Bioethanol production from red banana waste via Saccharomyces cerevisiae under optimized conditions

Abstract

Red banana waste (Musa acuminata) residue (leaves, pseudostems, banana peels, banana stalks, and flowers) was used to produce bioethanol via Saccharomyces cerevisiae. The banana waste was dried and pretreated individually with sodium hydroxide and sulfuric acid. Alkaline treatment increased the reducing sugar content more than acid hydrolysis. Bioethanol production from banana residue via enzyme saccharification and fermentation was performed via the filter paperase enzyme and fermentation via S. cerevisiae. The bioethanol production was temperature dependent. Maximum production was achieved at 36 °C (27.3 ± 0.3 g/L), after 96 h (30.4 ± 1.1 g/L), at a 7.5% substrate concentration (33.5 ± 0.91 g/L) and at pH 5.0 (36.2 ± 0.48 g/L). The maximum bioethanol production was achieved by the immobilized S. cerevisiae cells after 96 h of fermentation (39.8 ± 0.55 g/L). Moreover, at this stage, bioethanol production was 35.9 ± 0.51 g/L in S. cerevisiae-free fermentation. The bioethanol yield was 11% greater in the immobilized culture than in the free-cell fermentation after four days. Beyond its renewable energy role, bioethanol reduces fossil fuel emissions associated with neurological and developmental disabilities, a connection that will be evaluated in forthcoming research.

Full Article

Bioethanol Production from Red Banana Waste via Saccharomyces cerevisiae under Optimized Conditions

Dhas Sugatha Deepa Dhas,^a,* Arokya Glory Pushpa Thiraviam,^b Muthumareeswaran Muthuramamoorthy,^c,d,* Abdullah Alodhayb,^c,d and Selvaraj Arokiyaraj ^e

Red banana waste (Musa acuminata) residue (leaves, pseudostems, banana peels, banana stalks, and flowers) was used to produce bioethanol via Saccharomyces cerevisiae. The banana waste was dried and pretreated individually with sodium hydroxide and sulfuric acid. Alkaline treatment increased the reducing sugar content more than acid hydrolysis. Bioethanol production from banana residue via enzyme saccharification and fermentation was performed via the filter paperase enzyme and fermentation via S. cerevisiae. The bioethanol production was temperature dependent. Maximum production was achieved at 36 °C (27.3 ± 0.3 g/L), after 96 h (30.4 ± 1.1 g/L), at a 7.5% substrate concentration (33.5 ± 0.91 g/L) and at pH 5.0 (36.2 ± 0.48 g/L). The maximum bioethanol production was achieved by the immobilized S. cerevisiae cells after 96 h of fermentation (39.8 ± 0.55 g/L). Moreover, at this stage, bioethanol production was 35.9 ± 0.51 g/L in S. cerevisiae-free fermentation. The bioethanol yield was 11% greater in the immobilized culture than in the free-cell fermentation after four days. Beyond its renewable energy role, bioethanol reduces fossil fuel emissions associated with neurological and developmental disabilities, a connection that will be evaluated in forthcoming research.

DOI: 10.15376/biores.20.4.10685-10705

Keywords: Banana waste; Biomass; 2G ethanol; Saccharification; Fermentation; Neurological disabilities

Contact information: a: Bioprocess Engineering Division, Smykon Biotech, Nagercoil, Kanniyakumari, India; b: Department of Zoology, Holy Cross College (Autonomous) Nagercoil, Affiliated to Manonmaniam Sundaranar University, Tirunelveli, India; c: King Abdullah Institute for Nanotechnology, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; d: King Salman Center for Disability Research, Riyadh 11614, Saudi Arabia; e: Department of Food Science and Biotechnology, Sejong University-05006, Korea;

*Corresponding authors: mramamoorthy@ksu.edu.sa;deepadhasds@gmail.com

Graphical Abstract

INTRODUCTION

The demand for renewable energy has increased worldwide in recent years. This trend can be attributed to a continuous decline in available resources of fossil fuel energy due to the increasing population and rapid industrialization. The relatively low price of biofuels, especially biodiesel, bioethanol, and biohydrogen, has attracted considerable attention. Biofuels are broadly classified into first- and second-generation biofuels. First-generation biofuels are obtained from agroindustrial wastes, lipids, oil, and carbohydrates. In contrast, second-generation biofuels are obtained from lignocellulosic biomass, including cellulose-rich plant biomass such as stems, stalks, and wood (Naik et al. 2010). Second-generation biofuels (2G), such as biomethanol, biohydrogen, and mixed alcohols, are being commercialized. 2G biofuels, also referred to as advanced biofuels, reduce the use of food and minimize the competition between food and fuels. In addition, the use of 2G biofuels reduces carbon emissions. Among the nonfood feed stocks used in bioprocesses to produce 2G biofuels are manure, food waste, spent cooking oil, sawdust, wood, leftovers from agriculture, garbage, food processing wastes and energy crops (Balasubramanian et al. 2021; Bello et al. 2022; Bhatia et al. 2022). Lignocellulosic materials from crops are the most promising biomass due to their widespread availability in most countries throughout the year and their low cost. The lignocellulosic material composition from the leftover layer is heterogeneous, and an additional pretreatment method is warranted during 2G-biological production (Marraiki et al. 2020).

Bioethanol is obtained by physical, chemical, or enzymatic hydrolysis and fermentation of lignocellulosic biomass. Generally, a three-step procedure is utilized in bioethanol production, which includes pretreatment of biomass, saccharification, and microbial fermentation. Pretreatment is one of the important steps and is achieved via chemical (strong acid or alkali), physical (heat treatment or steaming), biological (enzymatic), or a combination of these three methods. The major objective of the pretreatment method is to change the structure of the lignocellulosic biomass and improve the cellulose content. Microbes utilize this pretreated biomass more easily than untreated biomass does. The yield of cellulose is affected by the selected chemical and physical treatment. The pretreatment procedure affects the lignin structure of the plant biomass, increases the available surface area, alters the solubility of the biomass, depolymerizes, and decomposes cross-links between macromolecules (Al Farraj et al. 2020). Alkali treatment has been found to disrupt the lignin component of the biomass. Therefore, pretreatment methods are recommended to make the biomass digestible to produce cellulase by microorganisms to release glucose (Desvaux 2005; Rajendran et al. 2018). The second step is microbial fermentation of cellulose and conversion of cellulose into glucose by microbial cellulases (saccharifications). The third step is the conversion of sugars into ethanol via the use of yeasts such as Saccharomyces cerevisiae (Irfan et al. 2016; Maceiras et al. 2021).

Microbial fermentation processes are widely used to produce ethanol. In the saccharification process, sugar is produced by cellulases, and the yeast S. cerevisiae is involved in the conversion of sugar into ethanol (Gupta and Verma 2015; Kamzon et al. 2016). This method is cost-effective, has improved saccharification efficacy, requires fewer enzymes, has a reduced operational time, has a high yield of ethanol, has a lower chance of inhibition and microbial contamination, and has a simple operational procedure (Sun and Cheng 2002; Chen and Fu 2016; Hakim et al. 2017).

Agro-industrial residues, including crop residues, are widely used to produce 2G biofuels. Approximately 130 countries cultivate banana plants worldwide, which are considered the second most common crop species, followed by citrus fruits. The share of banana fruit on the global market is 19%, and 114.08 million metric tons of banana waste is generated, which has caused environmental problems (Alzate Acevedo et al. 2021; Vijayaraghavan et al. 2021). In several countries, banana leaves are used to serve foods and act as natural packing agents. The moisture content of banana leaves protects foods and results in a fragrant aroma (Raji et al. 2017). Traditionally, banana waste has been used as animal feed and as organic compost for crops. Banana fruit can be used for several purposes and in biorefineries because of its lignocellulosic nature. They are used for the preparation of organic chemicals, gases, and other thermochemical compounds (Rambo et al. 2015). In addition, the remaining banana fruit residues can be used for heat and biogas production. Banana peels can be used for the degradation of carbohydrates, and significant amounts of xylose (9.9/100 g) and glucose (8.2/100 g) can be obtained (Pereira et al. 2021).

Agro-industrial wastes generated from banana processing industries are used in bioplastic production, biofuel production, and wastewater treatment (Vijayaraghavan et al. 2017; Alzate Acevedo et al. 2021). However, poor utilization of these wastes has a severe impact on the environment, contaminating water bodies, generating greenhouse gases, and causing environmental pollution (Martínez-Ruano et al. 2018). Biorefinery approaches are considered suitable alternatives for reducing waste volume, reducing environmental pollution, and producing high value-added products. In addition to plant-based biomass, several sectors have shown interest in the use of biorefinery methods for waste, such as sewage sludge (Banerjee et al. 2011), food waste (Fava et al. 2015), municipal solid waste (Pérez et al. 2020), and nonedible oils (Rojas Alfaro et al. 2018). Biorefinery methods are useful for the effective utilization of agroindustrial wastes and the reduction of environmental pollution (Fava et al. 2015). The moisture content of banana residues is high, which promotes their degradation of organic matter before production, thus affecting their transportation, handling, storage, and biorefinery technologies (Pathak et al. 2016). In banana rachises, the lignin content is high, whereas hemicelluloses, holocellulose, and lignin contents are high in banana leaves, which are considered reservoirs for biorefinery processing (Guerrero and Muñoz 2018). In banana, fruit peel residue composition varies according to variety, species, geographical location, seasonal variation, and maturation stage (Pathak et al. 2016). Banana rachises and peels are rich in several polymers, such as cellulose, pectin, lignin, proteins, fiber, water-soluble sugars, phenolic compounds, chlorophylls, and various minerals (Lavoine et al. 2012). Banana stalks have very low levels of lignin and high holocellulose contents; these can be regarded as favorable attributes relative to use of the resulting fibers in the pulp and paper industry. The material can also be used to produce ethanol after pretreatment (Ingale et al. 2014). In recent years, much attention has been given to the use of banana waste for bioenergy and to improving the circular economy (Rincón-Catalán et al. 2022). Among several banana varieties, red banana is rich in several essential nutrients, including vitamins, amino acids, and minerals (Tan et al. 2024). Bioethanol as a renewable fuel offers significant advantages in mitigating emissions of fine particulate matter, nitrogen oxides, and other toxic by-products of fossil fuel combustion. Exposure to these pollutants has been strongly associated with neurological impairments, developmental delays, and long-term disability outcomes in vulnerable populations. Thus, valorization of red banana waste for bioethanol production not only advances sustainable energy but also provides a potential pathway to reduce disability-related health risks (Landrigan et al. 2018). Thus, the conversion of red banana waste into bioethanol advances sustainable energy while providing a scientifically grounded basis for further investigations into its role in public health protection. In this study, red banana waste was used as a low-cost substrate to produce ethanol using Saccharomyces cerevisiae.

EXPERIMENTAL

Substrate

Red banana (Musa acuminata) waste, including leaves, pseudostems, banana peels, banana stalks, and flowers, was collected from agricultural fields. These wastes were dried in an oven at 60 ± 1 °C until a constant weight was reached, ground into powder, and sieved through a 1 mm sieve. The powdered sample (0.5 to 1.0 mm in size) was stored in an airtight container for future use. The moisture content and ash content were estimated as described by Dziedzic et al. (2015). The amounts of hemicelluloses, cellulose, and lignin were analyzed via the standard NREL method. The amount of pectins in the banana waste was estimated as described by Oberoi et al. (2012). Each experiment was performed three times, and the mean value was used for data analysis (Sluiter et al. 2010). The total protein content of each sample was estimated via the AOAC method (Latimer 2012).

Pretreatment

Banana waste was pretreated with acid, alkali, or hydrogen peroxide separately. To perform acid treatment, 10 g of fine powder was mixed with 2, 4, 6, 8, and 10% sulfuric acid. The mixture was autoclaved at 121 °C for 2 h (Chiranjeevi et al. 2018), and the optimum acid concentration was determined. To analyze the optimum alkaline pretreatment (NaOH), various concentrations of sodium hydroxide (1, 2, 3, 4, and 5%)were mixed with the biomass and treated for 30 min at 121 °C (Fuertez et al. 2021). All pretreatment experiments were performed in 250 mL round bottom flasks, and the solid‒liquid ratio was 1:10. The solid phase was subsequently collected, washed with distilled water three times, and dehydrated at 40 °C. The reducing sugars of the pretreated waste were determined (Brummer et al. 2014).

Enzyme Hydrolysis of Pretreated Banana Waste

A total of 10 g of pretreated banana waste (dry weight) was mixed with 100 mL of citrate buffer (pH 4.8, 0.05 M). Commercial β-glucosidase (>250 IU/g) and cellulases (>125 IU/g) were used for enzymatic hydrolysis. The pretreated sample was incubated at 40 °C for 42 h with shaking at 100 rpm. Enzymatic hydrolysis was initiated by loading 50 U/g cellulase and 25 U/g β-glucosidase. The samples were withdrawn every 12 h (0 h, 12 h, 24 h, 36 h, and 48 h) and centrifuged at 10,000 rpm for 10 min. The reducing sugars in the culture supernatant were determined (Brummer et al. 2014). The level of hydrolysis was determined via Eq. 1:

 (1)

Inoculum

Saccharomyces cerevisiae (ATCC 7754) was used as an inoculum to produce ethanol in banana waste medium. Briefly, S. cerevisiae was cultivated on yeast medium agar and incubated at 28 °C. The selected yeast strain was maintained at 4 °C and subcultured every 15 days on a Petri dish before being subjected to the experiment. The yeast strain was inoculated into a 500-mL Erlenmeyer flask consisting of 400 mL of yeast extract peptone dextrose (DPY) medium containing 0.5% (w/v) yeast extract, 1% (w/v) peptone, and 2% (w/v) dextrose and incubated for 48 h at 28 °C. Yeast growth was monitored via a UV/visible spectrophotometer at 550 nm (Bahry et al. 2017).

Bioethanol Production and Analysis

The culture medium (150 mL) was composed of 3 g of yeast extract, 3 g of peptone, and 10% reducing sugars (enzymatic hydrolyzed banana waste). The culture medium (150 mL) was sterilized for 15 min at 121 °C. Then, 0.5 mL of inoculum was added, and the mixture was incubated for 72 h at 30 °C. After every 24 h, the ethanol concentration in the culture medium was determined (Mendes et al. 2016). The amount of ethanol produced by the yeast cells was detected via a Waters 2695 Alliance high-performance liquid chromatography system (Waters Inc., Milford, CT, USA).

Effect of Temperature on Bioethanol Production

The production medium comprised 10% reducing sugar from banana waste, which was sterilized and cooled, and 50 U/g cellulase and 25 U/g β-glucosidase were added. Then, 5% inoculum was introduced, and the culture was incubated at 32, 34, 36, 38, and 40 °C. The mixture was placed on a rotary shaker incubator at 150 rpm. The fermentation experiment was performed under oxygen-limited conditions. Sampling was performed, and the amount of ethanol was detected via high-performance liquid chromatography (HPLC).

Effect of pH on Bioethanol Production

The optimum pH of the culture medium was analyzed by varying the pH value of the culture medium from 3.5 to 6.0. The other variables were kept at optimum levels. A fermentation experiment was performed, and the amount of bioethanol used was determined.

Effect of Fermentation Period on Bioethanol Production

The production medium was composed of 10% reducing sugar from banana waste, which was sterilized and cooled. Then, 50 U/g cellulase and 25 U/g β-glucosidase were added. Then, 5% inoculum was added, and the culture was incubated for 24 to 144 h. The mixture was placed on a rotary shaker incubator at 150 rpm at 36 °C under oxygen-limited conditions. The amount of ethanol was detected by high-performance liquid chromatography (HPLC) up to 144 h.

Effect of Sugar Concentration on Bioethanol Production Prepared from Banana Waste

In this study, an experiment was performed to determine bioethanol production at various concentrations of sugar. Briefly, banana waste reducing sugars were added at 2.5, 5.0, 7.5, and 10% in a production medium. S. cerevisiae (5%) was inoculated after sterilization and cooling. The culture was subsequently maintained at 36 °C for 120 h.

Effect of Inoculum Concentration on Bioethanol Production

The enzymatically hydrolyzed reducing sugar (7.5%) was added to the production medium. The other culture conditions included shaking at 150 rpm, incubation at 36 °C, and 120 h of yeast fermentation. S. cerevisiae was inoculated at various concentrations (2 to 10%) after sterilization and cooling. The amount of bioethanol generated was determined after 120 h.

Immobilization of S. cerevisiae Cells

S. cerevisiae was cultivated in YPD broth medium, the viable cells were counted, and the cell density of the culture used for the immobilization process was 1 × 10⁸ CFU/mL. The culture was subsequently centrifuged at 5,000 rpm for 10 min, after which the cells were harvested. The harvested cells were washed with double distilled water several times, followed by 0.85% (w/v) sodium chloride (NaCl), and was further added to 2.5% (w/v) sodium alginate solution at 28 ± 1 °C. The yeast cells were mixed with sodium alginate solution for 5 min and loaded into a 10 mL syringe. The mixture was slowly added to a 0.1 M CaCl₂ solution, and the immobilized beads were obtained. The diameter of the formed bead was approximately 6 mm. The Ca-alginate beads were slowly stirred via a magnetic stirrer for 1 h and stored in 0.1 M calcium chloride solution until further use (Waluyo et al. 2017).

Ethanol Production by Immobilized and Free Saccharomyces cerevisiae

The optimized culture medium was prepared to produce ethanol from immobilized and free Saccharomyces cerevisiae. The culture medium comprised 7.5% enzymatically treated banana waste reducing sugars, and the other parameters were 96 h of fermentation, pH 5.0, and a 36 °C cultivation temperature. A total of 50 mL of 7.5% banana waste reducing sugar was prepared, and free yeast cells were inoculated after sterilization. In another experiment, 50 mL (immobilized bead volume) of immobilized cells was added to culture medium comprising 7.5% reducing sugars, and the experiment was performed for 96 h (Sirisansaneeyakul et al. 2013). The amount of ethanol generated was determined.

Reusability of Alginate Beads in Successive Fermentation Cycles

At the end of each reuse, to determine the amount of viable cells in the beads with 2.5% sodium alginate, the beads were liquefied via a chemical method adapted from Göksungur et al. (2005). The 10 beads were weighed and dissolved in 50 mL of a sterile 50 mM sodium citrate solution (Merck, Darmstadt, Germany), with continuous stirring for approximately 1 h at room temperature. The cell viability was determined by quantifying the number of CFUs in YPD media incubated at 25 °C for 3 to 5 days. The viability of S. cerevisiae cells was determined via the spread plate method (Waluyo et al. 2017). The amount of bioethanol produced in all five cycles was determined.

Statistical Analysis

Analysis of variance (ANOVA) was performed with Excel and SPSS 23 (Chicago, IL, USA). The results are expressed as the means ± standard deviations.

RESULTS AND DISCUSSION

Chemical Composition of Banana Waste

The chemical composition of banana waste before pretreatment is provided in Table 1. The ash content of the banana waste was 13.3 ± 0.15%, and the moisture content was 70.3 ± 1.5%.The amounts of cellulose, hemicelluloses, and lignin were 24.3 ± 1.1%, 19.2 ± 0.9%, and 6.45 ± 0.4%, respectively. The pectin content of the banana waste was 9.42 ± 0.1%.

Table 1. Chemical Characterization of Raw Banana Waste Utilized for the Production of Bioethanol

Notes: Banana waste comprises leaves, pseudo stems, banana peels, banana stalks, and flowers. The means ± standard deviations are the averages of three replicate analyses.

Pretreatment of Banana Waste

Two different chemical pretreatment methods were used for the pretreatment of banana waste. The dried banana waste was pretreated with concentrated sulfuric acid (2, 4, 6, 8, and 10%), and the yield was high at 6%.The reducing sugar content obtained in the acid treatment reached a maximum at 6% sulfuric acid (13.5 ± 0.10%), which was lower than that in the alkaline treatment. After 4% NaOH treatment, 14.5 ± 0.10% reducing sugars were detected (Table 2).

Acid and alkaline treatments are considered reliable methods for the treatment of plant biomass. However, alkaline treatment is considered a most suitable method for improving product yield, and the present findings are consistent with those of previous studies (Nascimento et al. 2023). The present findings are consistent with previous findings on banana waste (Fiallos-Cárdenas et al. 2022), banana flower waste (Thokchom et al. 2023), banana pseudostems (Merais et al. 2022), and banana sheaths (Subramanian et al. 2022).

Table 2. Acid and Alkaline Pretreatment of Plant Biomass and Reducing Sugar Yield

Enzymatic Hydrolysis of Pretreated Banana Waste

After the delignification step with sulfuric acid and sodium hydroxide, the pretreated banana waste samples were further treated with enzymes (β-glucosidase and cellulases). This enzymatic reaction aimed to release sugars from cellulose. This step is critical for the conversion of banana waste to the desired products. Microorganisms utilize these sugars for growth and product formation (Gonzales et al. 2016). The supplemented enzymes acted on cellulose and released glucose monomers. As shown in Fig. 1, after 12 h of enzymatic treatment, the reducing sugar content of the acid-treated banana waste was 14.4 ± 0.9%, and the reducing sugar content increased after 24 h (40.3 ± 1.1%).

In the alkaline-treated biomass, the reducing sugar content reached a maximum after 24 h of enzymatic hydrolysis (41.5 ± 0.9%). However, the reducing sugar content decreased after 48 h of enzymatic hydrolysis. In banana pseudostems, the glucose yield was 28.9% after acid pretreatment and enzymatic hydrolysis (Hossain et al. 2019). The free sugar yield was high when banana waste was pretreated with the optimum concentration of enzymes because lignocelluloses are generally heterogeneous. Hence, pretreatment methods may improve or affect product yield. The success of enzymatic saccharification is based on the type of biomass and pretreatment method. Acids generally disrupt lignin and improve cellulose susceptibility to biocatalytic hydrolysis (Łukajtis et al. 2018).

Fig. 1. Enzymatic hydrolysis of pretreated red banana waste for the production of bioethanol by yeast fermentation. The error bar represents the standard deviation.

Effect of Temperature on Bioethanol Production by S. cerevisiae

The optimum temperature for improving bioethanol production was determined. Bioethanol production was temperature dependent, and maximum production was achieved at 36 °C (27.3 ± 0.3 g/L) and was lower at higher incubation temperatures (Fig. 2).

Fig. 2. Effect of temperature on bioethanol production. The error bars represent the standard deviation.

Temperature is one of the critical factors affecting membrane turgidity of yeast cells, and enzymatic activity and thermostable yeast cells are preferred for industrial processing. The amount of bioethanol produced by S. cerevisiae in this study was compared with that in previous studies. The yeast S. cerevisiae ITV-01 has been isolated from sugarcane molasses and produces the maximum amount of bioethanol at 30 °C, and the yield was 58.4 g/L (Ortiz-Muñiz et al. 2010), which was higher than that in this study. The optimum incubation temperature increased yeast growth and improved product formation. Lin et al. (2012) optimized ethanol production via S. cerevisiae BY4742 and reported maximum ethanol production between 30 °C and 40 °C. Moreover, ethanol production was reduced significantly at 50 °C, which was consistent with the results of this study. The variation in ethanol production at higher temperatures might be due to changes in the transport system, which might increase the accumulation of ethanol in yeast cells. The optimum temperature for ethanol production by Saccharomyces cerevisiae was 30 °C when yeast was cultivated in sugarcane molasses substrate (Rasmey et al. 2018), 41 °C in 10% glucose medium (Benjaphokee et al. 2012), and 45 °C in pretreated wheat straw substrate (Ruiz et al. 2012). The present investigation and previous studies revealed that the optimum incubation conditions for the growth of S. cerevisiae may vary on the basis of the strain type, culture medium, fermentation type, and ethanol concentration in the medium.

Effect of pH on Bioethanol Production

Bioethanol production reached a maximum at pH 5.0 (36.2 ± 0.48 g/L), and a slight reduction in ethanol yield was observed at higher pH values (Fig. 3). The pH is an important factor for yeast growth and metabolism, and the amount of H⁺ determines the pH of the culture medium, which affects the permeability of nutrients into yeast cells through the membrane (Lee et al. 2011; Zabed et al. 2014). Alterations in the pH of the culture medium affect the vacuolar and cytosolic pH of S. cerevisiae, and a moderate bioethanol yield was observed above pH 5.5. Changes in the pH of the culture medium affect the ATPase activity of the plasma membrane (Peña et al. 2015); hence, an optimum pH value is required for yeast growth and ethanol fermentation. At acidic pH values, the intracellular pH reduces the permeability of the plasma membrane to protons and the consumption of adenosine triphosphate, which significantly reduces the glucose uptake of microorganisms and glycolytic activity (Woo et al. 2014).

Fig. 3. Effect of pH on bioethanol production. The error bars represent the Standard Deviation.

Effect of Fermentation Period on Bioethanol Production by S. cerevisiae

The bioethanol production was 3.1 ± 0.92 g/L after 24 h at 36 °C. The bioethanol content was highest (30.4 ± 1.1 g/L) after 96 h when initial inoculum level was 5%. The bioethanol production decreased to 23.2 ± 0.75 g/L after 144 h of fermentation (Fig. 4). In an earlier study, ethanol production reached a maximum at the 38 h incubation period, and the initial inoculum concentration was 2% (Nikolić et al. 2009). The type of culture medium, amount of glucose, incubation temperature, and initial inoculum level may affect the fermentation process. Sudhakar et al. (2021) recently reported increased ethanol yield after four days of incubation, and the final yield was 1.57 g/L when Ganoderma lucidum was used as the test organism to meet industrial demand. The yield obtained in this study was greater than that reported previously. The bioethanol conversion efficacy of S. cerevisiae was greater than that of G. lucidum. Banana peels were used as the culture medium to produce ethanol, and the yield was 13 g/L by S. cerevisiae after 10 to 12 h, whereas it was lower (13 g/L) in the culture media containing K. marxianus at 10% inoculum and at 41 °C. Conversely, presaccharification, simultaneous saccharification and fermentation increased ethanol production (32.6 g/L) within 64 h of incubation in banana peel medium (Palacios et al. 2021).

Fig. 4. Effect of fermentation period on bioethanol production. The error bar represents the standard deviation.

Effect of Sugar Concentration on Bioethanol Production Prepared from Banana Waste

The effect of sugar concentration on bioethanol production was determined, and the results are shown in Fig. 5. The bioethanol yield was 14.9 ± 0.53 g/L at a 2.5% substrate concentration after 96 h of incubation. During the same incubation period, bioethanol production reached a maximum at a 7.5% substrate concentration (75 g L⁻¹) (33.5 ± 0.91 g/L). Moreover, bioethanol production decreased at a 10% substrate concentration (30.1 ± 0.88 g/L). The initial sugar concentration in the culture medium increased the specific growth rate of the yeast cells, and the average ethanol productivity increased. In this study, ethanol production was 2.24-fold greater with the 7.5% substrate than with the 2.5% substrate, which was consistent with a previous report. Le Man et al. (2011) used reducing sugars from food waste and achieved a 2.3-fold ethanol yield. The reported optimum culture conditions were pH − 4.0, temperature − 40 °C, and a reducing sugar concentration of 7.5% (75 g L⁻¹). In the present study, bioethanol production was greater than that reported by Le Man et al. (2011), and the ethanol conversion ratio was greater in this study. The variation in bioethanol in the medium may be due to substrate inhibition and the tolerance of the yeast to relatively high ethanol concentrations. Yeasts utilize sugars anaerobically and ferment into ethanol and carbon dioxide. Yeast can convert 1 kilogram of glucose into 500 g of ethanol on the basis of theoretical estimation. However, it may vary on the basis of the organism and culture conditions (Righelato 1980; Borges Filho and Dettmer 2025).

Fig. 5. Effect of sugar concentration (2.5 to 10%) on bioethanol production. The error bar represents the standard deviation.

Effect of Inoculum Concentration on Bioethanol Production

Five concentrations of inoculums (2, 4, 6, 8, and 10%, v/v)were used to determine the effect of the inoculums on bioethanol production. At the 6% inoculum concentration, glucose was rapidly consumed by the yeast and improved the production of bioethanol (35.9 ± 0.55 g/L) (Fig. 6). Moreover, the bioethanol amount detected declined at increased inoculum levels.

Fig. 6. Effect of inoculum concentration on bioethanol production. The error bar represents the standard deviation.

A smaller inoculum size is preferable for fermentation to reduce the production cost of ethanol. However, increasing the concentration of inoculum affected ethanol production. The inoculum dosage requirement varies on the basis of the substrate and composition of the medium. In coffee husk medium (5 g/L), 3 to 4% was considered the optimum ethanol production (Gouvea et al. 2009), and 10% was suggested to improve the ethanol yield in cassava starch medium without any nitrogen source (Akaracharanya et al. 2011). The optimum inoculum size for ethanol production was 40 g/L yeast dry cell mass. The ethanol production rate was increased from 1.29 to 2.35 g/L/h when the yeast concentration increased from 0.5 to 5 g/L (Laluce et al. 2009). In fermentation technology, inoculum concentration is an important factor that improves bioethanol production potential (Altınışık et al. 2025).

Production of Bioethanol via Immobilized S. cerevisiae and Free Cells

The bioethanol production of free and immobilized S. cerevisiae cells was analyzed, and the results are depicted in Fig. 7. The maximum bioethanol production was achieved by the immobilized S. cerevisiae cells after 96 h of fermentation (39.8 ± 0.55 g/L). Moreover, at this stage, bioethanol production was 35.9 ± 0.51 g/L in S. cerevisiae-free fermentation. After four days, bioethanol production was 11% greater in the immobilized culture than in the free-cell fermentation. This bioethanol yield was similar to bioethanol production by the thermotolerant Kluyveromyces marxianus (Khumsupan et al. 2025). After six days of fermentation, bioethanol production decreased in both the immobilized and the free S. cerevisiae cultures when 7.5% sugar was used. Ethanol productivity is greater in immobilized cultures than in S. cerevisiae-free cell cultures because of its potential to tolerate diverse environmental conditions, such as acidic environments, high temperatures, and high sugar concentrations (Krisch and Szajani 1997; Najafpour et al. 2004; Zhu 2007).

Fig. 7. Bioethanol production by immobilized Saccharomyces cerevisiae and free yeast cells. The error bar represents the Standard Deviation.

The small size of the immobilized beads affected the ability of Saccharomyces cerevisiae SC90, which was previously reported by Najafpour et al. (2004). The immobilization method is considered a suitable method for ethanol production by S. cerevisiae via pretreated banana waste sugars under optimum culture conditions. The continuous search for potent Saccharomyces cerevisiae strains to improve bioethanol yield is a challenge that involves several factors, such as temperature, initial pH, osmotic stress, and viable yeast cell concentration (Techaparin et al. 2017). The thermotolerant yeast Saccharomyces cerevisiae SC90 has been used to produce ethanol, and the maximum product yield was achieved at 40 °C (Tareen et al. 2021). Saccharomyces cerevisiae was found to be suitable for producing ethanol via the immobilization technique by entrapment in a porous matrix, mechanical containment behind a barrier, and attachment on a support surface (Moreno-Garcia et al. 2018). In the present study, entrapment was used for the preparation of immobilized cells. The immobilization method has several advantages over free-cell cultivation, including minimal risk of contamination, high product yield, and improved overall fermentation process and reuse of immobilized beads. In this sense, the calcium alginate entrapment method used in this study effectively served as an important protective barrier against ethanol toxicity, similar to previous studies (Milessi et al. 2020). The immobilization technique is useful because it significantly reduces the lag phase of microbial growth and effectively improves stress tolerance, thus improving product yield (Khumsupan et al. 2025).

Reusability of Yeast Immobilized with Alginate Beads

The mortality of yeast cells and ethanol yield were estimated for five successive fermentation cycles. At the end of the first fermentation cycle, the yeast cell count was 2.3 ± 0.14 ×10⁸/bead, and the yeast population and ethanol production improved during the second cycle. This can be attributed mainly to the adaptation of yeast cells to the alginate beads and the ethanol content. After the fourth cycle, rupture was observed on the surface of the immobilized cells during fermentation. During this cycle, the yeast cell population and subsequent bioethanol production decreased. Table 3 shows the profile of yeast growth and ethanol concentration during the first and fifth fermentation cycles. The present findings are similar to previous results (Clementz et al. 2015). In addition, ethanol production was greater during the second cycle than during the first fermentation cycle. The increased production in the second and third cycles was due mainly to the increased population of yeast cells within the sodium alginate beads. This finding is in line with previous reports that microbial cells can grow within alginate beads, and increased production has been reported previously in sodium alginate beads entrapped with S. cerevisiae (Clementz et al. 2015). In the present study, a significant amount of bioethanol was detected up to the third cycle, and this result was consistent with that of a previous study. The immobilization method has been used for the production of bioethanol by Saccharomyces cerevisiae using cellulose from wood waste. Bioethanol production was consistent up to the 3^rd cycle and declined after the fourth and fifth cycles (Pratama et al. 2023).

Table 3. Saccharomyces cerevisiae Population and Bioethanol Production in the Immobilized Sodium Alginate Beads

CONCLUSIONS

1. Banana waste was pretreated with either sulfuric acid or sodium hydroxide. The reducing sugar yield in the acid treatment was greatest at 6% (13.5 ± 0.10%), and it was 14.5 ± 0.10% in the 4% sodium hydroxide treatment. The reducing sugar content reached a maximum after 24 h of enzymatic hydrolysis (41.5 ± 0.9%).
2. The reducing sugar was used as the substrate for ethanol production by Saccharomyces cerevisiae in anaerobic fermentation. Bioethanol production was optimal at 36 °C, pH 5.0, and after 96 h, the inoculum concentration was 6%, and the substrate concentration was 7.5%.
3. The immobilization method was used to reuse yeast cells in fermentation cycles. In this work, a significant amount of bioethanol was detected up to the third cycle and declined after the fourth and fifth cycles. The present findings revealed that immobilized S. cerevisiae may be useful for achieving ecofriendly ethanol production, while contributing to ongoing research on reducing pollutant emissions and associated disability-related health risks.

ACKNOWLEDGMENTS

The authors acknowledge Research Institute Supporting Program (RICSP-25-1), King Saud University, Riyadh, Saudi Arabia.

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Article submitted: January 13, 2025; Peer review completed: August 1, 2025; Revised version received: September 4, 2025; Accepted: September 5, 2025; Published: October 27, 2025.

DOI: 10.15376/biores.20.4.10685-10705