*4.1. Chemical Composition of Raw and Pretreated Solids*

Although different issues related to hydrothermal pretreatment of wheat straw for bioconversion have already been well investigated, there are other aspects that remain to be addressed. The effects of pretreatment temperature, time and equipment configuration on the yield of solids, solubilization of the main components, and recovery of sugars have been widely discussed [32–35], but important details linking pretreatment conditions with byproduct formation, enzymatic digestibility, and inhibition of biochemical conversion are still lacking. The current study assesses the effects of different pretreatment temperatures and catalytic approaches on the formation of bioconversion inhibitors, the enzymatic digestibility of pretreated solids, and the inhibition of enzymatic saccharification and fermentation by pretreatment liquids.

Increasing the pretreatment temperature from 160 to 205 ◦C while holding the reaction time at 15 min resulted in increased solubilization of wheat straw constituents and led to a reduction of the gravimetric yield of pretreated solids (Figure 1). The high pretreatment yield (almost 88%) under low-severity pretreatment (160 ◦C, SF 2.9) indicates that under these conditions, wheat straw was only marginally affected, while the low yield (around 64%) under the most severe pretreatment (205 ◦C, SF 4.3) revealed major solubilization of the raw material. These values are within the typical range for wheat straw. Min et al. [33] showed a decrease in the yield of pretreated solids from 86% to 80% while increasing the SF from 3.2 to 3.9, and Chen et al. [32] reported yields of approximately 60% in pretreatment at 200 ◦C for 30 min (SF 4.4).

The analysis of the composition of pretreated solids and pretreatment liquids indicates that the main factor contributing to the reduction of the gravimetric yield is the solubilization of hemicelluloses. The increase of solubilization of hemicelluloses with the temperature range studied is in agreement with previous reports of hydrothermal pretreatment of wheat straw [34]. The fact that the yield of solids was more affected by the stepwise increase of temperature than by the use of sulfuric acid can be explained by the rather low acid-loading that was used.

The observed temperature-related increase of the glucan content in the pretreated solids was a consequence of hemicellulose solubilization (Table 1). Cellulose was not affected to any major extent by the different pretreatment conditions, as indicated by the high glucan recoveries achieved (Figure 2). The lack of substantial differences in glucan recovery between A-HTP and SA-HTP for similar temperatures might be explained by the rather low sulfuric acid-loading used in SA-HTP. In previous studies on hydrothermal pretreatment of sugarcane bagasse using a higher sulfuric acid loading (0.5 g/100 g of reaction mixture instead of 0.5 g/100 g dry biomass), glucan recovery was higher for A-HTP than for SA-HTP under the same temperature and time [35,36].

The decrease of xylan content with the increase of the temperature is a logical result, considering that a temperature-dependent increase of hemicellulose solubilization is normally expected for hydrothermal pretreatment [32]. In contrast to previous reports on studies in which higher sulfuric acid-loadings or longer reaction times were used [37], pretreatment at 160 ◦C was not effective for xylan solubilization, even when sulfuric acid was used (Table 1). At 190 ◦C, there was some sulfuric-acid-promoted xylan solubilization, as can be interpreted from the decrease of xylan recovery from around 41% with A-HTP to nearly 30% with SA-HTP (Figure 2).

The observed arabinose solubilization at 160 ◦C (Table 1) matches well with previous information on the hydrothermal deconstruction of different hemicellulosic components. Under mild conditions, arabinose side-chain moieties are cleaved faster than xylose units [38]. Xylan backbone is initially fragmented to a minor degree, and only after subsequent scissions, a major release of xylo-oligosaccharides and xylose occurs [39]. However, the solubilization of galactose and mannose at 160 ◦C is surprising considering that mannan and galactan typically exhibit comparable dissolution kinetics to xylan [40], and it would have been expected that they behave similarly under HTP at 160 ◦C.

The gradual increase of lignin content in the pretreated solids with the increase of the temperature (Table 1) is primarily a consequence of hemicellulose solubilization, as typically observed for hydrothermal processing and acid-based pretreatment methods [13]. As confirmed by combining TSSA and Py-GC/MS analysis, lignin recovery of over 100%, detected for A-HTP at 205 ◦C and for both SA-HTP experiments (Figure 1), was due to pseudolignin formation, a phenomenon that has not been well-studied previously and which is typically difficult to quantify. The lower lignin fraction found by using Py-GC/MS (Table 2) in comparison with the fraction determined by TSSA (Table 1) for A-205, SA-160, SA-190, and A-190 is due to the fact that TSSA does not distinguish differences between real lignin and Klason-positive partially degraded carbohydrates, i.e., pseudolignin. Py-GC/MS analysis will detect Klason-positive partially degraded carbohydrates as carbohydrates. The lignin recoveries exceeding 100% in Figure 1 can, with certainty, be attributed to pseudolignin formation. For estimating pseudolignin formation, we used the ∆Lignin value, a factor we recently introduced based on TSSA analyses of lignin and biomass characterization using Py-GC/MS [35,36]. As shown by the negative value for raw wheat straw, TSSA and Py-GC/MS do not give identical values, and it is, therefore, a possibility that also some of the pretreated samples, with low negative ∆Lignin values, contain small fractions of pseudolignin. As hydrothermal pretreatment mainly targets hemicelluloses, the lignin showed minor compositional changes, as shown by small changes observed for the G:S:H ratio. There was an evident decrease in the relative abundance of syringyl units (Table 2).

The high ash content of the raw material is within the range previously determined for wheat straw [33,34]. The ash content increased slightly with the temperature as other components were removed and minerals remained in the pretreated solids. These results are in agreement with previous studies showing an increase in ash content with the temperature in HTP of wheat straw [33,34].

The increase of the mass fraction of extractives observed at 190 and 205 ◦C (Table 1) might be due to the fact that the solvent used in the analytical procedure extracted not only native extractives present in untreated wheat straw but also lignin fragments deposited on the solid phase during pretreatment. This is further strengthened by the finding that at similar temperatures, the content of extractives was higher for SA-HTP, the pretreatment approach that is expected to cause more extensive lignin fragmentation, condensation, and redeposition [41] than for A-HTP, and that the difference was more obvious at 190 ◦C than at 160 ◦C.
