Factors Affecting Detoxification of Softwood Enzymatic Hydrolysates

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Article Factors Affecting Detoxiﬁcation of Softwood Enzymatic Hydrolysates Using Sodium Dithionite

Dimitrios Ilanidis 1, Stefan Stagge 1 , Björn Alriksson 2 and Leif J. Jönsson 1,*

1 Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden; [email protected] (D.I.); [email protected] (S.S.) 2 RISE Processum AB, SE-891 22 Örnsköldsvik, Sweden; [email protected] * Correspondence: [email protected]

Abstract: Conditioning of lignocellulosic hydrolysates with sulfur oxyanions, such as dithionite, is one of the most potent methods to improve the fermentability by counteracting effects of inhibitory by-products generated during hydrothermal pretreatment under acidic conditions. The effects of pH, treatment temperature, and dithionite dosage were explored in experiments with softwood hydrolysates, sodium dithionite, and Saccharomyces cerevisiae yeast. Treatments with dithionite at pH 5.5 or 8.5 gave similar results with regard to ethanol productivity and yield on initial glucose, and both were always at least ~20% higher than for treatment at pH 2.5. Experiments in the dithionite concentration range 5.0–12.5 mM and the temperature range 23–110 ◦C indicated that treatment at around 75 ◦C and using intermediate dithionite dosage was the best option (p ≤ 0.05). The investigation indicates that selection of the optimal temperature and dithionite dosage offers great benefits for the efficient fermentation of hydrolysates from lignin-rich biomass, such as softwood residues. Citation: Ilanidis, D.; Stagge, S.; Alriksson, B.; Jönsson, L.J. Factors Keywords: lignocellulose biorefining; conditioning; detoxification; cellulosic ethanol; inhibitors; Affecting Detoxification of Softwood sodium dithionite Enzymatic Hydrolysates Using Sodium Dithionite. Processes 2021, 9, 887. https://doi.org/10.3390/ pr9050887 1. Introduction Biorefining of lignocellulosic biomass offers a route for the production of bio-based Academic Editors: commodities, to reduce the dependency on fossil fuels, and reduce greenhouse gas emis- Francesca Raganati and sions [1,2]. Coniferous trees, principally Norway spruce and Scots pine, are predominant Alessandra Procentese (82%) in Swedish forests [3], and softwood is therefore the main form of lignocellulosic biomass. Mandatory reforestation after harvesting was introduced in Sweden in the Received: 27 April 2021 Forestry Act of 1903, and active forest management has led to an increase in standing Accepted: 14 May 2021 3 Published: 18 May 2021 volume from roughly 1700 million m in 1926 (when measurements were initiated) to around 3500 million m3 today [3].

Publisher’s Note: MDPI stays neutral Lignocellulosic biomass consists mainly of cellulose, hemicellulose, and lignin [1]. One with regard to jurisdictional claims in of the main biorefining routes is biochemical conversion, in which lignocellulosic biomass published maps and institutional affil- is converted by pretreatment, enzymatic saccharification of cellulose, microbial fermenta- iations. tion of sugars, and separation and refining of hydrolysis lignin, an approach sometimes referred to as a sugar-platform process. Pretreatment is necessary to make the cellulose in lignocellulosic biomass more accessible to enzymatic saccharification [2,4]. Enzymatic saccharification and microbial fermentation can be performed separately (SHF, separate hydrolysis and fermentation) or simultaneously (SSF, simultaneous saccharification and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. fermentation) [5]. Other approaches include consolidated bioprocessing (CBP) [6], in which This article is an open access article the fermenting microorganism contributes to the enzymes required for saccharification, distributed under the terms and and hybrid hydrolysis and fermentation (HHF) [7], in which the saccharification is initiated conditions of the Creative Commons prior to addition of the fermenting microorganisms. Attribution (CC BY) license (https:// Among numerous pretreatment methods, hydrothermal pretreatment with or without creativecommons.org/licenses/by/ externally added acid catalyst is one of the most studied [2,8,9]. Regardless of whether 4.0/). an acid catalyst, such as sulfuric acid or sulfur dioxide, is added, the pretreated material

Processes 2021, 9, 887. https://doi.org/10.3390/pr9050887 https://www.mdpi.com/journal/processes Processes 2021, 9, 887 2 of 13

will become acidic due to formation of carboxylic acids when hemicelluloses are degraded. Hydrothermal pretreatment targets hemicellulose, but cellulose and lignin will also be affected [2,10]. For feedstocks such as wood, and particularly softwood, harsh pretreatment conditions must be used to remove the main part of the hemicelluloses, which is imperative for getting high sugar yields in the ensuing enzymatic saccharification. Hydrothermal pretreatment under harsh acidic conditions is a good approach for removing hemicelluloses, but also leads to the formation of by-products, particularly from hemicellulose but also from cellulose, lignin, and extractives. The by-products may reach concentrations that are inhibitory for both enzymes that are used for saccharification [7,10–12] and microorganisms that are used for the fermentation process [10,13,14]. Certain groups of by-products that inhibit microorganisms have been extensively studied, and these include aliphatic carboxylic acids (such as acetic acid, formic acid, and levulinic acid), furan aldehydes (such as furfural and 5-hydroxymethylfurfural [HMF]), and phenolic and other phenylic substances [10,13,14]. More recently, small aliphatic aldehydes (such as formaldehyde and acetaldehyde) [15] and benzoquinones (such as p-benzoquinone) [16] were also found to be commonly occurring in pretreated biomass. Martín et al. [17] reported that formaldehyde was the most important inhibitor of Saccha- romyces cerevisiae yeast in a set of slurries of pretreated Norway spruce. p-Benzoquinone was found only in very low concentrations (micromolar range), but on the other hand it was by far the most toxic inhibitor on the basis of concentration. Enzyme inhibitors are less well characterized than microbial inhibitors. Apart from feedback inhibition by sugars [18], aromatic substances also cause inhibition [10]. Ximenes et al. [12] found that phenols occurring in lignocellulosic hydrolysates inhibit cellulases. Malgas et al. [19] investigated inhibition by lignin derivatives of mannanolytic enzymes. Zhai et al. [20] found that monomeric sugars and phenolics were the main enzyme inhibitors in steam-pretreated biomass, whereas oligomeric sugars had little influence. Conditioning of slurries and hydrolysates is used to make them suitable for biocatalysts, such as enzymes and microbes. Detoxification is a form of conditioning that is used to minimize inhibition of microorganisms [13,21]. The drawbacks of many detoxification methods include the requirement of an additional process step, the use of large amounts of chemicals, and that they only address inhibition of microorganisms and not inhibition of enzymes. These drawbacks have been addressed by the introduction of conditioning using reducing agents [22–24]. This approach relies on the use of relatively low concentrations of reducing agents, preferably industrial chemicals such as sodium sulfite and sodium dithionite, and their addition to the bioreactor just prior to or simultaneously with the biocatalysts [22]. Further investigations have shown that sulfur oxyanions, such as sulfite and dithionite, cause hydrophilization of inhibitors by sulfonation [23], which alleviates inhibition of cellulolytic enzymes in a way that other reducing agents, such as sodium borohydride, do not do [24]. Sodium dithionite has been shown to be very potent for the conditioning of softwood hydrolysates, but despite the potential benefits very little is known about fundamental aspects of the treatment. The current work addresses that lack of knowledge by investigation of the effects of pH, method of administration of dithionite, treatment temperature, and dosage of dithionite in experiments with a hydrolysate of Norway spruce. The effects were evaluated using a standard S. cerevisiae industrial yeast strain and by analyzing productivities and yields. Research on the efficiency of the process is important for industrial implementation of new technology for the biorefining of lignocellulosic feedstocks, an industrial enterprise in which high yields and efficient use of expensive biocatalysts are among the keys to high competitiveness.

2. Materials and Methods 2.1. Pretreatment The SF (Severity Factor) (Equation (1)) of the pretreatments was determined as the logarithm of the reaction ordinate according to Overend and Chornet [25] (Equation (2)), Processes 2021, 9, 887 3 of 13

and the CSF (Combined Severity Factor) of the acid-catalyzed pretreatments was calculated as outlined by Chum et al. [26] (Equation (3))

SF = Log Ro (1)

Tr − 100 Ro = t x exp (2) 14.75 CSF = Log (Ro) − pH (3)

where t is the holding time of pretreatment in minutes, and Tr is the pretreatment temperature in ◦C. The pretreatment of sawdust from Norway spruce was performed by SEKAB E- Technology in the Bioreﬁnery Demonstration Plant (BDP), Örnsköldsvik, Sweden. The demonstration unit has a pre-treatment capacity of approx. two tons of feedstock per day and is equipped with a continuous steam explosion pretreatment reactor. The Norway spruce was impregnated with sulfuric acid prior to pretreatment and the subsequent pretreatment was performed at a temperature, residence time and reactor pH that resulted in a CSF of approx. 2.6. The pretreatment conditions were: load of sulfuric acid, 1% (w/w) per dry weight biomass; temperature, 210 ◦C; residence time, 7–8 min. The resulting pH was 1.5. The pretreated biomass was collected and stored at 4 ◦C until further use. For determination of the dry-matter content, the slurry was separated through vacuum ﬁltration (membrane diameter equal to 0.50 mm and pore size 0.20 µm) (VWR International AB, Spånga, Sweden) and the solid phase was washed with deionized water and analyzed using an HG63 moisture analyzer (Mettler-Toledo, Greifensee, Switzerland). The WIS (water-insoluble solids) content of the slurry was around 17.5% (w/v) and was determined according to Sluiter et al. [27].

2.2. Enzymatic Hydrolysis Each of three 2 L shake flasks was filled with 750 g of slurry, and the pH of the slurry was adjusted to 5.5 with a 10 M aqueous solution of sodium hydroxide. Enzymatic saccharification was initiated by addition of 14 g Cellic CTec2 solution (obtained from Sigma- Aldrich, Steinheim, Germany) to each flask. The flasks were incubated for 48 h at 50 ◦C and 140 rpm in an orbital shaker (Kühner Lab-Therm LT-X, Adolf Kühner AG, Birsfelden, Switzerland). After enzymatic saccharification, the slurry was filtered by vacuum filtration and the enzymatic hydrolysates were pooled and used in fermentation experiments. The concentration of glucose after pretreatment and enzymatic saccharification was measured by high-performance liquid chromatography (HPLC). The HPLC instrument was a 1260 Infinity system equipped with a refractive index detector (RID), an autoinjector, and a column oven (Agilent Technologies, Santa Clara, CA, USA). The column was an Aminex HPX-87P column (300 mm × 7.8 mm) equipped with a guard column, 125-0131 Standard Cartridge Holder (30 mm × 4.6 mm) (Bio-Rad, Irvine, CA, USA). The temperature of the column oven was set to 80 ◦C and the temperature of the detector was set to 55 ◦C. The injection volume was 10 µL. The eluent consisted of ultrapure water and the flow rate was 0.6 mL/min. Glucose calibration standards were used for the determination of released glucose by converting the peak heights in chromatograms to concentrations. The software was OpenLAB CDS Chem Station Edition for LC and LC/MS Systems Rev.C.01.07 Edition 27. The analysis was performed in triplicates.

2.3. Conditioning of Hydrolysate Three different experimental series were performed. In the ﬁrst experimental series (A, Table1) the pH and the temperature of conditioning were varied. The pH was adjusted to 5.5 prior to conditioning using a 10 M aqueous solution of sodium hydroxide, and then the hydrolysates were conditioned with sodium dithionite, which was added as a powder (technical grade 93%) for 10 min at 23 ◦C with magnetic mixing. For reactions at 75 ◦C, the sodium dithionite was added when the hydrolysates reached a temperature of 75 ◦C, Processes 2021, 9, 887 4 of 13

and the reaction mixtures were incubated for 10 min. Experiments included triplicates of each reaction mixture.

Table 1. Experimental conditions for series with varying pH and conditioning temperature.

◦ Codiﬁcation Na2S2O4 (mM) T ( C) Time (min) pH A1 - 23 10 5.5 A2 5 23 10 2.5 A3 5 23 10 5.5 A4 5 23 10 8.5 A5 5 a 23 10 5.5 A6 5 75 10 5.5 AR Glucose reference B1 5 75 10 5.5 B2 - 75 10 5.5 B3 - 23 10 5.5 B4 5 23 10 5.5 BR Glucose reference C1 5 50 10 5.5 C2 8.7 50 10 5.5 C3 12.5 50 10 5.5 C4 5 75 10 5.5 C5 8.7 75 10 5.5 C6 12.5 75 10 5.5 C7 5 95 10 5.5 C8 8.7 95 10 5.5 C9 12.5 95 10 5.5 C10 5 110 10 5.5 C11 8.7 110 10 5.5 C12 12.5 110 10 5.5 CR Glucose reference a Dissolved in deionized water before addition.

In the second experimental series (B, Table1) the temperatures were decided based on the results of the ﬁrst experimental series and investigated in order to better understand and evaluate the impact of the temperature under the conditioning step. In the third experimental series (C, Table1) the concentration of sodium dithionite was varied from 5 mM to 12.5 mM, while the conditioning temperature was set at 50 ◦C, 75 ◦C, 95 ◦C, and 110 ◦C. The conditioning at 95 ◦C and 110 ◦C was performed in an oil bath. The experiments were performed using reagent bottles with screw caps. The bottles with the enzymatic hydrolysates were placed in the oil bath and the sodium dithionite was added when the sample reached the right temperature (95 ◦C or 110 ◦C) under magnetic stirring. Experiments included triplicates of each reaction mixture.

2.4. Fermentation For assessing the effects of conditioning, fermentation of media based on hydrolysate was performed with the industrial S. cerevisiae yeast Ethanol Red (Fermentis, Marcq-en- Baroeul, France). After the enzymatic sacchariﬁcation, the monosaccharide composition of the hydrolysate (in g/L) was: glucose, 97; galactose, 7.7; mannose, 37; xylose, 20; arabinose, 3.2. A medium based on a synthetic glucose solution was used as a reference. Reference fermentations were included in all series to compensate for potential differences between inocula used on different occasions. The glucose medium consisted of 97 g/L glucose in ultrapure water (the same glucose concentration as in the hydrolysate). Fermentations were performed in 30 mL glass ﬂasks containing 25 mL culture medium. The culture medium contained 23.5 mL hydrolysate, or, alternatively, glucose reference solution, and 0.5 mL of a nutrient solution (containing 150 g/L yeast extract, 75 g/L (NH4)2HPO4, 3.75 g/L MgSO4·7 H2O, and 238.2 g/L NaH2PO4·H2O). The pH of the media was adjusted to Processes 2021, 9, 887 5 of 13

5.5 before inoculation. Before inoculum, freeze-dried Ethanol Red was rehydrated by suspension in five times its weight of sterilized water for 30 min at 35 ◦C. One mL of yeast suspension was added to each flask to an initial yeast biomass concentration of 2 g/L. The flasks were sealed with rubber plugs pierced with cannulas for letting out carbon dioxide and were then incubated for 48 h or 60 h in a water bath, with magnetic stirring at 180 rpm and 30 ◦C. The glucose levels during the fermentation were monitored using a glucometer (Glucometer Elite XL, Bayer AG, Leverkusen, Germany). Fermentations were performed in duplicates.

2.5. Analysis of Ethanol and Glucose The Agilent 1260 Inﬁnity HPLC system was used for determination of ethanol and glucose in fermentation samples. The column was an Aminex HPX-87H column (300 mm × 7.8 mm) equipped with a 125-0131 Standard Cartridge Holder guard column (30 mm × 4.6 mm) (Bio-Rad). The temperature of the column oven was set to 80 ◦C and the temperature of the detector was set to 55 ◦C. The injection volume was 10 µL and the ﬂow rate was 0.6 mL/min. The eluent consisted of a 5 mM aqueous solution of H2SO4. The software used for the analysis was OpenLAB CDS Chem Station Edition for LC and LC/MS Systems Rev.C.01.07 Edition 27. Calibration standards were used for both glucose and ethanol. The concentrations were calculated by converting the peak heights in chromatograms to concentrations. Analysis was performed in triplicates.

2.6. Analysis of Monosaccharides Analysis of monosaccharides was performed using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The instrument used was an ICS-5000 system equipped with a CarboPac PA1 4 × 250 mm separation column and a 4 × 50 mm guard column (all from Dionex, Sunnyvale, CA, USA). Elution was performed with ultra-pure water using a ﬂow rate of 1 mL/min for 25 min. Post- column addition of a 300 mM aqueous solution of NaOH (0.5 mL/min) was used to amplify the signal. The column was regenerated by washing for 11 min with a mixture consisting of 60% of the 300 mM NaOH solution and 40% of a 200 mM NaOH solution with 170 mM sodium acetate, and equilibration with ultra-pure water for 3 min. PAD was performed on gold electrode applying a Gold Standard PAD waveform with Ag/AgCl as reference electrode. Prior to analysis all samples were diluted with ultra-pure water and ﬁltered through 0.2 µm nylon membranes.

2.7. Determination of Microbial Inhibitors Determination of furfural and 5-hydroxymethylfurfural (HMF) was performed using an HPLC system (Dionex UltiMate 3000) equipped with a diode-array detector and a 3 × 50 mm, 1.8 µm Zorbax RRHT SB-C18 column (Agilent Technologies). The temperature of the column oven was set to 40 ◦C. Gradient elution was performed using a mixture of Eluent A (an aqueous solution of 0.1% (v/v) formic acid) and Eluent B (acetonitrile with 0.1% (v/v) formic acid). The ﬂow rate was 0.5 mL/min. Separation was achieved with 3% Eluent B for 3 min. The ensuing cleaning step was done with 20% Eluent B for 4 min. Finally, the column was equilibrated for 4 min with 3% Eluent B. The absorption at 282 nm was monitored. Analysis of commonly occurring aliphatic carboxylic acids (acetic acid, formic acid, and levulinic acid) was performed by MoRe Research Örnsköldsvik AB, Sweden. The analysis was done using HPAEC. Ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry (UHPLC-ESI-QqQ-MS) was used for determination of formaldehyde, acetaldehyde, p-benzoquinone, vanillin, coniferyl aldehyde, syringaldehyde, p-hydroxybenzaldehyde, p-coumaraldehyde, and acetovanillone. Derivatization with 2,4-dinitrophenylhydrazine (DNPH) and calibration was performed as previously described [15,16]. Brieﬂy, UHPLC-ESI-QqQ-MS analysis was carried out using an Agilent Processes 2021, 9, 887 6 of 13

1290 Infinity system coupled to an Agilent 6490 TripleQuad mass spectrometer (QqQ-MS) used in negative electrospray ionization (ESI) mode and equipped with Agilent Jetstream technology. The QqQ-MS source parameters were optimized for each analyte using the following settings: gas temperature 290 ◦C, gas flow 20 L/min, nebulizer 30 psi, sheath gas temperature 400 ◦C, sheath gas flow 12 L/min, capillary voltage −3000 V, nozzle voltage −2000 V. The separation was performed on a 2.1 mm × 50 mm Kinetex 1.7 µm biphenyl 100 Å column (Phenomenex, Torrance, CA, USA) operating at 30 ◦C and using a flow rate of 0.3 mL/min. The eluent consisted of a mixture of aqueous 0.1% (v/v) formic acid (Eluent C) and a three-to-one (by volume) solution of acetonitrile and 2-propanol mixed with 0.1% formic acid (Eluent D). Elution was performed using a gradient profile containing the following fractions of Eluent D: 0.0–4.5 min 30–40%, 4.5–9.0 min 40–50%, 9.0–11.0 min 50–70%, 11.0–11.01 min 70–95%, 11.01–15.0 min 95%, 15.0–15.01 min 95–30%, 15.01–18.0 min 30%, and, at the end, two min post-time with 30% for further re-equilibration. Data evaluation was done with MassHunter quant software (Agilent Technologies). For determination of total phenolics, the Folin–Ciocalteu method [28] was used with vanillin as calibration standard. The color generated after 2 h at about 23 ◦C was read at 760 nm in a microtiter plate using SpectraMax i3x (Molecular Devices, San Jose, CA, USA). Reactions were performed in triplicates. Total aromatic content (TAC) [29] in pretreatment liquids was determined by mea- suring the absorbance at 280 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Total carboxylic acid content (TCAC) [29] was determined by titration from pH 2.8 to pH 7.0 using an aqueous solution of sodium hydroxide (200 mM).

2.8. Calculations

The ethanol yield on consumed sugar (Ycon) was calculated as the amount of ethanol formed per gram of consumed glucose (g/g). The ethanol yield on initial sugar (Yini) was calculated as the amount of ethanol formed per gram of initial glucose (g/g). The volumetric productivity (Q) was based on grams of ethanol produced per L of culture medium during the ﬁrst 24 h, 36 h, and 48 h of the fermentation [g/(L h−1)]. The speciﬁc productivity (q) was based on the volumetric productivity divided by the initial cell dry weight concentration [g/(g h−1)].

3. Results and Discussion Three series of fermentations, using different detoxiﬁcation conditions, were performed to investigate how pH, temperature, and loading of the reducing agent affected the fermentability of hydrolysates of wood of Norway spruce. The concentrations of microbial inhibitors were also determined (Table2). With regard to aliphatic aldehydes, the hydrolysate contained a high concentration of formaldehyde (6.1 mM) whereas the concentration of acetaldehyde was rather low (1.7 mM). Cavka et al. [15] and Martin et al. [17] found that 1.0 mM of formaldehyde and 5.0 mM of acetaldehyde was sufﬁcient to inhibit S. cerevisae. Thus, the formaldehyde concentration found in the hydrolysate would be expected to be strongly inhibitory, while the acetaldehyde concentration would not be inhibitory. The observed concentrations are in line with previous studies of pretreated spruce showing higher concentrations of formaldehyde than of acetaldehyde [15,17]. It is probable that formaldehyde primarily comes from lignin, and that acetaldehyde primarily comes from acetyl groups in hemicelluloses. As softwood has comparatively high lignin content and low acetyl content, this can explain the predominance of formaldehyde. Processes 2021, 9, 887 7 of 13

Table 2. Concentration of bioconversion inhibitors in pretreatment liquid a.

Substance Concentration Aliphatic aldehydes (mM) Acetaldehyde 1.7 (0.2) Formaldehyde 6.1 (0.4) Aliphatic carboxylic acids (mM) Acetic acid 95 (3) Formic acid 21 (2) Levulinic acid 19 (2) Benzoquinones (µM) p-Benzoquinone ND b Furan aldehydes (mM) Furfural 19.3 (0.9) HMF 20.1 (0.9) Group analyses TAC (AU) c 0.26 (0.01) TCAC (mM) d 153 (2) Total phenolics (g/L) e 1.6 (0.1) Phenols (µM) Acetovanillone 9.8 (1.0) Coniferyl aldehyde 121 (10) p-Coumaraldehyde 1.6 (0.1) 4-Hydroxybenzaldehyde 35.1 (9.4) Syringaldehyde 0.12 (0.01) Vanillin 430 (120) a Mean values of three replicates with standard deviations in parentheses. b Not detected. c Total Aromatic Content, absorbance units. d Total Carboxylic Acid Content. e Determined using vanillin as calibration standard.

The sum of the concentrations of acetic acid, formic acid, and levulinic acid (Table2) was 135 mM. As expected, the TCAC value was higher than that, viz. 153 mM. Lignocel- lulosic hydrolysates typically contain other aliphatic acids as well as aromatic carboxylic acids [30]. Individual carboxylic acids are not covered by the mass spectrometric method used in this investigation, as DNPH derivatization targets aldehydes and ketones. Studies by Larsson et al. [31] showed an inhibitory impact of aliphatic acids on S. cerevisiae yeast when the concentrations were above 100 mM. Thus, it is likely that carboxylic acids (Table2 ) contributed to the toxicity. However, detoxiﬁcation with sodium dithionite would not be expected to decrease the concentrations of aliphatic acids in the hydrolysates, as has been previously reported [22]. p-Benzoquinone was not detected (Table2). Previous studies have shown the presence of p-benzoquinone in steam-pretreated Norway spruce, even when less severe pretreatment conditions were used [17]. The concentration of p-benzoquinone is closely related to lignin degradation, especially at high temperature. The concentration of furan aldehydes (HMF and furfural) was slightly lower than in spruce hydrolysates in a previous study [17]. Although common in lignocellulosic hydrolysates due to dehydration of sugars, the molar toxicity of HMF and furfural is low [17]. According to a recent study by Wang et al. [32], concentrations of furfural and HMF above 31 mM and 39 mM, respectively, inhibit yeast. Therefore, concentrations around 20 mM (Table2) would hardly be inhibitory. Based on previous studies, conditioning of hydrolysates with reducing agents, such as sodium dithionite, would not be expected to cause any signiﬁcant changes in the concentrations of HMF and furfural [22,33]. In model experiments with only one inhibitor present at the time, the concentrations of HMF and Processes 2021, 9, 887 8 of 13

furfural decreased after treatment with dithionite [23], so the reaction may occur even if it is not favored in complex media such as lignocellulosic hydrolysates. The concentrations of six individual phenols (acetovanillone, coniferyl aldehyde, p-coumaraldehyde, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) are shown in Table2. Vanillin and coniferyl aldehyde were the most abundant of the phenols. This can be explained by the fact that both are guaiacyl-type substances, as is softwood lignin [34]. The concentration of total phenolics, estimated as vanillin equivalents using the Folin–Ciocalteu method, was 1.6 g/L, which corresponds to 11 mM. The combined concentration of the six phenols in Table2 is 0.6 mM. The reason for the discrepancy is that lignocellulosic hydrolysates contain a wide variety of phenolic and other aromatic substances [13,30], and that the subset shown in Table2 is just a small fraction of the total. Furthermore, common analytical procedures used (for example by Du et al. [30] and in this work) result in reports on mononuclear aromatics, whereas dimeric and oligomeric phenolic lignin fragments would also contribute to total phenolics as determined using the Folin–Ciocalteu method. The concentration of total phenolics would not be expected to be affected by conditioning with dithionite [22], as that treatment does not necessarily lead to a decrease in the content of phenolic hydroxyl groups although it does affect many individual phenols and causes detoxification by sulfonation [23]. TAC (Table2), which is based on UV absorption at 280 nm, covers both aromatic substances, such as phenols, and heteroaromatic substances, such as furans [29]. Thus, TAC reflects both lignin-degradation products and products of dehydration of sugars formed during the pretreatment. The first experimental series (Table1, Figure1) was aimed at the questions whether the pH (2.5, 5.5 or 8.5), the temperature (23 ◦C or 75 ◦C), or the mode of addition of dithionite (as a salt or as an aqueous solution) had any impact on the detoxification effect. For 24 and 36 h all combinations with dithionite (A2–A6) performed better than the combinations without dithionite (A1) (p ≤ 0.05) (Figure1). That was evident with regard to volumetric productivity (Q), specific productivity (q), and yield on initial sugar (Yini). It was somewhat less obvious with regard to the yield on consumed sugar (Ycon), which is reasonable considering that inhibitors might not necessarily affect how sugar is utilized by the cell. After 48 h, the values (Q, q, Yini,Ycon) for A1 were close to the values of the worst-performing of A2–A6, but they never came close to the values of the best-performing of A2–A6. The detoxification effect observed for dithionite agrees with the results of previous studies [22,24]. The results of A2–A4 indicate the impact of the pH when dithionite is added. A2 (pH 2.5) reflects the pH of a slurry after pretreatment, A3 (pH 5.5) the pH after conditioning with alkali to a pH that is suitable for enzymes and yeast, and A4 (pH 8.5) an alkaline pH, as alkaline pH improves the stability of dithionite [35]. At the beginning (24 h), there were no clear differences, but by the end (36 h, 48 h) A3 and A4 were always superior compared to A2 with regard to Q, q, and Yini (Table2). After 48 h, the superiority of A3 and A4 compared to A2 amounted to at least 20% for Q, 19% for q, and 19% for Yini. For Ycon, there were no clear differences. Thus, it is advantageous to wait with dithionite treatment until the pH has been adjusted to around 5.5 to be appropriate for biocatalysts, and there is no reason to raise the pH to an alkaline value. Processes 2021, 9, 887 9 of 13 ProcessesProcessesProcesses 2021 20212021,, , ,9 99,, , ,x x xx FOR FOR FORFOR PEER PEER PEERPEER REVIEW REVIEW REVIEWREVIEW 999 of ofof 13 1313

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QQQ48h48h48h 0.640.640.64 (0.05) (0.05)(0.05) 0.610.610.61 (0.06) (0.06)(0.06) 0.790.790.79 (0.06) (0.06)(0.06) 0.730.730.73 (0.08) (0.08)(0.08) 0.780.780.78 (0.09) (0.09)(0.09) 0.850.850.85 (0.08) (0.08)(0.08) 0.890.890.89 (0.05) (0.05)(0.05)

qqq24h24h24h 0.240.240.24 (0.01) (0.01)(0.01) 0.480.480.48 (0.02) (0.02)(0.02) 0.420.420.42 (0.02) (0.02)(0.02) 0.440.440.44 (0.03) (0.03)(0.03) 0.430.430.43 (0.03) (0.03)(0.03) 0.460.460.46 (0.01) (0.01)(0.01) 0.880.880.88 (0.09) (0.09)(0.09)

qqq36h36h36h 0.210.210.21 (0.01) (0.01)(0.01) 0.320.320.32 (0.01) (0.01)(0.01) 0.470.470.47 (0.02) (0.02)(0.02) 0.460.460.46 (0.02) (0.02)(0.02) 0.460.460.46 (0.01) (0.01)(0.01) 0.510.510.51 (0.06) (0.06)(0.06) 0.680.680.68 (0.06) (0.06)(0.06)

qqq48h48h48h 0.320.320.32 (0.01) (0.01)(0.01) 0.310.310.31 (0.01) (0.01)(0.01) 0.400.400.40 (0.02) (0.02)(0.02) 0.370.370.37 (0.01) (0.01)(0.01) 0.390.390.39 (0.01) (0.01)(0.01) 0.430.430.43 (0.01) (0.01)(0.01) 0.450.450.45 (0.01) (0.01)(0.01)

YYYini24hini24hini24hini24h 0.120.120.12 (0.01) (0.01)(0.01) 0.250.250.25 (0.01) (0.01)(0.01) 0.210.210.21 (0.01) (0.01)(0.01) 0.230.230.23 (0.01) (0.01)(0.01) 0.220.220.22 (0.01) (0.01)(0.01) 0.240.240.24 (0.01) (0.01)(0.01) 0.440.440.44 (0.02) (0.02)(0.02)

YYYini36hini36hini36hini36h 0.160.160.16 (0.01) (0.01)(0.01) 0.250.250.25 (0.01) (0.01)(0.01) 0.360.360.36 (0.01) (0.01)(0.01) 0.350.350.35 (0.01) (0.01)(0.01) 0.360.360.36 (0.01) (0.01)(0.01) 0.410.410.41 (0.01) (0.01)(0.01) 0.510.510.51 (0.02) (0.02)(0.02)

YYYini48hini48hini48hini48h 0.310.310.31 (0.01) (0.01)(0.01) 0.320.320.32 (0.01) (0.01)(0.01) 0.400.400.40 (0.01) (0.01)(0.01) 0.380.380.38 (0.01) (0.01)(0.01) 0.410.410.41 (0.01) (0.01)(0.01) 0.450.450.45 (0.01) (0.01)(0.01) 0.470.470.47 (0.01) (0.01)(0.01)

YYYcon24hcon24hcon24hcon24h 0.240.240.24 (0.01) (0.01)(0.01) 0.470.470.47 (0.01) (0.01)(0.01) 0.370.370.37 (0.01) (0.01)(0.01) 0.400.400.40 (0.01) (0.01)(0.01) 0.410.410.41 (0.02) (0.02)(0.02) 0.340.340.34 (0.01) (0.01)(0.01) 0.440.440.44 (0.02) (0.02)(0.02)

YYYcon36hcon36hcon36hcon36h 0.310.310.31 (0.01) (0.01)(0.01) 0.470.470.47 (0.02) (0.02)(0.02) 0.410.410.41 (0.03) (0.03)(0.03) 0.450.450.45 (0.01) (0.01)(0.01) 0.460.460.46 (0.02) (0.02)(0.02) 0.410.410.41 (0.02) (0.02)(0.02) 0.510.510.51 (0.03) (0.03)(0.03)

YYYcon48hcon48hcon48hcon48h 0.380.380.38 (0.02) (0.02)(0.02) 0.480.480.48 (0.03) (0.03)(0.03) 0.400.400.40 (0.01) (0.01)(0.01) 0.380.380.38 (0.01) (0.01)(0.01) 0.410.410.41 (0.02) (0.02)(0.02) 0.450.450.45 (0.03) (0.03)(0.03) 0.450.450.45 (0.02) (0.02)(0.02)

FigureFigureFigure 1. 1. 1. Fermentation FermentationFermentationFermentation results resultsresults results from fromfrom from fi fifirstrstrst ﬁrst experimental experimentalexperimental experimental series seriesseries series (A1–A (A1–A(A1–A (A1–AR).R).R).R). Codes CodesCodes Codes refer referrefer refer to toto conditions conditionsconditions to conditions detailed detaileddetailed detailed in inin Table TableTable in Table 1. 1.1. Cal- Cal-Cal-1. culationsculationsCalculationsculations are areare basedare basedbased based on onon the onthethe the results resultsresults results obtained obtainedobtained obtained after afterafter after 24 2424 24 h, h,h, h,36 3636 36 h, h,h, h, and andand and 48 4848 48 h hh hof ofof of fermentation. fermentation.fermentation. fermentation. Standard StandardStandard Standard deviations deviationsdeviations deviations are areare shown shown shown in in in parentheses.parentheses.parentheses. Color Color Color scheme: scheme: scheme: ,, , ,reference; reference;reference;reference; , , ,≥ ,≥ ≥≥67%67%67% of ofof reference; reference;reference; , , , ,<67% <67% <67%<67% to toto ≥ ≥≥33%33%33% of of of reference; reference; reference; ,, , ,<33% <33%<33%<33% of ofof reference.reference. reference. Q, Q, Q, g g g EtOH/(LEtOH/(LEtOH/(L × ×× × h); h);h);h); q, q,q, q, g gg EtOH/(gg EtOH/(gEtOH/(g EtOH/(g biomass biomassbiomass biomass × ×× h); h);×h); Yh); YYiniiniiniini,, Y, , gg ginig EtOH/gEtOH/g EtOH/g,EtOH/g g EtOH/g initialinitial initialinitial initial glucose;glucose; glucose;glucose; glucose; YY YYconconconcon,,, Y, gg ggcon EtOH/gEtOH/g EtOH/gEtOH/g, g EtOH/g consumedconsumed consumedconsumed consumed glucose.glucose. glucose.glucose. glucose.

◦ ◦ AAA comparison comparison comparison of ofof A3 A3A3A3 (23 (23(23 °C) °C)°C)C) and andand A6A6 A6A6 (75 (75 (75(75 °C) °C)C)°C) would would wouldwould indicate indicate indicateindicate whether whether whetherwhether the the thethe temperature temperature temperaturetemperature is isisrelevantis relevant relevantrelevant for for forfor dithionite dithionite dithionitedithionite treatment. treatment. treatment.treatment. In In InIn previous previo previoprevioususus studies studies studiesstudies [22 [22,24], [22,24],[22,24],,24], dithionite dithionite dithionitedithionite and and andand other other otherother reducing reduc- reduc-reduc- ingingagentsing agents agentsagents that that thatthat were were werewere investigated investigated investigatedinvestigated were were werewere added added addedadded at room at atat room roomroom temperature, temperature, temperature,temperature, but, but, but,but, asthe as asas the pretreatedthethe pretreated pretreatedpretreated ma- materialmaterialterialmaterial is warm is isis warm warmwarm after after afterafter the the thethe pretreatment, pretreatment, pretreatment,pretreatment, it might it itit mi mimightght beght anbe bebe optionan anan option optionoption to conduct to toto conduct conductconduct dithionite dithionite dithionitedithionite treatment treat- treat-treat- mentmentpriorment prior prior toprior cooling to toto cooling coolingcooling the pretreated the thethe pretreated pretreatedpretreated material material materialmaterial to a temperature to toto a aa temperature temperaturetemperature that is notthat thatthat harmful is isis not notnot harmful harmfulharmful for biocatalysts. for forfor bio- bio-bio- Furthermore, enzyme preparations used for saccharification of cellulose are often used catalysts.catalysts.catalysts. Furthermore, Furthermore,Furthermore, enzyme enzymeenzyme◦ preparations preparationspreparations used usedused for forfor saccharification saccharificationsaccharification of ofof cellulose cellulosecellulose are areare ofof-of- tentenatten temperatures used usedused at atat temperatures temperaturestemperatures around50 around aroundaroundC[9], 50 5050 or °C °C°C even [9], [9],[9], higher or oror even eveneven if thermostablehigher higherhigher if ifif thermostable thermostablethermostable cellulases cellulases cellulasescellulases are used [are 36areare]. Several industrial yeasts exhibit high vigor at 40 ◦C[37]. Even if both A3 (23 ◦C) and A6 usedusedused◦ [36]. [36].[36]. Several SeveralSeveral industrial industrialindustrial yeasts yeastsyeasts exhibit exhibitexhibit high highhigh vigorvigor vigorvigor atat atat 4040 4040 °C°C °C°C [37].[37]. [37].[37]. EvenEven EvenEven ifif ifif bothboth bothboth A3A3 A3A3 (23(23 (23(23 °C)°C) °C)°C) (75 C) performed well (Figure1), A6 was always slightly better for Q, q, and Y ini. For Ycon, andandand A6 A6A6 (75 (75(75 °C) °C)°C) performed performedperformed well wellwell (Figure (Figure(Figure 1), 1),1), A6 A6A6 was waswas always alwaysalways slightly slightlyslightly better betterbetter for forfor Q, Q,Q, q, q,q, and andand Y YYiniiniiniini... . the outcome of the comparison was variable. ForForFor Y YYconconconcon,,, , thethe thethe outcomeoutcome outcomeoutcome ofof ofof thethe thethe comparisoncomparison comparisoncomparison waswas waswas variable.variable. variable.variable. A comparison of A3 (addition as salt) and A5 (addition as aqueous solution) would AAA comparison comparisoncomparison of ofof A3 A3A3 (addition (addition(addition as asas salt) salt)salt) and andand A5 A5A5 (addition (addition(addition as asas aqueous aqueousaqueous solution) solution)solution) would wouldwould indicate whether the mode of addition of dithionite is relevant for dithionite treatment. indicateindicateindicate whether whetherwhether the thethe mode modemode of ofof addition additionaddition of ofof dith dithdithioniteioniteionite is isis relevant relevantrelevant for forfor dithionite dithionitedithionite treatment. treatment.treatment. A5 exhibited slightly higher values than A3 for Ycon, but otherwise A3 and A5 performed A5A5A5 exhibited exhibitedexhibited slightly slightlyslightly higher higherhigher values valuesvalues than thanthan A3 A3A3 for forfor Y YYconconconcon,,, , butbut butbut otherwiseotherwise otherwiseotherwise A3A3 A3A3 andand andand A5A5 A5A5 performedperformed performedperformed similarly. Thus, the mode of addition was of no significance (p > 0.05). similarly.similarly.similarly. Thus, Thus,Thus, the thethe mode modemode of ofof addition additionaddition was waswas of ofof no nono significance significancesignificance ( (p(pp >> >> 0.05).0.05). 0.05).0.05). At the end of the fermentation (48 h), A6 was always closest to the reference (AR) AtAtAt the thethe end endend of ofof the thethe fermentation fermentationfermentation (48 (48(48 h), h),h), A6 A6A6 was waswas always alwaysalways closest closestclosest to toto the thethe reference referencereference (AR) (AR)(AR) (Figure1). Compared to untreated (A1), the volumetric productivity of A6 was 33% higher, (Figure(Figure(Figure 1).1).1). ComparedComparedCompared tototo untreateduntreateduntreated (A1),(A1),(A1), thththeee volumetricvolumetricvolumetric productivityproductivityproductivity ofofof A6A6A6 waswaswas 33%33%33% the specific productivity was 34% higher, the yield on initial glucose was 45% higher, higher,higher,higher, thethethe specificspecificspecific productivityproductivityproductivity waswaswas 34%34%34% hihihigher,gher,gher, thethethe yieldyieldyield ononon initialinitialinitial glucoseglucoseglucose waswaswas 45%45%45% and the yield on consumed sugar was 18% higher. This demonstrates the efficiency of higher,higher, andand thethe yieldyield onon consumedconsumed sugarsugar wawass 18%18% higher.higher. ThisThis demonstratesdemonstrates thethe effi-effi- higher,higher,dithionite andand as thethe a detoxification yieldyield onon consumedconsumed agent, sugar evensugar at wawa asss low18%18% a higher.higher. concentration ThisThis demonstratesdemonstrates as 5 mM, which thethe effi-effi- was ciency of dithionite as a detoxification agent, even at as low a concentration as 5 mM, ciencyciencyused in ofof experimental dithionitedithionite asas series aa detoxificationdetoxification A. agenagent,t, eveneven atat asas lowlow aa concentrationconcentration asas 55 mM,mM, whichwhichwhichThe was waswas results used usedused in frominin experimental experimentalexperimental Series A encouraged series seriesseries A. A.A. further investigations of the significance of the treatmentTheTheThe results resultsresults temperature. from fromfrom Series SeriesSeries The A AA differences encouraged encouragedencouraged between further furtherfurther investigations A3investigationsinvestigations and A6 were of ofof thethe ratherthe significance significancesignificance small, and of ofof theone thethe treatmenttreatmentquestiontreatment was temperature. temperature.temperature. if treatment The TheThe at differences differences 75differences◦C without between betweenbetween dithionite A3 A3A3A3 and andandand addition A6 A6A6A6 were werewerewere had rather ratherrather anyrather effect. small, small,small,small, In and andandand Series one oneoneone B questionquestion(Tablequestion1), was dithionitewaswas if ifif treatment treatmenttreatment treatments at atat 75 7575 at °C °C°C 23 without withoutwithout◦C (B4) di didi andthionitethionitethionite 75 ◦C addition additionaddition (B1) were had hadhad again any anyanycompared, effect. effect.effect. In InIn Series SeriesSeriesbut this B BB (Table(Tabletime(Table with 1), 1),1), dithionite dithionitedithionite control fermentations treatments treatmentstreatments at atat without 23 2323 °C °C°C (B4) (B4)(B4) dithionite and andand 75 7575 °C both °C°C (B1) (B1)(B1) for were werewere 23 ◦C again againagain (B3) compared,and compared,compared, 75 ◦C (B2). but butbut this thisThethis timetimetime with withwith control controlcontrol fermentations fermentationsfermentations without withoutwithout dithionite dithionitedithionite both bothboth for forfor 23 2323 °C °C°C (B3) (B3)(B3) and andand 75 7575 °C °C°C (B2). (B2).(B2). The TheThe result (Figure2) showed that dithionite-treated cultures (B1 and B4) always (Q, q, Y ini, resultresultresult (Figure (Figure(Figure 2) 2)2) showed showedshowed that thatthat dithionite-treated dithionite-treateddithionite-treated cultures culturescultures (B1 (B1(B1 and andand B4) B4)B4) always alwaysalways (Q, (Q,(Q, q, q,q, Y YYiniiniiniini,,, , andand andand and Ycon) performed better than the corresponding control (B2 or B3). For Q, q, and Yini, B2 YYYconconconcon)) ) performed performedperformed better betterbetter than thanthan the thethe corresponding correspondingcorresponding control controlcontrol (B2 (B2(B2 or oror B3). B3).B3). For ForFor Q, Q,Q, q, q,q, and andand Y YYiniiniiniini,,, , B2B2 B2B2 andand andand

ProcessesProcessesProcessesProcesses2021 2021 20212021,,,9 , 9 9,9,, 887 , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 10101010 of of ofof 13 13 1313

andB3B3B3 performed performedperformed B3 performed similarly, similarly,similarly, similarly, and andand and the thethe the values valuesvalues values were werewere were always alwaysalways always <33% <33%<33% <33% of ofof of the thethe the values valuesvalues of of of the thethe referencereferencereference (Figure(Figure(Figure(Figure2 2).).2).2). With WithWithWith regard regardregardregard to tototo Y YYYconconconcon,, , B2B2B2B2 was waswaswas better betterbetterbetter than thanthanthan B3 B3B3B3 ( (p((ppp≤ ≤≤≤ 0.05). 0.05).0.05).0.05). B1B1B1 and andandand B4 B4B4B4 performed performedperformedperformed similarly.similarly.similarly. Importantly, Importantly, Importantly,Importantly, B4 B4 B4B4 performed performed performedperformed much much muchmuch better better betterbetter than than thanthan B2 B2 B2B2 ( p( ((ppp≤ ≤ ≤≤ 0.05), 0.05),0.05), demonstrating demonstratingdemonstrating that that thatthat it it itit ◦ waswaswaswas thethe thethe dithionitedithionite dithionitedithionite additionaddition additionaddition that that thatthat was was waswas important, important, important,important, and and andand not not notnot incubating incubating incubatingincubating the the thethe mixture mixture mixturemixture at at atat 75 75 7575 °C. °C.C.°C. Processes 2021, 9, x FOR PEER REVIEW 9 of 13

B1B1B1 B2B2B2 B3B3B3 B4B4B4 BRBRBR

QQQ24h24h24h 0.220.220.22 (0.02) (0.02)(0.02) 0.100.100.10 (0.01) (0.01)(0.01) 0.100.100.10 (0.01) (0.01)(0.01) 0.340.340.34 (0.01) (0.01)(0.01) 2.142.142.14 (0.19) (0.19)(0.19) A1 A2 A3 A4 A5 A6 AR QQQ36h36h36h 0.340.340.34 (0.01) (0.01)(0.01) 0.110.110.11 (0.01) (0.01)(0.01) 0.090.090.09 (0.01) (0.01)(0.01) 0.350.350.35 (0.02) (0.02)(0.02) 1.441.441.44 (0.12) (0.12)(0.12) Q24h 0.48 (0.08) 0.95 (0.10) 0.85 (0.07) 0.88 (0.09) 0.85 (0.09) 0.92 (0.10) 1.75 (0.18) QQQ48h48h48h 0.460.460.46 (0.02) (0.02)(0.02) 0.100.100.10 (0.01) (0.01)(0.01) 0.090.090.09 (0.01) (0.01)(0.01) 0.440.440.44 (0.02) (0.02)(0.02) 1.021.021.02 (0.11) (0.11)(0.11) Q36h 0.43 (0.07) 0.64 (0.07) 0.95 (0.08) 0.91 (0.09) 0.91 (0.10) 1.03 (0.17) 1.36 (0.18) qqq24h24h24h 0.110.110.11 (0.01) (0.01)(0.01) 0.060.060.06 (0.01) (0.01)(0.01) 0.050.050.05 (0.01) (0.01)(0.01) 0.170.170.17 (0.02) (0.02)(0.02) 1.111.111.11 (0.12) (0.12)(0.12) Q48h 0.64 (0.05) 0.61 (0.06) 0.79 (0.06) 0.73 (0.08) 0.78 (0.09) 0.85 (0.08) 0.89 (0.05) qqq36h36h36h 0.170.170.17 (0.01) (0.01)(0.01) 0.060.060.06 (0.01) (0.01)(0.01) 0.040.040.04 (0.01) (0.01)(0.01) 0.170.170.17 (0.01) (0.01)(0.01) 0.730.730.73 (0.03) (0.03)(0.03) q24h 0.24 (0.01) 0.48 (0.02) 0.42 (0.02) 0.44 (0.03) 0.43 (0.03) 0.46 (0.01) 0.88 (0.09) qqq48h48h48h 0.230.230.23 (0.01) (0.01)(0.01) 0.060.060.06 (0.01) (0.01)(0.01) 0.040.040.04 (0.01) (0.01)(0.01) 0.220.220.22 (0.01) (0.01)(0.01) 0.570.570.57 (0.07) (0.07)(0.07) q36h 0.21 (0.01) 0.32 (0.01) 0.47 (0.02) 0.46 (0.02) 0.46 (0.01) 0.51 (0.06) 0.68 (0.06) YYYini24hini24hini24h 0.050.050.05 (0.01) (0.01)(0.01) 0.030.030.03 (0.01) (0.01)(0.01) 0.020.020.02 (0.01) (0.01)(0.01) 0.070.070.07 (0.01) (0.01)(0.01) 0.510.510.51 (0.03) (0.03)(0.03) q48h 0.32 (0.01) 0.31 (0.01) 0.40 (0.02) 0.37 (0.01) 0.39 (0.01) 0.43 (0.01) 0.45 (0.01) YYYini36hini36hini36h 0.120.120.12 (0.01) (0.01)(0.01) 0.050.050.05 (0.01) (0.01)(0.01) 0.030.030.03 (0.01) (0.01)(0.01) 0.120.120.12 (0.01) (0.01)(0.01) 0.510.510.51 (0.05) (0.05)(0.05) Yini24h 0.12 (0.01) 0.25 (0.01) 0.21 (0.01) 0.23 (0.01) 0.22 (0.01) 0.24 (0.01) 0.44 (0.02) YYYini48hini48hini48h 0.220.220.22 (0.01) (0.01)(0.01) 0.050.050.05 (0.01) (0.01)(0.01) 0.040.040.04 (0.01) (0.01)(0.01) 0.210.210.21 (0.01) (0.01)(0.01) 0.510.510.51 (0.04) (0.04)(0.04) Yini36h 0.16 (0.01) 0.25 (0.01) 0.36 (0.01) 0.35 (0.01) 0.36 (0.01) 0.41 (0.01) 0.51 (0.02) YYYcon24hcon24hcon24h 0.470.470.47 (0.02) (0.02)(0.02) 0.400.400.40 (0.03) (0.03)(0.03) 0.340.340.34 (0.01) (0.01)(0.01) 0.390.390.39 (0.01) (0.01)(0.01) 0.510.510.51 (0.02) (0.02)(0.02) Yini48h 0.31 (0.01) 0.32 (0.01) 0.40 (0.01) 0.38 (0.01) 0.41 (0.01) 0.45 (0.01) 0.47 (0.01) YYYcon36hcon36hcon36h 0.510.510.51 (0.02) (0.02)(0.02) 0.410.410.41 (0.02) (0.02)(0.02) 0.330.330.33 (0.01) (0.01)(0.01) 0.510.510.51 (0.04) (0.04)(0.04) 0.510.510.51 (0.0.3) (0.0.3)(0.0.3) Ycon24h 0.24 (0.01) 0.47 (0.01) 0.37 (0.01) 0.40 (0.01) 0.41 (0.02) 0.34 (0.01) 0.44 (0.02) YYYcon48hcon48hcon48h 0.510.510.51 (0.02) (0.02)(0.02) 0.410.410.41 (0.02) (0.02)(0.02) 0.370.370.37 (0.02) (0.02)(0.02) 0.500.500.50 (0.03) (0.03)(0.03) 0.510.510.51 (0.02) (0.02)(0.02) Ycon36h 0.31 (0.01) 0.47 (0.02) 0.41 (0.03) 0.45 (0.01) 0.46 (0.02) 0.41 (0.02) 0.51 (0.03)

Ycon48h 0.38 (0.02) 0.48 (0.03) 0.40 (0.01) 0.38FigureFigureFigure (0.01) 2. 2.2. Results ResultsResults0.41 (0.02) of of of the thethe second secondsecond second0.45 experimental experimental(0.03)experimental experimental 0.45 series seriesseries (0.02) series (B) (B)(B) (B) of ofof of the thethe the fermentation fermentationfermentation fermentation of ofof detoxified ofdetoxifieddetoxified detoxiﬁed and andand and unde- unde-unde- un- detoxiﬁedtoxifiedtoxifiedtoxified Norway NorwayNorway Norway spruce sprucespruce spruce hydrolysates hydrolysateshydrolysates hydrolysates with withwith with S. S.S. cerevisiae cerevisiaecerevisiaeS. cerevisiae .. . The TheThe. codes The codescodes codes refer referrefer refer to toto conditions conditionsconditions to conditions detailed detaileddetailed detailed in inin Figure 1. Fermentation results from first experimental series (A1–AinTableTableTable TableR). 1.Calculations 1.Calculations1.CalculationsCodes1.Calculations refer to are areareconditions are based basedbased based on onon detailed on the thethe the results resultsresults results in Table obta obtaobta obtainedinedined ined1. Cal- after afterafter after 24 2424 24h, h,h, 36 h,3636 36h, h,h, and h,andand and 48 4848 48h hh of ofof h fermentation. offermentation.fermentation. fermentation. culations are based on the results obtained after 24 h, 36 h, andStandardStandard Standard48 h of fermentation. deviations deviationsdeviations are areare Standard shown shownshown in inindeviations pare pareparentheses.parentheses.ntheses.ntheses. are Colorshown Color Color sche sche schescheme:in me:me:me: ,, , reference; reference;reference; ,, , ≥ ≥≥67%67%67% of ofof reference; reference;reference; parentheses. Color scheme: , reference; , ≥67% of reference; ,,, , <67%<67% <67%<67% toto toto ≥≥ ≥33%33%33% of of of reference; reference; reference; , , , <33%<33% <33% of ofof reference. reference.reference. Q, Q,Q, g gg EtOH/(L EtOH/(LEtOH/(L × ×× h); h);h);h); q, q,q, q, g gg g EtOH/(g EtOH/(gEtOH/(g EtOH/(g × ×× h); h);×h); h); Y YYiniiniiniini Y,, , inig gg , g EtOH/(L × h); q, g EtOH/(g biomass × h); Yini, g EtOH/g initial glucose;EtOH/gEtOH/gEtOH/g Y con initial initialinitial, g EtOH/g glucose; glucose;glucose; consumed Y YYconconcon,, , ,g gg g EtOH/g EtOH/gEtOH/g EtOH/g glucose. consumed consumedconsumed consumed glucose. glucose.glucose. glucose.

◦ ◦ A comparison of A3 (23 °C) and A6TheTheThe (75 thirdthird third °C) experimental experimentalwouldexperimental indicate series,series, series, whether C,C, C, coveredcovered coveredcovered the temperature fourfo fofoururur treatmenttreatment treatmenttreatment temperaturestemperatures temperaturestemperatures (50(50 (50(50 °C, °C,C,°C, 7575 75 °C, °C,°C,C, ◦ ◦ is relevant for dithionite treatment.959595 In °C,°C,°C,C, previo andandand us 110110110 studies °C)°C)°C)C) andandand [22,24], threethreethree dithionite dithionitedithionitedithionite and concentrationsconcentrationsconcentrations other reduc- (5.0(5.0(5.0 mM,mM,mM, 8.78.78.7 mM,mM,mM, andandand 12.512.512.5 mM)mM)mM) ing agents that were investigated(Table(Table (Tablewere(Table 1added 1). ).1).1). The The TheThe at results results results resultsroom (Figure temperature,(Figure (Figure(Figure3 )3) 3)3) show show showshow but, that that thatthat as for thefo fofor therr pretreatedthe thethe lowest lowest lowestlowest dithionite dithionite dithionitedithionite concentration concentration concentrationconcentration (C1, (C1, (C1,(C1, C4, C4, C4,C4, C7, and C10), treatments at lower temperatures (50 ◦C and 75 ◦C) worked better (p ≤ 0.05) material is warm after the pretreatment,C7,C7,C7, and andand it C10), C10),C10), might treatments treatmentstreatments be an option at atat lower lowerlower to conduct temperatures temperaturestemperatures dithionite◦ (50 (50(50 treat- °C °C°C and and◦and 75 7575 °C) °C)°C) worked workedworked better betterbetter ( ((ppp ≤ ≤≤ 0.05) 0.05)0.05) than treatments at higher temperatures (95 C and 110 C), except for Ycon, which varied ment prior to cooling the pretreatedthanthanthan material treatments treatmentstreatments to a temperatureat atat higher higherhigher temperatur temperaturtemperatur that is noteseses harmful(95 (95(95 °C °C°C and andand for 110110bio-110 °C), °C),°C), except exceptexcept for forfor Y YYconconcon,, , which whichwhich◦ varied variedvaried catalysts. Furthermore, enzyme preparationswithinwithinwithinwithin aa aa comparativelycomparatively comparativelycomparatively used for saccharification low low lowlow range range rangerange (0.14–0.35 (0.14–0.35 (0.14–0.35(0.14–0.35 of cellulose g/g). g/g). g/g).g/g). are For ForFor of- the thethe lowest lowestlowest temperature temperaturetemperature (50 (50(50 °C, °C,°C,C, C2),C2),C2), ten used at temperatures aroundtherethere there50 °C waswaswas was[9], no nonoorno benefit benefitevenbenefitbenefit higher of ofofof raising raisinraisinraisin if thermostableggg the thethethe dithionite dithionitedithionitedithionite cellulases concentration concentrationconcentrationconcentration are tototo 8.78.78.7 mM,mM,mM,mM, butbutbutbut forforforfor all allallall other otherotherother temperatures there was a clear improvement when the concentration was raised from 5.0 to used [36]. Several industrial yeaststemperaturestemperaturestemperatures exhibit high there therevigorthere was wasatwas 40 a aa °C clear clearclear [37]. improvement improvementimprovement Even if both when whenA3when (23 the the the°C) concentration concentrationconcentration was waswas raised raisedraised from fromfrom 5.0 5.05.0 8.7 mM (p ≤ 0.05). After 48 h and for all parameters (Q, q, Yini,Ycon), C5 outperformed C8 and A6 (75 °C) performed well (Figuretototo 8.7 8.78.7 mM1), mMmM A6 ( ((pp pwas ≤ ≤≤ 0.05). 0.05).0.05). always After AfterAfter slightly 48 4848 h hh and andandbetter for forfor for all allall Q, parameters parametersparameters q, and Yini (Q, .(Q,(Q, q, q,q, Y YYiniiniiniini,, , Y YYconconcon),),), C5 C5C5 outperformed outperformedoutperformed and C11 (p ≤ 0.05), which in turn outperformed C2 (p ≤ 0.05). For the lowest temperature For Ycon, the outcome of the comparisonC8C8C8 and andand was C11 C11C11 variable. ( ((ppp ≤ ≤≤ 0.05), 0.05),0.05), which whichwhich in inin turn turnturn outperformed outperformedoutperformed C2 C2C2 ( ((ppp ≤ ≤≤ 0.05). 0.05).0.05). For ForFor the thethe lowest lowestlowest tempera- tempera-tempera- (50 ◦C, C3), there was a clear benefit of raising the dithionite concentration to 12.5 mM A comparison of A3 (additionturetureture as (50(50salt)(50 °C,°C,°C, and C3),C3),C3), A5 therethere there(addition waswaswas aasaa clearclear clearaqueous benefitbenefitbenefit solution) ofofof raisingraisingraising would thethethe dithionitedithionitedithionite concentrationconcentrationconcentration tototo 12.512.512.5 (p ≤ 0.05), but for all other temperatures there were no or relatively small benefits of raising indicate whether the mode of additionmMmMmM ( ((ppp of≤ ≤≤ 0.05), 0.05),0.05),dithionite but butbut for for foris all allrelevantall other otherother temperatures fortemperaturestemperatures dithionite there theretreatment.there were werewere no nono or oror relatively relativelyrelatively small smallsmall benefits benefitsbenefits of ofof the concentration from 8.7 to 12.5 mM (p > 0.05). At the beginning (24 and 36 h), the highest A5 exhibited slightly higher valuesraisingraisingraising than the A3thethe forconcentration concentrationconcentration Ycon, but otherwise from fromfrom 8.7 8.78.7 A3 to toto and12.5 12.512.5 A5mM mMmM performed ( ((ppp > >> 0.05). 0.05).0.05). At AtAt the thethe◦ beginning beginningbeginning (24 (24(24 and andand 36 3636 h), h),h), values for Q, q, and Yini were observed for C11 (8.7 mM and 110 C) and C12 (12.5 mM and similarly. Thus, the mode of additionthethethe highest highest highestwas of values novaluesvalues significance for forfor Q, Q,Q, q, q,q, (and andpand > 0.05). Y YYiniiniiniini were werewere observed observedobserved for forfor C11 C11C11 (8.7 (8.7(8.7 mM mMmM and andand 110 110110 °C) °C)°C) and andand C12 C12C12 110 ◦C). However, the highest values for hydrolysate-based cultures after 48 h were always At the end of the fermentation(12.5(12.5(12.5 (48 mM mMmM h), and andandA6 was110 110110 °C).°C).always°C). However, However,However, closest◦ thethetheto thehighest highesthighest reference values valuesvalues (AR) for forfor hydrolysate-based hydrolysate-basedhydrolysate-based cultures culturescultures after afterafter observed for C5 (8.7 mM and 75 C): Q, 0.85 g/(L × h); q, 0.43 g/(g × h); Yini, 0.39 g/g; (Figure 1). Compared to untreated484848 h h h (A1), were werewere thalways alwaysalwayse volumetric observed observedobserved productivity for forfor C5 C5C5 (8.7 (8.7(8.7 mMmMmMof A6 and andand was 75 7575 °C): °C):33%°C): Q, Q, Q, 0.85 0.850.85 g/(L g/(Lg/(L × ×× h); h);h); q, q,q, 0.43 0.430.43 ◦g/(g g/(gg/(g × ×× h); h);h); Ycon, 0.44 g/g. The second highest values were observed for C3 (12.5 mM and 50 C) (Q, q, higher, the specific productivity YYYwasiniiniiniini,, , 0.39 0.39 0.3934% g/g; g/g;g/g; hi gher, Y YYconconcon,, , 0.44 0.440.44the g/g. g/g.yieldg/g. The TheThe on second secondsecondinitial◦ highestglucose highesthighest values valueswasvalues 45% were werewere observed observedobserved for forfor C3 C3C3 (12.5 (12.5(12.5 mM mMmM and andand and Yini) and C9 (12.5 mM and 95 C) (Ycon). The lowest values after 48 h were observed higher, and the yield on consumed sugar was 18%iniiniiniini higher. This demonstrates the coneffi-concon for505050 °C) °C) C7°C) (Q, (5.0(Q,(Q, q, q,q, mM and andand and Y YY ) 95)) and andand◦C). C9 C9C9 The (12.5 (12.5(12.5 differences mM mMmM and andand 95 95 between95 °C) °C)°C) (Y (Y(Y the).).). The TheThe highest lowest lowestlowest and values valuesvalues lowest after afterafter values 48 4848 h hh were were werewere ciency of dithionite as a detoxification agent, even at as low a concentration as 5 mM, which was used in experimental series A. The results from Series A encouraged further investigations of the significance of the treatment temperature. The differences between A3 and A6 were rather small, and one question was if treatment at 75 °C without dithionite addition had any effect. In Series B (Table 1), dithionite treatments at 23 °C (B4) and 75 °C (B1) were again compared, but this time with control fermentations without dithionite both for 23 °C (B3) and 75 °C (B2). The result (Figure 2) showed that dithionite-treated cultures (B1 and B4) always (Q, q, Yini, and Ycon) performed better than the corresponding control (B2 or B3). For Q, q, and Yini, B2 and

ProcessesProcessesProcesses 2021 20212021,, , 9, 9,9, , x, x xx FOR FOR FORFOR PEER PEER PEERPEER REVIEW REVIEW REVIEWREVIEW 111111 of ofof 13 1313

Processes 2021, 9, 887 11 of 13

observedobservedobserved for forfor C7 C7C7 (5.0 (5.0(5.0 mM mMmM and andand 95 9595 °C). °C).°C). The TheThe di didifferencesfferencesfferences between betweenbetween the thethe highest highesthighest and andand lowest lowestlowest val- val-val- uesueslarge,ues were werewere as C7large, large,large, reached as asas C7 C7C7 12–13% reached reachedreached of 12–13% 12–13%12–13% the values of ofof the thethe for values values Q,values q, and for forfor Y Q, Q,Q,ini q, q,shownq, and andand Y YY byiniiniiniini shownshown C5,shownshown and byby byby 32% C5,C5, C5,C5, of andand andand the 32%32%value32% of ofof of the thethe Y con value valuevalueshown of ofof Y YYconcon byconcon shownshown shown C5.shown byby byby C5.C5. C5.C5.

C1C1C1 C2C2C2 C3C3C3 C4C4C4 C5C5C5 C6C6C6 C7C7C7 C8C8C8 C9C9C9 C10C10C10 C11C11C11 C12C12C12 CRCRCR

QQQ24h24h24h24h 0.160.160.16 0.170.170.17 0.240.240.24 0.220.220.22 0.340.340.34 0.230.230.23 0.110.110.11 0.240.240.24 0.210.210.21 0.170.170.17 0.740.740.74 0.620.620.62 1.571.571.57 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03) (0.03)(0.03)(0.03)(0.03) (0.11)(0.11)(0.11)(0.11) QQQ36h36h36h36h 0.500.500.50 0.180.180.18 0.640.640.64 0.460.460.46 0.530.530.53 0.340.340.34 0.130.130.13 0.430.430.43 0.330.330.33 0.190.190.19 0.740.740.74 0.830.830.83 1.091.091.09 (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03) (0.02)(0.02)(0.02)(0.02) (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) (0.12)(0.12)(0.12)(0.12) QQQ48h48h48h48h 0.350.350.35 0.300.300.30 0.700.700.70 0.610.610.61 0.850.850.85 0.640.640.64 0.100.100.10 0.570.570.57 0.440.440.44 0.180.180.18 0.590.590.59 0.670.670.67 0.870.870.87 (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03) (0.03)(0.03)(0.03)(0.03) (0.03)(0.03)(0.03)(0.03) (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.04)(0.04)(0.04)(0.04) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03) qqq24h24h24h24h 0.080.080.08 0.090.090.09 0.120.120.12 0.110.110.11 0.170.170.17 0.120.120.12 0.060.060.06 0.120.120.12 0.110.110.11 0.090.090.09 0.370.370.37 0.310.310.31 0.780.780.78 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) qqq36h36h36h36h 0.250.250.25 0.090.090.09 0.320.320.32 0.230.230.23 0.270.270.27 0.170.170.17 0.070.070.07 0.210.210.21 0.160.160.16 0.100.100.10 0.360.360.36 0.440.440.44 0.540.540.54 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) (0.03)(0.03)(0.03)(0.03) (0.03)(0.03)(0.03)(0.03) qqq48h48h48h48h 0.170.170.17 0.150.150.15 0.350.350.35 0.310.310.31 0.430.430.43 0.320.320.32 0.050.050.05 0.290.290.29 0.220.220.22 0.090.090.09 0.290.290.29 0.340.340.34 0.430.430.43 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) YYYini24hini24hini24hini24hini24h 0.040.040.04 0.040.040.04 0.060.060.06 0.050.050.05 0.080.080.08 0.050.050.05 0.030.030.03 0.060.060.06 0.050.050.05 0.040.040.04 0.180.180.18 0.150.150.15 0.340.340.34 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) YYYini36hini36hini36hini36hini36h 0.190.190.19 0.060.060.06 0.240.240.24 0.160.160.16 0.180.180.18 0.120.120.12 0.050.050.05 0.160.160.16 0.120.120.12 0.070.070.07 0.260.260.26 0.310.310.31 0.360.360.36 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) YYYini48hini48hini48hini48hini48h 0.170.170.17 0.140.140.14 0.360.360.36 0.270.270.27 0.390.390.39 0.300.300.30 0.050.050.05 0.290.290.29 0.220.220.22 0.080.080.08 0.290.290.29 0.320.320.32 0.380.380.38 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) YYYcon24hcon24hcon24hcon24h 0.280.280.28 0.290.290.29 0.270.270.27 0.150.150.15 0.250.250.25 0.200.200.20 0.200.200.20 0.250.250.25 0.270.270.27 0.220.220.22 0.300.300.30 0.350.350.35 0.400.400.40 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.02)(0.02)(0.02)(0.02) YYYcon36hcon36hcon36hcon36h 0.300.300.30 0.250.250.25 0.320.320.32 0.260.260.26 0.240.240.24 0.230.230.23 0.180.180.18 0.300.300.30 0.400.400.40 0.350.350.35 0.290.290.29 0.360.360.36 0.410.410.41 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03) YYYcon48hcon48hcon48hcon48h 0.280.280.28 0.280.280.28 0.380.380.38 0.300.300.30 0.440.440.44 0.340.340.34 0.140.140.14 0.330.330.33 0.430.430.43 0.260.260.26 0.300.300.30 0.340.340.34 0.440.440.44 (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.01)(0.01)(0.01)(0.01) (0.03)(0.03)(0.03)(0.03)

FigureFigureFigure 3. 3.3. Fermentation FermentationFermentation results results results from from from the the the third third third ex ex experimentalexperimentalperimentalperimental series series series (C1–CR). (C1–CR). (C1–CR). Codes Codes Codes refer referrefer to toto conditions conditions conditions detailed detaileddetailed in inin Table TableTable 1. 11.1.. CalculationsCalculationsCalculations are areare based basedbased on onon the thethe results resultsresults obtained obtainedobtained after afterafter 24 2424 h, h,h, 36 3636 h, h,h, and andand 48 4848 h hh of ofof fermentation. fermentation.fermentation. St St StandardStandardandardandard deviations deviations deviations are areare shown shownshown in inin ≥ ≥ parentheses.parentheses.parentheses. Color ColorColor scheme: scheme: scheme: ,, , ,reference; reference;reference;reference; , , , ,≥ ≥≥67%67%67% of ofof reference; reference;reference; , , , <67%, <67% <67%<67% to toto ≥ ≥≥33%33%33% of of of reference; reference; reference; ,, , ,<33% <33% <33%<33% of ofof reference. reference.reference. Q, Q, Q, g g g × × EtOH/(LEtOH/(LEtOH/(L × ×× h); h);h);h); q, q,q, q, g gg EtOH/(gg EtOH/(gEtOH/(g EtOH/(g × ×× h); h);h); Yh); YYiniiniiniini,, Y, , gg ginig EtOH/g EtOH/g EtOH/g,EtOH/g g EtOH/g initialinitial initialinitial initial glucose;glucose; glucose;glucose; glucose; YY YYconconconcon,,, Y, gg ggcon EtOH/gEtOH/g EtOH/gEtOH/g, g EtOH/g consumedconsumed consumedconsumed consumed glucose.glucose. glucose.glucose. glucose.

WithinWithinWithin the the the concentration concentration concentration and and and temperature temperature temperature ranges ranges ranges investigated, investigated, investigated, the the the results results results obtained obtainedobtained after 48 h indicate that both the dithionite concentration and the temperature have a large afterafterafter 48 4848 h hh indicate indicateindicate that thatthat both bothboth the thethe dithionite dithionitedithionite concentration concentrationconcentration and andand the thethe temperature temperaturetemperature have havehave a aa large largelarge impact on the effect, that using high temperatures (>75 ◦C) combined with a low dithionite impactimpactimpactimpact on on onon the the thethe effect, effect, effect,effect, that that thatthat using using usingusing high high highhigh temperatures temperatures temperaturestemperatures (>75 (>75 (>75(>75 °C) °C) °C)°C) combined combined combinedcombined with with withwith a a aa low low lowlow dithionite dithionite dithionitedithionite dosage can result in a very poor effect, that high temperatures (such as 95 ◦C and 110 ◦C) dosagedosagedosage can cancan result resultresult in inin a aa very veryvery poor poorpoor effect, effect,effect, that thatthat high highhigh temperatures temperaturestemperatures (such (such(such as asas 95 9595 °C °C°C and andand 110 110110 °C) °C)°C) are not needed to achieve an optimal effect, that the best effect is not necessarily achieved areareare not notnot needed neededneeded to toto achieve achieveachieve an anan optimal optimaloptimal effect, effect,effect, that thatthat thethe thethe bestbest bestbest effecteffect effecteffect isis isis notnot notnot necessarilynecessarily necessarilynecessarily achievedachieved achievedachieved with high dithionite dosage, and that high dithionite dosage might have adverse effects at withwithwith high highhigh dithionite dithionitedithionite dosage, dosage,dosage, and andand that thatthat high highhigh dithionite dithionitedithionite dosage dosagedosage might mightmight have havehave adverse adverseadverse effects effectseffects higher temperatures, such as 75 ◦C and 95 ◦C. Consequently, modulating dithionite dosage atatat higher higherhigher temperatures, temperatures,temperatures, such suchsuch as asas 75 7575 °C °C°C and andand 95 9595 °C. °C.°C. Consequently, Consequently,Consequently, modulating modulatingmodulating dithionite dithionitedithionite and treatment temperature emerges as a better alternative than consistently using high dosagedosagedosage and andand treatment treatmenttreatment temperature temperaturetemperature emerges emergesemerges as asas a aa better betterbetter alternative alternativealternative than thanthan consistently consistentlyconsistently using usingusing dithionite dosage or high temperature. highhighhigh dithionite dithionitedithionite dosage dosagedosage or oror high highhigh temperature. temperature.temperature. After 48 h, C4 (5.0 mM and 75 ◦C) performed clearly better (p ≤ 0.05) than C1 (5.0 mM AfterAfterAfter 48 4848 h, h,h, C4 C4C4 (5.0 (5.0(5.0 mM mMmM and andand 75 7575 °C) °C)°C) performed performedperformed clearly clearlyclearly better betterbetter ( (p(pp ≤ ≤≤ 0.05) 0.05) 0.05)0.05) than than thanthan C1 C1 C1C1 (5.0 (5.0 (5.0(5.0 mM mM mMmM and 50 ◦C) and C2 (8.7 mM and 50 ◦C), and C5 (8.7 mM and 75 ◦C) performed clearly andandand 50 5050 °C) °C)°C) and andand C2 C2C2 (8.7 (8.7(8.7 mM mMmM and andand 50 5050 °C), °C),°C), an ananddd C5 C5C5 (8.7 (8.7(8.7 mM mMmM and andand 75 7575 °C) °C)°C) performed performedperformed clearly clearlyclearly better (p ≤ 0.05) than C2 and C3 (12.5 mM and 50 ◦C). Thus, the results also indicate betterbetter ((pp ≤≤ 0.05)0.05)0.05) thanthanthan C2C2C2 andandand C3C3C3 (12.5(12.5(12.5 mMmMmM andandand 505050 °C).°C).°C). Thus,Thus,Thus, thethethe resultsresultsresults alsoalsoalso indicateindicateindicate that,that,that, that,betterat (p least ≤ 0.05)to some than extent,C2 and increased C3 (12.5 mM temperature and 50 °C). can Thus, compensate the results for aalso lower indicate dosage that, of atat leastleast toto somesome extent,extent, increasedincreased temperaturetemperature cancan compensatecompensate forfor aa lowerlower dosagedosage ofof so-so- sodiumat least to dithionite. some extent, increased temperature can compensate for a lower dosage of so- diumdiumdium dithionite. dithionite.dithionite. 4. Conclusions 4.4. ConclusionsConclusions 4. ConclusionsConditioning with dithionite was investigated with regard to the inﬂuence of pH, modeConditioningConditioningConditioning of addition with with ofwith dithionite, dithionite dithionitedithionite concentration was waswas investigat investigatinvestigat ofededed dithionite, with withwith regard regardregard and to toto temperature. the thethe influence influenceinfluence of Theofof pH, pH,pH, re- modemodesultsmode showedof ofof addition additionaddition clearly of ofof dithionite, dithionite,dithionite, that conditioning concentration concentrationconcentration should of ofof be dithionite, dithionite,dithionite, done after and andand the temperature. temperature.temperature. pH has been The TheThe raised results resultsresults to a showedshowedslightlyshowed clearly acidicclearlyclearly value that thatthat conditioning conditioningconditioning suitable for biocatalysts, should shouldshould be bebe done donedone and after after thatafterafter usingthe the thethe pH pH pHpH mildly has has hashas been been beenbeen alkaline raised raised raisedraised pH to to toto to a a aa slightly stabilizeslightly slightlyslightly acidicacidicdithioniteacidic value valuevalue is suitable suitablesuitable of no advantage. for forfor biocatalysts, biocatalysts,biocatalysts, The temperature and andand that thatthat usingusing usingusing and mildlymildly mildly themildly dosage alkalinealkaline alkalinealkaline of dithionite pHpH pHpH toto toto stabilizestabilize stabilizestabilize can be dithi-dithi- dithi-dithi- used oniteonitetoonite modulate is isis of ofof no nono the advantage. advantage.advantage. effect. The The TheThe ﬁnding temperature temperaturetemperature that increased an ananddd the thethe temperature dosage dosagedosage of ofof dithionite dithionite candithionite compensate can cancan be bebe for used usedused using to toto modulatemodulatelowermodulate concentrations the thethe effect. effect.effect. The TheThe of dithionite finding findingfinding that thatthat can incr incrincr potentiallyeasedeasedeased temperature temperaturetemperature be advantageous can cancan compensate compensatecompensate from an economical for forfor using usingusing lowerlowerlowerpointlower concentrationsconcentrations ofconcentrationsconcentrations view, as the costofof ofof dithionitedithionite dithionitedithionite for dithionite cancan cancan potentpotent potentpotent can beiallyiallyiallyially minimized bebe bebe advantageousadvantageous advantageousadvantageous and as the fromfrom fromfrom pretreated anan anan economicaleconomical economicaleconomical biomass pointpointispoint warm of ofof view, view,view, after as asas the the thethe pretreatment cost costcost for forfor dithionite dithionitedithionite and using can cancan be bebe temperatures minimized minimizedminimized and andand at as orasas the the aroundthe pretreated pretreatedpretreated 75 ◦C, biomass biomassbiomass therefore, is isis

Processes 2021, 9, 887 12 of 13

does not necessarily require heating. Further research in the ﬁeld can facilitate industrial implementation of new technology suitable for bioconversion of lignin-rich lignocellulosic biomass, such as softwood residues.

Author Contributions: Conceptualization, L.J.J., B.A. and D.I.; methodology, D.I. and S.S.; validation, D.I. and S.S.; formal analysis, D.I. and S.S.; investigation, D.I. and S.S.; resources, L.J.J. and B.A.; writing—original draft preparation, D.I.; writing—review and editing, L.J.J.; visualization, D.I. and L.J.J.; supervision, B.A. and L.J.J.; project administration, L.J.J.; funding acquisition, L.J.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Swedish Energy Agency (P41285-1, P47516-1), Kempe Foun- dations, and the Bio4Energy research environment (www.bio4energy.se, accessed on 23 April 2021). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: We thank SEKAB E-Technology for supplying the pretreated Norway spruce. Mass spectrometry was carried out using resources at the Swedish Metabolomics Centre Umeå. Conﬂicts of Interest: Three of the authors (L.J.J., B.A., and D.I.) are co-inventors of patents/patent applications in the area biochemical conversion of biomass. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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