*2.8. Statistical Processing of the Results*

Two-way analysis of variance (ANOVA) and Tukey's posthoc test were applied to evaluate the statistical difference between the results of enzymatic saccharification experiments corresponding to different pretreatment conditions and different liquid media (either sodium citrate buffer or pretreatment liquids) used in the assays. Levene's test for homogeneity of variance and Shapiro's test for normal distribution of residuals were used for verifying that the data met the model assumptions. Statistical tests were run using R package stat [30].

### **3. Results**

### *3.1. Chemical Composition of Raw and Pretreated Solids*

The wheat straw used in this study contained 39.8% glucan, 24.3% hemicellulosic carbohydrates, 22.8 ± 0.1% lignin, 5.1 ± 0.3% ash, and 4.7 ± 0.1% ethanol extractives (Table 1). That composition is in agreement with previous reports on wheat straw, showing similar values [31].

The pretreatment led to different degrees of reduction of the gravimetric yield of the resulting solid material with respect to the starting amount of wheat straw (Figure 1). Depending on the pretreatment conditions, the yields of pretreated solids ranged between 64.3% and 87.6%, and a steady decrease was observed with increasing temperature and increasing severity factor. The yield of pretreated solids resulting from the most severe autocatalyzed pretreatment (the one performed at 205 ◦C) was 27% lower than that of the least severe one (at 160 ◦C). The yields were lower for SA-HTP than for A-HTP, but the differences were relatively small. The most noticeable difference was found for 190 ◦C (69.4% for SA-HTP and 73.0% for A-HTP), while the values for 160 ◦C (85.5% for SA-HTP and 87.6% for A-HTP) were closer. It is noteworthy that the differences in yield between the pretreatment approaches (A-HTP and SA-HTP) at a given temperature were smaller than the differences in yield between two consecutive temperatures in the A-HTP series, for instance, 190 ◦C (73.0%) and 205 ◦C (64.3%) or 175 ◦C (80.3%) and 190 ◦C (73.0%). Evidently, a temperature increase of 15 ◦C had a stronger effect than the addition of sulfuric acid.


**Table 1.** Chemical composition of raw wheat straw and pretreated solids, mass fractions in % (dry weight) <sup>a</sup> .

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‐

‐ ‐ ‐

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‐

‐

‐

‐

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<sup>a</sup> Average values of solid fractions resulting from hydrothermal pretreatment of wheat straw (mass fractions in % of dry weight). Numbers in parenthesis indicate standard deviation. <sup>b</sup> The letters in the codification of the experimental conditions stand for the pretreatment approach (A for A-HTP or SA for SA-HTP), and the numerals (160, 175, 190, and 205) stand for temperatures. ND, not detected. ‐ ‐

The glucan content was higher in the pretreated solids than in raw wheat straw, and, for both pretreatment approaches, it increased with the increase in temperature (Table 1). For A-HTP, the glucan content increased from 42.5% at 160 ◦C to 57.3% at 205 ◦C, while for SA-HTP, it increased from 43.5% at 160 ◦C to 54.8% at 190 ◦C. For the same pretreatment temperature, A-HTP and SA-HTP did not exhibit any major difference in glucan content. The same applies for glucan recovery, which was comparable for both pretreatment approaches, and displayed values above 92% for all experimental conditions (Figure 2).

The xylan content decreased with increasing temperature (Table 1). Independently of the catalytic approach, the xylan content of solids pretreated at 160 ◦C was comparable to that of raw wheat straw. After pretreatment at 205 ◦C, the xylan content was below 3 ± 0.1%. The xylan recovery for pretreatments at 160 ◦C corresponded to approx. 87 ± 0.1%, whereas the recovery at 205 ◦C was below 9% (Figure 2). At 160 ◦C, the xylan recovery was not affected by acid addition, whereas at 190 ◦C, the value for A-HTP (41.1%) differed substantially from that of SA-HTP (30.3%). Other hemicellulosic carbohydrates were readily solubilized already at 160 ◦C as no arabinose, galactose, or mannose were detected in the analytical acid hydrolysates of the pretreated solids (Table 1).

‐ **Figure 2.** Recovery of glucan, xylan, and total lignin (Klason lignin plus acid-soluble lignin (ASL)) in the pretreated solids. The codification of the experimental conditions is the same as in Table 1.

‐ A gradual increase of Klason lignin content in the pretreated solids was observed with increasing temperature. For experiments performed at the same temperature, the Klason lignin content was higher for SA-HTP than for A-HTP (Table 1). The content of acid-soluble lignin (ASL) did not display a clear trend regarding pretreatment temperature, but it was lower for SA-HTP than for A-HTP.

‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ For the autocatalyzed pretreatment at 205 ◦C and for both acid-catalyzed pretreatments, the recovery of total lignin was higher than the theoretically possible value (Figure 2), indicating the formation of pseudolignin. To clarify this issue, Py-GC/MS analysis was performed. For the A-HTP experiments at 190 and 205 ◦C and for the SA-HTP experiments, Py-GC/MS revealed lower lignin content than the fraction of total lignin determined by the TSSA procedure (Klason lignin + acid-soluble lignin; Table 2). Higher ∆Lignin factor values (Table 2) than for raw wheat straw indicate pseudolignin formation. The increase in ∆Lignin factor value was especially apparent after pretreatment at 205 ◦C. At the same temperature, the ∆Lignin factor was higher for SA-HTP than for A-HTP, indicating that increasing acidity stimulated pseudolignin formation.

‐ ‐ ‐ Δ **Table 2.** Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) analysis of raw wheat straw and pretreated solids.


‐ ‐ <sup>a</sup> Mean values from triplicates. Standard deviations are shown in parentheses. <sup>b</sup> The ∆Lignin factor was calculated by subtracting the peak area fraction assigned to lignin in Py-GC/MS analysis (this table) from the mass fraction of total lignin (Klason lignin and ASL; Table 1). <sup>c</sup> Relative proportion of lignin guaiacyl (G), syringyl (S), and *p*-hydroxyphenyl (H) units. Same codification of experimental conditions as in Table 1.

Negative ∆Lignin factor values, particularly for raw wheat straw (Table 2), can be explained by the fact that the wheat straw contains relatively large fractions of ash (5.1%) and extractives (4.7%) (Table 1). While these substances are included in initial mass values used for TSSA calculations (Table 1), they (especially ash/inorganic substances) might not be covered by total peak areas used for Py-GC/MS calculations (Table 2). This explanation is supported by the fact that for raw wheat straw, it was not only the TSSA value for total

lignin (22.8%) that was lower than the Py-GC/MS counterpart (27.8%); the TSSA value for the sum of carbohydrates (64.1%) was also lower than the Py-GC/MS counterpart (70.4%).

Py-GC/MS analysis of the G:S:H ratio (Table 2) indicated that pretreatment caused a relative increase of G (guaiacyl) units, a relative decrease of S (syringyl) units, and a relative increase of H (*p*-hydroxyphenyl) units. The relative decrease of S units might be associated with the cleavage of β-*O*-4 linkages in lignin, whereas corresponding increases for G and H units might be indirect effects.

The ash content was not much affected by the pretreatment but was slightly higher for the highest pretreatment temperature (A-HTP at 205 ◦C; Table 1). The increase in ash content at high temperatures can be an indirect effect of the decrease in hemicellulosic carbohydrates.

The mass fraction of extractives, which was 4.7% in raw wheat straw, decreased slightly for pretreatments at low temperature (160/175 ◦C; Table 1). At higher temperatures (190/205 ◦C), there was an increase. The decrease at lower temperatures can be explained by the solubilization of original extractives during pretreatment, whereas the increase at higher temperatures can be explained by the formation of new extractives due to the fragmentation of carbohydrates and lignin.
