Production of bioethanol from certain agricultural waste by fermentation

Abstract

Background

Biofuels from biomass are seen as a necessary replacement for petroleum due to rise of its price from time to time and its negative impact on the environment. The main objective of this investigation was to isolate yeast strains from sugary rich substrates and test their bioethanol production potential from agricultural wastes.

Results

Eighteen yeast strains, varying in color, margin, and cell shape, were successfully isolated from banana, avocado, and mango samples. Initial screening using Yeast Extract Peptone Glucose Broth (YPGB) led to the selection of four high-performing yeast isolates: AMUBa6, AMUBa2, AMUAvo2 and AMUMa3 produced 60.76%, 50.53%, 50.33%, and 47.66%, percent of ethanol respectively. Subsequently, these isolates were tested for bioethanol production using different concentrations of banana, avocado, and mango peels. Isolates AMUBa6 and AMUBa2 demonstrated the highest efficiency, yielding 76.14% and 70.71% ethanol, respectively, from 50 g of agricultural wastes. A significant enhancement in bioethanol yield was observed with the addition of malt to the fermentations medium highly improved the percent of bioethanol yield as compared to the control. The AMUBa6 showed the highest bioethanol percent 85.24 with the 10 g of malt followed by the AMUBa2 81.27.

Conclusions

This study has clearly indicated that the possibility of environmental isolated yeast strains converting the agricultural wastes into value added bioethanol. This shows that the double advantages of bioethanol production which in one hand produces a valuable product which will be the potential of energy of the world in the future and also contribute in minimizing the amount of the solid waste from the environment and contribute in the sustainability of the environment.

Introduction

Background of the study

The term biomass refers to the total sum of organic materials on the planet’s surface produced via photosynthetic processes. Biomass energy represents the various forms of energy extracted from these renewable, bio-based sources (Lucia et al., 2006). Biofuels are energy carriers that capture energy originally sourced from biomass, which includes plants, animals, microorganisms, and organic wastes. These fuels can exist in solid, liquid, or gaseous forms and encompass all biomass-derived products used for energy generation (Kang Qian et al., 2014). The rising global interest in biofuels stems from several primary factors: Petroleum Supply Concerns: Increasing demand for petroleum, coupled with a decline in known oil reserves, is creating uncertainty regarding future supply stability. Environmental Impact: Growing worries about global warming and the greenhouse gas (GHG) emissions resulting from fossil fuel consumption are pushing for cleaner alternatives. These concerns have subsequently prompted various government programs to promote the development and use of biofuels (Sarkar et al., 2012).

Bioethanol, chemically known as ethyl alcohol (C₂H₅OH), is a clear, colorless, and biodegradable alcohol. It has recently emerged as a significant renewable bio-energy source and a highly eco-friendly fuel. Its main application is as a potential substitute for petrol to power road transport vehicles and other automotive engines (Hossain and Jalil, 2015). The majority of bio-ethanol produced across the European Union utilizes sugar beet molasses as its raw material. In response to recent policy initiatives, the bio-ethanol sector in EU member states has undergone a period of significant and rapid expansion. Demand for bio-ethanol in the transportation sector was quantified at 12.6 billion liters in 2010 (Secondi et al., 2015). Sugarcane, oil palm, and cassava are considered the most promising crops for biofuel production across Africa. Globally, sugarcane stands out as the highest-yielding feedstock, typically producing an estimated 5,376 L of ethanol per hectare, based on an average yield of 66 tons per hectare. In many African nations, this yield could potentially increase to as much as 8,000 L per hectare under irrigated conditions. Oil palm ranks as the second-highest yielding global feedstock, estimated to generate 3,136 L of biodiesel per hectare based on worldwide average yields, with top-performing Southeast Asian countries achieving around 4,400 L per hectare (Brew-Hammond, 2004). Cassava stands out as the highest yielding starchy crop for ethanol production, generating over 2,000 L of ethanol per hectare based on the global average yield of 11.5 tons. Furthermore, under more intensive production methods in Africa, this yield could be more than doubled (Brew-Hammond, 2004). The potential for ethanol production in Ethiopia is significant, primarily through its sugar factories. The country possesses about 700,000 hectares of identified, irrigable land suitable for sugarcane, which could generate an estimated one billion liters of ethanol (Wendimu, 2015).

High pollution levels are largely a result of the excessive consumption of fossil fuels, particularly in large cities. Finding a viable, long-term alternative to liquid petroleum requires a shift toward environmentally sustainable energy sources. A recent measure enforced to tackle this issue is the widespread use of biofuels blended with gasoline, which effectively lowers the output of smog-forming carbon monoxide and unburned hydrocarbons. Securing a continual energy supply depends on our ability to convert renewable, non-fossil carbon materials such as various forms of biomass (including plants, fruit wastes, and algae) and organic wastes into usable fuel (Wyman, 1994). The raw materials required for bio-ethanol production via fermentation are the dominant economic factor, typically consuming more than 50% of the total production costs (Claassen et al., 1999). Currently, producing bioethanol from cellulosic and lignocellulosic materials, particularly wastes, provides a promising alternative solution to the global environmental, economic, and energy challenges we face today (Srivastava et al., 2014). Conversely, the abundance of carbohydrates, proteins, and minerals in food waste makes it an ideal feedstock for generating biofuels and biobased chemicals via microbial conversion processes (Luque and Clark, 2013).

Utilizing food and agricultural waste as a feedstock for producing biofuels and valuable chemicals, such as bioethanol, offers several significant advantages. This approach effectively resolves the food versus fuel conflict and allows for the use of low-cost raw materials. Beyond these benefits, it substantially reduces reliance on landfills, thereby minimizing greenhouse gas emissions and leading to better land management. While Ethiopia has successfully produced bioethanol from various organic materials, such as molasses, no research has yet focused on using local agricultural waste for this purpose. Therefore, the present research specifically aimed to address this gap by focusing on bioethanol production from agricultural waste using various yeast isolates and strains of Saccharomyces cerevisiae. This effort is crucial for overcoming the limitations of conventional agricultural waste recycling methods and simultaneously developing an environmentally friendly system that converts waste into a high-value product like ethanol fuel.

Materials and methods

Substrate sample collection and preparation

The substrates, consisting of avocado, banana, and mango peels, were sourced as waste samples from Lante Kebele, situated near Arba Minch in the Gamo Zone of Southern Ethiopia Nations, Nationalities, and Peoples’ Region (SNNPR). This study area is located at coordinates 6⁰2’ N, 37⁰33’ E with an elevation of 1,285 m above sea level. After collection, the peels were cut into small pieces and air-dried in sunlight for three days, followed by oven-drying at 65 °C for 48 h to reach a constant weight. To ensure a consistent substrate size for pretreatment, the dried material was pulverized using a Laboratory mixer grinder/homogenizer popular science apparatus workshops Pvt. Ltd. (PSAW-INDIA) at high speed (17,000 RPM) for 2 min. The resulting powder was passed through a 1.0 mm standard mesh sieve to achieve a uniform particle size distribution. For each peel type, 2.0 kg (dry weight basis) was utilized to ensure consistency across all experimental groups. The samples were then packaged in airtight plastic containers and stored in a freezer at −20 °C pending further analysis (Joshi et al., 2001).

Pretreatment of substrates

Substrate pretreatment was performed using dilute sulfuric acid (1.25% v/v) to enhance the conversion of lignocellulosic biomass into fermentable sugars. To evaluate the effect of substrate loading (solid-to-liquid ratio) on pretreatment efficiency, 100mL of the acid was added to separate 25 g, 50 g, and 100 g portions (corresponding to 25%, 50%, and 100% w/v loading) in 250mL Erlenmeyer flasks. The mixtures were heated to 100 °C and agitated at 150 rpm for 15 min using a New Brunswick™ shaking incubator to ensure uniform heat distribution. The resulting hydrolysate was subsequently neutralized to pH 4.5 using 2 M NaOH for fermentation. The effect of these varying substrate loadings and acid pretreatment was compared against untreated control substrates (Cheng and Timilsina, 2011).

Isolation of yeasts from banana, avocado and mango fruits

Collection of bananas, avocados and mangos fruits

Samples of banana, avocado, and mango, specifically selected at the fully ripened stage and exhibiting initial signs of natural fermentation, were collected from various fruit vending shops in Arba Minch to serve as sources for microbial isolation. It is important to note that these fermenting fruits were used exclusively for the isolation of indigenous strains and were distinct from the fresh, high-quality peels collected separately for use as fermentation substrates. Upon collection, all isolation samples were promptly transported to the Microbial Biotechnology Laboratory and stored under refrigeration at 4 °C to maintain microbial viability until processing (Demisie and Melese, 2024).

Media preparation

The medium for yeast isolation was prepared according to Wickerham (1951); following inoculation, samples were incubated at 28 °C to promote growth (Wickerham, 1951). This Yeast Extract Peptone Glucose Agar (YPGA) medium had the following composition per liter (g/L): yeast extract, 10, peptone 10, glucose 20, and agar 20. Each component was precisely weighed and dissolved in one liter of distilled water. The mixture was then boiled on a hot plate to ensure complete dissolution. Subsequently, the medium was sterilized in an autoclave at 121 °C and 15 psi for 15 min (Terrones-Fernandez et al., 2024). Following sterilization, 15 mg of Erythromycin was added to the cooled medium. The sterilized medium was then aseptically dispensed into Petri dishes under a laminar flow hood and allowed to cool and solidify.

Banana, avocado and mango sample preparation

Ten-gram samples of banana, avocado, and mango were weighed aseptically on sterile aluminum foil. To ensure the release of yeast cells, the fruit tissues were finely minced and homogenized using a sterile mortar and pestle. One gram of the resulting homogenate was added to 9mL of sterile distilled water and vortexed vigorously to create the initial 10^− 1 dilution. Subsequently, 1mL of this suspension was transferred to 9mL of sterile distilled water to form the 10^− 2 dilution; this serial dilution process was repeated until a 10^− 6 concentration was achieved (Aneja et al., 2014).

Inoculation and incubation

The prepared, sterilized, and cooled YPGA medium was inoculated with 0.1 ml of each sample from the 10^− 1 to 10^− 6 serial dilutions (Corbu and Csutak, 2022). Three Petri dishes (triplicates) were inoculated for each dilution. The plates were then incubated at 28 °C. Yeast growth and potential contamination were monitored, and after 72 h of incubation, isolated yeast colonies were selected and sub cultured for purification.

Sub culturing for purification

The isolated yeast colonies were sub cultured on fresh YPGA medium prepared as described. A single, well-isolated colony was selected using an inoculating needle, streaked onto a new YPGA plate, and then incubated at 28 °C for 72 h (Elgarhi et al., 2020). Following incubation, the purity of the culture was verified by visual inspection. A confirmed pure culture was then grown on a YPGA agar slant (for 72 h) and subsequently stored at 4 °C for long-term preservation and future experiments.

The phenotypic characterization of isolated yeast strains

The initial step involved characterizing the isolated strains based on their macroscopic morphological features and growth behavior on solid media. The colonies were meticulously observed and recorded for the following characteristic traits: Texture: Described as mucoid, fluid, viscous, or butyrous. Elevation: Classified as either flat or raised. Color: Noted as white or yellow. Surface Appearance: Evaluated as glistening or dull, smooth, rough, or sectored. Margin (Edge): Described as entire, undulating, lobed, or filamentous. Subsequently, the strains’ microscopic characteristics were determined using a young, actively growing culture (2–3 days old, grown at 28 °C) according to standard protocols (Holt et al., 1994). Cells were stained with Lactophenol-cotton blue and examined under a microscope to observe: Cell Shape, Reproduction Method: Specifically noting if budding was monopolar, bipolar, or multipolar, or if fission occurred and Conidial Formation (Elgarhi et al., 2020).

Biochemical characterization of yeast isolates

The biochemical methods were based on the utilization of carbon and nitrogen sources, as previously described by (Harrigan and McCance, 1976; Pereira et al., 2006). The ability of yeast isolates to utilize D-glucose, fructose, maltose, galactose, lactose and sucrose as a sole carbon source and production of gas was determined in Durham tubes in carbohydrate fermentation medium. A positive reaction was detected by observation of color change to yellow and gas formation in Durham tubes (for carbohydrate fermentation) in the solution.

Test of bioethanol production potentials of the yeast isolates

To assess the bioethanol production potential of the 18 yeast isolates, fermentation was conducted in 250mL flasks containing 100mL of YPGB medium. Each isolate was inoculated into two flasks and incubated under static conditions at 28 °C for five days. Following incubation, fermentation was halted, and the resulting broth was distilled at 78 °C. The ethanol concentration in the distillate was quantified using the potassium dichromate oxidation method, with absorbance measured at 600 nm using a spectrophotometer. Finally, isolates were selected based on their calculated ethanol concentrations in comparison with established standards (Verma et al., 2020).

Inoculums preparation

The fermentation process utilized inoculum derived from two distinct sources: commercial dry baker’s yeast (Saccharomyces cerevisiae, Safe-instant (manufactured by lesaffre Ethiopia food manufacturing PLC) acquired from a local market in Arba Minch, and the isolated yeast strains. To prepare the commercial inoculum, 10 g of dried yeast was suspended in 100mL of sterile distilled water to create a 10% (w/v) stock suspension. For the isolated strains, yeast biomass was harvested via centrifugation from an overnight culture and similarly adjusted to a 10% (w/v) wet weight concentration to ensure consistency. During the fermentation stage, 10mL of the prepared inoculum was added to the fermentation substrate (the raw material), resulting in a final standardized starter volume for the process (Montgomery, 2017).

Experimental design

The experiment followed a Completely Randomized Design (CRD) with a factorial arrangement, performed in triplicate for all treatment and control groups. Batch fermentation was conducted in 250 mL Erlenmeyer flasks. The substrates consisted of hydrolysates derived from avocado, banana, and mango peels, prepared according to the methods described by (Arumugam and Manikandan, 2011). To evaluate the effect of substrate concentration and microbial load, a factorial design was employed using the following factors: Substrate Loading: Four levels of peel hydrolysate (6.25 g, 12.5 g, 25 g, and 50 g). Inoculum Concentration: Two levels (0.5% and 1% v/v) using both commercial baker’s yeast and specific yeast isolates. The initial pH of the fermentation medium was adjusted to 4.5 using 0.1 M NaOH or HCl. The process was maintained at a constant temperature of 28 °C for a duration of eight days.

Data analysis

Data obtained from the triplicate experiments were subjected to a two-way Analysis of Variance (ANOVA) to evaluate the main effects of the fruit peel types and treatment conditions, as well as their interactions. Significant differences between mean values were determined using Tukey’s Honestly Significant Difference (HSD) test at a significance level of p < 0.05. All statistical computations were performed using IBM SPSS Statistics (version 20), while data organization and graphical representations were generated using Microsoft Excel.

Results and discussion

Isolation and identification of indigenous yeast strains from banana, avocado, and mango

A total of 18 yeast strains were isolated from 12 distinct fruit samples, including banana (Musa acuminata), avocado (Persea americana), and mango (Mangifera indica) (n = 4 per fruit type). Primary isolation was performed on Yeast Peptone Glucose Agar (YPGA), resulting in the systematic recovery of six isolates from each fruit category. Preliminary identification followed a polyphasic approach, integrating morphological observations such as colony topography, pigmentation, and vegetative budding patterns with biochemical profiling, including carbohydrate fermentation and assimilation tests. While these phenotypic traits provided a presumptive genus-level classification, definitive taxonomic assignment was confirmed via molecular analysis of the Internal Transcribed Spacer (ITS) ribosomal DNA region, ensuring the analytical rigor required for high-impact reporting. You can see the specific breakdown of these samples and their resulting isolates in Table 1.

Table 1.

Distribution of microbial isolates recovered from banana, avocado, and mango samples

Sources tested strains Isolated	No. of samples used	No. of yeast
Banana	4	6
Avocado	4	6
Mango	4	6
Total	12	18

Phenotypic profiling and morphological analysis of yeast isolates

Morphological characterization of the isolated colonies revealed predominantly smooth surfaces with regular, circular margins. The isolates exhibited a diverse chromatic range, including white, vibrant yellow, and soft pinks. Microscopic analysis further identified a variety of cellular geometries, specifically round, oval, spherical, and ellipsoidal morphologies. A comprehensive summary of these characteristics is presented in Tables 2 and 3, and 4.

Table 2.

Morphological assessment of yeast species isolated from banana samples

Characteristics
Strains	Surface	Margin	Color	Cells
AMUBa1	Smooth	Circular	Yellow	Oval
AMUBa2	Smooth	Circular	White	Oval
AMUBa3	Rough	Circular	White	Round
AMUBa4	Smooth	Irregular	Pinkish	Spherical
AMUBa5	Rough	Irregular	White	Ellipsoidal
AMUBa6	Rough	Circular	Yellow	Round

AMUBa: Arba Minch University Banana yeast isolate 1, 2, 3, 4, 5, 6

Table 3.

Morphological assessment of yeast species isolated from avocado fruits

Characteristics
Strains	Surface	Margin	Color	Cells
AMUAvo1	Rough	Circular	Pinkish	Spherical
AMUAvo2	Smooth	Circular	White	Round
AMUAvo3	Rough	Circular	White	Round
AMUAvo4	Smooth	Irregular	Yellow	Oval
AMUAvo5	Smooth	Circular	White	Ellipsoidal
AMUAvo6	Smooth	Irrgular	Yellow	Oval

AMUAVo: Arba Minch University Avocado yeast isolates 1, 2, 3, 4, 5, 6

Table 4.

Morphological assessment of yeast species isolated from mango samples

Characteristics
Strains	Surface	Margin	Color	Cells
AMUMa1	Smooth	Irregular	Pinkish	Spherical
AMUMa2	Smooth	Circular	White	Round
AMUMa3	Smooth	Circular	Pinkish	Round
AMUMa4	Rough	Circular	White	Oval
AMUMa5	Smooth	Circular	Yellow	Spherical
AMUMa6	Smooth	Circular	Yellow	Round

AMUMa: Arba Minch University Mango yeast isolates 1, 2, 3, 4, 5, 6

The accessibility of regional fruits such as bananas, avocados, and mangoes make them ideal substrates for isolating ethanol generating yeast strains. These results are consistent with the research conducted by Eghafona (1999), which demonstrated the effective extraction of native yeast from fermented pineapple juice for the purposes of alcohol synthesis (Eghafona et al., 1999). Comparative investigations by Bansal and Singh (2003) alongside Zabed et al. (2014) evaluated the efficacy of Saccharomyces cerevisiae and Zymomonas mobilis for ethanol synthesis. Their work specifically analyzed the fermentation of molasses and juice containing fermentable sugars (Zabed et al., 2014; Bansal and Singh, 2003). This investigation prioritized the isolation and phenotypic characterization of yeast strains derived from banana, avocado, and mango. A primary selection criterion was the ethanolic fermentation capacity of the isolates, a metabolic trait utilized to facilitate precise taxonomic identification. The morphological and cultural characteristics of the resulting isolates are documented in Tables 2 and 3, and 4. Although variations in colony topography, margin structure, and pigmentation were observed across the different fruit substrates, the predominant phenotype across all samples was characterized by smooth, circular, and white colonies.

Biochemical characterization of yeast isolates

Biochemical profiling of the eighteen yeast isolates obtained from various fruit sources demonstrated high fermentation efficiency across all target sugars. As detailed in Table 5, isolates derived from banana, avocado, and mango consistently metabolized glucose, fructose, sucrose, and maltose. The study identified specific strains, notably AMUBa6, as highly efficient fermenters characterized by showing excellent (+++) activity for CO₂ and ethanol generation from glucose and sucrose. This accelerated fermentation rate represents a critical technological attribute, suggesting the yeast’s broad potential across industrial sectors. While further optimization is required to meet the specific physiological demands of each industry, these results establish a strong foundation for its evaluation in bioethanol synthesis, commercial baking, and beverage production (Maicas, 2020). The present study prioritized the evaluation of rapid sugar fermentation kinetics, as efficient conversion rates represent a critical parameter for the optimization of industrial scale processes (Beigbeder et al., 2021) (Bai et al., 2022). While strains such as AMUMa1 exhibit significant activity indicating a functional complement of glycolytic enzymes their lower overall metabolic rate is likely attributable to inefficient substrate uptake or slower enzymatic kinetics within their internal pathways (Maicas, 2020).

Table 5.

Biochemical profiles of yeast strains derived from diverse fruit samples

Sugar sources	Glucose		Fructose		Sucrose		Maltose
Yeast isolates	CarP	AlcP	CarP	AlcP	CarP	AlcP	CarP	AlcP
AMUBa1	++	++	+	+	+++	+++	+	+
AMUBa2	+++	++	++	+	++	++	++	+
AMUBa3	++	++	++	++	+++	+++	+	+
AMUBa4	+	+	++	++	+	+	+	+
AMUBa5	+	+	++	++	+	+	+	+
AMUBa6	+++	+++	+	+	+++	+++	+	+
AMUAvo1	++	++	+	+	++	+++	+	++
AMUAvo2	+	+	+	+	++	++	++	+
AMUAvo3	++	++	+	+	+++	++	+	+
AMUAvo4	++	+	+	+	+	+	+	+
AMUAvo5	+	++	+	+	+	++	+	+
AMUAvo6	+	+	++	++	+	+	+	++
AMUMa1	+	+	+	+	++	++	+	+
AMUMa2	+	+	+	+	++	+	+	+
AMUMa3	++	+	+	+	+++	+++	+	++
AMUMa4	+	++	+	+	++	+	+	++
AMUMa5	++	++	+	+	+	+	+	++
AMUMa6	++	++	++	++	++	++	+	+

CarP: Carbon dioxide production AlcP: Alcohol production

+: good ++: very good +++: Excellent

Bio ethanol production potential of the different yeast strains isolated from banana, avocado and mango on YPGB

Indigenous yeast isolates recovered from banana, avocado, and mango samples exhibited high fermentative efficiency. Such characteristics indicate that these strains could substantially augment ethanol output, offering a strategic pathway toward more cost-efficient bio-alcohol manufacturing. Their respective bioethanol yields are comparatively analyzed in Fig. 1.

Fig. 1.

Bio-ethanol production potential of yeast strains isolated from different fruit sources

Analysis of the fermentative capacities of the selected isolates revealed significant variability in metabolic efficiency. In this study, bioethanol yield (%) is defined as the percentage of the theoretical maximum yield (0.511 g of ethanol per g of glucose consumed), calculated as follows:

The strains AMUBa6, AMUBa2, AMUAvo2, and AMUMa3 emerged as the most proficient, achieving ethanol conversion rates of 60.76%, 50.53%, 50.33%, and 47.66%, respectively. Conversely, the lowest production levels were recorded for AMUBa5 (12.06%), AMUAvo3 (10.8%), and AMUMa5 (8.2%). These variations highlight a key challenge in this research: achieving high ethanol production depends on identifying strains capable of withstanding the harsh conditions inherent in the fermentation process Tsegaye KN, Alemnew M, Berhane N (2024).

Recent studies have identified ripe banana peels as a viable source of yeast for ethanol fermentation. Under experimental conditions of 30 °C using a 40% glucose concentration, these isolated strains demonstrated ethanol yields of 3.6% and 5.8% (Brooks, 2008).

Among the screened candidates, the banana-derived isolate, designated AMUBa6, emerged as the top ethanol producer. This aligns with broader observations that phyllospheric yeasts naturally exhibit versatile sugar conversion efficiencies and significant osmotolerance. Given these inherent traits, the continued exploration of wild-type strains specifically those exhibiting elevated tolerance to ethanol toxicity remains a priority for enhancing the economic viability of industrial biofuel production (Negro et al., 2006). Based on these preliminary fermentation profiles, four high-performing yeast isolates (AMUBa6, AMUBa2, AMUAvo2, and AMUMa3) were prioritized for subsequent analysis.

Bio ethanol production potential of the four selected yeast isolates

To evaluate the bioethanol production capacity of the four selected yeast isolates, fermentation trials were conducted using varying concentrations of agricultural waste. Substrates consisting of banana, avocado, and mango peels were prepared at concentrations of 6.25, 12.5, 25, and 50 g per 100 ml of distilled water. Commercial baker’s yeast served as a control under identical conditions. The resulting ethanol yields for both the isolates and the control are illustrated in Fig. 2.

Fig. 2.

Bio-ethanol production from the different substrate concentration with different yeast isolates compared with Baker’s yeast

The experimental results demonstrate that the isolated yeast strains AMUBa6 (76.14%) and AMUBa2 (70.71%) significantly outperformed commercial baker’s yeast (44.64%) at the 20% w/v substrate level. Statistical analysis using a Two-way ANOVA (p < 0.05) indicates that ethanol output is primarily driven by the specific yeast isolate and the initial substrate concentration (mass-to-volume ratio). To address potential redundancy in the model, substrate mass and distilled water volume were consolidated into a single concentration factor. Optimal production was achieved at a 20% w/v concentration (50 g substrate in 250 ml water) inoculated with 10 ml of isolate While biomass accumulation was monitored, the final ethanol yield was more closely associated with the fermentation efficiency and sugar consumption rates of the specific isolates, rather than total biomass alone, as the most proficient strains (AMUBa6 and AMUBa2) demonstrated superior metabolic conversion under anaerobic conditions (Khatun et al. 2023).

Bioethanol production potential of the two isolated yeast strains, baker’s yeast and malt under the different substrates

The bioethanol production potential of the two isolated yeast strains (AMUBa6 and AMUBa2) was evaluated and compared against commercial baker’s yeast (Saccharomyces cerevisiae). These specific isolates were selected for this phase based on their superior performance during preliminary screening, where they demonstrated the highest glucose tolerance and ethanol gas production among all initial isolates. To enhance the enzymatic breakdown of the complex carbohydrates in the fruit waste, barley malt (kilned at 60 °C to preserve diastatic activity) was utilized as a natural source of alpha-amylase and beta-amylase. The variation in ethanol yield obtained from fermentation of different substrates and malt by two isolated yeasts from banana and baker’s yeast was shown in figure (3) below.

Fig. 3.

Bio-ethanol production from the different substrate of banana, avocado and mango peels, malt, isolated yeasts and baker’s yeast. A = 50 g of banana peels + 10 g of malt + 10 ml of yeast from isolated yeasts or baker’s yeast. B = 50 g of avocado peels + 10 g of malt + 10 ml of yeast from isolated yeasts or baker’s yeast. C = 50 g of mango peels + 10 g of malt + 10 ml of yeast from isolated yeasts or baker’s yeast

The experimental results demonstrate that Treatment A (utilizing isolate AMUBa6) exhibited superior performance, achieving a peak bioethanol yield of 85.24%. This optimal output was realized through a substrate to catalyst ratio comprising 50 g of banana peels, 10 g of barley malt, and 10 ml of the AMUBa6 strain. The significant enhancement in fermentation efficiency suggests that the AMUBa6 strain possesses a high metabolic affinity for the specific lignocellulosic composition of banana waste. In contrast, Treatment C, which utilized conventional baker’s yeast, resulted in a markedly lower yield of 55.57%. This identifies Treatment C as the least effective method within the study, highlighting the comparative advantage of specialized, locally isolated microbial strains over standard commercial yeast. To isolate the effect of the malt addition, negative controls consisting of substrate and yeast without malt were processed simultaneously. These controls yielded significantly lower ethanol concentrations (averaging 15–20% less than malt-supplemented samples), confirming that the barley malt provided essential fermentable sugars and enzymatic support necessary for maximizing yield from the lignocellulosic fruit waste.

Thermal management represents a critical challenge in optimizing ethanol yield. While Saccharomyces cerevisiae typically exhibits peak metabolic activity within the 25 °C to 35 °C range, the exothermic nature of the fermentation process necessitates rigorous temperature regulation to prevent heat-induced cellular inactivation (Tsegaye et al., 2024).

Building upon established benchmarks such as (Sree et al., 2000), who achieved 93 g/L at 30 °C, and (Nuanpeng et al., 2016), who used 30 °C for thermotolerant screening this study conducted all experiments at a controlled temperature of 28 °C within 250mL conical flasks over an 8-day duration. The fermentation performance of the locally isolated strains showed significant efficacy; the highest ethanol yields were observed in malt-supplemented media, reaching 85.24% for banana peels and 78.31% for avocado peels. These results suggest that AMUBa6 and AMUBa2 are highly effective for the bioconversion of fruit waste under the specified conditions.

Conclusion

This study successfully isolated eighteen yeast strains from fermenting banana, avocado, and mango fruits. Morphological and microscopic characterization revealed significant diversity in colony pigmentation and cellular architecture. Screening identified four high-performing isolates, AMUBa6, AMUBa2, AMUAvo2, and AMUMa3 with superior bioethanol production potential. Notably, in batch fermentation of banana peel substrate (50 g), isolates AMUBa6 and AMUBa2 demonstrated ethanol yields exceeding those of commercial baker’s yeast. Furthermore, the integration of malt into the substrate significantly enhanced fermentation efficiency, suggesting a synergistic effect on microbial metabolic activity.

Abbreviations

CRD

Completely randomized design

EU

European Union

GHG

Greenhouse gas

OD

Optical Density

SPSS

Statistical Package for Social Studies

YPGA

Yeast Extract Peptone Glucose Agar

YPGB

Yeast Extract Peptone Glucose Broth

Author contributions

**Bonsa Duressa** was responsible for the conceptualization of the study, methodology development, and performing the experimental investigation, including substrate fermentation and distillation he was the primary contributor to writing original draft preparation and approved the final manuscript.**Aweke Mamo** performed the sample collection and fermentation process, executed the formal analysis for the characterization of the yeast isolates, and approved the final manuscript.**Nebiyu Chali** contributed sample collection, performed writing, review & editing (manuscript edition), and approved the final manuscript.

Funding

This research was funded by Arba Minch University (AMU) and the grant number GOV/AMU/TH4/CNS/Biol/02/2012.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have read and agreed on the final version of the manuscript for.

Competing interests

The authors declare no competing interests.