*2.5. Analytical Enzymatic Saccharification of Pretreated Solids*

The enzymatic digestibility of pretreated solids and the inhibitory effect of the pretreatment liquids on the cellulolytic enzymes were evaluated following an analytical enzymatic saccharification procedure [15]. In one set of assays, aliquots of 250 mg (DW) of pretreated solids or raw wheat straw were suspended in 50 mM citrate buffer (pH 5.2) in 15-mL Falcon tubes, with a total reaction mixture of 5 mL. In another set of assays, the pretreated solids were instead suspended in pretreatment liquids, whose pH had previously been adjusted to pH 5.2. No citrate buffer was included in the assays with pretreatment liquids as carboxylic acids present in the pretreatment liquid make the addition of buffer unnecessary. In all assays, cellulolytic enzymes (Cellic CTec2 enzyme blend, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were added so that the loading was 100 CMCase units/g biomass. The reaction mixtures were incubated at 50 ◦C and 180 rpm for 72 h in an Ecotron orbital incubator (INFORS HT, Bottmingen, Switzerland). After 72 h, the tubes were centrifuged, and the supernatants were collected for further analysis using HPLC. Enzymatic digestibility was determined using the following equation:

$$\text{Enzyme digestibility} \left( \text{\textquotedblleft} \right) = \frac{\text{G}\_{H}}{\text{G}\_{\text{APS}}} \times 100 \tag{7}$$

Here, *G<sup>H</sup>* is the amount of hydrolyzed glucan based on the glucose concentration in the enzymatic hydrolysate, and *GAPS* is the amount of glucan in pretreated solids.

The degree of inhibition of enzymatic saccharification of glucan by the pretreatment liquids was determined using Equation (8).

$$\text{Degree of inhibition (\%)} = \frac{\left(ED\_{buf} - ED\_{PL}\right)}{ED\_{buf}} \times 100\tag{8}$$

Here, *EDbu f* is the enzymatic digestibility of glucan (%) resulting from enzymatic hydrolysis of pretreated solids suspended in sodium citrate buffer, and *EDPL* is the enzymatic digestibility of glucan (%) resulting from enzymatic hydrolysis of pretreated solids suspended in pretreatment liquid.
