As has been demonstrated previously with pretreated substrates [30], the rates and extents of hydrolysis of pulp fibers have also been directly corre­lated to their initial specific surface area. This is not surprising, since the very existence of a substrate pretreatment step stems from the necessity to increase the accessibility of reaction sites on substrates to cellulases, as lig — nocellulosic substrates such as wood possess limited reactive surface area available to cellulases prior to pretreatment. Coincidentally, both the chem­ical and mechanical pulping processes that have been applied to produce pulp for the formation of paper also result in an increase in accessibility to cellulases compared to the starting lignocellulosic furnish. During pulping, wood chips are subjected to either physical or physiochemical action, re­sulting in the breakdown of the lignocellulosic matrix into fiber cells [48]. Consequently, the breakdown of the wood yields fibers with various phys­ical attributes such as length, coarseness, width, kink and curl [102,103]. Surface area of pulp fibers can be divided into exterior surface area affected mainly by fiber length and width, or interior surface area, which is gov­erned by the size of the lumen and the number of fiber pores and cracks. The varying fiber lengths and widths produced during pulping can be viewed in a similar manner as the array of particle sizes produced during the pre­treatment oflignocellulosic substrates for bioconversion. The specific surface area of a mixture of particles is inversely proportional to their average diam­eter, therefore, a smaller average particle size results in an increase in surface area. Indeed, cellulases have been shown to act on the surface of pulp fibers, resulting in a “peeling effect” [104]. Therefore, smaller particle sizes with a greater amount of specific surface area would be expected to hydrolyze at a faster rate.

In an investigation assessing the hydrolysis of Douglas-fir Kraft and me­chanical pulps, Mooney et al. showed that at equal lignin contents, the “fines” (small particles) of a delignified mechanical pulp were hydrolyzed faster than the longer fibers (large particles) of the Kraft pulp [17]. When each fiber length fraction was hydrolyzed separately, it was shown that the isolated long fiber fraction hydrolyzed slower and consequently adsorbed a lower amount of cellulases than the whole pulp [17]. The increased hydrolysis rate of the whole pulp was attributed to the greater amount of specific surface available for the adsorption of cellulases provided by the pulp fines and short fibers. Although it is apparent that particle size has a significant effect on cellulose hydrolyzability, it has also been shown that the fiber delamination and en­hanced swelling that results from mechanical treatment of Kraft and recycled pulp fibers has a greater effect on hydrolysis by cellulases than does a de­crease in particle size [105]. Since recycled pulps originate from fiber sources that undergo irreversible changes in their structure upon drying [106], their swelling properties must be regenerated by employing a mechanical treat­ment referred to as “refining” or “beating”. The swelling properties of pulps can be measured using the water retention value measurement [107]. After beating, the pulp sample usually drains at an inadequate rate to be used on a high-speed paper machine. Consequently, cellulases have been shown to improve the drainage of recycled pulps. Oksanen et al. [108] applied sepa­rate EG1, EG2 and CBH1 cellulase components to pulps during each recycling round. As each pulp was beaten after recycling, the water retention value (WRV) increased and the pulp became more responsive to cellulases, espe­cially EG1 and EG2. Although the particle size and swelling properties of pulps have been shown to be related to the ease of hydrolysis of lignocellulosic substrates, it has been shown that a greater amount of information related to the action of cellulases can be obtained from measurements of the pores or “interior” surface area of pulp fibers available for penetration by cellulases.

Direct correlations have been found between the initial pore volume or interior surface area of lignocellulosic substrates and their extent of hydro­lysis [30,83,90]. It has been proposed that the efficacy of cellulose hydrolysis is enhanced when the pores of the substrate are large enough to accommo­date both large and small enzyme components to maintain the synergistic action of the cellulase enzyme system [11]. From extensive studies, Grethlein et al. [83] and others [109-111] have found that the rate-limiting pore size for the hydrolysis of lignocellulosic substrates was 5.1 nm, thus the solute exclu­sion technique utilizing molecular probes in this size range has been shown to be effective for assessing the pore volume of substrates. Mooney et al. [68] utilized dextran molecular probes in the solute exclusion method to measure the pore volume of refiner mechanical pulp (RMP), sulfonated RMP, sodium chlorite delignified RMP and Kraft pulp from Douglas-fir to assess the ease of these pulps to subsequent hydrolysis by cellulases. As mentioned earlier, the delignification of the RMP resulted in a greater rate and extent of hydrolysis than the Kraft pulp sample, which may be attributed to the smaller particle size of the RMP. The sulfonation of the RMP dramatically increased swelling. Unlike delignification, however, this did not translate into either enhanced access to the 5.1 nm probe or hydrolysis performance. The most feasible ex­planation for these results is that the lignin content of the sulfonated pulp (30.9%) inhibited hydrolysis, regardless of the greater swelling of the pores, thus demonstrating the detrimental effect of substrate lignin on hydrolysis as mentioned earlier.

Since it is well known that the pore volumes of pulps undergo significant reductions upon drying [106], Esteghlalian et al. [112] innovatively applied the Simons’ stain technique to measure changes in pore volume imparted by air, oven and freeze drying prior to enzymatic hydrolysis. As expected, drying significantly reduced the number of larger pores in the pulp sample, which most likely restricted the access of the fibers to cellulases and thus de­creased hydrolysis rate over 12 h [112]. Although the specific surface area of the substrate provided by decreased particle size and increased swelling and pore volume plays a significant role in facilitating hydrolysis by cellulases, the interconnecting role of other substrate factors such as crystallinity and DP should also be considered.

5.2