Isolation and Identification of Cellulase-Producing Bacteria from Landfill Soil

Khoo, Er Zhao (2019) Isolation and Identification of Cellulase-Producing Bacteria from Landfill Soil. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Cellulase (EC 3.2.1.4) catalyses hydrolysis of cellulose. Besides, it is also able to convert biomass into biofuels. Hence, researchers are interested in this enzyme since fossil fuels are running out and biofuels are one of the substitutions. Cellulase is mainly produced by microbes such as bacteria, fungi and protozoans. However, researchers are more interested in celluloytic bacteria since bacteria has higher growth rate which leads to higher yield and more complex enzymes capable of adapting to extreme environment. Celluloytic bacteria can be found in landfill, feces of herbivores and even in hot springs. The main objective of this project was to improve the production of cellulase enzyme by screening and identifying new types of celluloytic bacteria from landfill soil. The reasons for choosing landfill soil as source are because (i) there is a lot of cellulosic materials in the soil and (ii) it is easy to obtain the soil. Another objective of the project was to investigate the effect of factors such as temperature, pH and substrate concentration on enzyme activity. The bacteria from the soil were isolated using serial dilution and spread plate technique. Congo red screening method was used to identify celluloytic bacteria by observing the presence of clear zone around the bacteria. DNS enzyme assays were used to determine enzyme activity. The enzyme activity of crude and partial purified enzyme were 0.0204 and 0.0043 umol/min/mL respectively. The specific enzyme activity of crude and partial purified enzyme were 0.0102 and 0.0905 umol/min/mg respectively. The optimum temperature and pH for enzyme was 55 OC and pH10 respectively. Kinetic studies showed that the enzyme had a Vmax of 0.0345 umol/min/mL and Km of 0.632 mg/mL. The crude enzyme showed bands of 97.2, 66.4, 55.6 and 42.7 KDa. Genome DNA isolation, PCR, gel electrophoresis and DNA sequencing were carried out to identify the isolated bacteria. Blast analysis of the DNA sequence showed the isolated bacteria to be Aeromonas caviae.