compared this pathway with other 1,4-BDO biosynthesis pathways and illustrated its potential advantages regarding cellular energy conservation and theoretical yield. Then, the feasibility of 1,4-BDO production in this way was tested by introducing a single 4HBD in Clostridium acetobutylicum that natively produced the C4 intermediate and a variety of aldehyde/alcohol dehydrogenases (AdhE). Five different 4HBD genes were screened and the Cbei-2100 gene from Clostridium beijerinckii was the most effective, producing 0.066 mg/mL of 1,4-BDO. To block the metabolic flux towards the main product butanol, disruption of butyryl-CoA dehydrogenase (Bcd) was tried but failed, while inactivation of its homologue (FAD/FMN-containing dehydrogenase, Fcd) obtained little effect. Alternatively, the electron-transferring flavoprotein EtfA coupled with Bcd was inactivated, and 1,4-BDO production was greatly increased to 0.182 mg/mL. In conclusion, this study demonstrated the feasibility of 1,4-BDO production through the Clostridia C4 pathway. Further blocking of the competing flux towards butanol would be effective to improve the production of in the future.

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.

The NLRP3 inflammasome is a key mediator of the innate immune response to sterile tissue injury and is involved in many chronic and acute diseases. Physically and chemically diverse agents activate the NLRP3 inflammasome. Here, we show that NLRP3 binds non-oxidized and Ox-mtDNA differentially, with a half maximum inhibitory concentration (IC50) for non-oxidized and Ox-mtDNA of 4 nM and 247.2 nM, respectively. The NLRP3 Neonatal-Onset Multisystem Inflammatory Disease (NOMIDFCAS) gain of function mutant could bind non-oxidized mtDNA but had higher affinity for Ox-mtDNA compared to WT with an IC50 of 8.1 nM. NLRP3 lacking the pyrin domain can bind both oxidized and non-oxidized mtDNA. Isolated pyrin domain prefers Ox-mtDNA. The NLRP3 pyrin domain shares a protein fold with DNA glycosylases and generate a model for DNA binding based on the structure and sequence alignment to Clostridium acetobutylicum and human OGG1, an inhibitor of Ox-mtDNA generation, 8-oxoguanine DNA glycosylases. We provide a new model for how NLRP3 interacts with Ox-mtDNA supported by DNA binding in the presence of a monoclonal antibody against the pyrin domain. These results give new insights into the mechanism of inflammasome assembly, and into the function of reactive oxygen species in establishing a robust immune response.

This study investigated how Clostridium butyricum affected rumen fermentation and the microbial communities of rumen and feces in beef cattle. Twenty beef cattle were divided into two groups: the control group (CK) and the C. butyricum group (CB, fed 2.5 x 108 CFU/kg of dry matter intake per day). The results showed that C. butyricum increased rumen pH, ammonia-N concentration, and microbial crude protein (MCP) concentration (P<0.05). Ruminal propionate and butyrate concentration increased, while the ruminal acetate to propionate ratio decreased (P <0.05). For rumen microbiota, observed species, Chao 1, and ACE indices were higher (P<0.05) with supplemented C. butyricum. At the phyla level, the C. butyricum enhanced the proportion of Firmicutes and decreased Bacteroidota (P<0.01). Christensenellaceae R-7 group, Methanobrevibacter, Oscillospiraceae NK4A214 group, Desulfovibrio, Streptococcus, and C. butyricum were increased (P<0.05) at the genus and species levels in the CB group. The proportion of Prevotella, Christensenellaceae R-7 group, Blautia, and Megasphaera elsdenii increased, while Escherichia coli decreased (P<0.05) in feces. E. coli and Salmonella populations were significantly reduced (P<0.01). These results indicated that diets supplemented with C. butyricum could improve rumen fermentation by increasing the diversity and altering the microbial community structure of the rumen. Additionally, the supplemented C. butyricum changed the fecal microbiota and decreased the harmful bacteria population in beef cattle. [ABSTRACT FROM AUTHOR]

Acetone-Butanol-Ethanol (ABE) fermentation has regained interest due to the environmental problems generated by fossil fuels and the need to modify production chains including considerations of sustainability and circular economy. This work focuses on the modelling of ABE fermentation, with a Clostridium acetobutylicum mutated strain, to support the synthesis and improvement of butanol and acetone production process. To reach this objective, bioreactor fermentations with different glucose concentrations were developed. Results show that a two phases model (with a set of equations for the acidogenesis and another for the solventogenesis) generates satisfactory process predictions. In acidogenesis, the main effect on growth and acid production is due to the inhibition by acids, which can be quantified as a function of the total concentration of acids. While in the solventogenesis, inhibition must be quantified as a function of the total concentration of solvents. This model agrees with metabolic analysis that considers solvents generation being produced, simultaneously, from acids and sugars. For the studied system, acetone and butanol production is based on re-assimilated acids and pyruvate from glycolysis, with a distribution that changes with glucose availability. [ABSTRACT FROM AUTHOR]

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production. [ABSTRACT FROM AUTHOR]

Endometritis occurs frequently in humans and animals, which can negatively affect fertility and cause preterm parturition syndrome. Orally administered Clostridium butyricum, a butyrate-producing gram-positive anaerobe, exhibits anti-inflammatory effects. However, the precise mechanism by which Clostridium butyricum attenuates endometritis remains unclear. This in vivo study evaluated the anti-inflammatory effects of orally administered Clostridium butyricum on uterine tissues. In addition, we conducted uterine microbiome and lipid metabolome analyses to determine the underlying mechanisms. Female Balb/c mice were divided into the following four groups (n = 5-20): (1) mock group, (2) only operation group (mice only underwent operation to exposed uterine horns from the side), (3) control group (mice underwent the same operation with the operation group + perfusion of lipopolysaccharide solution from uterine horns), and (4) Clostridium butyricum administration group (mice underwent the same operation with the control group + oral Clostridium butyricum administration from days 0 to 9). Clostridium butyricum was administered via oral gavage. On day 10, we investigated protein expression, uterine microbiome, and lipid metabolism in uterine tissues. Consequently, orally administered Clostridium butyricum altered the uterine microbiome and induced proliferation of Lactobacillus and Limosilactobacillus species. The effects can contribute to show the anti-inflammatory effect through the interferon-β upregulation in uterine tissues. Additionally, oral Clostridium butyricum administration resulted in the upregulations of some lipid metabolites, such as ω-3 polyunsaturated fatty acid resolvin D5, in uterine tissues, and resolvin D5 showed anti-inflammatory effects. However, the orally administered Clostridium butyricum induced antiinflammatory effect was attenuated with the deletion of G protein-coupled receptor 120 and 15-lipooxgenase inhibition. In conclusion, Clostridium butyricum in the gut has anti-inflammatory effects on uterine tissues through alterations in the uterine microbiome and lipid metabolism. This study revealed a gut-uterus axis mechanism and provided insights into the treatment and prophylaxis of endometritis. [ABSTRACT FROM AUTHOR]

The aim of this work was to give as much information as possible on Rosa canina dried fruit that is commercially available in Serbia. In order to provide the chemical composition, the UHPLC-DAD-ESI-MS method was employed for both polar and non-polar extracts of samples obtained with a solvent mixture consisting of hexane, acetone, and ethanol in a volume ratio of 2:1:1, respectively, and 0.05% (w/v) butylated hydroxytoluene. In addition, the total content levels of lycopene, β-carotene, total polyphenols, and flavonoids were determined by means of UV-vis spectrophotometry. The antioxidant activity was tested by applying four different methods: ABTS, DPPH, FRAP, and CUPRAC. Overall, nine compounds were identified. The results of chemical composition analysis were used as the basis for the interpretation of the calculated results for the antioxidant and antimicrobial activity. The obtained results for R. canina dried fruit extract are as follows: β-carotene—7.25 [mg/100 g fruit weight]; lycopene—2.34 (mg/100 g FW); total polyphenol content (TPC)—2980 [mg GAE/kg FW]; total flavonoid content (TFC)—1454 [mg CE/kg FW]; antioxidant activity—ABTS 12.3 [μmol/100 g FW], DPPH 6.84, FRAP 52.04, and CUPRAC 15,425; and antimicrobial activity—Staphylococcus aureus MIC/MMC 4/0 [mg∙mL−1], Enterococcus faecalis 4/0, Bacillus cereus 4/0, Escherichia coli 4/0, Salmonella enteritidis 4/4, Enteroabacter aerogenes 4/0, Pseudomonas aeruginosa 2/0, and Candida albicans 2/0. [ABSTRACT FROM AUTHOR]

Clostridia are interesting candidates for biotechnological applications due to their diverse and unique metabolic abilities. Particularly in continuous fermentation processes, productivity-decreasing metabolic oscillations have been reported in many species. The resulting process instability and reduced productivity can be a serious hurdle for the development of industrially feasible processes. This review highlights the current state of knowledge about oscillatory metabolic phenomena in Clostridia, including the mechanisms, assumed and proven, behind those oscillations and methods to mitigate the phenomena if applicable. The nature of observed metabolic oscillations in Clostridia is diverse, including a wide range of periods of oscillation and different parameters in which the oscillation is observed. Some phenomena remain to be investigated further, while others are already well understood. However, knowledge of mechanisms is a very valuable asset in overcoming the metabolic oscillation to create a stable process. [ABSTRACT FROM AUTHOR]

Abstract Background Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism’s vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. Results We utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. Conclusions This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the “raiA motif” has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor. [ABSTRACT FROM AUTHOR]

At present, the production of the majority of valuable chemicals is dependent on the microbial fermentation of carbohydrate substrates. However, direct competition is a potential problem for microbial feedstocks that are also used within the food/feed industries. The use of alternative carbon sources, such as acetate, has therefore become a research focus. As a common organic acid, acetate can be generated from lignocellulosic biomass and C1 gases, as well as being a major byproduct in microbial fermentation, especially in the presence of an excess carbon source. As a model microorganism, Escherichia coli has been widely applied in the production of valuable chemicals using different carbon sources. Recently, several valuable chemicals (e.g., succinic acid, itaconic acid, isobutanol, and mevalonic acid) have been investigated for synthesis in E. coli using acetate as the sole carbon source. In this review, we summarize the acetate metabolic pathway in E. coli and recent research into the microbial production of chemical compounds in E. coli using acetate as the carbon source. Although microbial synthetic pathways for different compounds have been developed in E. coli, the production titer and yield are insufficient for commercial applications. Finally, we discuss the development prospects and challenges of using acetate for microbial fermentation. [ABSTRACT FROM AUTHOR]

Humans have used fermentation processes since the Neolithic period, mainly to produce beverages. The turning point occurred in the 1850s, when Louis Pasteur discovered that fermentation resulted from the metabolism of living microorganisms. This discovery led to the fast development of fermented food production. The importance of industrial processes based on fermentation significantly increased. Many branches of industry rely on the metabolisms of bacteria, for example, the dairy industry (cheese, milk, yogurts), pharmaceutical processes (insulin, vaccines, antibiotics), or the production of chemicals (acetone, butanol, acetic acid). These are the mass production processes involving a large financial outlay. That is why it is essential to minimize threats to production. One major threat affecting bacteria-based processes is bacteriophage infections, causing substantial economic losses. The first reported phage infections appeared in the 1930s, and companies still struggle to fight against phages. This review shows the cases of phage infections in industry and the most common methods used to prevent phage infections. [ABSTRACT FROM AUTHOR]

Background: Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism's vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. Results: We utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis' CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis' primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. Conclusions: This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars. [ABSTRACT FROM AUTHOR]

Background: Clostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering. Results: We were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAMβ1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (102–103 CFU/µg) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters. Conclusions: The presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration. [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]

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]

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]

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]

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]

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.