*3.3. Effects of Pretreatment Conditions on Byproduct Formation*

For both the A-HTP and SA-HTP series, the furan aldehyde concentrations always increased with increasing pretreatment temperature (Table 4). The furfural concentrations (≤77.1 ± 2.4 mM) in the pretreatment liquids were always higher than the corresponding HMF concentrations (≤2.4 mM). The very low values after pretreatment at 160 ◦C are difficult to compare, but furfural values after pretreatment at 190 ◦C show clearly higher concentration for SA-HTP (24.1 ± 0.5 mM) than for A-HTP (14.4 mM). As acid conditions promote hydrolysis of hemicelluloses to pentose sugars, the precursors of furfural, this is an expected result. The condensate of A-205 was found to contain furfural that had been volatilized during the pretreatment.

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**Table 4.** Concentration of bioconversion inhibitors in the pretreatment liquids <sup>a</sup>


<sup>a</sup> Mean values from triplicates. The standard deviations are shown in parentheses. Same codification of experimental conditions as in Table 1. b–d Values given in mM (<sup>b</sup> ), µM (<sup>c</sup> ), or g/L (<sup>d</sup> ). <sup>e</sup> Furfural concentration in the condensate of the gas phase. Total aromatic content (TAC) was determined as UV absorption at 280 nm, with a dilution factor of 500. Total carboxylic acid content (TCAC; mM) was determined by titration. ND, not detected.

> The main aliphatic carboxylic acid in the pretreatment liquids was acetic acid (11.7–66.4 mM). Pretreatment liquids also contained formic acid (≤24.1 ± 0.1 mM) and levulinic acid (≤0.4 mM). The concentrations of acids increased with the pretreatment temperature, and there were only minor differences between the A-HTP and SA-HTP series. As expected, TCAC values were higher than the sum of concentrations of acetic acid, formic acid, and levulinic acids and followed the same trend.

> The formaldehyde concentrations were mostly below the detection level. A reason for this can be that during hydrothermal pretreatment, formaldehyde is probably formed mainly from lignin, and wheat straw has rather low lignin content compared to, for example, softwood [19,20].

> The concentration of total phenolics ranged from 1.0–7.2 g/L (Table 4). The SA-HTP series exhibited slightly higher values than corresponding pretreatment liquids in the A-HTP series. The concentrations of total phenolics and individual phenolics with a onecarbon side chain (i.e., vanillin, syringaldehyde, and *p*-hydroxybenzaldehyde) increased

with increasing pretreatment temperature (Table 4). Phenolics with two- or three-carbon side chains, such as acetovanillone, coniferyl aldehyde, and *p*-coumaraldehyde, increased with the pretreatment temperature up to 190 ◦C, but then decreased for A-205. This suggests that phenolics with longer side chains are more susceptible to degradation at higher temperatures, whereas phenolics with one-carbon side chains are more stable.

*p*-Benzoquinone was not detected in any of the pretreatment liquids (Table 4). Studies of yeast have shown that relevant toxic concentrations of *p*-benzoquinone are several orders of magnitude lower than for other inhibitors in Table 4, as concentrations in the micromolar range have clearly inhibitory effects [21]. The methodology used would have also revealed concentrations in that range.

The TAC measurement covers a wide range of aromatics, including phenylic compounds, such as phenolic and nonphenolic aromatics, and heteroaromatics, such as furan aldehydes. The TAC values increased with the temperature (Table 4). At corresponding temperatures, the TAC values for the SA-HTP series were identical to those of the A-HTP series.

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

Since the susceptibility to enzymatic saccharification is a key issue in the bioconversion of lignocellulosic biomass into bio-based products, the impact of pretreatment conditions on enzymatic saccharification was carefully examined using reaction mixtures with both buffer and pretreatment liquids. Reaction mixtures with buffer provide a clean analytical view of the susceptibility of pretreated solids to enzymatic saccharification. Reaction mixtures with pretreatment liquid provide a more complex and, perhaps, more industrially relevant view, where both the susceptibility of the solid phase and the influence of potential enzyme inhibitors in the pretreatment liquid are taken into account.

All pretreatment conditions resulted in a clear enhancement of saccharification of glucan, as revealed by the increase of enzymatic digestibility from 18% for raw wheat straw to 45–98% for pretreated solids (Figure 3). For A-HTP solids suspended in sodium citrate buffer, the enzymatic digestibility increased with the pretreatment temperature (from 46 ± 0.5% for A-160 to 98 ± 0.8% for A-205). For SA-HTP, the enzymatic digestibility values were comparable with those from A-HTP at corresponding temperatures. Xylan contained in the pretreated solids was also hydrolyzed during saccharification trials (data not shown).

‐ **Figure 3.** Enzymatic digestibility of glucan in cut raw wheat straw (WS) in buffer (blue bar) and pretreated solids suspended in either sodium citrate buffer (light green bars) or pretreatment liquid (dark green bars). Yellow bars represent the degree of inhibition (DI) of enzymatic saccharification by the pretreatment liquids. Same codification of experimental conditions as in Table 1.

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In the experiments with the pretreated solids suspended in pretreatment liquids, the enzymatic digestibility of glucan was lower than in the series with the buffer (Figure 3). The enzymatic digestibility ranged from 44 ± 0.8% for SA-160 to 90 ± 1.6% for A-205. Based on the differences between the hydrolysis series, the degree of inhibition (DI) by the pretreatment liquids was calculated. For A-HTP, the DI was 2.4 ± 0.1% for A-160, and it gradually increased with the temperature, reaching up to 7.3 ± 0.3% for A-205. At 160 ◦C, the DI was slightly higher for SA-HTP than for A-HTP, but at 190 ◦C, no discernible difference was found.

The statistical significance of the differences between enzymatic digestibility values achieved in saccharification trials of pretreated solids from different pretreatment conditions (*p* < 0.001), suspended in different liquid media (*p* < 0.001), and their interactions (*p* = 0.0155) was confirmed by two-way analysis of variance (Table 5) and Tukey's posthoc test. Levene's test for homogeneity of variance (*F* = 1.210, *p* = 0.333), and Shapiro's test for normal distribution of residuals (*W* = 0.957, *p* = 0.173) confirmed that the data met the model assumptions.

**Table 5.** Two-way ANOVA for enzymatic digestibility in saccharification trials with pretreatment solids suspended in either sodium citrate buffer or pretreatment liquids for all the pretreatment conditions.


Potential correlations between the enzymatic convertibility of glucan and the contents of xylan and lignin in the pretreated solids were investigated (Figure 4). For both the A-HTP and SA-HTP series, enzymatic digestibility displayed an obvious negative correlation (*R* <sup>2</sup> 0.92) with xylan content (Figure 4a). For lignin content, there was a slightly positive trend (Figure 4b) that can be attributed to indirect effects (hemicellulose removal leading to both higher lignin content and better digestibility). ‐

‐ ‐ ‐ **Figure 4.** Correlation between enzymatic digestibility of glucan with the content of xylan (**a**) and lignin (**b**) in the pretreated solids. The green markers are for A-HTP and the blue markers are for SA-HTP. R<sup>2</sup> = 0.92 (xylan) and R<sup>2</sup> = 0.45 (lignin). The plotted lignin content was determined using Py-GC/MS.

‐ ‐ ‐ In order to have a better picture of the effectiveness of different pretreatment conditions on the saccharification of wheat straw, the yield of sugars per ton of raw material was calculated. For A-HTP, the combined glucose yield (after pretreatment and enzymatic saccharification) increased almost proportionally with the temperature, giving the highest value (407 ± 13 kg/t) at 205 ◦C (Figure 5). The xylose yield reached a peak (127 ± 8 kg/t) at 190 ◦C, including the amount solubilized during pretreatment and that resulting from

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enzymatic hydrolysis. The xylose yield sharply decreased to only 21 ± 1 kg/ton raw wheat straw at 205 ◦C, mostly due to losses in the pretreatment step. For SA-HTP, glucose and xylose yields at 160 ◦C were higher than those observed for A-HTP, whereas at 190 ◦C, they were slightly lower. Putting together glucose and xylose, the highest total sugar yield, 480 ± 12 kg per ton of wheat straw, was observed for A-HTP at 190 ◦C. That yield was higher than the one achieved by SA-HTP at the same temperature. ‐ ‐ ‐ ‐ ‐

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‐ **Figure 5.** Yield of xylose (yellow bars), glucose (orange bars), and total sugar (brown bars) after pretreatment and enzymatic hydrolysis. Values are given in kg per ton raw wheat straw. Same codification of experimental conditions as in Table 1.
