ABSTRACT. Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of cellulosic biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to break down complex polysaccharides, notably cellulose, found in plant cell walls. The attachment of bacterial cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth. Here, we utilized both super resolution imaging and machine learning approaches to study the distribution of C. thermocellum cellulosomes at different stages of growth. We show that C. thermocellum initially retains its cellulosomes primarily on the cell surface but then relocates large cellulosome clusters to the interface with biomass therefore depleting its cell surface of cellulosomes. These results indicate dynamic redistribution of cellulosomes during growth, with a functional shift toward substrate-associated degradation later during growth on biomass.

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. Clostridium kluyveri is a microorganism capable of biosynthesise medium chain fatty acids (MCFA), such as butyrate and caproate by the r-βOx pathway, concurrently releasing hydrogen. This study investigated the impact of initial ethanol and acetate concentrations and ethanol-to-acetate (E:A) molar ratios on batch culture performance. A central composite design was used to comprehensively analyse ethanol and acetate concentration influence on product distribution and hydrogen generation. At a constant ethanol to-acetate (E:A) ratio, increasing initial ethanol concentrations reduces the caproate-to-butyrate ratio, which contrasts with the general observation that higher E:A ratios lead to a greater proportion of carbon directed toward caproate. The highest specific productivity for both butyrate and caproate was observed at an initial ethanol concentration of 340 mM. Hydrogen production demonstrated a balance between carboxylate elongation and biomass formation, with maximum specific hydrogen productivity achieved at initial concentrations of 340 mM ethanol and 170 mM acetate. The C. kluyveri strain in this study exhibited a specific hydrogen productivity of 9.56 mmol H2·gCDW−1·h−1, a rate that exceeds those reported for other Clostridium species. This work provides insights into tailoring initial substrate conditions for targeted product formation in C. kluyveri batch fermentations.

ABSTRACT. An overview of research relevant to consolidated bioprocessing (CBP) will be presented encompassing selected work from the institutions listed above. Topics addressed include: • Ability of Clostridium thermocellum and other thermophilic anaerobes to deconstruct lignocellulose without added enzymes or thermochemical pretreatment; • Metabolic engineering of thermophilic cocultures to achieve commercially-feasible ethanol yields and titers from model cellulose-xylose mixtures; • Physiological insights, including atypical glycolysis, in situ pathway thermodynamics, and protein cost; • Genetic tool development; • Terragia, a start-up devoted to commercializing CBP using engineered thermophiles which received venture investment in March 2024.

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 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.