Md. Sarower Hossain
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. Present address: Department of Microbiology, International Institute of Applied Science and Technology, Rangpur 5400, Bangladesh
Md. Ahasun Habib
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. Present address: Department of Microbiology, International Institute of Applied Science and Technology, Rangpur 5400, Bangladesh
Sumitra Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
Fahmina Akhtar
Department of Mathematics and Natural Sciences, BRAC University, Dhaka, Bangladesh
Sabina Yasmin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
Fahmida Khatun
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
Renewable energy source, Biofuel, Agricultural wastes, Saccharomyces cerevisiae, Fermentation conditions
Different areas of Mymensingh division, Bangladesh
Farm Mechanization
Bio fertilizer
Collection and processing of agricultural wastes: Agricultural wastes such as rice straw, wheat straw and sugarcane bagasse were collected from different areas of Mymensingh division. Rice straw and wheat straw were collected from Bangladesh Institute of Nuclear Agriculture (BINA), and sugarcane bagasse was collected from Ganginar par, Mymensingh using sterile plastic bags and brought to the laboratory for further processing. After collection, the raw samples were cleaned, oven-dried, and pH of the raw materials was recorded. Here, original gravity was measured by hydrometer. Pre-treatment of the substrates for fermentation: To reduce the biomass size and cellulose content, the raw samples were chipped, ground, milled, and finally pre-treated with acid hydrolysis following the protocol of Braide et al. (2016). Acid pre-treatment was done by dissolving 25gm of each substrate into 250ml of 5% H2SO4 using a 500ml conical flask to achieve delignification. The mixtures were hydrolysed by autoclaving at 121oC for 15minutes. Then the pre-treated samples were filtered using a 24cm pleated filter paper into a 500 ml conical flask. The filtrates were incubated in a water bath at 50oC for 30 minutes. The residue was washed with 1% NaOH to neutralise the acid and then with distilled water and finally dried in an oven at 70oC for 24h. Enzymatic hydrolysis: Enzymatic hydrolysis was done following the protocol of Braide et al. (2016). The cellulosic substrate was autoclaved for 15 minutes, followed by hydrolysis with the commercial enzyme Termamyl (Alpha-amylase) at 500C. This enzyme helps to break down the cellulose into simple-sugar (glucose) for yeast action. Then the mixtures of the cellulosic substrates and termamyl enzyme were placed into a programmed thermostatic mashing bath at 700C with stirring condition. After that, the mixture was allowed to boil for 30 minutes. Wort production: The volume of the samples in each beaker was made up to 250ml by the addition of distilled water. It was then boiled at 900C for one hour to halt enzymatic activity. The resultant sample (also called mash) was then cooled to 450C, and the addition of distilled water made up the volume of each mash. The mash was then filtered into a measuring cylinder by the use of 24cm pleated filter paper placed in a funnel. 250 ml of the resultant liquid called wort was then added into 500 ml sterile conical flask. Inoculation of S. cerevisiae source: The yeast S. cerevisiae was used from the collection of the Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. Before measuring bioethanol production from different agricultural wastes, six S. cerevisiae strains such as SC-O, SC-B, SC-P, SC-G, SC-Pp, and SC-L were sub-cultured and checked for their viability and growth at a higher temperature. The strain, which showed fast and active growth at 37°C was selected for further study. Inoculum development for fermentation process. A loop-full of the yeast colony was transferred from the agar plate into 100ml of the 5% yeast extract peptone dextrose (YEPD) broth and incubated at 30°C on a shaker at 120 rpm for 24 hrs. 7ml of the broth was centrifuged at 4500 rpm for 5min. The supernatant was decanted, and the pellet was re-suspended in 10ml of sterile distilled water, centrifuged and the supernatant decanted. The pellet was re-suspended in 1/10th of 50ml citrate buffer of working solution for each flask and was used as its inoculums. This process was performed in a centrifuge tube to obtain pure S. cerevisiae yeast. Determination of the effect of pH, temperature and incubation time on bioethanol production. To find out the optimum pH for bioethanol production, substrates were fermented in the fermentation broth having different pH values from 3.7, 3.9, 4.0, and 4.6 while the temperature and incubation period were kept constant at 400C and 96 hours, respectively. Samples were fermented at different temperatures condition, i.e., 370C, 400C and 450C having constant pH and incubation time (pH 4 and 96 hours) to determine the optimum temperature. Furthermore, samples were incubated for the different incubation periods of 72, 96, and 120 hours at pH 4.0 and temperature 400C to find the optimum incubation time for bioethanol production. Calculation for bioethanol concentration: The following formula was used for calculation of bioethanol yield: Bioethanol conc. (%) = [(Original gravity – Final gravity)/100] * 131. Here, 131= A mandated specific factor to calculate bioethanol concentration (Spedding et al., 2016). Statistical analysis: All analyses were carried out in three replicates, and statistical analysis was performed by one-way ANOVA followed by the posthoc test using the Statistical Package for Social Sciences (SPSS, 2007, version 16.0). All results were presented as mean ± SD, and P-values <0.05 were considered significant.
J Bangladesh Agril Univ 18(4): 975–981, 2020 ISSN 1810-3030 (Print) 2408-8684 (Online)
Journal