Enhancing bioethanol production using sodium microcrystalline cellulose sulfate (Na-MCS) encapsulated Saccharomyces cerevisiae in the presence of aldehyde and acid inhibitors

Enhancing bioethanol production using sodium microcrystalline cellulose sulfate (Na-MCS) encapsulated Saccharomyces cerevisiae in the presence of aldehyde and acid inhibitors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing bioethanol production using sodium microcrystalline cellulose sulfate (Na-MCS) encapsulated Saccharomyces cerevisiae in the presence of aldehyde and acid inhibitors Sri Peni Wijayanti, Muhammad Ma’ruf This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8133957/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Developing renewable energy sources, such as bioethanol, is crucial for mitigating greenhouse gas emissions. However, inhibitors such as aldehydes and organic acids generated during biomass pretreatment significantly hinder fermentation performance. Saccharomyces cerevisiae , the most widely used yeast in industrial bioethanol production, is particularly sensitive to these inhibitory compounds. While genetic engineering can enhance yeast tolerance, it often involves complex and time-consuming processes. As an alternative, encapsulation of S. cerevisiae within a partially permeable membrane offers improved stability and protection under stressful fermentation conditions. This study investigates the encapsulation of S. cerevisiae using sodium microcrystalline cellulose sulfate (Na-MCS) combined with a synthetic polycation to form a stable membrane matrix. The results demonstrate that encapsulated S. cerevisiae cells exhibit significantly higher glucose consumption and ethanol productivity compared to free cells when exposed to aldehydes and acids. Encapsulation effectively enhances yeast tolerance to inhibitors such as furfural, vanillin, and various organic acids, resulting in improved fermentation efficiency. Overall, Na-MCS encapsulation presents a promising strategy for increasing the robustness and productivity of S. cerevisiae in bioethanol fermentation under inhibitory conditions. bioethanol production yeast encapsulation sodium microcrystalline cellulose sulfate inhibitor tolerance Saccharomyces cerevisiae fermentation efficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Bioethanol has emerged as a viable alternative to fossil fuels due to its renewable nature and ability to reduce greenhouse gas emissions [ 1 , 2 ]. However, inhibitors such as aldehydes and acids, which arise during biomass pretreatment, reduce bioethanol production efficiency, decreasing ethanol yields and increasing costs [ 3 ]. Saccharomyces cerevisiae , a common yeast used in bioethanol production, is particularly vulnerable to these inhibitors. Strategies to enhance S. cerevisiae resistance, such as genetic modification and adaptive evolution, are often time-consuming and complex [ 4 ]. An alternative approach involves encapsulating yeast cells to protect them and increase their resilience under harsh conditions [ 5 ]. Encapsulation involves surrounding yeast cells with a partially permeable membrane that allows substance exchange while shielding the cells from harmful molecules [ 6 ]. This technology improves yeast stability, vitality, and reuse in multiple fermentation cycles, enhancing efficiency and cost-effectiveness [ 7 ]. Natural or synthetic polymers such as calcium alginate are generally used for encapsulation. This research, however, focuses on sodium microcrystalline cellulose sulfate (Na-MCS), a unique linear polymer combined with alg-chit. In Na-MCS, the cellulose sulfate (CS) chain is an analog of polysaccharide sulfates, with hydroxyl groups replaced by sulfate groups at positions 2, 3, and 6 of the anhydroglucose unit (AGU). Combining cellulose sulfate with a synthetic polycation forms stable polyelectrolyte complex membranes. Certain organic acids, such as formic acid, levulinic acid, and acetic acid, which are typically found in hydrolysates obtained from lignocellulosic materials, can have a major impact on bioethanol synthesis. These acids can act as inhibitors during the fermentation process, affecting the efficiency of bioethanol production [ 8 , 9 ]. Formic acid is recognized to be a key inhibitory component present in hydrolysates, preventing the conversion of sugars into bioethanol [ 8 , 10 ]. Weak acids, such as formic and acetic acid, are powerful yeast inhibitors during bioethanol synthesis [ 9 ]. Formic acid, when combined with other inhibitors such as acetic acid, can reduce ethanol yield and cell survival during bioethanol synthesis [ 11 ]. Formic acid can effectively penetrate cellulose molecules, disrupting their crystalline structure and facilitating hydrolysis, which is essential for bioethanol production [ 12 ]. The presence of fermentation inhibitors like furfural and vanillin is one of the main problems in producing bioethanol from lignocellulosic biomass. These compounds can significantly hinder yeast growth and fermentation efficiency. The use of encapsulated yeast offers a promising solution to this problem. Encapsulation has been shown to increase yeast resistance to fermentation inhibitors, allowing for a more robust fermentation process [ 13 ]. Encapsulated yeast strains exhibit enhanced sugar co-utilization, particularly in consuming xylose, which is crucial for efficient bioethanol production from lignocellulosic biomass [ 14 ]. Previous research used Na-MCS to evaluate its impact on bioethanol production from lignocellulosic hydrolysates [ 15 ]. This investigation aims to examine the effect of Na-MCS in media containing only aldehydes and acids as inhibitors, providing a deeper understanding of these inhibitors' impact on bioethanol production. Materials and Methods Materials The chemicals used in this research include Na-MCS, Tween 20, CaCl 2 , carboxymethylcellulose (CMC), chitosan, Na-alginate (molecular weight 250,000 g/mol), acetate buffer, (NH 4 ) 2 SO 4 , KH 2 PO 4 , MgSO 4 ·7H 2 O, EDTA, CaCl 2 ·6H 2 O, Na 2 MoO 4 ·2H 2 O, CoCl 2 ·2H 2 O, CuSO 4 ·5H 2 O, KI, d-biotin, p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine·HCl, thiamine·HCl, m-inositol, antifoam, ergosterol, and Tween 80, all of which were purchased from Sigma-Aldrich. The Na-MCS utilized in this study was developed based on empirical investigations conducted in prior studies [15]. The yeast Saccharomyces cerevisiae CBS 8066 was obtained from Centraalbureau Voor Schimmelcultures (Delft, Netherlands). All other compounds and solvents were analytical grade. Methods Preparation of media and culture stock Yeast culture stocks were grown in agar media made from 10 g/L yeast extract, 20 g/L soy peptone, and 20 g/L agar with 20 g/L D-glucose added as a carbon source. The synthetic media was prepared by mixing several components, including carbon sources, trace metals, vitamins, antifoam, ergosterol, and Tween 20. The trace metal solution was composed of 7.5 g (NH 4 ) 2 SO 4 , 3.5 g KH 2 PO 4 , 0.75 g MgSO 4 ·7H 2 O, 30 mg EDTA, 13 mg CaCl 2 ·6H 2 O, 0.8 mg Na 2 MoO 4 ·2H 2 O, 0.6 mg CoCl 2 ·2H 2 O, 0.6 mg CuSO 4 ·5H 2 O, and 0.2 mg KI. The vitamin solution included 50 µg d-biotin, 0.2 mg p-aminobenzoic acid, 1.0 mg nicotinic acid, 1.0 mg calcium pantothenate, 1.0 mg pyridoxine hydrochloride, 1.0 mg thiamine hydrochloride, and 25 mg m-inositol. Both glucose and trace metals were sterilized separately using an autoclave, while the vitamin solution was sterilized using a sterile filter. Ergosterol and Tween 80 were not sterilized but were dissolved in ethanol and evaporated for 10 minutes before use. The sterile solutions of glucose, trace metals, and vitamins were stored at 4°C, whereas the mixed solution of ergosterol and Tween 80 was stored at -20°C. Preparation solution for encapsulation The encapsulation process was carried out to form alg-chit-microcrystalline cellulose sulfate (alg-chit-MCS) and alg-chit (alg-chit) capsules. The encapsulation solution was prepared in three polymer solutions with different compositions. To make chitosan-microcrystalline cellulose sulfate alginate capsules, Solution I was made from a mixture of Na-alginate, NaMCS, and Tween 20 dissolved in distilled water. Meanwhile, to make chitosan alginate capsules, Solution I was made from a mixture of Na-alginate and Tween 20 dissolved in distilled water. Solution II consists of a mixture of carboxymethylcellulose (CMC) and CaCl 2 dissolved in distilled water. Solution III was prepared by dissolving chitosan and CaCl 2 in a 0.04 M acetate buffer with different pH levels. All three types of solutions were sterilized using an autoclave at 121°C for 20 minutes. Preparation of alg-chit and alg-chit-MCS capsules Capsule production was carried out using the method like in Figure 1. Solution II, containing yeast, was dropped into Solution I for 10 minutes while being stirred with a magnetic stirrer. The capsules were rinsed with distilled water after 10 minutes and then hardened in a 1.3% CaCl 2 solution for 20 minutes. The capsules were then rinsed once more with distilled water. Next, the capsules were soaked in Solution III at a ratio of 1:5 and placed in a shaker bath at 30°C with a speed of 130 rpm for 24 hours. The coated capsules were washed with 0.9 M NaCl solution to remove excess chitosan. Capsules containing yeast were transferred into 200 mL of synthetic media for aerobic propagation in a synthetic growth medium containing 50 g/L glucose. The material composition of the capsules' membrane varies significantly across different types of capsules. For instance, the A1 capsule, classified as alg-chit, comprises 0.60% alginate, 1.3% CaCl 2 , and 0.2% chitosan with a pH buffer acetate level of 5. In comparison, the A2 capsule, which is an alg-chit-MCS blend, includes 0.54% alginate, 0.06% MCS, 1.3% CaCl 2 , and 0.2% chitosan, also buffered at pH 5. Variations in CaCl 2 concentration and pH are observed across other capsules. For example, A3 and A4 maintain similar compositions to A1 and A2, respectively, but differ in their CaCl 2 concentrations, using only 25 mM compared to the standard 300 mM. Meanwhile, capsules A5 and A6, also alg-chit and alg-chit-MCS respectively, utilize 50 mM CaCl 2 with a slightly lower pH buffer of 4.5. Lastly, A8 is composed of suspended cells without specific percentages for alginate or MCS, though it includes 1.3% CaCl 2 and maintains a pH buffer of 5 [15]. Anaerobic fermentation Five milliliters of propagated yeast capsules (425 ± 25 capsules) were added to 50 milliliters of synthetic media. The 100-milliliter conical flasks containing capsule cells and synthetic media were incubated in a shaker incubator at 30°C with a shaking speed of 130 rpm. Fermentation was conducted in three different synthetic media: defined media, synthetic media containing aldehydes with 3 g/L furfural and 0.2 g/L vanillin; and synthetic media containing acids with 2.5 g/L formic acid, 2.5 g/L levulinic acid, and 10 g/L acetic acid. Throughout the tests, samples were removed from the flask, centrifuged, and kept at -20°C. HPLC The eluent was ultrapure water flowing at a rate of 0.5 mL/min. The Aminex HPX-87H column (Bio-Rad) was used at 60°C with 5 mM H2SO4 as the eluent and a flow rate of 0.5 mL/min to quantify glucose, ethanol, glycerol, vanillin, and furfural. The quantities of furfural and vanillin were assessed using UV chromatograms at 210 nm, whereas the remaining metabolites were determined using RI chromatograms. The glucose consumption rate (r S ), ethanol production rate (r E ), and ethanol yield (Y SE ) were also calculated [15]. Result and Discussions Fermentation in defined media Anaerobic cultivation of encapsulated and free-cell S. cerevisiae in defined growth media was characterized and compared. The most important results are presented in Figure 2 and 3 . Glucose was used as carbon and energy source throughout these experiments. The initial cell mass was provided aerobically by cultivation for 24 h in shake flasks. The experiments with the encapsulated cells contained 425 ± 25 capsules in a total volume of ca. 55 mL, and two batches were carried out per experiments. The CO 2 bubbles formed during the fermentation diffused easily through the capsules membrane to the media and left the flasks via the loop-trap. Since anaerobic condition was maintained in all the batches, the influence of mass transfer of oxygen on the growth of cells is negligible. According to Figure 2 and 3, the ethanol production, glucose consumption rate and yield ethanol for free cells was lower than encapsulated cells in 6 hours cultivation. Free cells take up the sugars at 2.91 g/l.h and produce ethanol at 0.50 g/l.h and encapsulated cells take up the sugars approximately two times faster than the free cells. There is relationship between the amounts of ethanol production with availability of cells. The high biomass inside capsule membrane will produce high concentration of ethanol. From the experiment we observed the ethanol production from encapsulated cells in 6 hours cultivation was high which has correlated to high cells concentration inside the capsules. The viability cells inside the capsule were more prevented by membrane layer both of alginate-chitosan (alg-chit) and alginate-chitosan-MCS (alg-chit-MCS) than free cells. The different composition of membrane capsules influenced the ethanol production rate but not the glucose consumption rate. Chitosan solution with 300 mM CaCl 2 in pH 5 produced 1.73 g/l ethanol per hours in 6 hours cultivation by yeast inside alg-chit capsules and 1.68 g/l.h glucose by yeast inside alg-chit-MCS. Reducing the CaCl 2 concentration from 300 mM to 25 mM in pH 5 increased productivity of ethanol in 6 hours cultivation, 2.6 g/l.h (alg-chit) and 2.25 g/l.h (alg-chit-MSC). pH 5 was adjusted in chitosan solution to turn the NH 2 to NH 3 + . On the other hand, the presence of Cl - in the chitosan solution will increase the counterbalancing between positive charge (NH 3 + ) and negative charge (Cl - ). A high concentration of CaCl 2 on chitosan solution would reduce electrostatic interaction between the protonated amino groups to carboxylic groups on the alginate. Thus decrease the chemical and mechanical stability of alg-chit and alg-chit-MCS, therefore directly impacting the ethanol production rate. The purpose of adding Na-MCS to alginate solution was to increase the mechanical and chemical stability of alg-chit capsule membrane. In this experiment, it was observed that adding Na-MCS to alg-chit reduced ethanol production rate. The negative charge in alginate-MCS solution should be present from carboxylate group (-COO) and sulfate group ( –SO 3 - ). Since the DS of Na-MCS was low, the amount of sulfate groups in the alginate-MCS solution was low. Because of this reason the ethanol production of alg-chit-MCS (2.25 g/l.h) was lower than algi-chit (2.60 g/l.h) in pH 5 of acetate buffer solution. Nevertheless by adding Na-MCS increased the ethanol productivity in pH 4.5. The idea to reduce the pH of acetate buffer in the chitosan solution was to incresae the electrostatic interaction by the presence higher amount of the protonated amino groups than at pH 5. Therefore the electrostatic interaction of protonated amino groups with negative charged (carboxylic and sulfate groups) would increase, eventhough the amount of negative charge was lower than in the alginate solution without Na-MCS. Figure 4 shows the changes in glucose, ethanol, and glycerol concentrations during anaerobic fermentation of Saccharomyces cerevisiae in defined media using both free and encapsulated cells. In all treatments, glucose concentration sharply decreased within the first 6 hours, indicating the active fermentation phase. Free cells (A8) exhibited the fastest glucose consumption, with nearly complete substrate depletion within 12 hours, whereas encapsulated cells in Alg-Chit (A1, A3, A5) and Alg-Chit-MCS (A2, A4, A6) matrices showed a slower decrease, likely due to substrate diffusion limitations through the capsule wall. Ethanol concentration increased concomitantly with glucose depletion, reaching its maximum level after 12–18 hours of incubation. Free cells produced slightly higher final ethanol concentrations than encapsulated cells but exhibited less stability in the later fermentation phase. In contrast, encapsulated cells—particularly those in the Alg-Chit-MCS system—displayed a more stable and consistent ethanol accumulation profile, suggesting that the encapsulation matrix could maintain cellular metabolic activity for a longer period under anaerobic conditions. Glycerol production remained relatively low and constant after the first 6 hours across all treatments, confirming its role as a minor metabolite involved in maintaining the cellular redox balance during anaerobic metabolism. Compared with the Alg-Chit system, the addition of MCS to form Alg-Chit-MCS appeared to improve metabolic stability without significantly reducing fermentative capability. The incorporation of inert materials such as silica or carbon microspheres is known to enhance the mechanical strength and permeability of polymer matrices, improving mass transfer and cell resistance against ethanol-induced stress. These results indicate that S. cerevisiae encapsulated within the Alg-Chit-MCS system exhibits improved tolerance to environmental stress during anaerobic fermentation, including ethanol accumulation and nutrient limitation. The protective effect provided by the polymeric matrix plays a key role in maintaining cell viability and enzymatic activity, suggesting that this encapsulation approach can be a promising alternative to enhance the efficiency and stability of industrial-scale bioethanol fermentation. Fermentation in aldehyde media The fermentation performance of free and encapsulated S. cerevisiae cells in aldehyde media, depicted in Figures 5 and 6, highlights the efficiency of encapsulated cells. Key parameters such as glucose consumption rate (r S ), ethanol production rate (r E ), and ethanol yields (Y SE ) were measured over 6 and 24 hours of fermentation. Encapsulated cells demonstrated significantly higher glucose consumption rates compared to free cells. Capsules A1, A2, A3, A4, A5, and A6 exhibited r S values ranging from 4.10 to 6.24 g/L·h, whereas free cells (A8) showed a rate of just 3.66 g/ L·h. Similarly, ethanol production rates were remarkably higher in encapsulated cells, with values between 1.45 and 2.65 g/L·h, compared to a mere 0.16 g/L·h in free cells. Ethanol yield over 6 hours was higher for encapsulated cells (0.34 to 0.51 g/g) compared to free cells (0.04 g/g). Over 24 hours, encapsulated cells showed yields ranging from 0.38 to 0.43 g/g, unequivocally demonstrating that encapsulation enhances ethanol production efficiency. Encapsulated cells in an aldehyde medium exhibited markedly higher glucose consumption and ethanol production rates than free cells (Figure 3). Different encapsulation compositions influence ethanol production rates and glucose consumption rates. Reducing the CaCl 2 concentration from 300 to 25 mM at pH 5 in chitosan solution significantly increased the ethanol production rate from 1.54 to 2.53 g/L·h. The ethanol productivity of alg-chit-MCS was higher than alg-chit when coating the alg-MCS with chitosan at pH 4.5 and 50 mM CaCl 2 . Figure 6 illustrates the ethanol yield (Y SE ) from both free and encapsulated yeast cells during fermentation over 6 hours and 24 hours. For free cells (A8), the yield is very low at 6 hours, close to 0 g/g, but increases significantly to approximately 0.35 g/g after 24 hours. In contrast, encapsulated cells (A1 - A6) show generally higher ethanol yields. At 6 hours, the yields for encapsulated cells A1, A2, A4, A5, and A6 are around 0.3 to 0.4 g/g, with A3 achieving the highest yield of approximately 0.5 g/g. After 24 hours, the yields for encapsulated cells remain high, with slight increases or decreases. A3 remains the highest, around 0.45 g/g, while others (A1, A2, A4, A5, A6) range from 0.35 to 0.4 g/g. Encapsulation not only boosts ethanol yield but also that different encapsulation methods can further optimize this process. Extending fermentation time improves yield for both free and encapsulated cells, with encapsulated cells demonstrating a clear advantage throughout. Figure 7 presented the profiles of glucose, ethanol, and glycerol concentrations during anaerobic batch fermentation in aldehyde media, clearly delineating the advantages of encapsulated cells over free cells. Encapsulated cells exhibited a markedly more efficient glucose consumption profile, with glucose levels dropping significantly faster than in free cells. Ethanol production was substantially higher in encapsulated cells, peaking much earlier than in free cells. For instance, capsules A5 and A6 achieved considerable ethanol production within the first 6 hours, which was sustained throughout the fermentation period. Higher glycerol levels in encapsulated cells further suggest a more robust and efficient fermentation process. Figure 8 demonstrated that S. cerevisiae, both free and encapsulated, efficiently metabolizes furfural and vanillin within the first 6 hours of fermentation, reducing their concentrations to near zero, where they remain stable for the next 18 hours. Specifically, furfural is completely reduced by the cells to furfuryl alcohol, while vanillin is bio-converted to vanillyl alcohol by the yeast strains. This indicates that encapsulation methods (alg-chit/A5 and alg-chit-MCS/A6) do not hinder the yeast's ability to process these inhibitory compounds. The rapid and sustained reduction highlights the potential of using S. cerevisiae in industrial fermentation processes where these inhibitors are present, ensuring effective fermentation without the adverse impact of these compounds. Fermentation in acidic media Figures 9 and 10 show a complete comparison of the glucose consumption rate (r S ), ethanol production rate (r E ), and ethanol yields (Y SE ) of free and encapsulated Saccharomyces cerevisiae cells in acidic conditions. The study tested various capsule compositions (A1 to A6) for the encapsulated cells, with free cells represented by A8. The glucose consumption rate (rS) for free cells (A8) is 1.88 g/L.h over a 6-hour fermentation period. In contrast, the encapsulated cells exhibit varying rates, with A1 (alg-chit) and A2 (alg-chit-MCS) showing significantly higher rS values of 4.35 g/L.h and 4.40 g/L.h, respectively. Encapsulation generally enhances glucose consumption, with A6 (alg-chit-MCS) achieving the highest r S of 5.54 g/L.h. This suggests a potentially optimized composition for maximum glucose uptake. The enhancement is likely due to the protective environment provided by the encapsulation, which helps maintain cell viability and metabolic activity in acidic conditions, as supported by previous studies on cell encapsulation technology [6]. The ethanol production rate (r E ) also shows considerable variation. For free cells, r E is relatively low at 0.26 g/L.h. Encapsulated cells, particularly A1 and A2, exhibit much higher ethanol production rates of 1.67 g/L.h and 1.50 g/L.h, respectively. Again, A6 stands out with the highest r E of 2.64 g/L.h, emphasizing the benefits of specific encapsulation compositions in enhancing ethanol production. This finding aligns with research indicating that encapsulation can improve the efficiency of bioconversion processes by stabilizing the cells and enhancing their metabolic functions [16]. Ethanol yields (Y SE ) calculated over 24 hours are more consistent across all samples, including free and encapsulated cells. Free cells show a Y SE of 0.41 g/g, while encapsulated cells range narrowly from 0.42 g/g to 0.44 g/g. This consistency suggests that over longer fermentation periods, the initial advantages of encapsulation may stabilize, leading to more uniform ethanol yields across different compositions. The performance of alg-chit capsules varied with the concentration of calcium chloride used for coating. Capsules with 50 mM CaCl 2 (A5) showed higher ethanol production rates and yields compared to those with 25 mM CaCl 2 (A3). This indicates that higher calcium chloride concentrations in the coating process enhance the structural integrity and performance of the capsules. Encapsulation of yeast cells using different compositions of alginate, chitosan, and MCS significantly improves fermentation performance in acidic media. Encapsulated cells exhibit higher glucose consumption rates, ethanol production rates, and yields than free cells. The protective environment provided by encapsulation materials enhances the viability and metabolic activity of yeast cells, enabling more efficient bioethanol production. The optimal formulation identified in this study involves alg-chit-MCS with 50 mM CaCl 2 , which shows the highest rates of glucose consumption and ethanol production. Cellulose sulfate is emerging as a promising material for encapsulating yeast in bioethanol production. Encapsulation in cellulose sulfate microspheres can enhance the survival of yeast cells in low-pH environments and protect them from the harmful effects of stomach acid and other environmental stresses. The negatively charged sulfate groups in cellulose sulfate buffer the encapsulated cells from acidic conditions, enhancing their viability during the fermentation process. This improved resistance can lead to higher bioethanol production efficiency. Encapsulated yeast in cellulose sulfate has shown promising results in maintaining cell viability and fermentative capacity, contributing to better bioethanol yields from lignocellulosic hydrolysates. Figure 11 shows the ethanol production profiles for free and encapsulated cells over a 24-hour fermentation period. Encapsulated cells produced significantly higher ethanol concentrations compared to free cells. For instance, A6 encapsulated cells reached an ethanol concentration of 20.06 g/L after 24 hours, compared to 19.15 g/L for free cells. This consistent and higher ethanol production aligns with the enhanced metabolic rates observed in encapsulated cells. Glucose consumption was more efficient in encapsulated cells, as indicated by the rapid decline in glucose levels in the media. For example, glucose levels dropped to zero within 15 hours for formulations A3, A4, A5, and A6, whereas free cells (A8) took 24 hours to achieve similar glucose depletion. Glycerol production, a byproduct of yeast metabolism, was also monitored. Encapsulated cells produced slightly higher glycerol concentrations, indicative of the stress response and redox balancing mechanisms in yeast cells during fermentation. Comparative Growth of Saccharomyces cerevisiae in Defined and Inhibitor-Containing Media Figure 12 illustrates the growth profile of suspended Saccharomyces cerevisiae cultivated in three different media: defined, aldehyde-containing, and acid-containing media, as indicated by the optical density (OD) at 610 nm over 72 hours of fermentation. The yeast exhibited the highest growth in the defined medium, where the OD value rapidly increased during the first 20 hours and reached a stable phase around 5.5–6.0, indicating efficient cell proliferation under optimal nutrient conditions. In contrast, cells grown in aldehyde-containing medium showed a slower increase in OD, achieving a maximum value of approximately 4.5 after 24 hours, suggesting partial inhibition of yeast metabolism by aldehydes. Aldehydes such as furfural and hydroxymethylfurfural are known to interfere with glycolytic enzymes and NADH regeneration, thereby reducing the rate of biomass formation [17, 18]. The lowest growth was observed in the acid-containing medium, with OD values stabilizing around 3.5. Organic acids, particularly acetic and formic acids, can diffuse into the cytoplasm and lower the intracellular pH, resulting in energy depletion due to ATP consumption for proton efflux and overall inhibition of cell metabolism [19]. These results clearly demonstrate that the presence of inhibitory compounds such as aldehydes and acids negatively affects the growth of S. cerevisiae compared to the defined control medium. Nevertheless, the yeast maintained measurable growth in both inhibitor-containing media, implying a degree of tolerance and metabolic adaptability to stress conditions. This finding supports the potential for developing resistant or encapsulated yeast systems to enhance fermentation performance in lignocellulosic hydrolysates that typically contain such inhibitory compounds [20]. Conclusion This study demonstrates the significant benefits of encapsulating Saccharomyces cerevisiae using sodium microcrystalline cellulose sulfate (Na-MCS) for bioethanol production in media containing aldehyde and acid inhibitors. Compared to free yeast cells, encapsulated yeast cells showed higher rates of glucose consumption, ethanol production, and ethanol yields. The protective encapsulation shielded the yeast from the toxic effects of inhibitors such as furfural, vanillin, formic acid, levulinic acid, and acetic acid, allowing for more robust fermentation. Among the various formulations tested, capsules with a combination of 0.54% alginate, 0.02% chitosan, 0.06% Na-MCS, 50 mM CaCl 2 , and pH buffer acetate 4.5, particularly at optimized conditions, showed the highest efficiency. These findings point to the possibility of Na-MCS encapsulation to enhance bioethanol production. This makes it an attractive method for industrial applications where fermentation inhibitors are prevalent. Further research should explore scaling up this technology and evaluating its performance in diverse industrial settings. Declarations Acknowledgment The authors would like to express their sincere gratitude to Associate Prof. Carl Johan Franzén and Prof. Mohammad J. Taherzadeh for their valuable supervision, guidance, and support throughout this work. The authors also thank Eva Alberts for providing dilute acid hydrolysates, Rakesh Kophram for assistance with HPLC analysis. Special thanks are extended to Esteban for laboratory assistance in media preparation, and to all members of the Industrial Biotechnology and Systems Biology groups in Chalmers University of Technology for their collaboration. The authors also acknowledge the kind help of Marianne Sognell. Author contributions Sri Peni Wijayanti was responsible for investigation, data curation, manuscript writing, and critical revision. Muhammad Ma’ruf contributed to the preparation of figures and critical revision. All authors reviewed and approved the final version of the manuscript. Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Funding declaration This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors Conflict of interest The authors declare no competing interests References Balat M, Balat H, Öz C (2008) Progress in bioethanol processing. Prog Energy Combust Sci 34:551–573. https://doi.org/10/b627cb Nigam PS, Singh A (2011) Production of liquid biofuels from renewable resources. Prog Energy Combust Sci 37:52–68. https://doi.org/10/cmxcdt Jönsson LJ, Martín C (2016) Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects. Bioresour Technol 199:103–112. https://doi.org/10/f3pcp6 Almeida JR, Modig T, Petersson A, et al (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae . J Chem Technol Biotechnol 82:340–349. https://doi.org/10/fwwfrw Verbelen PJ, De Schutter DP, Delvaux F, et al (2006) Immobilized yeast cell systems for continuous fermentation applications. Biotechnol Lett 28:1515–1525. https://doi.org/10/b7jxgr Nedovic V, Willaert R (2013) Fundamentals of Cell Immobilisation Biotechnology. Springer Science & Business Media Ullah MW, Khattak WA, Ul-Islam M, et al (2015) Encapsulated yeast cell-free system: A strategy for cost-effective and sustainable production of bio-ethanol in consecutive batches. Biotechnol Bioprocess Eng 20:561–575. https://doi.org/10/gt39nx Oshoma CE, Obueh HO (2018) Influence of nitrogen sources on ethanol production by Saccharomyces Spp in the presence of formic acid. Bayero J Pure Appl Sci 11:1–7. https://doi.org/10/gt5dtf Fosso-Kankeu E, Marx S, Meyer A (2015) Simulated Inhibitory Effects of Typical Byproducts of Biomass Pretreatment Process on the Viability of Saccharomyces cerevisiae and Bioethanol Production Yield. Afr J Biotechnol 14:2383–2394. https://doi.org/10/gt5dtg Oshoma CE, Greetham D, Louis EJ, et al (2015) Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation. PLOS ONE 10:e0135626. https://doi.org/10/f726bk de Klerk C, Fosso-Kankeu E, Du Plessis L, Marx S (2018) Assessment of the viability of Saccharomyces cerevisiae in response to synergetic inhibition during bioethanol production. Curr Sci 115:1124–1132. https://doi.org/10/gt5dth Li M-F, Yu P, Li S-X, et al (2017) Sequential two-step fractionation of lignocellulose with formic acid organosolv followed by alkaline hydrogen peroxide under mild conditions to prepare easily saccharified cellulose and value-added lignin. Energy Convers Manag 148:1426–1437. https://doi.org/10/ghhnmd Westman JO, Franzén CJ (2015) Current progress in high cell density yeast bioprocesses for bioethanol production. Biotechnol J 10:1185–1195. https://doi.org/10/f3cwcm Westman JO, Bonander N, Taherzadeh MJ, Franzén CJ (2014) Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules. Biotechnol Biofuels 7:102. https://doi.org/10/f23nc8 Wijayanti SP (2024) Evaluation of an alginate-chitosan-microcrystalline cellulose sulfate encapsulation system for efficient fermentation of lignocellulosic hydrolyzate. Bogor, Indonesia, p 040003 Divyashri G, Tulsi NP, Murthy TPK, et al (2024) Valorization of coffee bean processing waste for bioethanol production: comparison and evaluation of mass transfer effects in fermentations using free and encapsulated cells of Saccharomyces cerevisiae . Bioprocess Biosyst Eng 47:169–179. https://doi.org/10/gt5jj7 Almeida JR, Bertilsson M, Gorwa-Grauslund MF, et al (2009) Metabolic effects of furaldehydes and impacts on biotechnological processes. Appl Microbiol Biotechnol 82:625–638 Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol 74:25–33 Hasunuma T, Kondo A (2012) Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering. Biotechnol Adv 30:1207–1218 Zhao X, Bai F (2009) Mechanisms of yeast stress tolerance and its manipulation for efficient fuel ethanol production. J Biotechnol 144:23–30 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8133957","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":546196342,"identity":"68effb18-b9ce-49a2-982a-50eb5e23f9f8","order_by":0,"name":"Sri Peni Wijayanti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACxgaGBIN/cgYgBgiwMSQQoeVDxQFjUrQANc04cyBxA4JPQIv87MNtj3nb7qRvlz7c9oDhlw0DHzsBLQbnEtuNedue5e7sS2w3YOxLY2DjeUBACw9jmzRvG3PuhjOMbRKMPYcZ2CQIOawHoiXdgGgtDECVkjPOHE4Aa2H4QYQWoMp2gw8VaYY7gdZJJDak8RD0i3wP+7MHCQY28uY87M8kPvyxkZNvJ+QwUNzBQWIbAw9B9ahaGP4Qo2EUjIJRMApGGgAAZVNBh6FKpEkAAAAASUVORK5CYII=","orcid":"","institution":"National Research and Innovation Agency (BRIN), KST BJ Habibie","correspondingAuthor":true,"prefix":"","firstName":"Sri","middleName":"Peni","lastName":"Wijayanti","suffix":""},{"id":546196343,"identity":"6a0a1bfe-9262-4ea6-a283-83396297cadb","order_by":1,"name":"Muhammad Ma’ruf","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN), KST BJ Habibie","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Ma’ruf","suffix":""}],"badges":[],"createdAt":"2025-11-17 10:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8133957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8133957/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96699567,"identity":"e8717a02-a93d-45bb-986c-a4a1a32f9559","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":317038,"visible":true,"origin":"","legend":"","description":"","filename":"enkapsulasi.docx","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/c262d7c27468990735b8c820.docx"},{"id":96699555,"identity":"c83586de-6d61-4103-990a-6872a6c017f5","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4968,"visible":true,"origin":"","legend":"","description":"","filename":"4ab1c2e45b6b4e2282c766b003c0286e.json","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/cd194af8ec77d011c944428f.json"},{"id":96711632,"identity":"125d540a-8092-4920-9b6d-969c32501488","added_by":"auto","created_at":"2025-11-25 10:12:23","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76923,"visible":true,"origin":"","legend":"","description":"","filename":"4ab1c2e45b6b4e2282c766b003c0286e1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/e584951fcfde9644560cb4f0.xml"},{"id":96699558,"identity":"6e7f22ee-271c-4377-b643-dafade88c462","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69450,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage1.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/4f6883e7b8e48e033d7917bd.eps"},{"id":96711255,"identity":"6820311c-a514-4bf7-98e0-c85fd1aba570","added_by":"auto","created_at":"2025-11-25 10:11:48","extension":"eps","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76737,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage10.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/1408990baa564021a6c2f234.eps"},{"id":96699561,"identity":"e7658463-e12d-4fe5-986e-3d5981e40fe6","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46951,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage11.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/d02f421558a5f0e2a2acb7ab.eps"},{"id":96711313,"identity":"da429fa1-a174-4009-832d-a19f8e590fcf","added_by":"auto","created_at":"2025-11-25 10:11:52","extension":"eps","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46872,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage12.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/784a0ef3b6865add4ca42e23.eps"},{"id":96711002,"identity":"49ce88ed-6c4c-4859-8829-ebf9c6ab8e66","added_by":"auto","created_at":"2025-11-25 10:11:30","extension":"eps","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134149,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage13.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/9c53a47e6dcf9d17505e7f1d.eps"},{"id":96711173,"identity":"187d7872-8a22-4c2e-8b29-99a493b6aa13","added_by":"auto","created_at":"2025-11-25 10:11:43","extension":"eps","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76716,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage14.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/28683bac20d259538e72a4ef.eps"},{"id":96710989,"identity":"881b741d-d316-47ed-a808-04d6458f9923","added_by":"auto","created_at":"2025-11-25 10:11:29","extension":"eps","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47176,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage15.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/8d44eee5c025fed833d13b50.eps"},{"id":96699572,"identity":"4018c7d8-20c2-478a-95ac-60735d91a8ca","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46944,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage16.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/ebe7677f4b904c1391b3f8fa.eps"},{"id":96711619,"identity":"a824c56c-5b0f-4051-aabe-be42e36eab1b","added_by":"auto","created_at":"2025-11-25 10:12:21","extension":"eps","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53320,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage17.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/8ce4232ed88fb3708f408ca6.eps"},{"id":96711359,"identity":"c06234d7-5ca3-4ee5-858d-4095b51199a4","added_by":"auto","created_at":"2025-11-25 10:11:54","extension":"eps","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53320,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage18.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/bb84adf96f28d627fb76d433.eps"},{"id":96711417,"identity":"29d2eb57-b1a1-4b58-9edc-e71122cf3d67","added_by":"auto","created_at":"2025-11-25 10:11:59","extension":"eps","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138918,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage19.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2498b9fc83c35576ca9bae4f.eps"},{"id":96711278,"identity":"b1ddb3bd-880a-4603-b3d4-d0ada97fef7f","added_by":"auto","created_at":"2025-11-25 10:11:50","extension":"eps","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81498,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage20.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/9d23d61f90998793450a0ff2.eps"},{"id":96711368,"identity":"c4be8510-471f-4793-b189-678e972508ce","added_by":"auto","created_at":"2025-11-25 10:11:55","extension":"eps","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47252,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage24.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/bcbb9ac75303fde8bc47f599.eps"},{"id":96699582,"identity":"fcddb50c-3572-40c0-b77b-6fc27ba9a1a5","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125842,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage25.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/363aa006ed46f6e3a7d5371c.eps"},{"id":96699579,"identity":"3919dfb4-5025-4e62-b4c3-6c49ef326b55","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46926,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage26.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/0681e3f535be4b0164f43397.eps"},{"id":96699580,"identity":"3e0c1ee2-a1bd-48d1-b231-9a2d11f20101","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46688,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage27.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/4a8099b3c9fc4f8b3a7fb46b.eps"},{"id":96711435,"identity":"0c07b933-22df-471a-b5d0-ed4bde03376f","added_by":"auto","created_at":"2025-11-25 10:12:01","extension":"eps","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121099,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage28.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/c11942a5d4ea233e5a54b584.eps"},{"id":96699583,"identity":"7e261339-e145-4852-91d7-09a50942e51e","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65445,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage29.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/f39ce7303e014623fc817c74.eps"},{"id":96699581,"identity":"33c81ffc-c9bc-4e36-9c2a-8992ed33a1ca","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46683,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage30.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/dd4799a8f9cd3f3bb37645d5.eps"},{"id":96711384,"identity":"61c99ac3-97c9-4e1b-8b0f-673916e13add","added_by":"auto","created_at":"2025-11-25 10:11:56","extension":"eps","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47128,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage31.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/7f0f9894bda9b3fd1bcc53cf.eps"},{"id":96699615,"identity":"2bf5c9fe-f695-4d87-b790-cfc96fec4823","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121366,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage32.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/a926393c2e740413d537cb6e.eps"},{"id":96711309,"identity":"06cccdf7-c609-4b62-a984-af8d7c2a3fd4","added_by":"auto","created_at":"2025-11-25 10:11:51","extension":"eps","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":62539,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage33.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/0fdf5229b7b70cc71c0b60bb.eps"},{"id":96699585,"identity":"20789f4a-5922-49f5-87e0-dd471c2a8b36","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47357,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage34.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/70fed4b0a211e427f53f4310.eps"},{"id":96711408,"identity":"5bf8e42e-1340-438c-b553-80b73856f7c9","added_by":"auto","created_at":"2025-11-25 10:11:58","extension":"eps","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46949,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage35.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/ac3f30120f2a941f597a13dc.eps"},{"id":96711430,"identity":"ed829094-1ef0-44fb-a451-0f1f2a77d92d","added_by":"auto","created_at":"2025-11-25 10:12:01","extension":"eps","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125922,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage36.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/a065f6e7ed204034f64cc0bf.eps"},{"id":96699584,"identity":"fc47269c-3cab-461e-aff9-34ed13bfd817","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"eps","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69602,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage37.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/d9b4233ca7db1bdc5986354d.eps"},{"id":96699600,"identity":"1a01229d-f054-4342-aaf0-41c94a736750","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131495,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage41.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/318b206b1394532e46f99af8.eps"},{"id":96711092,"identity":"5253b61a-b72a-4ba3-a2a1-98b80ac5ad00","added_by":"auto","created_at":"2025-11-25 10:11:38","extension":"eps","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46682,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage42.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/474e6482c53c6c075617bd61.eps"},{"id":96699591,"identity":"30ee5477-a4c1-4d4e-84c2-a9d947c16d9a","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46920,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage43.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/a6a00773379898ad5b4840de.eps"},{"id":96699602,"identity":"16076ecc-e611-4aae-95a5-467062afe9be","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120702,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage44.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2087f58ba45ae5df270e58fa.eps"},{"id":96710975,"identity":"7b402b20-146a-42c4-87af-a5347bdafb16","added_by":"auto","created_at":"2025-11-25 10:11:28","extension":"eps","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65287,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage45.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/65b6f8aefd5ef511992f1d71.eps"},{"id":96711251,"identity":"b4942fed-7b45-4717-af51-19abbe3fd1c2","added_by":"auto","created_at":"2025-11-25 10:11:48","extension":"eps","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46693,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage46.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/207ff70948cf9d947d674231.eps"},{"id":96699588,"identity":"564c9a23-af26-4e76-947f-d0d8aed01882","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46939,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage47.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2f932a0ee006da73f3877ff0.eps"},{"id":96711061,"identity":"4b511a42-9449-4493-bd02-eebbe2e258b3","added_by":"auto","created_at":"2025-11-25 10:11:34","extension":"eps","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133042,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage48.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/60ba889daac679daf7bdf40f.eps"},{"id":96710800,"identity":"cd215bb6-554b-4493-b2df-92df40566946","added_by":"auto","created_at":"2025-11-25 10:11:12","extension":"eps","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65189,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage49.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/a06e2e479625100ebd949dc4.eps"},{"id":96711120,"identity":"7754f03d-a97d-48ea-bcbc-693c8f241fbe","added_by":"auto","created_at":"2025-11-25 10:11:40","extension":"eps","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53320,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage18.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/39acac2b25995a9a6b53c1a4.eps"},{"id":96699599,"identity":"a7d045fb-7cbc-4f16-a74e-161cde5f2cc8","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51558,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage50.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/43538d85eefcdb44049326d8.eps"},{"id":96699628,"identity":"94145cc0-cc47-404b-b28a-76b1fe115686","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"eps","order_by":49,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51546,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage51.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/7eb16dad09079634212305ab.eps"},{"id":96711552,"identity":"83020264-ba65-4f52-a138-0d2def01288d","added_by":"auto","created_at":"2025-11-25 10:12:12","extension":"eps","order_by":50,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47174,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage52.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/687eb17be009fa229199af12.eps"},{"id":96699596,"identity":"098ddd37-cc31-4546-8256-9a7ba7eaee57","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"eps","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46953,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage53.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/e17e36c45ffe50808a605d7f.eps"},{"id":96699625,"identity":"67193a43-0108-4fce-9fc2-ddf2e41dc88a","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"eps","order_by":52,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125890,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage54.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/8a8e1990949b61bbc4fc75df.eps"},{"id":96711186,"identity":"e0b4defd-e895-4bc5-874b-57b2ab1199f2","added_by":"auto","created_at":"2025-11-25 10:11:45","extension":"eps","order_by":53,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69852,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage55.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/bc1bd40391bbd94966495a37.eps"},{"id":96699619,"identity":"cc1a766a-2afe-4d91-b521-c269d1295210","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"eps","order_by":55,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143729,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage6.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/b8c3adf6018194f787d96d90.eps"},{"id":96699622,"identity":"4d8e0997-1e1e-4bcc-ad54-a98f09b8ac1e","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"eps","order_by":56,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47109,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage7.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/3ede01f0aadaa217db4b32ca.eps"},{"id":96711402,"identity":"738f9840-774d-40b6-96b8-8447c641d1a1","added_by":"auto","created_at":"2025-11-25 10:11:57","extension":"eps","order_by":57,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46683,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage8.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/5a8dc61c9a7b3df799fcfdd7.eps"},{"id":96699626,"identity":"5549a286-c29e-4fec-b002-4ec1f59f187b","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"eps","order_by":58,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133892,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage9.eps","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/6d7ea1a8743628c46481c84b.eps"},{"id":96711459,"identity":"75c03f71-e59b-4232-8c3d-3eabe68a6dd5","added_by":"auto","created_at":"2025-11-25 10:12:02","extension":"jpeg","order_by":59,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48680,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/9201bf3b1f82a3dc76d34579.jpeg"},{"id":96699593,"identity":"f72f6f51-94da-4e4b-b9c6-e02c1f3fdf4e","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"jpeg","order_by":60,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7585,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/82dfbe85ff296878281bcd05.jpeg"},{"id":96699605,"identity":"1175645a-6f61-4e88-a042-ebe6e12830c0","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"jpeg","order_by":61,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132629,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/3cfb5b891261108b3b89ebdf.jpeg"},{"id":96699633,"identity":"c37fc9cd-3175-48f2-ba0f-c5047a6e70c2","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"jpeg","order_by":62,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2afe840fdc0e5526dca88a05.jpeg"},{"id":96711264,"identity":"c019eee9-38d8-41f3-852c-e10cd0001284","added_by":"auto","created_at":"2025-11-25 10:11:49","extension":"jpeg","order_by":63,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/1d49d61a8489263326acd1b8.jpeg"},{"id":96699611,"identity":"adda31f2-5e77-4bcd-904d-3da0b7100311","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"jpeg","order_by":64,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28661,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/e94e280ec3a2db85b0521b90.jpeg"},{"id":96711372,"identity":"48b5c2fe-4cc7-46c5-bcc8-54f264d8d278","added_by":"auto","created_at":"2025-11-25 10:11:55","extension":"jpeg","order_by":65,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2b170ff47f20294db287b93e.jpeg"},{"id":96710938,"identity":"bde8bbe8-456e-4a9f-a4a0-afed057e2988","added_by":"auto","created_at":"2025-11-25 10:11:26","extension":"jpeg","order_by":66,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128335,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/15961135cc109a26803ff77e.jpeg"},{"id":96699597,"identity":"12b525e0-798d-4873-8f6c-5b837711ed01","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"jpeg","order_by":67,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1074,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/1e589c2448eb9a5f9429705f.jpeg"},{"id":96699627,"identity":"1f303ec8-c07a-4d01-8781-9f80889e0402","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"jpeg","order_by":68,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19144,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/63b2bf9d7beaa5e2ffc75a97.jpeg"},{"id":96699629,"identity":"b093bad8-8cb6-401a-b630-1943d9a212dd","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"png","order_by":69,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11393,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/cdb7ffd101605c58ddd322b2.png"},{"id":96711498,"identity":"8f4effc4-832e-49b8-9ee0-ddf276e7ad5f","added_by":"auto","created_at":"2025-11-25 10:12:05","extension":"png","order_by":70,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2377,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/9765485c6f6293138bcabe08.png"},{"id":96699598,"identity":"ae67b0dc-dc8d-459e-bcb7-b7600f87b3a7","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"png","order_by":71,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29452,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/e064cbab33eb0fe64d52e052.png"},{"id":96711565,"identity":"0ff6cf2e-e56e-48ec-8ea1-d81b4ce3fc2a","added_by":"auto","created_at":"2025-11-25 10:12:13","extension":"png","order_by":72,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/6a6885cc3439922480e9568a.png"},{"id":96711238,"identity":"cc0ee2dc-0263-4f76-a2a6-0b86856c8834","added_by":"auto","created_at":"2025-11-25 10:11:47","extension":"png","order_by":73,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/4928a3cf0a0e419059a3708b.png"},{"id":96711469,"identity":"dd7b913e-c01a-4be8-ae65-d7960880935c","added_by":"auto","created_at":"2025-11-25 10:12:02","extension":"png","order_by":74,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6012,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/73452ebe78f1dc53ec690f80.png"},{"id":96699618,"identity":"17fe0bfd-1883-4ff3-bf05-97c157ba8e16","added_by":"auto","created_at":"2025-11-25 08:11:29","extension":"png","order_by":75,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/4094fe28fb25867c9e5c1e98.png"},{"id":96711323,"identity":"ef39a1f0-99ae-4e43-93d6-9b74aa9f1ca5","added_by":"auto","created_at":"2025-11-25 10:11:53","extension":"png","order_by":76,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26641,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2369f482cbf32bbd6c23c206.png"},{"id":96711149,"identity":"e025919c-23d6-4a5d-a460-a3f4a8952095","added_by":"auto","created_at":"2025-11-25 10:11:42","extension":"png","order_by":77,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/ffa84652047dcc5666776641.png"},{"id":96711287,"identity":"8e977cb6-eb49-42f0-a68e-a0438094f6c3","added_by":"auto","created_at":"2025-11-25 10:11:51","extension":"png","order_by":78,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9335,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/2a19188e9a64983de9f86e6e.png"},{"id":96711399,"identity":"dd5bb823-422e-4cf8-83d5-fae76b8194b2","added_by":"auto","created_at":"2025-11-25 10:11:57","extension":"xml","order_by":79,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76395,"visible":true,"origin":"","legend":"","description":"","filename":"4ab1c2e45b6b4e2282c766b003c0286e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/be730d66daa1e806b08b0e9b.xml"},{"id":96699631,"identity":"ffe590b0-c943-4cd9-b324-8cb718f00dba","added_by":"auto","created_at":"2025-11-25 08:11:30","extension":"html","order_by":80,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96623,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/f6f9ba78d98ecdcb5bef5047.html"},{"id":96699553,"identity":"059f8a56-7e51-4754-ba00-cd962b377d9c","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77283,"visible":true,"origin":"","legend":"\u003cp\u003eA process diagram for preparing encapsulated yeast [15].\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/0acc7704b1f63272b8a508e0.png"},{"id":96699552,"identity":"b078fb50-4bb0-4304-8b59-042887c4ea0d","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26079,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e) and ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e) during 6 hours of free cells and encapsulated cell fermentation in defined media.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/b617607b31a8651669646298.png"},{"id":96699551,"identity":"35ccb426-77ff-4163-8083-09cdc9661a96","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34049,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) during 6 (a) and 24 (b) hours of fermentation in defined media.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/ec6281d32cf11ff7a5290fe6.png"},{"id":96711400,"identity":"837f6fd9-c594-42fc-ad8f-b68956d7f090","added_by":"auto","created_at":"2025-11-25 10:11:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106160,"visible":true,"origin":"","legend":"\u003cp\u003eAnaerobic batch cultivation \u003cem\u003eS. cerevisiae \u003c/em\u003ein defined media with free cells (A8), encapsulated cells Alg-Chit (A1, A3, A5), encapsulated cells Alg-Chit-MCS (A2, A4, A6) yielded (●) ethanol, (■) glucose, and (x) glycerol as metabolic products.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/44cc039df7ef7f5298b20ac3.png"},{"id":96699556,"identity":"312e476a-aa5b-4f8c-bf78-29d561bb3123","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24432,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e) and ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e) during 6 hours of free cells and encapsulated cell fermentation in aldehyde media.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/06a5a867c173b0fe1bfa9b73.png"},{"id":96711407,"identity":"db21797f-0898-4223-9803-ec4bbee6a087","added_by":"auto","created_at":"2025-11-25 10:11:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26137,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) during 6 (a) and 24 (b) hours of fermentation in aldehyde media.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/6187969452749ae42bd8ec94.png"},{"id":96699564,"identity":"c1246fc7-bded-4984-b8f0-4d4a21ae1e17","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":163942,"visible":true,"origin":"","legend":"\u003cp\u003eAnaerobic fermentation of S. cerevisiae in aldehyde media with free cells (A8), encapsulated cells in alg-chit (A1, A3, A5), and encapsulated cells in alg-chit-MCS (A2, A4, A6) yielded (●) ethanol, (■) glucose, and (x) glycerol as metabolic products.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/7ade33ed238c860227f3d75d.png"},{"id":96711557,"identity":"d059f3e6-ed70-4da7-aab8-664b724caae1","added_by":"auto","created_at":"2025-11-25 10:12:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":93259,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of furfural and vanillin on the fermentation of free and encapsulated S. cerevisiae yeast. The symbols represent (■) suspended cells/A8, (▲) alg-chit/A5 (● ) alg-chit-MCS/A6.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/afe62847c99432478e8b9d60.png"},{"id":96699559,"identity":"71b272a9-68d4-47f9-acb8-adedd887d845","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24556,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e) and ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e) during 6 hours of fermentation in acidic media.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/17e7cba97b1be47981ba79af.png"},{"id":96913100,"identity":"0dff5f7b-7d63-4396-bf5d-5d3443374f65","added_by":"auto","created_at":"2025-11-27 13:52:36","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":25043,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) during 6 (a) and 24 (b) hours of fermentation in acidic media.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/7671af6d26ea2e4241e08231.png"},{"id":96699571,"identity":"bef760e5-d62c-4102-a2c7-9fe15f988687","added_by":"auto","created_at":"2025-11-25 08:11:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":87199,"visible":true,"origin":"","legend":"\u003cp\u003eAnaerobic fermentation of S. cerevisiae in acidic media with free cells (A8), encapsulated cells in alg-chit (A1, A3, A5), and encapsulated cells in alg-chit-MCS (A2, A4, A6) yielded (● ) ethanol, ( ■) glucose, and (x) glycerol as metabolic products.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/df70418bbea51fe830742689.png"},{"id":96710767,"identity":"3a896fd1-3871-492c-a778-13884b6f2350","added_by":"auto","created_at":"2025-11-25 10:11:10","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":35994,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different media (defined, aldehyde and acid media) on the growth of suspended cells \u003cem\u003eS. cerevisiae\u003c/em\u003e as a function of time.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/1c5df7694740958c4f9a4967.png"},{"id":105729004,"identity":"87d5ab6c-cf95-45ec-b9c9-8baeced78a36","added_by":"auto","created_at":"2026-03-30 11:13:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1243718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8133957/v1/1d21551c-f505-42d9-9a7d-ea09f2be7db3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing bioethanol production using sodium microcrystalline cellulose sulfate (Na-MCS) encapsulated Saccharomyces cerevisiae in the presence of aldehyde and acid inhibitors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBioethanol has emerged as a viable alternative to fossil fuels due to its renewable nature and ability to reduce greenhouse gas emissions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, inhibitors such as aldehydes and acids, which arise during biomass pretreatment, reduce bioethanol production efficiency, decreasing ethanol yields and increasing costs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, a common yeast used in bioethanol production, is particularly vulnerable to these inhibitors. Strategies to enhance S. cerevisiae resistance, such as genetic modification and adaptive evolution, are often time-consuming and complex [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. An alternative approach involves encapsulating yeast cells to protect them and increase their resilience under harsh conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEncapsulation involves surrounding yeast cells with a partially permeable membrane that allows substance exchange while shielding the cells from harmful molecules [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This technology improves yeast stability, vitality, and reuse in multiple fermentation cycles, enhancing efficiency and cost-effectiveness [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Natural or synthetic polymers such as calcium alginate are generally used for encapsulation. This research, however, focuses on sodium microcrystalline cellulose sulfate (Na-MCS), a unique linear polymer combined with alg-chit. In Na-MCS, the cellulose sulfate (CS) chain is an analog of polysaccharide sulfates, with hydroxyl groups replaced by sulfate groups at positions 2, 3, and 6 of the anhydroglucose unit (AGU). Combining cellulose sulfate with a synthetic polycation forms stable polyelectrolyte complex membranes.\u003c/p\u003e\u003cp\u003eCertain organic acids, such as formic acid, levulinic acid, and acetic acid, which are typically found in hydrolysates obtained from lignocellulosic materials, can have a major impact on bioethanol synthesis. These acids can act as inhibitors during the fermentation process, affecting the efficiency of bioethanol production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Formic acid is recognized to be a key inhibitory component present in hydrolysates, preventing the conversion of sugars into bioethanol [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Weak acids, such as formic and acetic acid, are powerful yeast inhibitors during bioethanol synthesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Formic acid, when combined with other inhibitors such as acetic acid, can reduce ethanol yield and cell survival during bioethanol synthesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Formic acid can effectively penetrate cellulose molecules, disrupting their crystalline structure and facilitating hydrolysis, which is essential for bioethanol production [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe presence of fermentation inhibitors like furfural and vanillin is one of the main problems in producing bioethanol from lignocellulosic biomass. These compounds can significantly hinder yeast growth and fermentation efficiency. The use of encapsulated yeast offers a promising solution to this problem. Encapsulation has been shown to increase yeast resistance to fermentation inhibitors, allowing for a more robust fermentation process [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Encapsulated yeast strains exhibit enhanced sugar co-utilization, particularly in consuming xylose, which is crucial for efficient bioethanol production from lignocellulosic biomass [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious research used Na-MCS to evaluate its impact on bioethanol production from lignocellulosic hydrolysates [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This investigation aims to examine the effect of Na-MCS in media containing only aldehydes and acids as inhibitors, providing a deeper understanding of these inhibitors' impact on bioethanol production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemicals used in this research include Na-MCS, Tween 20, CaCl\u003csub\u003e2\u003c/sub\u003e, carboxymethylcellulose (CMC), chitosan, Na-alginate (molecular weight 250,000 g/mol), acetate buffer, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, EDTA, CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, KI, d-biotin, p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine\u0026middot;HCl, thiamine\u0026middot;HCl, m-inositol, antifoam, ergosterol, and Tween 80, all of which were purchased from Sigma-Aldrich. The Na-MCS utilized in this study was developed based on empirical investigations conducted in prior studies [15]. The yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e CBS 8066 was obtained from Centraalbureau Voor Schimmelcultures (Delft, Netherlands). All other compounds and solvents were analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of media and culture stock\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast culture stocks were grown in agar media made from 10 g/L yeast extract, 20 g/L soy peptone, and 20 g/L agar with 20 g/L D-glucose added as a carbon source. The synthetic media was prepared by mixing several components, including carbon sources, trace metals, vitamins, antifoam, ergosterol, and Tween 20. The trace metal solution was composed of 7.5 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 3.5 g KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.75 g MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 30 mg EDTA, 13 mg CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, 0.8 mg Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.6 mg CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.6 mg CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, and 0.2 mg KI. The vitamin solution included 50 \u0026micro;g d-biotin, 0.2 mg p-aminobenzoic acid, 1.0 mg nicotinic acid, 1.0 mg calcium pantothenate, 1.0 mg pyridoxine hydrochloride, 1.0 mg thiamine hydrochloride, and 25 mg m-inositol. Both glucose and trace metals were sterilized separately using an autoclave, while the vitamin solution was sterilized using a sterile filter. Ergosterol and Tween 80 were not sterilized but were dissolved in ethanol and evaporated for 10 minutes before use. The sterile solutions of glucose, trace metals, and vitamins were stored at 4\u0026deg;C, whereas the mixed solution of ergosterol and Tween 80 was stored at -20\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation solution for encapsulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe encapsulation process was carried out to form alg-chit-microcrystalline cellulose sulfate (alg-chit-MCS) and alg-chit (alg-chit) capsules. The encapsulation solution was prepared in three polymer solutions with different compositions. To make chitosan-microcrystalline cellulose sulfate alginate capsules, Solution I was made from a mixture of Na-alginate, NaMCS, and Tween 20 dissolved in distilled water. Meanwhile, to make chitosan alginate capsules, Solution I was made from a mixture of Na-alginate and Tween 20 dissolved in distilled water. Solution II consists of a mixture of carboxymethylcellulose (CMC) and CaCl\u003csub\u003e2\u003c/sub\u003e dissolved in distilled water. Solution III was prepared by dissolving chitosan and CaCl\u003csub\u003e2\u003c/sub\u003e in a 0.04 M acetate buffer with different pH levels. All three types of solutions were sterilized using an autoclave at 121\u0026deg;C for 20 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of alg-chit and alg-chit-MCS capsules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCapsule production was carried out using the method like in Figure 1. Solution II, containing yeast, was dropped into Solution I for 10 minutes while being stirred with a magnetic stirrer. The capsules were rinsed with distilled water after 10 minutes and then hardened in a 1.3% CaCl\u003csub\u003e2\u003c/sub\u003e solution for 20 minutes. The capsules were then rinsed once more with distilled water. Next, the capsules were soaked in Solution III at a ratio of 1:5 and placed in a shaker bath at 30\u0026deg;C with a speed of 130 rpm for 24 hours. The coated capsules were washed with 0.9 M NaCl solution to remove excess chitosan. Capsules containing yeast were transferred into 200 mL of synthetic media for aerobic propagation in a synthetic growth medium containing 50 g/L glucose. The material composition of the capsules\u0026apos; membrane varies significantly across different types of capsules. For instance, the A1 capsule, classified as alg-chit, comprises 0.60% alginate, 1.3% CaCl\u003csub\u003e2\u003c/sub\u003e, and 0.2% chitosan with a pH buffer acetate level of 5. In comparison, the A2 capsule, which is an alg-chit-MCS blend, includes 0.54% alginate, 0.06% MCS, 1.3% CaCl\u003csub\u003e2\u003c/sub\u003e, and 0.2% chitosan, also buffered at pH 5. Variations in CaCl\u003csub\u003e2\u003c/sub\u003e concentration and pH are observed across other capsules. For example, A3 and A4 maintain similar compositions to A1 and A2, respectively, but differ in their CaCl\u003csub\u003e2\u003c/sub\u003e concentrations, using only 25 mM compared to the standard 300 mM. Meanwhile, capsules A5 and A6, also alg-chit and alg-chit-MCS respectively, utilize 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e with a slightly lower pH buffer of 4.5. \u0026nbsp;Lastly, A8 is composed of suspended cells without specific percentages for alginate or MCS, though it includes 1.3% CaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand maintains a pH buffer of 5 [15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnaerobic fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive milliliters of propagated yeast capsules (425 \u0026plusmn; 25 capsules) were added to 50 milliliters of synthetic media. The 100-milliliter conical flasks containing capsule cells and synthetic media were incubated in a shaker incubator at 30\u0026deg;C with a shaking speed of 130 rpm. Fermentation was conducted in three different synthetic media: defined media, synthetic media containing aldehydes with 3 g/L furfural and 0.2 g/L vanillin; and synthetic media containing acids with 2.5 g/L formic acid, 2.5 g/L levulinic acid, and 10 g/L acetic acid.\u003c/p\u003e\n\u003cp\u003eThroughout the tests, samples were removed from the flask, centrifuged, and kept at -20\u0026deg;C. HPLC The eluent was ultrapure water flowing at a rate of 0.5 mL/min. The Aminex HPX-87H column (Bio-Rad) was used at 60\u0026deg;C with 5 mM H2SO4 as the eluent and a flow rate of 0.5 mL/min to quantify glucose, ethanol, glycerol, vanillin, and furfural. The quantities of furfural and vanillin were assessed using UV chromatograms at 210 nm, whereas the remaining metabolites were determined using RI chromatograms. The glucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e), ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e), and ethanol yield (Y\u003csub\u003eSE\u003c/sub\u003e) were also calculated [15].\u003c/p\u003e"},{"header":"Result and Discussions","content":"\u003cp\u003e\u003cstrong\u003eFermentation in defined media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnaerobic cultivation of encapsulated and free-cell \u003cem\u003eS. cerevisiae\u003c/em\u003e in defined growth media was characterized and compared. The most important results are presented in Figure 2 and 3 . Glucose was used as carbon and energy source throughout these experiments. The initial cell mass was provided aerobically by cultivation for 24 h in shake flasks. The experiments with the encapsulated cells contained 425 \u0026plusmn; 25 capsules in a total volume of ca. 55 mL, and two batches were carried out per experiments. The CO\u003csub\u003e2\u003c/sub\u003e bubbles formed during the fermentation diffused easily through the capsules membrane to the media and left the flasks via the loop-trap. Since anaerobic condition was maintained in all the batches, the influence of mass transfer of oxygen on the growth of cells is negligible.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to Figure 2 and 3, the ethanol production, glucose consumption rate and yield ethanol for free cells was lower than encapsulated cells in 6 hours cultivation. Free cells take up the sugars at 2.91 g/l.h and produce ethanol at 0.50 g/l.h and encapsulated cells take up the sugars approximately two times faster than the free cells. There is relationship between the amounts of ethanol production with availability of cells. The high biomass inside capsule membrane will produce high concentration of ethanol. \u0026nbsp;From the experiment we observed the ethanol production from encapsulated cells in 6 hours cultivation was high which has correlated to high cells concentration inside the capsules. The viability cells inside the capsule were more prevented by membrane layer both of alginate-chitosan (alg-chit) and alginate-chitosan-MCS (alg-chit-MCS) than free cells. The different composition of membrane capsules influenced the ethanol production rate but not the glucose consumption rate. Chitosan solution with 300 mM CaCl\u003csub\u003e2\u003c/sub\u003e in pH 5 produced 1.73 g/l ethanol per hours in 6 hours cultivation by yeast inside alg-chit capsules and 1.68 g/l.h glucose by yeast inside alg-chit-MCS. Reducing the CaCl\u003csub\u003e2\u003c/sub\u003e concentration from 300 mM to 25 mM in pH 5 increased productivity of ethanol in 6 hours cultivation, 2.6 g/l.h (alg-chit) and 2.25 g/l.h (alg-chit-MSC). pH 5 was adjusted in chitosan solution to turn the NH\u003csub\u003e2\u003c/sub\u003e to NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. On the other hand, the presence of Cl\u003csup\u003e-\u003c/sup\u003e in the chitosan solution will increase the counterbalancing between positive charge (NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) and negative charge (Cl\u003csup\u003e-\u003c/sup\u003e). A high concentration of CaCl\u003csub\u003e2\u003c/sub\u003e on chitosan solution would reduce electrostatic interaction between the protonated amino groups to carboxylic groups on the alginate. Thus decrease the chemical and mechanical stability of alg-chit and alg-chit-MCS, therefore directly impacting the ethanol production rate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe purpose of adding Na-MCS to alginate solution was to increase the mechanical and chemical stability of alg-chit capsule membrane. \u0026nbsp;In this experiment, it was observed that adding Na-MCS to alg-chit reduced ethanol production rate. The negative charge in alginate-MCS solution should be present from carboxylate group (-COO)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand sulfate group ( \u0026ndash;SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e). Since the DS of Na-MCS was low, the amount of sulfate groups in the alginate-MCS solution was low. Because of this reason the ethanol production of alg-chit-MCS (2.25 g/l.h) was lower than algi-chit (2.60 g/l.h) \u0026nbsp;in pH 5 of acetate buffer solution. Nevertheless by adding Na-MCS increased the ethanol productivity in pH 4.5. The idea to reduce the pH of acetate buffer in the chitosan solution was to incresae the electrostatic interaction by the presence higher amount of the protonated amino groups than at pH 5. Therefore the electrostatic interaction of protonated amino groups with negative charged (carboxylic and sulfate groups) would increase, eventhough the amount of negative charge was lower than in the alginate solution without Na-MCS.\u003c/p\u003e\n\u003cp\u003eFigure 4 shows the changes in glucose, ethanol, and glycerol concentrations during anaerobic fermentation of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in defined media using both free and encapsulated cells. In all treatments, glucose concentration sharply decreased within the first 6 hours, indicating the active fermentation phase. Free cells (A8) exhibited the fastest glucose consumption, with nearly complete substrate depletion within 12 hours, whereas encapsulated cells in Alg-Chit (A1, A3, A5) and Alg-Chit-MCS (A2, A4, A6) matrices showed a slower decrease, likely due to substrate diffusion limitations through the capsule wall.\u003c/p\u003e\n\u003cp\u003eEthanol concentration increased concomitantly with glucose depletion, reaching its maximum level after 12\u0026ndash;18 hours of incubation. Free cells produced slightly higher final ethanol concentrations than encapsulated cells but exhibited less stability in the later fermentation phase. In contrast, encapsulated cells\u0026mdash;particularly those in the Alg-Chit-MCS system\u0026mdash;displayed a more stable and consistent ethanol accumulation profile, suggesting that the encapsulation matrix could maintain cellular metabolic activity for a longer period under anaerobic conditions.\u003c/p\u003e\n\u003cp\u003eGlycerol production remained relatively low and constant after the first 6 hours across all treatments, confirming its role as a minor metabolite involved in maintaining the cellular redox balance during anaerobic metabolism. Compared with the Alg-Chit system, the addition of MCS to form Alg-Chit-MCS appeared to improve metabolic stability without significantly reducing fermentative capability. The incorporation of inert materials such as silica or carbon microspheres is known to enhance the mechanical strength and permeability of polymer matrices, improving mass transfer and cell resistance against ethanol-induced stress.\u003c/p\u003e\n\u003cp\u003eThese results indicate that S. cerevisiae encapsulated within the Alg-Chit-MCS system exhibits improved tolerance to environmental stress during anaerobic fermentation, including ethanol accumulation and nutrient limitation. The protective effect provided by the polymeric matrix plays a key role in maintaining cell viability and enzymatic activity, suggesting that this encapsulation approach can be a promising alternative to enhance the efficiency and stability of industrial-scale bioethanol fermentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFermentation in aldehyde media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fermentation performance of free and encapsulated S. cerevisiae cells in aldehyde media, depicted in Figures 5 and 6, highlights the efficiency of encapsulated cells. Key parameters such as glucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e), ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e), and ethanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) were measured over 6 and 24 hours of fermentation. Encapsulated cells demonstrated significantly higher glucose consumption rates compared to free cells. Capsules A1, A2, A3, A4, A5, and A6 exhibited r\u003csub\u003eS\u003c/sub\u003e values ranging from 4.10 to 6.24 g/L\u0026middot;h, whereas free cells (A8) showed a rate of just 3.66 g/ L\u0026middot;h. Similarly, ethanol production rates were remarkably higher in encapsulated cells, with values between 1.45 and 2.65 g/L\u0026middot;h, compared to a mere 0.16 g/L\u0026middot;h in free cells. Ethanol yield over 6 hours was higher for encapsulated cells (0.34 to 0.51 g/g) compared to free cells (0.04 g/g). Over 24 hours, encapsulated cells showed yields ranging from 0.38 to 0.43 g/g, unequivocally demonstrating that encapsulation enhances ethanol production efficiency.\u003c/p\u003e\n\u003cp\u003eEncapsulated cells in an aldehyde medium exhibited markedly higher glucose consumption and ethanol production rates than free cells (Figure 3). Different encapsulation compositions influence ethanol production rates and glucose consumption rates. Reducing the CaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003econcentration from 300 to 25 mM at pH 5 in chitosan solution significantly increased the ethanol production rate from 1.54 to 2.53 g/L\u0026middot;h. The ethanol productivity of alg-chit-MCS was higher than alg-chit when coating the alg-MCS with chitosan at pH 4.5 and 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eFigure 6 illustrates the ethanol yield (Y\u003csub\u003eSE\u003c/sub\u003e) from both free and encapsulated yeast cells during fermentation over 6 hours and 24 hours. For free cells (A8), the yield is very low at 6 hours, close to 0 g/g, but increases significantly to approximately 0.35 g/g after 24 hours. In contrast, encapsulated cells (A1 - A6) show generally higher ethanol yields. At 6 hours, the yields for encapsulated cells A1, A2, A4, A5, and A6 are around 0.3 to 0.4 g/g, with A3 achieving the highest yield of approximately 0.5 g/g. After 24 hours, the yields for encapsulated cells remain high, with slight increases or decreases. A3 remains the highest, around 0.45 g/g, while others (A1, A2, A4, A5, A6) range from 0.35 to 0.4 g/g. Encapsulation not only boosts ethanol yield but also that different encapsulation methods can further optimize this process. Extending fermentation time improves yield for both free and encapsulated cells, with encapsulated cells demonstrating a clear advantage throughout.\u003c/p\u003e\n\u003cp\u003eFigure 7 presented the profiles of glucose, ethanol, and glycerol concentrations during anaerobic batch fermentation in aldehyde media, clearly delineating the advantages of encapsulated cells over free cells. Encapsulated cells exhibited a markedly more efficient glucose consumption profile, with glucose levels dropping significantly faster than in free cells. Ethanol production was substantially higher in encapsulated cells, peaking much earlier than in free cells. For instance, capsules A5 and A6 achieved considerable ethanol production within the first 6 hours, which was sustained throughout the fermentation period. Higher glycerol levels in encapsulated cells further suggest a more robust and efficient fermentation process.\u003c/p\u003e\n\u003cp\u003eFigure 8 demonstrated that S. cerevisiae, both free and encapsulated, efficiently metabolizes furfural and vanillin within the first 6 hours of fermentation, reducing their concentrations to near zero, where they remain stable for the next 18 hours. Specifically, furfural is completely reduced by the cells to furfuryl alcohol, while vanillin is bio-converted to vanillyl alcohol by the yeast strains. This indicates that encapsulation methods (alg-chit/A5 and alg-chit-MCS/A6) do not hinder the yeast\u0026apos;s ability to process these inhibitory compounds. The rapid and sustained reduction highlights the potential of using S. cerevisiae in industrial fermentation processes where these inhibitors are present, ensuring effective fermentation without the adverse impact of these compounds. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFermentation in acidic media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigures 9 and 10 show a complete comparison of the glucose consumption rate (r\u003csub\u003eS\u003c/sub\u003e), ethanol production\u0026nbsp;rate (r\u003csub\u003eE\u003c/sub\u003e), and ethanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) of free and encapsulated \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e cells in acidic conditions. The study tested various capsule compositions (A1 to A6) for the encapsulated cells, with free cells represented by A8. The glucose consumption rate (rS) for free cells (A8) is 1.88 g/L.h over a 6-hour fermentation period. In contrast, the encapsulated cells exhibit varying rates, with A1 (alg-chit) and A2 (alg-chit-MCS) showing significantly higher rS values of 4.35 g/L.h and 4.40 g/L.h, respectively. Encapsulation generally enhances glucose consumption, with A6 (alg-chit-MCS) achieving the highest r\u003csub\u003eS\u003c/sub\u003e of 5.54 g/L.h. This suggests a potentially optimized composition for maximum glucose uptake. The enhancement is likely due to the protective environment provided by the encapsulation, which helps maintain cell viability and metabolic activity in acidic conditions, as supported by previous studies on cell encapsulation technology [6].\u003c/p\u003e\n\u003cp\u003eThe ethanol production rate (r\u003csub\u003eE\u003c/sub\u003e) also shows considerable variation. For free cells, r\u003csub\u003eE\u003c/sub\u003e is relatively low at 0.26 g/L.h. Encapsulated cells, particularly A1 and A2, exhibit much higher ethanol production rates of 1.67 g/L.h and 1.50 g/L.h, respectively. Again, A6 stands out with the highest r\u003csub\u003eE\u003c/sub\u003e of 2.64 g/L.h, emphasizing the benefits of specific encapsulation compositions in enhancing ethanol production. This finding aligns with research indicating that encapsulation can improve the efficiency of bioconversion processes by stabilizing the cells and enhancing their metabolic functions [16].\u003c/p\u003e\n\u003cp\u003eEthanol yields (Y\u003csub\u003eSE\u003c/sub\u003e) calculated over 24 hours are more consistent across all samples, including free and encapsulated cells. Free cells show a Y\u003csub\u003eSE\u003c/sub\u003e of 0.41 g/g, while encapsulated cells range narrowly from 0.42 g/g to 0.44 g/g. This consistency suggests that over longer fermentation periods, the initial advantages of encapsulation may stabilize, leading to more uniform ethanol yields across different compositions.\u003c/p\u003e\n\u003cp\u003eThe performance of alg-chit capsules varied with the concentration of calcium chloride used for coating. Capsules with 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e (A5) showed higher ethanol production rates and yields compared to those with 25 mM CaCl\u003csub\u003e2\u003c/sub\u003e (A3). This indicates that higher calcium chloride concentrations in the coating process enhance the structural integrity and performance of the capsules.\u003c/p\u003e\n\u003cp\u003eEncapsulation of yeast cells using different compositions of alginate, chitosan, and MCS significantly improves fermentation performance in acidic media. Encapsulated cells exhibit higher glucose consumption rates, ethanol production rates, and yields than free cells. The protective environment provided by encapsulation materials enhances the viability and metabolic activity of yeast cells, enabling more efficient bioethanol production. The optimal formulation identified in this study involves alg-chit-MCS with 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e, which shows the highest rates of glucose consumption and ethanol production.\u003c/p\u003e\n\u003cp\u003eCellulose sulfate is emerging as a promising material for encapsulating yeast in bioethanol production. Encapsulation in cellulose sulfate microspheres can enhance the survival of yeast cells in low-pH environments and protect them from the harmful effects of stomach acid and other environmental stresses. The negatively charged sulfate groups in cellulose sulfate buffer the encapsulated cells from acidic conditions, enhancing their viability during the fermentation process. This improved resistance can lead to higher bioethanol production efficiency. Encapsulated yeast in cellulose sulfate has shown promising results in maintaining cell viability and fermentative capacity, contributing to better bioethanol yields from lignocellulosic hydrolysates.\u003c/p\u003e\n\u003cp\u003eFigure 11 shows the ethanol production profiles for free and encapsulated cells over a 24-hour fermentation period. Encapsulated cells produced significantly higher ethanol concentrations compared to free cells. For instance, A6 encapsulated cells reached an ethanol concentration of 20.06 g/L after 24 hours, compared to 19.15 g/L for free cells. This consistent and higher ethanol production aligns with the enhanced metabolic rates observed in encapsulated cells. Glucose consumption was more efficient in encapsulated cells, as indicated by the rapid decline in glucose levels in the media. For example, glucose levels dropped to zero within 15 hours for formulations A3, A4, A5, and A6, whereas free cells (A8) took 24 hours to achieve similar glucose depletion. Glycerol production, a byproduct of yeast metabolism, was also monitored. Encapsulated cells produced slightly higher glycerol concentrations, indicative of the stress response and redox balancing mechanisms in yeast cells during fermentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative Growth of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in Defined and Inhibitor-Containing Media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 12 illustrates the growth profile of suspended \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e cultivated in three different media: defined, aldehyde-containing, and acid-containing media, as indicated by the optical density (OD) at 610 nm over 72 hours of fermentation. The yeast exhibited the highest growth in the defined medium, where the OD value rapidly increased during the first 20 hours and reached a stable phase around 5.5\u0026ndash;6.0, indicating efficient cell proliferation under optimal nutrient conditions.\u003c/p\u003e\n\u003cp\u003eIn contrast, cells grown in aldehyde-containing medium showed a slower increase in OD, achieving a maximum value of approximately 4.5 after 24 hours, suggesting partial inhibition of yeast metabolism by aldehydes. Aldehydes such as furfural and hydroxymethylfurfural are known to interfere with glycolytic enzymes and NADH regeneration, thereby reducing the rate of biomass formation\u0026nbsp;[17, 18].\u003c/p\u003e\n\u003cp\u003eThe lowest growth was observed in the acid-containing medium, with OD values stabilizing around 3.5. Organic acids, particularly acetic and formic acids, can diffuse into the cytoplasm and lower the intracellular pH, resulting in energy depletion due to ATP consumption for proton efflux and overall inhibition of cell metabolism\u0026nbsp;[19].\u003c/p\u003e\n\u003cp\u003eThese results clearly demonstrate that the presence of inhibitory compounds such as aldehydes and acids negatively affects the growth of \u003cem\u003eS. cerevisiae\u003c/em\u003e compared to the defined control medium. Nevertheless, the yeast maintained measurable growth in both inhibitor-containing media, implying a degree of tolerance and metabolic adaptability to stress conditions. This finding supports the potential for developing resistant or encapsulated yeast systems to enhance fermentation performance in lignocellulosic hydrolysates that typically contain such inhibitory compounds [20].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the significant benefits of encapsulating \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e using sodium microcrystalline cellulose sulfate (Na-MCS) for bioethanol production in media containing aldehyde and acid inhibitors. Compared to free yeast cells, encapsulated yeast cells showed higher rates of glucose consumption, ethanol production, and ethanol yields. The protective encapsulation shielded the yeast from the toxic effects of inhibitors such as furfural, vanillin, formic acid, levulinic acid, and acetic acid, allowing for more robust fermentation. Among the various formulations tested, capsules with a combination of 0.54% alginate, 0.02% chitosan, 0.06% Na-MCS, 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e, and pH buffer acetate 4.5, particularly at optimized conditions, showed the highest efficiency. These findings point to the possibility of Na-MCS encapsulation to enhance bioethanol production. This makes it an attractive method for industrial applications where fermentation inhibitors are prevalent. Further research should explore scaling up this technology and evaluating its performance in diverse industrial settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Associate Prof. Carl Johan Franz\u0026eacute;n and Prof. Mohammad J. Taherzadeh for their valuable supervision, guidance, and support throughout this work. The authors also thank Eva Alberts for providing dilute acid hydrolysates, Rakesh Kophram for assistance with HPLC analysis. Special thanks are extended to Esteban for laboratory assistance in media preparation, and to all members of the Industrial Biotechnology and Systems Biology groups in Chalmers University of Technology for their collaboration. The authors also acknowledge the kind help of Marianne Sognell.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSri Peni Wijayanti was responsible for investigation, data curation, manuscript writing, and critical revision. Muhammad Ma\u0026rsquo;ruf contributed to the preparation of figures and critical revision. All authors reviewed and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBalat M, Balat H, \u0026Ouml;z C (2008) Progress in bioethanol processing. Prog Energy Combust Sci 34:551\u0026ndash;573. https://doi.org/10/b627cb\u003c/li\u003e\n\u003cli\u003eNigam PS, Singh A (2011) Production of liquid biofuels from renewable resources. Prog Energy Combust Sci 37:52\u0026ndash;68. https://doi.org/10/cmxcdt\u003c/li\u003e\n\u003cli\u003eJ\u0026ouml;nsson LJ, Mart\u0026iacute;n C (2016) Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects. Bioresour Technol 199:103\u0026ndash;112. https://doi.org/10/f3pcp6\u003c/li\u003e\n\u003cli\u003eAlmeida JR, Modig T, Petersson A, et al (2007) Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. J Chem Technol Biotechnol 82:340\u0026ndash;349. https://doi.org/10/fwwfrw\u003c/li\u003e\n\u003cli\u003eVerbelen PJ, De Schutter DP, Delvaux F, et al (2006) Immobilized yeast cell systems for continuous fermentation applications. Biotechnol Lett 28:1515\u0026ndash;1525. https://doi.org/10/b7jxgr\u003c/li\u003e\n\u003cli\u003eNedovic V, Willaert R (2013) Fundamentals of Cell Immobilisation Biotechnology. Springer Science \u0026amp; Business Media\u003c/li\u003e\n\u003cli\u003eUllah MW, Khattak WA, Ul-Islam M, et al (2015) Encapsulated yeast cell-free system: A strategy for cost-effective and sustainable production of bio-ethanol in consecutive batches. Biotechnol Bioprocess Eng 20:561\u0026ndash;575. https://doi.org/10/gt39nx\u003c/li\u003e\n\u003cli\u003eOshoma CE, Obueh HO (2018) Influence of nitrogen sources on ethanol production by Saccharomyces Spp in the presence of formic acid. Bayero J Pure Appl Sci 11:1\u0026ndash;7. https://doi.org/10/gt5dtf\u003c/li\u003e\n\u003cli\u003eFosso-Kankeu E, Marx S, Meyer A (2015) Simulated Inhibitory Effects of Typical Byproducts of Biomass Pretreatment Process on the Viability of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and Bioethanol Production Yield. Afr J Biotechnol 14:2383\u0026ndash;2394. https://doi.org/10/gt5dtg\u003c/li\u003e\n\u003cli\u003eOshoma CE, Greetham D, Louis EJ, et al (2015) Screening of Non- \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e Strains for Tolerance to Formic Acid in Bioethanol Fermentation. PLOS ONE 10:e0135626. https://doi.org/10/f726bk\u003c/li\u003e\n\u003cli\u003ede Klerk C, Fosso-Kankeu E, Du Plessis L, Marx S (2018) Assessment of the viability of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in response to synergetic inhibition during bioethanol production. Curr Sci 115:1124\u0026ndash;1132. https://doi.org/10/gt5dth\u003c/li\u003e\n\u003cli\u003eLi M-F, Yu P, Li S-X, et al (2017) Sequential two-step fractionation of lignocellulose with formic acid organosolv followed by alkaline hydrogen peroxide under mild conditions to prepare easily saccharified cellulose and value-added lignin. Energy Convers Manag 148:1426\u0026ndash;1437. https://doi.org/10/ghhnmd\u003c/li\u003e\n\u003cli\u003eWestman JO, Franz\u0026eacute;n CJ (2015) Current progress in high cell density yeast bioprocesses for bioethanol production. Biotechnol J 10:1185\u0026ndash;1195. https://doi.org/10/f3cwcm\u003c/li\u003e\n\u003cli\u003eWestman JO, Bonander N, Taherzadeh MJ, Franz\u0026eacute;n CJ (2014) Improved sugar co-utilisation by encapsulation of a recombinant \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain in alginate-chitosan capsules. Biotechnol Biofuels 7:102. https://doi.org/10/f23nc8\u003c/li\u003e\n\u003cli\u003eWijayanti SP (2024) Evaluation of an alginate-chitosan-microcrystalline cellulose sulfate encapsulation system for efficient fermentation of lignocellulosic hydrolyzate. Bogor, Indonesia, p 040003\u003c/li\u003e\n\u003cli\u003eDivyashri G, Tulsi NP, Murthy TPK, et al (2024) Valorization of coffee bean processing waste for bioethanol production: comparison and evaluation of mass transfer effects in fermentations using free and encapsulated cells of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Bioprocess Biosyst Eng 47:169\u0026ndash;179. https://doi.org/10/gt5jj7\u003c/li\u003e\n\u003cli\u003eAlmeida JR, Bertilsson M, Gorwa-Grauslund MF, et al (2009) Metabolic effects of furaldehydes and impacts on biotechnological processes. Appl Microbiol Biotechnol 82:625\u0026ndash;638\u003c/li\u003e\n\u003cli\u003ePalmqvist E, Hahn-H\u0026auml;gerdal B (2000) Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol 74:25\u0026ndash;33\u003c/li\u003e\n\u003cli\u003eHasunuma T, Kondo A (2012) Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering. Biotechnol Adv 30:1207\u0026ndash;1218\u003c/li\u003e\n\u003cli\u003eZhao X, Bai F (2009) Mechanisms of yeast stress tolerance and its manipulation for efficient fuel ethanol production. J Biotechnol 144:23\u0026ndash;30\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bioethanol production, yeast encapsulation, sodium microcrystalline cellulose sulfate, inhibitor tolerance, Saccharomyces cerevisiae, fermentation efficiency","lastPublishedDoi":"10.21203/rs.3.rs-8133957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8133957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDeveloping renewable energy sources, such as bioethanol, is crucial for mitigating greenhouse gas emissions. However, inhibitors such as aldehydes and organic acids generated during biomass pretreatment significantly hinder fermentation performance. \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the most widely used yeast in industrial bioethanol production, is particularly sensitive to these inhibitory compounds. While genetic engineering can enhance yeast tolerance, it often involves complex and time-consuming processes. As an alternative, encapsulation of \u003cem\u003eS. cerevisiae\u003c/em\u003e within a partially permeable membrane offers improved stability and protection under stressful fermentation conditions. This study investigates the encapsulation of \u003cem\u003eS. cerevisiae\u003c/em\u003e using sodium microcrystalline cellulose sulfate (Na-MCS) combined with a synthetic polycation to form a stable membrane matrix. The results demonstrate that encapsulated \u003cem\u003eS. cerevisiae\u003c/em\u003e cells exhibit significantly higher glucose consumption and ethanol productivity compared to free cells when exposed to aldehydes and acids. Encapsulation effectively enhances yeast tolerance to inhibitors such as furfural, vanillin, and various organic acids, resulting in improved fermentation efficiency. Overall, Na-MCS encapsulation presents a promising strategy for increasing the robustness and productivity of S. cerevisiae in bioethanol fermentation under inhibitory conditions.\u003c/p\u003e","manuscriptTitle":"Enhancing bioethanol production using sodium microcrystalline cellulose sulfate (Na-MCS) encapsulated Saccharomyces cerevisiae in the presence of aldehyde and acid inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 08:11:23","doi":"10.21203/rs.3.rs-8133957/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"627b5689-edfc-4c69-b50a-0d1e1ffb39d1","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-29T22:38:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 08:11:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8133957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8133957","identity":"rs-8133957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}