1. Introduction

As the global demand for the agricultural economy is accelerating, the production of biofuel offers solutions for creating a carbon-neutral society (Mekunye et al., 2024). The agricultural sector is one of the leading generators of waste, which has created significant sustainability concerns; therefore, utilizing agricultural waste offers a solution to this problem (Ufitikirezi et al., 2024).

The rapid growth in global population and energy consumption has astonishingly increased the demand for alternative energy resources (Bhattacharjee et al., 2024). Although efforts toward achieving a sustainable and carbon-neutral society are accelerating, the world continues to face challenges such as climate change, resource depletion, and escalating energy requirements. One promising strategy to address these issues is the development of renewable, low-carbon bioethanol derived from agricultural waste, which can slightly replace fossil fuels while simultaneously contributing to sustainable agricultural waste management. Agricultural waste and byproducts offer several feedstock solutions as these materials are composed up of lignocellulosic materials and may be utilized for the production of bioethanol (Mekunye et al., 2024; Pattanaik et al., 2019; Verma et al., 2020).

This experiment explores the utilization of waste biomass for beneficial purposes, including feed for livestock and biofuel production as organic waste can serve as a valuable substrate for renewable energy generation (Kaushik and Jadhav 2022). Sugarcane cultivation generates approximately 1.6 billion tons of waste annually, reflecting the substantial volume of residues produced as a result of large-scale sugarcane production (Ajala et al., 2021). Fermentation plants have utilized baker’s yeast for bioethanol production in modern industrial processes. Bioethanol production must be significantly expanded to enable a transition away from dependence on fossil fuels. This study emphasizes the prospective role of yeast-based systems in advancing future biofuel production (Attfield et al., 2025).

In this experiment, we performed a process whereby agricultural waste residues like sugarcane bagasse and rice straw are pretreated using sulfuric acid and sodium hydroxide to disrupt the lignocellulosic structure, followed by hydrolysis to release simple sugars, which were subsequently fermented by Saccharomyces cerevisiae under anaerobic conditions to produce bioethanol (Rajput and Shrivastav 2025; Tavares et al., 2025). Lignocellulose biomass is composed of cellulose, hemicellulose, and lignin, which are low-energy-density compounds, so the pre-treatment step is necessary to release the sugars to produce bioethanol (Barciela et al., 2023).

Bioethanol production involves two primary pathways: first-generation processes based on sugar substrates such as glucose, and second-generation processes that utilize lignocellulosic biomass (Irfan et al., 2014). The present work focuses on the utilization of agricultural waste as a feedstock for bioethanol production (a sustainable biofuel source) (Nisar et al., 2024). Simultaneously, the residual biomass is used for the generation of nitrogen-rich manure through inoculation with nitrogen-fixing cyanobacteria, thereby promoting soil fertility and completing a circular bioeconomy loop by integrating both bioethanol generation and soil amendment from the same feedstock. The system advances environmental sustainability, waste valorization, and carbon neutrality goals. Conversion of agricultural waste into biofuel and biodiesel has emerged as a sustainable and eco-friendly approach (Dhir et al., 2025).

Thus, this study aims to demonstrate the efficient conversion of agricultural waste to bioethanol, followed by the conversion of residual biomass into a value-added soil fertilizer to minimize waste disposal and improve soil health. The present study emphasizes various treatment strategies for altering biowaste into sustainable products, encompassing pretreatment, enzymatic hydrolysis, and fermentation, highlighting the potential of these methods to transform waste into valuable resources (Kumar et al., 2009; Prakash et al., 2025; Sikiru et al., 2024). The enormous scale of agricultural waste generation indicates that even modest conversion efficiencies can translate into significant volumes of sustainable biofuel and soil-enhancing products, making this an especially attractive approach for academic and industrial applications. Although agricultural waste presents significant potential to be converted into a bioresource, effective methods are required to transform this waste into useful products (Egwuatu et al., 2024).

2. Materials and Methods

2.1 Reagents and Materials:

Analytical-grade chemicals, including potassium permanganate (KMnO₄), sodium hydroxide (NaOH), potassium dichromate (K₂Cr₂O₇), sulfuric acid (H₂SO₄, 95%), boric acid (H₃BO₃), methyl red indicator and  hydrochloric acid (HCl) were used in the experiments. BG-11 (HiMedia) medium was employed for the cultivation of cyanobacteria. Cellulase (HiMedia) was employed for enzymatic hydrolysis, while Saccharomyces cerevisiae (commercial baker’s yeast) was utilized for fermentation. Sugarcane bagasse and rice straw were obtained from local agricultural sites in Raipur, Chhattisgarh (Table 1).

Table 1: Samples used for bioethanol production

2.2 Production of Algal Biomass

Cyanobacteria were cultivated solely for the purpose of nitrogen enrichment of the residual biomass after fermentation and were not involved in the bioethanol production process.

Isolation of cyanobacteria

A serial dilution technique was employed to obtain varying concentrations of cyanobacterial cultures. Six dilutions ranging from 10¹ to 10⁶ were prepared and used for microscopic examination. BG-11 medium, a standard culture medium for cyanobacterial growth as described by Stanier et al., (1971), was prepared for cultivation; four selected dilutions were aseptically plated onto sterile Petri plates. Aliquots from the serially diluted cyanobacterial suspensions were subsequently inoculated into BG-11 medium flasks under aseptic conditions. The inoculated plates and flasks were incubated at 28°C for several days to weeks to facilitate cyanobacterial growth. Upon observation of visible growth, biomass from each flask was carefully transferred into separate conical flasks corresponding to their respective dilution concentrations for further analysis.

Figure 2: A) Isolation and B) Cultivation of cyanobacteria by serial dilution and mass multiplication in BG-11 medium

Identification of Cyanobacteria

Microscopic examination of the cyanobacterial culture revealed the presence of actively dividing cells with characteristic green pigment. The cyanobacteria predominantly exhibit filamentous morphology with well-defined cellular structure. The presence of differentiated cells within certain filaments indicated healthy growth conditions and suggested potential nitrogen-fixing capability. Overall, the observed cellular morphology and structural integrity confirmed the characteristic features of the cyanobacteria and their functional potential.

 Harvest of biomass

The inoculated flasks were kept under sunlight for continued growth and monitored over several days to weeks. Upon completion of the growth period, the cyanobacterial biomass was harvested, and the culture exhibiting the highest biomass yield was chosen for subsequent analysis. The harvested biomass was dried, finely powdered, and stored for subsequent experimental use.

Figure 3: Light micrograph of filamentous cyanobacteria exhibiting chlorophyll pigmentation, uniform cellular organization, and heterocyst differentiation.

2.3 Pretreatment of Agricultural Biomass

The collected sugarcane bagasse and rice straw were pretreated to disrupt the lignocellulosic structure and improve enzymatic accessibility (Siqueira et al., 2013). Lignocellulosic biomass possesses a complex structural matrix composed mainly of cellulose, hemicellulose, and lignin, which limits enzymatic accessibility and reduces hydrolysis efficiency. Pretreatment is therefore considered a crucial step in lignocellulosic bioethanol production because it disrupts the rigid lignin structure, reduces cellulose crystallinity, and increases the surface area available for enzymatic action. Acid pretreatment using sulfuric acid primarily hydrolyzes hemicellulose and facilitates the release of soluble sugars, whereas alkaline pretreatment using sodium hydroxide is more effective in removing lignin and improving cellulose accessibility. These structural modifications significantly enhance subsequent enzymatic hydrolysis and fermentation efficiency. Similar pretreatment strategies have been widely reported to improve lignocellulosic biomass digestibility and fermentable sugar release in bioethanol production processes (Sun and Cheng 2002; Mosier et al., 2005; Alvira et al., 2010).

·     Acid pretreatment: Biomass was treated with 95 % (v/v) sulfuric acid at a 1:10 ratio at 60 °C for 1 hour. The resulting mixture was filtered, washed with distilled water, and dried at 50 °C.

·     Alkali pretreatment: Another portion of the biomass was subjected to treatment with 2 % NaOH solution at 80 °C for 2 hours. The residues were washed thoroughly and dried for subsequent hydrolysis.

For each pretreatment experiment, 10 g of dried biomass was treated with 100 mL of chemical solution (H₂SO₄ or NaOH), maintaining a biomass-to-solution ratio of 1:10 (w/v).

Figure 4: A) Before acid and alkali treatment of Sugarcane bagasse and Rice straw B) After acid and alkali treatment of Sugarcane bagasse and Rice straw

2.4 Enzymatic hydrolysis

Pretreated biomass samples were transferred to a flask containing cellulase enzyme and incubated at 50 °C for 48 hours to facilitate enzymatic hydrolysis. The resulting hydrolysate was subsequently filtered to obtain fermentable sugars. Cellulase enzymes catalyze the breakdown of cellulose present in lignocellulosic biomass, which primarily consists of cellulose, hemicellulose, and lignin (Ali et al., 2024). Commercial cellulase enzyme was applied at an approximate loading of 15–20 FPU per gram of dry biomass to enhance cellulose degradation and promote the release of fermentable sugars.

2.5 Fermentation

The hydrolysate produced during enzymatic hydrolysis was used as the substrate for fermentation. The fermentable sugars obtained from pretreated agricultural biomass were fermented using Saccharomyces cerevisiae to produce bioethanol. Saccharomyces cerevisiae was inoculated and incubated at 30 °C under anaerobic conditions for 48–72 hours (Hor et al., 2022). Fermentation was carried out in batch culture under anaerobic conditions maintained by sealing fermentation flasks with airtight stoppers to minimize oxygen exposure.  The fermentation process followed a batch mode to allow continuous monitoring of CO₂ evolution and ethanol formation during the incubation period. The conversion of sugars to bioethanol was monitored periodically by observing CO₂ production. All fermentation experiments were performed in triplicate to ensure reproducibility of the observed results.

In the present laboratory-scale study, ethanol production was primarily evaluated using qualitative chemical confirmation tests (potassium dichromate oxidation, iodoform, and alkaline permanganate tests) along with observation of CO₂ evolution during fermentation. Absolute quantitative determination of ethanol concentration (e.g., g/L or % v/v using chromatographic or spectrophotometric methods) was not performed because the study aimed to demonstrate proof-of-concept bioconversion rather than industrial yield optimization. Similar qualitative analytical approaches for ethanol detection have been reported in previous studies (Sultan et al., 2025).

The following qualitative tests were performed for ethanol confirmation:
• Potassium dichromate oxidation method
• Iodoform test
• Alkaline permanganate test

2.5.1 Confirmatory Tests for Ethanol

Potassium Dichromate Oxidation

Ethanol in the sample was confirmed using the potassium dichromate oxidation method. An acidified potassium dichromate solution was prepared by adding 5 mL of concentrated H₂SO₄. Subsequently, 20 mL of the ethanol-containing sample was added and mixed gently. The mixture was then heated in a water bath at 50–60 °C for 5–10 minutes. A color change from orange to green indicated the reduction of dichromate ions to chromium (III), confirming the presence of ethanol.

Iodoform test

The iodoform test was performed to confirm the presence of ethanol in the sample. Two milliliters of the ethanol-containing sample were mixed with 2 mL of 10 % sodium hydroxide (NaOH) and heated at 50-60 °C for a few minutes. The formation of a yellow precipitate, along with a characteristic antiseptic odor, indicated a positive reaction, thereby confirming the presence of ethanol.

Alkaline permanganate test

The alkaline permanganate test was performed to confirm the presence of ethanol in the sample. Two to three milliliters of the sample were added to 2-3 mL of 0.1 % potassium permanganate (KMnO₄) solution, followed by 1 mL of 1 M sodium hydroxide (NaOH). The mixture was gently heated at 40–60 °C for 2-5 minutes. A positive result was indicated by the disappearance of the purple color of KMnO₄ and the appearance of a brown/black precipitate of manganese dioxide (MnO₂), confirming ethanol oxidation. Ethanol was used as a positive control, and water served as a negative control to validate the results.

2.6 Nitrogen Enrichment

Residual biomass obtained after fermentation was inoculated with cyanobacteria and incubated under light at 28 °C for 10-12 days. Agricultural waste residues (rice straw and sugarcane bagasse) were mixed with soil and supplemented with cyanobacterial biomass. The mixtures were kept under ambient conditions for up to four weeks, during which variations in biomass degradation and nitrogen enrichment were periodically monitored.

2.7 Nitrogen content determination

Soil nitrogen content was estimated using the alkaline permanganate method. Residual biomass was mixed with soil and supplemented with cyanobacterial biomass, followed by incubation under laboratory conditions for several days to weeks to allow decomposition. After incubation, the soil-cyanobacteria mixture was transferred to a 250 mL conical flask, and 10 mL each of potassium permanganate (KMnO₄) and sodium hydroxide (NaOH) solutions were added. The flask was immediately sealed and gently heated to liberate ammonia, which was trapped in a separate flask containing boric acid with methyl red indicator. The trapped ammonia was quantified by titration with 0.02 N hydrochloric acid until a color change from green to pink was observed. The procedure was adapted from Sahrawat et al., (1982).

3. Results

Bioethanol production in this study was achieved exclusively from pretreated agricultural residues (rice straw and sugarcane bagasse) through enzymatic hydrolysis and fermentation using Saccharomyces cerevisiae, whereas cyanobacteria were utilized only for nitrogen enrichment of the residual biomass.

Due to the qualitative nature of the study, ethanol production was evaluated primarily through comparative observations of CO₂ evolution and confirmatory chemical tests rather than precise quantitative measurements.

This study demonstrated the efficient conversion of agricultural residues, specifically rice straw and sugarcane bagasse, into sustainable bioethanol using sequential pretreatment methods, enzymatic hydrolysis, and microbial fermentation with Saccharomyces cerevisiae. Both acidic (H₂SO₄) and alkaline (NaOH) pretreatments effectively disrupted the lignocellulosic structure of the biomass, enhancing enzyme accessibility and the release of fermentable sugars, which were subsequently fermented to produce bioethanol demonstrated by increased CO₂ production and stronger positive responses in ethanol confirmatory tests.

Comparative Effects of H₂SO₄ and NaOH Pretreatments

The fermentation trends are presented using relative observational (arbitrary) units to indicate comparative fermentation activity rather than absolute ethanol concentration. Accordingly, the effect of acidic and alkaline pretreatments on bioethanol production over four weeks was monitored through relative ethanol production trends and CO₂ evolution.

·         H₂SO₄-treated biomass:

A gradual increase in ethanol production and CO₂ release was observed. In the first week, minimal production was recorded at one arbitrary unit, increasing moderately over the following weeks to a maximum of three arbitrary units by week four. The gradual increase indicates partial hydrolysis of lignocellulosic components and limited but steady availability of fermentable sugars (Figure 5).

Figure 5: Ethanol production and CO₂ release from H₂SO₄-treated biomass

·         NaOH-treated biomass:

Alkaline pretreatment resulted in a higher and more rapid increase in both ethanol production and CO₂ evolution. The fermentation activity is presented using relative observational units to compare pretreatment efficiency rather than absolute ethanol concentration. Initial CO₂ release was higher than ethanol production, and by the third week, ethanol and CO₂ reached four and five arbitrary units, respectively. By week four, both parameters peaked at six arbitrary units, indicating enhanced microbial activity and fermentation efficiency. Overall, alkaline pretreatment proved more effective than acidic pretreatment, likely due to delignification by NaOH, which improved cellulose and hemicellulose accessibility for enzymatic hydrolysis and subsequent fermentation (Figure 6).

Figure 6: Ethanol production and CO₂ release from NaOH-treated biomass

Fermentable Sugar Formation and Microbial Activity

Enzymatic hydrolysis of pretreated substrates generated fermentable sugars, as evidenced by active fermentation upon inoculation with Saccharomyces cerevisiae. During the fermentation process, a notable increase in CO₂ evolution was observed, indicating active microbial metabolism and effective sugar conversion. Alkali-treated samples exhibited higher fermentation efficiency, demonstrated by increased CO₂ production and stronger positive responses in ethanol confirmatory tests (Figure 7).

Figure 7: Fermentation of Pretreated Biomass

Ethanol Confirmation

Bioethanol production was confirmed qualitatively using potassium dichromate oxidation, alkaline permanganate, and the iodoform test. Distinct color changes-including orange to green in potassium dichromate, decolorization of alkaline permanganate, and formation of a yellow iodoform precipitate-validated the presence of ethanol in both rice straw and sugarcane bagasse samples (Figure 8 a-c).

Figure 8: Confirmatory tests showing positive change towards the presence of bioethanol (A. Potassium dichromate test, B. Alkaline permanganate test, C. Iodoform test)

Valorization of Residual Biomass

The residual biomass remaining after ethanol fermentation was subsequently treated with nitrogen-fixing cyanobacteria to evaluate its potential for soil nitrogen enrichment. Incubation of residual biomass with cyanobacteria enhanced soil nitrogen, as evidenced by visible decomposition and increased ammonia release. The alkaline permanganate assay confirmed nitrogen enrichment, indicated by the color change from green to pink (Figure 9, 10). The nitrogen enrichment was assessed qualitatively through the alkaline permanganate method based on color change during titration.

Figure 9: Production of Nitrogen-rich manure

Figure 10: Soil nitrogen enrichment in cyanobacteria-treated samples

These findings demonstrate the dual benefits of this approach: efficient bioethanol production and the generation of nitrogen-rich manure from residual biomass. This integrated strategy supports waste minimization, renewable energy generation, and soil fertility enhancement, highlighting the potential of agricultural waste-based biofuel systems within a circular bioeconomy framework.

4. Discussion

The present investigation demonstrates the effective conversion of agricultural residues into bioethanol and provides a comparative evaluation of the findings in relation to previously published studies. The findings emphasize the growing relevance of lignocellulosic biomass as a renewable and sustainable feedstock for biofuel production. By assessing pretreatment strategies, enzymatic hydrolysis, and microbial fermentation, this study contributes to the optimization of bioethanol yield and process efficiency. The results obtained aligned with earlier reports, thereby reinforcing the technical feasibility and environmental relevance of agricultural residue-based bioethanol production.

Agricultural residues arise in large quantities during plant harvesting as well as post-harvest handling activities (Dhanya et al., 2022). Among various residue categories, lignocellulosic biomass constitutes one of the most abundant and underutilized agricultural byproducts (Isikgor and Becer 2015). Improper disposal practices such as open field burning lead to environmental pollution and loss of valuable biomass resources. Consequently, the conversion of these residues through biochemical routes has gained considerable attention as a sustainable waste management strategy (Ramesh et al., 2019). The present study supports this approach by demonstrating the effective conversion of agricultural residues into usable bioenergy.

The growing focus on sustainability has driven researchers and industries towards greener and more resource-efficient technologies. Over the past decade, the concept of a regenerative bioeconomy gained momentum, encouraging the transformation from residual streams into value-added products (Gregg et al., 2020). Traditional refuse disposal methods are not only economically demanding but also environmentally unsustainable. Conversely, bioethanol production from agricultural residues provides a dual benefit by alleviating residue accumulation while generating renewable energy (Machineni et al., 2020). This study aligns with global sustainability goals by demonstrating an environmentally benign approach to waste valorization.

Microbial fermentation is a critical step in bioethanol production, and among the available fermentative organisms, Saccharomyces cerevisiae remains the most extensively employed due to its high ethanol productivity, metabolic stability, and tolerance to stress conditions such as ethanol toxicity and temperature fluctuations (Maean et al., 2022). The present findings confirm that optimal ethanol production occurs within a temperature range of approximately 30–35 °C, which is consistent with earlier studies reporting that Saccharomyces cerevisiae exhibits efficient fermentation and ethanol productivity within an optimal temperature range of approximately 26–35 °C under controlled fermentation conditions (Almeida et al., 2022).

Pretreatment of lignocellulosic biomass significantly influences sugar recovery and subsequent fermentation efficiency. In the present study, alkali pretreatment resulted in enhanced fermentable sugar release and improved ethanol production. These observations are consistent with previous reports demonstrating that alkaline pretreatment facilitates lignin removal and increases cellulose accessibility, thereby improving enzymatic hydrolysis and fermentation efficiency (Alvira et al., 2010; Mosier et al., 2005; Sun and Cheng 2002). The effectiveness of alkaline pretreatment observed in the present study may be attributed to its ability to disrupt lignin structure and enhance cellulose availability, which ultimately facilitates improved enzymatic hydrolysis and fermentation efficiency (Kumar et al., 2009). Enzymatic hydrolysis further enabled the conversion of complex polysaccharides into fermentable sugars, confirming its role as an effective and environmentally friendly approach for biomass bioconversion (Bilal et al., 2020; Vasic et al., 2021; Dukare et al., 2025).

Overall, the integration of alkali pretreatment, enzymatic hydrolysis, and fermentation using Saccharomyces cerevisiae proved to be a reliable approach for maximizing bioethanol production from agricultural waste. These results agree with recent studies reporting improved ethanol yields through similar process combinations (Ibiam et al., 2025). The successful transformation of agricultural residues into bioethanol highlights their potential as sustainable industrial resources and supports the advancement of renewable energy technologies while reducing environmental pollution (Sarkar et al., 2012). These observations are in agreement with earlier studies that employed qualitative chemical assays to confirm ethanol formation during laboratory-scale fermentation experiments. Such methods are commonly used for preliminary validation of bioethanol production prior to quantitative instrumental analysis. The utilization of residual biomass for nitrogen enrichment through cyanobacterial inoculation further supports the concept of integrated biomass valorization. Similar approaches have been reported in sustainable bioprocessing studies where fermentation residues were subsequently converted into nutrient-enriched biofertilizers, contributing to circular bioeconomy strategies (Jayakumar et al., 2023).

A limitation of the present study is that ethanol production was validated primarily through qualitative analytical tests and fermentation indicators such as CO₂ evolution, although all experiments were conducted in triplicate to ensure reproducibility. Quantitative estimation of ethanol concentration using advanced analytical techniques such as gas chromatography, HPLC, or spectrophotometric calibration was beyond the scope of this laboratory-scale investigation. Future studies will focus on precise ethanol yield determination and process optimization for industrial scalability.Top of Form

5.

Bottom of Form

Conclusion

Bioethanol production was successfully demonstrated and qualitatively confirmed using rice straw and sugarcane bagasse, as acidic and alkaline pretreatments effectively disrupted their structural recalcitrance, making them more amenable to enzymatic hydrolysis. Subsequent fermentation with Saccharomyces cerevisiae led to bioethanol production, which was confirmed using potassium dichromate, iodine, and alkaline permanganate tests.

Furthermore, the remaining residues/ biomass were utilized to produce nitrogen rich manure through cyanobacterial cultivation. This approach is significant, as it not only makes use of organic waste but also valorizes the leftover biomass after pretreatment, thereby contributing to sustainable waste management.

Overall, this integrated process demonstrates a sustainable and eco-friendly bioconversion strategy that valorizes agricultural waste into environmentally friendly bioethanol. This dual approach not only minimizes waste but also supports circular bioeconomy principles.

Furthermore, the integrated production of bioethanol and biofertilizer from agricultural residues highlights its practical applicability for rural waste management, renewable energy generation, and soil fertility improvement, thereby offering an environmentally sustainable and economically beneficial solution for agro-based industries.

Acknowledgements
The authors are thankful to ITM University, Raipur, Chhattisgarh, India, for providing the necessary laboratory facilities to carry out this work.

Conflict of interest Author declares that there is no conflict of interest.

Funding information not applicable.

Ethical approval not applicable.

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