Encapsulation in ZIF-8 as a Biopreservation Strategy and Enhancement of Thermal Tolerance of Saccharomyces cerevisiae

Encapsulation in ZIF-8 as a Biopreservation Strategy and Enhancement of Thermal Tolerance of Saccharomyces cerevisiae | 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 Encapsulation in ZIF-8 as a Biopreservation Strategy and Enhancement of Thermal Tolerance of Saccharomyces cerevisiae Luana Cristina Camargos Gomes, Gabriel Luis Castiglioni, Carlos Alberto Galeano Suarez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8926210/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract This study evaluated ZIF-8 as an immobilization matrix for Saccharomyces cerevisiae CAT-1 to determine its effectiveness as a biopreservation strategy for enhancing robustness and performance in ethanol fermentation under thermal and chemical stress. Encapsulated and free cells were compared in sequential batch fermentations to assess operational stability and reusability. Thermal tolerance assays were conducted in YPD medium at 25–50°C, and protective performance was further examined under inhibitory conditions representative of lignocellulosic hydrolysates derived from passion fruit peel. Biomass formation, cell viability, glucose consumption, substrate-to-product conversion yield, volumetric productivity, and fermentation efficiency were evaluated. ZIF-8 encapsulation significantly enhanced thermotolerance and resistance to inhibitory compounds. Encapsulated cells maintained consistently higher viability across the tested temperature range, reaching 96.7% at 50°C compared to 82.9% for free cells. Within the mesophilic range (25–35°C), immobilized cultures demonstrated superior fermentative performance, achieving fermentation efficiency above 93% at 30°C, whereas free cells reached 78.86%. Under hydrolysate stress, encapsulated cells preserved 99.42% viability after 48 h and retained metabolic activity in a subsequent fermentation cycle. Overall, ZIF-8 immobilization improved cellular stability and process resilience, supporting its application in intensified, inhibitor-rich bioprocesses for industrial ethanol production. Saccharomyces cerevisiae immobilization ZIF-8 thermal stability viability fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The use of microorganisms to produce industrially valuable compounds represents a highly relevant biotechnological strategy (Taylor, 2001 ). For such processes to be effective, factors such as high yield, productivity, operational stability, thermal resistance, and other parameters that ensure process viability and efficiency must be considered. Among the various applications, alcoholic fermentation stands out, with yeasts being the most widely employed microorganisms. Saccharomyces cerevisiae is the predominant species used due to its high fermentation efficiency, adaptability to different environments, and robustness under varying process conditions (Lima et al., 2001 ). The utilization of agro-industrial residues represents a strategic source of raw materials for multiple industrial sectors. There is an increasing demand for high-quality products and improved production efficiency, combined with the adoption of practices that promote environmental protection and process sustainability. In this context, waste reuse offers significant economic and ecological potential (Velvizhi et al., 2022 ). Residues generated from passion fruit processing, particularly the peels, are promising feedstocks for biorefineries, especially for ethanol production. These materials are rich in lignocellulosic sugars and represent one of the most advantageous technological routes for the sustainable production of biofuels (Oliveira, 2018 ). Several factors can influence fermentative performance, with fermentation efficiency being a key parameter. Fermentation efficiency is defined as the percentage of sugar converted into ethanol relative to the theoretical maximum predicted by the Gay-Lussac equation (Fermentec, 1978 ). Efficient conversion of lignocellulosic biomass into bioethanol involves three main steps: pretreatment, enzymatic hydrolysis, and fermentation. However, during the hydrolysis stage, a major challenge is the formation of byproducts resulting from the degradation of sugars and lignin. Many of these compounds are toxic to fermentative microorganisms and negatively affect their metabolism (Chandel et al., 2007 ). In general, the fermentation of lignocellulosic hydrolysates without prior detoxification is associated with slow fermentation kinetics, as well as reduced yields and productivities (Mussatto & Roberto, 2004 ). Among the key factors influencing alcoholic fermentation, temperature plays a fundamental role, as it directly affects microbial metabolic activity and the formation of volatile compounds that impact the quality of the final product (Lima, 2009 ). Moreover, ethanol production is closely associated with fermentation temperature, since carbon dioxide release during fermentation is accompanied by heat generation, which can favor alcohol formation (Mallouchos, 2003). However, elevated temperatures may act as stress factors, impairing yeast growth and viability, reducing ethanol production efficiency, promoting the proliferation of contaminating bacteria, and increasing yeast susceptibility to the toxic effects of ethanol accumulated in the culture medium (Reis & Ribeiro, 2009 ; Shen et al., 2020 ). Ethanol toxicity in yeast becomes more pronounced at higher temperatures due to increased membrane fluidity, which facilitates the influx of toxic compounds into the cell (Phisalaphong et al., 2006 ; Fernandes, 2008). In response to elevated temperatures, yeast cells modify the fatty acid composition of their cell membranes as an adaptive mechanism (Wanderley, 1997). According to Amorim et al. (1996), the toxic effects of ethanol on Saccharomyces cerevisiae remain relatively constant within the temperature range of 12 to 28°C but intensify at higher temperatures. Therefore, establishing an optimal temperature range prior to fermentation is essential to ensure suitable conditions for microbial growth and to maximize ethanol yield (Ballesteros et al., 2004 ; Phisalaphong et al., 2006 ). For ethanol production, the optimal temperature range generally lies between 26 and 35°C. Cell viability declines at temperatures above 35°C, as these conditions trigger a thermal stress response in yeast cells and disrupt normal physiological functions, including carbohydrate flux and membrane composition (Caspeta et al., 2014 ). At lower temperatures, higher ethanol yields may be achieved; however, overall productivity tends to decrease due to prolonged fermentation times (Lima et al., 2001 ; Torija et al., 2003 ). During fermentation, several stress factors—such as elevated temperature, toxic hydrolysates, high ethanol concentrations, and osmotic pressure—can negatively impact process efficiency (Patrascu et al., 2009 ). Lin et al. ( 2012 ) reported that the highest cell growth rates and ethanol productivity for the Saccharomyces cerevisiae BY4742 strain were observed between 30 and 45°C. However, at 50°C, a marked decline in both cell growth and ethanol yield was detected. Industrial strains of Saccharomyces cerevisiae exhibit a certain degree of tolerance to elevated temperatures. However, this tolerance can be compromised under conditions of high ethanol concentration or low pH, negatively affecting cell viability (Reis & Ribeiro, 2009 ). Yeasts are classified as mesophilic microorganisms, and in industrial practice, the optimal temperature range typically lies between 30 and 35°C (Caldas et al., 2012). In industrial fermentation processes, yeast immobilization is widely applied in the production of beer, wine, and cider. This technological strategy involves encapsulating intact and metabolically active yeast cells within a defined matrix, thereby prolonging metabolite production, including aromatic compounds. In this context, metal–organic frameworks (MOFs) and their derivatives, such as zeolitic imidazolate frameworks (ZIFs), have emerged as promising materials for yeast immobilization. These systems may enhance cellular stability and strain control, while also enabling cell recovery and reuse, facilitating continuous fermentation processes, and supporting other technological advancements (Nedović et al., 2014 ). The wide variety of substrates available for MOF synthesis, along with the broad range of possible topologies, enable these materials to be tailored for specific applications (Rogacka and Labus, 2025 ). MOFs have been applied in the chemical, food, pharmaceutical, and environmental sectors, serving as materials for the capture of toxic industrial chemicals (Islamoglu et al., 2020 ), gas storage (Ghanbari et al., 2020 ), chemical catalysis (Gascon et al., 2014 ), drug delivery systems (Horcajada et al., 2006, 2008 ), and the immobilization of enzymes and other biocompounds (Rogacka and Labus, 2025 ). MOFs can be modified through the introduction of different functional groups into their ligands (Deria et al., 2014 ), by structural modification via ligand exchange or incorporation of new ligands (Tanabe & Cohen, 2011 ), or by increasing the inertness of the central metal ion. The thermal stability of a MOF depends largely on the strength of the bond between the metal center and the ligand oxygen; as this bond strength increases, so does the material’s thermal stability (Tanabe & Cohen, 2011 ). Among the various MOF families, zeolitic imidazolate frameworks (ZIFs) are particularly notable for their high porosity, well-defined cavity size, strong interactions with organic polymers, exceptional thermal stability, and outstanding chemical resistance. These features make them especially suitable for addressing key challenges in fermentation processes, such as temperature fluctuations and the presence of toxic compounds (Yang and Chung, 2013 ). Therefore, ZIF-8 can be regarded as a promising protective matrix capable of limiting the diffusion of denaturing agents and dissipating heat gradients within the cellular microenvironment. This stress-mitigating effect may contribute to preserving the structural integrity of the cell membrane and organelles, maintaining intracellular homeostasis, and ultimately improving kinetic parameters and overall fermentation productivity. Accordingly, this study proposed the use of ZIF-8 for the immobilization of S. cerevisiae cells to evaluate its potential as a biopreservative agent under conditions of thermal stress and in the presence of inhibitory compounds derived from lignocellulosic hydrolysates. The effects of this strategy on cell viability and overall fermentative performance were also investigated. Despite the well-recognized importance of S. cerevisiae and immobilization strategies in enhancing fermentative processes, there remains a need to investigate support materials capable of providing more robust and efficient protection against thermal stress and inhibitory compounds. Such protection is essential to maintain cell viability and alcoholic fermentation performance under elevated temperature conditions. In this context, zeolitic imidazolate frameworks, such as ZIF-8, have emerged as promising candidates. The novelty of this work lies in the in-depth evaluation of ZIF-8 both as an immobilization support and as a biopreservative agent for S. cerevisiae cells. This study assessed the capacity of ZIF-8 to enhance cellular protection and optimize fermentative performance over an extended temperature range. Additionally, the efficiency of encapsulation was examined in the presence of inhibitory compounds found in lignocellulosic biomass fermentations derived from passion fruit peel, which may exert toxic effects on yeast cells. Materials and Methods MEV/EDS analysis Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) analyses were performed using a HITACHI TM3030Plus microscope. Yeast samples were vacuum-dried, mounted on aluminum stubs with conductive carbon tape, and placed in the microscope’s vacuum chamber. After adjusting the operating conditions, high-resolution images were captured at various magnifications. The analyses were performed at CAITEC, Universidade Estadual de Goiás. Microorganisms The microorganism used in this study was the Saccharomyces cerevisiae CAT-1 strain. The yeast was supplied by the Biochemical Engineering Laboratory of the Universidade Federal de Goiás. The strain was maintained on solid YPD medium composed of 10 g L⁻¹ yeast extract, 20 g L⁻¹ glucose, 20 g L⁻¹ bacteriological peptone, and 20 g L⁻¹ agar. Inoculum Preparation Cells were aseptically removed from YPD agar plates using a sterile inoculation loop and transferred into Erlenmeyer flasks containing 17.5 mL of liquid YPD medium composed of 10 g L⁻¹ yeast extract, 20 g L⁻¹ glucose, and 20 g L⁻¹ bacteriological peptone. The pH was adjusted to 5.5. Subsequently, 2.5 mL of a concentrated glucose solution (100 g L⁻¹) was added to the medium. The culture medium was sterilized by autoclaving at 121°C and then incubated at 30°C under agitation at 250 rpm for 12 hours, until the exponential growth phase was reached. ZIF 8 Preparation and Cell Encapsulation Yeast immobilization by encapsulation in ZIF-8 capsules was carried out according to the method described by Sun et al. ( 2019 ). After inoculum preparation, yeast cells corresponding to 4.5 mg of dry biomass were harvested from the YPD medium by centrifugation and washed three times with deionized water at 3000 rpm for 5 minutes each. The washed cells were then resuspended in 1 mL of an aqueous zinc nitrate solution (0.05 mol L⁻¹), followed by the addition of 1 mL of an aqueous 2-methylimidazole solution (0.2 mol L⁻¹). The reaction mixture was maintained under orbital agitation at 300 rpm and 30°C for 1 hour. Finally, the resulting encapsulated cells were collected and washed three times with deionized water. Ethanolic Fermentation with Free and Immobilized Cells at Different Temperatures Encapsulated yeast cells were cultivated under anaerobic conditions in a nutrient medium consisting of 17.5 mL of YPD medium supplemented with 2.5 mL of a concentrated glucose solution (100 g L⁻¹). The initial pH was adjusted to 5.5. Cultivation was performed in Erlenmeyer flasks under agitation at 250 rpm for 24 hours at different temperatures: 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C. Samples were collected at predetermined time intervals, appropriately diluted, and analyzed to monitor metabolite production and substrate consumption. Ethanolic Fermentation with Free and Immobilized Yeast Cells Using Lignocellulosic Biomass Derived from Passion Fruit Peel The lignocellulosic biomass underwent acid pretreatment, enzymatic hydrolysis, and fermentation. Pretreatment was performed with 2% (v/v) sulfuric acid at a 1:10 solid-to-liquid ratio and autoclaved at 121°C. After cooling, the pH was adjusted to 4.8 with citrate buffer. The suspension was filtered through cotton cloth, and the recovered solid fraction was dried in a vacuum oven at 50°C for 16 hours, reaching 7.71% final moisture content. Enzymatic hydrolysis was performed in three 250 mL Erlenmeyer flasks containing dried passion fruit peel (5.4 g), citrate buffer (45 mL, pH 4.8), and diluted enzyme solution (5 mL). The flasks were incubated at 50°C and 250 rpm for 24 hours, with samples collected between 0 and 24 hours. After centrifugation at 3000 rpm for 5 minutes, the supernatant was recovered for glucose determination. After hydrolysis, the slurry was filtered through cotton cloth to remove solids. The liquid hydrolysate (~ 120 mL) was concentrated to 20 mL in a rotary evaporator, adjusted to pH 5.5 with citrate buffer, and sterilized at 121°C. The resulting acid–enzyme hydrolysate was used as the substrate for ethanol production with free and immobilized Saccharomyces cerevisiae CAT-1 cells. Fermentations were performed in 11 mL glass flasks containing 9 mL of hydrolysate at 30°C without agitation. Free yeast fermentation lasted 24 hours, with samples collected at 12 and 24 hours. Immobilized yeast fermentations were conducted for 48 hours, with samples taken at 24 and 48 hours for analysis. After 48 hours, immobilized cells were recovered by centrifugation and washed four times with deionized water (3000 rpm, 5 min). Fresh YPD medium (10 g L⁻¹ yeast extract, 80 g L⁻¹ glucose, and 20 g L⁻¹ peptone) was added, the pH adjusted to 5.5, and the volume set to 9 mL to start a new fermentation cycle. This step evaluated cell viability and metabolic activity over an additional 30 hours, as well as the effectiveness of encapsulation after exposure to inhibitory compounds from passion fruit peel hydrolysate. Validation of Cell Viability To validate the viability assessment method, free yeast cells were treated with 25% NaCl for 24 hours to induce cell death, which was confirmed using a Neubauer counting chamber. These nonviable cells were then subjected to the immobilization process, and viability was reassessed using methylene blue staining to ensure they remained nonviable. This approach confirmed that ZIF-8 encapsulation did not interfere with the accuracy of viability determination. Cell concentration Cell biomass concentration was determined using a calibration curve established by correlating dry cell mass with absorbance at 600 nm. Eq. (1), described by Santos ( 2017 ), was applied for the CAT-1 strain: $$\:\left({X}_{CAT-1}=0.3379.ABS-0.0266\right)\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(1\right)$$ Cell viability Cell viability was determined using a Neubauer counting chamber and the methylene blue staining method (0.025%). Viable cells do not absorb the dye, whereas nonviable cells appear blue. Samples were diluted in the methylene blue solution, and a 10 µL aliquot was transferred to the Neubauer chamber for accurate cell counting. Live and dead cells were counted under an optical microscope at 40× magnification. Cell viability was calculated according to Eq. (2), as described by Ceccato-Antonini (2012). $$\:Viabilidade\:celular\:\left(\%\right)=\:\frac{número\:de\:células\:viáveis}{número\:de\:células\:contadas}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(2\right)$$ Glucose and ethanol concentration Glucose and ethanol levels were quantified by HPLC. Samples were centrifuged (1075 × g, 2 min), and the supernatant was diluted and filtered through a 0.22 µm nylon membrane before analysis. Separation was performed on a Shimadzu Prominence system equipped with an SCR-102HG Shim-pack column and pre-column. Compounds were detected using refractive index (RID-20A) and UV–Vis (SPD-20A) detectors. The column temperature was set at 50°C, and 5 mM perchloric acid was used as the mobile phase at a flow rate of 0.600 mL min⁻¹. Fermentation kinetic parameters The substrate-to-product conversion factor (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η) were calculated according to Equations ( 3 ), (4), and (5), respectively. $$\:{Y}_{p/s}\:=\:\frac{Pf-Po}{So-Sf}$$ 3 $$\:{Q}_{p}=\:\frac{Pf-Po}{tf}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(4\right)$$ $$\:{\eta\:}=\:\frac{{Y}_{p/s}\:}{\text{0,511}}\:x\:100\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left(5\right)$$ where Yₚ/ₛ (g ethanol·g substrate⁻¹) represents the substrate-to-product conversion factor; S₀ (g substrate·L⁻¹) is the initial substrate concentration; S f (g substrate·L⁻¹) is the final substrate concentration; P f (g ethanol·L⁻¹) is the final ethanol concentration; P 0 (g ethanol·L⁻¹) is the initial ethanol concentration; Qₚ (g ethanol·L⁻¹·h⁻¹) is the volumetric productivity; t f (h) is the total fermentation time; and η (%) corresponds to the fermentation efficiency. Results and discussion The results allowed to evaluate the performance of free and ZIF-8-immobilized yeasts under different fermentation conditions. The samples were collected at predetermined intervals, and the analyses were performed in triplicate. Cell growth, viability, glucose consumption, substrate-to-product conversion, ethanol yield, and fermentation efficiency were assessed for both free and immobilized yeast cultures. Morphology and composition of ZIF-8 encapsulated cells (SEM/EDS) Figure 1 a displays the typical ellipsoidal morphology of free Saccharomyces cerevisiae cells, with visible budding scars characteristic of asexual reproduction. In contrast, Fig. 1 b shows successful encapsulation with a ZIF-8 layer, forming a core–shell structure composed of yeast cells coated with ZIF-8. The micrographs reveal a uniform coating, with no partial or uncoated cells, indicating high encapsulation homogeneity. Morphological analysis identified polyhedral particles with a predominantly cubic tendency, including near-cubic and irregular shapes (Fig. 1 c). Furthermore, chemical mapping demonstrated a homogeneous distribution of zinc, oxygen, and carbon on the cell surface, confirming the presence of a continuous ZIF-8 layer surrounding individual yeast cells (Figs. 2 – 4 ). These results confirm the effective immobilization of S. cerevisiae in ZIF-8, yielding core–shell biohybrid structures with high structural integrity and surface uniformity. Effect of Temperature on Growth and Glucose Consumption The growth kinetics of yeast cells during 24 hours of fermentation are presented in Figs. 3 a and 3 b, allowing evaluation of cellular behavior under different temperatures and immobilization conditions. Prior inoculation was used as a preadaptation strategy, in which the microorganism was cultivated in a medium like that of the main process. This approach stimulates the activation of specific metabolic pathways and the synthesis of adaptation-related proteins, thereby reducing the lag phase. As a result, the onset of exponential growth is accelerated, contributing to improved growth kinetics and a more consistent fermentative process (Cunha, 2023; Hiss, 2001). This strategy facilitated a faster transition to the active growth phase, as indicated by the marked increase in cell density at the beginning of the stationary phase. The findings suggest that prior inoculation not only shortened the adaptation period but also enhanced the specific growth rate (µ). Such an effect is particularly important in industrial applications, where reduced processing time and increased productivity are highly desirable (Schuler, 2012 ). Growth kinetics (Figs. 5 a and 5 b) were further evaluated to compare the performance of free and immobilized yeast cells at different temperatures. As shown in Fig. 5 a, free yeast cells cultivated between 25 and 40°C did not exhibit a distinct lag phase, corroborating the observations reported by Pires ( 2017 ). The lag phase is typically characterized by enzyme synthesis and cellular adaptation to the cultivation medium, occurring without measurable cell division (Schmidell et al., 2001 ). Its absence in this study can be attributed to the use of a medium identical to that employed during inoculum preparation, which facilitated a rapid transition to the exponential phase and promoted an increase in the specific growth rate (µ). For immobilized yeast cells (Fig. 5 b), a delay in process onset was observed. This effect may be attributed to stress imposed by immobilization, which alters the cellular microenvironment and restricts space for growth (Pajić Lijaković et al., 2017). Despite this initial delay, essential metabolic activities were not compromised, as cell growth and the production of ethanol and carbon dioxide still occurred during fermentation. The observed lag likely corresponds to the period required for cellular adaptation to the new microenvironment and to the specific immobilization method applied. A delayed onset of cell multiplication was consistently observed in immobilized yeasts, likely resulting from physiological stress imposed by the immobilization matrix. This environment reduces the available space for growth and alters mass transfer conditions, limiting the diffusion of substrates and metabolites (Pajić Lijaković et al., 2017; Verbelen et al., 2006 ). Furthermore, modifications in the microenvironment may influence gene expression and membrane organization, triggering adaptive responses such as stress-hardening mechanisms (Gasch, 2002). The impact of the support material on cellular physiology should also be considered, as physicochemical interactions between the support surface and the cell wall can alter membrane permeability and metabolic dynamics (Loo et al., 2000 ). Bolaños Barbosa et al. (2023) investigated yeast encapsulation in alginate using 0.1 mol L⁻¹ calcium chloride as a crosslinking agent. Batch experiments demonstrated a significant reduction in cell viability and mechanical stability, which limited the reuse of the encapsulated systems. In addition, cell leakage from the polymeric matrix through pores, cell proliferation within the capsules, and gas production may further weaken capsule integrity (Simó et al., 2017 ). Immobilization may also modify the physiological and metabolic properties of cells, as well as the characteristics of the surrounding microenvironment, thereby influencing cellular kinetic behavior. In this regard, the local microenvironment is affected by solute partitioning between the liquid phase and the solid matrix, external mass transfer resistance in the liquid film, and internal diffusion limitations within the immobilization matrix (Merchant, 1987). These phenomena are described by the solute partition coefficient (Kp), the liquid film mass transfer coefficient (kL), and the effective solute diffusivity within the immobilization matrix (De). The substrate must first diffuse through the bulk liquid and cross the external liquid film surrounding the immobilized particle until it reaches the liquid–solid interface. It then diffuses through the liquid phase inside the matrix, where additional resistance arises from microcolony formation, and finally reaches the interior of the yeast cells, where metabolic reactions occur (Pilkington et al., 1998 ). Using a model of cell growth and substrate consumption of N. agilis immobilized in carrageenan gel spheres, Gooijer et al. ( 1991 ) demonstrated that external mass transfer resistance was the most sensitive parameter affecting the growth of entrapped cells. Similarly, Wang et al. ( 2020 ) showed that Candida antarctica lipase B immobilized in ZIF-8 exhibited enhanced activity and stability, while the ZIF-8 layer also regulated substrate accessibility to the enzyme interior. Several studies have reported that immobilized cells behave differently from free cells in solutions, including enhanced metabolic efficiency (Navarro & Durand, 1977 ) and alterations in metabolic pathways (Brodelius et al., 1979 ). Mattiasson et al. ( 1982 ) proposed that environmental factors such as water activity and oxygen availability play a central role in regulating the metabolism of immobilized cells. One possible explanation for the metabolic differences between immobilized and free cells is the reduced water activity within gel matrices, likely caused by increased osmotic pressure (Mattiasson et al., 1984 ). Immobilized systems generally exhibit lower water activity and limited oxygen availability, conditions that can suppress cell growth, metabolic activity, and reproduction. Galazzo and Bailey ( 1990 ) reported enhanced polyphosphate synthesis in immobilized yeasts, possibly induced by reduced water activity. Polyphosphate, an inorganic phosphate polymer, plays multiple biological roles, including energy storage, regulation of metabolic pathways, and participation in stress response and survival mechanisms in microorganisms such as bacteria and yeasts (Júnior et al., 2024). Accordingly, Shinmyo et al. ( 1982 ) reported that Bacillus amyloliquefaciens entrapped in κ-carrageenan operated under oxygen-deficient conditions, resulting in reduced cell growth and enhanced α-amylase production. When immobilized cells sustain high maintenance metabolism at the expense of growth due to reduced water activity, higher yields can be expected for reactions linked to maintenance metabolism and for oxygen-independent processes. Doran and Bailey ( 1986 ) reported that, in alcoholic fermentation with immobilized yeast, substrate consumption was higher than in fermentations with free cells, whereas cell growth and product formation (e.g., ethanol and glycerol) were lower. These findings suggest that immobilization affects microbial growth through interactions between solid support and the cells, as well as cell-to-cell interactions. According to the authors, although the microenvironment of immobilized yeasts—characterized by concentration gradients and osmotic pressure—affects cellular metabolism, reduced growth is primarily attributed to direct physical contact with the immobilization material, whether solid or gel. This contact induces morphological changes and alters cell wall protein expression, thereby impacting overall metabolism. From a kinetic perspective, the transition from free to immobilized cells can alter the relationship between the maximum specific growth rate (µ max ) and the saturation constant (Ks), often reducing µ max due to additional diffusional resistances imposed by the support. In the present study, free cells-initiated growth immediately after inoculation, whereas immobilized cells exhibited a lag phase of up to 8 hours before significant growth, particularly at 25–35°C. These findings suggest that immobilization affects not only kinetic parameters but also physiological aspects of yeast cells, directly influencing their metabolic performance and growth (Kovaleski, 2019 ). During the exponential phase, rapid cell multiplication and biomass increase were observed. Free yeast cells cultivated at 25, 30, and 35°C reached concentrations of 2.83, 3.49, and 3.32 g L⁻¹ after 24 h, respectively. In contrast, temperatures ≥ 40°C significantly reduced the specific growth rate, leading to cell death at 45 and 50°C. For immobilized cells, although growth ceased at these temperatures, viability was maintained due to the protective effect of ZIF-8. Similar findings were reported by Lin et al. ( 2012 ) for Saccharomyces cerevisiae BY4742, which showed markedly reduced growth and ethanol production at 50°C. This effect is attributed to thermal stress, as high temperatures induce structural alterations in proteins and plasma membranes, impairing growth, viability, and fermentative capacity (Naves et al., 2010 ). For immobilized yeast cells (Fig. 5 b), the exponential phase began after approximately 8 h of fermentation, with significant growth observed at 25–35°C. After 24 h, cell concentrations reached 3.30, 3.56, and 3.44 g L⁻¹, respectively. Substrate consumption was maximal during this phase, resulting in complete glucose depletion by 24 h, as it was utilized to meet metabolic demands. Each glucose molecule yields two ATP molecules, supplying the energy required for cellular functions (Schmidell et al., 2001 ). Accordingly, glucose consumption reached 100% after 24 h of fermentation (Figs. 6 a and 6 b). Analysis of Figs. 6 a and 6 b shows glucose depletion by the end of the exponential phase for both free and immobilized cells, increasing linearly up to 24 h at 25–35°C (Figs. 5 a and 5 b). In contrast, glucose consumption was markedly reduced at 45 and 50°C in both systems. This reduction reflects the direct effect of temperature on microbial metabolic activity, a key factor in fermentative performance (Lima, 2009 ). Caspeta et al. ( 2014 ) reported that at temperatures above 36°C, yeasts activate the heat stress response, a transcriptional defense mechanism that disrupts carbohydrate metabolism and plasma membrane composition. Although Saccharomyces cerevisiae , a mesophilic yeast, can grow at temperatures up to approximately 42°C, prolonged exposure to higher temperatures compromises cell viability (Yamamoto et al., 2008 ). Neves ( 2018 ) reported that glucose consumption is reduced during the lag phase, as yeast cells adapt to the medium and reorganize their enzymatic machinery in preparation for logarithmic growth. Similarly, Brito ( 2019 ) observed negligible initial sugar consumption in S. cerevisiae immobilized in calcium alginate, indicating a lag period required for adaptation to the new system. This behavior aligns with the classical microbial growth curve, in which the lag phase represents the time needed for metabolic adjustment to new environmental conditions. Although the lag period is inherently related to cellular adaptation, mass transfer of substrates and products through polymeric support is another key factor influencing immobilization efficiency. Coutinho ( 2020 ) highlighted limited diffusion as a critical parameter in biocatalyst encapsulation, since the porous barrier between the microorganism and the fermentative medium may restrict compound transport into and out of the matrix, impairing fermentation performance. Nevertheless, the present results indicate that despite the initial adaptation phase, immobilization did not compromise overall fermentation efficiency as shown in Table 1 . In addition to diffusion limitations imposed by the polymeric matrix, cell density within the support is a crucial factor affecting immobilization efficiency (Kovaleski, 2019 ). In that study, Saccharomyces cerevisiae immobilized in calcium alginate at densities of 1 × 10⁶, 8 × 10⁶, and 2 × 10⁷ cells mL⁻¹ showed distinct sucrose consumption profiles over 54 h of alcoholic fermentation. Beads with the lowest density reached 71% sucrose consumption, with a lag phase during the first 6 h. In contrast, higher densities resulted in lower consumption rates (11% and 29%), with a lag phase observed in one case. These findings suggest that increased cell density promotes internal nutrient competition, limiting growth and impairing substrate consumption and ethanol production (Cha et al., 2014 ). Although high cell density may impose metabolic constraints, immobilization also induces physiological changes in yeast cells. Some of these changes enhance fermentative performance, whereas others may be detrimental. Given that S. cerevisiae naturally adheres to surfaces in its native habitats, immobilization can be considered a growth mode closer to natural conditions, potentially providing protection against environmental stresses (Verstrepen and Klis, 2006 ). However, even under conditions closer to the natural environment, the negative effects observed are mainly related to stress caused by restricted mass transfer. In gel entrapment and inert supports, diffusional limitations depend on the immobilization method. In adsorption-based systems, resistance occurs primarily at the carrier–medium interface, with minimal internal limitations. In encapsulation methods, internal mass transfer is governed by the chemical and mechanical properties of the matrix, such as size, porosity, and texture. Verbelen et al. ( 2006 ) cautioned that physiological changes should not be attributed solely to immobilization, as these effects are difficult to separate from mass transfer limitations. Accordingly, immobilization strongly influences yeast plasma membrane properties and may alter specific solute transport systems (Shen et al., 2003 ). Comparisons between immobilized and free cells show activation of energy metabolism in immobilized systems, along with increased accumulation of storage polysaccharides (trehalose and glycogen) and structural polysaccharides (glucan and mannan) (Galazzo & Bailey, 1990 ; Jirku et al., 2000 ). Their accumulation enhances resistance to environmental stress, supports the production of industrially relevant metabolites, and improves viability under adverse conditions. In S. cerevisiae , trehalose and glycogen accumulate in response to unfavorable growth conditions, serving as energy reserves and stress protectants. These compounds are essential for cell cycle progression under reduced growth rates and carbon limitation. Singer and Lindquist ( 1998 ) demonstrated that trehalose protects cells during thermal shock by preventing protein denaturation and aggregation, underscoring its key role in cellular protection. Fermentation Performance and Kinetic Parameters The ethanol concentrations and principal kinetic parameters obtained during fermentation are summarized in Table 1 . Comparative analysis of these parameters highlights significant differences in the fermentative performance of Saccharomyces cerevisiae under the tested conditions, for both free and ZIF-8-immobilized cells. Overall, immobilized yeasts exhibited superior performance within the temperature range of 25–35°C, particularly regarding final ethanol concentration, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η). Table 1 Fermentation kinetic parameters, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η), of free Saccharomyces cerevisiae cells and cells immobilized in ZIF-8 after 24 hours of fermentation. Temperature 25ºC 30ºC 35ºC 40ºC 45ºC 50ºC Final ethanol (g.L − 1 ) Lev. Free 10,23 12,89 12,05 7,54 0 0 Final ethanol (g.L − 1 ) Lev. Immobilized 12,29 15,71 13,32 0,32 0 0 Y P/S (g.L − 1 ) Lev. Free 0,306 0,403 0,377 0,236 0 0 Y P/S (g.L − 1 ) Lev. Immobilized 0,371 0,479 0,401 0,075 0 0 Qp (g.L −1 .h −1 ) Lev. Free 0,426 0,537 0,482 0,314 0 0 Qp (g.L −1 .h −1 ) Lev. Immobilized 0,512 0,655 0,555 0,133 0 0 η (%) Lev. Free 59,88 78,86 73,77 46,18 0 0 η (%) Lev. Immobilized 72,6 93,74 78,47 14,68 0 0 At temperatures considered optimal for yeast metabolism (25 and 30°C), immobilization significantly increased the final ethanol concentration, with gains ranging from 10.54% to 21.89%. This improvement was accompanied by higher substrate-to-product yields (Yₚ/ₛ), which increased by 6.4% to 21.2% under these conditions. These findings suggest that the ZIF-8 matrix enhances cell viability and metabolic performance, possibly by reducing environmental stress and allowing greater operational stability and cell reuse. According to Nedović et al. ( 2014 ), immobilized cells often exhibit higher glycolytic flux than free cells, resulting in faster glucose consumption and more efficient channeling of substrate toward biomass and ethanol production. The present results support this observation, as the increased ethanol production by immobilized yeasts indicates a shift in fermentative metabolism. This shift may stem from growth limitations imposed by the ZIF-8 matrix, which restricts cell expansion and redirect carbon flux preferentially toward fermentative pathways rather than biosynthesis. Such microenvironmental constraints associated with immobilization may be related to polymer network relaxation around cell aggregates, mechanical forces generated during cell growth within the capsule, and physicochemical interactions among the solvent, matrix, and cells (Pajić Lijaković et al., 2012). This metabolic modulation is reflected in the average fermentative productivity (Qₚ), which showed a similar improvement and reached a maximum of 0.655 g L⁻¹ h⁻¹ for immobilized yeasts at 30°C, approximately 22% higher than for free cells. From an industrial perspective, this increase is significant, as productivity determines the rate of product formation and directly impacts cycle time and process economics. Consistent with these findings, fermentation efficiency further confirms the superiority of immobilized yeasts under the evaluated conditions. While free cells showed efficiencies of 59.88–78.86% at 25–35°C, immobilized cells reached 72.60-93.74%. At 30°C, efficiency (~ 91%) approached the average value reported for industrial operations (Abreu Cavalheiro & Monteiro, 2013), underscoring the robustness and operational stability of the ZIF-8 system. Overall, these results indicate that immobilization enhances conversion efficiency and productivity while increasing biocatalyst resilience. Conversely, increasing the temperature to 40°C sharply reduced fermentative performance, with significant declines in final ethanol concentration, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and efficiency (η) in both systems. This result indicates that temperatures above the physiological optimum impair plasma membrane integrity, enzymatic stability, and intracellular homeostasis, leading to metabolic inhibition and loss of viability. At 45 and 50°C, fermentative activity was completely suppressed, indicating that these temperatures exceed the thermal tolerance of the strain, regardless of immobilization. Mattiasson et al. ( 1982 ) reported improved ethanol productivity and yield using immobilized cells. For free cells in solution, the theoretical maximum ethanol yield from glucose is 51%; however, adsorbed cells have achieved substantially higher yields (Navarro & Durand, 1977 ), likely due to alterations in metabolic pathways. In the immobilized state, cells are confined within a polymer-rich environment whose macromolecules can organize water and reduce its availability. Even moderate polymer concentrations can induce distinct metabolic effects. For example, adding only 0.5% sodium alginate to batch fermentation with Saccharomyces cerevisiae markedly altered cellular productivity (Holcberg & Margalith, 1981 ). To evaluate the effect of reduced water activity on S. cerevisiae metabolism, batch fermentations were performed at water activities as low as 0.986 by adding increasing concentrations of dextran and polyethylene glycol (Hahn-Hägerdal et al., 1982 ). Under these conditions, the initial ethanol production rate increased by 50%. Similarly, yeast cells adsorbed onto porous glass showed a 25% increase in ethanol production rate. Cell Viability and Thermal Stress The encapsulation process aims to preserve the viability and metabolic functionality of microorganisms by creating a favorable microenvironment for fermentation development. In this context, the use of encapsulation proved to be promising in the present study, as yeasts immobilized in ZIF 8 exhibited superior performance compared to free cells. This improvement was particularly evident in terms of enhanced thermal stability, which allowed satisfactory productivity and fermentation efficiency. This operational advantage may be associated with the intrinsic properties of ZIF 8, whose thermal stability and behavior have been well documented. Fang et al. ( 2015 ) investigated the thermal stability and degradation behavior of ZIF 8 nanocrystals using thermogravimetric analysis under a nitrogen atmosphere and demonstrated that pure ZIF 8 crystals exhibit degradation temperatures above 600°C. This characteristic is especially relevant for applications involving temperature fluctuations, such as gas flow in reactors or industrial ducts, as it ensures durability and structural integrity under severe conditions. Other studies corroborate this thermal robustness. Sung et al. (2019) reported stability up to 550°C, while Yang et al. (2013) observed excellent thermal and separation stability of ZIF 8 PBI nanocomposites under elevated temperatures and hot vapor exposure of up to 230°C during ten days of continuous operation. This stability was a determining factor in the results obtained in the present study, as illustrated in Figs. 7 a and 7 b, which show the evolution of cell viability of free and ZIF 8 immobilized Saccharomyces cerevisiae over time at different temperatures. Immobilization was effective in maintaining stable cell viability throughout the fermentative process, even under more severe thermal conditions. It is important to note that while the capsule protects cellular integrity, it does not necessarily ensure maximum metabolic activity under highly stressful conditions. After 24 hours of cultivation, immobilized yeasts exhibited cell viability values of 98.4 percent, 97.7 percent, and 96.7 percent, respectively. In contrast, free cells showed more pronounced reductions, reaching 93.7 percent, 88.9 percent, and 82.9 percent. This behavior was expected, considering that the thermal stability of ZIF 8 greatly exceeds the tolerance limits of the yeast itself, allowing it to function as a physical and protective barrier against thermal stress. These findings are consistent with the observations of Torija et al. ( 2003 ), who reported that the initial fermentation rate is maximized between 25°C and 31°C, while cell viability declines significantly above 35°C, particularly in unprotected cultures. Thus, the thermal resistance provided by the ZIF 8 matrix proved sufficient to mitigate the negative effects of heat on encapsulated cells under thermal stress. The protective effect of immobilization against thermal shock is also supported by physiological studies on yeast. Scientific evidence indicates that cells in the logarithmic phase of rapid growth are more sensitive to thermal stress than slowly dividing cells or those in the stationary phase (Washburne et al., 1993 ; Lu et al., 2009 ). Nagarajan et al. ( 2014 ) compared the thermotolerance of S. cerevisiae confined within or released from calcium alginate matrices with planktonic cells at different physiological stages following exposure to 48°C for two hours. The authors observed that planktonic and immobilized cells in the stationary phase were significantly more tolerant to thermal shock than planktonic cells in the logarithmic phase. The greater resistance observed in immobilized cells resulted not only from their physiological state but also from the physical protection provided by the matrix, as evidenced by the intermediate survival of cells released from the beads. Furthermore, when immobilized, cells ceased division but remained metabolically active and highly fermented, maintaining viability for up to seventeen weeks with viability levels exceeding 95 percent. The relationship between growth rate and heat resistance was further explored by Lu et al. ( 2009 ), who demonstrated that genes involved in the heat stress response and previously associated with thermotolerance exhibit higher expression in slowly growing cells. To test this hypothesis, the authors cultivated cells at different growth rates under steady state conditions in chemostats at 50°C, a lethal temperature for yeast cells growing in rich media. Cells with a doubling time of 2.8 hours exhibited high sensitivity, whereas cells with a doubling time of 13.8 hours were resistant. Cultures with intermediate growth rates, corresponding to a doubling time of 4.6 hours, showed intermediate mortality. As a control, cells cultivated at the intermediate growth rate but at 36°C exhibited resistance like that of slow growing cells. These results indicate that growth deceleration, a characteristic observed in immobilized cells, promotes gene expression patterns that contribute to the enhanced thermal resistance observed in the present work. Performance in Lignocellulosic Hydrolysate and Tolerance to Inhibitors After acid pretreatment, passion fruit peels underwent enzymatic hydrolysis at time intervals of 0, 0.5, 1, 2, 4, 8, and 24 hours. As shown in Fig. 8 , glucose concentration progressively increased during the process, reaching 32.53 g L⁻¹ after 24 hours. Following hydrolysis, the filtrate (approximately 120 mL) was concentrated using a rotary evaporator to enhance sugar content, reducing the volume to 20 mL. Citrate buffer (pH 5.5) was then added to adjust the pH, resulting in a final glucose concentration of 62.05 g L⁻¹. The hydrolysate was subsequently sterilized by autoclaving at 121°C. The passion fruit peel hydrolysate was used as the fermentation medium for alcoholic fermentation by Saccharomyces cerevisiae CAT-1, employing both free and immobilized cells. The results for cell growth, substrate consumption, ethanol production, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η) are summarized in Tables 2 and 3 . This procedure was designed to evaluate, over an additional 30-hour period, the metabolic activity and viability of the immobilized yeasts, as well as the effectiveness of the ZIF-8 capsule after exposure to inhibitory compounds commonly present in lignocellulosic hydrolysates derived from passion fruit peel, which may exert toxic effects on yeast cells. The results of these tests are shown in Table 4 . Edwards and Doran-Peterson ( 2012 ) reviewed the main pectin-rich residues—such as those generated from citrus, sugar beet, and apple processing—that can be used for ethanol production. According to the authors, this type of biomass is characterized by low lignin content and a high pectin fraction, typically ranging from 12% to 35%. However, a major limitation in using these residues for ethanol production is that galacturonic acid and arabinose are not metabolized by microorganisms traditionally employed in alcoholic fermentation. This constraint helps explain the low fermentative efficiency observed with Saccharomyces cerevisiae , since passion fruit peel contains, on average, approximately 25% pectin (Júnior et al., 2006 ). In addition to its inability to ferment pentoses, this yeast is also incapable of metabolizing the galacturonic acid released during biomass hydrolysis, further limiting the conversion efficiency of pectin-rich substrates into ethanol. Table 2 Cell growth, substrate consumption, ethanol production, and fermentation kinetic parameters of Saccharomyces cerevisiae immobilized in ZIF-8 during fermentation using lignocellulosic biomass derived from passion fruit peel. Time (h) Cell growth (g/L) Glucose (g/L) Final ethanol (g/L) Y P/S (g.L − 1 ) Qp (g.L −1 .h −1 ) η (%) Viability (%) 0 hours 2,5 62,05 0 0 0 0 100 12 hours 2,66 51,92 2,70 0,266 0,225 52,05 68,59 24 hours 2,67 44,24 6,80 0,381 0,283 74,56 19,89 Table 3 Cell growth, substrate consumption, ethanol production, and fermentation kinetic parameters (substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η)) of Saccharomyces cerevisiae immobilized in ZIF-8 during fermentation using lignocellulosic biomass derived from passion fruit peel. Time (h) Cell growth (g/L) Glucose (g/L) Final ethanol (g/L) Y P/S (g.L − 1 ) Qp (g.L −1 .h −1 ) η (%) Viability (%) 0 hours 2,5 62,05 0 0 0 0 100 24 hours 2,5 57,63 0 0 0 0 100 48 hours 2,5 56,09 0 0 0 0 99,42 Table 4 Cell growth, substrate consumption, ethanol production, and fermentation kinetic parameters (substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η)) of Saccharomyces cerevisiae immobilized in ZIF-8 during fermentation in YPD medium. Time (h) Cell growth (g/L) Glucose (g/L) Final ethanol (g/L) Y P/S (g.L − 1 ) Qp (g.L −1 .h −1 ) η (%) Viability (%) 0 hours 2,5 85,00 0 0 0 0 100 30 hours 3,5 61,03 10,60 0,442 0,353 86,54 100 When lignocellulosic hydrolysates are used as feedstock, significant amounts of pentose sugars, such as D-xylose and L-arabinose, are released from hemicellulose (Azhar et al., 2017 ). Ndubuisi et al. (2020), working with the yeast Pichia kudriavzevii LC375240, reported that almost no ethanol was produced in pentose-based media due to limited cell growth. Their findings suggest that although the strain was able to assimilate pentose sugars to support growth, it was unable to efficiently ferment them into ethanol. Acid hydrolysis generates not only sugars but also toxic byproducts that inhibit fermentative microorganisms. These include organic acids (acetic, formic, and levulinic acids), furan derivatives (furfural and 5-hydroxymethylfurfural), and phenolic compounds (Chandel et al., 2007 ). Acetic acid originates from hemicellulose, furans from pentose and hexose degradation, and formic and levulinic acids from further furan breakdown (Martín et al., 2007 ). Lignin is the main source of phenolic compounds, which are considered stronger inhibitors than sugar degradation products (Jönsson et al., 2013 , Palmqvist and Hahn Hägerdal, 2000; Rasmussen et al., 2014 , Jönsson et al., 1998 ). Inhibitory compounds formed during biomass hydrolysis reduce ethanol yield and productivity by impairing microbial metabolism and carbohydrate uptake (Almeida et al., 2007 ). Furans such as hydroxymethylfurfural and furfural damage DNA, inhibit RNA and protein synthesis, and interfere with key enzymes involved in ethanol production (Modig et al., 2002 ). Their toxicity is mainly linked to reactive aldehyde groups that disrupt cellular components and membranes (Miller et al., 2010 ). Organic acids (acetic, formic, and levulinic) inhibit cells in their undissociated forms by diffusing across membranes, while lignin-derived phenolic compounds compromise membrane integrity and enzyme function (Mills et al., 2009, Parawira et al., 2011). The type and concentration of inhibitory compounds formed during pretreatment depend on the feedstock and the severity of processing conditions (Jönsson et al., 2013 ; Ko et al., 2015 ). As a result, purification strategies to reduce inhibitor levels before fermentation are highly important. In general, fermentation of untreated hydrolysates leads to slow kinetics, low productivity, and reduced ethanol yields (Mussatto et al., 2004). Fermentation converts sugars released during pretreatment and hydrolysis into ethanol. However, the absence of microorganisms capable of efficiently fermenting both hexoses and pentoses into ethanol limits the economic viability of cellulosic ethanol production (Nichols et al., 2014 ). Although research has advanced in developing wild-type and genetically engineered strains to address this issue, challenges such as incomplete pentose utilization, low ethanol tolerance, and sensitivity to pretreatment-derived inhibitors persist (Kumar et al., 2016 ). According to Milessi et al. ( 2020 ), cell immobilization on solid supports is a promising strategy to overcome fermentation challenges, as it creates a protective microenvironment that shields yeast from inhibitors present in hydrolysates. This concept supports the use of ZIF-8 immobilization applied in the present study. In addition to enhanced protection, immobilization enables easy recovery and reuse of cells, operation at high cell densities for extended periods, and reduced biomass handling requirements. It also facilitates the industrial application of genetically modified microorganisms, particularly in regions with strict biosafety regulations (Kourkoutas et al., 2004 , Milessi et al., 2020 ). Fermentations using passion fruit, banana, and coconut hydrolysates commonly show lower ethanol production compared to media containing pure glucose. For example, Zymomonas mobilis produced less ethanol when hydrolysates were used as the carbon source. This reduction is attributed to the presence of inorganic compounds and other inhibitory substances in hydrolysates, which can interfere with microbial metabolism (Doelle and Doelle, 1990 ). Non-purified sugar media may contain impurities that inhibit ethanol production and suppress enzymatic activity, thereby reducing overall fermentation efficiency (Khoja et al., 2015 ). Rosa (2021) evaluated fermentation of orange bagasse hydrolysate using Saccharomyces cerevisiae G2 104 immobilized in calcium alginate and observed low ethanol yields. After 12 hours, ethanol concentrations were 2.57 g L⁻¹ (enzymatic hydrolysate), 1.69 g L⁻¹ (acid hydrolysate), and 4.9 g L⁻¹ (enzymatic hydrolysate without hydrothermal pretreatment). These findings indicate that enzymatic hydrolysates provide better fermentative performance, likely due to the absence of inhibitory compounds commonly formed during acid hydrolysis. Acid hydrolysates showed reduced sugar consumption and lower ethanol production, probably due to inhibitor formation and generation of non-fermentable sugars. However, acid hydrolysis can still be effective for sugar release if combined with detoxification and supplementation strategies. To assess the protective effect of the ZIF-8 capsule against inhibitory compounds present in passion fruit peel hydrolysate, encapsulation was evaluated as a protective strategy. The results (Tables 2 and 3 ; Figs. 9 a and 9 b and 10 ) show clear differences between free and immobilized S. cerevisiae . Immobilization effectively preserved cell viability throughout fermentation. After 48 hours at 30°C without agitation, immobilized cells maintained 99.42% viability, while free cells showed a sharp decline to 69.59% at 12 hours and 19.89% at 24 hours. As a result, free-cell fermentations produced low ethanol concentrations (maximum of 6.8 g L⁻¹). In contrast, immobilized cells consumed less glucose, and ethanol productivity was not determined, suggesting that glucose was mainly used for cell maintenance rather than ethanol production. The enhanced tolerance of immobilized yeasts to toxic compounds may be linked to osmotic stress responses, which stimulate intracellular polyol production for osmotic regulation. This mechanism reduces water activity and increases resistance to harmful chemical substances (Norton and D’Amore, 1994 ). Furthermore, data from Tables 3 and 4 confirm the effectiveness of encapsulation as a protective strategy. Immobilized cells remained metabolically active and viable after medium replacement for an additional 30 hours. Fermentation efficiency values reinforced this observation, with immobilized yeasts reaching an efficiency of 86.54%, demonstrating superior stability under the evaluated conditions. Conclusion Fermentations conducted at 25–50°C showed clear differences between free and ZIF-8-immobilized yeasts. ZIF-8 acted as a protective barrier, improving cell integrity and fermentative performance at optimal temperatures (25–35°C), with higher Yₚ/ₛ, Qₚ, and η values compared to free cells. The capsule also maintained high viability at elevated temperatures, reaching 96.7% at 50°C after 24 hours, although ethanol production was suppressed above 40°C in both conditions. In fermentations using passion fruit peel hydrolysate, ZIF-8 preserved cell viability against inhibitory compounds; however, ethanol yield remained limited due to the hydrolysate composition, including pentoses, phenolics, organic acids, furfural, and hydroxymethylfurfural. Overall, immobilization in ZIF-8 proved to be an effective biopreservation strategy, enhancing cell protection, robustness, and potential reuse. Declarations Author Contribution C.N.P. conceived and designed the research project and supervised the M.Sc. student L.C.C.G., particularly regarding the immobilization of Saccharomyces cerevisiae in ZIF-8 for application in alcoholic fermentation of lignocellulosic biomass derived from passion fruit peel. C.A.G.S. co-supervised the student and directly guided most of the experimental work involving the yeast. L.C.C.G. performed all laboratory experiments and contributed to data organization and manuscript preparation. G.L.C. conducted and interpreted all high-performance liquid chromatography (HPLC) analyses. All authors reviewed and approved the final version of the manuscript. Acknowledgement The authors gratefully acknowledge the financial support provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG). 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Int Sugar J 109:33–39 Miller EN, Turner PC, Jarboe LR, Ingram LO (2010) Genetic changes that increase 5-hydroxymethylfurfural resistance in ethanol-producing Escherichia coli LY180. Biotechnol Lett 32:661–667 Milessi TS, Perez CL, Zangirolami TC, Corradini FAS, Sandri JP, Foulquié-Moreno MR, Thevelein JM, Giordano RC, Giordano RLC (2020) Repeated batches as a strategy for high-concentration 2G ethanol production from non-detoxified hemicellulose hydrolysate using immobilized recombinant Saccharomyces cerevisiae cells in a fixed-bed reactor. Biotechnol Biofuels 13:85 Modig T, Lidén G, Taherzadeh MJ (2002) Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem J 363:769–776 Mussatto SI, Roberto IC (2004) Evaluation of different types of activated carbon in the detoxification of rice straw hydrolysate for xylitol production. 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Master’s Dissertation, Federal University of Grande Dourados, Faculty of Exact Sciences and Technology, Dourados, Brazil Nichols NN, Hector RE, Saha BC, Frazer SE, Kennedy GJ (2014) Biological reduction of inhibitors in rice husk hydrolysate and fermentation to ethanol using conventional and engineered microbes. Biomass Bioenergy 67:79–88 Norton S, D’Amore T (1994) Physiological effects of yeast cell immobilization: Applications in brewing. Enzym Microb Technol 16:365–375. https://doi.org/10.1016/0141-0229(94)90150-3 Oliveira AP (2018) Food industry waste for flour preparation: A strategy for utilization. Undergraduate Thesis, Federal University of Paraíba, João Pessoa, Brazil Pajić-Lijaković I, Bugarski B, Plavšić M, Lević S, Kalušević A, Nedović V (2012) Microenvironmentally restricted yeast cell growth on calcium alginate microspheres. Engineering 4:180–183. https://doi.org/10.4236/eng.2012.410B047 Pajić-Lijaković I, Milivojević M, Lević S, Trifković K, Stevanović-Dajić Z, Radošević R, Bugarski B (2017) Matrix resistance stress: A key parameter for immobilized cell growth regulation. Process Biochem 52:30–43 Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition. Bioresour Technol 74:25–33 Patrascu E, Rapeanu G, Hopulele T (2009) Current approaches to efficient biotechnological production of ethanol. Innovative Romanian Food Biotechnol 4:1–11 Phisalaphong M, Srirattana N, Tanthapanichakoon W (2006) Mathematical modeling to investigate temperature effect on kinetic parameters of ethanol fermentation. Biochem Eng J 28:36–43. https://doi.org/10.1016/j.bej.2005.08.039 Pilkington PH, Margaritis A, Mensour NA (1998) Mass transfer characteristics of immobilized cells used in fermentation processes. Crit Rev Biotechnol 18:237–255. https://doi.org/10.1080/07388559891224239 Pires JMC (2017) Crabtree effect in Saccharomyces cerevisiae and its modulation by titanium dioxide nanoparticles. Doctoral Thesis, University of Évora, Évora, Portugal Rasmussen H, Sørensen HR, Meyer AS (2014) Formation of degradation compounds from lignocellulosic biomass in the biorefinery: Sugar reaction mechanisms. Carbohydr Res 385:45–57 Ribeiro E, Reis H (2009) Joint influence of pH, temperature and sulfite concentration in alcoholic fermentation of sucrose musts. In: Proceedings of the IX Internal Meeting and XIII Seminar of Scientific Initiation , Federal University of Uberlândia, Uberlândia, Brazil. Available at: https://repositorio.ufu.br/handle/123456789/15135 Rogacka J, Labus K (2025) Metal-organic frameworks as highly effective platforms for enzyme immobilization – current developments and future perspectives. Braz J Chem Eng 42:1273–1301. https://doi.org/10.1007/s43153-024-00513-4 Santos MV (2017) Study of the competence of Saccharomyces cerevisiae in co-culture for ethanol production. Master’s Dissertation, Federal University of Goiás, Graduate Program in Chemical Engineering, Goiânia, Brazil Schmidell W, Facciotti MCR, Reginatto V (2001) Bioreactors and fermentative processes. In: Schmidell W, Lima UA, Aquarone E, Borzani W (eds) Industrial Biotechnology: Biochemical Engineering. Edgard Blücher, São Paulo, Brazil, pp 179–192 Shen H-Y, Moonjai IN, Verstrepen KJ, Delvaux FR (2003) Impact of attachment immobilization on yeast physiology and fermentation performance. J Am Soc Brew Chem 61:79–87 Shen L, Wang Y-T, Tang X-X, Wang K, Wang PM, Sui Y, Zheng DQ (2020) Heat shock causes genomic instability and phenotypic variations in yeast. AMB Express 10:101. https://doi.org/10.1186/s13568-020-01091-7 Shinmyo A, Kimura H, Okada H (1982) Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens . Eur J Appl Microbiol Biotechnol 14:7–12 Schuler MM, Marison IW (2012) Real-time monitoring and control of microbial bioprocesses focusing on specific growth rate: Current status and perspectives. Appl Microbiol Biotechnol 94:1469–1482. https://doi.org/10.1007/s00253-012-4095-z Simó G, Fernández-Fernández E, Vila-Crespo J, Ruipérez V, Rodríguez-Nogales JM (2017) Progress in research on alginate gel polymer coating techniques for cell encapsulation. Carbohydr Polym 170:1–14 Singer MA, Lindquist S (1998) Multiple effects of trehalose on protein folding, in vitro and in vivo. Mol Cell 1:639–648 Sun C, Chang L, Hou K, Liu S, Tang Z (2019) Encapsulation of live cells by metal-organic frameworks for viability protection. Sci China Mater 62:885–891. https://doi.org/10.1007/s40843-018-9384-8 Tanabe KK, Cohen SM (2011) Post-synthetic modification of metal-organic frameworks – a progress report. Chem Soc Rev 40:498–519. https://doi.org/10.1039/C0CS00031K Taylor J (2001) Microorganisms and Biotechnology. Nelson Thornes, Cheltenham, UK Torija MJ, Poblet M, Novo M, Beltran G, Guillamón JM, Mas A, Rozès N (2003) Effects of fermentation temperature and Saccharomyces species on the cell fatty acid composition and presence of volatile compounds in wine. Int J Food Microbiol 85:127–136 Velvizhi G et al (2022) Integrated biorefinery processes for conversion of lignocellulosic biomass to value-added materials: Paving a path towards circular economy. Bioresour Technol 343:126151. https://doi.org/10.1016/j.biortech.2021.126151 Verbelen PJ, De Schutter DP, Delvaux F, Verstrepen KJ, Delvaux FR (2006) Immobilized yeast cell systems for continuous fermentation applications. Biotechnol Lett 28:1515–1525 Verstrepen KJ, Klis FM (2006) Flocculation, adhesion and biofilm formation in yeasts. Mol Microbiol 60:5–15 Washburne MW, Braun E, Johnston GC, Singer RA (1993) Stationary phase in the yeast Saccharomyces cerevisiae . Microbiol Rev 57:383–401 Wang Y, Zhang N, Tan D, Qi Z, Wu C (2020) Facile synthesis of enzyme-embedded metal-organic frameworks for size-selective biocatalysis in organic solvent. Front Bioeng Biotechnol 8:714. https://doi.org/10.3389/fbioe.2020.00714 Yamamoto N, Maeda Y, Ikeda A, Sakurai H (2008) Regulation of thermotolerance by stress-induced transcription factors in Saccharomyces cerevisiae . Eukaryot Cell 7:783–790. https://doi.org/10.1128/EC.00029-08 Yang T, Chung T-S (2013) High-performance ZIF-8/PBI nanocomposite membranes for high-temperature hydrogen separation consisting of carbon monoxide and water vapor. Int J Hydrog Energy 38:229–239. https://doi.org/10.1016/j.ijhydene.2012.10.045 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 26 Mar, 2026 Reviews received at journal 25 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 02 Mar, 2026 Editor assigned by journal 02 Mar, 2026 Submission checks completed at journal 21 Feb, 2026 First submitted to journal 20 Feb, 2026 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. 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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-8926210","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600824904,"identity":"922af1c6-fe3a-4cb5-838d-9c4c19e733c8","order_by":0,"name":"Luana Cristina Camargos Gomes","email":"","orcid":"","institution":"Federal University of Goiás","correspondingAuthor":false,"prefix":"","firstName":"Luana","middleName":"Cristina Camargos","lastName":"Gomes","suffix":""},{"id":600824905,"identity":"37af4aa2-4b5f-4833-bc65-d9fb1490f737","order_by":1,"name":"Gabriel Luis Castiglioni","email":"","orcid":"","institution":"Federal University of Goiás","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"Luis","lastName":"Castiglioni","suffix":""},{"id":600824906,"identity":"ff077c0f-2a4e-4d13-8654-aa7785fbaaa0","order_by":2,"name":"Carlos Alberto Galeano Suarez","email":"","orcid":"","institution":"Federal University of Goiás","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Alberto Galeano","lastName":"Suarez","suffix":""},{"id":600824907,"identity":"99f0dc47-c3b6-4291-af0e-b53ec22d7d22","order_by":3,"name":"Caridad Noda Perez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYPACZgZ+ZuYDDBUMDAnEa5Fsb0tgOEOSFoOeMwbEaeFvP/tMuqDCWt5AIuebxIFfDHnmDQS0SJxJN5OecSbdcLtE7jaJg30MxTIHCGgxYEhjk+ZtO8y4c0buNumPPQyJMwg5zID/GViL/YYbOc8kDhKlRQJiS+KGM2fYJA78IEKLxI1nzNY8Z9KTZ7a3GVscbJAoliCkhb8/jfE2T4W1bT8z88MbB/7Y5BHUAgQsCEWMbcRoAEbjBwT7D1E6RsEoGAWjYIQBAOAFQJTqPUK1AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Goiás","correspondingAuthor":true,"prefix":"","firstName":"Caridad","middleName":"Noda","lastName":"Perez","suffix":""}],"badges":[],"createdAt":"2026-02-20 13:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8926210/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8926210/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103972965,"identity":"3c36d460-13f1-4acf-b828-2bc09744be09","added_by":"auto","created_at":"2026-03-05 08:04:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106795,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) images: (a) free yeast cells, UD10.1x3.0 k, 30 mm; (b) yeast cells immobilized in ZIF-8, UD10.3x2.5k, 30 mm; and (c) ZIF-8 capsules, UD10.3x600k, 100 mm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/42d3185a41b4b82ead884a02.png"},{"id":103972967,"identity":"683c4836-c440-408d-b58b-3db3012499da","added_by":"auto","created_at":"2026-03-05 08:04:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104081,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy-dispersive spectroscopy (EDS) analysis of free yeast cells.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/c1931f7308010a90db46f10b.png"},{"id":104402275,"identity":"b5beac0a-57b1-4c59-8b6e-1140e895dc9c","added_by":"auto","created_at":"2026-03-11 12:14:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109892,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy-dispersive spectroscopy (EDS) analysis of yeast cells immobilized in ZIF-8.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/c350ec9fe650504dea71b776.png"},{"id":104402505,"identity":"ee8cf930-a644-49ad-b180-6668693789f0","added_by":"auto","created_at":"2026-03-11 12:15:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130519,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy-dispersive spectroscopy (EDS) analysis of ZIF-8 capsules.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/8b4c52fe9d33d2846121d66e.png"},{"id":103972973,"identity":"0d218521-7529-419b-9b1b-f7e8604dc2e1","added_by":"auto","created_at":"2026-03-05 08:04:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45982,"visible":true,"origin":"","legend":"\u003cp\u003eCell growth profile during aerobic cultivation as a function of time at different temperatures for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: (a) free cells; (b) cells immobilized in ZIF 8.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/8f5c8895a9e3c3b4c4ff0bbd.png"},{"id":104401608,"identity":"d6192c01-3207-49ec-9345-60189dbf6d37","added_by":"auto","created_at":"2026-03-11 12:13:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37248,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose consumption profile during aerobic cultivation as a function of time at different temperatures for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: (a) free cells; (b) cells immobilized in ZIF 8.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/71c3a162960984f359b26931.png"},{"id":103972971,"identity":"8c9a65cc-d033-4422-9600-ff7f819de4d1","added_by":"auto","created_at":"2026-03-05 08:04:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":27709,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability as a function of time and temperature for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: (a) free cells; (b) cells immobilized in ZIF 8.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/896a54d0a93a7171b3948468.png"},{"id":103972974,"identity":"ff2bbe04-958f-4071-ab26-1bd2c1b5a51b","added_by":"auto","created_at":"2026-03-05 08:04:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":102360,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose concentration during enzymatic hydrolysis of pretreated passion fruit peel over time.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/e6fde035332136489e21e2be.png"},{"id":104402516,"identity":"36d3fcb8-2c66-47c6-b56b-11ce8e18428d","added_by":"auto","created_at":"2026-03-11 12:15:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":454700,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of free and ZIF-8-immobilized \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e: a) Free cells after 24 hours of fermentation b) Immobilized cells after 48 hours of fermentation\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/364d3919be3c880bb7f6529e.png"},{"id":104401847,"identity":"8521078a-8d59-4005-be9e-4c3be2ef04e1","added_by":"auto","created_at":"2026-03-11 12:13:42","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":47165,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic image of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e immobilized in ZIF-8 after 30 hours of fermentation.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/900b1e93db77ed9bf93c8e33.png"},{"id":104408416,"identity":"fd7d0f93-faa1-4d0c-9a3f-f5584dc78c24","added_by":"auto","created_at":"2026-03-11 12:42:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2574849,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8926210/v1/34a5291f-be81-48d8-9652-6e0800d32e7e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Encapsulation in ZIF-8 as a Biopreservation Strategy and Enhancement of Thermal Tolerance of Saccharomyces cerevisiae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use of microorganisms to produce industrially valuable compounds represents a highly relevant biotechnological strategy (Taylor, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). For such processes to be effective, factors such as high yield, productivity, operational stability, thermal resistance, and other parameters that ensure process viability and efficiency must be considered. Among the various applications, alcoholic fermentation stands out, with yeasts being the most widely employed microorganisms. \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is the predominant species used due to its high fermentation efficiency, adaptability to different environments, and robustness under varying process conditions (Lima et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe utilization of agro-industrial residues represents a strategic source of raw materials for multiple industrial sectors. There is an increasing demand for high-quality products and improved production efficiency, combined with the adoption of practices that promote environmental protection and process sustainability. In this context, waste reuse offers significant economic and ecological potential (Velvizhi et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Residues generated from passion fruit processing, particularly the peels, are promising feedstocks for biorefineries, especially for ethanol production. These materials are rich in lignocellulosic sugars and represent one of the most advantageous technological routes for the sustainable production of biofuels (Oliveira, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several factors can influence fermentative performance, with fermentation efficiency being a key parameter. Fermentation efficiency is defined as the percentage of sugar converted into ethanol relative to the theoretical maximum predicted by the Gay-Lussac equation (Fermentec, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1978\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEfficient conversion of lignocellulosic biomass into bioethanol involves three main steps: pretreatment, enzymatic hydrolysis, and fermentation. However, during the hydrolysis stage, a major challenge is the formation of byproducts resulting from the degradation of sugars and lignin. Many of these compounds are toxic to fermentative microorganisms and negatively affect their metabolism (Chandel et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In general, the fermentation of lignocellulosic hydrolysates without prior detoxification is associated with slow fermentation kinetics, as well as reduced yields and productivities (Mussatto \u0026amp; Roberto, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the key factors influencing alcoholic fermentation, temperature plays a fundamental role, as it directly affects microbial metabolic activity and the formation of volatile compounds that impact the quality of the final product (Lima, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, ethanol production is closely associated with fermentation temperature, since carbon dioxide release during fermentation is accompanied by heat generation, which can favor alcohol formation (Mallouchos, 2003). However, elevated temperatures may act as stress factors, impairing yeast growth and viability, reducing ethanol production efficiency, promoting the proliferation of contaminating bacteria, and increasing yeast susceptibility to the toxic effects of ethanol accumulated in the culture medium (Reis \u0026amp; Ribeiro, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEthanol toxicity in yeast becomes more pronounced at higher temperatures due to increased membrane fluidity, which facilitates the influx of toxic compounds into the cell (Phisalaphong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fernandes, 2008). In response to elevated temperatures, yeast cells modify the fatty acid composition of their cell membranes as an adaptive mechanism (Wanderley, 1997). According to Amorim et al. (1996), the toxic effects of ethanol on \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e remain relatively constant within the temperature range of 12 to 28\u0026deg;C but intensify at higher temperatures. Therefore, establishing an optimal temperature range prior to fermentation is essential to ensure suitable conditions for microbial growth and to maximize ethanol yield (Ballesteros et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Phisalaphong et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor ethanol production, the optimal temperature range generally lies between 26 and 35\u0026deg;C. Cell viability declines at temperatures above 35\u0026deg;C, as these conditions trigger a thermal stress response in yeast cells and disrupt normal physiological functions, including carbohydrate flux and membrane composition (Caspeta et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). At lower temperatures, higher ethanol yields may be achieved; however, overall productivity tends to decrease due to prolonged fermentation times (Lima et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Torija et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). During fermentation, several stress factors\u0026mdash;such as elevated temperature, toxic hydrolysates, high ethanol concentrations, and osmotic pressure\u0026mdash;can negatively impact process efficiency (Patrascu et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Lin et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) reported that the highest cell growth rates and ethanol productivity for the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e BY4742 strain were observed between 30 and 45\u0026deg;C. However, at 50\u0026deg;C, a marked decline in both cell growth and ethanol yield was detected.\u003c/p\u003e \u003cp\u003eIndustrial strains of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e exhibit a certain degree of tolerance to elevated temperatures. However, this tolerance can be compromised under conditions of high ethanol concentration or low pH, negatively affecting cell viability (Reis \u0026amp; Ribeiro, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Yeasts are classified as mesophilic microorganisms, and in industrial practice, the optimal temperature range typically lies between 30 and 35\u0026deg;C (Caldas et al., 2012). In industrial fermentation processes, yeast immobilization is widely applied in the production of beer, wine, and cider. This technological strategy involves encapsulating intact and metabolically active yeast cells within a defined matrix, thereby prolonging metabolite production, including aromatic compounds. In this context, metal\u0026ndash;organic frameworks (MOFs) and their derivatives, such as zeolitic imidazolate frameworks (ZIFs), have emerged as promising materials for yeast immobilization. These systems may enhance cellular stability and strain control, while also enabling cell recovery and reuse, facilitating continuous fermentation processes, and supporting other technological advancements (Nedović et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe wide variety of substrates available for MOF synthesis, along with the broad range of possible topologies, enable these materials to be tailored for specific applications (Rogacka and Labus, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). MOFs have been applied in the chemical, food, pharmaceutical, and environmental sectors, serving as materials for the capture of toxic industrial chemicals (Islamoglu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), gas storage (Ghanbari et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), chemical catalysis (Gascon et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), drug delivery systems (Horcajada et al., 2006, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and the immobilization of enzymes and other biocompounds (Rogacka and Labus, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). MOFs can be modified through the introduction of different functional groups into their ligands (Deria et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), by structural modification via ligand exchange or incorporation of new ligands (Tanabe \u0026amp; Cohen, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), or by increasing the inertness of the central metal ion. The thermal stability of a MOF depends largely on the strength of the bond between the metal center and the ligand oxygen; as this bond strength increases, so does the material\u0026rsquo;s thermal stability (Tanabe \u0026amp; Cohen, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the various MOF families, zeolitic imidazolate frameworks (ZIFs) are particularly notable for their high porosity, well-defined cavity size, strong interactions with organic polymers, exceptional thermal stability, and outstanding chemical resistance. These features make them especially suitable for addressing key challenges in fermentation processes, such as temperature fluctuations and the presence of toxic compounds (Yang and Chung, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, ZIF-8 can be regarded as a promising protective matrix capable of limiting the diffusion of denaturing agents and dissipating heat gradients within the cellular microenvironment. This stress-mitigating effect may contribute to preserving the structural integrity of the cell membrane and organelles, maintaining intracellular homeostasis, and ultimately improving kinetic parameters and overall fermentation productivity. Accordingly, this study proposed the use of ZIF-8 for the immobilization of \u003cem\u003eS. cerevisiae\u003c/em\u003e cells to evaluate its potential as a biopreservative agent under conditions of thermal stress and in the presence of inhibitory compounds derived from lignocellulosic hydrolysates. The effects of this strategy on cell viability and overall fermentative performance were also investigated.\u003c/p\u003e \u003cp\u003eDespite the well-recognized importance of \u003cem\u003eS. cerevisiae\u003c/em\u003e and immobilization strategies in enhancing fermentative processes, there remains a need to investigate support materials capable of providing more robust and efficient protection against thermal stress and inhibitory compounds. Such protection is essential to maintain cell viability and alcoholic fermentation performance under elevated temperature conditions. In this context, zeolitic imidazolate frameworks, such as ZIF-8, have emerged as promising candidates. The novelty of this work lies in the in-depth evaluation of ZIF-8 both as an immobilization support and as a biopreservative agent for \u003cem\u003eS. cerevisiae\u003c/em\u003e cells. This study assessed the capacity of ZIF-8 to enhance cellular protection and optimize fermentative performance over an extended temperature range. Additionally, the efficiency of encapsulation was examined in the presence of inhibitory compounds found in lignocellulosic biomass fermentations derived from passion fruit peel, which may exert toxic effects on yeast cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMEV/EDS analysis\u003c/h2\u003e \u003cp\u003eScanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) analyses were performed using a HITACHI TM3030Plus microscope. Yeast samples were vacuum-dried, mounted on aluminum stubs with conductive carbon tape, and placed in the microscope\u0026rsquo;s vacuum chamber. After adjusting the operating conditions, high-resolution images were captured at various magnifications. The analyses were performed at CAITEC, Universidade Estadual de Goi\u0026aacute;s.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicroorganisms\u003c/h3\u003e\n\u003cp\u003eThe microorganism used in this study was the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e CAT-1 strain. The yeast was supplied by the Biochemical Engineering Laboratory of the Universidade Federal de Goi\u0026aacute;s. The strain was maintained on solid YPD medium composed of 10 g L⁻\u0026sup1; yeast extract, 20 g L⁻\u0026sup1; glucose, 20 g L⁻\u0026sup1; bacteriological peptone, and 20 g L⁻\u0026sup1; agar.\u003c/p\u003e\n\u003ch3\u003eInoculum Preparation\u003c/h3\u003e\n\u003cp\u003eCells were aseptically removed from YPD agar plates using a sterile inoculation loop and transferred into Erlenmeyer flasks containing 17.5 mL of liquid YPD medium composed of 10 g L⁻\u0026sup1; yeast extract, 20 g L⁻\u0026sup1; glucose, and 20 g L⁻\u0026sup1; bacteriological peptone. The pH was adjusted to 5.5. Subsequently, 2.5 mL of a concentrated glucose solution (100 g L⁻\u0026sup1;) was added to the medium. The culture medium was sterilized by autoclaving at 121\u0026deg;C and then incubated at 30\u0026deg;C under agitation at 250 rpm for 12 hours, until the exponential growth phase was reached.\u003c/p\u003e\n\u003ch3\u003eZIF 8 Preparation and Cell Encapsulation\u003c/h3\u003e\n\u003cp\u003eYeast immobilization by encapsulation in ZIF-8 capsules was carried out according to the method described by Sun et al. (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After inoculum preparation, yeast cells corresponding to 4.5 mg of dry biomass were harvested from the YPD medium by centrifugation and washed three times with deionized water at 3000 rpm for 5 minutes each. The washed cells were then resuspended in 1 mL of an aqueous zinc nitrate solution (0.05 mol L⁻\u0026sup1;), followed by the addition of 1 mL of an aqueous 2-methylimidazole solution (0.2 mol L⁻\u0026sup1;). The reaction mixture was maintained under orbital agitation at 300 rpm and 30\u0026deg;C for 1 hour. Finally, the resulting encapsulated cells were collected and washed three times with deionized water.\u003c/p\u003e\n\u003ch3\u003eEthanolic Fermentation with Free and Immobilized Cells at Different Temperatures\u003c/h3\u003e\n\u003cp\u003eEncapsulated yeast cells were cultivated under anaerobic conditions in a nutrient medium consisting of 17.5 mL of YPD medium supplemented with 2.5 mL of a concentrated glucose solution (100 g L⁻\u0026sup1;). The initial pH was adjusted to 5.5. Cultivation was performed in Erlenmeyer flasks under agitation at 250 rpm for 24 hours at different temperatures: 25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C, 40\u0026deg;C, 45\u0026deg;C, and 50\u0026deg;C. Samples were collected at predetermined time intervals, appropriately diluted, and analyzed to monitor metabolite production and substrate consumption.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthanolic Fermentation with Free and Immobilized Yeast Cells Using Lignocellulosic Biomass Derived from Passion Fruit Peel\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe lignocellulosic biomass underwent acid pretreatment, enzymatic hydrolysis, and fermentation. Pretreatment was performed with 2% (v/v) sulfuric acid at a 1:10 solid-to-liquid ratio and autoclaved at 121\u0026deg;C. After cooling, the pH was adjusted to 4.8 with citrate buffer. The suspension was filtered through cotton cloth, and the recovered solid fraction was dried in a vacuum oven at 50\u0026deg;C for 16 hours, reaching 7.71% final moisture content.\u003c/p\u003e \u003cp\u003eEnzymatic hydrolysis was performed in three 250 mL Erlenmeyer flasks containing dried passion fruit peel (5.4 g), citrate buffer (45 mL, pH 4.8), and diluted enzyme solution (5 mL). The flasks were incubated at 50\u0026deg;C and 250 rpm for 24 hours, with samples collected between 0 and 24 hours. After centrifugation at 3000 rpm for 5 minutes, the supernatant was recovered for glucose determination.\u003c/p\u003e \u003cp\u003eAfter hydrolysis, the slurry was filtered through cotton cloth to remove solids. The liquid hydrolysate (~\u0026thinsp;120 mL) was concentrated to 20 mL in a rotary evaporator, adjusted to pH 5.5 with citrate buffer, and sterilized at 121\u0026deg;C. The resulting acid\u0026ndash;enzyme hydrolysate was used as the substrate for ethanol production with free and immobilized \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e CAT-1 cells.\u003c/p\u003e \u003cp\u003eFermentations were performed in 11 mL glass flasks containing 9 mL of hydrolysate at 30\u0026deg;C without agitation. Free yeast fermentation lasted 24 hours, with samples collected at 12 and 24 hours. Immobilized yeast fermentations were conducted for 48 hours, with samples taken at 24 and 48 hours for analysis.\u003c/p\u003e \u003cp\u003eAfter 48 hours, immobilized cells were recovered by centrifugation and washed four times with deionized water (3000 rpm, 5 min). Fresh YPD medium (10 g L⁻\u0026sup1; yeast extract, 80 g L⁻\u0026sup1; glucose, and 20 g L⁻\u0026sup1; peptone) was added, the pH adjusted to 5.5, and the volume set to 9 mL to start a new fermentation cycle. This step evaluated cell viability and metabolic activity over an additional 30 hours, as well as the effectiveness of encapsulation after exposure to inhibitory compounds from passion fruit peel hydrolysate.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eValidation of Cell Viability\u003c/h2\u003e \u003cp\u003eTo validate the viability assessment method, free yeast cells were treated with 25% NaCl for 24 hours to induce cell death, which was confirmed using a Neubauer counting chamber. These nonviable cells were then subjected to the immobilization process, and viability was reassessed using methylene blue staining to ensure they remained nonviable. This approach confirmed that ZIF-8 encapsulation did not interfere with the accuracy of viability determination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell concentration\u003c/h3\u003e\n\u003cp\u003eCell biomass concentration was determined using a calibration curve established by correlating dry cell mass with absorbance at 600 nm. Eq.\u0026nbsp;(1), described by Santos (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), was applied for the CAT-1 strain:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\left({X}_{CAT-1}=0.3379.ABS-0.0266\\right)\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCell viability\u003c/h3\u003e\n\u003cp\u003eCell viability was determined using a Neubauer counting chamber and the methylene blue staining method (0.025%). Viable cells do not absorb the dye, whereas nonviable cells appear blue. Samples were diluted in the methylene blue solution, and a 10 \u0026micro;L aliquot was transferred to the Neubauer chamber for accurate cell counting. Live and dead cells were counted under an optical microscope at 40\u0026times; magnification. Cell viability was calculated according to Eq.\u0026nbsp;(2), as described by Ceccato-Antonini (2012).\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Viabilidade\\:celular\\:\\left(\\%\\right)=\\:\\frac{n\u0026uacute;mero\\:de\\:c\u0026eacute;lulas\\:vi\u0026aacute;veis}{n\u0026uacute;mero\\:de\\:c\u0026eacute;lulas\\:contadas}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGlucose and ethanol concentration\u003c/h2\u003e \u003cp\u003eGlucose and ethanol levels were quantified by HPLC. Samples were centrifuged (1075 \u0026times; g, 2 min), and the supernatant was diluted and filtered through a 0.22 \u0026micro;m nylon membrane before analysis. Separation was performed on a Shimadzu Prominence system equipped with an SCR-102HG Shim-pack column and pre-column. Compounds were detected using refractive index (RID-20A) and UV\u0026ndash;Vis (SPD-20A) detectors. The column temperature was set at 50\u0026deg;C, and 5 mM perchloric acid was used as the mobile phase at a flow rate of 0.600 mL min⁻\u0026sup1;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFermentation kinetic parameters\u003c/h2\u003e \u003cp\u003eThe substrate-to-product conversion factor (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η) were calculated according to Equations (\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e3\u003c/span\u003e), (4), and (5), respectively.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{Y}_{p/s}\\:=\\:\\frac{Pf-Po}{So-Sf}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:{Q}_{p}=\\:\\frac{Pf-Po}{tf}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:{\\eta\\:}=\\:\\frac{{Y}_{p/s}\\:}{\\text{0,511}}\\:x\\:100\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(5\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere Yₚ/ₛ (g ethanol\u0026middot;g substrate⁻\u0026sup1;) represents the substrate-to-product conversion factor; S₀ (g substrate\u0026middot;L⁻\u0026sup1;) is the initial substrate concentration; S\u003csub\u003ef\u003c/sub\u003e (g substrate\u0026middot;L⁻\u0026sup1;) is the final substrate concentration; P\u003csub\u003ef\u003c/sub\u003e (g ethanol\u0026middot;L⁻\u0026sup1;) is the final ethanol concentration; P\u003csub\u003e0\u003c/sub\u003e (g ethanol\u0026middot;L⁻\u0026sup1;) is the initial ethanol concentration; Qₚ (g ethanol\u0026middot;L⁻\u0026sup1;\u0026middot;h⁻\u0026sup1;) is the volumetric productivity; t\u003csub\u003ef\u003c/sub\u003e (h) is the total fermentation time; and η (%) corresponds to the fermentation efficiency.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe results allowed to evaluate the performance of free and ZIF-8-immobilized yeasts under different fermentation conditions. The samples were collected at predetermined intervals, and the analyses were performed in triplicate. Cell growth, viability, glucose consumption, substrate-to-product conversion, ethanol yield, and fermentation efficiency were assessed for both free and immobilized yeast cultures.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMorphology and composition of ZIF-8 encapsulated cells (SEM/EDS)\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea displays the typical ellipsoidal morphology of free \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e cells, with visible budding scars characteristic of asexual reproduction. In contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows successful encapsulation with a ZIF-8 layer, forming a core\u0026ndash;shell structure composed of yeast cells coated with ZIF-8. The micrographs reveal a uniform coating, with no partial or uncoated cells, indicating high encapsulation homogeneity. Morphological analysis identified polyhedral particles with a predominantly cubic tendency, including near-cubic and irregular shapes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, chemical mapping demonstrated a homogeneous distribution of zinc, oxygen, and carbon on the cell surface, confirming the presence of a continuous ZIF-8 layer surrounding individual yeast cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results confirm the effective immobilization of \u003cem\u003eS. cerevisiae\u003c/em\u003e in ZIF-8, yielding core\u0026ndash;shell biohybrid structures with high structural integrity and surface uniformity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Temperature on Growth and Glucose Consumption\u003c/h2\u003e \u003cp\u003eThe growth kinetics of yeast cells during 24 hours of fermentation are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, allowing evaluation of cellular behavior under different temperatures and immobilization conditions. Prior inoculation was used as a preadaptation strategy, in which the microorganism was cultivated in a medium like that of the main process. This approach stimulates the activation of specific metabolic pathways and the synthesis of adaptation-related proteins, thereby reducing the lag phase. As a result, the onset of exponential growth is accelerated, contributing to improved growth kinetics and a more consistent fermentative process (Cunha, 2023; Hiss, 2001).\u003c/p\u003e \u003cp\u003eThis strategy facilitated a faster transition to the active growth phase, as indicated by the marked increase in cell density at the beginning of the stationary phase. The findings suggest that prior inoculation not only shortened the adaptation period but also enhanced the specific growth rate (\u0026micro;). Such an effect is particularly important in industrial applications, where reduced processing time and increased productivity are highly desirable (Schuler, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGrowth kinetics (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) were further evaluated to compare the performance of free and immobilized yeast cells at different temperatures. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, free yeast cells cultivated between 25 and 40\u0026deg;C did not exhibit a distinct lag phase, corroborating the observations reported by Pires (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The lag phase is typically characterized by enzyme synthesis and cellular adaptation to the cultivation medium, occurring without measurable cell division (Schmidell et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Its absence in this study can be attributed to the use of a medium identical to that employed during inoculum preparation, which facilitated a rapid transition to the exponential phase and promoted an increase in the specific growth rate (\u0026micro;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor immobilized yeast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), a delay in process onset was observed. This effect may be attributed to stress imposed by immobilization, which alters the cellular microenvironment and restricts space for growth (Pajić Lijaković et al., 2017). Despite this initial delay, essential metabolic activities were not compromised, as cell growth and the production of ethanol and carbon dioxide still occurred during fermentation. The observed lag likely corresponds to the period required for cellular adaptation to the new microenvironment and to the specific immobilization method applied.\u003c/p\u003e \u003cp\u003eA delayed onset of cell multiplication was consistently observed in immobilized yeasts, likely resulting from physiological stress imposed by the immobilization matrix. This environment reduces the available space for growth and alters mass transfer conditions, limiting the diffusion of substrates and metabolites (Pajić Lijaković et al., 2017; Verbelen et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, modifications in the microenvironment may influence gene expression and membrane organization, triggering adaptive responses such as stress-hardening mechanisms (Gasch, 2002). The impact of the support material on cellular physiology should also be considered, as physicochemical interactions between the support surface and the cell wall can alter membrane permeability and metabolic dynamics (Loo et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBola\u0026ntilde;os Barbosa et al. (2023) investigated yeast encapsulation in alginate using 0.1 mol L⁻\u0026sup1; calcium chloride as a crosslinking agent. Batch experiments demonstrated a significant reduction in cell viability and mechanical stability, which limited the reuse of the encapsulated systems. In addition, cell leakage from the polymeric matrix through pores, cell proliferation within the capsules, and gas production may further weaken capsule integrity (Sim\u0026oacute; et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Immobilization may also modify the physiological and metabolic properties of cells, as well as the characteristics of the surrounding microenvironment, thereby influencing cellular kinetic behavior. In this regard, the local microenvironment is affected by solute partitioning between the liquid phase and the solid matrix, external mass transfer resistance in the liquid film, and internal diffusion limitations within the immobilization matrix (Merchant, 1987).\u003c/p\u003e \u003cp\u003eThese phenomena are described by the solute partition coefficient (Kp), the liquid film mass transfer coefficient (kL), and the effective solute diffusivity within the immobilization matrix (De). The substrate must first diffuse through the bulk liquid and cross the external liquid film surrounding the immobilized particle until it reaches the liquid\u0026ndash;solid interface. It then diffuses through the liquid phase inside the matrix, where additional resistance arises from microcolony formation, and finally reaches the interior of the yeast cells, where metabolic reactions occur (Pilkington et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsing a model of cell growth and substrate consumption of \u003cem\u003eN. agilis\u003c/em\u003e immobilized in carrageenan gel spheres, Gooijer et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) demonstrated that external mass transfer resistance was the most sensitive parameter affecting the growth of entrapped cells. Similarly, Wang et al. (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed that \u003cem\u003eCandida antarctica\u003c/em\u003e lipase B immobilized in ZIF-8 exhibited enhanced activity and stability, while the ZIF-8 layer also regulated substrate accessibility to the enzyme interior.\u003c/p\u003e \u003cp\u003eSeveral studies have reported that immobilized cells behave differently from free cells in solutions, including enhanced metabolic efficiency (Navarro \u0026amp; Durand, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) and alterations in metabolic pathways (Brodelius et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Mattiasson et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) proposed that environmental factors such as water activity and oxygen availability play a central role in regulating the metabolism of immobilized cells.\u003c/p\u003e \u003cp\u003eOne possible explanation for the metabolic differences between immobilized and free cells is the reduced water activity within gel matrices, likely caused by increased osmotic pressure (Mattiasson et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Immobilized systems generally exhibit lower water activity and limited oxygen availability, conditions that can suppress cell growth, metabolic activity, and reproduction.\u003c/p\u003e \u003cp\u003eGalazzo and Bailey (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) reported enhanced polyphosphate synthesis in immobilized yeasts, possibly induced by reduced water activity. Polyphosphate, an inorganic phosphate polymer, plays multiple biological roles, including energy storage, regulation of metabolic pathways, and participation in stress response and survival mechanisms in microorganisms such as bacteria and yeasts (J\u0026uacute;nior et al., 2024).\u003c/p\u003e \u003cp\u003eAccordingly, Shinmyo et al. (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) reported that Bacillus \u003cem\u003eamyloliquefaciens\u003c/em\u003e entrapped in κ-carrageenan operated under oxygen-deficient conditions, resulting in reduced cell growth and enhanced α-amylase production. When immobilized cells sustain high maintenance metabolism at the expense of growth due to reduced water activity, higher yields can be expected for reactions linked to maintenance metabolism and for oxygen-independent processes.\u003c/p\u003e \u003cp\u003eDoran and Bailey (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) reported that, in alcoholic fermentation with immobilized yeast, substrate consumption was higher than in fermentations with free cells, whereas cell growth and product formation (e.g., ethanol and glycerol) were lower. These findings suggest that immobilization affects microbial growth through interactions between solid support and the cells, as well as cell-to-cell interactions. According to the authors, although the microenvironment of immobilized yeasts\u0026mdash;characterized by concentration gradients and osmotic pressure\u0026mdash;affects cellular metabolism, reduced growth is primarily attributed to direct physical contact with the immobilization material, whether solid or gel. This contact induces morphological changes and alters cell wall protein expression, thereby impacting overall metabolism.\u003c/p\u003e \u003cp\u003eFrom a kinetic perspective, the transition from free to immobilized cells can alter the relationship between the maximum specific growth rate (\u0026micro;\u003csub\u003emax\u003c/sub\u003e) and the saturation constant (Ks), often reducing \u0026micro;\u003csub\u003emax\u003c/sub\u003e due to additional diffusional resistances imposed by the support. In the present study, free cells-initiated growth immediately after inoculation, whereas immobilized cells exhibited a lag phase of up to 8 hours before significant growth, particularly at 25\u0026ndash;35\u0026deg;C. These findings suggest that immobilization affects not only kinetic parameters but also physiological aspects of yeast cells, directly influencing their metabolic performance and growth (Kovaleski, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the exponential phase, rapid cell multiplication and biomass increase were observed. Free yeast cells cultivated at 25, 30, and 35\u0026deg;C reached concentrations of 2.83, 3.49, and 3.32 g L⁻\u0026sup1; after 24 h, respectively. In contrast, temperatures\u0026thinsp;\u0026ge;\u0026thinsp;40\u0026deg;C significantly reduced the specific growth rate, leading to cell death at 45 and 50\u0026deg;C. For immobilized cells, although growth ceased at these temperatures, viability was maintained due to the protective effect of ZIF-8. Similar findings were reported by Lin et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e BY4742, which showed markedly reduced growth and ethanol production at 50\u0026deg;C. This effect is attributed to thermal stress, as high temperatures induce structural alterations in proteins and plasma membranes, impairing growth, viability, and fermentative capacity (Naves et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor immobilized yeast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), the exponential phase began after approximately 8 h of fermentation, with significant growth observed at 25\u0026ndash;35\u0026deg;C. After 24 h, cell concentrations reached 3.30, 3.56, and 3.44 g L⁻\u0026sup1;, respectively. Substrate consumption was maximal during this phase, resulting in complete glucose depletion by 24 h, as it was utilized to meet metabolic demands. Each glucose molecule yields two ATP molecules, supplying the energy required for cellular functions (Schmidell et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Accordingly, glucose consumption reached 100% after 24 h of fermentation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eAnalysis of Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb shows glucose depletion by the end of the exponential phase for both free and immobilized cells, increasing linearly up to 24 h at 25\u0026ndash;35\u0026deg;C (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, glucose consumption was markedly reduced at 45 and 50\u0026deg;C in both systems. This reduction reflects the direct effect of temperature on microbial metabolic activity, a key factor in fermentative performance (Lima, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Caspeta et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) reported that at temperatures above 36\u0026deg;C, yeasts activate the heat stress response, a transcriptional defense mechanism that disrupts carbohydrate metabolism and plasma membrane composition. Although \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, a mesophilic yeast, can grow at temperatures up to approximately 42\u0026deg;C, prolonged exposure to higher temperatures compromises cell viability (Yamamoto et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNeves (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that glucose consumption is reduced during the lag phase, as yeast cells adapt to the medium and reorganize their enzymatic machinery in preparation for logarithmic growth. Similarly, Brito (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) observed negligible initial sugar consumption in \u003cem\u003eS. cerevisiae\u003c/em\u003e immobilized in calcium alginate, indicating a lag period required for adaptation to the new system. This behavior aligns with the classical microbial growth curve, in which the lag phase represents the time needed for metabolic adjustment to new environmental conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the lag period is inherently related to cellular adaptation, mass transfer of substrates and products through polymeric support is another key factor influencing immobilization efficiency. Coutinho (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) highlighted limited diffusion as a critical parameter in biocatalyst encapsulation, since the porous barrier between the microorganism and the fermentative medium may restrict compound transport into and out of the matrix, impairing fermentation performance. Nevertheless, the present results indicate that despite the initial adaptation phase, immobilization did not compromise overall fermentation efficiency as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn addition to diffusion limitations imposed by the polymeric matrix, cell density within the support is a crucial factor affecting immobilization efficiency (Kovaleski, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In that study, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e immobilized in calcium alginate at densities of 1 \u0026times; 10⁶, 8 \u0026times; 10⁶, and 2 \u0026times; 10⁷ cells mL⁻\u0026sup1; showed distinct sucrose consumption profiles over 54 h of alcoholic fermentation. Beads with the lowest density reached 71% sucrose consumption, with a lag phase during the first 6 h. In contrast, higher densities resulted in lower consumption rates (11% and 29%), with a lag phase observed in one case. These findings suggest that increased cell density promotes internal nutrient competition, limiting growth and impairing substrate consumption and ethanol production (Cha et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough high cell density may impose metabolic constraints, immobilization also induces physiological changes in yeast cells. Some of these changes enhance fermentative performance, whereas others may be detrimental. Given that \u003cem\u003eS. cerevisiae\u003c/em\u003e naturally adheres to surfaces in its native habitats, immobilization can be considered a growth mode closer to natural conditions, potentially providing protection against environmental stresses (Verstrepen and Klis, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, even under conditions closer to the natural environment, the negative effects observed are mainly related to stress caused by restricted mass transfer. In gel entrapment and inert supports, diffusional limitations depend on the immobilization method. In adsorption-based systems, resistance occurs primarily at the carrier\u0026ndash;medium interface, with minimal internal limitations. In encapsulation methods, internal mass transfer is governed by the chemical and mechanical properties of the matrix, such as size, porosity, and texture. Verbelen et al. (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) cautioned that physiological changes should not be attributed solely to immobilization, as these effects are difficult to separate from mass transfer limitations.\u003c/p\u003e \u003cp\u003eAccordingly, immobilization strongly influences yeast plasma membrane properties and may alter specific solute transport systems (Shen et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Comparisons between immobilized and free cells show activation of energy metabolism in immobilized systems, along with increased accumulation of storage polysaccharides (trehalose and glycogen) and structural polysaccharides (glucan and mannan) (Galazzo \u0026amp; Bailey, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Jirku et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Their accumulation enhances resistance to environmental stress, supports the production of industrially relevant metabolites, and improves viability under adverse conditions. In \u003cem\u003eS. cerevisiae\u003c/em\u003e, trehalose and glycogen accumulate in response to unfavorable growth conditions, serving as energy reserves and stress protectants. These compounds are essential for cell cycle progression under reduced growth rates and carbon limitation. Singer and Lindquist (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) demonstrated that trehalose protects cells during thermal shock by preventing protein denaturation and aggregation, underscoring its key role in cellular protection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFermentation Performance and Kinetic Parameters\u003c/h2\u003e \u003cp\u003eThe ethanol concentrations and principal kinetic parameters obtained during fermentation are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Comparative analysis of these parameters highlights significant differences in the fermentative performance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e under the tested conditions, for both free and ZIF-8-immobilized cells. Overall, immobilized yeasts exhibited superior performance within the temperature range of 25\u0026ndash;35\u0026deg;C, particularly regarding final ethanol concentration, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFermentation kinetic parameters, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η), of free \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e cells and cells immobilized in ZIF-8 after 24 hours of fermentation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal ethanol (g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) Lev. Free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10,23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal ethanol \u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e Lev. Immobilized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12,29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15,71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0,32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY\u0026nbsp;\u003csub\u003eP/S\u003c/sub\u003e\u0026nbsp;\u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e Lev. Free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0,403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0,236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY\u0026nbsp;\u003csub\u003eP/S\u003c/sub\u003e\u0026nbsp;\u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e Lev. Immobilized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0,479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0,075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQp (g.L\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e.h\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e) Lev. Free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0,537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0,314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQp (g.L\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e.h\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e) Lev. Immobilized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0,655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0,133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eη (%) Lev. Free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59,88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78,86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73,77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46,18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eη (%) Lev. Immobilized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e93,74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78,47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14,68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAt temperatures considered optimal for yeast metabolism (25 and 30\u0026deg;C), immobilization significantly increased the final ethanol concentration, with gains ranging from 10.54% to 21.89%. This improvement was accompanied by higher substrate-to-product yields (Yₚ/ₛ), which increased by 6.4% to 21.2% under these conditions. These findings suggest that the ZIF-8 matrix enhances cell viability and metabolic performance, possibly by reducing environmental stress and allowing greater operational stability and cell reuse.\u003c/p\u003e \u003cp\u003eAccording to Nedović et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), immobilized cells often exhibit higher glycolytic flux than free cells, resulting in faster glucose consumption and more efficient channeling of substrate toward biomass and ethanol production. The present results support this observation, as the increased ethanol production by immobilized yeasts indicates a shift in fermentative metabolism. This shift may stem from growth limitations imposed by the ZIF-8 matrix, which restricts cell expansion and redirect carbon flux preferentially toward fermentative pathways rather than biosynthesis. Such microenvironmental constraints associated with immobilization may be related to polymer network relaxation around cell aggregates, mechanical forces generated during cell growth within the capsule, and physicochemical interactions among the solvent, matrix, and cells (Pajić Lijaković et al., 2012).\u003c/p\u003e \u003cp\u003eThis metabolic modulation is reflected in the average fermentative productivity (Qₚ), which showed a similar improvement and reached a maximum of 0.655 g L⁻\u0026sup1; h⁻\u0026sup1; for immobilized yeasts at 30\u0026deg;C, approximately 22% higher than for free cells. From an industrial perspective, this increase is significant, as productivity determines the rate of product formation and directly impacts cycle time and process economics.\u003c/p\u003e \u003cp\u003eConsistent with these findings, fermentation efficiency further confirms the superiority of immobilized yeasts under the evaluated conditions. While free cells showed efficiencies of 59.88\u0026ndash;78.86% at 25\u0026ndash;35\u0026deg;C, immobilized cells reached 72.60-93.74%. At 30\u0026deg;C, efficiency (~\u0026thinsp;91%) approached the average value reported for industrial operations (Abreu Cavalheiro \u0026amp; Monteiro, 2013), underscoring the robustness and operational stability of the ZIF-8 system. Overall, these results indicate that immobilization enhances conversion efficiency and productivity while increasing biocatalyst resilience.\u003c/p\u003e \u003cp\u003eConversely, increasing the temperature to 40\u0026deg;C sharply reduced fermentative performance, with significant declines in final ethanol concentration, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and efficiency (η) in both systems. This result indicates that temperatures above the physiological optimum impair plasma membrane integrity, enzymatic stability, and intracellular homeostasis, leading to metabolic inhibition and loss of viability. At 45 and 50\u0026deg;C, fermentative activity was completely suppressed, indicating that these temperatures exceed the thermal tolerance of the strain, regardless of immobilization.\u003c/p\u003e \u003cp\u003eMattiasson et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) reported improved ethanol productivity and yield using immobilized cells. For free cells in solution, the theoretical maximum ethanol yield from glucose is 51%; however, adsorbed cells have achieved substantially higher yields (Navarro \u0026amp; Durand, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), likely due to alterations in metabolic pathways. In the immobilized state, cells are confined within a polymer-rich environment whose macromolecules can organize water and reduce its availability. Even moderate polymer concentrations can induce distinct metabolic effects. For example, adding only 0.5% sodium alginate to batch fermentation with \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e markedly altered cellular productivity (Holcberg \u0026amp; Margalith, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1981\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the effect of reduced water activity on \u003cem\u003eS. cerevisiae\u003c/em\u003e metabolism, batch fermentations were performed at water activities as low as 0.986 by adding increasing concentrations of dextran and polyethylene glycol (Hahn-H\u0026auml;gerdal et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Under these conditions, the initial ethanol production rate increased by 50%. Similarly, yeast cells adsorbed onto porous glass showed a 25% increase in ethanol production rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability and Thermal Stress\u003c/h2\u003e \u003cp\u003eThe encapsulation process aims to preserve the viability and metabolic functionality of microorganisms by creating a favorable microenvironment for fermentation development. In this context, the use of encapsulation proved to be promising in the present study, as yeasts immobilized in ZIF 8 exhibited superior performance compared to free cells. This improvement was particularly evident in terms of enhanced thermal stability, which allowed satisfactory productivity and fermentation efficiency. This operational advantage may be associated with the intrinsic properties of ZIF 8, whose thermal stability and behavior have been well documented.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFang et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) investigated the thermal stability and degradation behavior of ZIF 8 nanocrystals using thermogravimetric analysis under a nitrogen atmosphere and demonstrated that pure ZIF 8 crystals exhibit degradation temperatures above 600\u0026deg;C. This characteristic is especially relevant for applications involving temperature fluctuations, such as gas flow in reactors or industrial ducts, as it ensures durability and structural integrity under severe conditions. Other studies corroborate this thermal robustness. Sung et al. (2019) reported stability up to 550\u0026deg;C, while Yang et al. (2013) observed excellent thermal and separation stability of ZIF 8 PBI nanocomposites under elevated temperatures and hot vapor exposure of up to 230\u0026deg;C during ten days of continuous operation.\u003c/p\u003e \u003cp\u003eThis stability was a determining factor in the results obtained in the present study, as illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, which show the evolution of cell viability of free and ZIF 8 immobilized \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e over time at different temperatures. Immobilization was effective in maintaining stable cell viability throughout the fermentative process, even under more severe thermal conditions. It is important to note that while the capsule protects cellular integrity, it does not necessarily ensure maximum metabolic activity under highly stressful conditions. After 24 hours of cultivation, immobilized yeasts exhibited cell viability values of 98.4 percent, 97.7 percent, and 96.7 percent, respectively. In contrast, free cells showed more pronounced reductions, reaching 93.7 percent, 88.9 percent, and 82.9 percent.\u003c/p\u003e \u003cp\u003eThis behavior was expected, considering that the thermal stability of ZIF 8 greatly exceeds the tolerance limits of the yeast itself, allowing it to function as a physical and protective barrier against thermal stress. These findings are consistent with the observations of Torija et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), who reported that the initial fermentation rate is maximized between 25\u0026deg;C and 31\u0026deg;C, while cell viability declines significantly above 35\u0026deg;C, particularly in unprotected cultures. Thus, the thermal resistance provided by the ZIF 8 matrix proved sufficient to mitigate the negative effects of heat on encapsulated cells under thermal stress.\u003c/p\u003e \u003cp\u003eThe protective effect of immobilization against thermal shock is also supported by physiological studies on yeast. Scientific evidence indicates that cells in the logarithmic phase of rapid growth are more sensitive to thermal stress than slowly dividing cells or those in the stationary phase (Washburne et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Nagarajan et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) compared the thermotolerance of \u003cem\u003eS. cerevisiae\u003c/em\u003e confined within or released from calcium alginate matrices with planktonic cells at different physiological stages following exposure to 48\u0026deg;C for two hours. The authors observed that planktonic and immobilized cells in the stationary phase were significantly more tolerant to thermal shock than planktonic cells in the logarithmic phase. The greater resistance observed in immobilized cells resulted not only from their physiological state but also from the physical protection provided by the matrix, as evidenced by the intermediate survival of cells released from the beads. Furthermore, when immobilized, cells ceased division but remained metabolically active and highly fermented, maintaining viability for up to seventeen weeks with viability levels exceeding 95 percent.\u003c/p\u003e \u003cp\u003eThe relationship between growth rate and heat resistance was further explored by Lu et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), who demonstrated that genes involved in the heat stress response and previously associated with thermotolerance exhibit higher expression in slowly growing cells. To test this hypothesis, the authors cultivated cells at different growth rates under steady state conditions in chemostats at 50\u0026deg;C, a lethal temperature for yeast cells growing in rich media. Cells with a doubling time of 2.8 hours exhibited high sensitivity, whereas cells with a doubling time of 13.8 hours were resistant. Cultures with intermediate growth rates, corresponding to a doubling time of 4.6 hours, showed intermediate mortality. As a control, cells cultivated at the intermediate growth rate but at 36\u0026deg;C exhibited resistance like that of slow growing cells. These results indicate that growth deceleration, a characteristic observed in immobilized cells, promotes gene expression patterns that contribute to the enhanced thermal resistance observed in the present work.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePerformance in Lignocellulosic Hydrolysate and Tolerance to Inhibitors\u003c/h2\u003e \u003cp\u003eAfter acid pretreatment, passion fruit peels underwent enzymatic hydrolysis at time intervals of 0, 0.5, 1, 2, 4, 8, and 24 hours. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, glucose concentration progressively increased during the process, reaching 32.53 g L⁻\u0026sup1; after 24 hours. Following hydrolysis, the filtrate (approximately 120 mL) was concentrated using a rotary evaporator to enhance sugar content, reducing the volume to 20 mL. Citrate buffer (pH 5.5) was then added to adjust the pH, resulting in a final glucose concentration of 62.05 g L⁻\u0026sup1;. The hydrolysate was subsequently sterilized by autoclaving at 121\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe passion fruit peel hydrolysate was used as the fermentation medium for alcoholic fermentation by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e CAT-1, employing both free and immobilized cells. The results for cell growth, substrate consumption, ethanol production, substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η) are summarized in Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThis procedure was designed to evaluate, over an additional 30-hour period, the metabolic activity and viability of the immobilized yeasts, as well as the effectiveness of the ZIF-8 capsule after exposure to inhibitory compounds commonly present in lignocellulosic hydrolysates derived from passion fruit peel, which may exert toxic effects on yeast cells. The results of these tests are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eEdwards and Doran-Peterson (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) reviewed the main pectin-rich residues\u0026mdash;such as those generated from citrus, sugar beet, and apple processing\u0026mdash;that can be used for ethanol production. According to the authors, this type of biomass is characterized by low lignin content and a high pectin fraction, typically ranging from 12% to 35%.\u003c/p\u003e \u003cp\u003eHowever, a major limitation in using these residues for ethanol production is that galacturonic acid and arabinose are not metabolized by microorganisms traditionally employed in alcoholic fermentation. This constraint helps explain the low fermentative efficiency observed with \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, since passion fruit peel contains, on average, approximately 25% pectin (J\u0026uacute;nior et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition to its inability to ferment pentoses, this yeast is also incapable of metabolizing the galacturonic acid released during biomass hydrolysis, further limiting the conversion efficiency of pectin-rich substrates into ethanol.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell growth, substrate consumption, ethanol production, and fermentation kinetic parameters of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e immobilized in ZIF-8 during fermentation using lignocellulosic biomass derived from passion fruit peel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003e(h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell growth (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlucose (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFinal ethanol (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u0026nbsp;\u003csub\u003eP/S\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eQp\u003c/p\u003e \u003cp\u003e(g.L\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e.h\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eη\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eViability (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51,92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e52,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e68,59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6,80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e74,56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e19,89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell growth, substrate consumption, ethanol production, and fermentation kinetic parameters (substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η)) of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e immobilized in ZIF-8 during fermentation using lignocellulosic biomass derived from passion fruit peel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003e(h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell growth (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlucose (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFinal ethanol (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u0026nbsp;\u003csub\u003eP/S\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eQp\u003c/p\u003e \u003cp\u003e(g.L\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e.h\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eη\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eViability (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57,63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e48 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56,09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e99,42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell growth, substrate consumption, ethanol production, and fermentation kinetic parameters (substrate-to-product yield (Yₚ/ₛ), volumetric productivity (Qₚ), and fermentation efficiency (η)) of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e immobilized in ZIF-8 during fermentation in YPD medium.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003cp\u003e(h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell growth (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlucose (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFinal ethanol (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u0026nbsp;\u003csub\u003eP/S\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(g.L\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eQp\u003c/p\u003e \u003cp\u003e(g.L\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e.h\u0026nbsp;\u003csup\u003e\u003cem\u003e\u0026minus;1\u003c/em\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eη\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eViability (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61,03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10,60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e86,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen lignocellulosic hydrolysates are used as feedstock, significant amounts of pentose sugars, such as D-xylose and L-arabinose, are released from hemicellulose (Azhar et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ndubuisi et al. (2020), working with the yeast \u003cem\u003ePichia kudriavzevii\u003c/em\u003e LC375240, reported that almost no ethanol was produced in pentose-based media due to limited cell growth. Their findings suggest that although the strain was able to assimilate pentose sugars to support growth, it was unable to efficiently ferment them into ethanol.\u003c/p\u003e \u003cp\u003eAcid hydrolysis generates not only sugars but also toxic byproducts that inhibit fermentative microorganisms. These include organic acids (acetic, formic, and levulinic acids), furan derivatives (furfural and 5-hydroxymethylfurfural), and phenolic compounds (Chandel et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Acetic acid originates from hemicellulose, furans from pentose and hexose degradation, and formic and levulinic acids from further furan breakdown (Mart\u0026iacute;n et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Lignin is the main source of phenolic compounds, which are considered stronger inhibitors than sugar degradation products (J\u0026ouml;nsson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Palmqvist and Hahn H\u0026auml;gerdal, 2000; Rasmussen et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, J\u0026ouml;nsson et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInhibitory compounds formed during biomass hydrolysis reduce ethanol yield and productivity by impairing microbial metabolism and carbohydrate uptake (Almeida et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Furans such as hydroxymethylfurfural and furfural damage DNA, inhibit RNA and protein synthesis, and interfere with key enzymes involved in ethanol production (Modig et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Their toxicity is mainly linked to reactive aldehyde groups that disrupt cellular components and membranes (Miller et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Organic acids (acetic, formic, and levulinic) inhibit cells in their undissociated forms by diffusing across membranes, while lignin-derived phenolic compounds compromise membrane integrity and enzyme function (Mills et al., 2009, Parawira et al., 2011).\u003c/p\u003e \u003cp\u003eThe type and concentration of inhibitory compounds formed during pretreatment depend on the feedstock and the severity of processing conditions (J\u0026ouml;nsson et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ko et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As a result, purification strategies to reduce inhibitor levels before fermentation are highly important. In general, fermentation of untreated hydrolysates leads to slow kinetics, low productivity, and reduced ethanol yields (Mussatto et al., 2004).\u003c/p\u003e \u003cp\u003eFermentation converts sugars released during pretreatment and hydrolysis into ethanol. However, the absence of microorganisms capable of efficiently fermenting both hexoses and pentoses into ethanol limits the economic viability of cellulosic ethanol production (Nichols et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although research has advanced in developing wild-type and genetically engineered strains to address this issue, challenges such as incomplete pentose utilization, low ethanol tolerance, and sensitivity to pretreatment-derived inhibitors persist (Kumar et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Milessi et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), cell immobilization on solid supports is a promising strategy to overcome fermentation challenges, as it creates a protective microenvironment that shields yeast from inhibitors present in hydrolysates. This concept supports the use of ZIF-8 immobilization applied in the present study. In addition to enhanced protection, immobilization enables easy recovery and reuse of cells, operation at high cell densities for extended periods, and reduced biomass handling requirements. It also facilitates the industrial application of genetically modified microorganisms, particularly in regions with strict biosafety regulations (Kourkoutas et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Milessi et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFermentations using passion fruit, banana, and coconut hydrolysates commonly show lower ethanol production compared to media containing pure glucose. For example, \u003cem\u003eZymomonas mobilis\u003c/em\u003e produced less ethanol when hydrolysates were used as the carbon source. This reduction is attributed to the presence of inorganic compounds and other inhibitory substances in hydrolysates, which can interfere with microbial metabolism (Doelle and Doelle, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Non-purified sugar media may contain impurities that inhibit ethanol production and suppress enzymatic activity, thereby reducing overall fermentation efficiency (Khoja et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRosa (2021) evaluated fermentation of orange bagasse hydrolysate using \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e G2 104 immobilized in calcium alginate and observed low ethanol yields. After 12 hours, ethanol concentrations were 2.57 g L⁻\u0026sup1; (enzymatic hydrolysate), 1.69 g L⁻\u0026sup1; (acid hydrolysate), and 4.9 g L⁻\u0026sup1; (enzymatic hydrolysate without hydrothermal pretreatment). These findings indicate that enzymatic hydrolysates provide better fermentative performance, likely due to the absence of inhibitory compounds commonly formed during acid hydrolysis. Acid hydrolysates showed reduced sugar consumption and lower ethanol production, probably due to inhibitor formation and generation of non-fermentable sugars. However, acid hydrolysis can still be effective for sugar release if combined with detoxification and supplementation strategies.\u003c/p\u003e \u003cp\u003eTo assess the protective effect of the ZIF-8 capsule against inhibitory compounds present in passion fruit peel hydrolysate, encapsulation was evaluated as a protective strategy. The results (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) show clear differences between free and immobilized \u003cem\u003eS. cerevisiae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImmobilization effectively preserved cell viability throughout fermentation. After 48 hours at 30\u0026deg;C without agitation, immobilized cells maintained 99.42% viability, while free cells showed a sharp decline to 69.59% at 12 hours and 19.89% at 24 hours. As a result, free-cell fermentations produced low ethanol concentrations (maximum of 6.8 g L⁻\u0026sup1;). In contrast, immobilized cells consumed less glucose, and ethanol productivity was not determined, suggesting that glucose was mainly used for cell maintenance rather than ethanol production.\u003c/p\u003e \u003cp\u003eThe enhanced tolerance of immobilized yeasts to toxic compounds may be linked to osmotic stress responses, which stimulate intracellular polyol production for osmotic regulation. This mechanism reduces water activity and increases resistance to harmful chemical substances (Norton and D\u0026rsquo;Amore, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, data from Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e confirm the effectiveness of encapsulation as a protective strategy. Immobilized cells remained metabolically active and viable after medium replacement for an additional 30 hours. Fermentation efficiency values reinforced this observation, with immobilized yeasts reaching an efficiency of 86.54%, demonstrating superior stability under the evaluated conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFermentations conducted at 25\u0026ndash;50\u0026deg;C showed clear differences between free and ZIF-8-immobilized yeasts. ZIF-8 acted as a protective barrier, improving cell integrity and fermentative performance at optimal temperatures (25\u0026ndash;35\u0026deg;C), with higher Yₚ/ₛ, Qₚ, and η values compared to free cells. The capsule also maintained high viability at elevated temperatures, reaching 96.7% at 50\u0026deg;C after 24 hours, although ethanol production was suppressed above 40\u0026deg;C in both conditions.\u003c/p\u003e \u003cp\u003eIn fermentations using passion fruit peel hydrolysate, ZIF-8 preserved cell viability against inhibitory compounds; however, ethanol yield remained limited due to the hydrolysate composition, including pentoses, phenolics, organic acids, furfural, and hydroxymethylfurfural. Overall, immobilization in ZIF-8 proved to be an effective biopreservation strategy, enhancing cell protection, robustness, and potential reuse.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.N.P. conceived and designed the research project and supervised the M.Sc. student L.C.C.G., particularly regarding the immobilization of Saccharomyces cerevisiae in ZIF-8 for application in alcoholic fermentation of lignocellulosic biomass derived from passion fruit peel. C.A.G.S. co-supervised the student and directly guided most of the experimental work involving the yeast. L.C.C.G. performed all laboratory experiments and contributed to data organization and manuscript preparation. G.L.C. conducted and interpreted all high-performance liquid chromatography (HPLC) analyses. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the financial support provided by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), and the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Goi\u0026aacute;s (FAPEG). The authors also thank the CAM (IQ-UFG) and CAiTEC (UEG) laboratories and their technical staff for experimental support, as well as colleagues and collaborators who contributed directly or indirectly to the development of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbreu-Cavalheiro GM (2013) Solving ethanol production problems with genetically modified yeast strains. 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Int J Hydrog Energy 38:229\u0026ndash;239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijhydene.2012.10.045\u003c/span\u003e\u003cspan address=\"10.1016/j.ijhydene.2012.10.045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"brazilian-journal-of-chemical-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bjce","sideBox":"Learn more about [Brazilian Journal of Chemical Engineering](http://link.springer.com/journal/43153)","snPcode":"43153","submissionUrl":"https://www.editorialmanager.com/bjce/default2.aspx","title":"Brazilian Journal of Chemical Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Saccharomyces cerevisiae, immobilization, ZIF-8, thermal stability, viability, fermentation","lastPublishedDoi":"10.21203/rs.3.rs-8926210/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8926210/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated ZIF-8 as an immobilization matrix for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e CAT-1 to determine its effectiveness as a biopreservation strategy for enhancing robustness and performance in ethanol fermentation under thermal and chemical stress. Encapsulated and free cells were compared in sequential batch fermentations to assess operational stability and reusability. Thermal tolerance assays were conducted in YPD medium at 25\u0026ndash;50\u0026deg;C, and protective performance was further examined under inhibitory conditions representative of lignocellulosic hydrolysates derived from passion fruit peel. Biomass formation, cell viability, glucose consumption, substrate-to-product conversion yield, volumetric productivity, and fermentation efficiency were evaluated. ZIF-8 encapsulation significantly enhanced thermotolerance and resistance to inhibitory compounds. Encapsulated cells maintained consistently higher viability across the tested temperature range, reaching 96.7% at 50\u0026deg;C compared to 82.9% for free cells. Within the mesophilic range (25\u0026ndash;35\u0026deg;C), immobilized cultures demonstrated superior fermentative performance, achieving fermentation efficiency above 93% at 30\u0026deg;C, whereas free cells reached 78.86%. Under hydrolysate stress, encapsulated cells preserved 99.42% viability after 48 h and retained metabolic activity in a subsequent fermentation cycle. Overall, ZIF-8 immobilization improved cellular stability and process resilience, supporting its application in intensified, inhibitor-rich bioprocesses for industrial ethanol production.\u003c/p\u003e","manuscriptTitle":"Encapsulation in ZIF-8 as a Biopreservation Strategy and Enhancement of Thermal Tolerance of Saccharomyces cerevisiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 08:04:40","doi":"10.21203/rs.3.rs-8926210/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-26T14:57:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-25T20:34:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T14:14:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247188921773269204438105215906623268565","date":"2026-03-04T18:58:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1363144689880814193202424475712214597","date":"2026-03-04T13:23:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T20:00:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313698183102000037798096246682937468186","date":"2026-03-02T18:42:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-02T18:18:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-02T18:04:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T15:25:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Brazilian Journal of Chemical Engineering","date":"2026-02-20T12:51:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"brazilian-journal-of-chemical-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bjce","sideBox":"Learn more about [Brazilian Journal of Chemical Engineering](http://link.springer.com/journal/43153)","snPcode":"43153","submissionUrl":"https://www.editorialmanager.com/bjce/default2.aspx","title":"Brazilian Journal of Chemical Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"60e52705-8de6-4227-8e8e-c68b346ec37a","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T15:11:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 08:04:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8926210","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8926210","identity":"rs-8926210","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}