Enzymatic hydrolysis of lignocellulose : cellulase enzyme adsorption and recycle

Producing ethanol from the bioconversion of lignocellulosic substrates is one of the most promising technologies to decrease fossil fuel utilization. However, the current economics of the bioconversion process prohibit its commercialization due to the high cost of cellulase enzymes. One potential means to decrease enzyme costs is to recycle enzymes during the bioconversion process. The initial work focused on comparing the distribution of cellulases among the solid and liquid phases after a typical enzymatic hydrolysis of Avicel and an organosolv pretreated Douglas fir substrate. It was shown that 50% of the applied cellulases desorbed into the liquid phase after the hydrolysis of an ethanol pretreated D. fir substrate compared to 76% in the case of Avicel. By exploiting the natural affinity of cellulases for cellulosic substrates, the free enzymes were recovered via readsorption onto fresh substrates. Using this approach, 85% of the free enzymes could be recovered, compared to an 82% recovery predicted by the Langmuir isotherm model. A novel recycling strategy for recovering both the free and bound enzymes was developed where Tween 80 was added at the beginning of the hydrolysis, followed by the addition of fresh substrate to recover free enzymes after hydrolysis. The cellulases from T. reesei ( Hypocrea jecorina ) could be recycled for four consecutive rounds of hydrolysis of an ethanol pretreated (EPLP) substrate with the addition of 0.2% Tween 80, compared to one round with a steam exploded (SELP) substrate, presumably due to the higher lignin content of the SELP substrate. Comparing isolated lignin preparations from SELP and EPLP, it was shown that CEL-SELP lignin exhibited a greater capacity to bind cellulases than CEL-EPLP lignin. A reduction in the adsorption of cellulases to lignin was achieved by the addition of Tween 80. The recycling of β-glucosidase was achieved by immobilization on an inert carrier, Eupergit C. The immobilized β-glucosidase exhibited improved operational stability and an increase in the apparent K[sub m] and V[sub max]. Overall, the results demonstrated that enzyme recycling using a combination of surfactants, readsorption onto substrates and enzyme immobilization could potentially decrease enzyme costs in the hydrolysis of softwoods during the bioconversion process.Forestry, Faculty ofGraduat