Improvement of Bacillus subtilis PI agarase production, hydrolysate scavenging capability assessment, and saccharification of algal biomass for green ethanol generation.

The goal of the current work was to optimize the growth parameters needed to manufacture agarase enzyme from a non-marine PI strain of Bacillus subtilis on an agar-based medium. Using Plackett-Burman design (PBD), nine process parameters were evaluated, and agar, peptone, and yeast-extract were identified as the most significant independent factors influencing agarase production with confidence levels more than 90%. To evaluate the optimal concentrations of the indicated process parameters on agarase production, the Box-Behnken design (BBD) was applied. After optimization, B. subtilis strain PI produced 119.8 U/ml of agarase, representing a 1.36-fold increase. In addition the agar hydrolysate fermented products contain the liberated oligosaccharide acts as strong antioxidant which has 62.4% scavenging activity. Also, the agarase yields increased (1141.12, 1350.253, 1684.854 and 1921.863 U/ml) after substitution the agar with algal biomass of Carolina officinalis at different concentrations (2, 5, 10 and 15%), respectively. After completing the saccharification process, the resulted hydrolysate was used to produce ethanol through fermentation with Pichia pastoris yeast strain as an economical method giving yields (6.68317, 7.09748, 7.75648 and 8.22332 mg/ml), that are higher than using yeast extract peptone dextrose (YPD) medium (4.461 mg/ml).

Influence of different stress factors during the elaboration of grape must's pieddecuve on the dynamics of yeast populations during alcoholic fermentation.

The pieddecuve (PdC) technique involves using a portion of grape must to undergo spontaneous fermentation, which is then used to inoculate a larger volume of must. This allows for promoting autochthonous yeasts present in the must, which can respect the typicality of the resulting wine. However, the real impact of this practice on the yeast population has not been properly evaluated. In this study, we examined the effects of sulphur dioxide (SO2), temperature, ethanol supplementation, and time on the dynamics and selection of yeasts during spontaneous fermentation to be used as PdC. The experimentation was conducted in a synthetic medium and sterile must using a multi-species yeast consortium and in un-inoculated natural grape must. Saccharomyces cerevisiae dominated both the PdC and fermentations inoculated with commercial wine yeast, displaying similar population growth regardless of the tested conditions. However, using 40 mg/L of SO2 and 1% (v/v) ethanol during spontaneous fermentation of Muscat of Alexandria must allowed the non-Saccharomyces to be dominant during the first stages, regardless of the temperature tested. These findings suggest that it is possible to apply the studied parameters to modulate the yeast population during spontaneous fermentation while confirming the effectiveness of the PdC methodology in controlling alcoholic fermentation.

Effect of Hanseniaspora vineae and Saccharomyces cerevisiae co-fermentations on aroma compound production in beer.

In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.

[PMA-ddPCR method for detecting high ethanol-producing klebsiella pneumoniae in viable but non-culturable state].

Objective: To establish an absolute quantitative method for high ethanol-producing klebsiella pneumoniae in a viable non-culturable (VBNC) state. Methods: High ethanol-producing Klebsiella pneumonia was induced to enter the VBNC state and then the ethanol production was evaluated. A PMA-ddPCR method was established to count the copies of live cell genes in the VBNC state of high ethanol-producing Klebsiella pneumoniae using single-copy genes. Further, the sensitivity and adaptability of ddPCR for detecting low-concentration samples were evaluated in VBNC fecal simulation. Results: The lower detection limit of ddPCR for quantitative analysis of high ethanol-producing Klebsiella pneumoniae gradient diluent was 10 times that of qPCR. At low temperature and low nutritional state, high ethanol-producing Klebsiella pneumoniae entered the VBNC state on the 45th day. The quantitative results of PMA-ddPCR on VBNC state cells were (5.46±0.05) log10 DNA copies/ml. The ethanol production in the VBNC state was<2.2 mmol/L and the ability to produce ethanol was restored after recovery. The minimum detection limit for ddPCR in fecal simulated samples with VBNC state was 3.2 log10 DNA copies/ml. Conclusion: The ddPCR detection method for high ethanol-producing Klebsiella pneumoniae with VBNC state has good sensitivity and adaptability, and can be used for the detection of VBNC state cells in clinical samples.

Engineered Chlorella vulgaris improves bioethanol production and promises prebiotic application.

Microalgal biomass for biofuel production, integration into functional food, and feed supplementation has generated substantial interest worldwide due to its high growth rate, non-competitiveness for agronomic land, ease of cultivation in containments, and presence of several bioactive molecules. In this study, genetic engineering tools were employed to develop transgenic lines of freshwater microalga Chlorella vulgaris with a higher starch content, by up-regulating ADP-glucose pyrophosphorylase (AGPase), which is a rate-limiting enzyme in starch biosynthesis. Expression of the Escherichia coli glgC (AGPase homolog) gene in C. vulgaris led to an increase in total carbohydrate content up to 45.1% (dry cell weight, DCW) in the transgenic line as compared to 34.2% (DCW) in the untransformed control. The starch content improved up to 16% (DCW) in the transgenic alga compared to 10% (DCW) in the control. However, the content of total lipid, carotenoid, and chlorophyll decreased differentially in the transgenic lines. The carbohydrate-rich biomass from the transgenic algal line was used to produce bioethanol via yeast fermentation, which resulted in a higher ethanol yield of 82.82 mg/L as compared to 54.41 mg/L from the untransformed control. The in vitro digestibility of the transgenic algal starch revealed a resistant starch content of up to 7% of total starch. Faster growth of four probiotic bacterial species along with a lowering of the pH of the growth medium indicated transgenic alga to exert a positive prebiotic effect. Taken together, the study documents the utilization of genetically engineered C. vulgaris with enriched carbohydrates as bioethanol feedstock and functional food ingredients.

Deletions of conserved extracytoplasmic function sigma factors-encoding genes in Streptomyces have a major impact on secondary metabolism.

BACKGROUND: Ethanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function. RESULTS: To establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism. CONCLUSIONS: These findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Analysis of alcohol-metabolizing enzymes genetic variants and RAR/RXR expression in patients diagnosed with fetal alcohol syndrome: a case-control study.

Understanding the mechanisms underlying alcohol metabolism and its regulation, including the effect of polymorphisms in alcohol-metabolizing enzymes, is crucial for research on Fetal Alcohol Spectrum Disorders. The aim of this study was to identify specific single nucleotide polymorphisms in key alcohol-metabolizing enzymes in a cohort of 71 children, including children with fetal alcohol syndrome, children prenatally exposed to ethanol but without fetal alcohol spectrum disorder, and controls. We hypothesized that certain genetic variants related to alcohol metabolism may be fixed in these populations, giving them a particular alcohol metabolism profile. In addition, the difference in certain isoforms of these enzymes determines their affinity for alcohol, which also affects the metabolism of retinoic acid, which is key to the proper development of the central nervous system. Our results showed that children prenatally exposed to ethanol without fetal alcohol spectrum disorder traits had a higher frequency of the ADH1B*3 and ADH1C*1 alleles, which are associated with increased alcohol metabolism and therefore a protective factor against circulating alcohol in the fetus after maternal drinking, compared to FAS children who had an allele with a lower affinity for alcohol. This study also revealed the presence of an ADH4 variant in the FAS population that binds weakly to the teratogen, allowing increased circulation of the toxic agent and direct induction of developmental abnormalities in the fetus. However, both groups showed dysregulation in the expression of genes related to the retinoic acid pathway, such as retinoic acid receptor and retinoid X receptor, which are involved in the development, regeneration, and maintenance of the nervous system. These findings highlight the importance of understanding the interplay between alcohol metabolism, the retinoic acid pathway and genetic factors in the development of fetal alcohol syndrome.

Experimental and numerical investigation of microdialysis probes for ethanol metabolism studies.

Microdialysis is an important technique for in vivo sampling of tissue's biochemical composition. Understanding the factors that affect the performance of the microdialysis probes and developing methods for sample analysis are crucial for obtaining reliable results. In this work, we used experimental and numerical procedures to study the performance of microdialysis probes having different configurations, membrane materials and dimensions. For alcohol research, it is important to understand the dynamics of ethanol metabolism, particularly in the brain and in other organs, and to simultaneously measure the concentrations of ethanol and its metabolites - acetaldehyde and acetate. Our work provides a comprehensive characterization of three microdialysis probes, in terms of recovery rates and backpressure, allowing for interpretation and optimization of experimental procedures. In vivo experiments were performed to measure the time course concentration of ethanol, acetaldehyde, and acetate in the rat brain dialysate. Additionally, the combination of in vitro experimental results with numerical simulations enabled us to calculate diffusion coefficients of molecules in the microdialysis membranes and study the extent of the depletion effect caused by continuous microdialysis sampling, thus providing additional insights for probe selection and data interpretation.

Effect of different inoculation strategies of mixed culture Saccharomyces cerevisiae/Oenococcus oeni on the aroma quality of Chardonnay wine.

There has been growing interest in the use of mixed cultures comprised of Oenococcus oeni and Saccharomyces cerevisiae to produce wine with local style and typicality. This study has investigated the influence of the inoculation protocol of O. oeni on the fermentation kinetics and aromatic profile of Chardonnay wine. The one selected autochthonous O. oeni strain (ZX-1) inoculated at different stages of the alcoholic fermentation process successfully completed malolactic fermentation (MLF). Co-inoculum of S. cerevisiae and O. oeni enabled simultaneous alcoholic fermentation and MLF, leading to at least a 30 % reduction in the total fermentation time when compared to the sequential inoculation process, which was attributed to the lower ethanol stress. Meanwhile, co-inoculum stimulated the accumulation of volatile aroma compounds in Chardonnay wine. In particular, the mixed modality where the O. oeni strain ZX-1 was inoculated 48 h after S. cerevisiae allowed higher levels of terpenes, acetates, short-chain, and medium-chain fatty acid ethyl esters to be produced, which may result in the enhanced floral and fruity attributes of wine. Aroma reconstitution and omission models analysis revealed that the accumulation of linalool, geraniol, isoamyl acetate, ethyl hexanoate, and ethyl caprylate during the mixed fermentation process enhanced the stone fruit, tropical fruit, and citrus aromas in Chardonnay wine. Therefore, the simultaneous fermentation of S. cerevisiae and autochthonous O. oeni ZX-1 has a positive effect on MLF and contributes to producing wines with distinctive style.

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance.

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Wild Patagonian yeast improve the evolutionary potential of novel interspecific hybrid strains for lager brewing.

Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options.

Gaining insights into the responses of individual yeast cells to ethanol fermentation using Raman tweezers and chemometrics.

Saccharomyces cerevisiae is the most common microbe used for the industrial production of bioethanol, and it encounters various stresses that inhibit cell growth and metabolism during fermentation. However, little is currently known about the physiological changes that occur in individual yeast cells during ethanol fermentation. Therefore, in this work, Raman spectroscopy and chemometric techniques were employed to monitor the metabolic changes of individual yeast cells at distinct stages during high gravity ethanol fermentation. Raman tweezers was used to acquire the Raman spectra of individual yeast cells. Multivariate curve resolution-alternating least squares (MCR-ALS) and principal component analysis were employed to analyze the Raman spectra dataset. MCR-ALS extracted the spectra of proteins, phospholipids, and triacylglycerols and their relative contents in individual cells. Changes in intracellular biomolecules showed that yeast cells undergo three distinct physiological stages during fermentation. In addition, heterogeneity among yeast cells significantly increased in the late fermentation period, and different yeast cells may respond to ethanol stress via different mechanisms. Our findings suggest that the combination of Raman tweezers and chemometrics approaches allows for characterizing the dynamics of molecular components within individual cells. This approach can serve as a valuable tool in investigating the resistance mechanism and metabolic heterogeneity of yeast cells during ethanol fermentation.

Short-term adaptation as a tool to improve bioethanol production using grass press-juice as fermentation medium.

Grass raw materials collected from grasslands cover more than 30% of Europe's agricultural area. They are considered very attractive for the production of different biochemicals and biofuels due to their high availability and renewability. In this study, a perennial ryegrass (Lolium perenne) was exploited for second-generation bioethanol production. Grass press-cake and grass press-juice were separated using mechanical pretreatment, and the obtained juice was used as a fermentation medium. In this work, Saccharomyces cerevisiae was utilized for bioethanol production using the grass press-juice as the sole fermentation medium. The yeast was able to release about 11 g/L of ethanol in 72 h, with a total production yield of 0.38 ± 0.2 gEthanol/gsugars. It was assessed to improve the fermentation ability of Saccharomyces cerevisiae by using the short-term adaptation. For this purpose, the yeast was initially propagated in increasing the concentration of press-juice. Then, the yeast cells were re-cultivated in 100%(v/v) fresh juice to verify if it had improved the fermentation efficiency. The fructose conversion increased from 79 to 90%, and the ethanol titers reached 18 g/L resulting in a final yield of 0.50 ± 0.06 gEthanol/gsugars with a volumetric productivity of 0.44 ± 0.00 g/Lh. The overall results proved that short-term adaptation was successfully used to improve bioethanol production with S. cerevisiae using grass press-juice as fermentation medium. KEY POINTS: • Mechanical pretreatment of grass raw materials • Production of bioethanol using grass press-juice as fermentation medium • Short-term adaptation as a tool to improve the bioethanol production.

Transcriptomic-metabolomic analysis reveals the effect of copper toxicity on fermentation properties in Saccharomyces cerevisiae.

Copper is one of the unavoidable heavy metals in wine production. In this study, the effects on fermentation performance and physiological metabolism of Saccharomyces cerevisiae under copper stress were investigated. EC1118 was the most copper-resistant among the six strains. The ethanol accumulation of EC1118 was 26.16-20 mg/L Cu2+, which was 1.90-3.15 times higher than that of other strains. The fermentation rate was significantly reduced by copper, and the inhibition was relieved after 4-10 days of adjustment. Metabolomic-transcriptomic analysis revealed that amino acid and nucleotide had the highest number of downregulated and upregulated differentially expressed metabolites, respectively. The metabolism of fructose and mannose was quickly affected, which then triggered the metabolism of galactose in copper stress. Pathways such as oxidative and organic acid metabolic processes were significantly affected in the early time, resulting in a significant decrease in the amount of carboxylic acids. The pathways related to protein synthesis and metabolism under copper stress, such as translation and peptide biosynthetic process, was also significantly affected. In conclusion, this study analyzed the metabolite-gene interaction network and molecular response during the alcohol fermentation of S. cerevisiae under copper stress, providing theoretical basis for addressing the influence of copper stress in wine production.

Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792.

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying acetone-butanol-ethanol fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived the interest in research on this bacterium. The recent development of efficient genetic tools allows to better understand the physiology of this micro-organism, aiming at improving its fermentation capacities. Knowledge about gene essentiality would guide the future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing method to generate large mutant libraries containing millions of independent mutants that allowed us to identify a core group of 418 essential genes needed for in vitro development. Future research on this significant biocatalyst will be guided by the data provided in this work, which will serve as a valuable resource for the community. IMPORTANCE: Clostridium acetobutylicum is a leading candidate to synthesize valuable compounds like three and four carbons alcohols. Its ability to convert carbohydrates into a mixture of acetone, butanol, and ethanol as well as other chemicals of interest upon genetic engineering makes it an advantageous organism for the valorization of lignocellulose-derived sugar mixtures. Since, genetic optimization depends on the fundamental insights supplied by accurate gene function assignment, gene essentiality analysis is of great interest as it can shed light on the function of many genes whose functions are still to be confirmed. The data obtained in this study will be of great value for the research community aiming to develop C. acetobutylicum as a platform organism for the production of chemicals of interest.

Continuous chain elongation process for carbon resource recovery from excess sludge: Enhanced n-caprylate production and specific microbial functionalities.

Thermal hydrolyzed sludge (THS) exhibits considerable promise in generating medium-chain fatty acids (MCFAs) through chain elongation (CE) technology. This study developed a novel continuous CE process using THS as the substrate, achieving an optimal ethanol loading rate (5.8 g COD/L/d) and stable MCFA production at 10.9 g COD/L, with a rate of 3.6 g COD/L/d. The MCFAs primarily comprised n-caproate and n-caprylate, representing 41.5 % and 54.3 % of the total MCFAs, respectively. Utilization efficiencies for ethanol and acetate were nearly complete at 100 % and 92.8 %, respectively. Key microbial taxa identified under these optimal conditions included Alcaligenes, SRB2, Sporanaerobacter, and Kurthia, which were instrumental in critical pathways such as the generation of acetyl-CoA, the initial carboxylation of acetyl-CoA, the fatty acid biosynthesis cycle, and energy metabolism. This research provides a theoretical and technical blueprint for converting waste sludge into valuable MCFAs, promoting sustainable waste-to-resource strategies.

Upcycling CO2 into succinic acid via electrochemical and engineered Escherichia coli.

Converting CO2 into value-added chemicals still remains a grand challenge. Succinic acid has long been considered as one of the top building block chemicals. This study reported efficiently upcycling CO2 into succinic acid by combining between electrochemical and engineered Escherichia coli. In this process, the Cu-organic framework catalyst was synthesized for electrocatalytic CO2-to-ethanol conversion with high Faradaic efficiency (FE, 84.7 %) and relative purity (RP, 95 wt%). Subsequently, an engineered E. coli with efficiently assimilating CO2-derived ethanol to produce succinic acid was constructed by combining computational design and metabolic engineering, and the succinic acid titer reached 53.8 mM with the yield of 0.41 mol/mol, which is 82 % of the theoretical yield. This study effort to link the two processes of efficient ethanol synthesis by electrocatalytic CO2 and succinic acid production from CO2-derived ethanol, paving a way for the production of succinic acid and other value-added chemicals by converting CO2 into ethanol.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.