Thermophilic SSF (tSSF) allows for cellulose hydrolysis at a temperature optimal for cellulase enzymes without the interference of soluble sugars, which have a well-documented inhibitory effect [3–5]. However, despite the advantage of reduced enzyme loading , tSSF also shows a decrease in final conversion with increasing solids concentration. The trend has also been demonstrated in both SSF and hydrolysis systems, where it has been attributed to several causes, including product inhibition and enzyme inactivation. One of the potential causes of this heightened enzyme inactivation at the higher solids concentration is the concurrent presence of higher ethanol concentrations. Despite excess substrate remaining, hydrolysis stops at a lower conversion in the presence of higher ethanol concentrations. However, the predictions from the tSSF model  which take into account both inhibition and inactivation of cellulase activity by ethanol as measured by standard procedures [3–5, 8] cannot predict this loss of activity. The 32% conversion measured after 96 hours from the tSSF with an initial ethanol concentration of 32.17 g/L shows that inactivation of cellulase enzymes are occurring faster and to a greater extent than has previously been measured and thus predicted by the model. Based on this discrepancy between experimental and model results from the added ethanol tSSF experiments our understanding embodied in the former model is incomplete.
The initial assessment of cellulase stability as a function of ethanol concentration was carried out by initial rate measurements in aerobic conditions. Since these correlations did not predict the extent of inactivation measured from tSSF directly, the conditions specific to tSSF were further evaluated. The effect of the reducing conditions was assessed by comparing hydrolysis in spent medium under a nitrogen headspace to an aerated control. Prior controls had shown no difference between anaerobic conditions setup with uninoculated, anaerobic medium versus spent medium, thus ruling proteases or other T. saccharolyticum enzymes as the source of this reduced activity. Subsequent reactions were performed in spent medium to best mimic tSSF conditions. Regardless of incubation time and exposure to anaerobic conditions, by transferring the reaction to a headspace filled with air faster hydrolysis was achieved, thus reactivating the enzymes. Given longer incubation periods, we predict that all aerobic samples would reach the same total glucose production, indicative that there is a given fraction of the substrate for which this activity is vital.
The inhibitory effect of a nitrogen environment was confirmed in the aerated tSSF experiment. In contrast to the spent medium used in the hydrolysis experiments, the aerated tSSF samples have actively growing cells and successively higher ethanol concentrations. The data further show that the inhibitory effect of the reduced environment and nitrogen headspace is a reversible phenomenon and that it limits further hydrolysis.
The commercial cellulase mixture used these experiments is derived from T. reesei, an aerobic fungus. Cellobiohydrolase I, the most abundant protein produced by T. reesei, contains 12 disulfide bonds [18, 19]. We hypothesized that the reduced state of the medium achieved by fermentation with T. saccharolyticum leads to the reduction of these disulfide bonds. Once the disulfide bonds are reduced to sulfhydryls, the protein is less stable, thus escalating the effects of ethanol, a denaturant. A difference in hydrolysis between oxygen and nitrogen environments was demonstrated several decades ago by Eriksson and co-workers . Using culture supernatants from several cellulolytic species, an increase in hydrolysis was measured in an aerobic environment. In the case of Trichoderma viride, they reported a 2-fold increase in hydrolysis under an oxygen containing environment. Rather than an effect on the disulfide bonds in the proteins hypothesized above, their work indicated the activity of an oxidizing enzyme. This enzyme was thought to use an oxidative mechanism to promote swelling of the crystalline cellulose by breaking hydrogen bonds. Since this early work, several oxidative enzymes including cellobiose quinine oxidoreductase, lactonase and cellobiose oxidase have been described. In addition, several wood degrading fungi utilize an oxidative enzyme, cellobiose dehydrogenase (CDH) [21, 22]. However, while the presence of CDH activity has been reported for T. reesei by Dekker , it was later questioned by Henriksson and co-workers . At present the presence of CDH activity in commercial T. reesei cellulase preparations remains to be definitively demonstrated.
Another redox-active class of enzymes, GH61s, has recently received much publicity and their mechanism and function are still under investigation. Preliminary results indicated that GH61 enzymes, like several of the enzymes described above may utilize an oxidative mechanism to cleave cellulose . Novozymes reported that GH61 enzymes increase the enzyme efficiency on pretreated substrates, but do not do so on Avicel . Further work determined that this discrepancy was due to the absence of redox-active co-factors which were present in the pretreated biomass. When the soluble fraction of dilute acid pretreated biomass was added to pure cellulosic substrates, an increase in cellulose degradation was observed . Thus, the reactivation of enzyme activity upon exposure to air, and thus a higher redox state, led us to investigate the potential role of GH61 enzymes in our system. Since copper is also necessary for GH61 stimulation [17, 25, 26], the addition of EDTA is a suggestive, though not definitive, means to test for GH61 activity. As shown in Figure 5, in our system the addition of EDTA prevented the reactivation of cellulase activity upon aeration. Since EDTA chelates metal ions, this result likely indicates that GH61 enzymes or the cellulase components they interact with are likely being inactivated in the anaerobic, reducing conditions of tSSF, though further work is necessary to confirm this hypothesis. In addition, the previously demonstrated ethanol inhibition, which the model could not predict, may also be a result of GH61 dependent inactivation.
Overall, while the precise mechanism underlying the loss of cellulase activity under nitrogen conditions is not known, the increase in activity upon exposure to air suggests that a redox dependent change is occurring. Whether the enzyme itself is altered due to the low redox state and/or hydrolysis itself utilizes an oxidative mechanism and thus does not function in a reduced environment, is still unknown. The opportunity to achieve higher conversion makes this phenomenon important to pursue.