Identification and Quantification of Volatile Compounds Found in Vinasses from Two Different Processes of Tequila Production

Vinasses are the main byproducts of ethanol distillation and distilled beverages worldwide and are generated in substantial volumes. Tequila vinasses (TVs) could be used as a feedstock for biohydrogen production through a dark fermentative (DF) process due to their high content of organic matter. However, TV components have not been previously assayed in order to evaluate if they may dark ferment. This work aimed to identify and quantify volatile compounds (VC) in TV and determine if the VC profile depends upon the type of production process (whether the stems were initially cooked or not). TVs were sampled from 3 agave stems with a not-cooking (NC) process, and 3 agave stems with a cooking (C) process, and volatile compounds were determined by gas chromatography coupled with mass spectrometry (GC–MS). A total of 111 volatile compounds were identified, the TV from the cooking process (C) showed the higher presence of furanic compounds (furfural and 5-(hydroxymethyl) furfural) and organic acids (acetic acid and butyric acid), which have been reported as potential inhibitors for DF. To our knowledge, this is the first description of the VC composition from TVs. This study could serve as a base for further investigations related to vinasses from diverse sources.


Introduction
Vinasses are produced as byproducts of the fermentation and distillation of ethanol [1,2] from sugarcane (Saccharum officinarum L.) in South America [3], beet molasses (Beta vulgaris ssp. vulgaris var. altissima Döll) in Europe [4,5], or from the distillation of fermented beverages, such as mezcal, bacanora, and tequila in Mexico [6][7][8]. Large quantities of vinasses are produced worldwide; on average, 12-15 L vinasses are obtained for each liter of ethanol produced [9]. According to the Renewable Fuel Association (http://www.ethanolrfa.org), global ethanol production was 25.7 billions of gallons in 2016, leading to approximately 2.56 × 10 12 L of vinasses, which are released without any treatment into agricultural soils or water bodies [9,10]. Regardless of the ethanol production process or the sugar source, these may have similar characteristics. They are complex wastewaters and have high biological oxygen demand (BOD) and chemical oxygen demand (COD), 16-45 g/L and 26-91 g/L, respectively, pH ranges from 3 to 5, high concentrations of suspended solids (2-8.4 g/L), volatile solids (1.1-9 g/L), and phenols (0.04-0.08 g/L). Occasionally they may contain heavy metals, and display a dark brown color [2,4,[11][12][13]. The toxic and recalcitrant nature of this effluent can produce negative environmental impacts like anoxia, eutrophication, and the death of aquatic microorganism and wildlife [13,14].
Tequila distilleries are one of the most important agro-industries [15] producing a popular alcoholic beverage called tequila, which is obtained from the fermentation of hydrolyzable sugars from the stems of Agave tequilana Weber var. Azul [10,14]. The agave juice or "mieles de agave" (as locals call it) extraction process requires fructans to be transformed by thermic treatments into fermentable sugars (fructose and glucose), so they can be subsequently fermented by yeast [16,17]. Currently, two processes are used to accomplish sugar hydrolysis. Some distilleries employ traditional methods, i.e., (cooking (C) of agave stems, whereby the mature stems (with no leaves) of agave, so-called "piñas", are cooked in ovens or autoclaves with steam injection (0.5-1.4 kg/cm 2 ) at 95-120 • C for approximately 48 h or 8-12 h. Once the agave stems are cooked, they are transferred on conveyor belts to a mill with rotatory knives where they are shredded and washed under pressure with potable water to dissolve the sugars. Finally, the stems are placed in a press to extract the agave juice, containing hydrolyzable sugars, soluble, and insoluble compounds which are generated during the cooking process [6,[18][19][20]. However, most producers use a "not-cooking" (NC) process, i.e., [20,21], they used equipment called a "diffuser" to obtain the raw agave juice with hot water (80 • C) directly from previously shredded raw agave stems. Afterwards, the raw agave juice is hydrolyzed through heat (80-85 • C) in acidic conditions (pH 1.8-3) and thermal conditions [18,20,22]. The agave juice obtained either from cooking or not-cooking are submitted to a fermentation process, where hydrolizable sugars are biotransformed to ethanol, carbon dioxide, and other organoleptic compounds such as volatile compounds (esters, aldehydes, ketones, and furans, among others) [23]. Once fermentation is completed, the fermented juice, "must", is transferred to a distiller, where two tandem distillations are performed to obtain tequila. After the final distillation, vinasses are generated as the residual liquid [22,23].
Both processes (cooking or not-cooking the agave stems) might influence the composition of distillation wastewater. Like other vinasses, tequila vinasses (TVs) can also be used as a feedstock for a dark fermentative process (DF) to produce hydrogen, due to its high organic matter content. However, the vinasses' organic matter is not entirely used for biohydrogen production during DF [7,[24][25][26]. Nevertheless, there is not sufficient information about the presence of volatile or complex organic compounds in vinasses, and even less in tequila vinasses. Some work has tried to determine the composition of molasses and sugarcane vinasses to predict their toxicity. Fagier et al. [27] identified phenolic compounds and some carboxylic acids as volatile compounds in sugarcane vinasses, some of which are highly toxic for Daphnia magna (IC50 = 0.9 mg/L). Additionally, Lima et al. [28] identified some fatty acids, alcohols, and esters in hydrolyzed sugarcane vinasses.
There is still a gap in the knowledge of vinasse composition from various sources regarding the presence of volatile compounds, which could inhibit downstream processes or uses, such as biohydrogen production. Furthermore, the extent of the effect of the initial process for tequila production (cooking or not-cooking of agave stems) on the final vinasse composition have not yet been described. Thus, the aim of this work was to identify and quantify the profile of volatile compounds in vinasses, obtained from two different tequila-production processes: (i) cooking (C) and not-cooking (NC) agave stems.

Identification and Concentration of Volatile Compounds
The vinasse samples have different profiles and volatile compound concentration. It was possible to identify 104 compounds belonging to different chemical families ( Table 1). The most frequent chemical families were alcohols (20), acids (16), and furans (11); also, alkanes, aldehydes, esters, ketones, phenols, and pyrans were identified.
Acetic acid was found in all studied vinasses, its concentration (13.20-181.25 mg/L) showed significant differences (p < 0.05), between C and NC processes, where the vinasse C2 showed the highest acetic acid concentration (181.25 mg/L). Butyric acid was identified in all samples too, with significant differences (p < 0.05) among vinasse samples, ranging from 7.84 to 38.86 mg/L, NC2 vinasse showed the highest concentration than other vinasses ( Figure 1).
Energies 2018, 11, x FOR PEER REVIEW 3 of 17 been described. Thus, the aim of this work was to identify and quantify the profile of volatile compounds in vinasses, obtained from two different tequila-production processes: (i) cooking (C) and not-cooking (NC) agave stems.

Identification and Concentration of Volatile Compounds
The vinasse samples have different profiles and volatile compound concentration. It was possible to identify 104 compounds belonging to different chemical families ( Table 1). The most frequent chemical families were alcohols (20), acids (16), and furans (11); also, alkanes, aldehydes, esters, ketones, phenols, and pyrans were identified.
Acetic acid was found in all studied vinasses, its concentration (13.20-181.25 mg/L) showed significant differences (p < 0.05), between C and NC processes, where the vinasse C2 showed the highest acetic acid concentration (181.25 mg/L). Butyric acid was identified in all samples too, with significant differences (p < 0.05) among vinasse samples, ranging from 7.84 to 38.86 mg/L, NC2 vinasse showed the highest concentration than other vinasses ( Figure 1). Figure 1. The total concentration of volatile compounds found in tequila vinasses from both processes (not-cooking (NC) and cooking (C)) by functional group. Bars represents mean ± standard deviation. Different lowercase letters indicate significant differences for each compound among the analyzed vinasses (p < 0.05, an analysis of variance (ANOVA)). The total concentration of volatile compounds found in tequila vinasses from both processes (not-cooking (NC) and cooking (C)) by functional group. Bars represents mean ± standard deviation. Different lowercase letters indicate significant differences for each compound among the analyzed vinasses (p < 0.05, an analysis of variance (ANOVA)).
In this research, furfuryl alcohol (39), benzyl alcohol (42) and phenylethyl alcohol (43) were identified and quantified in all vinasse samples (Figure 1). Furfuryl alcohol in vinasses from the C process presented significantly higher concentrations (3.76-16.25 mg/L) than those from the NC process (p < 0.05). The vinasse C1 showed the highest concentration (16.26 mg/L). Benzyl alcohol was present in all the samples, showing significant differences (p < 0.05) between vinasses from the NC and C processes. The concentrations ranged from 3.42-46.6 mg/L. Among them, the NC2 vinasse had the highest concentration of benzyl alcohol. Phenylethyl alcohol was found in both processes ranging from 366.41-470.92 mg/L, with significant differences (p < 0.05) between both kinds of vinasse evaluated. The highest concentration was found in the C3 vinasse ( Figure 1).

Ketones
Thirteen ketones were identified in the vinasses. Ten were mostly found in the C vinasses, while only seven were found in some NC. γ-butyrolactone (78) was identified and quantified in all TVs, showing significantly higher concentration in the C vinasses (p < 0.05) than in the NC vinasses. The concentration range was 1.5-56.78 mg/L, where vinasse C1 presented the highest amount ( Figure 1).

Principal Component Analysis (PCA)
Principal component analysis (PCA) was used to determine the main sources of variability of the data sets and establish the relationship between tequila vinasses (objects) and volatile compounds (variables) [35]. PCA facilitated the interpretation in this study. The analysis allowed us to identify what volatile compounds were correlated with the kind of vinasse (NC or C). The two principal components (PCs) were enough to explain 60% of total variability from the data set, 32.29% and 28.09% by PC1 and PC2, respectively ( Figure 2).

Principal Component Analysis (PCA)
Principal component analysis (PCA) was used to determine the main sources of variability of the data sets and establish the relationship between tequila vinasses (objects) and volatile compounds (variables) [35]. PCA facilitated the interpretation in this study. The analysis allowed us to identify what volatile compounds were correlated with the kind of vinasse (NC or C). The two principal components (PCs) were enough to explain 60% of total variability from the data set, 32.29% and 28.09% by PC1 and PC2, respectively ( Figure 2).

Discussion
The identification of approximately 37% of the compounds in the present work is in agreement with previous work published with similar matrices, such as cooked agave juice, tequila beverage, and sugarcane vinasses [27][28][29][30]. This work focuses on the volatile compounds found in higher concentrations or with inhibitory potential for the biological process in the vinasse treatment.

Discussion
The identification of approximately 37% of the compounds in the present work is in agreement with previous work published with similar matrices, such as cooked agave juice, tequila beverage, and sugarcane vinasses [27][28][29][30]. This work focuses on the volatile compounds found in higher concentrations or with inhibitory potential for the biological process in the vinasse treatment.
The presence of organic acids in high concentrations confer toxicity to the vinasses, hindering their treatment by biological process or their exploitation via biohydrogen production. Acetic and butyric acids have negatives effects in the DF process of acetic acid-decreased Hmax (maximum potential of H2 production) and Rmax (maximum H2 production rate) in batch production [36]. In addition, Wang, Wan, and Wang [37] reported inhibitory effects for Hmax and Rmax in batch conditions, using 6 g/L and 8 g/L of acetic and butyric acids, respectively. In this work, these acids were found in both processes studied, NC (146.14 mg/L) and C (512.47 mg/L), at lower concentration than those reported in the literature [34,36,37]. Nonetheless, a synergistic effect might occur, as both are present in the vinasses, which can boost their potential individual inhibitory effects. Franden et al. (2013) demonstrated that the concomitant presence of acetic and formic acids (IC25 = 50.3 mg/L) inhibited the growth of Zymomonas mobilis [38].
Organic acids may be either protonated or unprotonated, both species inhibiting biohydrogen production [34]. They can uncouple hydrogen-producing bacteria (HPB) growth in two ways: (i) on the one hand, the nonpolar un-dissociated form can penetrate cell membrane HPB whereby they dissociate due to higher intracellular pH, releasing protons in the cell cytoplasm; as a result, this creates a pH imbalance and decreases intracellular pH, producing a reduction in the available energy used in HPB growth; (ii) on the other hand, if the polar-dissociated part of organic acids is present in the fermentative hydrogen production system at a high concentration, the ionic strength in the solution will be increased, causing HPB growth inhibition and cell lysis [34,36,37,39].
It is known that furfuryl alcohol might cause a significant membrane leakage in some bacteria (i.e., Escherichia coli) and exhibits synergism when present along with other inhibitors of microbial growth [40]. Currently, there is no available information about the inhibitory nature of the identified alcohols on the DF process.
It is known that the presence of some aldehydes (i.e., 4-hydroxybenzaldehyde) has been reported to impair the growth of an ethanologenic Escherichia coli B (LY01) at concentrations of 600 mg/L, where a 50% growth inhibition was observed [41]. Aldehydes inhibit some pathways such as glycolysis and fermentation [41][42][43]. Therefore, their presence in tequila vinasses could be considered as a potential inhibitor for DF in hydrogen production.
The chemical group of alkanes has been not reported as potential inhibitors of the DF process. However, the presence of some hydrocarbons (i.e., alkanes, branched alkanes, among others) in soils can alter soil enzymatic activities and microbial biomass carbon [44,45]. Also, they may inhibit some bacterial populations [46].
Furanic compounds such as furfural and HMF have been reported as inhibitors during fermentative hydrogen production [32,34,36,47]. The presence of furfural and HMF can inhibit several enzymes (i.e., alcohol dehydrogenase and pyruvate dehydrogenase) in Saccharomyces cerevisiae at concentrations of 192-480 mg/L for furfural and 252-630 mg/L for HMF [48]. Even furfural is known to be more toxic than HMF for industrial catalysts, in E. coli and S. cerevisiae. Furthermore, ethanol production by E. coli B LYO1 is inhibited at concentrations ≥ 2.6 g/L of furfural. This may be due to the fact that this compound has a direct effect on glycolytic and fermentative enzymes [41].
In agreement with previous studies performed in batch conditions for biohydrogen production, concentrations higher than 250 and 100 mg/L of furfural and HMF, respectively, directly affected Hmax and Rmax [32,36]. In this study, furfural concentration in vinasses was lower than the inhibitory concentration previously reported, but HMF concentration in tequila vinasses (C) was higher than that reported [32,34,36]. Also, both compounds in vinasses could increase the inhibitory effects.
Furfural and HMF are known to suppress cell growth, induce DNA damage, and inhibit several enzymes of the glycolytic and fermentative pathways (i.e., hexokinase, phosphofructokinase, triosephosphate, dehydrogenase, aldolase, among others) [32,34,48,49]. Furfural may decrease the activities of NADH-dependent enzymes involved in fermentation reactions; moreover, HMF and furfural reduction to furfuryl alcohol requires NADH in yeasts, which decrease H 2 production. Additionally, NADH may reduce furan derivatives as it is oxidized to NAD + , leading to lower levels of NADH and lowering H 2 production. Furthermore, HMF bears a hydroxyl group, which can bind to DNA nitrogen bases (specifically, adenine and thymine), causing DNA damage and mutations in many HPB [34,36,42,43]. In addition, it is known that furfural can inhibit fermentation and growth synergistically with phenols in E. coli and, when applied with acetic acid, can inhibit growth in S. cerevisiae [38].
There are no reports about ketones with potential inhibition for the DF process. In fact, Mills, Sandoval and Gill [40] state that ketones can be generated during the acidogenic stage, but are not considered as potential inhibitors because they are produced at low concentrations (<0.05 g/L). However, there is still a lack of knowledge about the effect of ketones on the DF process.
Several studies have shown the inhibitory effects on the DF process caused by other phenolic compounds such as hydroxybenzoic acid, syringaldehyde and vanillin [32,34,36,49,50]. However, those phenols were not identified in tequila vinasses, and other phenols were found that may have other adverse effects. High concentrations (0.12-50 mg/L) of phenolic compounds can inhibit fungior bacteria-fermenting carbohydrates into ethanol, by deactivation of lignocellulose-hydrolyzing enzymes [51]. Phenolic compounds are more inhibitory than furan compounds and organic acids, as phenols can lead to precipitation and irreversible inhibition of lignocellulose-hydrolyzing enzymes [51,52]. Also, since phenols are the most abundant components in soils [53], these compounds may affect the pools and fluxes of soil nutrients by disturbing the communities of microbial decomposers. Generally, vinasses are disposed into soils without any treatment [10], which could cause a disturbance in soils by altering the natural concentration of phenols, thereby affecting soil microbiota and plants. Phenolics, depending on their concentration, have been found to either stimulate or inhibit spore germination. For instance, hyphal growth of saprophytic fungi and hyphal branching are altered by quercetin (30.22 mg/L), p-coumaric acid (16.4 mg/L), ferulic acid (19.41 mg/L), hydroxybenzoic acid (13.81 mg/L) [54][55][56]. Arbuscular mycorrhizal fungi colonization of asparagus roots is decreased by ferulic acid (>50 µg/g) [55]. Also, other phenolic compounds (trans-Cinnamic acid and p-coumaric acid) inhibit growth of the etiolated seedling and seeding growth of lettuce [57]. Moreover, phenolic compounds present antimicrobial activity against Escherichia coli [58], but also against other Gram-negative and positive bacteria such as Salmonella typhimurium, Shigella dysenteriae, Oenococcus oen and Lactobacillus hilgardii by changing the cell's membrane permeability DNA binding [59,60], and as a consequence, affecting their ability to serve as selective barriers causing leakage and intracellular damage, leaving cells vulnerable to extracellular toxic compounds [32,34,36,50]. In the tequila vinasses phenolic compounds were found at 108.13-206.32 g/moL, which suggests that p-guaiacol, p-methylguaiacol, phenol, 3-methyl and 4-2(hydroxyethyl)phenol may negatively affect HPB
There is no report about the impact of pyrans on the DF process. Although, 15 mg/L, 5,6 dihydro-2(H)-pyran-2-one has been shown to inhibit growth, and biofilm formation of the Gram-negative bacteria Ralstonia solanacearum [61,62]. All this suggest that the biological processes in all matrices where vinasses are disposed (soil and water) are negatively influenced.

Relationship between PCA and Not-Cooking (NC) and Cooking (C) Processes
According to PCA, it can be inferred from C1 and C3 that thermal hydrolysis carried out by cooking the agave stems with autoclave show a significant effect on furan compounds formation, (i.e., furfural and HMF), which may inhibit subsequently the fermentative process. Fructans are insoluble in water at room temperature (25 • C) but are soluble at >50 • C [63]. Therefore, hydrolysis is required to obtain fermentable sugars. When the cooking process is carried out, sugars are subjected to a series of complex reactions, mainly caramelization, Maillard and oxidation-dehydration reactions [22]. These reactions are influenced by several factors, such as temperature and period of the cooking process. Furfural is produced by xylose dehydration at elevated temperatures and pressures [34,64]. HMF is generated during the cooking process because of the dehydration of fructose in the initial stages of the Maillard reaction [16,65]. The generation of furfural and HMF is continuously increased throughout the cooking process of agave juice [29]. In our research, C3 vinasse showed a higher presence of acetic acid, which is known to be a degradation product of the Maillard reaction of glucose and fructose [66].
During the not-cooking agave process, acidic hydrolysis is carried out at 80 • C. NC2 vinasses showed a higher presence of phenolic compounds. They might be already present in the feedstock or generated during the initial stages of acid-thermal degradation (NC) of [29,30]. Also, the stems of Agave tequilana Weber var. Azul presented high contents of α-cellulose (65%), hemicellulose (5.5%) and lignin (16.8%) [66]; and when fructans hydrolysis is performed under acidic conditions, the splitting of β-O-4 ether and other acid-labile linkages in lignin forms phenolic compounds [67]. Also, vinasses NC1 and NC3 presented a higher presence of organic acids than might be formed during hemicellulose acid-hydrolysis [68], contributing to the formation of phenolic compounds.

Samples
Vinasses were obtained from six tequila distilleries located in Tequila, Jalisco, Mexico. The tequila factories were selected from two types of agave juice extraction process, three of them were manufactured by using the traditional process (cooking agave stems) and were coded from C1 to C3; the other three come from a not cooking process and were labeled as NC1 to NC3. Twenty liters of vinasses from each industry were sampled after the distillation process was accomplished. Vinasses were kept at 4 • C until their analysis.

Volatile Compound Extractions
Volatile compounds were extracted by the liquid-liquid extraction method proposed by Prado-Jaramillo et al. [30] using dichloromethane. Vinasses were placed in a centrifuge tube (40-mL) and 10 mL of CH 2 Cl 2 were added. Subsequently, the samples were shaken for 5 min and centrifuged for 10 min at 5000 rpm and 10 • C. The organic layer was separated and dried with anhydrous Na 2 SO 4 . All extracts were concentrated to a final volume of 1.5 mL with a rotatory evaporator IKA ® RV 10 basic (IKA, Wilmington, NC, USA). The extracts were settled in suitable amber vials and preserved at −19 • C, until chromatographic analysis. Each vinasse sample was extracted and analyzed in triplicate.

Separation and Identification of Volatile Compounds
The volatile compounds were analyzed by a gas chromatography (GC) 6890N Network system (Agilent Technology, Santa Clara, CA, USA), coupled to a selective mass spectrometer detector 5975 (Agilent Technology, USA). Compound separations were performed using an HP-FFAP capillary column of 25 m × 0.32 mm (i.d) , coated with a 0.52 µm film (Agilent Technologies, Santa Clara, CA, USA). Helium was used as carrier gas, using a 2 mL/min of flow with an initial temperature of 40 • C for 5 min. Followed by a temperature program of 20 • C/min to 100 • C for 1 min, and by a second rate of 3 • C/min to a final temperature of 230 • C kept for 40 min. The injector temperature was 220 • C, and the injection mode was splitless. The selective mass detector operated an electronic impact ionization system at 70 eV, and at 260 • C. The identification of compounds was based on three criteria: (1) by comparing the mass spectra with the National Institute of Standards and Technology (NIST) library mass spectra; (2) by comparing the retention index with literature data; and (3) whenever possible, the identification was confirmed by using pure standards. Chromatograms obtained from the GC-MS analysis were integrated, and the peak areas were recorded for each identified compound. Quantification was performed by standard curves, obtained by preparing a solution with the different standard compounds in concentrations of 0.5, 1, 5, 10, 15 and 20 mg/L. Interpolation within a linear regression equation (R 2 from 0.995 to 0.999) was used to calculate each compound concentration. When a commercial standard was not available, the quantification was achieved using a slope obtained for a standard of an analog compound that was structurally similar, but slightly different, as has been described elsewhere [69].

Statistical Analysis
All the statistical analyses were achieved with Statgraphics Centurion XVI (Statgraphics, The Plains, VA, USA). An analysis of variance (ANOVA) as well as Fisher's multiple-range tests of the minimal significant differences (LSD) were performed to find significant differences between C and NC processes in the volatile profile. A PCA was used with the whole data.

Conclusions
The profile of volatile compounds was similar in cooking vinasses (C) and not-cooking steam vinasses, but some differences were found in the concentration of volatile compounds, suggesting that the cooking process has an influence on the profile obtained in the vinasse composition. The cooking process increased the content of furanic compounds and organic acids, which are described as inhibitors of DF biohydrogen production from wastewater. The tequila vinasses obtained from a not-cooking process showed a higher presence of phenolic compounds. Although, the identified phenolic compounds have not been reported as inhibitors of the DF process, they may inhibit other biological processes in other matrices such as soil. Detailed description of the vinasses' composition could help in the development of any process to eliminate compounds that may interfere with biological processes to treat or use those or similar waste products around the world.