Fermentation of off-cut waste paper hydrolysed with cellulase produced by a bacterium isolated from decaying sawdust
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Date
2015
Authors
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Publisher
Microbiology, Obafemi Awolowo University
Abstract
This study investigated fermentation of off-cut waste paper hydrolysed with cellulase enzyme produced by a bacterium isolated from decaying sawdust; isolated a cellulolytic bacterium; identified and optimized conditions for cellulase production by the isolate. This was with a view to hydrolysing off-cut waste paper with the cellulase enzyme obtained for bioethanol production.
Samples of decaying sawdust were collected from a saw mill and were serially diluted. Aliquot of each dilution was appropriately plated and incubated. The bacterial isolates obtained from the plates were screened for cellulolytic ability on carboxymethylcellulose agar plates. The bacterial isolate with the highest cellulolytic activity was selected and identified by 16S rRNA gene sequencing. Conditions for cellulase production were optimized and cellulase was produced by submerged fermentation under the optimum conditions recorded. Partial purification of cellulase was achieved by ion exchange and gel filtration chromatography. The kinetics and other physicochemical parameters of cellulase were determined. Off-cut waste paper was soaked in distilled water to form a mash which was pretreated with alkaline, alkaline-peroxide and dilute acid pretreatment methods. The pretreated mashes were hydrolysed with both crude and purified cellulase, alongside untreated mash for 48 h and the reducing sugars released were monitored. The pH of the enzyme-hydrolysed mashes was adjusted to 5.5, sterilized, inoculated with standardized broth culture of 24 h old Saccharomyces cerevisiae and incubated at 30oC for 48 h. The mixture was analysed for alcohol production before and after distillation using acidified-dichromate method.
Twenty-six (26) bacterial isolates were obtained. Seven (7) of the isolates (26.92%) tested positive for cellulose hydrolysis with appreciable halozones. The best bacterial isolate with a halozone of 58 mm and an initial cellulase activity of 29.36 Units/ml was identified as Bacillus subtilis A8. Optimum conditions for cellulase production by Bacillus subtilis A8 were determined to be a temperature of 35 ± 2oC, pH 7, 0.8% substrate concentration, 4% inoculum size, carboxymethylcellulose and peptone as the more preferred carbon and nitrogen source respectively. Enzyme yields of 18.5 and 13.5% were obtained from purification by ion exchange and gel filtration chromatography respectively. The Michealis-Menten (km) was found to be 0.0108 ± 0.0032 mg/ml with a maximum velocity (Vmax) of 119.3 ± 7.4 µmol/min. Maximum activity for partially purified cellulase was recorded at pH 9.5 and 55oC with stability at 50oC; and pH 9, 35oC with stability at 45oC for crude cellulase. Hydrolysis of dilute acid pretreated waste paper with crude cellulase gave the highest quantity of reducing sugars at the 48th hour, and also gave the highest ethanol yield upon fermentation and distillation.
The study concluded that cellulase from Bacillus subtilis A8 could be utilized in the hydrolysis of waste paper to generate reducing sugars which can be fermented to bioethanol. Also, pretreatment of off-cut waste paper produced more ethanol.
Description
xvii,82
Keywords
Bacterium Isolated, Decaying Sawdust, Alcohol Production, Fermentation, Sawdust, Cellulase, Cellulase enzyme
Citation
Agbolade, A.E. (2015). Fermentation of off-cut waste paper hydrolysed with cellulase produced by a bacterium isolated from decaying sawdust. Obafemi Awolowo University