Production of Cellulolytic Enzymes by Chaetomium Globosum, Hypocrea Lexii and Gibberella Intermedia for Biomass Saccharification and Ethanol Production
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Date
2024-10
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Kenyatta University
Abstract
The world is staring at an energy crisis because of increase in population and utilization of non-renewable energy resources; petroleum and coal. Availability of these nonrenewable resources is abundant presently but will be exhausted in a limited period of time. Renewable energy resource provides an opportunity to serve as a substitute to the ever diminishing non-renewable energy resources, in this regard lignocellulosic plant biomass as an abundant renewable resource generated from plants and algae can be utilized. Exploration of plant biomass as a renewable energy resource not only allows for the conservation of non-renewable energy resources, but also for environmental and biodiversity protection. This study was aimed at isolation and identification of cellulase producing fungi from a decaying tree trunk, determining the effect of incubation time, moisture content level and initial medium pH levels on cellulase production using untreated maize cobs, rice husks and sugarcane bagasse as substrates under solid state fermentation thereafter, saccharifized untreated maize cobs, eucalyptus and sugarcane bagasse for ethanol production. Samples of wood were collected from Ngong forest, Nairobi County (1°19'13"S 36°44'54"E) and screened for isolation of cellulase producing fungi on carboxymethylcellulose agar stained in 1% Congo red. The isolated fungi were further grown to obtain pure cultures on Potato dextrose agar before DNA extraction and amplification by Polymerase Chain Reaction (PCR) was carried out to identify the isolated fungi. The PCR products were sent to Macrogen, Netherlands where sequencing was done by Sanger dideoxy sequencing technique. The isolated fungi were cultured on the untreated substrates in glass flasks for cellulase enzyme production in solid state fermentation. The crude cellulase obtained after fermentation was used to carry out cellulase assays; filter paper assay, exoglucanase assay and endoglucanase assay. Molecular data analysis was carried out using the National Center for Biotechnology Information (NCBI) and Basic Local Alignment Search Tool (BLAST) algorithm and Molecular Evolutionary Genetics Analysis (MEGA X version 10) software to identify the isolated fungi, while enzyme activity analysis was done using one-way Analysis of Variance (ANOVA) on R software at P≤0.05 significance level and the significant differences were determined by Tukeys Post Hoc test. The isolated fungi were identified as Chaetomium globosum, Hypocrea lexii and Gibberella intermedia. For effect of time of incubation on cellulase production on the three untreated substrates, the three fungi expressed high enzyme production on different days within the incubation period, highest cellulase enzyme activity was recorded at moisture content ratio of 1:2 and initial medium pH of 5. The three untreated plant biomass; maize cobs, eucalyptus and sugarcane bagasse were hydrolyzed with the crude cellulases produced from the three fungi, the saccharification studies showed that 7% (v/v) enzyme concentration, 12% (v/w) substrate concentration and a hydrolysis time of 72 hours were optimal for maximum yield of reducing sugars by the Dinitro salicylic acid method. The total reducing sugar produced maximum bioethanol at 72hours when Saccharomyces cerevisiae was used as a fermentation agent. Consequently, bioethanol was successfully produced from the cellulose of the three substrates using cellulases produced by the three fungi. Therefore, cellulase enzymes can be produced from microorganism found locally and by using readily available agricultural waste.
Description
A Thesis Submitted in Partial Fulfillment of the Requirement for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University, October 2024
Supervisor:
1.George Isanda Omwenga
2.Fredrick Mjomba Mwamburi