Feasibility of Bioethanol Waste Stream for Production of Laccase in Pichia pastoris

Farah Wahida, Mamat (2022) Feasibility of Bioethanol Waste Stream for Production of Laccase in Pichia pastoris. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Laccases are multipurpose enzymes that have wide biotechnological applications. The present study seeks to evaluate the feasibility of spent fermentation broth generated upon the bioethanol production from sago hampas, as a feedstock for production of laccases in recombinant Pichia pastoris GS115. Characterisation of the sago bioethanol liquid waste (SBLW) indicated glycerol as the main component, along with glucose and lactic acid. Evaluation of SBLW as a fermentation feedstock for laccase production in P. pastoris GS115 showed that the cell growth was generally feasible when SBLW was used as a feedstock. The activity of laccases reached the highest (0.00076 U mL-1 ± 3.5 × 10-5) in fermentations that employed 40% (v/v) SBLW. This represented 73% of that obtained using the standard synthetic medium. Supplementation of 40% (v/v) SBLW with 1.0% (w/v) yeast extract (YE) yielded enhancements of 1.2-fold and 1.5-fold of biomass concentration and laccase activity, respectively. The expression of laccases was further enhanced when the 40% (v/v) SBLW and 1.0% (w/v) YE was supplemented with 2.0% (v/v) glycerol. The highest laccase activity recorded was 0.00206 U mL-1 ± 5.8 × 10-5 . Both of the biomass and laccase production were increased by 1.9-fold and 2.1-fold in comparison to that achieved by the standard synthetic medium. The efficacy of laccases produced by the optimised SBLW medium was evaluated in terms of the capability of the enzymes to decolourise Remazol Brilliant Blue R (RBBR) dye. The enzyme showed a decolourisation percentage of 68.6%, which represented 91% of the decolourising capability of laccases produced using standard BMMH medium. This indicates the promising efficacy of laccases produced from SBLW. In summary, this work gives an important insight into exploitation of SBLW for production of value-added products. Moreover, this work contributes to the development of recombinant laccase production using low cost and eco-friendly feedstock.

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