Enzyme adsorption-desorption and evaluation of various cellulase recycling strategies for steam pre-treated Eucalyptus enzymatic degradation

Abstract

One of the biggest challenges hindering the successful commercialisation of biofuels is the high dosages of enzymes still required to hydrolyse lignocellulose into fermentable sugars. Enzyme recycling has been recognised as a promising approach to alleviate this problem by reducing enzyme costs and improving the profitability of the bioconversion process. However, developing an effective recycling system is a challenging process and requires an in-depth understanding of enzyme-substrate interactions. In this context, this study investigated the adsorption, desorption and re-adsorption of a cellulase mixture (CelMix) to various lignocellulosic components to gain insights into enzyme-substrate interactions, in order to develop an effective enzyme recycling strategy. Lignin-rich residues exhibited a higher adsorption capacity for CelMix (~ 38% adsorption) compared to Avicel cellulose (15.6% adsorption). The recovery of CelMix from lignin, steam pre-treated Eucalyptus and cellulose by various chemicals and alkaline washing was examined. Alkaline washing with Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption (90.7% activity) and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. Using this information, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pre-treated Eucalyptus was evaluated by two different approaches: (i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1-RA1) and (ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2—RA2). The recycling performance of RA1 and RA2 reached > 95% of the initial sugar liberation for three continuous rounds, whilst successful reduction in enzyme loading by 50% and 40% for RA1 and RA2, respectively, was achieved. Therefore, this study shows that enzyme recycling presents a simple and effective pathway for increasing enzyme productivity, improving the economic feasibility of fermentable sugar production for biofuel.