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ABSTRACT. Thermoanaerobacterium saccharolyticum is an anaerobic, thermophilic bacterium that has been proposed for use in consolidated bioprocessing in coculture with cellulolytic bacteria such as Clostridium thermocellum for ethanol production. Although it cannot break down cellulose, T. saccharolyticum is capable of fermenting C5 and C6 sugars and soluble C5 oligomers (i.e., xylan) from lignocellulosic biomass. Therefore, to enable cellulosic ethanol production, another strategy to be considered is transferring the ethanol production pathway from this organism to native cellulolytic organisms such as C. thermocellum. There is thus motivation to understand the mechanistic basis of the T. saccharolyctium robust ethanol pathway so that key features can be recapitulated in C. thermocellum. Previously, we characterized the individual role of the main genes responsible for electron transfer in the ethanol production of T. saccharolyticum (nfnA, nfnB, hfsD, and hydA), and we observed that they are necessary for high titer ethanol production. However, the consequences of the combined loss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of the ferredoxin nicotinamide oxidoreductases (nfnA and nfnB) and hydrogenases (hydA and hfsD), and investigated the effects on growth and fermentation. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and found a mutation in the alcohol dehydrogenase adhA gene. With a combination of genetic testing, enzyme assays, and molecular dynamics simulations, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings provide deeper insights into the adaptation of electron transfer pathways in this organism and consolidate our understanding of the functioning of its unique ethanol production pathway.

ABSTRACT. Reducing reliance on fossil resources and CO₂ emissions in the industrial production of commodity chemicals is an important step towards a more sustainable chemical industry. Acetone is a key industrial solvent and precursor for numerous specialty chemicals, yet its current production primarily depends on fossil-based processes. Although bio-based, industrial acetone production was historically established via the ABE fermentation process, traditional producers such as the solventogenic Clostridium acetobutylicum rely on carbohydrate feedstocks, leading to competition with food resources and limiting its economic viability. Gas-fermenting acetogens such as Clostridium ljungdahlii offer a promising alternative, as they couple carbon capture with the production of valuable chemicals from gaseous substrates. Utilizing H₂ and CO₂ as sole energy and carbon sources, wild-type C. ljungdahlii produces mainly acetate and ethanol. However, its available genetic toolbox enables metabolic engineering approaches to expand its product spectrum towards non-native compounds such as acetone and to improve production efficiency. In the BETA (BioEthanol To Acetone) project, a sustainable process is developed to produce acetone from bioethanol. First, ethanol is converted into acetone in a chemical process, in which H₂ and CO₂ are formed as side products. We focus on the second stage, where we engineer C. ljungdahlii to convert H₂ and CO₂ into acetone via heterologous expression of the acetone biosynthesis pathway. Basic acetone-expression plasmids have been successfully introduced into Clostridium ljungdahlii, resulting in the formation of isopropanol as the initial product, thus demonstrating the feasibility of the pathway. Further pathway optimization is being pursued through systematic evaluation of promoter combinations and gene variants to improve expression balance, while genome engineering is used to reduce side product formation and improve the general energy metabolism of C. ljungdahlii.

ABSTRACT. Sorghum, the fifth major global cereal, has potential as a source crop in temperate regions. To completely use sorghum bagasse, the ideal enzyme cocktail aims to identify and select the contributed enzymatic system. This study investigated the enzymatic system of Clostridium cellulovorans cellulosome and noncellulosomal enzymes using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry LC-MS/MS. Enzyme solutions from treated and untreated sorghum bagasse were prepared and compared based on carboxymethyl cellulase (CMCase) activity. As a result, the enzyme solution derived from untreated sorghum bagasse had the highest activity. Protein bands from each C. cellulovorans culture showed distinct patterns on SDS-PAGE examination: three enzyme fractions, including culture supernatants, crystalline cellulose (Avicel) bound, and unbound fractions. These results suggested that untreated sorghum bagasse induced a variety of cellulosomal and uncellulosomal proteins. On the other hand, 5% or 10% sorghum supernatants could not induce Avicel-bound proteins, including the cellulosome, although even 5% sorghum juice induced three major bands: 180 kilodalton (kDa), 100 kDa, and 70 kDa, respectively. In contrast, cellobiose induced three major bands, while the total number of all isolated proteins from the cellobiose medium was the most limited among all culture media. More intriguingly, our investigation detected one cellulosomal protein, hydrophobic protein A (HbpA) and three noncellulosomal enzymes, indicating that glycosyl hydrolase family 130 (GH130) was identified as a biomass-induced enzyme in good accord with previously published proteomic studies. Therefore, the proteomic dataset generated in this study provides us a foundation for future computational approaches, including machine learning-based prediction of optimal enzyme cocktails for target biomass degradation.

ABSTRACT. The efficient and effective conversion of lignocellulose into soluble carbohydrates to produce biofuels and chemical intermediates remains a central challenge. Biomass recalcitrance -the natural resistance of the plant cell wall to deconstruction- is a major obstacle in this process. While plants have evolved mechanisms to resist microbial and enzymatic deconstruction, the biosphere harbors biocatalysts capable of efficiently degrading plant biomass. Some of the best biocatalyst can be found in thermophilic anaerobic environments and seem to be naturally divided into two groups which heavily feature Clostridia: cellulolytic specialists and saccharolytic generalists. Cellulolytic specialists solubilize both cellulose and hemicellulose while only utilizing cellulose-derived sugars. Saccharolytic generalists are unable to solubilize carbohydrates directly but readily utilize soluble sugars, particularly those derived from hemicellulose. We will discuss the roles of these somewhat unique organisms. Central is Clostridium thermocellum, one of the most effective cellulolytic organisms due to its cellulosome-centered array of carbohydrate active enzymes (CAZymes). The addition of non-cellulolytic saccharolytic organisms like Thermoanaerobacterium thermosaccharolyticum creates particularly powerful cocultures or consortia. Proteomics indicate that T. thermosaccharolyticum has a wide array of CAZymes, some of which are organized in polysaccharide utilization loci. Using cocultures of thermophilic anaerobic saccharolytic and cellulolytic bacteria for processing unpretreated lignocellulose materials (corn stover, sugarcane bagasse, switchgrass) has resulted in the highest levels of solubilization and utilization reported in literature this far. The surprisingly persistent division of labor not only enhances deconstruction but also increases conversion. Experimental results suggest that metabolic codependency extends beyond a shared front-end metabolism. Using lignocellulosic microbiomes, creating an environment wholly dependent on the deconstruction of lignocellulose, combined with metaproteomics and metagenomics help us further understand and improve defined cultures performance. These microbiomes also guide the performance limits for defined cultures. Finally, different cultivation approaches and processing methods geared towards current shortcomings will be discussed.

ABSTRACT. Overall, Clostridia can ferment a wide range of substrates comprising soluble sugars (e.g., glucose, xylose, fructose, lactose, cellobiose), polysaccharides (e.g., starch, cellulose), glycerol and gaseous carbon compounds (CO, CO2). This enables to use them for the valorization of several waste feedstocks such as lignocellulosic biomass, (micro) algae biomass; food waste; municipal waste or agro-industrial effluents. Among dark fermentative microbes, Clostridia show the highest maximum theoretical H2 yield (that is 4 H2 mol/mol glucose). These characteristics make Clostridia promising candidates for cost-effective industrial production of H2 gas via fermentation. Here, a variety of paradigms involving waste biomass fermentation to H2 will be presented that include different feedstocks (grape stalks, wastewater sludge, livestock manure, olive mill wastewater), selected Clostridium strains (e.g., C. beijerinckii, C. thermocellum), strain improvement by metabolic engineering, various process configurations including cutting edge approaches (e.g., microbial electrolysis cells). Globally, these examples confirm the potential of bacteria belonging to the Clostridium genus for the development of sustainable biomass biorefining processes.

ABSTRACT. Future biomanufacturing of industrial products will use novel synthetic biology tools and advanced bioprocesses to convert abundant biomass and waste resources into value-added products with comparable or superior properties to replace current petroleum-based products, thus enabling circular bioeconomy with affordable energy, economic growth, and innovation in renewable energy and chemicals production. However, biomanufacturing faces many challenges in its development that requires fundamental research in synthetic biology and novel bioprocesses involving multidisciplinary teams and academic-industry partnerships. My research group has developed several bioprocesses for production of biofuels and bio-based chemicals, including butanol, short-chain fatty acids (e.g., acetic acid, propionic acid, and butyric acid), and dicarboxylic acids (e.g., malic acid and fumaric acid). These carboxylic acids and butanol are important chemicals with wide applications in food, pharmaceutical, and chemical industries. Butanol can also be used as an advanced biofuel with superior fuel properties compared to ethanol. Currently, these chemicals are almost exclusively produced via petrochemical routes, although they can also be produced from renewable biomass via microbial fermentation. The bioconversion (biorefinery) provides an environmentally friendly and sustainable route for chemicals and fuels production with minimal greenhouse gas emissions. In my talk, I’ll discuss some of the major challenges and opportunities in industrial production of biofuels and biobased chemicals from biomass and carbon dioxide using engineered Clostridia. Some of our technologies in engineering Clostridial cells for the biosynthesis of butanol and carboxylic acids and novel bioreactors and bioprocesses with in-situ product recovery for economical production of these chemicals from various carbon sources at an industrial scale will be highlighted.

ABSTRACT. The transition towards a circular and greener economy presents many challenges, including the need for biofuels (Lee & Lavoie, 2013). A promising route is through second-generation processes, specifically consolidated bioprocessing (CBP), where enzyme production, hydrolysis, and fermentation are combined in one step (Lynd et al., 2022). The thermophilic bacterium Acetivibrio thermocellus (previously Clostridium thermocellum) is a model CBP organism due to its extensive cellulolytic capabilities and native fermentative metabolism (Akinosho et al., 2014). However, A. thermocellus has limited capacity to metabolize hemicellulose derived carbohydrates, reducing overall yields from lignocellulosic biomass (Verbeke et al., 2017). Co-culturing with thermophilic saccharolytic generalists, such as Thermoanerobacterium thermosaccharolyticum (previously Clostridium thermosaccharolyticum), that are able to utilize a wide array of complex and simple C5 and C6 sugars, improves overall solubilization, utilization and product yield (Kubis et al., 2022; Zambello et al., 2024). We evaluated a fiber reject side-stream from the paper and pulp industry, consisting of 96 ± 3 wt.% cellulose with low impurity levels, which is currently underutilized and primarily incorporated as a low-value filling material. The high carbohydrate content makes this material a highly relevant substrate for ethanol production by CBP. In this study various solids loadings were assessed using monoculture and co-cultures in bioreactors, with a focus on carbohydrate solubilization, sugar utilization, and product formation. Notably, the fiber reject side-stream was used directly without additional processing. Results highlight similarities between the fibre reject stream and microcrystalline cellulose. The results demonstrate the differences between ethanologen and wild-type cultures, mono- versus co-culture systems, and the effects of solid loading on ethanol titer. Maximum ethanol titers were observed to be in excess of 27 g/L, with >98% solubilization of 100 g/L solids loadings, bringing this industrial substrate on par with crystalline cellulose (Holwerda et al., 2014; Tian et al., 2016).

ABSTRACT. Microbial electrosynthesis (MES) systems have emerged as a promising strategy for converting CO₂ into value-added compounds using microorganisms as biocatalysts. Clostridium carboxidivorans stands out for its ability to utilize CO₂ and H₂ or CO through the Wood–Ljungdahl pathway, producing acetyl-CoA and metabolites such as acetate, propionate, butyrate, and alcohols. This study evaluated the effect of cathodic potentials (-0.6, -0.7, and -0.8 V vs. Ag/AgCl) on the growth and metabolism of C. carboxidivorans DSM 15243 in a microbial electrolysis cell (MEC), compared to a control operated without applied potential. The strain was anaerobically activated in DSMZ 879 medium at 37 °C under a CO₂ atmosphere, and assays were conducted using NaHCO₃ (3 g L⁻¹) as the inorganic carbon source in a three-electrode configuration with a Gamry Interface 1010 potentiostat. At -0.7 V, higher initial cell growth was observed (OD₆₀₀ = 1.89) compared to the control. However, following shifts to -0.8 V and subsequently to -0.6 V, growth profiles became similar to the control, suggesting metabolic adaptation to the imposed electrochemical conditions. At -0.7 V, metabolite production was low — only 18 ppm acetate detected between days 4 and 5 — whereas the control exhibited greater fermentative activity, with propionic acid increasing from 73 to 242 ppm and acetic acid from 38 to 177 ppm. The application of -0.8 V enhanced the formation of more reduced products, reaching 347 ppm propionic acid and 184 ppm butyric acid, indicating greater availability of reducing equivalents and redistribution of metabolic flux. At -0.6 V, moderate butyric acid production was observed (53 ppm vs. 25 ppm in the control) alongside higher acetic acid formation (66 ppm vs. 3 ppm), suggesting lower accumulation of metabolic intermediates under less negative potentials. Cyclic voltammetry of the biofilm revealed redox peaks at approximately -0.65 V and -0.1 V vs. Ag/AgCl, potentially associated with catalytic centers involved in CO₂ reduction. These findings demonstrate that cathodic potentials directly influence C. carboxidivorans metabolism, favoring the formation of more reduced compounds and highlighting the potential of microbial electrosynthesis for bioelectrochemical CO₂ conversion.

ABSTRACT. 1,3-Propanediol (PDO) is an important chemical widely used in the fields of material science and cosmetics industry. The biomanufacturing of PDO offers numerous advantages such as the renewability of raw materials and environmental friendliness. Among various microorganisms, Clostridium pasteurianum stands out as an ideal choice for industrial PDO production due to its safety, non-pathogenic nature, rapid glycerol metabolism, swift growth, independence from expensive culture medium components, and its inherent efficient metabolic pathway for PDO production. This review begins by introducing the current state and challenges of PDO biomanufacturing, followed by an in-depth discussion of the methods for producing PDO using C. pasteurianum. Special attention is paid to the glycerol metabolism mechanism, strategies for glycerol fermentation, and the design of the fermentation process. It is worth mentioning that the C. pasteurianum mutant strains screened by our research group and the robust processes developed have, to a large extent, overcome the traditional sensitivities of C. pasteurianum to environmental conditions, especially regarding iron concentration and impurities of raw glycerol. In an electricity-aided fermentation process PDO concentration as high as 120.6 g/L was achieved with a productivity of 4.8 g/L/h and a yield reaching the theoretical maximum. We further discuss the natural limitations of genetic engineering in C. pasteurianum, elaborating on the exploration of strategies based on rational genomic modification and directed evolution. Finally, the development of efficient downstream processing technologies is emphasized as crucial for realizing the cost-effective microbial production of PDO from renewable resources, since the industrial application of PDO requires a very high purity (>99.9%). The discussion on PDO downstream processing mainly focuses on techniques based on evaporation and distillation as well as extraction-based purification. Through a comprehensive coverage of metabolic engineering, strain evolution, fermentation process optimization, and product separation technologies, this review discusses about the characteristics and advantages of PDO production from C. pasteurianum, and points out important issues for further development of this microorganism as a new industrial chassis.

ABSTRACT. The urgent need to mitigate carbon emissions and transition toward a circular bioeconomy has intensified interest in gas fermentation as a platform for carbon capture and utilization. In this context, acetogenic Clostridium species have emerged as robust microbial catalysts capable of converting C1 feedstocks such as CO, CO₂, and syngas into value-added chemicals through the Wood–Ljungdahl pathway. This lecture will present the advances of our research group in developing Clostridium-based bioprocesses for the efficient bioconversion of gaseous substrates into C2+ products, including organic acids and alcohols, as well as their integration into hybrid bioprocess platforms. Emphasis will be given to bioprocess engineering strategies encompassing medium formulation, gas–liquid mass transfer, operational mode selection, and process optimization to enhance productivity, robustness, and scalability. In addition, we will discuss the concept of integrated C1–C2+ platforms, in which gas fermentation is coupled to subsequent aerobic or anaerobic bioprocesses using complementary microbial cell factories to upgrade fermentation products into high-value biochemicals, such as biopolymers, lipids, and biosurfactants. This integrated approach expands the technological and economic potential of gas fermentation beyond conventional acetogenesis. Overall, this presentation will highlight how Clostridium-based gas fermentation can serve as a cornerstone technology for sustainable carbon recycling, bridging gaseous C1 resources and industrial biomanufacturing within future low-carbon biorefineries.

ABSTRACT. The transition from a fossil-based economy to a circular bioeconomy requires robust and scalable microbial platforms to valorize carbon-rich waste streams. In this context, the project GoodByO aims to develop a next-generation multi-commodity biorefinery integrating food waste, biogenic CO2, and process wastewaters into sustainable innovative value chains. Among these, biogenic CO2 represents a promising substrate for value-added chemicals production via acetogenic Clostridium species. Clostridium carboxidivorans P7 naturally produces 1-hexanol from syngas or carbon monoxide, a C6 alcohol commonly used as solvent but also in the flavor and fragrance industries. Through Adaptive Laboratory Evolution (ALE), our research group previously developed an evolved strain exhibiting enhanced growth on CO2/H2 mixtures and improved hexanol production. The strain achieved a hexanol cell-specific productivity of 0.8 g L-1 day-1 in serum bottle experiments and a selectivity of 60% in elevated-pressure stirred tank reactors, representing the highest performance so far reported in literature from CO2/H2 substrate. Within the GoodByO framework, this evolved strain is exploited to establish a continuous and stable 1-hexanol production process at TRL 5, upgrading the CO2 of raw biogas from the ChainCraft bioplant with green H2. Physiological characterization in close-batch systems demonstrated an efficient growth on unpurified biogas with H2, confirming the feasibility of using a real biogenic CO2 stream. Process development focused on simplifying the medium to reduce costs and contamination risk by selecting a novel strain (8T-CDM), obtained via ALE, able to grow in a chemically defined medium without yeast extract (YE) and requiring only three essential vitamins. The growth of 8T-CDM was evaluated in a 0.5L pressurized continuous stirred-tank reactor operated in chemostat, continuously feeding simulated biogas and H2 , showing performance comparable to the parental strain grown in YE-supplemented medium. For the first time, stable growth of C. carboxidivorans adapted strains were demonstrated under pressurized chemostat conditions using H2 and CO2. These findings provide a strong foundation for transition to pilot-scale operation and unpurified biogas integration, representing a concrete step toward scalable, carbon-negative hexanol production from biogenic CO2. GoodByO is supported by the Circular Bio-based Europe Joint Undertaking, and it is funded by the European Union.

ABSTRACT. A circular carbon bioeconomy requires the chemical industry to move away from fossil resources. As an example of unsustainable fossil-based process, the actual industrial hydrogenation process of carboxylic acids to alcohols remains costly and energy-intensive due to harsh operating conditions and expensive metal catalysts. Gas fermentation by acetogenic bacteria could offer a sustainable alternative, enabling the bio-hydrogenation of exogenous acids precursors into alcohols while valorizing CO₂ and renewable H₂ as gaseous feedstock.

The native metabolism of acetogenic Clostridium strains is thermodynamically constrained to even-chain metabolites, making the direct autotrophic production of odd-chain medium-chain alcohols (MCOHs) a significant challenge.

Here, we investigated a bio-hydrogenation process to produce 1-pentanol from a CO₂/H₂ gas mixture using Clostridium carboxidivorans_hex21, an acetogenic strain previously adapted though Adaptive Laboratory Evolution by our research group [1]. To overcome native metabolic limitations, the strain was exploited as a biocatalyst with propionate and valerate evaluated as exogenous precursors. Although both proved effective in enhancing pentanol production, valerate supported higher pentanol productivity and was therefore selected as the precursor of choice. Its toxicity was characterized revealing a dose-dependent inhibitory effect on growth and hydrogenation activity. Despite this trade-off, pentanol titers and specific productivity increased dose-dependently across the range tested. 1-pentanol emerged as an equally critical constraint, exhibiting inhibitory effects even at relatively low concentrations.

Building on these findings, a preliminary gas-fed batch strategy at a sub-inhibitory valerate concentration of 1 g L⁻¹ was first applied, yielding a pentanol specific productivity (qPentOH) of 0.026 g g_CDW⁻¹ h⁻¹. Since the product accumulation remained a critical bottleneck, the process was subsequently transitioned to a semi-continuous gas-fed batch configuration, simultaneously reducing product toxicity and maintaining elevated valerate availability. This optimized strategy proved decisive, doubling the qPentOH to 0.060 g g_CDW⁻¹ h⁻¹ and achieving a maximum 1-pentanol titer of 1.13 g L⁻¹ with an unprecedented carbon selectivity of 38%.

These results demonstrate that it is possible to leverage C. carboxidivorans as a versatile platform for the biohydrogenation of organic acids to alcohols, expanding the potential of gas fermentation as a sustainable route to high-value odd-chain alcohols.

[1] Antonicelli et al., Bioresour. Technol., 2023

ABSTRACT. The development of efficient and economically viable processes for biobutanol production remains a major challenge for industrial acetone–butanol–ethanol (ABE) fermentation. One of the main limitations of conventional clostridial fermentations is the strict requirement for anaerobic conditions, which typically requires nitrogen purging and chemical reducing agents, increasing operational complexity and cost. In this study, a microbial co-culture composed of Bacillus subtilis and Clostridium beijerinckii BA101 was investigated as a biological strategy to facilitate anaerobic conditions through oxygen scavenging while improving solvent production.

Growth kinetics of both microorganisms were first characterized in monoculture using P2 medium, obtaining maximum specific growth rates of 0.207 h⁻¹ for Bacillus subtilis and 0.093 h⁻¹ for Clostridium beijerinckii. Co-culture fermentations were then evaluated under different inoculation ratios (70:30, 50:50, and 30:70), inoculation delays (0, 4, and 8 h), and inoculum concentrations (10 and 20%). The best performance was achieved at a 30:70 inoculum ratio with a 4 h inoculation delay, resulting in a butanol concentration of 13.14 g L⁻¹ at flask scale, representing a 26.6% increase compared with monoculture fermentation. To understand the dynamic interactions within the microbial consortium, a structured kinetic model was developed based on a system of ordinary differential equations describing microbial growth, substrate consumption, dissolved oxygen dynamics, and solvent production. The formulation incorporates the biphasic metabolism of Clostridium beijerinckii, including the transition from acidogenic to solventogenic cells, as well as oxygen depletion driven by Bacillus subtilis, which establishes the anaerobic conditions required for solventogenesis.

The optimal co-culture configuration was further validated in a 1 L stirred bioreactor operated at 37 °C and pH 5.9. Dissolved oxygen was depleted within 53 min, enabling anaerobic conditions without external reducing agents. The fermentation achieved 18.6 g L⁻¹ butanol with a productivity of 0.194 g L⁻¹ h⁻¹ and a yield of 0.30 g g⁻¹ (92% of the theoretical maximum). Overall, this kinetic framework provides a useful tool for understanding microbial interactions and optimizing co-culture-based ABE fermentation for sustainable biobutanol production.