Sustainable Bioethanol Production Through Fermentation of Guava ( Psidium guajava L .) Fruit Waste Juice Supplemented With Sorghum

Abstract

Objective

Global reliance on costly and rapidly depleting fossil fuels worsens pollution and climate change, prompting a shift toward renewable bioenergy alternatives. Bioethanol offers a sustainable alternative that reduces fossil-fuel dependence and diversifies energy sources.

Methods

The present study investigated bioethanol production from guava waste juice supplemented with sorghum, using fermentation and distillation, offering a sustainable solution to competition with food supply and management of municipal solid waste. Supplementation with sorghum flour enhances the hydrolysis of complex sugars in guava fruit waste juice and improves fermentation efficiency after which the fermented juice broth was distilled at 78 °C to produce bioethanol.

Results

Total soluble solids (TSS) decreased throughout the fermentation period with a more pronounced reduction observed in the broth supplemented with sorghum. The mean TSS values for broths with and without sorghum supplementation were 2.443 ± 1.572 and 2.981 ± 1.817 °Brix, respectively, with the difference being statistically significant (p = 0.001). The sorghum-supplemented broth produced the higher alcohol content of 7.87% ABV than un-supplemented broth which gave 6.56% ABV as determined from specific gravity measurements before distillation. Distillation yielded 22%, 18%, and 12% alcohol content in the first three 100 mL aliquots with sorghum compared to 19%, 14%, and 9% without sorghum. Redistillation increased bioethanol purity from 9-22% to 50-84%, suitable grade for intermediate fuel, solvent, or a feedstock for other value-added bioproducts.

Conclusion

Overall, the study demonstrates the potential of guava fruit waste, complemented with sorghum as a sustainable feedstock for bioethanol production. This exploratory study shows that sorghum can enhance bioethanol yield and quality, while highlighting opportunities for process optimization and scale-up to support circular-economy and waste-to-energy initiatives.

Keywords

guava fruit waste bioethanol circular economy sustainable energy

1. Introduction

The demand for renewable energy sources, particularly bioenergy like bioethanol has surged due to growing interest in producing it from various clean and renewable biomass sources as an alternative to fossil fuels (FFs).^1,2 Researchers are exploring alternative biofuels from agricultural waste such as bioethanol to reduce the reliance on conventional fuels, take advantage of low emissions, and use it in vehicles, household appliances, and laboratories.^3,4 Currently, the globe is facing serious issues with energy security, economic stability, and environmental sustainability when FFs resources are overused.⁵ Thus, diversifying energy sources including the use of clean and renewable sources like biomass is essential as energy demand rises and FFs supplies run out.⁵ Agricultural residues, forestry waste, and organic waste are examples of biomass resources that provide sustainable alternative raw materials for energy production while mitigating the environmental effects of FFs exploitation.⁶ The best course of action is to develop second-generation bioethanol from primary agricultural waste materials. Recent studies highlights the importance of bioethanol production from food and agricultural wastes through bioprocessing.^7,8 Tanzania among other nations produces substantial quantities of fruit and has strong potential to expand production even further but the country continues to face major post-harvest challenges with an estimated 40-60% of harvested fruit lost before it reaches consumers due to inadequate storage, limited processing capacity, and inefficiencies along the supply chain.⁹ Besides, studies show that fruits produce high-quality alcoholic beverages due to their enhanced colour and flavour in their pulps.^10-12 Reports indicate that fruit wastes with their high sugar content and inexpensive collection costs are a viable resource for bioethanol production.^8,13-15 For instance, apples are commonly used for making cider while peaches and berries make specialized alcoholic beverages.^11,12,16 Historically, technologies have primarily focused on utilizing fruit and vegetable wastes to improve human and animal diets.^11,14 Still, studies indicate that sorghum, a carbohydrate-rich cereal grain has the potential to contribute to bioethanol generation.^17,18 The addition of sorghum to fruit waste juice enhances fermentation kinetics due to the presence of fermentable sugars.¹⁸ Still, sorghum contains key enzymes including amylases, glucoamylase, and proteases which enhance fermentation and bioethanol production by breaking down starch into simpler sugars.¹⁷ Glucoamylase is an enzyme that further breaks down maltose into glucose, enhancing the availability of glucose, a crucial substrate in the fermentation process.² Enzymes in sorghum improve fermentation efficiency, making it an ideal component for enhancing bioethanol quality and yield. Researches show that agricultural waste can be used as a feedstock for bioethanol production and combining it with sorghum enhances the conversion efficiency.^17,18 Guava (Psidium guajava L.), a high sugar content feedstock is an ideal substrate for bioethanol production through natural fermentation process.¹⁹ Ripe guava has high moisture content between 78-84%, high carbohydrate content, mostly glucose and fructose, dietary fibre between 5-6%, and little fat.^20-22 Along with noteworthy amounts of provitamin A, folate, potassium, and antioxidants like lycopene and polyphenols, it has an extraordinarily high vitamin C content.²⁰ While enzymatic activity decreases structural carbs and tannins, ripening improves sugars, fragrance chemicals, and bioactive.^20,23 However, guava fruits are highly perishable because they continue to ripen rapidly after harvest, making them vulnerable to spoilage. During peak production periods, this accelerated ripening leads to significant post-harvest losses, particularly when market volumes exceed available storage and handling capacity.²¹ Guava production is highest in countries such as India, Brazil, and Mexico, making them the leading global producers.²² However, commercial cultivation of guava in Tanzania is still minimal. Most production comes from naturalized guava trees that spread spontaneously into farmlands and uninhabited areas with widespread colonization occurring through seed dispersal by birds, mammals, and even human activity. As a result, guava production is largely unplanned and relies on unmanaged, wild-growing trees rather than organized orchards or improved cultivars. Because of limited commercial value, guava fruit in Tanzania remains unharvested in farms, leading to higher post-harvest loss. Still, there is limited documented information on guava production, utilization, and processing in Tanzania. This knowledge gap combined with various production and market constraints has significantly hindered the development of a sustainable and competitive guava value chain in the country.

In the present study, guava fruit waste was selected as a potential feedstock for bioethanol production due to its advantages, particularly its high content of fermentable sugars such as glucose, fructose, and sucrose which serve as suitable carbon sources for microbial fermentation. The fruit generates abundant by-products during processing, with approximately 80 kg of waste produced per metric ton of processed guava, making it readily available and relatively inexpensive.²⁰ In the absence of alternative uses, fruit growers often experience significant post-harvest losses, which negatively affect their socioeconomic livelihoods. Unlike food-grade substrates, this waste stream has limited direct economic value and therefore does not significantly compete with food or feed uses, instead, its utilization helps mitigate challenges associated with municipal solid waste. Reports indicate that guava-based substrates can support bioethanol production at natural sugar concentrations of around 10% (w/v), demonstrating their suitability for fermentation processes.²⁴ In contrast with other substrates such as molasses, one of the most widely used industrial feedstocks containing approximately 50-65% sugars and commonly used due to its high fermentable sugar content and fermentation efficiency.^25,26 Still, molasses has competing uses in food which can increase cost and limit accessibility in some regions.²⁶ Additionally, previous studies reveal the effective bioethanol yields from guava-based substrates, demonstrating their technical feasibility for fermentation.²⁷

The present study investigates the use of guava fruit waste juice supplemented with sorghum flour to enhance hydrolysis and fermentation efficiency, thereby improving bioethanol yield and quality while contributing to waste management and enhancing the economic value of guava processing for fruit growers. The study highlights the effectiveness of supplementing guava fruit waste juice with sorghum to enhance the fermentation process and ultimately improve the quality of bioethanol. The study further banks on a cost-effective, environmentally friendly bioethanol production method from guava fruit waste that does not use corrosive chemicals in the pre-treatment stages and its non-competitiveness to food chain, enhancing economic status, supporting local economies by transforming postharvest waste into valuable resources thus, supporting circular economy principals.

2. Material and Methods

2.1. Study Area and Sample Collection

The study was conducted in Iringa Municipality, Tanzania, at an elevation of 1554 meters and an average annual temperature of 20.8 °C. Guava fruit waste was collected from Mashine tatu markets, Mtwivila, Itamba, and Mkwawa wards in Iringa Municipal. Sorghum was collected from Soko Kuu market in Iringa Municipality. The experiments involving the juice extraction, fermentation, distillation processes, and bioethanol analysis were carried out in the Chemistry Laboratory.

2.2. The Study Design

The present study was conducted using a randomized experimental design to evaluate the effect of sorghum supplementation on bioethanol production from fermented guava fruit waste juice broth. Guava fruit waste was treated to obtain clear juice which served as the primary fermentation feedstock. Optimization experiment was conducted to determine the appropriate amount of sorghum supplementation. Different quantities of sorghum (10, 15, 30, 45, and 60 g) were each added to 1 L of fresh guava fruit waste juice, with 30 g yielding the most favourable hydrolysis and fermentation results. The study included a control setup without sorghum flour which served as the basis for comparison. All experiments were conducted in triplicate to ensure reliability and reproducibility of the results and fermentation was performed under controlled conditions at room temperature (22–27 °C), pH 3.64–4.08 for 14 days. During the fermentation process, key parameters including total dissolved solids (TSS), specific gravity (SG), temperature, and pH were monitored at 24-hour intervals.

2.3. Pretreatment of Guava Fruit Waste for Juice Production

A spring balance (Nops- CHINA) was used to accurately measure 49 kg of guava fruit wastes. The guava wastes were chopped them into smaller pieces, grinded, and blended with a blender (KENWOOD-BLPA-10) to obtain a highly viscous juice shown in Figure 1. The pretreatment process primarily aims to reduce particle size and disrupt the structural matrix of guava waste, thereby increasing the accessibility of cellulose and hemicellulose for subsequent hydrolysis and fermentation. After the extraction process, clean tap water was added in a 2:1 mass by volume ratio, 49 kg of guava with 24.5 litres of water, then stirred to obtain a homogeneous guava fruit waste juice solution. This step aimed at reducing the viscosity of guava fruit waste juice thus, ensuring easy fermentation and filtration of fermented broths for distillation step.

Figure 1.

Guava fruit waste pretreatment for juice production

2.4. Fermentation of Guava Fruit Waste Juice Broths

Fermentation media comprised of guava fruit waste juice augmented with sorghum flour and guava fruit waste juice without sorghum. Sorghum contains enzymes that hydrolyses complex sugars present in guava fruit waste juice hence, promoting the fermentation process and enhancing bioethanol yield. The fermentation was carried out in a 40 L container filled with 25 L of guava fruit waste juice and 25 L of guava fruit waste juice supplemented with 750 g of sorghum flour.

The fermentation broths were then sealed to allow effective natural fermentation at room temperature. The mixture of guava fruit waste juice with and without sorghum flour were left to ferment utilizing the natural microbes while the parameters including temperature, pH, TSS, SG, and percentage alcohol by volume (%ABV) were determined after each 24 hours over the entire 14 days fermentation period. After fermentation, the fermented broths were filtered from residues using a clean sterile sieve achieving 19 L and 21 L of guava fruit waste juice broths with sorghum and without sorghum, respectively, and stored in sterile plastic containers ready for distillation.

2.5. Distillation of Fermented Guava Fruit Waste Juice Broths

After the fermentation period, fermented guava fruit juice broths were distilled to obtain bioethanol of different concentrations. The filtrate was transferred into a laboratory distiller machine (VETA- Dar es Salaam) and distilled to extract bioethanol. The distillation process was carefully controlled via the connection of a distiller to an electricity source at a temperature of 78 °C being controlled using the temperature sensor (Delta Temperature Controller-DTA4848R0). The obtained bioethanol of different concentrations was analysed to determine its quality using alcoholmeter (Alcoholmeter Gay Lussac). The quantity was determined by measuring the volume of distillates using the measuring cylinder (AXIOM, Germany). During distillation, the first aliquot was collected after 20 minutes followed by the second aliquot at 25 minutes, and the third aliquot at 30 minutes each of 100 mL. Bioethanol purity was improved through redistillation at the same temperature conditions by reducing residual water content and refining the final product quality. Redistilled bioethanol was again collected in 100 mL increments and alcohol content analysed, ensuring its quality for potential biofuel or industrial applications. Figure 2 presents a schematic approach for bioethanol production process.

Figure 2.

Approach towards bioethanol generation

2.6. Analytical Assay

2.6.1. Determination of Total Soluble Solids for Guava Fruit Waste Juice

The concentration of sugars and other dissolved solids in guava fruit waste juice, TSS was established using a digital refractometer (Model: ERMA, TOKYO). Prior to measurement, the refractometer was calibrated using distilled water. A few drops of each juice sample were placed on the refractometer’s prism, and the refractive index was measured. The TSS values were expressed in degrees Brix (°Brix), where 1 °Brix represents 1 g of sucrose per 100 grams of solution (% w/w).²⁸ The measurement serves as a reliable and rapid indicator of the available fermentable sugars and is used to monitor sugar depletion during the fermentation process.

2.6.2. Determination of Specific Gravity

The SG of guava fruit waste juice was established to measure the concentration of fermentable sugars before fermentation, which relates to bioethanol yield. SG which reflects the liquid’s density relative to water, indicates TSS, primarily sugars in the juice. A glass hydrometer with a range of 0.980 to 1.160 was used for measurements. Each sample was placed in a 100 mL measuring cylinder, and the hydrometer was carefully immersed without touching the cylinder wall. Readings were taken at the lower meniscus of the liquid. Measurements were conducted on guava fruit waste juice with and without sorghum.

2.6.3. Determination of Percentage Alcohol by Volume

The %ABV during fermentation was established by measuring the change in SG of guava fruit waste juice prior and post fermentation. This method is based on the principle that a decrease in SG reflects the conversion of sugars into bioethanol. Measurements were taken using a hydrometer, and %ABV was calculated using equation (1).

% ABV = (Initial SG - Final SG) \times {131.25}^{30}

(1)

where, Initial SG represents the density of the juice prior to fermentation, corresponding to sugar content while the Final SG indicates residual sugars post-fermentation. The constant 131.25 is an empirically derived factor that converts SG difference into alcohol concentration by volume, accounting for density differences between bioethanol and water.²⁹ This procedure was applied to guava fruit waste juice with and without sorghum. The value of %ABV gives an important insight on the conversion of guava fruit waste juice into bioethanol.

2.6.4. pH Measurement

The pH of guava fruit waste juice broths was monitored throughout the fermentation process, assessing the changes in acidity which influences microbial activity and enzyme performance. A calibrated digital pH meter (Model HI98129, HANNA Instruments, USA and Romania) was applied. The pH meter was calibrated using standard buffer solutions of pH 4.0 and 7.0 and the readings were taken every 12 hours over 14 days fermentation duration. Regular monitoring allowed for observation of dynamic pH trends associated with sugar metabolism, organic acid formation, and microbial growth. Measurements were conducted on guava fruit waste juice with and without sorghum. The pH values give critical insights into the evolution of acidity and its impact on fermentation efficiency and microbial stability.

2.6.5. Determination of the Quality of Bioethanol

The quality of bioethanol was assessed by establishing the alcohol concentration of each sample using a Gay-Lussac alcoholmeter (Type 2110) which determines alcohol content based on liquid density. Alcohol levels were recorded as a percentage of alcohol by volume (v/v) for standardized comparison across samples. Assessments were undertaken on fermented guava fruit waste juice broths with and without sorghum.

2.6.6. Determination of Bioethanol Quantity

The quantification of bioethanol produced was carried out by measuring the total volume of bioethanol collected after the completion of fermentation and distillation phases. After distillation, bioethanol fractions were carefully collected in separate, clearly labelled bottles to avoid cross-contamination and ensure accurate tracking of yield from each batch. The quantity of bioethanol for each sample was measured using a measuring cylinder (AXIOM, Germany). The cumulative volume of distillates from all fractions was recorded in millilitres (mL), representing the final bioethanol yield for each fermentation setup. This method provides a straightforward and reliable approach to assessing the efficiency of the fermentation and distillation processes in converting guava fruit waste juice into bioethanol.

2.7. Bioethanol Confirmation With Gas Chromatography Flame Ionization Detector

Representative bioethanol samples were evaluated for quality using gas chromatography coupled with a flame ionization detector (GC-FID; Model 8890, Agilent Technologies, Inc., Wilmington, DE, USA). Aliquots of 250 µL were placed in 2 mL autosampler vials and injected directly into the GC system, operated with OpenLab CDS 2.2 software. Separation was achieved on the 8890 GC equipped with an FID maintained at 250 °C. The detector airflow, hydrogen, and nitrogen flow rates were set at 400, 30, and 25 mL/min, respectively. The injector, operated in split mode (50:1) at 250 °C, used helium as the carrier gas with a constant flow rate of 2.0 mL/min.

2.8. Microbial Identification

Microbial analysis of the fermenting pear fruit waste juice samples was performed using established culture-dependent and morphological characterization techniques, consistent with standard practice in food microbiology for yeasts and molds. Following aseptic sampling, aliquots of the fermented juice broths were serially diluted and inoculated onto solid agar media designed to support fungal growth such as yeast and mold selective media, and incubated at temperatures suitable for fungal propagation at 25 °C for 3-5 days to allow colony formation.³⁰ Colonies were grouped based on these visible characteristics, reflecting accepted methods whereby colony morphology serves as an initial phenotypic grouping criterion in culture-based microbial analysis of food and fermentation systems.³¹

2.9. Data Analysis

Data analysis was performed using functions in Microsoft Excel 365, including average, standard deviation, maximum, and minimum. Descriptive statistics were calculated, and the results were visualized using figures generated within the software.³² Measurements for guava fruit waste juice with and without sorghum enabled the comparison of fermentation performance and bioethanol yield. All treatments were performed in three independent fermentation replicates to ensure experimental reliability and reproducibility.

3. Results and Discussion

3.1. Pre-Treatment of Guava Fruit Waste

The physical pretreatment approach efficiently extracted the juice from guava fruit waste for fermentation and bioethanol generation. The average TSS, SG, pH, and temperature of guava fruit waste juice with and without sorghum flour were established prior, during, and after fermentation as presented in Table 1. Previous study show that TSS is an indicative measure of sugar availability in the substrate for microbial metabolism during fermentation, resulting in quality bioethanol.¹⁸ Results show that guava fruit waste juice supplemented with sorghum recorded the highest TSS as compared to that without sorghum. Statistical analysis reveals a significant difference in TSS with p-value of 0.0003 (<0.05) for the broth with sorghum, possibly due to fermentable sugar from sorghum. This reveals the contribution of sorghum in enhancing fermentable sugar availability for fermentation as its starch can be hydrolysed into simple sugars, mainly glucose and maltose. This observation is consistent with previous findings that sorghum is a source of fermentable sugar thus, enhances bioethanol generation.⁵ Besides, sorghum contains natural hydrolytic enzymes like amylases, proteases, and phytases which break down complex starches and protein, enhancing sugar release for fermentation.³³ It is critical to note that higher quantities of fermentable sugars leads to quality alcohol whereas lower sugar levels produce less quality alcohol.¹⁸ However, guava fruit waste juice broth with and without sorghum had the pH value between 4.08-3.64 which is consistent with earlier pH range of 2.0-4.5 favourable for microbial activity.¹⁸ Still, the pre-treatment technique employed in the present study does not involve the use of costly and environmentally hazardous chemicals like sulphuric acid, sodium hydroxide, and hydrochloric acid thus, it is both economical and environmentally benign.

Table 1.

Parameters for Guava Fruit Waste Juice With and Without Sorghum Prior and Post Fermentation

Sample broth	Before fermentation			After fermentation
Sample broth	TSS (^oBrix)	SG	pH	TSS (^oBrix)	SG	pH
Guava juice without sorghum	6.07±0.12	1.05±0.01	3.92±0.03	0.97±0.06	1.0±0.0	3.64±0.01
Guava juice with sorghum	6.10±0.10	1.06±0.01	4.08±0.03	0.97±0.06	1.0±0.0	3.65±0.01

3.2. Effect of Sorghum Flour on Total Soluble Solids and pH of Guava Fruit Broths

Fermentation of guava fruit waste juice was enhanced via sorghum supplementation. The addition of sorghum flour influenced the TSS, a key fermentation parameter by increasing from 5.07±0.12 to 6.10±0.10 °Brix. Studies indicate that sorghum is rich in starch and carbohydrates.³⁴ The changes in TSS observed after addition of sorghum is consistent with that experienced when sorghum was added in the mixture of fruit waste juice,¹⁷ suggesting a more favourable environment for microbial activities and subsequent higher bioethanol quality. Higher TSS offers a richer substrate for microbial fermentation, enhancing bioethanol output. Earlier work demonstrate that adding sorghum flour to diluted fruit waste juice considerably raised the juice’s TSS.¹⁷ Besides, inclusion of sorghum flour significantly boosted hydrolysis process due to its rich enzymatic content such as α-amylase, xanthine oxidase, α-glucosidase, and angiotensin-converting enzyme which assist the breakdown of complex sugars into simpler fermentable forms.⁵ This enzymatic support led to increased bioethanol quality, consistent with findings from previous studies that recognized the effectiveness of cereals like sorghum and millet in improving bioethanol production due to their fermentable sugars and enzymatic properties.^5,35

The effect of sorghum flour on pH of guava fruit waste juice of is evident in Table 1. The pH raised from 3.92±0.03 to 4.08±0.03. This is attributed to slightly alkaline nature of sorghum. Alkaline minerals including calcium, magnesium, and potassium found in sorghum can counteract the acids in fruit waste juice, raising the pH.³⁶ The alkaline character of sorghum flour is shown by the elevated pH, which may help to improve the environment for microbial activity and increase the production of bioethanol from guava fruit waste juice.

3.3. Effect of Fermentation on Total Soluble Solids of Guava Fruit Waste Juice Broths

The effect of fermentation on TSS of guava fruit wastes juice with and without sorghum is presented on Figure 3. During the fermentation process, TSS levels dropped, indicating that microbes were consuming fermentable carbohydrates. One of the most important aspects of fermentation is the decline in TSS, which shows that microbes are converting carbohydrates into bioethanol and weak acids.³⁷ Results show that fermentation reduced TSS from 6.10 ± 0.10 to 0.97 ± 0.06 and 6.07 ± 0.12 to 0.97 ± 0.06 °Brix for broths with and without sorghum supplementation, respectively, indicating the conversion of fermentable sugars into bioethanol, consistent with previous studies.³⁸ The mean TSS values for the broths with and without sorghum supplementation were 2.443 ± 1.572 and 2.981 ± 1.817 °Brix, respectively, showing a statistically significant difference (p = 0.001, p < 0.05). The analysis of TSS in guava waste juice reveals a noticeable decrease in TSS levels within the initial five days of the fermentation process, which implies extensive consumption of fermentable sugars by the microorganisms, leading to formation bioethanol and weak acids. The analysis of guava waste juice broths with and without sorghum flour indicates a significant decrease of TSS during the proceeds of fermentation process. The noticeable decrease in TSS levels is due to the utilization of fermentable sugars by microorganisms during fermentation, converting sugars into bioethanol and organic acids.¹⁷ The difference in TSS levels between the two broths samples indicates the availability of fermentable sugars thus, affects the quality of bioethanol from the fermentation process. Though a notable decrease in TSS was observed during fermentation, a portion of sugar remained at the end of fermentation process. The presence of complex carbohydrates or sugars that are difficult to ferment may be the reason why the fermentation process reduced the residual sugar level but did not bring it to zero ^oBrix.¹⁸ However, reports show that complete fermentation process of all sugars cannot be achieved due to the mixed composition of complex sugars and the fermentation limitations of specific microorganisms involved in fermentation.¹⁸ The reduction in TSS gives a concrete indicator of the progress and effectiveness of fermentation process. The decrease in TSS corresponds with the principles of alcoholic fermentation that involves sugars as the substrate for microorganisms in the formation of bioethanol and CO₂. The declining TSS not only confirms the occurrence of fermentation but also provides the insights into the extent of bioethanol yield. Biochemical transformation provides key aspect of the overall fermentation dynamics to facilitate the transformation of sugars into alcohol as a primary outcome of the microbial-driven process. Results indicate that the fermentation process was maintained from the 11^th-14^th day of fermentation, indicating that the fermentation has been completed. This implies that the consumption of substrates and production of fermentation products were at equilibrium.

Figure 3.

Trend of total soluble solids changes as fermentation progresses

3.4. Effect of Fermentation on pH of Guava Wastes Juice Broths

The pH of guava fruit waste juice broths was monitored over the fermentation duration. Findings indicate a decline in the average pH with fermentation progression. The pH declined from 4.08±0.03 to 3.65±0.01 for guava waste juice broth with sorghum and 3.92±0.03 to 3.64±0.01 for guava waste juice broth without sorghum as presented on Figure 4. The mean values of 3.826±0.133 and 3.771±0.094 for broths with and without sorghum, respectively, were significantly different, as indicated by statistical analysis (p = 0.00035, p < 0.05). The variation in pH values is attributed to specific composition of guava waste juice broths, their interaction with sorghum flour, and the microbial activities during fermentation. The trend of pH for guava waste juice broths with and without sorghum provides evidence that the pH range falls within the favourable acidic range during the fermentation process consistent with earlier work.³⁹ A drop in pH values of guava wastes juice broth with and without sorghum is attributed to microbial activity, which converts sugars into alcohol and weak acids such as lactic acids, acetic acid, and other by-products by fermenting microorganisms.¹⁸ This is supported by previous study where pH declined during the fermentation of pineapple fruit waste juice.⁴⁰

Figure 4.

Trend of pH changes as fermentation progresses

3.5. Effect of Fermentation on Specific Gravity of Guava Fruit Wastes Juice Broths

The effect of fermentation on SG of guava fruit wastes juice broths was evaluated as shown in Figure 5. Results show a decline in SG from 1.06±0.01 to 1.00±0.00 and 1.05±0.01 to 1.00±0.00 for guava fruit waste juice broths with and without sorghum, respectively. Statistical analysis revealed a significant difference in SG between the broth with and without sorghum supplementation, with mean values of 1.02 ± 0.024 and 1.01 ± 0.016, respectively (p = 0.018, p < 0.05). The trend in SG decline as fermentation progresses implies that fermentable sugars are being converted to bioethanol.⁴¹ Despite the higher initial value of SG for guava fruit waste juice broth with sorghum, the broths attained the same SG at the end of fermentation, implying that all fermentable sugars were converted into bioethanol for all broths. Generally, the decrease in SG directly corresponds to reduction in sugar content, reflecting the formation of bioethanol during fermentation.⁵ The calculated SG of the fermenting broth supplemented with sorghum revealed an alcohol content of 7.87% ABV higher than 6.56% ABV obtained from un-supplemented broth at the end of the fermentation process. The higher %ABV observed in the supplemented broth indicates improved hydrolysis and fermentation efficiency, resulting in increased bioethanol production due to the greater availability of fermentable sugars provided by the supplement.^42,43

Figure 5.

Trend of specific gravity changes as fermentation progresses

3.6. Distillation of Fermented Guava Fruit Wastes Juice Broths

Guava fruit wastes juice broth with and without sorghum were distilled after 14 days of fermentation for bioethanol extraction. Various aliquots from distillation were analysed for alcohol concentration. Results show that the first aliquot had the highest alcohol level while the third had the lowest as presented in Table 2, showing a distinct trend of declining bioethanol concentrations across the aliquots. This is in line with the fundamentals of simple distillation in which the more volatile ingredients like bioethanol is separated earlier.⁴⁴ With the maximum yield seen initially, the bioethanol concentrations from guava fruit waste juice broth with sorghum were 22% in the first aliquot against 19% (v/v) without sorghum with a total of 1.6 L of bioethanol extracted from 3 L of broth. Results show that guava wastes juice broth with sorghum produced bioethanol with higher concentration because sorghum efficiently increased sugars content ultimately enhancing bioethanol quality. These findings are in agreement with the previous findings that co-fermentation with starch-rich materials like sorghum enhances bioethanol production.¹⁷ Sorghum adds fermentable sugars and supports microbial activity thus, resulting in improved sugar-to-ethanol conversion.⁵ The amount of bioethanol in the residual solution falls with the length of the collection period, lowering the amount of bioethanol in the ensuing aliquots.^44,45 This implies that water and other less volatile substances make up the majority of the residual liquid in the distillation apparatus.⁴⁶ The declining bioethanol recovery at this point emphasizes how distillation efficiency declines as the amount of bioethanol in the feed broths drops. The data’s observed trend has multiple ramifications, including in the first place, it emphasizes how crucial it is to concentrate on the early fractions to maximize bioethanol recovery because later phases produce lower concentrations, which makes extended distillation less profitable. Still, the changing quantities of bioethanol in each aliquot offer the possibility of generating several types of bioethanol grades from the procedure.

Table 2.

Percentage Alcohol Content From Distillation of Guava Fruit Waste Juice Broths

Fermentation broth	1^st aliquot (%)	2^nd aliquot (%)	3^rd aliquot (%)
Guava fruit waste juice with sorghum	22	18	12
Guava fruit waste juice without sorghum	19	14	9

3.7. Redistillation of Bioethanol

Bioethanol with concentrations between 9-22% (v/v) were mixed to create 19% (v/v), which was redistilled to produce high-purity bioethanol with the concentrations between 50-84% (v/v), with 84% (v/v) suitable as biofuel, offering a clean energy substitute for FFs. The improvement in purity suggests that this method is an effective strategy for generating value-added products from guava fruit waste resources. The initial aliquot of high-purity bioethanol can be further purified and used in high-value applications such as biofuels for automobile engines.⁴⁷ Research indicates that redistilling bioethanol with initial alcohol concentrations between 30-50% (v/v) yields high-purity bioethanol between 86-90% (v/v), a potential biofuel.^17,39,41 Conversely, aliquots of lesser concentration can be used in other refining procedures or added to blended products like cleaning solutions, industrial solvents, or components for creation of bioplastics.⁴⁸ However, the produced bioethanol represents a preliminary product whose potential applications require further detailed purification and evaluation.

4. Sustainable and Clean Bioethanol Production From Guava Fruit Waste

The process of turning guava fruit waste into bioethanol with sorghum added complies with cleaner and sustainable production principles by using locally accessible, renewable feedstocks, cutting down on agricultural waste, and lowering the dependency on food crops like maize and sugar cane.⁴⁹ The method turns underutilized biomass into useful biofuels, improving resource efficiency and advancing a circular economy.⁴⁷

5. Gas Chromatography With Flame Ionization Detection

Samples were analysed using a GC-FID (Model 8890, Agilent Technologies, Inc., Wilmington, DE, USA) at the Tanzania Bureau of Standards (TBS). The analysis confirmed that bioethanol was the primary component in all distillate samples with only trace impurities present. The ethanol concentrations determined by GC-FID agree with those measured values using an alcoholmeter. A representative GC-FID chromatogram is presented in Figure 6.

Figure 6.

GC-FID chromatogram obtained from a representative sample

6. Microbial Identification

The microorganisms isolated from the fermenting guava fruit waste juice were characterized as yeasts and molds. Yeast colonies exhibited oval, budding morphologies, while mold colonies displayed a range of colours from white to creamy white, reflecting distinct colony morphologies as presented in Figure 7. The observed mold displayed a filamentous morphology characterized by elongated, branching hyphae, whereas yeast cells predominantly exhibited an oval or ellipsoidal shape.

Figure 7.

Yeast and mold colonies in petri dishes

The predominance of molds and yeasts in the fermenting guava fruit waste juice samples is consistent with previous studies examining microbial communities in fruit-based fermentations.⁵⁰ Yeasts and molds are frequently involved in the breakdown and fermentation of sugar-rich fruit substrates, owing to their capacity to grow in acidic environments with high sugar concentration.⁵⁰ These findings reinforce the well-established notion that yeasts and molds perform complementary functions in the natural fermentation of fruit waste.

7. Conclusion and Recommendations

The present study indicates the potential of guava fruit waste in bioethanol generation. Addition of sorghum to guava fruit waste juice increases bioethanol output through fermentation and distillation. Sorghum improves the yield by adding fermentable sugar and enhancing enzymatic hydrolysis. Over the course of 14 fermentation days, TSS dropped from 6.1 ± 0.1 to 0.97±0.06 ^oBrix with a maximum of 7.87% ABV. For the first aliquots of guava fruit waste juice broth with and without sorghum, distillation produced bioethanol concentrations of 22% and 19% (v/v), respectively, while redistillation produced 50-84% (v/v), appropriate for use as biofuel. According to results, guava fruit waste presents itself as a viable renewable feedstock for generation of sustainable bioethanol. The method enhances the financial standing of guava fruit growers and helps to lower postharvest losses of guava crops. Future studies should focus on optimizing bioethanol production from guava fruit waste and conducting detailed characterization of the microbial communities present in the fermenting broth. In addition, further investigations are required to evaluate bioethanol yield in terms of sugar consumption, volumetric productivity, conversion efficiency, and assess the techno-economic implications of sorghum supplementation relative to feedstock mass. Moreover, subsequent research should include comprehensive physicochemical analyses of the produced bioethanol and compare the results with relevant fuel quality standards to determine its suitability for industrial and fuel applications. Comprehensive physicochemical characterization and evaluation against established fuel quality standards will be essential to fully assess the potential of the product for large-scale or commercial utilization.

Footnotes

Acknowledgement

Authors are grateful to Mkwawa University College of Education for materials support.

ORCID iD

Jovine Kamuhabwa Emmanuel

Ethical Considerations

Ethical approval was not required for this study as it did not involve human participants, animals, or sensitive personal data.

Author Contributions

LAM participated in the conception, design, drafting and revising the paper critically for intellectual content, NMN participated in data collection and data analysis, JAK participated in data collection and data analysis, FJN participated in data collection and data analysis, JKE participated in the conception, design, data analysis and drafting and revising the paper critically for intellectual content. All authors approved the final manuscript for publication.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

This study did not involve the generation or use of any datasets, and therefore, no data is available.*

References

Zhao

Wang

Griffin

Roozeboom

Wang

. Bioconversion of industrial hemp biomass for bioethanol production: A review. Fuel. 2020;281:118725. doi:10.1016/j.fuel.2020.118725.

Saggi

Dey

. An overview of simultaneous saccharification and fermentation of starchy and lignocellulosic biomass for bio-ethanol production. Biofuels. 2019;10(3):287-299. doi:10.1080/17597269.2016.1193837.

Bhuvaneswari

Sivakumar

. Bioethanol production from fruit and vegetable wastes. Bioprocessing for biomolecules production. 2019; 417-427.

Tse

Wiens

Reaney

. Production of bioethanol—A review of factors affecting ethanol yield. Fermentation. 2021;7(4):268.

Mgeni

Gervas

Mtashobya

Emmanuel

. Quality of Bioethanol Produced from Concentrated Fruit Waste Juice Augmented with Sorghum as Additional Fermentable Sugar. Sugar Tech. 2025;27:1-11.

Al-Hamamre

Saidan

Hararah

Rawajfeh

Alkhasawneh

Al-Shannag

. Wastes and biomass materials as sustainable-renewable energy resources for Jordan. Renewable and Sustainable Energy Reviews. 2017;67:295-314.

Shah

Vyas

Patel

. Bioethanol production from pulp of fruits. Biosci Biotechnol Res Commun. 2019;12(2):464-471. doi:10.21786/bbrc/12.2/32.

Mgeni

Mero

Mtashobya

Emmanuel

. Utilizing fruit wastes as a sustainable feedstock for bioethanol production: A Review. Cleaner Energy Systems. 2025;10:100188.

Dube

Paremoer

Jahari

Kilama

. Growth and development of the fruit value chain in Tanzania and South Africa. 2018.

10.

Dubinina

Andrievskaya

Tomgorova

Nebezhev

. Innovative technologies of alcoholic beverages based on fruit distillates. Food systems. 2020;3(2):18-23.

11.

Arora

Ansari

Arora

. Nutritional beverages. Am J Pharmatech Res. 2019;9:1-28.

12.

Moreno

Curtis

Sarkhosh

, et al. Considerations when brewing with fruit juices: A review and Case study using peaches. Fermentation. 2022;8(10):567.

13.

Kandemir

Piskin

Xiao

Tomas

Capanoglu

. Fruit juice industry wastes as a source of bioactives. Journal of Agricultural and Food Chemistry. 2022;70(23):6805-6832.

14.

Emmanuel

Mtashobya

Mgeni

. Potential Contributions of Banana Fruits and Residues to Multiple Applications: An Overview. Natural Product Communications. 2025;20(2):1934578X251320151. doi:10.1177/1934578x251320151.

15.

Mgeni

Mero

Mtashobya

Emmanuel

. The prospect of fruit wastes in bioethanol production: A review. Heliyon. 2024;10(19):e38776.

16.

Miles

Alexander

Peck

Galinato

Gottschalk

van Nocker

. Growing apples for hard cider production in the United States—trends and research opportunities. HortTechnology. 2020;30(2):148-155.

17.

Mgeni

Mero

Gervas

Mtashobya

Emmanuel

. Bioethanol production from dilute fruit wastes using sorghum as additional fermentable sugar. Biofuels. 2025;16(1):1-10.

18.

Mgeni

Mtashobya

Emmanuel

. Bioethanol production from fruit wastes juice using millet and sorghum as additional fermentable sugar. Cleaner Energy Systems. 2025;10:100177.

19.

Dahiya

Nigam

. Bioethanol synthesis for fuel or beverages from the processing of agri-food by-products and natural biomass using economical and purposely modified biocatalytic systems. Aims Energy. 2018;6(6):979-992.

20.

Emam

Abdou

Younis

Farag

. Metabolomics in guava: a comprehensive review of quality, agricultural traits, composition, and nutraceutical applications of Psidium guajava fruits and byproducts. Phytochemistry Reviews. 2025;24:1-23. doi:10.1007/s11101-025-10153-2.

21.

Omayio

Abong’

Okoth

Gachuiri

Mwang’ombe

. Trends and constraints in guava (Psidium guajava l.) production, utilization, processing and preservation in Kenya. International Journal of Fruit Science. 2020;20(sup3):S1373-S1384.

22.

Rangare

Lal

Shiurkar

, et al. Impact of Climate Change on Guava Production: Challenges and Adaptive Strategies. Applied Fruit Science. 2025;67(5):404.

23.

Jain

Dhawan

Malhotra

Singh

. Biochemistry of Fruit Ripening of Guava (Psidium guajava L.): Compositional and Enzymatic Changes. Plant Foods for Human Nutrition. 2003;58(4):309-315. doi:10.1023/B:QUAL.0000040285.50062.4b.

24.

Harshini

Kavitha

. Valorization of Diverse Tropical Fruit Residues for Bioethanol Production: A Review of S. cerevisiae Strains and Simulation Models. Preprints. 2025. doi:10.20944/preprints202510.1696.v1.

25.

Akar

Canbaz

. Effect of molasses as an admixture on concrete durability. Journal of Cleaner Production. 2016;112:2374-2380. doi:10.1016/j.jclepro.2015.09.081.

26.

Geremew Kassa

Asemu

Belachew

Satheesh

Abera

Alemu Teferi

. Review on the application, health usage, and negative effects of molasses. CyTA-Journal of Food. 2024;22(1):2321984.

27.

Harshini

SSHK

. Valorization of Diverse Tropical Fruit Residues for Bioethanol Production: A Review of <em>S. cerevisiae</em> Strains and Simulation Models. Preprints: Preprints. 2025.

28.

Ames

Camp

Cox

Mathurin

. The Automated Laboratory for Sugar Processing. Journal of Sugar Beet Research. 2021;58:5-39.

29.

Bajaj

Markandeywar

Kaur

Singh

Gupta

. Determination of alcoholic content of Asava and Arishta by distillation method and specific gravity method. Pharmaspire. 2020;12:16-19.

30.

Kovács

Pomázi

Taczman-Brückner

, et al. Detection and Identification of Food-Borne Yeasts: An Overview of the Relevant Methods and Their Evolution. Microorganisms. 2025;13(5):981. doi:10.3390/microorganisms13050981.

31.

Signoroni

Ferrari

Lombardi

Savardi

Fontana

Culbreath

. Hierarchical AI enables global interpretation of culture plates in the era of digital microbiology. Nature Communications. 2023;14(1):6874. doi:10.1038/s41467-023-42563-1.

32.

Singh

. Use of Statistical Analysis Supporting Tools in Libraries: an overview of statistical package for social science. World Digital Libraries-An international journal. 2022;15(2):91-104.

33.

Ibitoye

. Physical, Functional, Pasting and Microbial Properties of Sorghum (Sorghum bicolor M.) Ogi as Influenced by Fermentation Methods. University of Ibadan; 2023.

34.

Jeevan Kumar

Sampath Kumar

Chintagunta

. Bioethanol production from cereal crops and lignocelluloses rich agro-residues: prospects and challenges. SN Applied Sciences. 2020;2(10):1673.

35.

Vasić

Knez

Leitgeb

. Bioethanol production by enzymatic hydrolysis from different lignocellulosic sources. Molecules. 2021;26(3):753.

36.

Odukoya

De Saeger

De Boevre

, et al. Influence of nixtamalization cooking ingredients on the minerals composition of nixtamalized maize and sorghum. Journal of Cereal Science. 2022;103:103373.

37.

Wang

Song

Zhang

. Anaerobic digestion of food waste and its microbial consortia: A historical review and future perspectives. International Journal of Environmental Research and Public Health. 2022;19(15):9519.

38.

Azhar

SHM

Abdulla

Jambo

, et al. Yeasts in sustainable bioethanol production: A review. Biochemistry and biophysics reports. 2017;10:52-61.

39.

Emmanuel

Mgeni

Gervas

Mtashobya

. Bioethanol production from concentrated fruit wastes’ juice enhanced with fermentable sugar from millet (Panicum miliaceum). Biofuels. 2025;16(5):445-451. doi:10.1080/17597269.2024.2432157.

40.

Efunwoye

Oluwole

. Bioethanol production from pineapple waste by solid state fermentation method. Nigerian Journal of Microbiology. 2019;33(2):4811-4820.

41.

Mgeni

Mtashobya

Emmanuel

. Bioethanol production from pineapple fruit waste juice using bakery yeast. Heliyon. 2024;10(19):e38172. doi:10.1016/j.heliyon.2024.e38172.

42.

Vázquez-Rodríguez

Heredia-Olea

Alamilla-Morales

Pérez-Carrillo

Perez-Perez

Serna-Saldívar

. Blasting extrusion pretreatment of sweet sorghum bagasse for enhanced enzymatic saccharification and ethanol production using Pichia kudriavzevii ATCC 20,381. Bioresources and Bioprocessing. 2025;12(1):65. doi:10.1186/s40643-025-00905-5.

43.

Usman

Zhang

Yang

Guo

Shen

. Lignocellulose degradation, enzymatic saccharification and bioethanol production from whole-crop sweet sorghum silage inoculated with feruloyl-esterase producing lactic acid bacteria. Int J Biol Macromol. 2025;311(Pt 4):143691. doi:10.1016/j.ijbiomac.2025.143691.

44.

Mwanyesya

Mtashobya

Emmanuel

. Enhanced bioethanol production from bamboo juice supplemented with banana fruit at the verge stage. Biofuels. 2025;17:1-14. doi:10.1080/17597269.2025.2524911.

45.

Janković

Straathof

McGregor

Kiss

. Bioethanol separation by a new pass-through distillation process. Separation and Purification Technology. 2024;336:126292.

46.

Thielmann

Cavalcante

Young

. Simulation and economic evaluation of different process alternatives for the fermentation and distillation steps of ethanol production. Energy Conversion and Management. 2022;265:115792.

47.

Mohanan

Yusuf

. Biodiesel from rubber seed oil. Department of Botany, University of Calicut; 2022.

48.

Ogwu

Izah

Alves

AAC

Babu

. Sustainable cassava: Strategies from production through waste management. Elsevier; 2024.

49.

Faisal

Saeed

. Sustainable approaches toward the production of bioethanol from biomass. In: Sustainable Ethanol and Climate Change: Sustainability Assessment for Ethanol Distilleries. Springer; 2020:15-38.

50.

Ciani

Comitini

. Yeast interactions in multi-starter wine fermentation. Current Opinion in Food Science. 2015;1:1-6.