The use of cellulase adsorption and recycle as a means of assessing and enhancing the hydrolysis of cellulosic substrates

In the past, two main approaches have been used to try to enhance the enzymatic hydrolysis step of a lignocellulosics residue-to-ethanol process. In one approach, cellulosic substrates can be pretreated to increase their susceptibility to cellulases. In an alternative approach, the cellulases are recovered and re-used in order to lower the cost of the enzymes required for the hydrolysis step. The increased susceptibility of pretreated substrates has been attributed to an increase in the surface area accessible to cellulases, as the enzymes must first adsorb to the substrate in order to carry out hydrolysis. However, it has not been clearly demonstrated that the amount of cellulase adsorbed to different substrates is the primary factor determining the hydrolysis rate. In the first part of this study, CBDcex, the nonhydrolytic cellulose-binding domain of a Cellulomonas fimi cellulase, was evaluated as a probe to measure the surface area available for cellulase adsorption. The adsorption isotherms of CBDcex and Celluclast, a commercial cellulase mixture, were determined using various cellulosic substrates. It was found that the adsorption of both protein preparations could be represented by the Langmuir isotherm. Pmax. t ne maximal adsorbed protein, was calculated in order to compare the amount of surface area in each substrate that was available to CBDcex or Celluclast. The surface areas of nine different substrates, as measured by CBDcex and Celluclast adsorption, were found to be different in all cases except one. In the second part of this study, the relationship between cellulase adsorption and the initial hydrolysis rate was examined. The accessibility of the various substrates to cellulases and the corresponding hydrolysis rates were measured. There was no apparent correlation between the amount of enzyme adsorbed and the initial hydrolysis rate. Specific hydrolysis rates were also found to differ among the various substrates. It was apparent that both accessibility to the cellulases and the specific hydrolysis rates were changed by chemical and physical pretreatment of the substrate. The third part of this study focussed on cellulase recycling as a means of reducing the amount of enzyme required for cellulose hydrolysis. Three cellulase recycling strategies were evaluated to determine their efficiencies after 5 rounds of hydrolysis. The cellulases were recovered from the residual substrate containing adsorbed enzymes, the reaction mixture consisting of both the residual substrate and the supernatant, and the reaction mixture consisting of the supernatant and the non-cellulosic residue obtained after complete hydrolysis of the cellulose in each substrate. The effect of lignin on recycling was assessed by using both steam-exploded birch (WB, 32% lignin) and delignified steam-exploded birch (PB, 4% lignin) as the substrates. The activity of the recovered enzymes was assessed by measuring the amount of reducing sugars obtained after each hydrolysis round. The only strategy that resulted in the complete recovery of all of the cellulase activity for 4 hydrolysis rounds was when the cellulases from the supernatant and the non-cellulosic residue were recycled together after complete hydrolysis of the PB substrate. When either of the other two recycling strategies was used, the recovered cellulase activity decreased with each recycling step. Also, when these two recycling strategies were used, the recovered activities did not correspond to the activities expected from the amount of cellulase protein recovered during recycling. In all 3 recycling strategies studied, lower cellulase activity was recovered from the substrate with the higher lignin content (WB).Science, Faculty ofMicrobiology and Immunology, Department ofGraduat