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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]

Degenerate strains of Clostridium acetobutylicum lack the ability to produce solvents and sporulate and remain in a permanent acidogenic state, allowing continuous hydrogen and organic acid production through anaerobic fermentation. Eichhornia crassipes, an invasive aquatic plant, emerges as a promising source of fermentable sugars for hydrogen production via anaerobic fermentation. In this study, a degenerated strain of Clostridium acetobutylicum was isolated and subsequently cultivated in the presence of a hydrolysate solution obtained from the alkaline pre-treatment and enzymatic hydrolysis of Eichhornia crassipes. The hydrolysate was mixed with a defined medium and served the dual purpose of providing essential nutrients and mitigating inhibitors, eliminating the need for an additional detoxification step. A pure defined culture medium served as a control. The extraction methods employed led to the release of low concentrations of inhibitors, reaching 0.1 g/L of furfural and 0.18 g/L of HMF. Kinetic characterization revealed that in the presence of Eichhornia crassipes hydrolysate, the degenerate strain exhibited lower specific growth rates ranging from 0.114 to 0.156 h−1, compared with the control medium which ranged from 0.131 to 0.179 h−1. This was accompanied by lower yields, ranging from 0.115 to 0.167 gDCW/g in the presence of hydrolysate versus 0.178 to 0.190 gDCW/g in the control medium, and diminished butyric acid production of 1.318 to 2.932 g/L in the presence of hydrolysate versus 1.749 to 3.471 g/L in control cultures. Despite reduced growth, high biohydrogen volumetric productivity was achieved, reaching 7.3 L/L·d, along with a significant yield of 2.642 mol of hydrogen per mole of glucose consumed. This represents 66.05% of the maximum stoichiometric yield calculated when acetic acid is the sole byproduct. Apparently, the presence of low concentrations of furfural and HMF released during the pre-treatment of Eichhornia crassipes not only negatively affects growth capacity but also diminishes butyric acid production, favoring biohydrogen production. [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.

The primary objective of this study was to assess the antimicrobial activities of various polar and nonpolar solvent extracts derived from the leaves of Viola odorata. The disc diffusion method was employed to test the antimicrobial susceptibility against a diverse range of pathogenic microorganisms, including Escherichia coli, Erwinia carotovora, Klebsiella pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and the fungus Candida albicans. The results indicated that isobutanol and ethyl acetate fractions exhibited significantly higher antimicrobial activity compared to petroleum ether, crude, chloroform and aqueous fractions. Among the tested bacteria, E. carotovora was the most susceptible bacterium to all the extracts of V. odorata, whereas S. typhi demonstrated the highest resistance. The isobutanol extract inhibited the proliferation of E. carotovora by 62 % at a concentration of 2 mg disc-1, while the ethyl acetate extract achieved 53% inhibition under the same conditions. Additionally, our findings revealed that the overall antimicrobial activity of V. odorata leaves was consistently effective against both Gram-positive and Gram-negative bacterial strains. [ABSTRACT FROM AUTHOR]

Summary: This study investigated the inhibition of Clostridium species, isolated from cheeses with late‐blowing defects, by lactic acid bacteria (LAB). Four batches of Turkish white cheeses were produced; control cheese, cheese sample containing Lactococcus lactis subsp. lactis (PY91K) and Lactiplantibacillus plantarum (Y48) as protective cultures (LAB), a cheese sample containing the protective culture and inoculated with Clostridium sporogenes spores (CL‐LAB) and finally a cheese sample containing only Cl. sporogenes spores (CL). White cheese's microbiological and physicochemical properties were also determined throughout the 90‐day storage period at 4 °C. Approximately 1 log reduction in Cl. sporogenes (ZMP‐1‐4) spores was observed in the LAB co‐inoculated Turkish white cheese samples. [ABSTRACT FROM AUTHOR]

Wheat straw is the second most abundant agricultural biomass. Conversion from wheat straw to butanol, the second-generation biofuel, offers an economically feasible and ecologically viable strategy to minimize environmental pollution. However, by-products generated during wheat straw pretreatment severely inhibit the subsequent fermentation by microbial strains and need to be removed prior to fermentation. In this study, three detoxification methods were evaluated for their ability to improve butanol fermentation with Clostridium acetobutylicum CICC 8012 from wheat straw hydrolysate. Strong acid cation exchange resin 001 × 7 exhibited the best comprehensive performance, yielding 2.03 g/L of acetic acid, 0.59 g/L of butyric acid, 7.42 g/L of butanol, and 12.97 g/L of acetone–butanol–ethanol. These values were 77.72% lower, 85.32% lower, 518.33% higher, and 582.63% higher, respectively, than those in the un-detoxified group. Further investigation revealed that treatment with 001 × 7 alleviated the depletion of ATP and NADH to ensure adequate supply of energy and reducing power for the shift from acidogenesis to solventogenesis. Transcriptome analysis revealed that detoxification reinforced the acid reassimilation route and the direct formation route for butanol formation, resulting in significantly improved fermentation performance by reducing acetic acid and butyric acid metabolic flux and increasing butanol metabolic flux. This study demonstrates a novel approach for detoxification of wheat straw hydrolysate with the cost-saving resin 001 × 7 and reveals the mechanism underlying the improvement in butanol fermentation performance observed following 001 × 7 treatment. [ABSTRACT FROM AUTHOR]

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]

Vitamin B 5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapC cae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products. [Display omitted] • A non-induced and non-plasmid E. coli for D-PA synthesis was obtained. • TCA was dynamically attenuated by self-regulatory promoter P fliC. • "PEACES" was developed to enhance NADPH sustainability in D-PA biosynthesis. • A newly discovered putative D-PA efflux system was demonstrated. • The D-PA titer reached 83.26 g/L in a 5-L fermenter. [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying acetone-butanol-ethanol fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived the interest in research on this bacterium. The recent development of efficient genetic tools allows to better understand the physiology of this micro-organism, aiming at improving its fermentation capacities. Knowledge about gene essentiality would guide the future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing method to generate large mutant libraries containing millions of independent mutants that allowed us to identify a core group of 418 essential genes needed for in vitro development. Future research on this significant biocatalyst will be guided by the data provided in this work, which will serve as a valuable resource for the community. [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]

This study investigated the effects of the multi-probiotics consisting of Bacillus subtilis and Clostridium butyricum with varying doses (0%, 0.05%, 0.1%, and 0.2%) on the growth performance, nutrient digestibility, meat quality, and cecal microbes of male broiler chickens. Seven hundred and twenty Ross 308, 1-day-old male broiler chicks were distributed into four dietary groups. Over 35 days of feeding, the average daily gain (ADG) was linearly elevated (P < 0.05) during days 1–21 and 1–35 as probiotic doses increased. The average daily feed intake (ADFI) tended to be linearly (P = 0.059) increased from day 22 to 35, and was improved from day 1 to 35 (P = 0.031). Ascending doses of multi-probiotics tended to (P = 0.060) reduce Clostridium perfringens counts on day 35 and prompted (P = 0.001) the proliferation of Lactobacillus. Moreover, broilers fed a 0.1% dose of multi-probiotics had a higher pH and water-holding capacity (P < 0.05) in the breast meat. In conclusion, the 0.2% multi-probiotics could boost ADG by improving ADFI and modulating the cecal microbe. The dietary 0.1% multi-probiotics contributed to better meat quality. [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]

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.

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]

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different β-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the β-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy. [Display omitted] • Second generation biofuel (biohydrogen) from consolidated bioprocessing of biomass. • Clostridium thermocellum is engineered to co-utilize cellulose and hemicellulose. • Growth on xylan by C. thermocellum is achieved by expression of β-xylosidase. • β-xylosidase reduces xylan degradation intermediates, xylo-oligomers and xylobiose. • Hemicellulose utilization increases hydrogen yield by 20%. [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]

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2‐to‐ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi‐omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi‐omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium. [ABSTRACT FROM AUTHOR]

Anaerobes dominate the microbiota of the gastrointestinal (GI) tract, where a significant portion of small molecules can be degraded or modified. However, the enormous metabolic capacity of gut anaerobes remains largely elusive in contrast to aerobic bacteria, mainly due to the requirement of sophisticated laboratory settings. In this study, we employed an in silico machine learning platform, MoleculeX, to predict the metabolic capacity of a gut anaerobe, Clostridium sporogenes, against small molecules. Experiments revealed that among the top seven candidates predicted as unstable, six indeed exhibited instability in C. sporogenes culture. We further identified several metabolites resulting from the supplementation of everolimus in the bacterial culture for the first time. By utilizing bioinformatics and in vitro biochemical assays, we successfully identified an enzyme encoded in the genome of C. sporogenes responsible for everolimus transformation. Our framework thus can potentially facilitate future understanding of small molecules metabolism in the gut, further improve patient care through personalized medicine, and guide the development of new small molecule drugs and therapeutic approaches. [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]

Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi-NT and Clostridium sporogenes, are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR–cas9 technology, can facilitate the genetic modification of Clostridium, allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium-directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi-NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment. [ABSTRACT FROM AUTHOR]

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

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]

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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]

Seaweed biomass in Korea is rich in galactose following hydrolysis, and leveraging this resource for enhancing the biohydrogen production is the aim of this study. The study investigates the biohydrogen production potential of a newly isolated pure strain, Clostridium sp. JH03, utilizing galactose and seaweed biomass as renewable feedstocks. The strain could utilize galactose as the sole carbon source for biohydrogen production, with a maximum hydrogen yield of 1.61 mol H2/mol galactose. The parameters included pH, temperature, and initial galactose concentration, which were varied to determine the optimal conditions for maximum biohydrogen production. The optimal conditions for biohydrogen production were pH 9 and a temperature of 25 °C, with an initial galactose concentration of 10 g/L. Moreover, hydrogen production from seaweed hydrolysate by Clostridium sp. JH03 resulted in maximum production of 1.71 mol H2/mol galactose. The study also investigated that combining sludge, a common practice in dark fermentation, with JH03 increased biohydrogen production by up to 34%. By addressing the need for clean energy and reducing raw materials price using biomass, this study contributes to the advancement of sustainable and cost-compatible energy solutions. [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]

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

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]

Molasses are a source of fermentable sugars, the substrate utilized for this study, as it is a subproduct from sugarcane processing for sucrose recovery. These sugars can be metabolized for bioH 2 production by bacterial strains, particularly strict-anaerobe Clostridium acetobutylicum (Clac) and its media preparation requires chemical of O 2 removal and N 2 flushing to promote anaerobiosis. Bacillus subtilis (Bsub) can deplete O 2 from culture media and promotes anaerobiosis, which is an aspect that would benefit from further research. Clac was grown in an anaerobic medium and produced 269 mL of hydrogen at 72 h. By co-culturing Clac and Bsub , 502 mL of bioH 2 were produced in a cysteine/N 2 flushed medium, while 203 mL of bioH 2 at 72 h were produced by aerobic co-culture. Additionally, in an untreated/aerobic conventional growth culture for Clac , no production of H 2 was detected, reinforcing the utility of Bsub for anaerobiosis generation. • Clostridium acetobutylicum consumes substrate approximately 6x faster than Bacillus subtilis. • Clostridium acetobutylicum produced 202 mL of bioH 2 in a culture with anaerobiosis assisted by Bacillus subtilis. • Bacillus subtilis CBB5555 can act as a biological O 2 depleter for anaerobic bioprocesses. [ABSTRACT FROM AUTHOR]

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.