By selectively choosing pretreatment conditions it should be possible to cre­ate substrates with characteristics (hemicellulose and lignin content, particle size, available surface area etc.) that greatly enhance subsequent enzyme — mediated hydrolysis. For example we can adjust the time, temperature and SO2 concentration of steam pretreatment with consequential effects on over­all product recovery, ease of hydrolysis, etc. In addition to adjusting pretreat­ment parameters, it has been shown that the inherent physical properties of the biomass can be used to anticipate the performance of the pretreated sub­strate in subsequent hydrolysis experiments [45-47]. The particle size, purity, moisture content, and the internal variations in the biomass feedstock, such as the presence of compression or tension wood can all have significant ef­fects on the efficiency of pretreatment. As mentioned earlier, a reduction in the substrate particle size prior to pretreatment is a common practice used in some pretreatment processes [22]. However, size reduction through milling or grinding requires a substantial input of energy, and adds significantly to the total cost of the pretreatment [47]. Therefore, in the case of woody biomass, it is desirable to utilize a biomass particle size that can be produced economically at a large scale with existing equipment, such as the wood chips used in the pulp and paper industry [48].

For the operation of a large-scale bioconversion process a suitable wood chip size should be selected based on a compromise between the energy/cost of producing the chips and the subsequent effectiveness of the pretreatment and product recovery. Some of our previous work has shown that chip size and moisture content have a profound effect on the ease of hydrolyzability of the resulting substrate [46]. For example, by increasing the chip size of Douglas-fir (Pseudotsuga menziesii (Mirb.)) from 0.42 mm2 to 5 cm2 prior to SO2-catalyzed steam pretreatment, greater recovery of the solids and a re­duction in the production of inhibitors could be observed. This could be attributed to a decrease in the “relative severity” of pretreatment undergone by the larger chip at equivalent pretreatment conditions. It was also shown that the lignin present in the larger chips (5 x 5 cm) experienced less con­densation and was therefore more amenable to subsequent alkaline peroxide delignification. This material from the larger chips consequently exhibited a 10-15% increase in hydrolysis yield over that obtained with the mate­rial originating from the smaller chips. Similarly, by increasing the moisture content of the chips from 12 to 30% an improved recovery of glucose and hemicellulose-derived sugars could be achieved. The increased recovery of sugars by raising the moisture content of the chips could be explained by a similar mechanism as it was observed with the increase in chip size. This was due to the additional moisture adsorbing the heat applied during steam pretreatment, resulting in a decrease in the severity of the treatment under­gone by the chips.

In addition to the inherent variations in the properties of incoming biomass, pretreatment schemes must also deal with the fluctuating “purity” of the lignocellulosic substrate, as woody biomass can be expected to contain “con­taminants” such as bark, needles, leaves, branches, etc, that differ significantly in their chemical composition from “white wood” [49]. It is likely that future wood-based bioconversion facilities would involve large amounts of biomass being sent to a chipping unit without careful control of debarking or branch removal. Similarly, it is unlikely that higher-value wood chips will be used extensively in commercial bioconversion facilities due to competition from traditional pulp and paper mills. Therefore a bioethanol facility’s feedstocks are likely to be either coppiced whole plants such as willow or, in the short term, residues from saw and pulp mills such as sawdust, shavings and hogfuel that contain significant amounts of bark, ash and lipophilic extractives. Tree thinnings such as branches have been pretreated using dilute acid during inves­tigations assessing their viability as a biomass feedstock for bioconversion [50], where a two-stage pretreatment was required to hydrolyze 50% of the cellulose while the remaining cellulose was readily hydrolyzed by cellulases.

In similar work, utilizing feedstocks with high bark content, the liquid stream from the steam pretreatment (SP) of a Douglas-fir chip furnish con­taining 30% bark (SP-DFB) was shown to contain lower amounts of total sugars, furfural and 5-hydroxymethylfurfural compared to the liquid stream (prehydrolyzate) isolated from the pure whitewood (SP-DF) [51]. Although the concentration of lipophilic extractives increased in the bark-laden water — soluble stream, subsequent fermentation by Saccharomyces cerevisiae re­sulted in a complete utilization of the hexose sugars within 48 h with compa­rable ethanol yields regardless of whether bark was present or not. Although these results looked encouraging, with regard to the “robustness” of the over­all SP process, the solid fraction of the pretreated Douglas-fir containing bark showed significant differences when compared to the whitewood. The addition of 30% bark to a Douglas-fir chip furnish prior to SP resulted in a significant increase in the lignin detected in the water-insoluble fraction. The SP-DFB solids fraction also contained 56% lignin compared to only 44% lignin in the case of the pure whitewood (SP-DF) sample. Consequently, the lignin content decreased to only 18% for the SP-DFB fraction as compared to 9% in the case of SP-DF upon subsequent alkaline peroxide delignifica — tion [52]. Although the alkaline peroxide delignified SP-DFB had a higher lignin and phenolic extractive content, it resulted in a similar hydrolysis yield to the SP-DF fraction, most likely due to the removal of surface lignin dur­ing the alkaline peroxide delignification stage [53]. Although hydrolysis was not performed in the absence of the delignification step, it can be expected that, due to its higher lignin content, the SP-DFB fraction would be more re­sistant to hydrolysis than was the SP-DF substrate. It is apparent that, during SP of lignocellulosic feedstocks such as Douglas-fir, the inherent properties of the wood have a significant effect on the downstream partitioning of the cellulose, hemicellulose and lignin components.

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