The enzymatic hydrolysis of pretreated pulp fibers predominantly involves "peeling/erosion" modes of action

Abstract

Background: There is still considerable debate regarding the actual mechanism by which a "cellulase mixture" deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their "peeling/delamination/erosion," or if both cutting and peeling mechanisms occurred simultaneously.

Results: Regardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis.

Conclusion: It appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action involving an initial rapid fragmentation followed by simultaneous swelling and peeling/erosion of the fragmented fibers. This latter mechanism is the predominant mode of action involved in effectively hydrolyzing the cellulose present in pretreated wood substrates.

Keywords: Biomass deconstruction; Cellulose hydrolysis; Mechanism of enzymatic deconstruction.

Figure 1

Time-course hydrolysis of size-fractionated steam and organosolv pretreated pulp fibers. Cellic CTec2 loading was 15 mg protein/g glucan unless labeled otherwise.

Figure 2

Changes in the average fiber length of the size-fractionated steam and organosolv pulp fibers during the course of enzymatic hydrolysis.

Figure 3

Relative changes in the population of short (100 to 200 μm) fibers over the course of hydrolysis of size-fractionated steam and organosolv pretreated pulp fibers relative to unhydrolyzed pulp fibers.

Figure 4

Changes in the average fiber width of pretreated woody biomass over the course of hydrolysis of size-fractionated steam and organosolv pretreated pulp fibers. The height of the bars represents the range/variability of the average fiber width in the sample (95% confidence interval).

Figure 5

The influence of shaking and glucose on fiber fragmentation and changes in fiber width during hydrolysis of size-fractionated pulp fibers SPP200 R20 at 15 mg/g glucan for 6 h at 2% solids loading (w/w). Left) Decrease in the extent of fiber fragmentation; right) change in the average fiber width represented as changes in the range/variability of the average fiber width in the sample (95% confidence interval). Blue bar: control sample (only substrate); green bar: 15 mg CTec2/g glucan for 6 h.