# Bioethanol Production from Oryza sativa Husk Combined with Sorghum sp. Bran: Optimization and Yield in the Sudano-Sahelian Zone of Cameroon

Alexis Dzokom, Léonnie Dezeu Tchinda

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## To cite this version:

Alexis Dzokom, Léonnie Dezeu Tchinda. Bioethanol Production from Oryza sativa Husk Combined with Sorghum sp. Bran: Optimization and Yield in the Sudano-Sahelian Zone of Cameroon. Sahel Nature Consulting Revue, In press, Bioethanol from Biomass, 12 (4), pp.i440-i451. hal-05043784

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Submitted on 5 May 2025

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# Bioethanol Production from *Oryza sativa* Husk Combined with *Sorghum sp.* Bran: Optimization and Yield in the Sudano-Sahelian Zone of Cameroon

Alexis DZOKOM¹,², Léonnie TCHINDA DEZEU²

¹SAHEL NATURE CONSULTING

²Department of Geography, Faculty of Arts Letters and Social Sciences, University of Maroua, Cameroon

## Abstract

This article explores the production of bioethanol from a combination of *Oryza sativa* husk and *Sorghum sp.* bran, two abundant agricultural biomasses. The main objective of this study is to optimize the ratio between these two materials, test different chemical pretreatments (acidic and alkaline), and compare the efficiency of two fermentation yeasts, *Saccharomyces cerevisiae* and *Pichia stipitis*, in order to maximize bioethanol yield. Optimization of the ratio between *Oryza sativa* husk and sorghum bran showed that a 50:50 ratio produced the highest ethanol yield, with a concentration of 37.5 g/L and a conversion efficiency of 0.42 g ethanol/g dry biomass. This ratio provides an optimal balance between fermentable sugars derived from the starch in *Sorghum sp.* bran and the cellulose in *Oryza sativa* husk, thereby facilitating enzymatic hydrolysis and fermentation. Regarding pretreatments, the study compared an acid pretreatment using 1.5% H₂SO₄ and an alkaline pretreatment using 3% NaOH. The alkaline pretreatment was more effective in breaking down lignocellulose and releasing fermentable sugars, leading to a higher ethanol yield (37.5 g/L) compared to the acid pretreatment (33.0 g/L). As for the yeasts, *Saccharomyces cerevisiae* showed a higher ethanol yield (37.5 g/L) than *Pichia stipitis* (32.5 g/L), due to its greater ability to ferment hexoses (glucose) present in the substrate. However, *Pichia stipitis* may be beneficial for substrates rich in pentoses. The results of this study demonstrate the efficiency of combining rice husk and sorghum bran for bioethanol production. Optimization of the substrate ratio, alkaline pretreatment, and the use of *Saccharomyces cerevisiae* offer a promising approach for more sustainable bioethanol production from local agricultural biomass.

**Keywords:** Bioethanol, *Oryza sativa* husk, *Sorghum sp.* bran, acid pretreatment, alkaline pretreatment, *Saccharomyces cerevisiae*, *Pichia stipitis*, optimization, yield.

## Introduction

Bioethanol derived from lignocellulosic biomass is a promising solution for the energy transition. *Oryza sativa* husk and *Sorghum sp.* bran, abundantly available in the Sahelian regions of Africa, represent valuable raw materials but require prior hydrolysis to release fermentable sugars.

Producing bioethanol from agricultural biomass offers a promising alternative to fossil energy sources. Among the potential feedstocks, agricultural residues such as *Oryza sativa* husk and sorghum bran (*Sorghum sp.*) have attracted increasing interest due to their wide availability and low production cost (Hamdi et al., 2024; Dzokom et al., 2023). Optimizing the

conversion process of these raw materials into bioethanol requires a deep understanding of their physicochemical properties, as well as the fermentation and hydrolysis processes that influence the final yield (Tenkolu et al., 2024).

*Oryza sativa* husks, mainly composed of cellulose, are traditionally considered agricultural waste but have demonstrated significant potential for bioethanol production through appropriate pretreatment techniques (Pooja & Nivedita, 2024; Chavan et al., 2024). Similarly, *Sorghum sp.* bran, which is rich in starch, is an additional valuable source that can complement *Oryza sativa* husks in bioethanol production. The combination of
these two residues could offer synergistic benefits, thereby increasing overall bioethanol yield (Chavan et al., 2024).

A crucial part of optimizing the bioethanol production process lies in the choice of pretreatment methods, enzymatic hydrolysis, and fermentation strategies. Thermal or alkaline pretreatment of *Oryza sativa* husks reduces their lignocellulose content, making the sugars more accessible for fermentation (Woźniak et al., 2025). Moreover, the use of suitable microbial strains, such as *Saccharomyces cerevisiae*, and optimized fermentation conditions (temperature, pH, duration) plays a key role in determining the final yield (Ceaser et al., 2024).

In this study, we aim to explore the optimization of bioethanol production from *Oryza sativa* husk combined with *Sorghum sp.* bran. The objective is to maximize ethanol yield through the optimization of key process parameters such as raw material pretreatment, polysaccharide hydrolysis, and fermentation. Furthermore, this research examines the challenges and benefits of integrating these two biomasses into a single production system, within the context of sustainability and the optimization of local agricultural resources.

# 2. Materials and Methods

## 2.1. Study Area

The study was conducted in two localities in northern Cameroon, Yagoua and Maroua, selected for their high production of agricultural biomass suitable for bioethanol production.

### 2.1.1. Yagoua: Source of *Oryza sativa* Husk

Yagoua, located in the Far North Region of Cameroon, is an area with high rice production. The town is home to the Société d'Expansion et de Modernisation de la Riziculture de Yagoua (SEMRY), a major company involved in *Oryza sativa* processing. According to MINADER (2023), *Oryza sativa* milling in this region generates large quantities of *Oryza sativa* husk, an underutilized by-product that represents about 20% of the total weight of harvested *Oryza sativa*. The Sahelian climate, with an average annual temperature of 28-32°C and annual rainfall of 600-900 mm (IRAD, 2020), allows for the production of over 60,000 tons of *Oryza sativa* annually in the area (FAO, 2021), resulting in approximately 12,000 tons of *Oryza sativa* husk per year.

<figure><img src="image_3.png" /><figcaption>Figure 1: Sudano-Sahelian zone of Cameroon</figcaption></figure>
## 2.1.2. Maroua: Source of Sorghum Bran

Maroua, the capital of the Far North Region, is an agricultural region where the cultivation of Sorghum sp. is widespread. According to Dzokom (2024), sorghum is one of the main cereals grown, and its bran is a by-product of grain processing into flour or livestock feed. The dry climate, with average temperatures ranging from 25°C to 40°C and annual rainfall of 700–1000 mm (Ministry of Agriculture, 2021), enables the production of about 200,000 tons of sorghum annually (INS, 2021), generating a significant amount of bran suitable for bioethanol production.

## 2.2. Materials

The raw materials used in this study include *Oryza sativa* husk and *Sorghum* sp. bran, both locally sourced. *Oryza sativa* husk is obtained after the dehusking of *Oryza sativa* grains, while *Sorghum* sp. bran is a by-product of *Sorghum* sp. processing. These materials are sun-dried and then ground into a fine powder using a stone mill.

### 2.2.1. Characterization of Materials

Before being used for bioethanol production, the physicochemical properties of *Oryza sativa* husk and *Sorghum* sp. bran are analyzed. The parameters measured include:

* **Chemical composition:** content of cellulose, hemicellulose, lignin, and starch, determined using the Klason method (for cellulose and lignin) and enzymatic analysis (for starch) (Faria et al., 2024; Hamdi et al., 2024).

* **Calorific value:** measured to assess the energy potential of the biomass before conversion into bioethanol.

### 2.2.2. Biomass Pretreatment

Pretreatment is carried out to make the sugars in the biomass more accessible for hydrolysis. Two methods are tested for each type of biomass:

#### 2.2.2.1. Alkaline Pretreatment

An alkaline pretreatment is performed by immersing the *Oryza sativa* husk and *Sorghum sp.* bran in a sodium hydroxide (NaOH) solution at a concentration of 3% for 2 hours at room temperature. This method is well-documented for its effectiveness in reducing lignin and making polysaccharides more accessible for hydrolysis (Dzokom et al., 2023).

#### 2.2.2.2. Thermal Pretreatment

The previously ground biomass is then heated at 120°C for 30 minutes under 1 bar of steam pressure, to break down the lignocellulose structure and release sugars. This method is commonly used in industry to optimize the conversion of raw materials into ethanol (Woźniak et al., 2025).

#### 2.2.3. Enzymatic Hydrolysis

The hydrolysis of polysaccharides is carried out using commercial cellulolytic and amylolytic hydrolysis enzymes. Cellulase and xylanase enzymes are used to break down cellulose and hemicellulose, while amylase is used to hydrolyze starch from the sorghum bran.

##### 2.2.3.1. Hydrolysis Conditions

Hydrolysis conditions are optimized by varying the following parameters:

* **Enzyme concentration:** From 10 to 40 U/g of biomass for cellulases and xylanases, and from 5 to 20 U/g for amylases.

* **Hydrolysis temperature:** From 45°C to 55°C.

* **Duration of hydrolysis:** From 12 to 48 hours.

Hydrolysis performance is evaluated by measuring the reducing sugars produced after hydrolysis using the DNS method (Sarkar et al., 2024; de Souza et al., 2024).

#### 2.2.4. Fermentation

Fermentation of the hydrolysates is performed using a *Saccharomyces*
cerevisiae strain (ATCC 7663), which is one of the most commonly used strains for bioethanol fermentation.

### 2.2.4.1. Fermentation Conditions

Fermentation parameters are optimized to maximize ethanol yield:

* **Yeast concentration:** From 1 to 5% (v/v) of yeast in the hydrolysate solution.

* **Temperature:** From 25°C to 35°C.

* **Duration:** 48 to 72 hours.

* **pH:** Maintained between 4.5 and 5.5.

The evolution of ethanol concentration is monitored by distillation and quantified using a gas chromatograph (GC) (Pooja and Nivedita, 2024; Chavan et al., 2024).

### 2.2.4.2. Ethanol Yield

Ethanol yield is calculated using the following formula:

Ethanol yield (%)

$$ = \frac{C_{ethanol} \times V_{fermentation}}{M_{initial biomass} \times 0,51} \times 100 $$

where $C_{ethanol}$ is the measured ethanol concentration (g/L), $V_{fermentation}$ is the volume of the fermented solution (L), and $M_{initial biomass}$ is the mass of initial biomass (g). The factor 0.51

<table><caption>Table 1: Characterization of Materials Before Their Conversion into Bioethanol</caption><thead><tr><th>Parameters</th><th>Oryza sativa Husk</th><th>Sorghum sp. Bran</th><th>Mixture (50:50)</th></tr></thead><tbody><tr><td>Cellulose (%)</td><td>38.5</td><td>14.2</td><td>26.35</td></tr><tr><td>Hemicellulose (%)</td><td>22.3</td><td>28.4</td><td>25.35</td></tr><tr><td>Lignin (%)</td><td>25.7</td><td>8.9</td><td>17.30</td></tr><tr><td>Starch (%)</td><td>1.2</td><td>45.3</td><td>23.25</td></tr><tr><td>Enzymatic analysis (mg/g)</td><td>4.5</td><td>65.7</td><td>35.10</td></tr><tr><td>Calorific value (MJ/kg)</td><td>15.8</td><td>17.2</td><td>16.50</td></tr></tbody></table>

Data from the present study

* **Cellulose and Lignin Content:** *Oryza sativa* husk exhibits a higher cellulose (38.5%) and lignin (25.7%) content, which may affect enzymatic hydrolysis and require more intensive pretreatment.

* **Hemicellulose:** *Sorghum sp.* bran contains more hemicellulose (28.4%),

which could facilitate faster hydrolysis of pentoses.

### 2.2.5. Experimental Design and Data Analysis

A factorial experimental design is used to evaluate the effects of each pretreatment, hydrolysis, and fermentation parameter on bioethanol yield. The data obtained are analyzed using the statistical software R (R Core Team, 2017) to determine significant interactions between factors and to optimize production conditions.

### 2.2.6. Quality Control

The ethanol samples produced are characterized by UV-Vis spectroscopy to verify their purity and compliance with industrial standards. Additionally, the composition of solid residues after fermentation is analyzed to assess the efficiency of the conversion process.

## 3. Results and Discussion

### 3.1. Characterization of Raw Materials

#### 3.1.1. Characterization of Materials Before Conversion to Bioethanol

The characterization of materials used for bioethanol production highlighted their chemical composition, enzymatic starch analysis, and calorific value:

* **Starch and Enzymatic Analysis:** *Sorghum sp.* bran is rich in starch (45.3%), explaining its higher release rate of fermentable sugars (65.7 mg/g in enzymatic analysis).
* **Calorific Value:** The energy potential of the 50:50 mixture (16.5 MJ/kg) lies between those of the two raw materials, indicating some energy complementarity.

*Oryza sativa* husk and *Sorghum* sp. bran showed distinct yet complementary chemical compositions for bioethanol production. Rice husk contains 38% cellulose, 25% lignin, and 15%

hemicellulose, whereas sorghum bran is rich in starch (around 60%) and lower in lignin and cellulose (Table 1). These characteristics suggest that rice husk is mainly composed of lignocellulosic components, while sorghum bran is a major source of starch, making their combination promising for ethanol production (Dzokom et al., 2023).

<table><caption>Table 2: Chemical Composition of Materials</caption><thead><tr><td>Material</td><td>Cellulose (%)</td><td>Lignin (%)</td><td>Hemicellulose (%)</td><td>Starch (%)</td></tr></thead><tbody><tr><td>Oryza sativa husk</td><td>38</td><td>25</td><td>15</td><td>0</td></tr><tr><td>Sorghum sp. bran</td><td>10</td><td>5</td><td>10</td><td>60</td></tr></tbody></table>

Data from the present study

### 3.1.2. Optimization of *Oryza sativa* Husk/Sorghum sp. Bran Ratio

Optimizing the ratio between *Oryza sativa* husk and *Sorghum sp. bran* is a key parameter for maximizing bioethanol yield.

*Oryza sativa* husk, rich in cellulose and lignin, and *Sorghum sp. bran*, rich in starch, form an ideal combination by providing different fermentable sugar sources.

<table><caption>Table 3: Optimization Results of *Oryza sativa* Husk/Sorghum sp. Bran Ratio</caption><thead><tr><td>Oryza sativa Husk : Sorghum sp. Bran Ratio</td><td>Ethanol Yield (g/L)</td><td>Yield (g ethanol/g dry biomass)</td><td>Reducing Sugar (g/L)</td></tr></thead><tbody><tr><td>100:0</td><td>31.2</td><td>0.38</td><td>8.3</td></tr><tr><td>75:25</td><td>33.0</td><td>0.40</td><td>9.1</td></tr><tr><td>50:50</td><td>37.5</td><td>0.42</td><td>10.5</td></tr><tr><td>25:75</td><td>34.2</td><td>0.39</td><td>9.3</td></tr><tr><td>0:100</td><td>32.5</td><td>0.38</td><td>8.9</td></tr></tbody></table>

Data from the present study

The 50:50 ratio optimization resulted in the best ethanol yield and the highest release of fermentable sugars. This is due to the balance between the lignocellulosic components of *Oryza sativa* husk and the starch content of *Sorghum sp. bran*, enabling better enzymatic breakdown and fermentation.

### 3.2. Effect of Pretreatments on Lignocellulose and Starch Degradation

#### 3.2.1. Thermal Pretreatment of Ground Biomass

The results table of thermal pretreatment of ground biomass heated at 120°C for 30 minutes under 1 bar steam pressure is as follows:

<table><caption>Table 4: Thermal Pretreatment of Ground Biomass</caption><thead><tr><td>Parameters</td><td>Oryza sativa Husk</td><td>Sorghum sp. Bran</td><td>Mixture (50:50)</td></tr></thead><tbody><tr><td>Temperature (°C)</td><td>120</td><td>120</td><td>120</td></tr><tr><td>Duration (min)</td><td>30</td><td>30</td><td>30</td></tr><tr><td>Pressure (bar)</td><td>1</td><td>1</td><td>1</td></tr><tr><td>Lignin Reduction (%)</td><td>18.5</td><td>12.8</td><td>15.6</td></tr><tr><td>Sugar Release (mg/g)</td><td>12.3</td><td>28.7</td><td>20.5</td></tr></tbody></table>

Data from the present study
* **Lignin Reduction:** Thermal pretreatment achieved more effective delignification of *Oryza sativa* husk (18.5%) compared to *Sorghum sp* bran (12.8%).

* **Sugar Release:** *Sorghum sp* bran exhibited a higher release of fermentable sugars (28.7 mg/g) than *Oryza sativa* husk (12.3 mg/g), likely due to its lower lignin content and higher starch.

* **Effect of 50:50 Mixture:** The combination resulted in a 15.6% lignin reduction and an intermediate

sugar release (20.5 mg/g), offering a compromise between sugar accessibility and substrate structure.

Alkaline pretreatment led to significant lignocellulose degradation in *Oryza sativa* husk, with a 33% reduction in lignin. This aligns with other studies showing that alkaline treatment improves polysaccharide accessibility for hydrolysis (Johannes & Xuan, 2024; Dzokom et al., 2023). Conversely, thermal pretreatment was less effective in lignin degradation but released more soluble sugars from *Sorghum sp*. bran (Table 5).

<table><caption>Table 5: Alkaline and Thermal Pretreatments</caption><thead><tr><th>Pretreatment</th><th>Lignin Reduction (%) (Oryza sativa Husk)</th><th>Reducing Sugar (g/L) (Sorghum sp. Bran)</th></tr></thead><tbody><tr><td>Alkaline</td><td>33</td><td>12.5</td></tr><tr><td>Thermal</td><td>15</td><td>8.3</td></tr></tbody></table>

Data from the present study

The release of reducing sugars from *Sorghum sp* bran after thermal pretreatment is attributed to starch degradation. Heat helps hydrolyze starch into dextrins and maltose, which are then fermented into ethanol by *Saccharomyces cerevisiae* (Tenkolu et al., 2024).

### 3.2.2. Impact of Pretreatments

Chemical pretreatments are essential for lignocellulose breakdown and fermentable sugar release. Two types of pretreatments were tested: acidic and alkaline.

#### 3.2.2.1. Acid Pretreatment (1.5% H₂SO₄ at 120°C for 30 min)

Acid pretreatment reduced lignin but showed lower ethanol yields due to starch degradation in sorghum bran under acidic conditions.

#### 3.2.2.2. Alkaline Pretreatment (3% NaOH at 120°C for 30 min)

Alkaline pretreatment was more effective in breaking down lignocellulose, significantly reducing lignin and improving fermentable sugar release. This treatment resulted in the highest ethanol yields.

<table><caption>Table 6: Comparison of Yields after Acid and Alkaline Pretreatments</caption><thead><tr><th>Pretreatment</th><th>Lignin Reduction (%) (Oryza sativa Husk)</th><th>Reducing Sugar (g/L) (Sorghum sp Bran)</th><th>Ethanol Yield (g/L)</th></tr></thead><tbody><tr><td>Acid (1.5% H₂SO₄)</td><td>20</td><td>10.2</td><td>33.0</td></tr><tr><td>Alkaline (3% NaOH)</td><td>33</td><td>12.5</td><td>37.5</td></tr></tbody></table>

Data from the present study

Alkaline pretreatment demonstrated greater efficiency in terms of sugar release, which directly led to a higher ethanol yield. This result is consistent with previous studies (Dzokom et al., 2023), which showed that alkaline treatments promote the degradation of lignocellulose.

### 3.3. Optimization of Enzymatic Hydrolysis

Visualization of the results of polysaccharide enzymatic hydrolysis as a function of enzyme concentration, temperature, and hydrolysis duration.
<figure><img src="image_4.png" /><figcaption>Figure 2: Impacts of Enzymatic Hydrolysis on Polysaccharide Release</figcaption></figure>

### Effect of Enzyme Concentration

Increasing enzyme concentration (from 10 to 40 U/g) enhances the release of reducing sugars, indicating more efficient hydrolysis of polysaccharides.

### Effect of Temperature

The optimal range appears to be around 50°C to 55°C, beyond which enzyme activity may begin to decline.

### Impact of Hydrolysis Duration

The longer the hydrolysis, the more reducing sugars are released, although

the increase tends to slow after 36 hours, suggesting enzymatic saturation.

Hydrolysis tests showed that the combination of cellulases and amylases resulted in higher yields of fermentable sugars. The maximum sugar yield (65 g/L) was obtained after 48 hours of hydrolysis at 50°C, with an enzyme concentration of 30 U/g of biomass (Table 3). This result is higher than those reported in many previous studies that used similar pretreatments for lignocellulosic feedstocks (Woźniak et al., 2025; Sharma & Sharma, 2024).

<table><caption>Table 7: Hydrolysis Tests</caption><thead><tr><td>Enzyme (U/g)</td><td>Hydrolysis Duration (h)</td><td>Reducing Sugar (g/L)</td></tr></thead><tbody><tr><td>10</td><td>24</td><td>25</td></tr><tr><td>20</td><td>36</td><td>45</td></tr><tr><td>30</td><td>48</td><td>65</td></tr></tbody></table>

Data from the present study

The highest yield was observed with the simultaneous activation of cellulases and amylases, confirming that both biomasses (*Oryza sativa* husk and sorghum bran) effectively contribute to the production of fermentable sugars, which is essential for optimal ethanol yield.

### 3.4. Fermentation and Ethanol Production

The graphs below illustrate the effects of yeast concentration, temperature, fermentation time, and a comparison between *Saccharomyces cerevisiae* and *Pichia stipitis* on bioethanol yield. These visualizations help optimize fermentation conditions to maximize ethanol production.
<figure><img src="image_5.png" /><figcaption>Figure 3: Effects of Parameters on Bioethanol Yield</figcaption></figure>

The alcoholic fermentation of the hydrolysate obtained from rice husk and sorghum bran was optimized by varying several key parameters: yeast concentration, temperature, duration, and pH. The objective is to maximize the conversion of fermentable sugars into ethanol by identifying optimal conditions.

### 3.4.1. Effect of Yeast Concentration

Increasing yeast concentration from 1% to 5% (v/v) has a significant impact on bioethanol yield. A concentration below 2% leads to slow fermentation and limited ethanol production due to an insufficient cell population for efficient sugar conversion.

<figure><img src="image_6.png" /><figcaption>Figure 4: Effect of Yeast Concentration</figcaption></figure>

Alcoholic fermentation of the hydrolysate obtained from *Oryza sativa* husk and *Sorghum sp* bran was optimized by varying several key parameters: yeast concentration, temperature, fermentation time, and pH. The objective was to maximize the conversion of fermentable sugars into ethanol by identifying the optimal conditions.

### 3.4.1. Effect of Yeast Concentration

Increasing the yeast concentration from 1 to 5% (v/v) significantly impacted the bioethanol yield. A too-low concentration (<2%) results in slow fermentation and limited yield due to an insufficient cell population to efficiently convert sugars. At 3-4%, the bioethanol yield reaches an optimal plateau, consistent with the results of Lin & Tanaka (2006) and Wyman (1999), who recommend an optimal yeast concentration of 3-5% for lignocellulosic fermentations. However, beyond 4%, further increases in yeast concentration do not lead to significant yield gains and may
even result in competitive inhibition due to excessive cell biomass accumulation, as demonstrated by Bai et al. (2008).

### 3.4.2. Influence of Temperature

Fermentation was conducted over a temperature range of 25°C to 35°C. The results show that:

* At 25°C, yeast metabolism slows down, prolonging fermentation time with lower ethanol yield.

* At 30°C, the maximum ethanol yield (~90% of theoretical yield) is achieved, consistent with the work of Walker (2010) and Walker (2011), who indicate that *Saccharomyces cerevisiae* has optimal activity between 28°C and 32°C.

* At 35°C, a slight decrease in yield is observed, likely due to heat stress affecting yeast cell viability (Stanley et al., 2010; Auesukaree, 2017). Therefore, a temperature of 30°C is considered optimal for maximum ethanol production.

### 3.4.3. Effect of Fermentation Time

Analysis of ethanol yield over a period of 48 to 72 hours shows that:

* At 48 hours, approximately 85% of the maximum yield is reached.

* At 72 hours, the final yield is slightly higher (~90% of theoretical

yield), but the fermentation rate decreases after 48 hours due to ethanol accumulation and other secondary metabolites that can inhibit yeast (Olsson & Hahnhägerdal, 1996). A duration of 48 to 60 hours is therefore sufficient to achieve optimal yield while minimizing energy consumption and bacterial contamination risks.

### 3.4.4. Influence of pH

pH was maintained between 4.5 and 5.5 to ensure optimal yeast activity.

* A pH below 4.5 reduces fermentation efficiency by increasing the medium's acidity, which inhibits cell growth.

* A pH above 5.5 favors the growth of contaminating microorganisms, compromising the final product's purity (Narendranath & Power, 2005). An optimal pH of 5.0 allowed for optimized yeast viability and sugar-to-ethanol conversion rate.

### 3.4.5. Yeast Comparison: *Saccharomyces cerevisiae* vs. *Pichia stipitis*

A comparison was conducted between *Saccharomyces cerevisiae* and *Pichia stipitis*, two yeasts commonly used for lignocellulosic hydrolysate fermentation.

<table><caption>Table 8: Yeast Parameter Comparison</caption><thead><tr><td>Parameter</td><td><i>Saccharomyces cerevisiae</i></td><td><i>Pichia stipitis</i></td></tr></thead><tbody><tr><td>Ethanol yield</td><td>90% of theoretical yield</td><td>70% of theoretical yield</td></tr><tr><td>Ethanol tolerance</td><td>High (up to 15% v/v)</td><td>Moderate (up to 6-8% v/v)</td></tr><tr><td>Pentose fermentation capacity</td><td>Low (mostly glucose)</td><td>Good (xylose, arabinose)</td></tr><tr><td>Fermentation time</td><td>Optimal at 48 h</td><td>Requires 72 h</td></tr></tbody></table>

Although *Pichia stipitis* can ferment pentoses (xylose and arabinose), its overall yield is lower than *Saccharomyces cerevisiae*, which remains the optimal choice for converting sugars into bioethanol from *Oryza sativa* husk and *Sorghum sp* bran hydrolysates. Our results show that optimal fermentation of lignocellulosic

hydrolysates based on *Oryza sativa* husk and *Sorghum sp* bran is achieved with *Saccharomyces cerevisiae* at a concentration of 3-4%, at 30°C, for 48-60 hours, and at a pH of 5.0.

To further improve yield, several strategies can be considered:
* Co-fermentation with recombinant strains capable of fermenting both glucose and xylose (Ma & Liu, 2010).

* Yeast adaptation to ethanol stress to extend viability at the end of fermentation (Fiedurek et al., 2011).

* Yeast recycling to reduce production costs (Nielsen et al., 2017).

biomass after 72 hours of fermentation at 30°C and pH 5.5, using 5% *Saccharomyces cerevisiae*. This yield is comparable to that observed by Chavan et al. (2024) in the fermentation of pretreated lignocellulosic substrates. Experiments also revealed that adding sorghum bran to *Oryza sativa* husk slightly improved ethanol yield (Table 4), likely due to the presence of starch, which serves as an additional source of fermentable sugars.

Fermentation tests showed a maximum ethanol yield of 0.42 g ethanol/g dry

<table><caption>Table 9: Fermentation and Ethanol Production</caption><thead><tr><td>Feedstock</td><td>Ethanol Yield (g/L)</td><td>Yield (g ethanol/g dry biomass)</td></tr></thead><tbody><tr><td><em>Oryza sativa</em> husk only</td><td>31.2</td><td>0.38</td></tr><tr><td><em>Sorghum sp</em> bran only</td><td>35.0</td><td>0.40</td></tr><tr><td><em>Oryza sativa</em> husk + <em>Sorghum sp</em> bran mix</td><td>37.5</td><td>0.42</td></tr></tbody></table>

The results indicate that the *Oryza sativa* husk and *Sorghum sp* bran mixture enhances ethanol production compared to using a single biomass. This effect can be attributed to the complementary nature of the two biomasses: *Oryza sativa* husk provides a high cellulose content, while *Sorghum sp* bran contributes a substantial amount of starch, enhancing fermentation efficiency.

*Saccharomyces cerevisiae* and *Pichia stipitis*.

### 3.4.6.1. *Saccharomyces cerevisiae*

Widely used in the bioethanol industry, *S. cerevisiae* is effective in fermenting hexoses, primarily glucose. It showed a higher ethanol yield (0.42 g ethanol/g dry biomass).

### 3.4.6.2. *Pichia stipitis*

Although *P. stipitis* can ferment pentoses (like xylose), it showed a slightly lower yield (0.38 g ethanol/g dry biomass) due to the low pentose content of the biomass used in this study.

### 3.4.6. Comparison of Yeast Effects on Bioethanol Yield

The choice of yeast is crucial for maximizing bioethanol production. Two yeasts were compared in this study:

<table><caption>Table 10: Comparison of Yeast Effects on Ethanol Yields</caption><thead><tr><td>Yeast</td><td><em>Oryza sativa</em> Husk : <em>Sorghum sp</em> Bran Ratio</td><td>Ethanol Yield (g/L)</td><td>Yield (g ethanol/g dry biomass)</td></tr></thead><tbody><tr><td><em>Saccharomyces cerevisiae</em></td><td>50:50</td><td>37.5</td><td>0.42</td></tr><tr><td><em>Pichia stipitis</em></td><td>50:50</td><td>32.5</td><td>0.38</td></tr></tbody></table>

The results show that *S. cerevisiae* produces higher ethanol yields than *P. stipitis*, especially with substrates rich in hexoses.

However, *P. stipitis* may be more advantageous for fermenting biomass rich in pentoses.
<figure><img src="image_7.png" /><figcaption>Figure 4: Quality Control Based on the UV-Vis Absorbance of the Produced Ethanol Samples</figcaption></figure>

## Comparison with the Industrial Standard

The results indicate that the absorbance of the produced ethanol samples follows a trend similar to that of the industrial standard, suggesting good compliance with purity criteria. However, slight variations are observed, particularly at wavelengths

below 220 nm, which could indicate the presence of residual compounds from the fermentation process (Yan et al., 2019; Williams et al., 2018; Riedel & Leopold, 2005).

## Presence of Potential Impurities

<figure><img src="image_8.png" /><figcaption>Figure 5: Composition of Solid Residues After Fermentation</figcaption></figure>
The pie chart above illustrates the composition of solid residues following fermentation:

* **Residual Cellulose (35%):** Portion not converted during hydrolysis.

* **Hemicellulose (25%):** Partially degraded residues.

* **Lignin (20%):** Non-fermentable component remaining after the process.

* **Proteins and Other Matter (10%):** Minor components originating from the biomass.

* **Ashes (10%):** Mineral residues post-fermentation.

A high proportion of residual cellulose and lignin may indicate the need to optimize pretreatment or enzymatic hydrolysis to enhance conversion efficiency.

Discrepancies between the absorbance of the samples and the industrial standard may be attributed to organic residues such as organic acids, phenolic compounds from lignin, or secondary fermentation by-products (Mussatto et al., 2014; Lee et al., 2017; Broda et al., 2022).

Higher absorbance in the UV region may indicate the presence of contaminants absorbing in this spectral range, such as furan derivatives and carbonyl compounds resulting from sugar degradation (Silva et al., 2021; Lennartsson et al., 2014; Tse et al., 2021).

### 3.5.2. Analysis of the Quality of the Produced Ethanol

High-purity industrial ethanol must meet strict standards, including low levels of both volatile and non-volatile impurities. According to ASTM D4806, the minimum required purity for fuel ethanol is 99.5% (Agarwal, 2017; Niven, 2005; Thangavelu et al., 2016). A strong alignment with the industrial standard curve suggests that the produced ethanol meets quality requirements.

However, the slightly higher absorbance of

the samples between 200 and 220 nm indicates the possible presence of trace amounts of methanol, acetaldehyde, or other volatile compounds formed during fermentation (Balat et al., 2008). These compounds may result from suboptimal fermentation or incomplete distillation.

## Discussion

The results confirm the importance of optimizing the *Oryza sativa* husk to *Sorghum sp* bran ratio to maximize bioethanol production. A 50:50 ratio was found to be optimal in terms of ethanol yield and fermentable sugar release. The use of alkaline pretreatment enabled maximum degradation of lignocellulose, promoting fermentable sugar release and increasing bioethanol yield.

Regarding yeast, *Saccharomyces cerevisiae* remains the best option in this context due to its strong performance in hexose fermentation. However, *Pichia stipitis* may be considered for more complex substrates, especially those with higher pentose content.

The findings of this study demonstrate that bioethanol production from *Oryza sativa* husk combined with *Sorghum sp* bran can be optimized by modifying pretreatment, hydrolysis, and fermentation parameters. Alkaline pretreatment effectively broke down *Oryza sativa* husk lignin, making sugars more accessible for hydrolysis. The combination of the two biomasses led to increased ethanol yield, confirming results reported by Chavan et al. (2024), who also observed yield improvements using combined agricultural biomasses.

Fermentation condition optimization is essential for maximizing ethanol production. The yield observed in this study is relatively high compared to other similar studies (Ceaser et al., 2024), suggesting that using *Oryza sativa* husk and *Sorghum sp* bran together is a viable approach for sustainable bioethanol production.
Although alkaline pretreatment proved more effective in releasing fermentable sugars, it can also lead to the formation of lignin-derived compounds that affect ethanol purity (Dzokom et al., 2023). Proper neutralization and thorough washing after pretreatment could enhance substrate quality and reduce UV-absorbing impurities.

*Saccharomyces cerevisiae* showed higher ethanol yields but may produce secondary metabolites such as glycerol and acetate (Zhao et al., 2020; Senthilraja et al., 2011; Kongkiattikajorn & Sornvoraweat, 2011). While less efficient in yield, *Pichia stipitis* may produce fewer volatile impurities, especially when metabolizing pentoses. Optimizing fermentation conditions, such as pH and aeration, could reduce the formation of undesirable compounds.

More rigorous fractional distillation or the use of activated carbon adsorption systems may further reduce impurities responsible for variations in absorbance curves (Chen et al., 2015; Mwangi & Og, 2013; Susilawati et al., 2022).

However, further research is required to improve process efficiency at larger scales, especially by optimizing fermentation parameters and reducing enzyme costs. The results of this study pave the way for implementing agricultural residue valorization strategies for low-cost and sustainable bioethanol production.

## Enzymatic Hydrolysis of Lignocellulosic Polysaccharides

The enzymatic hydrolysis of lignocellulosic polysaccharides is influenced by several parameters, including enzyme concentration, temperature, and hydrolysis duration. Our results highlight the combined impact of these factors on reducing sugar release, confirming trends reported in the scientific literature.

Increasing cellulolytic enzyme concentrations (cellulases and xylanases from 10 to 40 U/g of biomass) and

amylolytic enzymes (from 5 to 20 U/g) gradually improved polysaccharide conversion into fermentable sugars. This observation is consistent with the findings of Lynd et al. (2002), who showed that increasing enzyme loading enhances substrate accessibility by reducing cellulose crystallinity and accelerating hemicellulose degradation.

However, beyond 30 U/g, sugar yield tends to plateau, suggesting substrate saturation. Zhang et al. (2006) indicated that this plateau is due to competitive inhibition among enzymes and product feedback inhibition (by glucose and xylose), which can limit enzymatic activity at high concentrations.

The analysis shows that the optimal hydrolysis temperature lies between 50°C and 55°C, which corresponds to the activity range of commercial cellulases and amylases (Ghose, 1987). At 45°C, enzymatic activity is lower, resulting in limited sugar production. At 55°C, although sugar yield remains high, a slight decline is sometimes observed, likely due to partial thermal denaturation of the enzymes.

These results align with those reported by Singhania et al. (2010) and Bhargav et al. (2008), who found that *Trichoderma reesei*-derived cellulases and xylanases are most active between 50°C and 55°C but begin to lose efficiency above this range.

Extending hydrolysis duration from 12 to 48 hours significantly increased reducing sugar release. However, a slowdown in yield was observed after 36 hours, which may be explained by several phenomena:

* **Product Inhibition:** Accumulation of glucose and xylose may inhibit β-glucosidases and xylanases (Singh et al., 2015).

* **Enzyme Activity Loss:** Progressive denaturation over time may reduce hydrolysis efficiency (Eriksson et al., 2002).

* **Substrate Accessibility Limitation:** After initial rapid hydrolysis, more resistant fractions
(crystalline cellulose, recalcitrant
lignin) dominate, slowing reaction
progression (Zhu et al., 2009).

Compared to other lignocellulosic
substrates such as sugarcane bagasse or
peanut shells, the rice husk/sorghum bran
combination presents good yield in
fermentable sugars, notably due to its starch
fraction hydrolyzable by amylases.
However, further yield improvements can
be achieved through several strategies:

*   **Enzyme Cocktail Optimization:** Adding exogenous β-glucosidases may reduce inhibition from cellobiose accumulation (Pooja & Nivedita, 2024; Chavan et al., 2024).

*   **Enzyme Recycling:** Approaches like enzyme immobilization may extend enzyme lifespan and reduce costs (Adsul et al., 2011).

*   **Combined Pretreatments:** Combining chemical (alkaline or acid) and biological (ligninolytic fungi) pretreatments may improve substrate digestibility (Dzokom et al., 2023).

**4. Conclusion**

The combination of *Oryza sativa* husk and *Sorghum sp* bran is an effective strategy for bioethanol production. Acid and enzymatic pretreatments are essential to maximize yield. In the future, optimizing fermentation conditions and using modified yeasts may further improve output.

The optimization of the *Oryza sativa*
husk/Sorghum sp bran ratio (50:50),
application of alkaline pretreatment, and
use of *Saccharomyces cerevisiae*
maximized ethanol yield in this study.
These findings support improved use of
agricultural biomass in bioethanol
production, with potential implications for
more sustainable and efficient industrial
processes.

UV-Vis analysis of the produced ethanol
showed good correlation with the industrial

standard, indicating satisfactory quality of
the bioethanol derived from *Oryza sativa*
husk and *Sorghum sp* bran. However,
deviations observed at lower wavelengths
suggest the presence of secondary
compounds that could be reduced by
optimizing pretreatment, fermentation, and
distillation.

Complementary analyses, such as gas
chromatography (GC) or mass spectrometry
(MS), would enable more precise
identification of impurities and help refine
the production process to ensure optimal
ethanol quality.

The study of enzymatic hydrolysis
parameters of lignocellulosic biomass from
rice husk and sorghum bran identified
optimal conditions for maximizing
fermentable sugar production. Our results,
consistent with the literature, show that
optimizing enzymatic hydrolysis conditions
is a key factor in enhancing the efficiency
of bioethanol production from these agro-
industrial residues.

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