Bioethanol Production by Saccharomyces Cerevisiae Using Lignocellulose Substrates Saccharified by Fungal Isolates from Karura Forest Reserve, Nairobi County, Kenya
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Date
2025-01
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Kenyatta University
Abstract
Lignocellulose from plant biomass is the most available organic compound in nature that can be used by the microbes for the cellulases production, reducing sugars and bioethanol. However, its recalcitrant to hydrolysis and use of lignocellulolytic enzymes to convert this plant biomass into fermentable sugars and for biofuel production is paramount. Push for clean environment and renewable energy is health concerns emanating from fossil fuels The main goal of the study was to to investigate bioethanol production by saccharomyces cerevisiae using lignocellulose substrates saccharified by fungal isolates from karura forest reserve, nairobi county, kenya. A total of 20 of fungal samples were obtained from Karura forest reserve growing on decaying biomass based on their morphological variation. Six fungi from collected sample were selected after screening using 1% CMC- congo red agar for cellulolytic activities based on zonal inhibition and the isolates selected for the study. Characterization by molecular technique (ITS1 and ITS4 primers) was used and biological sequences analyzed using BLAST algorim and MEGA II software to identify the six isolates, as Xylaria sp. km01, Nemania sp. km02, Xylaria sp. km03, Cyathus sp. KM04, Podoscypha bolleana km05 and Podoscypha petalodes km06. The phylogenetical analysis revealed divergence of isolated fungi and classified them as either ascomycota or basidiomycota. The six isolates were cultured for cellulases production and enzymes used for saccharification under SSF process. Data obtained were recorded in triplicates and statistically analyzed using one-way ANOVA at P ≤ 0.05 significant level on R software. Any significant differences in the factors affecting enzyme and ethanol production was determined by Tukey's HSD Post Hoc test. Effect of time of incubation was investigated and maize cobs substrate produced the highest cellulase activity. Xylaria sp.km01 recorded the maximum FPase activity at 16.7±0.34 IU/ml on the 9th day of incubation. Xylaria sp. km03 produced the highest exoglucanase activity at 8.32±0.23 IU/ml while P. petalodes km06 produced the highest endoglucanase activity at 28.7±1.2 IU/ml on the 6th day of incubation. Podosycpha boleana km05 produced the highest β-glucosidase activity at 6468±210 IU/g on the 12th day of incubation. Effect of pretreatment on substrates was also investigated and maize cobs produced the highest cellulase activity. Xylaria sp.km01 produced the highest FPase at 20.1±1.31 IU/ml, exoglucanase activity at 9.35±0.77 IU/ml and endoglucanase activity 35.8±1.19 IU/ml while P. boleana km05 produced the highest β-glucosidase activity at 5111±101 IU/g when pretreated with 0.1M HCl at 121oc for 15 min. Saccharification and Fermentation of maize cob substrate of low and high loading concentration (4%,8%,18% and 20%). Hydrolysis by cellulase of P. petalodes km06 recorded the maximum of 10.63±0.70 g/l reducing sugars with 8%, whereas cellulase of P. bolleana km05 and Saccharomyces cerevisiae produced higher bioethanol of 37.3±0.72 g/l with 20% during fermentation period at 72 hours and 96 hours respectively. These findings show that both cellulolytic enzymes and bioethanol can be produced from local microbes using agro waste and the technology harnessed for creating income. Maize cobs, therefore performed as the best substrate for cellulases and bioethanol production
Description
A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University, January 2025.
Supervisors
1. George Isanda Omwenga
2. Mathew Piero Ngugi