*4.3. Effects of Pretreatment Conditions on the Formation of Bioconversion Inhibitors*

Degradation reactions leading to byproducts that are inhibitory to microorganisms and enzymes is a common problem for different pretreatment methods, including hydrothermal processing [14]. In the current work, the formation of inhibitory compounds was rather moderate for pretreatment temperatures up to 190 ◦C, but a clear increase was observed when the pretreatment temperature was raised to 205 ◦C or when sulfuric acid was used as a catalyst at 190 ◦C (Table 4).

Increased formation of furan aldehydes for SA-190 can partially explain the previously discussed (Section 4.2) sugars that were not accounted for by carbohydrate analyses. Dehydration of sugars to furan aldehydes is a typical phenomenon for pretreatments performed under acidic conditions and at high temperatures, and the furans can be further degraded if the pretreatment is very severe [14]. There was a sharp increase in sugar degradation when the A-HTP temperature was increased to 205 ◦C, as indicated by high concentrations of furfural and HMF after pretreatment at 205 ◦C. At 205 ◦C, using a method that has not been reported before for hydrothermal pretreatment of wheat straw, furfural was also detected in condensate from the gas phase. The increase of formic acid and levulinic acid at 205 ◦C indicates further degradation of furans, as is expected for harsh conditions [44]. Acetic acid is formed mainly by the hydrolysis of acetyl groups of hemicelluloses. For the A-HTP series, the concentration of acetic acid continued to increase even up to 205 ◦C (Table 4), which agrees with the observation that there was still plenty of xylan left in the solid fraction after treatment at 190 ◦C (Table 1).

The formation pattern of phenolic substances was found to be different depending on the length of the side chain, as phenols with one-carbon side chains increased over the whole temperature range, whereas phenols with two- or three-carbon side chains exhibited a maximum within the temperature range (Table 4). Thermal degradation of phenols with a two- or three-carbon side chain can contribute to increased formation of the corresponding phenolic benzaldehydes. For instance, degradation of coniferyl aldehyde and

*p*-coumaraldehyde can result in the formation of vanillin and *p*-benzaldehyde, respectively. Vanillin has been shown to be formed from cleavage of the Cα-C<sup>β</sup> bond of acetovanillone through radiolysis [45]. A similar trend was observed in studies of hydrothermal pretreatment of sugarcane bagasse, where the formation of vanillin, *p*-hydroxybenzaldehyde, and syringaldehyde increased with severity, while coniferyl aldehyde concentrations reached a maximum at intermediate severity (log R<sup>0</sup> = 3.8) and then decreased when the severity increased further [35].

### *4.4. Enzymatic Saccharification of Pretreated Wheat Straw*

The reports on enzymatic hydrolysis of pretreated wheat straw are often limited to studies where pretreated solids are suspended in a buffer [33,46], which does not provide information on the effect of the pretreatment liquids on enzymatic conversion. The experimental setup used in this work, with pretreated solids suspended in either buffer or pretreatment liquids, allowed us to investigate the enzymatic digestibility of glucan contained in the pretreated solids and evaluate the inhibitory effect of the pretreatment liquids on the saccharification of glucan.

The results showed that (i) all pretreatment conditions greatly (2.5- to 5.5-fold) enhanced the enzymatic saccharification of wheat straw glucan; (ii) the enzymatic digestibility of pretreated solids increased with the pretreatment temperature and was found to be inversely correlated with the xylan content; (iii) the use of 0.5% (on a DW basis) sulfuric acid in the pretreatment did not have any major impact on saccharification in comparison to autocatalyzed pretreatment; (iv) the pretreatment liquids had a small (2–7%) but clear inhibitory effect on enzymatic saccharification; (v) the degree of inhibition increased with temperature, with no clear differences between SA-HTP pretreatment liquids and A-HTP pretreatment liquids (Figure 3). While some of these results agree with previous studies [41], the finding on the inhibition of enzymatic hydrolysis by the pretreatment liquids is an important contribution of this work to the knowledge on hydrothermal pretreatment. The relatively high enzyme inhibition observed for the pretreatment liquid from A-HTP at 205 ◦C (7%) can be attributed to its higher content of solubilized aromatics [16], expressed as total phenolic compounds and TAC (Table 4).

The correlations between the enzymatic convertibility of glucan and the content of xylan and lignin in the pretreated solids revealed that for achieving efficient enzymatic hydrolysis of wheat straw glucan, it is important that the pretreatment removes as much xylan as possible, while the effect of lignin is less important. That is in agreement with previous studies of pretreated lodgepole pine, implicating that removal of hemicelluloses is more important than removal of lignin in order to achieve efficient enzymatic saccharification [47].

Since different biorefinery applications, with glucose-based routes and xylose-based routes, can be considered for wheat straw, the total yields of sugars per ton of raw material are an important indicator for a pretreatment method of relevance. This work evaluates the effect of HTP on total sugar yield, including glucose, mainly formed during enzymatic saccharification, and xylose, mainly formed during the pretreatment stage. Although A-HTP at 205 ◦C was very effective for producing glucose in enzymatic hydrolysis, it led to extensive xylose degradation in the pretreatment stage. Instead, moderate pretreatment conditions (A-HTP, 190 ◦C) resulted in the highest xylose yield and, even though the glucose yield in the enzymatic saccharification step was lower than after pretreatment at 205 ◦C, the combined sugar yield was the highest. Thus, the investigation has revealed that since the highest yield was obtained with A-HTP at 190 ◦C, no sulfuric acid and no temperatures over 200 ◦C are required for maximizing sugar production from wheat straw.
