2. Pretreatment/conditioning of agave bagasse and tequila vinasse

AD is the core technology for the treatment of several biodegradable organic wastes with concomitant bioenergy recovery in the form of biogas that is rich in bioCH4, although bioH2 may also be recovered. Besides bioCH4 recovery, AD is advantageous due to low energy and nutrient requirements, low sludge production, and high organic loading capacity (20–35 g-COD/L-d) [15]. From a biochemical point of view, AD consists of four successive steps, namely hydrolysis, acidogenesis, acetogenesis and methanogenesis [15, 16].

It is worth mentioning that in the case of AB, the low biodegradability due to its lignocellulosic structure constitutes one of the main barriers to accelerate hydrolysis and enhance the recovery of bioH2/bioCH4. In the case of TV, its complex composition such as high COD, high solids content, unbalanced nutrient, presence of putative toxicants (e.g. organic acids, phenols, melanoidins) and the negligible

process, namely agave juice (must) extraction, fermentation, and distillation. In the first stage, the agave juice containing fermentable sugars is first obtained either through cooking or not-cooking processes. In the former, agave stems are cooked in ovens or autoclaves at high temperatures (95–120°C) for a long time (usually 8–12 h). Once cooked, the water-soluble carbohydrates are extracted by simultaneous shredding and pressure washing followed by pressing. In the latter, raw agave juice is obtained from previously shredded raw agave stems using hot water (80°C)

At this point, it must be noted that enormous quantities of solid (Agave tequilana bagasse, hereinafter referred to as AB) and semi-liquid (tequila vinasse, hereinafter referred to as TV) by-products are generated each year during the process of tequila manufacturing, particularly after the stages of agave juice extraction and distillation, respectively (Figure 1). It has been estimated that 1.4 kg of AB and 10–12 L of TV are obtained by each liter of tequila produced [4, 5]. Considering the tequila production of 264.9 31.2 million liters reported in the last lustrum (2014–2018) by

through the use of equipment called diffuser. Afterward, the carbohydrates contained in the raw agave juice are hydrolyzed for 4–6 h under acidic conditions (pH 1.8–3) at high temperatures (80–85°C) [2, 3]. In the second stage, the agave juice is subjected to an alcoholic fermentation process, wherein agave sugars are transformed to ethanol, carbon dioxide, and other compounds (e.g. aldehydes, esters, furans, and ketones) by the action of different microorganisms, particularly yeasts [2, 3]. In the third stage, the fermented must is subjected to a two-step

distillation process to obtain tequila [2, 4].

New Advances on Fermentation Processes

Figure 1.

104

Tequila manufacturing process and generation of agave bagasse and tequila vinasse.

alkalinity along with the high concentration of components with a tendency to suffer very rapid acidification constitutes the major limitations for bioH2/bioCH4 production. Thus, in practice, before the feedstock (AB or TV) is sent to either the hydrogenogenic or the methanogenic stage, a pretreatment/conditioning step is commonly performed as a prerequisite to improve its biodegradability as well as to prevent DF/AD processes from potential toxicants, elevated solids, and organic overloading (Figure 2). Unlike AB, TV is only subjected to one or more conditioning steps. Commonly, they consist of lowering temperature, rising pH (adding alkalinity), diluting, adding complementary nutrients, and removing suspended solids (Figure 2).

on the chemical composition of hydrolysates of AB. Overall, results showed that the use of HCl induced higher sugar recoveries than the use of H2SO4, 0.39 versus 0.26 g-total sugars/g of AB. Furthermore, the H2SO4 hydrolysate contained higher concentrations of HAc and furans. To remove undesirable compounds derived from acid hydrolysis of AB (30 g AB, HCl 1.9%, 130°C, 132 min reaction time), Valdez-Guzmán et al. [19] performed detoxification of acid AB hydrolysates using 1% (w/v) powdered coconut shell-activated carbon. Under batch conditions (pH 0.6, 20 min reaction time, 150 rpm, room temperature), the highest removal of HAc and phenols obtained were 89 and 21%, respectively, with minimal losses of fermentable sugars (3.6%). Besides, during acid hydrolysis, a hydrolysis yield of almost 40% of total sugars, a delignification of 44%, complete hydrolysis of

A Comprehensive Overview of the Potential of Tequila Industry By-Products for Biohydrogen…

DOI: http://dx.doi.org/10.5772/intechopen.88104

hemicellulose, and no detection of furfural or HMF in the hydrolysate was obtained. In another study, Contreras-Dávila et al. [20] pretreated AB for bioH2 production using Celluclast 1.5 L during 10 h, obtaining sugar yields in the range of 0.19–0.38 gtotal sugars/g of AB. Montiel and Razo-Flores [21] also pretreated AB by enzymatic hydrolysis to produce bioH2 and bioCH4. The conditions were 3.5% (w/v) of AB with Celluclast 1.5 L at 18 FPU/g of AB at 40°C during 12 h. The resulting hydrolysate had 27.2 g/L of total COD with 5.3 0.8 g/L of total sugars (0.15 g-total sugars/ g of AB) which contributed to 20% of the total COD, citrate buffer with 26%, enzyme with 38%, and other non-determined components with 16%. In the same year, Galindo-Hernández et al. [22] used alkaline hydrogen peroxide (AHP) as a pretreatment to remove lignin before enzymatic hydrolysis of AB. Under the experimental conditions tested (5% w/v of AB, 2% w/v of AHP, 50°C, pH 11.5 using NaOH, 120 rpm, 1.5 h reaction time), 97% of the lignin was removed and 88% of holocellulose (cellulose and hemicellulose) was recovered, promoting that the polysaccharide fractions are more available or exposed to a further enzymatic attack. The authors also demonstrated, in delignification terms, that it is better to use hydrogen peroxide and NaOH solution in a combined form than in a separate or sequential way and that using binary enzymatic hydrolysis (cellulases and

hemicellulases) may improve the yield, percentage, and productivity of saccharification, which were 0.19 g-total sugars/g of AB, 26.7% and 17.1 g-total sugars/g of AB-h, respectively. The synergistic effect of using binary enzymatic hydrolysis was verified by Montoya-Rosales et al. [23], who compared the enzymatic hydrolysis of AB using a binary enzyme preparation that is composed of Celluclast 1.5 L and Viscozyme L with a single enzyme, that is, Stonezyme, which is a commercial cellulase preparation. The results showed that hydrolysis yields were higher with the binary enzymatic hydrolysis, 0.27 versus 0.22 g-carbohydrates/g of AB and

3. Biohydrogen production from agave bagasse and tequila vinasse

H2 is one of the most promising alternative energy carriers to partly fulfill the growing energy demands and overcome fossil fuel dependency and has attracted global attention for its highest energy content per unit weight (142 kJ/g) and carbon-free nature since it generates only water vapor during combustion. It can be used for a variety of purposes either alone to produce energy in fuel cells and combustion engines or blended with CH4 to produce a superior fuel known as hythane [24]. Comparing thermochemical, electrochemical, and biological ways of producing H2, the latter is considered the most sustainable because it is ecofriendlier and less energy intensive. Among biological processes, dark fermentation (DF) is thought to be practically applicable at large commercial scales in a near time horizon owing to its capability of producing bioH2 at higher rates and versatility of

0.5 versus 0.28 g-COD/g of AB.

107

In contrast, AB is exposed to a drying step to prevent fungal and bacterial growth, mainly for long-time storage. Once AB is dried, it is subjected to a mechanical milling step devoted to reducing particle size, thereby increasing surface area, which makes carbohydrates more easily available for downstream processes. The mechanical fractionation also makes AB more homogeneous and easier to handle. After milling, the pretreatment applied to AB for either bioH2 or bioCH4 production may differ. For such purposes, dilute acid, alkaline hydrogen peroxide, detoxification and enzymatic hydrolysis have been evaluated in detail. Arreola-Vargas et al. [8] pretreated cooked and uncooked AB through a dilute acid hydrolysis at 5% (w/v), 56.4–123.6°C, 1.2–2.8% HCl, and 0.3–3.7 h reaction time, finding temperature as the principal factor which could increase the hydrolysis yield. Total sugars concentrations obtained were 27.9 and 18.7 g/L for cooked and uncooked AB hydrolysates, respectively. The higher yield of cooked AB was attributed to the fact that during the elaboration of tequila using cooking process, agave stems receives an in situ thermal treatment. Nevertheless, high concentrations (up to 1200 mg/L) of hydroxymethylfurfural (HMF) were detected in the cooked AB. In a further study, Arreola-Vargas et al. [17] pretreated AB through either acid or enzymatic hydrolysis for bioCH4 and bioH2 production. Acid hydrolysis was carried out for 1.3 h at 5% (w/v) of AB, 2.7% HCl and 124°C, while enzymatic hydrolysis was performed at 4% (w/v) of AB in 50 mM citrate buffer at pH 4.5 with Celluclast 1.5 L at 40 filter paper units (FPU) for 10 h at 45°C. As a result, 17.3 and 8.9 g-total sugars/L were obtained from acid and enzymatic hydrolysis, respectively. However, unlike enzymatic hydrolysates, acid hydrolysates promoted the generation of potential inhibitors such as formic acid (HFor), acetic acid (HAc), and phenolic and furanic compounds. In another study, Breton-Deval et al. [18] compared the type of acid catalyst (HCl vs. H2SO4)


Flow chart of biohydrogen and biomethane production process from agave bagasse and tequila vinasse.

### A Comprehensive Overview of the Potential of Tequila Industry By-Products for Biohydrogen… DOI: http://dx.doi.org/10.5772/intechopen.88104

on the chemical composition of hydrolysates of AB. Overall, results showed that the use of HCl induced higher sugar recoveries than the use of H2SO4, 0.39 versus 0.26 g-total sugars/g of AB. Furthermore, the H2SO4 hydrolysate contained higher concentrations of HAc and furans. To remove undesirable compounds derived from acid hydrolysis of AB (30 g AB, HCl 1.9%, 130°C, 132 min reaction time), Valdez-Guzmán et al. [19] performed detoxification of acid AB hydrolysates using 1% (w/v) powdered coconut shell-activated carbon. Under batch conditions (pH 0.6, 20 min reaction time, 150 rpm, room temperature), the highest removal of HAc and phenols obtained were 89 and 21%, respectively, with minimal losses of fermentable sugars (3.6%). Besides, during acid hydrolysis, a hydrolysis yield of almost 40% of total sugars, a delignification of 44%, complete hydrolysis of hemicellulose, and no detection of furfural or HMF in the hydrolysate was obtained. In another study, Contreras-Dávila et al. [20] pretreated AB for bioH2 production using Celluclast 1.5 L during 10 h, obtaining sugar yields in the range of 0.19–0.38 gtotal sugars/g of AB. Montiel and Razo-Flores [21] also pretreated AB by enzymatic hydrolysis to produce bioH2 and bioCH4. The conditions were 3.5% (w/v) of AB with Celluclast 1.5 L at 18 FPU/g of AB at 40°C during 12 h. The resulting hydrolysate had 27.2 g/L of total COD with 5.3 0.8 g/L of total sugars (0.15 g-total sugars/ g of AB) which contributed to 20% of the total COD, citrate buffer with 26%, enzyme with 38%, and other non-determined components with 16%. In the same year, Galindo-Hernández et al. [22] used alkaline hydrogen peroxide (AHP) as a pretreatment to remove lignin before enzymatic hydrolysis of AB. Under the experimental conditions tested (5% w/v of AB, 2% w/v of AHP, 50°C, pH 11.5 using NaOH, 120 rpm, 1.5 h reaction time), 97% of the lignin was removed and 88% of holocellulose (cellulose and hemicellulose) was recovered, promoting that the polysaccharide fractions are more available or exposed to a further enzymatic attack. The authors also demonstrated, in delignification terms, that it is better to use hydrogen peroxide and NaOH solution in a combined form than in a separate or sequential way and that using binary enzymatic hydrolysis (cellulases and hemicellulases) may improve the yield, percentage, and productivity of saccharification, which were 0.19 g-total sugars/g of AB, 26.7% and 17.1 g-total sugars/g of AB-h, respectively. The synergistic effect of using binary enzymatic hydrolysis was verified by Montoya-Rosales et al. [23], who compared the enzymatic hydrolysis of AB using a binary enzyme preparation that is composed of Celluclast 1.5 L and Viscozyme L with a single enzyme, that is, Stonezyme, which is a commercial cellulase preparation. The results showed that hydrolysis yields were higher with the binary enzymatic hydrolysis, 0.27 versus 0.22 g-carbohydrates/g of AB and 0.5 versus 0.28 g-COD/g of AB.
