Bioethanol Production from Sugarcane Press-Mud: Assessment of the Fermentation Conditions to Reduce Fusel Alcohol

: Within a bioreﬁnery context, bioethanol is a promising platform molecule since it can be used as raw material to produce a wide spectrum of valuable industrial products such as H 2 and light oleﬁns. However, the presence of impurities limits the conversion of bioethanol in these products. Herein, we aimed to determine the proper pretreatment and fermentation conditions to yield bioethanol with a low content of impurities, such as 3-methyl-1-butanol, by using sugarcane press-mud as feedstock. To do so, a Box-Behnken methodology was employed to select proper pretreatment and fermentation conditions. Factors assessed were temperature, stirring, and pH during fermentation of hydrolysates coming from two different pretreatment methods named as hydrothermal and acid hydrolysis. Results showed that the fermentation temperature should be kept between 26–30 ◦ C to assure at least 91 g/L ethanol. The fusel alcohol content would be reduced by 22% at 30 ◦ C, pH = 4.5, and 200 rpm if sugarcane press-mud is pretreated under acid hydrolysis conditions (T = 130 ◦ C, t = 1 h, 16 g HNO 3 /kg solid). Further studies should aim to integrate these conditions within a bioreﬁnery concept to yield valuable products such as H 2 and ethylene.


Introduction
Decarbonizing the economy by using biomass and biofuels is necessary to meet the agenda stated by the United Nations (UN) associated with sustainable development goals (SDG) [1]. Bioethanol is an interested feedstock since it can be used as a building block to produce a wide variety of important products, making it attractive for shifting from traditional oil refineries into biorefineries [2]. These products include hydrogen (H 2 ), light olefins, gasoline, and acetonitrile [3][4][5]. Bioethanol can be produced from a wide variety of biomass feedstocks including sugarcane, cornstarch, sugar beet, wheat, rye, lignocellulosic material, potato, cassava, sugarcane press-mud, and pineapple industrial wastes [3,[6][7][8]. However, the complex composition of bioethanol affects the production of H 2 and light olefins. For instance, bioethanol conversion to H 2 by ethanol steam reforming (ESR) is strongly influenced by the presence of impurities such as fusel alcohols, sulfur components, and acetic acid [3]. Besides, the presence of those impurities affects the conversion of ethanol into light olefins through dehydration reactions [9]. Since impurities have a detrimental effect on bioethanol upgrade, it is pertinent to seek alternatives to mitigate their formation. Those alternatives might include controlling the formation of impurities in upstream processes such as pretreatment, fermentation, and/or distillation, used for producing beverage spirits like vodka, where the optimum operating temperature ranges between 25 and 33 • C. Strain C-70 is employed due to its high attenuation and ethanol tolerance. Besides, it is used for low gravity fermentation with a wide variety of feedstocks such as sugarcane juice and molasses for producing rum [25]. This strain could be employed for fermentation carried out between 25 and 33 • C. Lastly, strain M-1 is highly employed for producing scotch whisky, whose optimum temperature ranges between 20 and 31 • C [26]. Afterwards, fermentation conditions were selected by using the response surface methodology to seek possible interaction among factors tested (i.e., temperature, pH, and stirring). This study gives insights on the alternatives to produce bioethanol while reducing the content of fusel alcohol for further integration within a biorefinery.

Evaluation of Yeast Strains
A one-factor experimental design method at three different levels was employed to determine the suitable yeast strains. Experiments were performed by three independent replicates. HG-1, M-1, and C-70 yeast strains (Fermentis by Lesaffre, Marcq-en-Baroeul, France) were evaluated in terms of ethanol and fusel alcohol concentration after the fermentation of hydrolysates coming from sugarcane press-mud pretreatment. To do so, 0.53 ± 0.04 g/L of dry yeast was inoculated in YPD (Yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) at 30 • C and 200 rpm until the concentration was about 1 × 10 7 colony forming unit per mL (CFU/mL). Fermentation of sugarcane press-mud hydrolysates by using those yeast strains was performed as described in Section 2.4. Quantification of yeast cell viability, carbohydrates conversion, ethanol, and fusel alcohol concentration was done according to procedures described in Section 2.6. Data were analyzed with SPSS software V26 (IBM, Armonk, NY, USA). A Duncan test (p < 0.05) was performed to determine significant differences among the yeast strains.

Sample Preparation
Sugarcane press-mud samples collected from the Andean region had a total solid content of 30 wt.% according to the characterization described in Section 2.1. Total solid content was determined by the oven method described by the National Renewable Energy Laboratory (NREL), who report that total solids represent the amount of solids remaining after drying the sample at 105 • C. [27]. To assess the effect of the fermentation conditions, sugarcane press-mud samples were diluted with water until a solid concentration of 29 wt.% was obtained. Under this condition, a high ethanol content was assured [10]. Then, samples were hydrolyzed in an autoclave (Tomy SX 700, Tomy Digital-Biology Co., LTD., Tokyo, Japan) at 130 • C for 1 h. Samples were hydrolyzed under two different hydrolysis conditions, the former without HNO 3 , hereafter named as hydrothermal, and the latter with an addition of 16 g of HNO 3 per dry solid, hereafter named as acid hydrolysis. The reaction was stopped by placing the sample in an ice water bath. After that, sugarcane press-mud was sieved through a 70-mesh sieve (212 µm). The recovered liquid was supplemented with peptone, yeast extract, MgSO 4 ·7H 2 O, Ca 3 (PO 4 ) 2 , and (NH 4 ) 2 SO 4 [10]. The supplemented culture medium was split into 3 similar fractions for each hydrolysis condition. The pH of the samples was adjusted to 4.5, 5.5, and 6.5 by using either 3M HNO 3 or 3M NaOH aqueous solutions. All samples were sterilized in an autoclave (Tomy SX 700, Tomy Digital-Biology Co., LTD., Tokyo, Japan) at 121 • C for 15 min.

Fermentation
Samples were fermented in 15 mL centrifuge tubes with a total volume of 4 mL and an inoculum size of 10 vol.%. Inoculum was prepared according to previous studies [14]. Dry Saccharomyces cerevisiae (Ethanol Red ® , Fermentis by Lesaffre, Marcq-en-Baroeul, France) was activated in water at 30 • C for 20 min. After that, yeast was cultivated in Potato Dextrose Agar (PDA) (Oxoid, New Hampshire, England) at 30 • C during 48 h and propagated in YPD culture (i.e., glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L) until yeast concentration reached about 10 7 CFU/mL. Fermentation was performed in a shaker (Innova 42, Eppendorf, Hamburg, Germany), at the stirring and fermentation conditions described in Section 2.5, for 24 h. After fermentation time, samples were characterized in terms of yeast cell viability, carbohydrates conversion, ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and acetic acid concentration according to the methods described in Section 2.7.

Experimental Design
A Box-Behnken design method (BBD) was employed to determine the effects of selected fermentation factors such as pH (A), stirring (B), temperature (C), and hydrolysis (D). This method allows for defining whether the factors or their interaction have an effect over the response variables with less experimental units, compared to a factorial experiment [28]. Walker and Stewart [29] prompt that yeast such as S. cerevisiae grows well between 20 and 30 • C and pH 4.5 and 6.5. Besides, a stirring rate up to 200 rpm is advisable to produce ethanol by fermentation [30]. Hence, each numerical factor, such as pH, stirring, and temperature, was set to 3 levels according to the above parameters: pH (4.5, 5.5, and 6.5); stirring (0 rpm, 100 rpm, and 200 rpm); and temperature (20 • C, 25 • C, and 30 • C). Those values agree with the optimal growth temperature (25-35 • C) suggested by the supplier. Otherwise, hydrolysis conditions were set as categorical factors to 2 levels, named as hydrothermal and acid hydrolysis, according to previous results [7]. Therefore, 34 independent experiments were carried out. Experimental data was adjusted to a second-order polynomial model as shown in Equation (1), where Y represents the response variable (i.e., yeast cell viability, carbohydrates conversion, ethanol content, and fusel alcohol content) and β i , β ii , and β ij represent the regression coefficients for intercept, linearity, and quadratic terms, respectively. In addition, i and j represent the factors A, B, and C [31].
The statistical analysis was performed in the software design expert, version V.7.1.0 (Stat-Ease Inc., Minneapolis, MN, USA). Besides, the significance of the model was determined through an analysis of variance (ANOVA) and the fit model was assessed by the correlation coefficient (R 2 ) and the lack of fit values.

Validation Results
Villeta samples were employed as feedstock to validate the experimental conditions found previously. Samples were pretreated and fermented under the same procedure described in Sections 2.2 and 2.3. Validation was performed in terms of yeast cell viability, carbohydrates conversion, ethanol, and fusel alcohol content after fermentation at the selected conditions after the pretreatment stage.

Analytical Methods
Yeast cell viability was quantified through the counting plate agar method [32]. Yeast cells were grown on PDA after 48 h incubation. Yeast cell viability, expressed as log units of colony-forming units per mL (Log CFU/mL), was calculated before and after fermentation. Differences between both states were reported. Carbohydrates were quantified spectrophotometrically by the phenol-sulfuric method by using microwells [10]. Absorbance was read at 490 nm in a microwell absorbance reader (iMarkTM, Bio-Rad, Hercules, CA, USA). The amount of carbohydrates was estimated by employing glucose (Carlos Erba, Cornaredo, Italy) as standard (0-500 µg/mL) and expressed as glucose equivalent per liter (GE/L). Carbohydrate conversion was calculated according to Equation (2), where Xc is the carbohydrate conversion, G 0 is the initial carbohydrate content, and Gf is the carbohydrate content after fermentation.
This study aimed to identify the effect of hydrolysis conditions and fermentation parameters, such as pH, temperature, and stirring, on ethanol and fusel alcohol. To do so, an initial screen of the yeast was done following by a Box-Behnken design method to determine the effect of the above variables. The Box-Behnken design method was employed to minimize the number of experimental runs and to identify possible interactions among the tested factors and their influence, mainly on the production of ethanol and fusel alcohol content.

Yeast Screening
The strains used in a fermentation can yield different distributions of fermentation products depending on the application [21,22]. Herein, it is important to employ a yeast strain that will achieve the higher amount of ethanol while lessening the fusel alcohol content. In addition, larger sugar consumption is a desirable characteristics during ethanol production [33]. Table 1 shows the performance of three different yeast strains (i.e., HG-1, M-1, and C-70) in terms of yeast cell viability, carbohydrate conversion, ethanol, and fusel alcohol concentration when sugarcane press-mud was hydrolyzed and fermented according to previous experiments [7]. Yeast cell viability is an outstanding parameter for designing industrial processes [32]. According to Table 1, the M-1 strain showed the highest yeast cell viability (2.03 ± 0.01 Log CFU/mL), followed by the HG-1 strain (1.89 ± 0.05 Log CFU/mL) and C-70 strain (1.69 ± 0.12 Log CFU/mL), which implies that the M-1 strain adapts better to the sugarcane press-mud fermentation culture than the other strains. Concerning carbohydrate conversion, all strains showed a similar behavior and consequently, the ethanol concentration was similar among strains according to the ANOVA test (p < 0.05), as shown in Table 1. Similar results were observed by Kawa-Rygielska et al. [34], who reported an ethanol concentration of 92 g/L after 72 h fermentation of sugar beet juice at a temperature of 30 • C, pH of 5.0, and 150 rpm with C-70 and HG-1 strains. Likewise, Askarbekov et al. [35] reported an ethanol concentration of 12 vol.% after the fermentation of sweet sorghum juice at 26 • C with C-70, HG-1, and M-1 yeast strains. Therefore, those strains produce similar ethanol concentration. Concerning the fusel alcohol concentration, 3-methyl-1-butanol concentration was significantly different among strains according to Duncan test (p < 0.05). For instance, HG-1 produced less of this alcohol (176.01 mg/L), followed by C-70 (228.69 mg/L) and M-1 (244.94 mg/L). Askarbekov et al. [35] also compared the production of those yeast strains during the fermentation of sweet sorghum juice at 26 • C. They reported that M-1 produces less of 3-methyl-1-butanol (183 mg/L), followed by HG-1 (288 mg/L) and C-70 (338 mg/L). Differences between both studies show that fermentation conditions and feedstock affect the production of 3-methyl-1-butanol when yeast strains such as C-70, HG-1, and M-1 are employed. Those streams were commonly employed for producing beverage spirits such as rum, vodka, and whisky [26]. Therefore, it is expected that the ethanol concentration would be similar, but the formation of side products, which are related to the flavor of the beverages, would be different among the strains. Different letters indicate significant differences among yeast strains (p < 0.05).
It is well known that 3-methyl-1-butanol is one of the main harsher impurities during SR [3,10]. Hence, HG-1 yeast strain was selected to investigate the effect of pretreatment and fermentation conditions on the amount of fusel alcohol since it allows a reduction in the amount of 3-methyl-1-butanol by at least 30%. In the upcoming section, we explore the effect of pretreatment and the main fermentation variables, such as temperature, pH, and stirring.

Effect of Pretreatment and Fermentation Conditions
The effect of hydrolysis conditions and fermentation parameters, such as pH, temperature, and stirring, were carried out by using a Box-Behnken design model. Tables 2 and 3 show the results for yeast viability, carbohydrates conversion, ethanol, and fusel alcohol concentration for the hydrothermal and acid hydrolysis pretreatment conditions, respectively. Table 4 shows the statistical results of the assessed factors, i.e., pH (A), stirring (B), temperature (C), and hydrolysis (D), over several response variables (i.e., yeast cell viability, carbohydrates conversion, ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and acetic acid). Mostly all response variables were adjusted to a quadratic model (Equation (1)) (p < 0.01 and not significant lack of fits, p > 0.05). However, the conversion of both acetic acid and HMF showed a significant lack of fit (p < 0.05), indicating that those models have a poor prediction ability. Despite the significant lack of fit, the difference between the adjusted and predicted R 2 was higher than 0.2. Hence, those models represent the response surface well and could be used to give insights on the effect of the factors over both the acetic acid and HMF conversion during fermentation [36,37]. Furthermore, the R 2 for all the response variables was higher than 0.80. Therefore, the assessed factors (i.e., pH, stirring, temperature, and hydrolysis) can explain 80% of the variability of the response variables. Table 5 shows the β coefficients of the quadratic response shown in Equation (1). The sign of the β coefficients indicates whether the effect of the factor is negative or positive [16]. Table 2. Experimental design for the effect of fermentation factors such as pH (A), stirring (B), and temperature (C) over the final change in yeast viability (Log CFU/mL), carbohydrates conversion, ethanol (g/L), 1-propanol (mg/L), 2-methyl-1-propanol (mg/L), 3-methyl-1-butanol (mg/L), and acetic acid (g/L) concentration after 24 h fermentation of sugarcane press-mud by using a S. cerevisiae strain (Ethanol Red, Fermentis ® ). Hydrolysis conditions: no HNO 3 addition; 130 • C, 1 h.  Table 3. Experimental design for the effect of fermentation factors such as pH (A), stirring (B), and temperature (C) over the final change in yeast viability (Log CFU/mL), carbohydrates conversion, ethanol (g/L), 1-propanol (mg/L), 2-methyl-1-propanol (mg/L), 3-methyl-1-butanol (mg/L), and acetic acid (g/L) concentration after 24 h fermentation of sugarcane press-mud by using a S. cerevisiae strain (Ethanol Red, Fermentis ® ).

Inhibitory Compounds
Previous studies showed that two inhibitory compounds (i.e., acetic acid and HMF), were mainly detected during pretreatment of sugarcane press-mud [7]. Table 6 shows that the initial concentration of acetic acid was 5.64 ± 1.15 g/L and 6.51 ± 0.74 g/L under hydrothermal and acid hydrolysis, respectively. Besides, the initial concentration of HMF was 3.40 ± 1.16 g/L under hydrothermal conditions. Whilst under acidic conditions, the HMF concentration was 6.64 ± 1.16 g·L −1 , and ANOVA showed that there was not a significant difference between pretreatment conditions on the production of fermentation inhibitors (p < 0.05). However, fermentation conditions affected the uptake of acetic acid and HMF, as shown in Table 4.  Table 4 shows that acetic acid conversion was influenced by all the main effects (i.e., pH (A), stirring (B), temperature (C), and pretreatment (D)). Besides, the interaction between fermentation temperature and pretreatment condition (CD) and the square of the temperature (C 2 ) had a significant effect on the acetic acid conversion. Table 5 portrays that among the significant variables, C 2 had the highest effect (β = −0.14), followed by temperature (C) (β = 0.15) and pH (A) (β = −0.08). The negative effect of the square temperature indicates that acetic acid conversion reached a maximum [38]. Figure 1a shows that the maximum acetic acid conversion was 33% at 26 • C, regardless of the pretreatment conditions when sugarcane press-mud was fermented at a pH of 5.5 and 100 rpm. Besides, the effect of pretreatment on acetic acid conversion was clear when fermentation was carried out at 20 • C. Rising fermentation temperature hindered the effect of pretreatment conditions. An opposite behavior was observed by Shang et al. [39] who reported that the higher the temperature was, the higher the accumulation of acetic acid and the lower the conversion during fermentation of lychee juice and using S. cerevisiae 2137 strain would be. Differences were associated with the yeast strain and medium nutrients that could affect the acetic acid uptake [40]. Herein, the initial concentration of carbohydrates was 146.10 ± 17.47 GE/L and 134.45 ± 38.60 GE/L after the pretreatment of sugarcane pressmud under hydrothermal and acid hydrolysis conditions, respectively, as shown in Table 6. In addition, the initial concentration of acetic acid was above 5.0 g/L, regardless of the pretreatment conditions. Furthermore, the culture was supplemented with yeast extract, peptone, MgSO 4 ·7H 2 O, Ca 3 (PO 4 ) 2 , and (NH 4 ) 2 SO 4 . Herein, two carbon sources were identified: sugar and acetic acid. Different enzymes such as PDC (pyruvate decarboxylase), ADH (alcohol dehydrogenase), ALDH (acetaldehyde dehydrogenase), ACS (acetyl-CoA synthase), and ILC (isocitrate lyase) act during the metabolism of acetic acid [39]. In the presence of acetic acid, ALDH, which catalyzes the oxidation of acetaldehyde into acetic acid, is suppressed, while the activity of PDC and ADH increases. PDC catalyzes the decarboxylation of pyruvate into acetaldehyde and ADH catalyzes the reduction of acetaldehyde into ethanol [39]. In addition, acetic acid is also converted into acetyl CoA by ACS. Acetyl CoA could be converted into acetaldehyde via EutE using cytosolic NADH [41]. The high availability of acetaldehyde might result in a larger concentration of ethanol.  Figure 2 shows the Spearman correlation coefficient (RHO) among response variables. RHO is employed when there is a mixture that is normally and non-normally distributed [10,42]. According to the Shapiro−Wilk test, shown in Figure 2, the following variables were not normally distributed: carbohydrate conversion (CC), ethanol concentration (ET), 1-propanol concentration (1P), acetic acid concentration (AA), acetic acid conversion, and HMF conversion (p < 0.05). On the other hand, yeast, 2-methyl-1-propanol concentration (2M1P), 3-methyl-1-butanol concentration, and total fusel alcohol concentration were normally distributed. Here, a positive correlation between ethanol and acetic acid conversion was observed (RHO = 0.674). The positive correlation showed that acetic acid could act as substrate to produce ethanol [42], as explained above. The consumption of acetic acid along with an increment on the ethanol production was also observed by Wei et al. [43] during the co-fermentation of synthetic culture based on glucose and using S. cerevisiae strain at 30 • C. Concerning the effect of pH, Figure 1b shows that at pH = 4.0, acetic acid conversion was about 40%. However, an increment in the pH resulted in a drop off of acetic acid conversion. The higher acetic acid conversion at low pH was associated with the amount of undissociated forms of acetic acid that permeates the yeast membrane. The fraction of undissociated forms is determined by the pKa, whose value is 4.6. This means that acetic acid dissociates when pH > pKa and hence, higher undissociated forms of acetic acid are found at low pH values. For instance, at pH = 4.0, the fraction of undissociated acid is 64% [44]. The permeation of undissociated forms of acetic acid acidifies the cytosol, reduces the metabolic activity during fermentation, and under extreme conditions, inhibits the yeast activity [41]. Yang et al. [44] showed when acetic acid content was higher than 7.5 g/L at a pH of 4.5, a considerable drop off was observed for the cell number during fermentation of glucose synthetic culture by using a commercial strain TG1348 of S. cerevisiae at 30 • C without shaking. Herein, the maximum initial content of acetic acid was 6.51 ± 0.74 g/L, as shown in Table 6, concluding that the amount of acetic acid present in the sugarcane press-mud hydrolysates did not have an inhibitory effect, since the RHO value between yeast cell viability and acetic acid conversion was −0.023 according to Figure 2.
Moreover, as shown in Table 4, HMF conversion was affected by the temperature (C) and the pretreatment (D). Besides, the interaction between the fermentation pH and temperature (AC) and the square of temperature (C 2 ) had a significant effect on HMF conversion. According to Table 5, the highest effect was observed for the temperature, followed by AC and the pretreatment conditions, since the β values were −0.14, −0.04, and 0.01, respectively. The negative value of the β coefficient for the squared term of temperature (C 2 ), i.e., −0.14, represents a maximum conversion of HMF. This result can be clearly observed in Figure 1c, where at 27 • C, a maximum conversion of HMF was observed when sugarcane press-mud was fermented at a pH of 6.5. Akillioglu et al. [45] reported complete conversion of HMF by using ground malt, as raw material, and S. cerevisiae at 30 • C for 24 h. HMF is reduced by the enzymatic activity of alcohol dehydrogenase that is affected by fermentation parameters such as pH and temperature [39,46,47]. Besides, according to Figure 2, HMF conversion was better correlated with yeast cell viability than acetic acid conversion whose RHO were 0.308 and −0.023, respectively. Hence, HMF had a stronger inhibition effect than acetic acid. The positive value of RHO indicated that the lower the HMF conversion was, the lower the yeast cell viability would be. Lee et al. [48] reported a similar result during the fermentation of lignocellulosic hydrolysates by using S. cerevisiae K35 strains at 30 • C.

Yeast Cell Viability
The initial yeast cell viability for all the experiments was 6.56 ± 0.13 Log CFU/mL. According to statistical results described in Tables 4 and 5, yeast cell viability was mainly affected (p < 0.05) by the interaction between pH and temperature (AC, in Table 4), the interaction between pH and hydrolysis (AD, in Table 4) and the quadratic terms of both pH (A 2 , in Table 4) and temperature (C 2 , in Table 4). The β coefficients depicted in Table 5 show that the square term of temperature (C 2 , β = −0.21) had the strongest influence, followed by the square term of pH (A 2 , β = −0.16), the interaction between pH and temperature (AC, β = 0.11), and the interaction between pH and hydrolysis (AD, β = 0.06). Figure 3a shows that there is a maximum yeast cell viability at 25 • C and at a pH of 5.5 when sugarcane press-mud was pretreated under hydrothermal conditions. Thus, eliciting that significant effect of the square term of temperature reflects a maximum yeast cell viability at a specific temperature and pH value. A similar result was observed by Heard and Fleet [49] and Lu et al. [50]. Figure 3 shows an average increment of 2.14 ± 0.22 Log CFU/mL and 1.97 ± 0.23 Log CFU/mL when sugarcane press-mud was pretreated under hydrothermal and acid hydrolysis conditions, respectively. A decrease in the yeast cell viability was observed at higher temperatures, possibly associated with the accumulation of ethanol that has a toxic effect on the yeast cell [50]. However, a successful adaptation of the microorganism is represented by an increment in almost 2 Log units [51]. Therefore, S. cerevisiae was fruitfully adapted, regardless of the pretreatment conditions, to the classical yeast medium formed by peptone, yeast extract, MgSO 4 ·7H 2 O, Ca 3 (PO4) 2 , and (NH 4 ) 2 SO 4 , where the carbon source was sugarcane press-mud, an agro-industrial residue with high content of fermentable sugars such as sucrose and glucose.  Table 6 shows that the initial carbohydrate content was 146.10 ± 17.47 GE/L and 134.45 ± 38.60 GE/L after the pretreatment under hydrothermal and acid hydrolysis conditions, respectively. ANOVA depicted that there was not a significant difference between the carbohydrate content for both pretreatment conditions. This result agrees with previous results obtained during fermentation of sugarcane press-mud under different pretreatment conditions [7]. Sugarcane press-mud is a feedstock rich in carbohydrates (i.e., 22.95 wt.% carbohydrates), as shown in Section 2.1. Sucrose is the main sugar present in sugarcane press-mud, as has been previously reported [7,52]. The content of fiber is low in comparison with the carbohydrate content. Indeed, fiber concentration in sugarcane press-mud is about 2.0 wt.%, as shown in Section 2.1. Fiber refers to cellulose, hemicellulose, and lignin [53]. Under acid hydrolysis conditions, fiber fractions could decompose, thus producing hexoses such as glucose and galactose and pentoses such as xylose and arabinose [54]. However, due to the low content of fiber in comparison with other carbohydrates, the contribution of fermentable sugars from fiber decomposition will be negligible. The pretreatment of sugarcane press-mud increases the content of reducing sugar, such as glucose and fructose, to ease the fermentation process [7]. Under acid hydrolysis conditions, the amount of reducing sugar will be higher than under hydrothermal conditions due to the activity of H + . Figure 4 shows that carbohydrate conversion during fermentation at 20 • C of hydrolysates obtained through acid hydrolysis of sugarcane press-mud was higher than that observed after the fermentation of hydrolysates obtained through hydrothermal conditions. However, at temperatures above 25 • C, carbohydrate conversion was similar, regardless of the pretreatment condition. As described above, sucrose and glucose are the main sugars present in the hydrolysates from sugarcane press-mud, while sucrose must be converted into glucose and fructose by an invertase enzyme [55]. Glucose is directly metabolized by yeast through the glycolytic pathway [56]. Glycolytic enzymes such as phosphoglucose isomerase, phosphofructokinase, and pyruvate kinase are not strongly affected by temperature [57], while invertase activity is highly affected by temperature [58]. Therefore, at a higher glucose concentration and lower temperature, yeast will uptake carbohydrates faster than when there are at low glucose levels and high sucrose content. However, an increment on the fermentation temperature will rise the invertase activity, thus producing glucose and keeping similar carbohydrate conversion. On the other hand, during fermentation of sugarcane press-mud, acetic acid and carbohydrates served as carbon source to yield ethanol. However, carbohydrates conversion was always higher than acetic acid. Those sugars are firstly uptaken by yeast, and after glucose exhaustion, acetic acid serves as a carbon source [59]. Figure 2 shows that carbohydrate conversion was strongly correlated with ethanol production since RHO was higher than 0.9. Carbohydrates are the main substrate employed by yeast to produce ethanol.

Ethanol Concentration
Ethanol is the main metabolite produced during fermentation of feedstocks with high content of hexoses. According to Table 4, the following main effects, interaction effects, and quadratic effects had a significant influenced on the ethanol concentration (p < 0.05): stirring (B), temperature (C), pretreatment (D), the interaction between stirring and fermentation temperature (BC), the interaction between fermentation temperature and pretreatment conditions (CD), and the square of temperature (C 2 ). Among them, C 2 had the greatest influence, which is possibly ascribed to the enzyme activities that are sensitive to temperature [60]. Figure 5 shows that a maximum ethanol concentration was observed at 28 • C, regardless of the pretreatment conditions, which is within the optimum range temperature to carry out fermentation for free S. cerevisiae cells [61,62]. Moreover, at 20 • C, a clear effect of the pretreatment was observed. For instance, at said temperature, the ethanol concentration was 50 and 30 g/L for the hydrolysates obtained under hydrothermal and acid hydrolysis conditions, respectively. However, an increment of the fermentation temperature hindered the effect of the pretreatment conditions. The outstanding effect of temperature on the ethanol concentration was associated with the activity of key enzymes that are involved in the central metabolism of S. cerevisiae [50,63]. Concerning the effect of stirring, Figure 5a,b shows that stirring had a more notable effect at 20 • C, since an increment of the stirring speed caused a rise in the ethanol concentration. Stirring is an important fermentation variable because it promotes homogeneity and allows contact between the free yeast cells, suspended in the fermentation culture, and the substrate [62]. Poor stirring leads to a low mass transfer coefficient and consequently affects fermentation performance [64]. At higher fermentation temperatures (>26 • C), the effect of mechanical agitation is hampered, possibly ascribed to the formation of CO 2 . Under aerobic conditions and high glucose concentration, S. cerevisiae displays the Crabtree effect. This effect is associated with the use of both respiration and fermentation pathways to produce ATP [65]. Mixing the culture will improve mass transfer, consequently enhancing the dissolving of oxygen and boosting the Crabtree effect [66]. Besides, CO 2 bubbles, produced metabolically by yeast, create an agitation environment that might favor the mass transfer effects during fermentation [64,67]. Since CO 2 production is metabolically correlated with ethanol, the higher the fermentation temperature is, the higher the ethanol production, and consequently the higher the CO 2 will be. Hence, at temperatures higher than 26 • C, an agitation effect might be associated with CO 2 , and the effect of external agitation becomes negligible.

Fusel Alcohol Concentration
Fusel alcohols are 1-propanol, 2-methyl-1-propanol, and 3-methyl-1-butanol. These alcohols are mainly yielded during fermentation of sugarcane press-mud, 3-methyl-1butanol being the most representative [7]. According to Table 4, the main effects (i.e., pH (A), stirring (B), temperature (C), and pretreatment conditions (D)) were significant over all the tested fusel alcohols, except pH which did not have an impact over 1-propanol (p < 0.10). Moreover, the interaction between pH and pretreatment (AD) influenced the production of 1-propanol, and the square of temperature (C 2 ) had a significant effect over all tested alcohols (p < 0.05). The β coefficients in Table 5 indicate that the main effects have a positive effect on the production of fusel alcohol. For instance, the higher the pH was, the higher the fusel alcohol production would be. A similar behavior of pH fermentation on fusel alcohol was observed by Arsahd et al. [15], who reported a significant increment (p < 0.05) on the fusel alcohol concentration during the fermentation of blackstrap molasses at 30 • C under non-aerated conditions. The slight alkaline conditions of the culture medium boost the activity of the enzymes involved in the Ehrlich pathway, thus enhancing the production of fusel alcohol at higher pH values [68]. Table 5 shows that among the main factors, the square term of temperature (C 2 ) had the greatest influence on the production of fusel alcohol (β = −135), followed by pretreatment conditions (β = 51.1), stirring (β = 43.5), and pH (β = 35.7). Since pH did not influence the ethanol production during fermentation, as shown in Table 3, we plotted contour diagrams to describe the effect of pH, temperature, and pretreatment on the concentration of main fusel alcohol. Figure 6 shows that the highest production of main fusel alcohol was found at a temperature of about 26 • C. The effect of temperature on fusel alcohol has been previously described by other authors [16,63,69] and is associated with the gene expression of BAT1, which encodes for the enzymes involved in the transamination of amino acids, the first reaction in the Ehrlich pathway [12,13]. Moreover, the effect of pretreatment is clear on the fusel alcohol concentration. For instance, the production of higher alcohols was higher under hydrothermal conditions (Figure 6a-c) than under acid hydrolysis conditions (Figure 6d-f). This effect was previously reported during the fermentation of hydrolysates of sugarcane press-mud and was possibly linked to enzyme competition between the fusel aldehydes, which are intermediary in the Ehrlich metabolic pathway, and the HMF [7].

Condition Selection
Based on the above description and the quadratic models, a suitable fermentation condition to produce bioethanol without affecting the ethanol concentration but reducing the amount of fusel alcohol will be pH: 4.5, temperature: 30 • C, and 200 rpm. Furthermore, sugarcane press-mud must be pretreated under acid hydrolysis conditions. At these conditions, the ethanol concentration will be increased by almost 7%, while reducing the fusel alcohol concentration by almost 22%. In addition, the concentration of 3-methyl-1-butanol, the harsher component in ESR, would be dwindled by 29%. To validate this result, a second sugarcane press-mud sample was collected, pretreated, and fermented according to the selected conditions to validate the results. In the upcoming section, we present those results.

Model Validation
Sugarcane press-mud from Villeta was employed as feedstock to validate the model obtained from the previous experiment under laboratory conditions. Table 7 displays the initial concentration of carbohydrates on both fermentation samples, as well as the conditions of pH, temperature, and stirring involved in each experiment. For the columns labeled Model, the predicted value for yeast cell viability is expressed in Log CFU/mL, carbohydrate conversion, and 3-methyl-1-butanol concentration in mg/L, while Villeta columns express the experimental value obtained for each parameter in comparison to the expected result. According to Table 7, the yeast cell viability for the validation experiments were 1.89 Log CFU/mL and 1.74 Log CFU/mL, contrasted with the anticipated 2.08 Log CFU/mL and 1.57 Log CFU/mL for hydrothermal and acid hydrolysis pretreatment, respectively. The error between the model and experimental results was 9.1% and 10.8% for hydrothermal and acid hydrolysis, correspondingly. Despite the difference between the model and the experimental values, a similar trend was observed. This means that when sugarcane press-mud is pretreated under acid hydrolysis conditions, the yeast cell viability is more affected than when it is pretreated under hydrothermal conditions. Concerning carbohydrate conversion, the validation experiment showed best fit to the model. Indeed, for hydrothermal and acid hydrolysis pretreatment, the experimental value was 95.6% in both cases. Besides, the error associated for each pretreatment was 0.5% and 4.2%, respectively. Hence, the model was able to predict this parameter.
Ethanol concentration, as one of the most important parameters, was also assessed during validation, yet neither model was able to predict the result from Villeta's sugarcane press-mud, as the errors between the experiments and the model predictions were 20.2% and 7.4%, respectively. Furthermore, ethanol concentration after acid hydrolysis decreased in 7.7%, contrasted with hydrothermal pretreatment. However, according to the ANOVA test (p < 0.05), there was not a significant difference between both conditions when sugarcane press-mud was employed as feedstock to produce bioethanol.
On the contrary, the production of 3-methyl-1-butanol was significantly affected (p < 0.05), but the model also failed to predict 3-methyl-1-butanol concentration, showing errors of 40.7% and 40.0% in validation experiments for hydrothermal and acid pretreatment conditions, respectively. However, a reduction in 3-methyl-1-butanol concentration of 28.4% was observed in contrast to the expected decrease of 29.2%. This result confirms that acid pretreatment does reduce the fusel alcohol concentration after fermentation as expected.
Based on the above results, it is possible to elicit that the model is not able to accurately forecast the results from biomasses obtained in different locations. However, the model will predict the expected behavior between two different conditions. Hence, the model presented in this study might be employed as a tool to select suitable conditions to reduce the amount of 3-methyl-1-butanol, which is the main fusel alcohol obtained after fermentation and has a detrimental effect on ESR and even on dehydration reactions to produce ethylene.

Future Perspectives
A biorefinery is defined as facility where organic materials from renewable sources are converted into energy, fuels, chemicals, and materials for industrial purposes [70]. Due to the complexity of organic materials, living organisms, such as fungus, bacteria, and yeasts, are commonly employed to produce the aforecited goods. However, during the conversion of organic material by using those organisms, a wide spectrum of components could be also yielded. For example, bioethanol production from fermentable sugars yield ethanol as main product, but side products such as fusel alcohol, glycerol, and acetic acid are yielded during this process [3]. Those side products have a negative impact on the upgrading of bioethanol into more valuable products such as syngas and olefins. Therefore, mitigating those side products might increase the production of syngas and olefins. This study showed that it is possible to decrease and control the formation of those side products by using different pretreatment and fermentation conditions. The novelty of this study was to determine new alternatives that could be employed within process design to increase the production of valuable goods such as syngas and ethylene that are derived from bioethanol.
In addition, future studies should aim to integrate those strategies to reduce side products during the fermentation of feedstocks with high content of fermentable sugars such as sugarcane press-mud, sweet sorghum, and sugar beet, among others. The depletion of side products will increase syngas and olefins production and consequently boost the economic and social development of rural areas in Latin American countries, where biomass is an interesting alternative to decarbonize the economy.

Conclusions
A strategy to reduce the concentration of fusel alcohol during bioethanol production from sugarcane press-mud was developed by using the Box-Behnken response surface methodology. Accordingly, fermentation temperature had the greatest influence on the all the tested variables (p < 0.05). Moreover, the pH should be 4.5 because fusel alcohol can be reduced without affecting the ethanol concentration. Lastly, sugarcane press-mud should be pretreated under acid hydrolysis conditions to dwindle the concentration of fusel alcohol by almost 30% without affecting the ethanol concentration at 30 • C, 200 rpm, and pH = 4.5. Further studies must integrate these conditions along with the upgrading of bioethanol into H 2 and light olefins to develop a suitable biorefinery concept.