Ang, Teck Nam (2013) Production of laccase enzyme using rice husk as substrate in fungal solid-state fermentation / Teck Nam, Ang. PhD thesis, University of Malaya.
Abstract
To produce laccase enzyme in large volume at affordable prices, a fungal solid-state fermentation (SSF) process that uses lignocellulosic by-product from rice milling industry – rice husk was introduced. This research aims to provide deeper insights into major aspects of fungal solid-state fermentation from the selection and pretreatment of solid substrate, selection of fungi to assessment and optimization of the process parameters. The research assesses all the mentioned aspects of solid-state fermentation for the production of laccase enzyme using the oyster mushroom Pleurotus sajor-caju. No pretreatment reagent has been reported effective for pretreating rice husk, the research thus reports on various reagents, which include sulphuric acid, hydrochloric acid, phosphoric acid, acetic acid, nitric acid, sodium hydroxide, calcium hydroxide, 1- butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium acetate and 1-ethyl- 3-methylimidazolium diethyl phosphate on the pretreatment of rice husk. Dilute hydrochloric acid pretreatment was found to be most effective for pretreating rice husk by showing desirable chemical and structural characteristics that are favourable for fungal solid-state fermentation. The dilute hydrochloric acid pretreatment was further optimized, and the result showing a relatively mild pretreatment condition of 0.5% (w/v) acid loading, 125oC, and 1.5 hours. The screening and selection of fungi with ligninolytic enzyme activity was conducted on three pre-selected fungi, namely Pycnoporus sanguineus, Phlebia radiata, and Pleurotus sajor-caju, and the selection was done using screening plates containing chromogens like guaiacol and Remazol brilliant blue-R (RBB-R). Despite all the fungi possessed guaiacol oxidation and RBB-R decolourization activities, P. sajor-caju outperformed the other fungi with activities shown at both room temperature and 35oC. Therefore, P. sajor-caju was selected for the subsequent fungal solid-state fermentation. Prior to solid-state fermentation, an improved fungal inoculum preparation method – cellophane film culture (CFC) technique was developed in this study to overcome the limitations of the existing fungal inoculum preparation methods. This method was verified and validated to be able to produce actively growing fungal inoculum and it is feasible to be used in solid-state fermentation. With the pretreated rice husk and P. sajor-caju inoculum prepared using cellophane film culture (CFC) technique, the fungal solid-state fermentation was optimized, and the optimal fermentation condition was at 84 hours fermentation, 0.5 mM copper sulphate, 10 g/L glucose, and 0.1 g/L Tween 80. Laccase production at the optimized fermentation condition fits well into the logistic model. The model is thus used to simulate the increasing phase of laccase enzyme production. The characterization of laccase enzyme shows that the enzyme has a molecular mass of 60 kDa, and it possesses the ability to degrade 2,2‟-azino-bis (3- ethylbenzothiazoline)-6-sulphonic acid (ABTS), 2,6-dimethoxyphenol (DMP) and guaiacol. The research findings demonstrated that the described solid-state fermentation process for laccase production shown comparatively well performance to reported studies. Besides minimizing the „waste‟ generated from rice crops, rice husk can be transformed into added-value enzyme that has useful applications in the industries. This signifies the potential of up-scaling the solid-state fermentation for the production of laccase.
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