Introduction

The expanding world population and industrialization have dramatically increased global energy utilization a century ago and there are natural concerns related to the use of fossil fuels. There is an immediate need for alternatives sources like bioethanol, biomethane, biohydrogen, and biodiesel which can substitute the diminishing fossil fuels and reduce their environmental effect (Sanusi et al. 2020). Additionally, the burning of fossil fuels promotes the emission of greenhouse gases along with global warming that causes a rise in sea level, climate change, pollution and depletion of biodiversity. Bioethanol has arisen as one of the most promising fossil fuel substitutes. It's an odourless, flammable, and highly volatile liquid. Compared to traditional fuels, bioethanol has a number of advantages as it contains 35% oxygen, which helps in completing the combustion of the fuel and reduces particle and NOx emissions (Saini et al. 2014). Bioethanol has a greater compression ratio and shorter burn time than gasoline due to its higher flammability limits, higher octane number, higher heat of vaporization, and quicker flame speed. This translates to a higher compression ratio and shorter burn duration in an internal combustion engine (Balat et al. 2008)

Starch, sugars, algae, and lignocellulosic biomass are the most common renewable sources used to produce bioethanol. The first-generation bioethanol is made from starch and sugar, whereas the second and third generations are made from lignocellulosic biomass and algae, respectively. Third-generation bioethanol from algae is still in the early stages of development and is limited to laboratory research, whereas other types of biomasses have demonstrated commercial viability as bioethanol feedstocks. As there is a barrier in the case of first -generation biofuels above which they cannot generate enough biofuel without endangering food supplies and biodiversity, bioethanol production from first generation sources has a negative influence on food security, resulting in a conflict between "food vs. fuel." So, to avoid the conflict lignocellulosic biomass is the best replacement. These substances are also cheap in comparison than first-generation biomass. The cellulose, hemicelluloses, and lignin components of lignocellulosic biomass are the three main components. In fact, using lignocellulosic materials for bioethanol production can help reduce greenhouse gas emissions (Seung et al.2013).

The most abundant lignocellulosic crops in many parts of the world are rice straw.  Half of the world population uses this cereal as a principal food. Globally, there is a capacity to produce approximately 282 billion liters of bioethanol per year based on the quantity of rice straw produced. Pretreatment, enzymatic hydrolysis, and fermentation are all required for the conversion of rice straw to bioethanol. Pretreatment is one of the most important processes in bioethanol production because it regulates the range of hydrolysis that enzymes used in the next step can achieve. It can alter cellulose structure and crystallinity, as well as disrupt lignin-carbohydrate linkages, enhancing sugar yield in enzymatic hydrolysis and hence bioethanol production (Ashoor et al. 2020). The primary goal of pretreatment is to increase the surface area of biomass while disrupting hemicellulose and/or lignin and reducing biomass particle size.

As various microorganisms, including bacteria and algae, have been explored for the production of bioethanol, Saccharomyces cerevisiae, the yeast remains the most required species for bioethanol production. Yeasts like S. cerevisiae have been used in the production of alcohol for thousands of years, primarily in the wine and brewing industries. Saccharomyces cerevisiae is a facultative anaerobe that can efficiently convert glucose to ethanol under anaerobic circumstances. Because S. cerevisiae can tolerate a wide range of pH, it is the most commonly utilized microbe in commercial ethanol production. This makes the process less prone to infections. Acid tolerance, high-temperature tolerance, and high ethanol production are all critical features of strains that are needed for successful bioethanol production.

Materials and methods

Collection of Substrates

Rice straw were provided by the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (Chhattisgarh) for the Project Work. To remove the impurities, it was cleaned and sieved through flour sifter, before being stored in an air tight container.

Microbial Culture

Microorganism Saccharomyces cerevisiae was used for bioethanol production which was also provided by the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (Chhattisgarh). The microbial culture was revived in their respective nutrient media.

Medium Used

For Saccharomyces cerevisiae, Yeast Peptone Glucose (YPG) agar media was used. In 1000ml of distilled water, YPG media – Yeast Extract - 10gm, Peptone- 20gm, Glucose- 20gm, and Agar- 15gm were added.  The culture was inoculated, then incubated at 30ºC for 48 hours before being stored at 4ºC for future research.

Selection of Bioethanol Producing Yeast by Fermentation Test

To check the fermentation ability of yeast, fermentation test was performed. For this 3 ml of fermentation broth was taken whose composition for 1ml is Glucose (5g), Peptone (10g), Sodium Chloride (15g), Phenol Red (0.018g) by maintaining 7.3 pH in test tube containing Durham’s tube in inverted position and 1 ml of yeast culture was inoculated followed by incubation at 30ºC for 48 hrs. Due to formation of acid and gas production in Durham tube the colour change was observed from red to yellow.

Bioethanol Production from Rice straw

Bioethanol Fermentation

20 gm of Rice straw was added in 200 ml of distilled water in 250 ml of conical flask followed by autoclaving at 121 ºC at 15 psi for 15 min. After autoclaving, the substrate was inoculated with the yeast culture. Under aseptic conditions; the flasks were incubated at 30 ºC for 24 h, 48 h and 72 h respectively for fermentation. After fermentation, the fermented sample was distilled.

Distillation of Fermented sample

For Distillation, 150 ml of fermented sample was poured into the distillation flask and      distillation was conducted in distillation apparatus. 

Estimation of Bioethanol

Qualitative estimation for bioethanol

The Jones test was used to determine the presence of Bioethanol in the fermentation medium. (Jones et al, 1953). For this test, 1 ml of fermented sample was added in a test tube, and then 1ml of K₂Cr₂O₇and 0.5 ml of H₂SO₄ were added, the change in colour of the sample to bluish green color indicates the presence of bioethanol and hence confirms the test.

Quantitative estimation of bioethanol

The quantity of bioethanol was estimated by Specific Gravity Method. Specific Gravity (SG) is the ratio of density of liquid to the density of water at a specified temperature. Distilled rice straw was taken up to 90 ml and 100 ml of distilled water was added to it. The distilled sample and distilled water mixture were transferred to a 25 ml specific gravity bottle (Pharmacopoeia of India, 1985).

Optimization of Parameters

Production of bioethanol from Rice straw using Saccharomyces cerevisiae was done by optimizing the following parameters:

Optimization of Incubation period

The effect of incubation period plays a crucial role in the production of Bioethanol. Therefore, Incubation period of 24 h, 48 h, 72 h were optimized for the production of bioethanol from rice straw by Saccharomyces cerevisiae. For optimization, the rice straw hydrolysate was inoculated with Saccharomyces cerevisiae and was incubated at 30ºC and distilled on 24, 48, and 72h of fermentation respectively.

Optimization of Temperature

The enzymatic activity and cell maintenance are entirely dependent on the temperature.  The fermentation of rice straw sample was performed by inoculating 1% saccharomyces cerevisiae inoculum into rice straw and incubating the flask at 30°C, 35°C and 40°C respectively in the incubator for 24 h.

Optimization of pH

pH is a critical parameter in the regulation of microbial metabolism, with a well-defined influence in processes involving multiple end product formation (Bhatia and Johri, 2015). The pH has an impact on the biomass composition and the nature of microbial metabolism. The pH range of 7, 8 and 9 was used to optimize pH for maximum bioethanol production by Saccharomyces cerevisiae.

Statistical analysis of data

All data from the experiments were presented as means and standard errors of triplicate values, and they were analysed using one-way analysis of variance (ANNOVA) with significant differences between means determined at p<0.05 and measured with Duncan's multiple range tests using the statistical package for social science research (SPSS) version 16.

Results and Discussion

The main aim of this work was to study the production of bioethanol from rice straw as a substrate using Saccharomyces cerevisiae and optimization of different parameters affecting the production of Bioethanol. For this, the fermented sample was distilled in the distillation unit, and the amount of bioethanol produced was estimated qualitatively and quantitatively.

Bioethanol production from rice residues and its estimation

Rice straw was taken in a conical flask (20gm in 200 ml of distilled water) (Fig.1) and fermented for 24 to 72 h at 30^°C and the production of bioethanol were estimated at 24, 48, 72. Fermented sample was distilled in the distillation unit and bioethanol was estimated by qualitative (Jones Test) and quantitatively.

Optimization of different parameters for increasing production of Bioethanol

Different parameters were tested to optimize the production of bioethanol by Saccharomyces cerevisiae from Rice straw. After the optimization of each parameter, the fermented sample was distilled and the bioethanol content was quantified. The outcomes of parameter optimization for bioethanol production are shown below

Fig.1. Rice bran and its hydrolysate

Optimization of different parameters for increasing production of Bioethanol

Different parameters were tested to optimize the production of bioethanol by Saccharomyces cerevisiae from Rice straw. After the optimization of each parameter, the fermented sample was distilled and the bioethanol content was quantified. The outcomes of parameter optimization for bioethanol production are shown below

Effect of incubation period for bioethanol production

The amount of bioethanol obtained after incubation period was found to be 4.99±0.12^a % in 24 h, 6.13±0.0^b % in 48 h, 7.22±0.0^c % in 72 h, 6.14±0.13^b % in 96 h. Thus, the results showed that the maximum bioethanol production was obtained in 72 h of incubation (Fig.2).

Beliya et al. (2017) used deoiled rice bran for the production of bioethanol by Pichia stipitis NCIM 3497through various parametric optimizations. The highest ethanol concentration was 9.31±0.08 g/L which was obtained in 48 h. Hence concluded that ethanol concentration increased with increase in incubation period up to 48 h and decreased thereafter.

Choudhary et al. (2016) optimized various physical factors by using Shorea robusta (Sal) seeds for the bioethanol production from Zymomonas mobilis MTCC92. It was found that the production of bioethanol increased with the increase of incubation period. The highest bioethanol production was observed on 72 h of incubation period.

Fig.2. Bioethanol production from Rice straw by using Saccharomyces cerevisiae at different incubation period

Effect of temperature for bioethanol production

Fermentation of rice straw was carried out by Saccharomyces cerevisiae at different temperatures for optimizing the temperature for the maximum production of bioethanol. The amount of bioethanol obtained at 30ºC was 7.67±0.09^c %, at 35ºC was 7.022±0.0^b % and at 40ºC was 6.81±0.13^a %. Thus, the results showed that the maximum bioethanol production was obtained at 30ºC (Fig.3).

Chohan et al. (2020) found the optimum temperature of 40ºC for bioethanol production Saccharomyces cerevisiae BY4743 from potato peel waste. It was observed that maximum bioethanol concentration 22.54 g/L was obtained after optimization.

Tahir et al. (2010) studied the effect of temperature on bioethanol production sugarcane molasses by Saccharomyces cerevisiae and concluded that the bioethanol production was highest at temperature 30ºC. The above reported temperature used for bioethanol production was matched with the findings of this work.

Effect of inoculum size for bioethanol production

Fermentation of rice straw was carried out by Saccharomyces cerevisiae at different inoculum size for optimization and thus producing maximum bioethanol. Three different inoculum sizes (1%, 5%, 10 %(v/v)) were investigated to determine the effect of inoculum size for ethanol fermentation from rice straw. The maximum ethanol production 7.22±0.0^c % was obtained with 1% of inoculum. Although 5% inoculum sizes show a lower ethanol yield of 6.95±0.0^b % and with 10% inoculum sizes the bioethanol production was found to be 6.24±0.13^a% (Fig.4).

Fig.3. Bioethanol production from Rice straw by using Saccharomyces cerevisiae at different temperature

Zhang et al. (2011) produced bioethanol from sweet potato as a feedstock with S. cerevisiae strain CCTCC M206111 and concluded that out of different sizes of inoculum (3%, 7%, 10%, 12% and 15%) 7% inoculum size showed maximum bioethanol production of 112.4 (g/L).

Choi et al. (2010) used cassava starch as a feedstock with S. cerevisiae strain CHY1011 for production of bioethanol. They used 5% inoculum size with 4.5pH and concluded 89.1 (g/L) ethanol concentration.

Fig.4. Bioethanol production from Rice straw by using Saccharomyces cerevisiae at different inoculum size

Conclusion

This study shows how rice straw, an agricultural waste with a high cellulose content, may also be used to produce bioethanol under optimal conditions. Rice straw as a feedstock for bioethanol production could help with waste management, as well as maintaining an environmentally friendly atmosphere, being cost-effective, and reducing economic loss. Microorganism Saccharomyces cerevisiae has the capability to produce bioethanol. A maximum bioethanol concentration was achieved under the incubation period of 72 hrs, with the 1% inoculum size and optimum temperature of 30ºC.

Conflict of interest

Authors had no conflict of interest.

Acknowledgement

Whole hearted thanks to School of Studies in Biotechnology, Pandit Ravishankar Shukla University, Raipur (C.G.)

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