**1. Introduction**

Growing global energy demand and the need to replace fossil fuels with renewable fuels are the major challenges of modern society [1]. The current production of liquid biofuels, such as bioethanol, is dominated by biochemical conversion routes mainly based on food-related feedstocks, such as corn starch and sugarcane sugar. Lignocellulosic materials can serve as additional resources for the biofuel sector [1,2]. Agricultural and agroindustrial residues, e.g., wheat straw, corn stover, and sugarcane bagasse, are lignocellulosic materials of interest in many countries due to their availability [3,4]. Co-utilization of lignocellulosic residues with starch or sugar can further boost bioethanol production through integration into 1.5 G processes [5].

Wheat (*Triticum aestivum* L.) is the world's most widely cultivated crop [6]. Based on the data on wheat production worldwide (FAOSTAT) and on a residue/crop ratio of 1.3 [7], around 980 million tons of wheat straw are estimated to be available on a yearly basis. Dry wheat straw consists mainly of cellulose (30–40%), hemicelluloses (20–30%), and lignin

**Citation:** Ilanidis, D.; Stagge, S.; Jönsson, L.J.; Martín, C. Hydrothermal Pretreatment of Wheat Straw: Effects of Temperature and Acidity on Byproduct Formation and Inhibition of Enzymatic Hydrolysis and Ethanolic Fermentation. *Agronomy* **2021**, *11*, 487. https:// doi.org/10.3390/agronomy11030487

Academic Editors: Tony Vancov and Peter Langridge

Received: 19 January 2021 Accepted: 1 March 2021 Published: 5 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(10–20%) [8]. Theoretically, bioconversion processes can yield around 270 L of ethanol per ton of dry wheat straw [9]. Biochemical conversion of agricultural residues, such as corn stover and wheat straw, has been significantly improved [7,10], but further research is still required for designing highly competitive and sustainable processes for the production of biofuels and other bio-based commodities.

The production of biofuels from lignocellulosic biomass comprises several steps, such as pretreatment, enzymatic saccharification, and microbial fermentation [11,12]. For achieving effective enzymatic saccharification of cellulose and, consequently, a higher release of fermentable sugars, a pretreatment step is indispensable. By disrupting the lignocellulosic matrix, pretreatment exposes cellulose and makes it more reactive towards cellulases [13]. Among different existing pretreatment methods, hydrothermal processing is an attractive option for agricultural residues, and it has good potential for industrial implementation [14]. Hydrothermal pretreatment (HTP) can be catalyzed by either hydronium ions generated by water autoionization or externally added acid species [15]. In HTP, moist lignocellulosic biomass is heated to around 200 ◦C for a certain period of time. Under those conditions, degradation of hemicelluloses and relocation of lignin can occur, and that leads to better accessibility of cellulose, which facilitates enzymatic hydrolysis [15,16].

While aiming at producing pretreated biomass with highly digestible cellulose, most pretreatment methods unavoidably lead to the formation of byproducts, resulting mainly from partial degradation of polysaccharides and lignin [14]. The formed byproducts can negatively affect the efficiency of enzymatic saccharification and microbial fermentation [14,17]. Certain groups of substances that inhibit microorganisms, such as aliphatic carboxylic acids, furan aldehydes, and phenolic compounds, have been extensively studied [18], while the importance of other groups of inhibitors has only recently started to emerge. Recent studies [19,20] have shown the significance of small aliphatic aldehydes as inhibitors of microbes used for biochemical conversion of biomass. Furthermore, the presence of benzoquinones in pretreatment liquids of different materials and their inhibitory effects on *Saccharomyces cerevisiae* have recently been discovered [21]. With regard to the inhibition of enzymes, aromatic substances such as phenolics have been found to play a role [14]. Pseudolignin, another byproduct, consists of thermal degradation products of carbohydrates. Pseudolignin remains insoluble and is accounted for as Klason lignin in the analytical two-step treatment with sulfuric acid (TSSA). The formation of pseudolignin can affect the enzymatic saccharification process [22,23]. In previous studies on hydrothermal pretreatment of wheat straw, the inhibition problem has generally been limited to substances such as furfural, HMF (5-hydroxymethylfurfural), acetic acid, formic acid, and certain phenolic compounds [8,9,24], while newly discovered inhibitors, such as aliphatic aldehydes and benzoquinones, have not been considered.

There are still several issues about hydrothermal pretreatment of wheat straw that require further research and innovation efforts. Issues that have so far not received enough attention include (i) how autocatalyzed (A-HTP) and sulfuric acid-catalyzed (SA-HTP) hydrothermal pretreatments affect the formation of byproducts, including pseudolignin and newly discovered inhibitors, (ii) how the pretreatment liquids inhibit the enzymatic hydrolysis of cellulose, and (iii) how xylan and lignin affect the digestibility of pretreated solids. In the current study, hydrothermal pretreatment of wheat straw under different temperatures between 160 and 205 ◦C, using two catalytic approaches, auto-catalysis and catalysis with sulfuric acid, was investigated. The investigation covered the evaluation of the effects of pretreatment conditions on (i) release of sugars and bioconversion inhibitors, (ii) chemical composition and enzymatic digestibility of pretreated solids, and (iii) inhibitory effects of pretreatment liquids on enzymatic saccharification and yeast fermentation. The results were backed by advanced analytical techniques, such as pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), high-performance anion-exchange chromatography (HPAEC), and ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry (UHPLC-ESI-QqQ-MS).
