ABSTRACT. Microbial production of acetate from CO2 is catalyzed by the ecophysiologically as well as biotechnologically important group of acetogenic bacteria. CO2 is reduced by the Wood-Ljungdahl pathway (WLP), a two branched, linear pathway in which one CO2 is reduced to a methyl group and another to a carbonyl group that are combined by the key enzyme, the CODH/ACS to acetyl-CoA, the precursor of acetate. The carbon reduction pathway to acetate is not coupled to net ATP formation but nevertheless it allows acetogens to make a living. Net ATP synthesis is catalyzed by a respiratory chain that is hooked up to the carbon reduction pathway. The respiratory chains are different in different acetogens and are either of the Rnf- or Ech-type that are going to be discussed. Both respiratory chains use reduced ferredoxin as electron donor that is reduced with hydrogen as reductant by electron bifurcating hydrogenases. The concept in electron bifurcation in saving cellular energy will be discussed. Of critical importance is the nature of the electron carriers involved in the carbon reduction pathway: any use of reduced ferredoxin will reduce the amount of ATP synthesized. I will describe how different acetogens solved the problem of reducing CO2 to formic acid, the first step in the methyl branch of the WLP, without using reduced ferredoxin as reductant. Some use a hydrogen-dependent CO2 reductase, others an electron-bifurcating formate dehydrogenase. Acetogens are phylogenetically very different but have in common the basic chemistry of acetogenesis. However, they differ in (i) the respiratory chain used for ATP synthesis, (ii) the nature of the electron carriers used in the WLP and finally (iii) the amount of ATP synthesized. This is the more important since acetogens operate at the thermodynamic limit of life and the production of many value-added compounds from H2 + CO2 is thermodynamically restricted. At the end, I will present the entire biochemistry and bioenergetics of acetogenesis from H2 + CO2 or CO in model acetogens such as Acetobacterium woodii, Thermoanaerobacter kivui, Eubacterium limosum, Sporomusa ovata, Clostridium aceticum and Clostridium autoethanogenum and outline ways to improve the energetics for producing ATP-intensive valued-added products from H2 + CO2. "

ABSTRACT. Anaerobic acetogens convert C1 substrates very efficiently, but most of them have a limited product spectrum (acetate and ethanol). Only some are able to produce butyrate, hexanoate, or the respective alcohols via reverse-β-oxidation. In the projects CaproMix and CaproSyn, we established a synthetic co-culture composed of the acetogens Acetobacterium woodii and Clostridium drakei to produce hexanoate and butyrate via lactate-mediated H2 + CO2 fermentation. A. woodii is known for its ability to grow autotrophically and rapidly, utilising H2 + CO2 to produce acetate. In CaproMix, the product spectrum of A. woodii was extended to include lactate by establishing plasmid-borne expression of a D-lactate dehydrogenase (LdhD) from Leuconostoc mesenteroides. In CaproSyn, lactate production was further improved by additionally expressing the pyruvate formate lyase from Clostridium pasteurianum in A. woodii. The lactate produced by the recombinant A. woodii strains served as a substrate for C. drakei, which is capable of producing butyrate and hexanoate via reverse-β-oxidation. C. drakei and A. woodii have similar environmental requirements, making them suitable for a synthetic co-culture. In the CaproMix project, C. drakei SL1T was chosen to pair with A. woodii. During CaproSyn, C. drakei FP was established as a partner in the co-culture to produce butyrate, isobutyrate, and hexanoate. One challenge in cultivating a synthetic co-culture is developing a species monitoring strategy to analyse the dynamics and proportions of both partners. Therefore, in CaproMix, the gene encoding LdhD was fused with a gene encoding a fluorescence-activating and absorption-shifting tag (FAST) to determine the cell number of A. woodii via flow cytometry. During CaproSyn, the monitoring of cells from each species was performed using either fluorescence in-situ hybridisation (FISH) combined with flow cytometry or via qPCR. The qPCR method emerged as the preferred option for real-time monitoring of the partners in the co-culture, as it proved to be more reliable, precise, and time-saving compared to the FISH method. In summary, we successfully established a lactate-mediated co-culture producing butyrate, isobutyrate, and hexanoate from A. woodii and C. drakei and determined the proportions of both species during the respective growth experiments.

ABSTRACT. Given the current global environmental challenges, waste could be an attractive renewable resource within a circular economy. Gasification of solid organic waste streams yields syngas, which is primarily composed of CO, CO2, and H2, with its composition varying depending on feedstock and process conditions. Syngas is a suitable substrate for gas fermentation for the production of valuable chemicals using acetogens. It is known that the composition of syngas can strongly affect the process output. Therefore, our study aimed to simulate syngas compositions derived from relevant biomass feedstocks in Estonia and analyze the metabolic response of the model-acetogen Clostridium autoethanogenum in batch bioreactor cultivations. For this, seven synthetic syngas mixtures were tested to evaluate the impact of H2:CO ratios on growth, gas uptake, and by-product distribution during exponential growth. Indeed, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates (μmax) ranged from 0.05 to 0.13 h-1, with lower values observed under CO-limited conditions. Growth by-product distribution was dominated by acetate, ethanol, and CO2, with minor 2,3-butanediol formation. Acetate production yields from ~20 to ~133 mmol/gDCW and ethanol production yields from ~70 to ~353 mmol/gDCW were measured. As expected, syngas mixtures with higher H2 content favored ethanol production. However, the ethanol-to-acetate ratio did not positively correlate with higher H2-to-CO uptake rate ratios. In fact, positive trends suggested links between H2:CO uptake and both ethanol and acetate production (R2 ~ 0.5 and 0.8, respectively). Additionally, carbon balance showed significant carbon loss as CO2 under lower H2:CO uptake conditions (R2 ~ 0.7), possibly indicating enhanced CO oxidation to maintain redox balance. Taken together, higher H2:CO uptake rates increased the total flux through the Wood–Ljungdahl pathway rather than selectively favoring reduced products. Our findings quantitate the influence of syngas composition on carbon distribution, emphasizing the importance of process optimization to improve efficiency in industrial gas fermentation.

ABSTRACT. This talk will present a forward-looking perspective on how my laboratory, in collaboration with and supported by others, has applied quantitative metabolomics, isotope-based metabolic flux analysis, and computational modeling to uncover the unique metabolic constraints of Clostridium thermocellum. I will highlight evidence that its central metabolism, particularly glycolysis and ethanol fermentation as well as other pathways, operates near thermodynamic equilibrium and imposes unusually high enzyme and protein allocation costs. Using quantitative flux, thermodynamic, and resource balance analyses, we identify phosphofructokinase and pyrophosphate-dependent reactions as key bottlenecks shaping metabolic flux, yield, and product titers. Finally, I will discuss how thermodynamics-guided metabolic engineering, including redesign of glycolysis and cofactor usage, can overcome these constraints and improve ethanol production, providing general principles for engineering cellulolytic Clostridium species.

ABSTRACT. The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. In this presentation, we will discuss the importance of butyrate on C. acetobutylicum cell growth and health demonstrated through genetic modifications and metabolite complementation. Through clean deletion of the 3-hydroxybutyryl-CoA dehydrogenase (hbd) gene from the chromosome, we eliminated butyrate formation from the two C. acetobutylicum strains. Addition of butyrate and its precursor crotonate to the engineered strains revealed that butyrate is important for robust cell growth as well as sporulation. We further discovered that butyrate enhanced biomass accumulation of a non-butyrate forming C. saccharolyticum. While butyrate is not a substrate for cell growth, it promotes cell growth and the developmental program of sporulation. The mechanism of action is likely related to complicated regulatory machinery including Spo0A mediated gene expression. We propose butyrate as a potential microbial population modulator because cell growth and health of a wide range of microbes within microbiota could be differently influenced by butyrate. The findings provide significant insights into the design of metabolic pathways and fermentation process for Clostridium based production of metabolites.

ABSTRACT. Acetivibrio thermocellus (formerly Clostridium thermocellum) is a remarkable lignocellulose degrader making it interesting for production of biofuels and chemicals. Engineering of A. thermocellus has hitherto focused on improving yield and titre for ethanol production. However, limitations in metabolism and ethanol tolerance currently hinder ethanol titres required for industrial application. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. This study explores the relative contributions of these two aspects. Specifically, the native ethanol pathway (Pfor and adhE) is sensitive to accumulation of ethanol and NADH (1,2). Engineering A. thermocellus for high ethanol production likely requires replacement or engineering of its native ethanol pathway. To study metabolism independent of the native ethanol pathway, lactate formation can instead be used as the primary NADH sink. Natively, the lactate dehydrogenase (Ldh) requires activation by FBP (3), making lactate formation an overflow metabolite linked to limitations in glycolytic flux. However, a single point mutation (C483A leading to S161R substitution) can remove this need for allosteric activation leading to constitutive Ldh activity (4). In our work, the ability to shift from ethanol formation to lactate formation was investigated as a tool to study metabolism as well as ethanol inhibition. The results in this study revealed insights into the metabolism of A. thermocellus while allowing investigation of limitations in a simplified metabolism. The constructed strain provides a platform for further research for consolidated bioprocessing with A. thermocellus independent of the native metabolism.

1 Cui et al (2019): doi.org/10.1016/j.ymben.2018.09.006 2 Pech-Canul et al (2024): doi.org/10.1016/j.jbc.2024.107559 3 Özkan et al (2004): doi.org/10.1139/w04-071 4 Lo et al (2015): doi.org/10.1128/jb.02450-14

ABSTRACT. One of the lesser-known groups of bacteria in the Class of the Clostridia are the family of the Heliobacteriaceae. These organisms are not only the sole phototrophic members of the Clostridia, but they are also the only known phototrophic gram-positive bacteria. Our lab studies the electron transport chain and energy metabolism in these organisms, especially during anaerobic phototrophy in light but also during fermentative growth in darkness. Heliobacteria possess the most streamlined photosynthetic reaction center in which the terminal electron acceptor is the interpolypeptide [4Fe-4S] cluster FX. We have shown that three soluble, low molecular mass, 2[4Fe-4S] cluster ferredoxins accept electrons from reduced FX (Ferlez et al. 2016; Walters et al. 2023). Electrons were previously thought to be transferred from ferredoxins to nicotinamide cofactors via a FNR enzyme. Recent analysis of the genome failed to find a gene for an FNR but rather, revealed that polypeptides encoded by the genes HM1_0289 and HM1_0290 may be candidates for subunits of an NAD-dependent ferredoxin:NADP+ oxidoreductase electron-bifurcating enzyme, NfnAB (Walters et al manuscript in review). We propose that heliobacteria use NfnAB in the reverse direction (i.e., electron confurcation) to accomplish two functions: light-driven cyclic electron transport and transfer of reducing equivalents from NADH to NADP+. This hypothesis was tested by deletion of the nfnAB genes from the chromosome. The ΔnfnAB mutants were viable in all growth modes tested, but in vivo spectroscopic analysis indicates that light-driven electron transport was limited in the absence of NfnAB. Phototrophic energy conversion is a function not previously observed for this protein complex.

ABSTRACT. Synthesis gas conversion to products (“fermentation”) has been established with mesophilic bacteria including Acetobacterium woodii, Clostridium authoethanogenum and Clostridium ljungdahlii, and commercialized by the company LanzaTech. Alternatively, gas fermentation may be carried out at at higher temperatures, as the advantages of higher turnover rates and lower cooling costs may balance the low gas solubilities. The thermophilic acetogen Thermoanaerobacter kivui within the Clostridial order Thermoanaerobacterales (Topt 66°C) utilizes H2+CO2 or sugars as substrates (td <1.5 h) and has been adapted to thrive on CO, with acetate as metabolic product. Towards understanding its physiology and the development of an industrially-relevant platform strain, we developed a genetic system that allows for genome integration and plasmid-based protein overproduction. Recently, we added a reporter gene assay and some inducible promoters to the toolset. With collaboration partners from the Universities of Frankfurt (Prof. Volker Müller) and Göttingen (Dr. Anja Poehlein, Prof. Rolf Daniel), we studied the metabolism of T. kivui and its regulation during growth on different substrates, revealing surprises in the redox and energy metabolism. With regards to its bioenergetics, T. kivui is now the best understood thermophilic acetogen and serves as a model organism to better understand acetogenesis at high temperatures. Based on these fundamental findings, we then engineered several strains of T. kivui for ethanol production from synthesis gas. We are currently evaluating the potential of genetically modified strains and developing novel and improved methods toward a better understanding and application of T. kivui for conversion of synthesis gas at high temperature.

ABSTRACT. Clostridium autoethanogenum, a model gas-fermenting acetogen, is a promising microbial chassis for converting C1 gases into fuels and chemicals. Central to autotrophic metabolism are carbon monoxide dehydrogenase (CODH) enzymes, which couple carbon fixation to energy conservation and product formation. Interestingly, the primary CODH, AcsA, in the wild-type C. autoethanogenum JA1-1 strain is uniquely truncated, and this truncation is lost in the superior LAbrini strain following autotrophic adaptive laboratory evolution of JA1-1. In addition, protein expression of monofunctional CODH CooS1 has remained high and differentially regulated in C. autoethanogenum in various autotrophic conditions. Here, we investigated the functional significance of replacing acsA stop codon with leucine (Leu_SNP) or serine (Ser_SNP), and the effect of cooS1 deletion in C. autoethanogenum. Autotrophic batch and chemostat characterisation revealed significantly altered growth, carbon flux, and by-product distributions in SNP strains, whereas cooS1 deletion produced minimal and condition-dependent effects. Structural modelling of the SNP-derived AcsA variants identified no major conformational changes compared to wild-type AcsA. Transcriptomics revealed extensive transcriptional changes associated with reduced robustness and altered by-product profiles in Leu_SNP strain, whereas cooS1 deletion had limited transcriptional changes. Our study broadens current understanding of CODH function in acetogen metabolism and offers engineering targets for improving acetogen cell factories.

ABSTRACT. Clostridium acetobutylicum can produce several compounds of industrial interest such as acetone, n-butanol and ethanol, from a wide variety of sugars such as pentoses and hexoses. This model micro-organism is therefore an ideal candidate for synthetic biology due to its metabolic versatility and proven suitability for chemical production. To that extent, the engineering of chassis strains with streamlined metabolism is an important prerequisite for the efficient integration of heterologous metabolic pathways. Here, we report a newly engineered mutant strain of C. acetobutylicum featuring a minimized metabolism generated with a CRISPR-Cas9 tool. While the strain was still capable of producing ethanol, acetate and lactate, the production of butanol, butyrate and acetone has been entirely abolished. Subsequent complementation with homologous and heterologous genes allowed to evaluate the efficiency of their respective products. Overcoming the well-known challenges of engineering C. acetobutylicum, this newly developed strain has the potential to serve as a versatile microbial chassis for customized bioproductions. It might represent a critical technical milestone in expanding the synthetic biology toolkit for C. acetobutylicum.

ABSTRACT. Some anaerobic bacteria perform mixotrophic metabolism, assimilating both organic and inorganic carbon simultaneously. Inorganic carbon assimilation by mixotrophy is an interesting approach to improve product yields and reduce CO2 emissions. Our isolate Clostridium beijerinckii Br21 performs a heterotrophic lifestyle while detaining the CO2 originating from glycolysis. The strain harbors genetic markers associated with autotrophic pathways, including: the Wood–Ljungdahl pathway; carbonic anhydrase-mediated anaplerotic reactions; and reverse reaction of pyruvate:ferredoxin oxidoreductase (rPFOR). Previously, we improved endogenous CO2 capture of C. beijerinckii Br21 cells grown in complex Reinforced Clostridial Medium (RCM) by optimizing medium composition. The optimal condition yielded higher cell biomass and acetic acid concentrations and significantly recovered more carbon (97.43±0.34%), compared to a control without supplementation (92.03±0.27%) (Tukey’s post hoc test, p<0.001). Deciphering the metabolic pathways involved in this mixotrophy, absolute expression of target genes was quantified by RT-qPCR, while comparing optimized and control growth conditions. Cells in RCM were sampled at mid-exponential phase, centrifuged, and pellets stored at −80 °C. Total RNA was extracted, complementary DNA synthesized, and qPCR performed using custom made TaqMan™ probes, that were designed to target mRNA fragments of the C. beijerinckii Br21. Genes encoding β-carbonic anhydrase and phosphoenolpyruvate carboxykinase were the most upregulated (5.8-, and 2.3-fold), supporting CO2 incorporation into oxaloacetate via anaplerotic reactions. Increased PFOR gene expression (1.6-fold) under optimized condition suggests also the contribution of the rPFOR mechanism. In contrast, formate dehydrogenase and carbon monoxide dehydrogenase genes showed low gene expression, indicating a minor role for the Wood–Ljungdahl pathway under the evaluated conditions. Overall, C. beijerinckii Br21 efficiently detains and assimilate endogenous CO2 probably via carbonic anhydrase/phosphoenolpyruvate carboxykinase and rPFOR driven reactions. Future experiments will focus on assessing the growth capabilities of the Br21 strain in defined PETC medium without yeast extract for accurate calculation of glucose-based yields. Furthermore, β- and γ-carbonic anhydrase-coding genes will be overexpressed individually and simultaneously using plasmid-based strategies, for evaluation of the impact on this CO2 detaining anaerobic mixotrophic metabolism. This special metabolic feature makes C. beijerinckii Br21 an ideal platform strain for large scale fermentations and applications in respective biorefineries.

ABSTRACT. Eubacterium limosum is a metabolically versatile acetogen, capable of converting a wide range of single-carbon (C1) substrates as well as O- and N-methylated compounds into acetate and butyrate using the Wood-Ljungdahl pathway. Its high carbon- and energy-efficiency has garnered interest from metabolic engineers in developing E. limosum as a biocatalyst for the production of biofuels and biochemicals from C1 feedstocks. Likewise, its ability to metabolize a variety of disease-relevant nutrients in the gut microbiota has intrigued the microbiome community as to its role in human health. Like many acetogenic Clostridia, technology for the genetic manipulation of E. limosum has lagged behind conventional microbes. In this talk, I will present the work from my group over the last several years developing robust genetic systems for E. limosum. I will briefly discuss our early work expanding the range of genetic 'parts', including plasmids, antibiotic resistance markers and promoters. The bulk of the talk will focus on more recent work establishing modern genome engineering tools, including CRISPR-recombineering and high-efficiency Cre-mediated integration. Each of these topics will include example applications of these tools in metabolic engineering and natural products biosynthesis projects. Finally, the talk will conclude with unpublished work on plasmid-free genome engineering methodologies designed to avoid some of the traditional bottlenecks of developing genetic systems in recalcitrant microbes. Collectively, this work, along with contributions from others in the community, has established E. limosum as a highly tractable model acetogen for studying metabolism in the gut microbiota, and for metabolic engineering efforts focused on sustainable fuel and chemical biosynthesis.

ABSTRACT. Reliable genetic control systems are essential for engineering the thermophilic anaerobic bacterium Thermoanaerobacter saccharolyticum for lignocellulosic biofuel production, yet the repertoire of strictly regulated inducible promoters remains limited. Standard characterization methods relying on fluorescent reporters often fail under requisite high-temperature, anoxic conditions. Furthermore, many existing controllable promoters exhibit high levels of transcriptional leakage that precludes the use of toxic genes. To overcome this bottleneck, we developed a rapid, dual-reporter disk diffusion assay utilizing a synthetic operon encoding erythromycin resistance (erm) and thymidine kinase (tdk) genes. This survival-based platform allows for the simultaneous evaluation of promoter inducibility and basal leakiness by measuring growth inhibition zones in the presence of erythromycin (positive selection) and 5-fluoro-2'-deoxyuridine (FUDR, negative selection). By applying this assay to a library of candidate promoters, we classified functional phenotypes into four distinct categories: Always On, Leaky Inducible, Inducible, and Always Off. Our screen identified three high-performing Inducible constructs: pEKR053 (sodium fluoride-inducible), pEKR032 (xylose-inducible), and pEKR040 (anhydrotetracycline-inducible). Dose-response characterization revealed that constructs pEKR053 and 032 exhibited a titratable response, whereas 040 exhibited full induction at the lowest non-zero inducer concentration tested. We also established that the sensitivity disparity between the two reporters allows for a broader dynamic range, where erm captures low-level expression and tdk identifies strong induction. Finally, control experiments confirmed that while some inducers like fluoride slightly increase erythromycin inhibition zones in the absence of a plasmid, they do not inherently interfere with the functional readout of the system.

ABSTRACT. Clostridium butyricum is gaining significant traction as a powerful live biotherapeutic product (LBP) due to its robust butyrate production and clinical efficacy in gastrointestinal disorders. However, deploying genetically modified anaerobes introduces critical regulatory challenges, particularly regarding the risk of horizontal gene transfer (HGT) within the dense gut microbiome and environmental escape. Here, we introduce "Genetic Fortress," a biocontainment and genetic stabilisation strategy engineered directly into a ΔrecA chassis.

To prevent both environmental persistence and the acquisition of foreign antimicrobial resistance (AMR) genes, we developed a dynamic genetic firewall utilising a restriction-modification (R-M) system placed under the tight regulation of a native, primary bile acid-inducible (bai) promoter. In the cholic acid-rich environment of the upper mammalian GI tract, the circuit drives methyltransferase expression to safeguard the host genome, while simultaneously allowing the restriction endonuclease to actively destroy incoming, unmethylated foreign plasmids. Upon exit from the gut, the absence of primary bile acids halts methylation, triggering targeted genomic degradation and complete cell death. This approach establishes a regulator-friendly, Clostridium platform capable of delivering therapeutic payloads without the risk of environmental release or AMR dissemination.

ABSTRACT. Climate change remains one of the major challenges in modern society, mainly driven by increasing fossil fuel energy consumption, which rose by 1.5 % from 2024 to 2025.[1] Gas fermentation offers a promising approach to mitigate our climate crisis by converting CO2 emissions into biocomodities.[2] Acetobacterium woodii is a well-studied anaerobic acetogen and therefore represents a suitable and promising platform bacterium for gas fermentation.[3] Our new project (microbial carbon capture and utilization as a CO2 sink for wood coatings) aims to combine sustainable lactate production with carbon capture by utilizing H2 and CO2. This project is a collaboration of the Institute of Molecular Biology and Biotechnology of Prokaryotes (UULM), the Institute of Biochemistry and Systems Biology of Metabolism (Charité) and PNZ-Produkte GmbH. Our contribution to MCCU focuses on the development of an industrially relevant A. woodii strain for lactate and acetate production. Previous genetic modifications of A. woodii already enabled lactate formation from H2 and CO2 by the expression of a gene encoding a D-lactate dehydrogenase and the deletion of the native lctBCD genes to prevent lactate utilization and the deletion of pyrE to enable knock-out of lctBCD.[4] Building on this, we aim to increase lactate yields through “Design-Build-Test-Learn” cycles. One first aim is the restoration of the pyrE knock-out to exclude the uracil auxotrophy of the pyrE deficient strain. In addition, process optimization will focus on developing a defined medium by elimination of complex additives from existing media formulations such as yeast extract, L-cysteine-HCL and antibiotics. Fermentation conditions for continuous cultivation at scales up to 25 L will be optimized, with particular emphasis on seed train transfer and process stability. Overall, we aim to establish an efficient, scalable anaerobic autotrophic lactate production, reducing CO2 emissions through carbon capture and reducing dependence on fossil fuel based production of wood coatings.

[1] Ripple et al. 2025 https://doi.org/10.1093/biosci/biaf149 [2] Bengelsdorf, 2017, https:// doi. org/ 10. 1111/ 1751- 7915. 12763 [3] Stock et al. 2026 Engineering in Life Sciences, 26(2), e70072. [4] Mook, et al. 2022. Applied Microbiology and Biotechnology, 106(4), 1447-1458.

ABSTRACT. The gut microbiome is a critical determinant of human metabolic health, immune function, and nutritional status. Among its key constituents, butyrate-producing Clostridium species — notably Clostridium butyricum and members of Clostridial clusters IV and XIVa — occupy a central role in colonic fermentation of dietary fibers and resistant starches. The resulting short-chain fatty acid, butyrate, functions as the primary energy substrate for colonocytes, modulates inflammatory signaling pathways, and has been associated with reduced risk of obesity, type 2 diabetes, and colorectal cancer. These multifaceted bioactivities position butyrate-producing Clostridia as high-value candidates for industrial exploitation in the functional food and nutraceutical sectors.This perspective evaluates current and emerging industrial strategies to harness clostridial fermentation for the production of butyrate-enriched functional ingredients and postbiotic formulations. Key focus areas include the optimization of anaerobic bioprocesses using food-grade feedstocks such as inulin, pectin, and cereal brans, as well as encapsulation and delivery technologies that ensure butyrate stability through gastrointestinal transit. The application of C. butyricum-based next-generation probiotics in fermented food systems is also critically assessed against the backdrop of rising global consumer demand for gut-health-oriented products.Significant challenges persist, encompassing strain biosafety classification, fermentation scalability, and evolving regulatory standards for Clostridial-derived food ingredients. This work proposes an interdisciplinary framework integrating anaerobic microbiology, food processing technology, and human nutritional science to bridge the gap between laboratory-scale discovery and industrial-scale application, ultimately accelerating the clinical and commercial translation of butyrate-producing Clostridia as functional food agents.

ABSTRACT. Acetivibrio thermocellus is an efficient lignocellulose-degrading bacterium due to its ability to produce cellulosomes and a diverse repertoire of carbohydrate-active enzymes (CAZymes). Here, genomic, transcriptomic, and secretomic approaches were integrated to investigate the mechanisms employed by A. thermocellus B8, a cellulolytic isolate recovered from the rumen of Brazilian Moxotó goats adapted to semiarid environments, during the deconstruction of sugarcane bagasse and straw. Transcriptomic profiling was performed during growth on sugarcane bagasse and sugarcane straw using microcrystalline cellulose as a reference substrate. A total of 625 and 509 genes were upregulated in bagasse- and straw-grown cultures, respectively. Differentially expressed genes were mainly associated with CAZymes involved in cellulose and hemicellulose degradation, carbohydrate metabolism, chemotaxis, cell motility, quorum sensing, and regulatory pathways controlling glycoside hydrolase expression. Several hemicellulolytic enzymes, including esterases, mannanases, arabinofuranosidases, and xyloglucan-active enzymes, were among the most highly induced genes, highlighting the importance of hemicellulose deconstruction for sugarcane residue utilization. Secretome analysis by LC-MS/MS identified 112 proteins, of which 63% were associated with polysaccharide catabolism. The predominant group comprised cellulases together with a diverse set of holocellulolytic enzymes, including mannanases, acetyl xylan esterases, pectate lyases, galactanases, and arabinoxylan-degrading enzymes. The second most abundant protein group consisted of oxidases, including copper amine oxidases and other oxidoreductases, followed by proteins of unknown function. The presence of oxidoreductases suggests additional mechanisms involved in plant cell wall deconstruction that remain poorly understood in A. thermocellus. To further characterize this isolate, the genome of A. thermocellus B8 was sequenced and compared with publicly available genomes. Comparative analyses revealed the absence of several genes commonly found in environmental isolates and reference strains, indicating substantial genomic variability and suggesting alternative strategies for lignocellulose utilization and ecological adaptation. Overall, the integration of genomic, transcriptomic, and secretomic datasets demonstrates that A. thermocellus B8 dynamically modulates gene expression and extracellular enzyme production in response to lignocellulosic substrates. These findings provide new insights into cellulosome organization and biomass deconstruction and may support the development of improved enzyme cocktails and microbial platforms for sugarcane-based biorefineries.

ABSTRACT. The genus Clostridium is a key contributor to dark fermentation because of its metabolic diversity and its efficiency in transforming organic waste into value-added products. Among potential feedstocks, sewage sludge, an abundant byproduct of wastewater treatment, represents a significant but underutilized resource for sustainable biohydrogen production. However, maximizing yields often requires overcoming the inherent metabolic bottlenecks of wild-type microbial communities. In this study, the fermentative performance of several Clostridium species, selected for their well-established H2-producing capabilities and metabolic plasticity in degrading diverse substrates was evaluated. Clostridium strains C. perfringens, C. tyrobutyricum, C. saccharoperbutylacetonicum and C. beijerinckii, were tested using sterile and non-sterile sewage sludge, with Clostridial Nutrient Medium (CNM) employed as a benchmark. Our results highlight a standout strain capable of achieving H2 yields of 26.8 ± 6.33 mL H₂/g from non-sterile sludge and 15.39 ± 2.97 mL H₂/g from sterile sludge. Notably, the performance on raw waste was highly comparable to the 24.5 ± 1.18 mL H₂/g achieved in optimized commercial media, demonstrating the strain's robustness in complex environments. A targeted metabolic engineering strategy was developed to maximize H₂ production through the overexpression of an oxygen-resistant hydrogenase and distinct phosphorylating and non-phosphorylating GAPDHs, thereby enhancing carbon flux and increasing reducing equivalents availability for hydrogen biosynthesis. The metabolic shifts induced by these genetic interventions were characterized by correlating H2 kinetics with substrate uptake and soluble end-product profile. These findings highlight tailored Clostridial platforms as robust biocatalysts for sewage sludge valorization. Future work will focus on expanding the genetic toolbox and validating strain stability under large-scale, non-axenic conditions.

ABSTRACT. The valorization of residual biomasses and the reduction of CO2 emissions represent two major challenges in the development of sustainable biotechnological processes. In this context, the present project aims to develop an integrated process for biological hydrogen (H2) production through dark fermentation of microalgal biomass using bacteria belonging to the genus Clostridium. The algal biomass is produced by cultivating microalgae on liquid digestate derived from anaerobic digestion plants, with the dual purpose of bioremediation and valorization of agro-industrial waste. The study initially focuses on optimizing algal growth on anaerobic digestate-derived supernatants through chemical and biological screening of cultivation matrices. Subsequently, the microalgal biomass is used as fermentative substrate for selected Clostridium strains, including Clostridium beijerinckii, Clostridium tyrobutyricum, and Clostridium acetobutylicum, evaluating their ability to produce H2 from untreated or pretreated algal biomass. Pretreatments include autoclaving, acid/alkaline hydrolysis, enzymatic hydrolysis, or combinations of these approaches. Different strategies aimed at enhancing fermentative yields are also investigated, including controlled-pH cultures, reduction of H2 partial pressure, and sequential cultures involving cellulolytic microorganisms such as Clostridium thermocellum, in order to improve the degradation and accessibility of the microalgal biomass. In addition, metabolic engineering strategies may be applied to this microrganism to enhance polysaccharide degradation efficiency and improve substrate conversion toward hydrogen production. In parallel, metabolic engineering approaches are applied to increase H2 productivity through hydrogenase overexpression and deletion of competing metabolic pathways responsible for the formation of alternative reduced metabolites. Overall, the project contributes to the development of a sustainable and scalable platform for converting CO2 and agro-industrial residues into biohydrogen and high-value biomass, integrating circular economy principles, bioremediation, and renewable energy production.

ABSTRACT. The gut microbiome is a critical determinant of human metabolic health, immune function, and nutritional status. Among its key constituents, butyrate-producing Clostridium species — notably Clostridium butyricum and members of Clostridial clusters IV and XIVa — occupy a central role in colonic fermentation of dietary fibers and resistant starches. The resulting short-chain fatty acid, butyrate, functions as the primary energy substrate for colonocytes, modulates inflammatory signaling pathways, and has been associated with reduced risk of obesity, type 2 diabetes, and colorectal cancer. These multifaceted bioactivities position butyrate-producing Clostridia as high-value candidates for industrial exploitation in the functional food and nutraceutical sectors. This perspective evaluates current and emerging industrial strategies to harness clostridial fermentation for the production of butyrate-enriched functional ingredients and postbiotic formulations. Key focus areas include the optimization of anaerobic bioprocesses using food-grade feedstocks such as inulin, pectin, and cereal brans, as well as encapsulation and delivery technologies that ensure butyrate stability through gastrointestinal transit. The application of C. butyricum-based next-generation probiotics in fermented food systems is also critically assessed against the backdrop of rising global consumer demand for gut-health-oriented products. Significant challenges persist, encompassing strain biosafety classification, fermentation scalability, and evolving regulatory standards for Clostridial-derived food ingredients. This work proposes an interdisciplinary framework integrating anaerobic microbiology, food processing technology, and human nutritional science to bridge the gap between laboratory-scale discovery and industrial-scale application, ultimately accelerating the clinical and commercial translation of butyrate-producing Clostridia as functional food agents.

ABSTRACT. Clostridium thermocellum is a promising candidate organism for consolidated bioprocessing (CBP) due to its innate ability to directly deconstruct and ferment carbohydrates in lignocellulosic biomass using a highly efficient cellulosome multi-enzyme complex system. Advancing CBP strategies capable of utilizing diverse biomass feedstocks under industrially relevant conditions is critical for cost-effective renewable biofuel production. We evaluated the performance of C. thermocellum cocultured with Thermoanaerobacterium thermosaccharolyticum on untreated corn stover and switchgrass at high solids loadings (60-80 g/L). To further improve biomass solubilization and fermentation efficiency, particularly at high solids loadings, we tested a cotreatment strategy termed ferment–mill–ferment (FMF), which incorporates a short milling step between fermentation stages. This approach resulted in substantially improved lignocellulose solubilization compared with conventional fermentation. Using a purpose-built bioreactor system, the coculture grown on 120g/L lignocellulose achieved the highest solubilization rate reported thus far (0.856 g/L/h). In addition, we used engineered C. thermocellum and T. thermosaccharolyticum for the conversion of multiple pretreated biomass feedstocks resulting in ethanol titers of ~30 g/L, at near-to-complete solubilization of the solids substrates, demonstrating efficient utilization of pretreated biomass. These results demonstrate the versatility and robustness of C. thermocellum-based CBP strategies for processing untreated, cotreated, and pretreated lignocellulosic biomass toward sustainable biofuel production.

ABSTRACT. Recent advances in high-throughput technologies have exponentially expanded the data-generation capacity of biological research, providing the foundation for increasingly accurate data-driven approaches to modeling cellular behavior. In this context, constraint-based models (CBMs) have emerged as robust tools for metabolic modeling at genome-scale level. Through CBM resolution, commonly formulated as Linear Programming (LP) problems, complex metabolic behaviors can be predicted and systematically explored, facilitating metabolic engineering studies at multiple scales. This way, given the importance of the adopted constraints in model reconstruction and resolution, several CBM frameworks have been developed, with particular emphasis on the thermodynamics-based Max-min Driving Force (MDF) approach.By MDF utilization, aspects beyond reaction directionality can be elucidated, given thermodynamics’ importance for in vivo reaction kinetics. Indeed, by applying the MDF framework to the central metabolism of Escherichia coli, we investigated how biohydrogen production mediated by the heterologously expressed HydABC complex of Thermotoga maritima affects pathway behavior and determines the maximum feasible hydrogen yield before enzymes shift the reaction direction. Our analyses identified the HydABC reaction on H2 production direction as the major thermodynamic bottleneck of the pathway, restricting biohydrogen production to 3.66 mol per mol of glucose consumed, slightly below the theoretically maximal yield reported, 4 mol H2 per mol glucose. Building on these results, we propose the development of a computational pipeline for the automated reconstruction of a metabolic model for Clostridium thermocellum. Using the bacterial genome as input, the algorithm will generate the corresponding metabolic reconstruction based on gene-protein-reaction (GPR) associations. Model resolution will rely on iterative MDF simulations, in which intracellular metabolite concentrations will be dynamically updated according to changes in extracellular conditions. The resulting model is expected to correctly associate enzymes involved in commonly annotated pathways while supporting both local and global metabolic simulations. Finally, model accuracy will be evaluated through the integration of condition-specific transcriptomic data derived from experiments conducted at different growth temperatures. Through this approach, we expect the model to accurately predict the ethanol production profile under distinct temperature conditions, thereby highlighting regulatory mechanisms associated with ethanol biosynthesis and supporting future strategies for in vitro yield optimization in C. thermocellum.

ABSTRACT. The integration of gas fermentation with organic waste valorization offers a highly efficient route for sustainable biorefining. This study evaluates the metabolic flexibility of Clostridium luticellarii to produce platform organic acids, such as propionate, isobutyrate and caproate, using fructose as a proxy for representing sugar-rich agro-industrial effluents (e.g., winery lees). We conducted batch fermentations comparing heterotrophic growth against mixotrophic growth (1 g/L fructose plus 0.3 bar CO2 and 1.2 bar H2 gas headspace mixture), assessing the specific impact of targeted amino acid supplementation versus a standard peptone control. Subsequently, a second mixotrophic (5 g/L fructose) preliminary amino acid screening was performed using a dropout method, excluding glutamate-family amino acids (glutamine, glutamic acid, proline and arginine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), methionine or serine for each condition. Cultures were sampled on days 2, 4 and 7 and each culture condition was performed in triplicate. Sugars and organic acid in the culture were measured by HPLC, while gas headspace composition was measured by GC. Results of the first screening demonstrated that nutrient availability and growth regimes dictate distinct metabolic shifts. Mixotrophic conditions outperformed heterotrophy across all major growth and production parameters. Notably, the synthesis of propionic acid (0.17 and 0.26 g/L for mixotrophic and heterotrophic growth respectively) was exclusively triggered by the presence of amino acids. Under mixotrophy, targeted amino acid supplementation enhanced biomass concentration (1.5-fold, reaching up to 0.15 g/L at 48 hours) compared to non-supplemented and peptone-controlled cultures. Furthermore, this supplementation drove slight increases in the production of formic (1.6-fold, 0.48 g/L), acetic (1.75-fold, 0.28 g/L), and isobutyric acids (4.12-fold, 0.2 g/L). Additionally, chain elongation to caproic acid 0.12 g/L was selectively achieved only when utilizing peptone or a specific amino acid matrix excluding the glutamate-derived amino acids. These findings demonstrate that precise modulation of the amino acid profile under mixotrophic conditions can direct carbon flow in C. luticellarii, potentially unlocking customized metabolic outputs. This stoichiometric control establishes a critical foundation for optimizing scalable bioprocesses that convert complex agricultural waste and captured gases into high-value industrial chemicals.

ABSTRACT. The use of sugarcane-derived substrates in consolidated bioprocessing (CBP) strategies offers a promising route for cost-effective bioethanol production by integrating the conversion of structural solid and soluble carbohydrates. Cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum have been shown to be particularly effective in CBP including for sugarcane bagasse. However, high concentrations of soluble sugars may inhibit cellulolytic activity and compromise process performance. In addition, the performance of coculture fermentation under soluble sugar-rich conditions has not been well studied. This study evaluates the growth and fermentation profile of Thermoanaerobacterium thermosaccharolyticum with sugarcane juice as substrate. The effect of increasing sucrose concentrations on the growth and fermentative performance of Clostridium thermocellum under conditions relevant to CBP is also evaluated. We show that the cellulolytic capacity of C. thermocellum was not impaired by high concentrations of sucrose, while T. thermosaccharolyticum demonstrated low ethanol conversion at high concentrations of sugarcane juice.

ABSTRACT. Consolidated bioprocessing (CBP) for second-generation ethanol production has emerged as an alternative to thermochemical pretreatment, integrating enzymatic hydrolysis and fermentation in a single step using the cellulolytic thermophile Clostridium thermocellum. To further enhance CBP efficiency, cotreatment consolidated bioprocessing (C-CBP) combines biomass milling and microbial fermentation, improving carbohydrate solubilization and reducing process costs. With the addition of the saccharolytic bacterium, Thermoanaerobacterium thermosaccharolyticum, hemicellulose-derived pentose-based carbohydrates can additionally be converted to ethanol, improving carbohydrate solubilization and conversion. Different milling configurations were tested for sugarcane bagasse to increase carbohydrate solubilization, from low to high solid content in bioreactors. Results demonstrated that in-situ cotreatment increased carbohydrate solubilization from 40% to 75% after 172 h of milling at 10 g/L solid loading using cocultures of C. thermocellum and T. thermosaccharolyticum. In contrast, the ex-situ configuration achieved comparable solubilization (75%) with significantly shorter milling times (20 min) and higher solid loadings (30 g/L and 80 g/L). Characterization of residual solids showed that during milling wet biomass specific surface area is increased while particle size is reduced, contributing to increased access for microorganisms to act on the biomass. These findings suggest that even short milling sessions in between fermentations improve biomass deconstruction efficiency, particularly at elevated solid loadings, where process optimizations translate directly into cost reductions.

ABSTRACT. Aldehyde Alcohol dehydrogenase (AdhE) is an essential bi-functional enzyme conserved across bacteria, including Clostridium thermocellum. AdhE consists of both ALDH and ADH domains joined by a linker region and catalyzes the sequential conversion of acetyl-CoA to acetaldehyde to ethanol. Cryo-EM structures have revealed a channel between the two catalytic domains, suggesting the volatile intermediate acetaldehyde may be directly transferred without release into the surrounding environment. This process is known as substrate channeling. However, biochemical evidence is also required to support this structural hypothesis. Here we present the first biochemical evidence of substrate channeling in AdhE using two complementary assays. First, a UV-Vis-based acetaldehyde capture assay employing semicarbazide demonstrated that wildtype AdhE activity is insensitive to acetaldehyde sequestration. Second, we use quantitative ¹H NMR (qNMR) to directly monitor all reaction components in real time, revealing minimum acetaldehyde accumulation and tight kinetic coupling between acetyl-CoA consumption and ethanol production. Together, these orthogonal assays provide compelling evidence that AdhE channels acetaldehyde between its catalytic domains, with implications for enzyme engineering in Clostridium and related organisms.

ABSTRACT. Short- and medium-chain fatty acids (SCFAs and MCFAs) are widely applied in the food, pharmaceutical, and livestock industries. However, naturally occurring microorganisms capable of efficient synthesis of target products remain scarce, and the underlying metabolic mechanisms are largely unexplored. Anaerobic fermentation ecosystems harbor a rich reservoir of uncultured SCFA/MCFA producers. We systematically investigated three key aspects: functional microbe mining, metabolic mechanism elucidation, and high-efficiency fermentation technology development.

In terms of novel species discovery, several Clostridia species were identified and characterized, including a novel caproate-producing bacterium capable of utilizing both glucose and lactate (Caproicibacterium lactatifermentans), as well as a proteolytic specialist (Aminobacterium zhubaoyuanii). Regarding metabolic mechanisms, we revealed differential metabolic strategies of C. lactatifermentans under varying carbon sources and identified a novel regulatory mechanism in which glucose negatively modulates lactate metabolism via the lactate utilization repressor LldR.

For fermentation technology, given the current lack of effective genetic tools for these novel species, we employed a synthetic microbiome approach leveraging interspecies metabolic interactions to overcome bottlenecks in monoculture fermentation. Using a top-down strategy, we constructed a simplified microbial community dominated by caproate-producing bacteria, achieving stable caproate production from non-sterile liquor-making wastewater. Furthermore, we established a two-species co-culture system combining lactic acid bacteria and caproate-producing chain elongators. Unidirectional interspecies cross-feeding of lactate drove efficient operation of the reverse β-oxidation pathway, significantly enhancing caproate titer and yield.

Collectively, we establish an integrated research framework from functional microbe mining and metabolic network dissection to synthetic microbiome construction, providing important strain resources and technological support for green biomanufacturing of short- and medium-chain fatty acids.

ABSTRACT. In the production of butyric acid via sequential batch fermentation using brown sugar effluents, lactic acid bacteria (LAB) predominantly acted as allies to butyrate-producing bacteria through a mechanism known as metabolic cross-feeding. In this symbiotic relationship, LAB converted carbohydrates, such as sucrose or simple sugars from these effluents, into lactate, which served as an essential metabolic precursor for butyrigenic species as Clostridium tyrobutyricum. This cooperation was advantageous as it facilitated a more efficient conversion of the substrate: LAB initiated the degradation process, while butyrate producers finalized the synthesis of the target acid, often consuming acetate present in the medium as well. However, for this relationship to remain beneficial, rigorous control of operational parameters—especially pH—was fundamental. With the progression of sequential batch rounds, an excessive accumulation of lactic acid and/or the production of bacteriocins inhibited butyrate-producing bacteria, leading to metabolic failure. Therefore, in the sequential batch system, population dynamics were carefully balanced to ensure that the lactate produced in the initial phase was promptly converted, preventing drastic pH drops and maximizing the final yield of butyric acid.

ABSTRACT. The use of renewable biomass resources and biogenic CO 2 and other greenhouse gases as feedstock for sustainable production of chemicals and fuels contributes to the reduction of harmful emissions and to the replacement of fossil resources in industry. In this study, the use of acetate and lactate as substrates for production of the chemicals butyrate and iso-butyrate by an anaerobic co-culture is described. This co-culture, composed by the acetogen C. autoethanogenum and the solventogen C. beijerinckii was previously designed based on genome-scale models and validated experimentally (1). The feedstock use and product range from each individual strain under the conditions of the co- culture has been characterized. C. autoethanogenum converts CO 2 and H 2 into mainly acetic acid, with consumption of a small amount of lactic acid. The solventogen C. beijerinckii utilized acetic acid and lactic acid for the production of butyric acid, reaching titers of butyric acid of a 17 mM, after which sporulation and OD decrease was observed. After prolonged incubation, a second growth phase was observed in the culture, where lactate and acetate were consumed resulting in final product titers 28 mM butyrate and 43 mM isobutyrate after 18 days of cultivation. In this case, sporulation was also observed after the growth phase. Sequencing and transcriptomic analysis of these cultures revealed that an unexpected acetogenic strain was present in the culture, and was responsible for the production of the (iso-)butyrate found in the medium. The results described in this presentation give insights into the substrate use and product formation by solventogens and acetogens growing on CO 2 and lactate as co-substrate.