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

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

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.

solids (Figure 2).

New Advances on Fermentation Processes

Figure 2.

106

utilizing several different types of carbohydrate-rich wastes as substrate [25]. In this connection, since AB and TV are abundantly available, renewable, and have a high content of carbohydrates, they have been considered as suitable feedstocks for bioH2 production. In the following sections, the operational performance, metabolic pathways, and microbial communities of DF systems treating either AB or TV are extensively reviewed.

simultaneously enhanced VHPR and HY2, attaining values of 3.45 L-H2/L-d and 1.53 mol-H2/mol-substrate, respectively, with bioH2 concentrations of the produced gas between 26 and 52% (v/v). The observed bioH2 production performances were explained by differences in the liquid and gas flow rates, agitation speed, and liquidgas interface between the CSTR and TBR configurations, which in turn may have

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

In a further study which set up to assess the batch bioH2 production from

(hemicellulases + cellulases), Galindo-Hernández et al. [22] performed a series of experiments in the AMPTS II system at 37°C, 150 rpm, initial pH of 7.5, and using an organic load of 5 g-COD/L and 13.5 g-volatile solid (VS)/L of thermally treated anaerobic sludge. The results suggested that delignification of AB and subsequent hydrolysis with a synergistic enzymatic mixture had a beneficial effect on bioH2 production, obtaining a YH2 of 3 mol-H2/mol-hexose and a VHPR of 0.93

In an investigation on the effect of OLR and agitation speed on the continuous bioH2 production from enzymatic hydrolysates of AB, Montiel and Razo-Flores [21] operated for 84 days a mesophilic (35°C) CSTR reactor (with a working volume of 1 L) inoculated with 4.5 g-VS/L of heat-treated anaerobic granular sludge and operated at different OLRs (40–52 g-COD/L-d), which were achieved by varying hydrolysate concentration. The evaluated stirring speeds were in the range of 150–300 rpm, while the HRT was maintained at 6 h during the whole operation. The authors observed that the strategy of increasing the agitation speed from 150 to 300 rpm favored both the VHPR and bioH2 content in the gas phase, obtaining 6 NL-H2/L-d and 55% (v/v), respectively, at an OLR of 44 g-COD/L-d. Such results indicated that the increase of the agitation speed in the CSTR improved the transfer of dissolved bioH2 from the liquid to the reactor gas phase, overcoming one of the

In another study, Toledo-Cervantes et al. [26] addressed the bioH2 production from enzymatic hydrolysates of AB using an anaerobic sequencing batch reactor (AnSBR) with a working volume of 1.25 L. The reactor was inoculated with 10 g-VS/L of thermally treated anaerobic sludge and operated at 37°C, pH 4.8, and at four OLR (10.6–21.3 g-COD/L-d), which were modified by decreasing the cycle time (from 24 to 12 h) and increasing the COD concentration (from 8 to 12 and 16 g/L). Results showed that the highest OLR promoted the highest VHPR of 0.6 NL-H2/L-d. Conversely, the YH2 remained constant at 1.6 mol-H2/mol of

In a similar study, Valdez-Guzmán et al. [19] showed the importance not only of optimizing pretreatment but also of removing several compounds (e.g. furfural, HMF, phenolic compounds, and organic acids) that are generated during its application. They compared the bioH2 production potential of undetoxified and detoxified acid hydrolysates from AB. The authors reported 39 and 9% increases on YH2 and VHPR, respectively, comparing detoxified AB with activated carbon and undetoxified AB, 1.71 versus 1.23 mol-H2/mol of consumed sugar and 1.51 versus 1.38 NL-H2/L-d. Such increments were correlated to changes in the fermentation by-products suggesting the occurrence of different pathways or changes in the microbial community, since the detoxified hydrolysate produced HAc and butyric acid (HBu), while lactic acid (HLac) was found in the undetoxified hydrolysate. Most recently, Montoya-Rosales et al. [23] compared and evaluated the continuous bioH2 production from individual and binary enzymatic hydrolysates of AB in two different configurations, that is, CSTR and TBR. The experiments were carried out at 37°C and pH 5.5 and at various OLRs 36–100 g-COD/L-d, which were achieved by increasing the influent concentration, while keeping the HRT constant

pretreated AB with AHP followed by binary enzymatic saccharification

caused distinct bioH2 concentrations in the liquid phase.

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

limitations for bioH2 production previously observed by [21].

NL-H2/L-d.

consumed sugar.

109

### 3.1 Operational performance

Regarding the use of AB for bioH2 production (Table 1), the first systematic study dealing with bioH2 production from AB was conducted by Arreola-Vargas et al. (2016) [17], who assessed the use of AB hydrolysates obtained either from acid or enzymatic pretreatment for bioH2 production. To the end, different proportions of hydrolysate (20, 40, 60, 80, and 100% v/v) were tested in an automatic methane potential test system (AMPTS II provided by Bioprocess control) at 37°C, 120 rpm, initial pH of 7, and using 10 g-volatile suspended solids (VSS)/L of heat-pretreated anaerobic granular sludge. Overall, the best bioH2 production performance was achieved in the assays with enzymatic hydrolysate, obtaining the maximal bioH2 yield (HY2) and volumetric bioH2 production rate (VHPR) of 3.4 mol-H2/molhexose and 2.4 NL-H2/L-d, respectively, both with the hydrolysate at 40% (v/v). The lower values observed with the acid hydrolysate were attributed to the feedstock composition in terms of sugar profile, weak acids, furans, and phenolics.

In another work, Contreras-Dávila et al. [20] used an enzymatic AB hydrolysate for bioH2 production in a continuously stirred tank reactor (CSTR) and a trickling bed reactor (TBR), which were operated up to 87 days under different organic loading rates (OLR, 17–60 g-COD/L-d) obtained by varying hydrolysate concentration and/or hydraulic retention time (HRT). The reactor configurations showed different performances. In the CSTR, the VHPR and HY2 displayed an inverse correlation with maximum values of 2.53 L-H2/L-d and 1.35 mol-H2/mol-substrate, attained at OLR of 52.2 and 40.2 g-COD/L-d, respectively, both with 6 h HRT. The bioH2 concentrations of the produced gas were between 18 and 35% (v/v). In contrast, in the TBR, increasing OLR up to 52.9 g COD/L-d (4 h HRT)


Notes: All studies were conducted using thermally treated anaerobic granular sludge; <sup>a</sup> Initial pH value; <sup>b</sup> mol-H2/mol hexose; <sup>c</sup> mol-H2/mol of consumed sugar; <sup>d</sup> Value measured during the starting period; NR: not reported.

#### Table 1.

Comparison of the literature data on biohydrogen production efficiency using pretreated agave bagasse as feedstock.

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

simultaneously enhanced VHPR and HY2, attaining values of 3.45 L-H2/L-d and 1.53 mol-H2/mol-substrate, respectively, with bioH2 concentrations of the produced gas between 26 and 52% (v/v). The observed bioH2 production performances were explained by differences in the liquid and gas flow rates, agitation speed, and liquidgas interface between the CSTR and TBR configurations, which in turn may have caused distinct bioH2 concentrations in the liquid phase.

In a further study which set up to assess the batch bioH2 production from pretreated AB with AHP followed by binary enzymatic saccharification (hemicellulases + cellulases), Galindo-Hernández et al. [22] performed a series of experiments in the AMPTS II system at 37°C, 150 rpm, initial pH of 7.5, and using an organic load of 5 g-COD/L and 13.5 g-volatile solid (VS)/L of thermally treated anaerobic sludge. The results suggested that delignification of AB and subsequent hydrolysis with a synergistic enzymatic mixture had a beneficial effect on bioH2 production, obtaining a YH2 of 3 mol-H2/mol-hexose and a VHPR of 0.93 NL-H2/L-d.

In an investigation on the effect of OLR and agitation speed on the continuous bioH2 production from enzymatic hydrolysates of AB, Montiel and Razo-Flores [21] operated for 84 days a mesophilic (35°C) CSTR reactor (with a working volume of 1 L) inoculated with 4.5 g-VS/L of heat-treated anaerobic granular sludge and operated at different OLRs (40–52 g-COD/L-d), which were achieved by varying hydrolysate concentration. The evaluated stirring speeds were in the range of 150–300 rpm, while the HRT was maintained at 6 h during the whole operation. The authors observed that the strategy of increasing the agitation speed from 150 to 300 rpm favored both the VHPR and bioH2 content in the gas phase, obtaining 6 NL-H2/L-d and 55% (v/v), respectively, at an OLR of 44 g-COD/L-d. Such results indicated that the increase of the agitation speed in the CSTR improved the transfer of dissolved bioH2 from the liquid to the reactor gas phase, overcoming one of the limitations for bioH2 production previously observed by [21].

In another study, Toledo-Cervantes et al. [26] addressed the bioH2 production from enzymatic hydrolysates of AB using an anaerobic sequencing batch reactor (AnSBR) with a working volume of 1.25 L. The reactor was inoculated with 10 g-VS/L of thermally treated anaerobic sludge and operated at 37°C, pH 4.8, and at four OLR (10.6–21.3 g-COD/L-d), which were modified by decreasing the cycle time (from 24 to 12 h) and increasing the COD concentration (from 8 to 12 and 16 g/L). Results showed that the highest OLR promoted the highest VHPR of 0.6 NL-H2/L-d. Conversely, the YH2 remained constant at 1.6 mol-H2/mol of consumed sugar.

In a similar study, Valdez-Guzmán et al. [19] showed the importance not only of optimizing pretreatment but also of removing several compounds (e.g. furfural, HMF, phenolic compounds, and organic acids) that are generated during its application. They compared the bioH2 production potential of undetoxified and detoxified acid hydrolysates from AB. The authors reported 39 and 9% increases on YH2 and VHPR, respectively, comparing detoxified AB with activated carbon and undetoxified AB, 1.71 versus 1.23 mol-H2/mol of consumed sugar and 1.51 versus 1.38 NL-H2/L-d. Such increments were correlated to changes in the fermentation by-products suggesting the occurrence of different pathways or changes in the microbial community, since the detoxified hydrolysate produced HAc and butyric acid (HBu), while lactic acid (HLac) was found in the undetoxified hydrolysate.

Most recently, Montoya-Rosales et al. [23] compared and evaluated the continuous bioH2 production from individual and binary enzymatic hydrolysates of AB in two different configurations, that is, CSTR and TBR. The experiments were carried out at 37°C and pH 5.5 and at various OLRs 36–100 g-COD/L-d, which were achieved by increasing the influent concentration, while keeping the HRT constant

utilizing several different types of carbohydrate-rich wastes as substrate [25]. In this connection, since AB and TV are abundantly available, renewable, and have a high content of carbohydrates, they have been considered as suitable feedstocks for bioH2 production. In the following sections, the operational performance, metabolic pathways, and microbial communities of DF systems treating either AB or TV are

Regarding the use of AB for bioH2 production (Table 1), the first systematic study dealing with bioH2 production from AB was conducted by Arreola-Vargas et al. (2016) [17], who assessed the use of AB hydrolysates obtained either from acid or enzymatic pretreatment for bioH2 production. To the end, different proportions of hydrolysate (20, 40, 60, 80, and 100% v/v) were tested in an automatic methane potential test system (AMPTS II provided by Bioprocess control) at 37°C, 120 rpm, initial pH of 7, and using 10 g-volatile suspended solids (VSS)/L of heat-pretreated anaerobic granular sludge. Overall, the best bioH2 production performance was achieved in the assays with enzymatic hydrolysate, obtaining the maximal bioH2 yield (HY2) and volumetric bioH2 production rate (VHPR) of 3.4 mol-H2/molhexose and 2.4 NL-H2/L-d, respectively, both with the hydrolysate at 40% (v/v). The lower values observed with the acid hydrolysate were attributed to the feedstock composition in terms of sugar profile, weak acids, furans, and phenolics.

In another work, Contreras-Dávila et al. [20] used an enzymatic AB hydrolysate for bioH2 production in a continuously stirred tank reactor (CSTR) and a trickling bed reactor (TBR), which were operated up to 87 days under different organic loading rates (OLR, 17–60 g-COD/L-d) obtained by varying hydrolysate concentration and/or hydraulic retention time (HRT). The reactor configurations showed different performances. In the CSTR, the VHPR and HY2 displayed an inverse correlation with maximum values of 2.53 L-H2/L-d and 1.35 mol-H2/mol-substrate, attained at OLR of 52.2 and 40.2 g-COD/L-d, respectively, both with 6 h HRT. The bioH2 concentrations of the produced gas were between 18 and 35% (v/v). In contrast, in the TBR, increasing OLR up to 52.9 g COD/L-d (4 h HRT)

(°C)

Acid hydrolysis Batch 37 7<sup>a</sup> 1.6<sup>b</sup> 2.4 NR [17] Individual enzymatic hydrolysis Batch 37 7<sup>a</sup> 140, 3.4<sup>b</sup> 2.4 NR [17] Individual enzymatic hydrolysis Continuous 37 5.5 67 3.45 26–52 [20] Individual enzymatic hydrolysis Continuous 35 5.5 105 6 55 [21]

Acid hydrolysis + detoxification Batch 37 8.2<sup>a</sup> 56.2 1.51 NR [19] Binary enzymatic hydrolysis Continuous 37 5.5 117.8 13 51–60 [23]

Comparison of the literature data on biohydrogen production efficiency using pretreated agave bagasse as

continuous

Notes: All studies were conducted using thermally treated anaerobic granular sludge; <sup>a</sup>

pH YH2 (NL/ kg AB)

Value measured during the starting period; NR: not reported.

Batch 37 7.5a 215 0.93 NR [22]

37 4.8 1.6<sup>c</sup> 0.6 49.3<sup>d</sup> [26]

Initial pH value; <sup>b</sup>

VHPR (NL/L-d) H2 (% v/v)

Ref.

mol-H2/mol

extensively reviewed.

3.1 Operational performance

New Advances on Fermentation Processes

Pretreatment Feeding T

Alkaline hydrogen peroxide + binary enzymatic hydrolysis

hexose; <sup>c</sup>

Table 1.

feedstock.

108

Individual enzymatic hydrolysis Semi-

mol-H2/mol of consumed sugar; <sup>d</sup>

at 6 h. The results showed that the performance was highly dependent on the type of reactor and OLR. Regarding the CSTR configuration, in general, the higher OLR resulted in higher VHPR. Nonetheless, the bioH2 production efficiency using individual enzymatic hydrolysate (0.72–2.25 NL-H2/L-d and 11.8–20.4 NL-H2/kg of AB) was lower compared to that obtained with the binary enzymatic hydrolysate (3.9–13 NL-H2/L-d and 83.3–117.9 NL-H2/kg of AB), with the maximum VHPR and YH2 at 100 and 60 g-COD/L-d and 90 and 52 g-COD/L-d, respectively. Regarding the TBR configuration, the binary enzymatic hydrolysate also outperformed the individual one, obtaining the maximum VHPR of 5.76 NL-H2/L-d at an OLR of 81 g-COD/L-d and YH2 of 72.4 NL-H2/kg of AB at an OLR of 69 g-COD/L-d. The enhancement was attributed, on one hand, to the use of binary hydrolysis that could have contributed to produce a higher proportion of monomers of easy degradation by bioH2 producing bacteria (HPB) and to avoid the formation/release of potential inhibitors; on the other hand, to the differences of substrate availability given by the mode of growth in each reactor.

(iv) evaluating the feasibility of co-fermentation [11, 36]; and (v) exploring the

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

More particularly, Espinoza-Escalante et al. [27] evaluated the effect of three pretreatments, that is, alkalinization, cavitation, and thermal pretreatment, on the metabolic profile and the increments of COD and total reducing sugars (TRS) of TV, as well as on its bioH2 production potential. From that study, it can be concluded that the application of such pretreatments to raw TV resulted in different degrees of solubilization of COD and TRS, depending on the applied pretreatment and combinations thereof. However, there was no apparent relation in the consumption of TRS and COD with bioH2 production. Indeed, the optimal conditions that led to the highest solubilization of both COD and TRS did not result in a significant improvement in the YH2, which was about 2.8 NL-H2/L of reactor, indicating that compounds other than TRS could be involved in the mechanism of

In another report, Espinoza-Escalante et al. [28] studied the effect of pH (4.5, 5.5, and 6.5), HRT (1, 3, and 5 d), and temperature (35 and 55°C) on the semicontinuous production of bioH2 from TV. The experiments were performed in 1-L glass vessels inoculated with 10% (v/v) of mesophilic anaerobic digester sludge. The results showed that all factors studied had an important effect on bioH2 production. The highest efficiency in terms of bioH2 production was achieved at a pH of 5.5, an HRT of 5 d and a temperature of 55°C. Based on constructed mathematical models,

In a similar study, Buitrón and Carvajal [30] investigated the effect of tempera-

Later, Buitrón et al. [34] evaluated the performance of an FBR to produce bioH2 in a continuous mode from TV. The reactor had a working volume of 1.7 L and was packed with polyurethane rings for biomass immobilization. The temperature, pH, HRT, and OLR were kept constant at 35°C, 4.7, 4 h, and 2.15 g-COD/L-d (influent concentration of 8 g-COD/L), respectively. After an initial acclimatization period of HPB to TV, the FBR exhibited a VHPR of 1.7 NL-H2/L-d and a YH2 of 1.36 NL-H2/L of TV. In a follow-up study conducted by the same research group, by using a 0.6-L AnSBR operated under mesophilic and acidophilic conditions at an HRT of 6 h, it was observed that increasing substrate concentration from 2 to 16 g-COD/L increased the VHPR up to 1.4 NL-H2/L-d. Hence, the use of TV for bioH2 produc-

Another interesting advance was made by García-Depraect et al. [11], who studied the technical feasibility of using a co-fermentation approach to produce

Nixtamalization wastewater (NW) was chosen as the complementary substrate based on its wide availability in Mexico and high alkalinity. The TV:NW ratio of 80:20 (w/w) resulted in the highest VHPR of 2.6 NL-H2/L-d with a bioH2 content in the gas phase of 71% (v/v). Interestingly, the co-fermentation study allowed the identification of iron and nitrogen as essential nutrients which may be limiting in TV-fed DF reactors. This identification becomes significant to avoid nutrientlimited conditions and to prevent excessive nutrient supplementation that has been

bioH2 from TV in a well-mixed reactor operated under batch mode.

ture (25 and 35°C), HRT (12 and 24 h), and substrate concentration on bioH2 production from TV using a 7-L AnSBR, with a working volume of 6 L. The exchange volume was 50% with a reaction time of 11.3 or 5.3 h depending on the applied HRT, while pH and mixing were controlled at 5.5 and 153 rpm, respectively, in all cases. It was evidenced that all parameters studied affected the efficiency of bioH2 production. The HRT had a major influence on bioH2 production. It was found that the shorter the HRT, the higher the bioH2 production. Overall, the maximum VHPR of 2.2 NL-H2/L-d and an average bioH2 content in the biogas of

29.2 8.8% (v/v) were obtained at 35°C, 12 h HRT, and 3 g-COD/L OLR.

microbial ecology of the process [32, 36, 37].

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

pH was the most influential parameter.

tion did not result in inhibition [35].

111

bioH2 production.

Concerning the use of TV for bioH2 production (Table 2), there are a few studies in the literature, with a particular focus on (i) optimizing pretreatments to further enhance bioH2 production [27]; (ii) testing the effect of different operational conditions such as pH [28, 29], temperature [28, 30], substrate concentration [28, 30, 31], solid content [22, 31], nutrient formulation [22, 31], inoculum addition [22, 31], HRT [22, 30, 32], and OLR [22, 32]; (iii) producing bioH2 in different systems, such as serum bottle [33], fixed bed reactor (FBR) [34], and CSTR [35];


Notes: Inoculum: anaerobic digester sludge [27, 28], thermally treated anaerobic granular sludge [11, 29–31, 33–38]; \*Units: <sup>a</sup> mol-H2/mol glucose; <sup>b</sup> NL-H2/L of reactor; <sup>c</sup> NL-H2/L of TV; <sup>d</sup> NL-H2/g-COD; <sup>e</sup> NL-H2/g-VSfed; <sup>f</sup> Calculated from provided information; <sup>g</sup> Initial pH value; NR: not reported.

#### Table 2.

Comparison of the literature data on biohydrogen production efficiency using tequila vinasse as feedstock.

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

(iv) evaluating the feasibility of co-fermentation [11, 36]; and (v) exploring the microbial ecology of the process [32, 36, 37].

More particularly, Espinoza-Escalante et al. [27] evaluated the effect of three pretreatments, that is, alkalinization, cavitation, and thermal pretreatment, on the metabolic profile and the increments of COD and total reducing sugars (TRS) of TV, as well as on its bioH2 production potential. From that study, it can be concluded that the application of such pretreatments to raw TV resulted in different degrees of solubilization of COD and TRS, depending on the applied pretreatment and combinations thereof. However, there was no apparent relation in the consumption of TRS and COD with bioH2 production. Indeed, the optimal conditions that led to the highest solubilization of both COD and TRS did not result in a significant improvement in the YH2, which was about 2.8 NL-H2/L of reactor, indicating that compounds other than TRS could be involved in the mechanism of bioH2 production.

In another report, Espinoza-Escalante et al. [28] studied the effect of pH (4.5, 5.5, and 6.5), HRT (1, 3, and 5 d), and temperature (35 and 55°C) on the semicontinuous production of bioH2 from TV. The experiments were performed in 1-L glass vessels inoculated with 10% (v/v) of mesophilic anaerobic digester sludge. The results showed that all factors studied had an important effect on bioH2 production. The highest efficiency in terms of bioH2 production was achieved at a pH of 5.5, an HRT of 5 d and a temperature of 55°C. Based on constructed mathematical models, pH was the most influential parameter.

In a similar study, Buitrón and Carvajal [30] investigated the effect of temperature (25 and 35°C), HRT (12 and 24 h), and substrate concentration on bioH2 production from TV using a 7-L AnSBR, with a working volume of 6 L. The exchange volume was 50% with a reaction time of 11.3 or 5.3 h depending on the applied HRT, while pH and mixing were controlled at 5.5 and 153 rpm, respectively, in all cases. It was evidenced that all parameters studied affected the efficiency of bioH2 production. The HRT had a major influence on bioH2 production. It was found that the shorter the HRT, the higher the bioH2 production. Overall, the maximum VHPR of 2.2 NL-H2/L-d and an average bioH2 content in the biogas of 29.2 8.8% (v/v) were obtained at 35°C, 12 h HRT, and 3 g-COD/L OLR.

Later, Buitrón et al. [34] evaluated the performance of an FBR to produce bioH2 in a continuous mode from TV. The reactor had a working volume of 1.7 L and was packed with polyurethane rings for biomass immobilization. The temperature, pH, HRT, and OLR were kept constant at 35°C, 4.7, 4 h, and 2.15 g-COD/L-d (influent concentration of 8 g-COD/L), respectively. After an initial acclimatization period of HPB to TV, the FBR exhibited a VHPR of 1.7 NL-H2/L-d and a YH2 of 1.36 NL-H2/L of TV. In a follow-up study conducted by the same research group, by using a 0.6-L AnSBR operated under mesophilic and acidophilic conditions at an HRT of 6 h, it was observed that increasing substrate concentration from 2 to 16 g-COD/L increased the VHPR up to 1.4 NL-H2/L-d. Hence, the use of TV for bioH2 production did not result in inhibition [35].

Another interesting advance was made by García-Depraect et al. [11], who studied the technical feasibility of using a co-fermentation approach to produce bioH2 from TV in a well-mixed reactor operated under batch mode. Nixtamalization wastewater (NW) was chosen as the complementary substrate based on its wide availability in Mexico and high alkalinity. The TV:NW ratio of 80:20 (w/w) resulted in the highest VHPR of 2.6 NL-H2/L-d with a bioH2 content in the gas phase of 71% (v/v). Interestingly, the co-fermentation study allowed the identification of iron and nitrogen as essential nutrients which may be limiting in TV-fed DF reactors. This identification becomes significant to avoid nutrientlimited conditions and to prevent excessive nutrient supplementation that has been

at 6 h. The results showed that the performance was highly dependent on the type of reactor and OLR. Regarding the CSTR configuration, in general, the higher OLR resulted in higher VHPR. Nonetheless, the bioH2 production efficiency using individual enzymatic hydrolysate (0.72–2.25 NL-H2/L-d and 11.8–20.4 NL-H2/kg of AB) was lower compared to that obtained with the binary enzymatic hydrolysate (3.9–13 NL-H2/L-d and 83.3–117.9 NL-H2/kg of AB), with the maximum VHPR and YH2 at 100 and 60 g-COD/L-d and 90 and 52 g-COD/L-d, respectively. Regarding the TBR configuration, the binary enzymatic hydrolysate also outperformed the individual one, obtaining the maximum VHPR of 5.76 NL-H2/L-d at an OLR of 81 g-COD/L-d and YH2 of 72.4 NL-H2/kg of AB at an OLR of 69 g-COD/L-d. The enhancement was attributed, on one hand, to the use of binary hydrolysis that could have contributed

to produce a higher proportion of monomers of easy degradation by bioH2-

continuous

continuous

continuous

Dilution Batch 36 5.5g 0.7<sup>b</sup> 0.5 NR [33] Co-fermentation Batch 35 5.5 1.1b 2.6 71 [11]

Co-fermentation Batch 35 5.5 1.2<sup>b</sup> 2.4 68 [36]

Notes: Inoculum: anaerobic digester sludge [27, 28], thermally treated anaerobic granular sludge [11, 29–31, 33–38];

Comparison of the literature data on biohydrogen production efficiency using tequila vinasse as feedstock.

NL-H2/L of TV; <sup>d</sup>

growth in each reactor.

New Advances on Fermentation Processes

Pretreatment/conditioning Feeding T

None Semi-

Dilution, nutrient supplementation Semi-

Dilution, nutrient supplementation Semi-

Solid removal (centrifugation), nutrient supplementation

mol-H2/mol glucose; <sup>b</sup>

from provided information; <sup>g</sup>

\*Units: <sup>a</sup>

Table 2.

110

Alkalinization Batch 35 6.5–

Dilution Continuous 35 4.7 1.3a

Nutrient supplementation Batch 35 6.5–

Solid removal (centrifugation) Batch 35 6.5–

Co-fermentation Batch 35 6.5–

NL-H2/L of reactor; <sup>c</sup>

Initial pH value; NR: not reported.

producing bacteria (HPB) and to avoid the formation/release of potential inhibitors; on the other hand, to the differences of substrate availability given by the mode of

Concerning the use of TV for bioH2 production (Table 2), there are a few studies in the literature, with a particular focus on (i) optimizing pretreatments to further enhance bioH2 production [27]; (ii) testing the effect of different operational conditions such as pH [28, 29], temperature [28, 30], substrate concentration [28, 30, 31], solid content [22, 31], nutrient formulation [22, 31], inoculum addition [22, 31], HRT [22, 30, 32], and OLR [22, 32]; (iii) producing bioH2 in different systems, such as serum bottle [33], fixed bed reactor (FBR) [34], and CSTR [35];

(°C)

7.5

5.8

5.8

5.8

pH YH2 \* VHPR

1.5<sup>a</sup> , 2.8b,f

, 1.36c

4.8<sup>c</sup> , 0.12<sup>e</sup>

4.3<sup>b</sup> , 0.11<sup>e</sup>

2.5b , 2.7<sup>c</sup>

Continuous 35 5.8 3.4<sup>c</sup> 12.3 90 [38]

NL-H2/g-COD; <sup>e</sup>

(NL/L-d)

55 5.5 13.8b,f 2.8 NR [28]

35 5.5 NR 2.2 29.2 [30]

35 5.5 0.12<sup>d</sup> 1.4 NR [35]

H2 (% v/v)

NR NR [27]

1.7 64 [34]

3.8 70 [37]

5.4 71 [31]

3.7 73 [29]

NL-H2/g-VSfed; <sup>f</sup>

Calculated

Ref.

occurring in several studies at bench scale, but its practice may be prohibited on larger scales.

In this field of progressive research, the effect of pH on the bioH2 production efficiency was subsequently studied by García-Depraect et al. [29] through macroand micro-scale behavior analysis approaches. It was found that fixed pH of 5.8 showed a longer lag phase compared with fixed pH of 6.5, but the latter promoted bioH2 sink through propionogenesis. Based on the above observations, a two-stage pH-shift control strategy was devised to further increase bioH2 production. The strategy entailed the control of pH at 6.5 for first 29 h of culture to decrease the lag time, and then the pH was maintained at 5.8 to increase the bioH2 conversion efficiency by inhibiting the formation of propionic acid (HPr). The pH-shift strategy reduced running time and enhanced bioH2 production by 17%, obtaining 2.5 NL-H2/L of reactor. In a further study, the use of TV as the sole carbon source in the batch bioH2-yielding process was evaluated through a comprehensive approach entailing the operational performance, kinetic analysis, and microbial ecology [37]. A YH2 of 4.3 NL-H2/L of reactor and a peak VHPR of 3.8 NL-H2/L-d were obtained.

The effects of total solids content, substrate concentration, nutrient formulation, and inoculum addition on bioH2 production performance from TV have been also investigated in batch experiments [31]. It was observed a consistent bioH2 production which was primarily influenced by inoculum addition followed by substrate concentration, nutrient formulation, and solids content. Maximum VHPR (5.4 NL-H2/L-d) and YH2 (4.3 NL-H2/L of reactor) were achieved by removing suspended solids and enhancing nutrient content, respectively [31]. Finally, the highest VHPR (12.3 NL-H2/L-d, corresponding to 3.4 NL-H2/L of TV) up to date has been achieved via a novel multi-stage process operated under continuous mode for 6 h HRT, which also resulted in high stability (VHPR fluctuations <10%) and a high bioH2 content in the gas phase of 90% (v/v) [38].

HBu are the end-products, respectively. However, from published studies in the field of DF, it seems reasonable to conclude that, in mixed cultures, a high bioH2 production efficiency is rather related with the formation of HBu than HAc because

Metabolic reactions occurring in dark fermentation systems treating tequila processing by-products.

Glucose þ 2H2O ! 2HAc þ 2CO2 þ 4H2 (10) Glucose ! HBu þ 2CO2 þ 2H2 (11) HLac þ 0:5HAc ! 0:75HBu þ CO2 þ 0:5H2 þ 0:5H2O (12) HLac þ H2O ! HAc þ CO2 þ 2H2 (13) 2HLac ! HBu þ 2CO2 þ 2H2 (14) HFor ! H2 þ CO2 (15) Glucose þ H2O ! C2H5OH þ HAc þ 2CO2 þ 2H2 (16)

Competing reactions Reaction Glucose þ 2H2 ! 2HPr þ 2H2O (1) HLac þ H2 ! HPr þ H2O (2) 3HLac ! 2HPr þ H2O (3) 4H2 þ CO2 ! CH4 þ 2H2O (bioCH4-producing reaction) (4) Glucose ! 2HLac (5) Glucose ! HLac þ HAc þ CO2 (6) 2Glucose ! 2HLac þ 3HAc (7) Glucose ! 3HAc (8) 4H2 þ 2CO2 ! HAc þ 2H2O (9)

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

At this point, it must be noted that bioH2 can also come from the degradation of HLac, as shown in reactions 12–14 [37]. The HLac-type fermentation could provide the basis for the design of stable bioH2-producing reactors whose feedstocks are rich in HLac and HAc such as distillery wastewater (including TV), food waste, dairy wastewater, ensiled crops, lignocellulosic residues, and their hydrolysates (including AB), among others [36]. The amount of bioH2 obtained from the HLac-type fermentation may vary significantly depending on several factors such as pH, temperature, HRT, OLR, operation mode, substrate type, mixing, and prevailing microorganisms [31]. Also, it has been observed that the HLac-type fermentation in vinasse-fed DF reactors could be induced by low carbohydrate-available conditions [31, 36, 37]. On the other hand, the formation of HFor also can yield bioH2 (reaction 15) via the action of HFor hydrogenase complexes [37]. In addition, ethanol-type fermentation (reaction 16) generates ethanol, HAc, bioH2, and carbon dioxide. According to Ren et al. [39], the ethanol-type fermentation is favored by a pH of 4.0–5.0 and oxidation-reduction potential (ORP) of < �200 mV. In comparison to the HAc-HBu-mixed type fermentation, which has been ascertained as the most common bioH2-producing pathway, the latter two reactions have been less fre-

Another pertinent point is that the performance of bioH2-producing reactors strongly depends on the selection and maintenance of HPB. However, this is a

the latter may come from acetogenesis/homoacetogenesis.

quently found in DF reactors fed with AB/TV.

3.3 Microbial communities

113

BioH2-producing reactions

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

Table 3.

#### 3.2 Metabolic pathways

Following the by-products formed during fermentation is of utmost importance to understand, predict, control, and optimize the behavior of DF processes. It is well known that the distribution of the fermentation by-products may change depending on culture conditions. Low bioH2 productions matched with the presence of undesired electron sinks, such as HLac, HPr, iso-butyrate, valerate, iso-valerate, and solvents (e.g. ethanol, acetone, and butanol). For instance, the production of HPr reduces the amount of bioH2 that may be produced, as shown in reactions 1–3 (Table 3). Biomass growth also represents an electron sink. Commonly bioH2 production is growth-associated. However, higher biomass growth does not necessarily imply the achievement of the best bioH2 production [29]. Thus, a proper balance between biomass growth and bioH2 production is desirable. On the other hand, bioH2 sink through the formation of bioCH4 via the hydrogenotrophic pathway (reaction 4) seems to be less problematic in DF processes due to the application of inoculum pretreatments together with biokinetic control such as acidic pH and low HRT, even using attached-growth reactors [34]. The formation of HLac can also lead to stuck DF fermentations, as shown in reactions 5–7. Acetogenesis (reaction 8) and homoacetogenesis (reaction 9) may also occur during the process, decreasing the bioH2 production efficiency. It has been reported that the consumption of bioH2 and carbon dioxide due to homoacetogenesis depends on the type of reactor and OLR, being its occurrence accentuated in suspended growth systems and high OLR [20, 23].

Contrarily, bioH2 production via DF is typically related to HBu and HAc production from carbohydrates degradation, as shown in reactions 10 and 11, respectively. Theoretically, 4 and 2 mol of H2 derive from 1 mol of glucose when HAc and A Comprehensive Overview of the Potential of Tequila Industry By-Products for Biohydrogen… DOI: http://dx.doi.org/10.5772/intechopen.88104


#### Table 3.

occurring in several studies at bench scale, but its practice may be prohibited on

bioH2 content in the gas phase of 90% (v/v) [38].

3.2 Metabolic pathways

high OLR [20, 23].

112

In this field of progressive research, the effect of pH on the bioH2 production efficiency was subsequently studied by García-Depraect et al. [29] through macroand micro-scale behavior analysis approaches. It was found that fixed pH of 5.8 showed a longer lag phase compared with fixed pH of 6.5, but the latter promoted bioH2 sink through propionogenesis. Based on the above observations, a two-stage pH-shift control strategy was devised to further increase bioH2 production. The strategy entailed the control of pH at 6.5 for first 29 h of culture to decrease the lag time, and then the pH was maintained at 5.8 to increase the bioH2 conversion efficiency by inhibiting the formation of propionic acid (HPr). The pH-shift strategy reduced running time and enhanced bioH2 production by 17%, obtaining 2.5 NL-H2/L of reactor. In a further study, the use of TV as the sole carbon source in the batch bioH2-yielding process was evaluated through a comprehensive approach entailing the operational performance, kinetic analysis, and microbial ecology [37]. A YH2 of 4.3 NL-H2/L of reactor and a peak VHPR of 3.8 NL-H2/L-d were obtained. The effects of total solids content, substrate concentration, nutrient formulation, and inoculum addition on bioH2 production performance from TV have been also investigated in batch experiments [31]. It was observed a consistent bioH2 production which was primarily influenced by inoculum addition followed by substrate concentration, nutrient formulation, and solids content. Maximum VHPR (5.4 NL-H2/L-d) and YH2 (4.3 NL-H2/L of reactor) were achieved by removing suspended solids and enhancing nutrient content, respectively [31]. Finally, the highest VHPR (12.3 NL-H2/L-d, corresponding to 3.4 NL-H2/L of TV) up to date has been achieved via a novel multi-stage process operated under continuous mode for 6 h HRT, which also resulted in high stability (VHPR fluctuations <10%) and a high

Following the by-products formed during fermentation is of utmost importance to understand, predict, control, and optimize the behavior of DF processes. It is well known that the distribution of the fermentation by-products may change depending

undesired electron sinks, such as HLac, HPr, iso-butyrate, valerate, iso-valerate, and solvents (e.g. ethanol, acetone, and butanol). For instance, the production of HPr reduces the amount of bioH2 that may be produced, as shown in reactions 1–3 (Table 3). Biomass growth also represents an electron sink. Commonly bioH2 production is growth-associated. However, higher biomass growth does not

necessarily imply the achievement of the best bioH2 production [29]. Thus, a proper balance between biomass growth and bioH2 production is desirable. On the other hand, bioH2 sink through the formation of bioCH4 via the hydrogenotrophic pathway (reaction 4) seems to be less problematic in DF processes due to the application of inoculum pretreatments together with biokinetic control such as acidic pH and low HRT, even using attached-growth reactors [34]. The formation of HLac can also lead to stuck DF fermentations, as shown in reactions 5–7. Acetogenesis (reaction 8) and homoacetogenesis (reaction 9) may also occur during the process, decreasing the bioH2 production efficiency. It has been reported that the consumption of bioH2

on culture conditions. Low bioH2 productions matched with the presence of

and carbon dioxide due to homoacetogenesis depends on the type of reactor and OLR, being its occurrence accentuated in suspended growth systems and

Contrarily, bioH2 production via DF is typically related to HBu and HAc production from carbohydrates degradation, as shown in reactions 10 and 11, respectively. Theoretically, 4 and 2 mol of H2 derive from 1 mol of glucose when HAc and

larger scales.

New Advances on Fermentation Processes

Metabolic reactions occurring in dark fermentation systems treating tequila processing by-products.

HBu are the end-products, respectively. However, from published studies in the field of DF, it seems reasonable to conclude that, in mixed cultures, a high bioH2 production efficiency is rather related with the formation of HBu than HAc because the latter may come from acetogenesis/homoacetogenesis.

At this point, it must be noted that bioH2 can also come from the degradation of HLac, as shown in reactions 12–14 [37]. The HLac-type fermentation could provide the basis for the design of stable bioH2-producing reactors whose feedstocks are rich in HLac and HAc such as distillery wastewater (including TV), food waste, dairy wastewater, ensiled crops, lignocellulosic residues, and their hydrolysates (including AB), among others [36]. The amount of bioH2 obtained from the HLac-type fermentation may vary significantly depending on several factors such as pH, temperature, HRT, OLR, operation mode, substrate type, mixing, and prevailing microorganisms [31]. Also, it has been observed that the HLac-type fermentation in vinasse-fed DF reactors could be induced by low carbohydrate-available conditions [31, 36, 37]. On the other hand, the formation of HFor also can yield bioH2 (reaction 15) via the action of HFor hydrogenase complexes [37]. In addition, ethanol-type fermentation (reaction 16) generates ethanol, HAc, bioH2, and carbon dioxide. According to Ren et al. [39], the ethanol-type fermentation is favored by a pH of 4.0–5.0 and oxidation-reduction potential (ORP) of < �200 mV. In comparison to the HAc-HBu-mixed type fermentation, which has been ascertained as the most common bioH2-producing pathway, the latter two reactions have been less frequently found in DF reactors fed with AB/TV.

#### 3.3 Microbial communities

Another pertinent point is that the performance of bioH2-producing reactors strongly depends on the selection and maintenance of HPB. However, this is a

difficult task because DF processes treating unsterilized feedstocks under continuous conditions are open systems, meaning that several microbial interactions may take place. In the literature, it has been used defined mixed cultures to inoculate DF reactors treating complex feedstocks such as AB and TV. In most cases, heat-shock pretreatment has been used as the selective method for the enrichment of HPB (based on their ability in forming spores), while killing bioH2 consumers. However, other aspects such as biological/physiological (e.g. growth rate, microbial interactions, auto/allochthonous bacteria, adaptation to environmental stress conditions, and nutrients requirements), the composition of broth culture (e.g. availability of substrate/nutrients, organic acids, and toxicants), process parameters (e.g. pH, temperature, HRT, OLR, and ORP) and reactor configurations (e.g. suspended and attached biomass, mixing, and liquid-gas interface mass transfer capacity) are also selective pressure factors to determine prevailing microbial community structure during operation. At this point, it must be noted that the application of the heatshock pretreatment decreases the diversity eliminating not only microorganisms with a negative effect on the overall bioH2 production, but also with a potentially positive role. Besides having a high capacity to produce bioH2, the biocatalyst must be able to thrive on the presence of putative toxic by-products such as HFor, HAc, phenols, and furans which are commonly detected in pretreated AB and raw TV.

possible changes of metabolites and microbial communities through time have also been investigated to understand the potential mechanism of bioH2 production from HLac and HAc [36]. In this regard, the microbial structure showed coordinated dynamic behavior over time, identifying three stages throughout the process: (i) a first stage (corresponding to the lag phase in relation to bioH2 production) in which the major part of TRS were consumed by dominant LAB and AAB, (ii) a second stage (corresponding to the exponential bioH2 production phase) during which the HLac-type fermentation was catalyzed by emerging HPB, and (iii) a third stage (corresponding to the stationary bioH2 production phase) in which non-HPB regrown while HPB became subdominant [36]. Interestingly, it has been also shown that an operating strategy based on pH-control may stimulate the syntrophy between Clostridium and Lactobacillus, and reduced the proliferation of Blautia and

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

Propionibacterium (which are undesirable microorganisms due to their

tion to enhanced efficiency [29].

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

4.1 Operational performance

Acid hydrolysis Semi-

Acid hydrolysis Semi-

Individual enzymatic

Individual enzymatic

Individual enzymatic

Individual enzymatic

Alkaline hydrogen peroxide + binary enzymatic hydrolysis

hydrolysis

hydrolysis

hydrolysis

hydrolysis

Table 4.

feedstock.

115

NL-CH4/kg of AB; <sup>d</sup>

Pretreatment Feeding Stage T

continuous

Semicontinuous

continuous

Notes: All studies were conducted using anaerobic granular sludge; <sup>a</sup>

homoacetogenic and propionogenic activity, respectively), trending bioH2 produc-

The operational performance, metabolic pathways, and microbial communities

In recent years, there have been several efforts to improve the AD performance of AB and TV (Table 4). Regarding the use of AB, the first study reported in this

(°C)

Acid hydrolysis Batch Single 37 8<sup>a</sup> 0.16<sup>b</sup> 0.78d NR [17]

Acid hydrolysis Batch Two 37 8<sup>a</sup> 0.24<sup>b</sup> 0.75d NR [17]

Batch Single 37 7.5a 0.2<sup>b</sup>

Continuous Two 22–25 7.5 0.32b

Comparison of the literature data on biomethane production efficiency using pretreated agave bagasse as

Calculated from provided information; NR: not reported.

NL-CH4/g-CODremoved, <sup>c</sup>

Single 35 7 0.28b

pH YCH4 \* VMPR

Single 32 7.5 0.26b 0.3 70–74 [8]

Two 37 7 NR 0.41 NR [7]

Initial pH value; \*Units: <sup>b</sup>

Batch Single 37 8<sup>a</sup> 0.09b 0.6<sup>d</sup> NR [17]

Batch Two 37 8<sup>a</sup> 0.24<sup>b</sup> 0.96 NR [17]

(NL/L-d)

, 130<sup>c</sup> NR NR [18]

, 393<sup>c</sup> 0.67 NR [22]

, 225c 6.4 70–76 [21]

CH4 (% v/v) Ref.

4. Biomethane production from agave bagasse and tequila vinasse

of the AD of AB and TV are extensively reviewed in the following sections.

Interestingly, molecular biology tools reveal that HPB (e.g. Clostridium, Klebsiella, and Enterobacter) are, in almost all DF systems, accompanied by lactic acid bacteria (LAB) (e.g. Lactobacillus and Sporolactobacillus) [40]. This co-occurrence could be attributed to the fact that LAB are ubiquitous in the environment, the physicochemical characteristics of feedstocks could sustain the proliferation of LAB, and LAB possess complex adaptation mechanisms that confer their ecological advantages over other bacteria [31]. Streptococcus and Lactobacillus have actually been detected in TV [31]. Bearing in mind such explanations, it is reasonable to assume that DF reactors fed with TV will naturally undergo the proliferation of LAB. Indeed, this assumption was verified by [11, 29, 31, 36, 37].

Except for capnophilic HLac pathway, it is well known that HLac is produced through zero-bioH2-producing pathways. Moreover, the proliferation of LAB is commonly associated with the deterioration of bioH2 production, mainly due to substrate competition, acidification of cultivation broth, and excretion of antimicrobial peptides known as bacteriocins [41]. At this point, another important constraint to be mentioned is that methods devoted to preventing the growth of LAB such as pretreatment of inoculum and sterilization of feedstock may be expensive, thus imposing a high economic burden on the process. Besides, the application of pretreatments does not always hinder the proliferation of LAB [42]. Therefore, there is an urgent need for novel technical solutions to ensure a maximum VHPR and YH2.

Fortunately, the activity of LAB may also have positive effects on the overall DF process, mainly through the aforementioned HLac-type fermentation (HLac-driven bioH2 production). Indeed, it is noteworthy mentioning that, under certain conditions, a DF process mediated by beneficial trophic links between HPB and LAB may be highly stable and consequently of high relevance for practical applications. In this case, LAB may help in the production of bioH2 by pH regulation, substrate hydrolysis, biomass retention, oxygen depletion, and substrate detoxification [36]. Nevertheless, to exploit these advantages, a thorough understanding of the mechanisms underlying the HLac-type fermentation is essential. In this context, molecular analyses have depicted a possible syntrophy between LAB, acetic acid bacteria (AAB) and HPB [11, 29, 31, 36, 37]. For instance, Illumina MiSeq sequencing has revealed that Clostridium beijerinckii, Streptococcus sp., and Acetobacter lovaniensis were the most abundant species at the highest bioH2 production activity [37]. The

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

possible changes of metabolites and microbial communities through time have also been investigated to understand the potential mechanism of bioH2 production from HLac and HAc [36]. In this regard, the microbial structure showed coordinated dynamic behavior over time, identifying three stages throughout the process: (i) a first stage (corresponding to the lag phase in relation to bioH2 production) in which the major part of TRS were consumed by dominant LAB and AAB, (ii) a second stage (corresponding to the exponential bioH2 production phase) during which the HLac-type fermentation was catalyzed by emerging HPB, and (iii) a third stage (corresponding to the stationary bioH2 production phase) in which non-HPB regrown while HPB became subdominant [36]. Interestingly, it has been also shown that an operating strategy based on pH-control may stimulate the syntrophy between Clostridium and Lactobacillus, and reduced the proliferation of Blautia and Propionibacterium (which are undesirable microorganisms due to their homoacetogenic and propionogenic activity, respectively), trending bioH2 production to enhanced efficiency [29].

## 4. Biomethane production from agave bagasse and tequila vinasse

The operational performance, metabolic pathways, and microbial communities of the AD of AB and TV are extensively reviewed in the following sections.

#### 4.1 Operational performance

difficult task because DF processes treating unsterilized feedstocks under continuous conditions are open systems, meaning that several microbial interactions may take place. In the literature, it has been used defined mixed cultures to inoculate DF reactors treating complex feedstocks such as AB and TV. In most cases, heat-shock pretreatment has been used as the selective method for the enrichment of HPB (based on their ability in forming spores), while killing bioH2 consumers. However, other aspects such as biological/physiological (e.g. growth rate, microbial interactions, auto/allochthonous bacteria, adaptation to environmental stress conditions, and nutrients requirements), the composition of broth culture (e.g. availability of substrate/nutrients, organic acids, and toxicants), process parameters (e.g. pH, temperature, HRT, OLR, and ORP) and reactor configurations (e.g. suspended and attached biomass, mixing, and liquid-gas interface mass transfer capacity) are also selective pressure factors to determine prevailing microbial community structure during operation. At this point, it must be noted that the application of the heatshock pretreatment decreases the diversity eliminating not only microorganisms with a negative effect on the overall bioH2 production, but also with a potentially positive role. Besides having a high capacity to produce bioH2, the biocatalyst must be able to thrive on the presence of putative toxic by-products such as HFor, HAc, phenols, and furans which are commonly detected in pretreated AB and raw TV. Interestingly, molecular biology tools reveal that HPB (e.g. Clostridium, Klebsiella, and Enterobacter) are, in almost all DF systems, accompanied by lactic acid bacteria (LAB) (e.g. Lactobacillus and Sporolactobacillus) [40]. This co-occurrence could be attributed to the fact that LAB are ubiquitous in the environment, the physicochemical characteristics of feedstocks could sustain the proliferation of LAB, and LAB possess complex adaptation mechanisms that confer their ecological advantages over other bacteria [31]. Streptococcus and Lactobacillus have actually been detected in TV [31]. Bearing in mind such explanations, it is reasonable to assume that DF reactors fed with TV will naturally undergo the proliferation of

New Advances on Fermentation Processes

LAB. Indeed, this assumption was verified by [11, 29, 31, 36, 37].

and YH2.

114

Except for capnophilic HLac pathway, it is well known that HLac is produced through zero-bioH2-producing pathways. Moreover, the proliferation of LAB is commonly associated with the deterioration of bioH2 production, mainly due to substrate competition, acidification of cultivation broth, and excretion of antimicrobial peptides known as bacteriocins [41]. At this point, another important constraint to be mentioned is that methods devoted to preventing the growth of LAB such as pretreatment of inoculum and sterilization of feedstock may be expensive, thus imposing a high economic burden on the process. Besides, the application of pretreatments does not always hinder the proliferation of LAB [42]. Therefore, there is an urgent need for novel technical solutions to ensure a maximum VHPR

Fortunately, the activity of LAB may also have positive effects on the overall DF process, mainly through the aforementioned HLac-type fermentation (HLac-driven bioH2 production). Indeed, it is noteworthy mentioning that, under certain conditions, a DF process mediated by beneficial trophic links between HPB and LAB may be highly stable and consequently of high relevance for practical applications. In this case, LAB may help in the production of bioH2 by pH regulation, substrate hydrolysis, biomass retention, oxygen depletion, and substrate detoxification [36]. Nevertheless, to exploit these advantages, a thorough understanding of the mechanisms underlying the HLac-type fermentation is essential. In this context, molecular analyses have depicted a possible syntrophy between LAB, acetic acid bacteria (AAB) and HPB [11, 29, 31, 36, 37]. For instance, Illumina MiSeq sequencing has revealed that Clostridium beijerinckii, Streptococcus sp., and Acetobacter lovaniensis were the most abundant species at the highest bioH2 production activity [37]. The


In recent years, there have been several efforts to improve the AD performance of AB and TV (Table 4). Regarding the use of AB, the first study reported in this

Notes: All studies were conducted using anaerobic granular sludge; <sup>a</sup> Initial pH value; \*Units: <sup>b</sup> NL-CH4/g-CODremoved, <sup>c</sup> NL-CH4/kg of AB; <sup>d</sup> Calculated from provided information; NR: not reported.

#### Table 4.

Comparison of the literature data on biomethane production efficiency using pretreated agave bagasse as feedstock.

field was conducted by Arreola-Vargas et al. [8], who evaluated the feasibility of producing bioCH4 from acid uncooked AB hydrolysates under two conditions, that is, with and without nutrient addition. The experiments were conducted in a mesophilic (32°C) AnSBR (with recirculation) at an OLR of 1.3 g-COD/L-d (influent concentration of 5 g-COD/L). The reactor had a working volume of 3.6 L and was inoculated with 5.8 g-VSS/L of anaerobic granular sludge collected from a fullscale UASB reactor treating brewery wastewater. The total cycle time was 72 h with a reaction time of 71 h and an exchange ratio of 80% (v/v). Unexpectedly, the best performance was obtained without additional supplementation of nutrients, achieving a volumetric bioCH4 production rate (VMPR) of 0.3 NL-CH4/L-d and a bioCH4 yield (YCH4) of 0.26 NL-CH4/g-CODremoved with a CH4 content in the biogas of 70–74% (v/v).

(0.39 NL-CH4/g of AB) and 0.67 NL-CH4/L-d, respectively, indicating the potential advantage of integrating a delignification pretreatment and the use of synergistic

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

of OLR on the VMPR using a mesophilic (23–25°C) 1.5-L UASB reactor (with a working volume of 1.25 L) feeding with diluted (and supplemented with nutrients) acidogenic effluent generated during the DF of enzymatic hydrolysates of AB. The reactor was inoculated with 20 g-VS/L of anaerobic granular sludge from a full-scale UASB reactor treating TV and operated for 80 d to achieve OLRs between 1.35 and 24 g-COD/L-d by increasing the COD concentration of the influent and then by decreasing the HRT from 21 to 10 h. The highest VMPR and YCH4 of 6.4 NL-CH4/L-d and 0.32 NL-CH4/g-CODfed (225 NL-CH4/kg of AB) were achieved at an OLR of 20 g-COD/L-d (14 h HRT). Under such conditions, the COD removal efficiency was above 90% and the CH4 content in the gas phase was of 73% (v/v). Regarding the use of TV for bioCH4 production (Table 5), Méndez-Acosta et al. [43] assessed the mesophilic AD of TV in a lab-scale CSTR reactor for 250 d at HRTs of 14–5 d corresponding to increments in the OLR from 0.7 to 6 g-COD/L-d (influent COD concentrations of 10–33 g/L). The highest YCH4 of 0.32 L-CH4/g-CODremoved and VMPR of 2.8 L-CH4/L-d with bioCH4 concentrations in the biogas greater than 65% (v/v) and COD removal efficiencies over 90% were obtained, even with an unbalanced COD/N/P ratio, at 6 g-COD/L-d OLR. However, a relatively long start-up of 50 d and continuous supplementation of external alkalinity were

Regarding continuous processes, Montiel and Razo-Flores [21] studied the effect

With the aim of enhancing the stability of the AD of TV, López-López et al. [44] investigated the influence of alkalinity and volatile fatty acids (VFAs) on the performance of a 2-L UASB reactor. The UASB reactor was inoculated with anaerobic granular sludge and operated under mesophilic conditions during 235 d at OLRs from 2.5 to 20 g-COD/L-d with recirculation of the treated effluent at recycling flow rate to influent flow rate ratios of 1:1 to 10:1 in one-unit increments. In that study, it was found that, by maintaining a VFAs to alkalinity ratio ≤ 0.5 with recirculation 1:10, the recirculation of the effluent could induce stable performances by reducing the impact of VFAs and organic matter concentration present in the effluent, attaining a COD removal efficiency higher than 75% with a YCH4 of 0.33

enzymatic mixtures before the AD process.

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

needed in order to provide stability to the process.

Feeding Stage T

Semicontinuous

continuous

Notes: All studies were conducted using anaerobic granular sludge; <sup>a</sup>

(°C)

Two 35 6.8–

Dilution Continuous Single 35 7.4 0.32a 1.7<sup>a</sup> 65 [43] Dilution Continuous Single 35 7.4 0.32 1.9a 75 [45]

Dilution Continuous Single 35 7 0.24 3.03 65 [47] Dilution Continuous Two 35 7.7 0.29 2.3<sup>a</sup> 80 [7]

Comparison of the literature data on biomethane production efficiency using tequila vinasse as feedstock.

7.5

pH YCH4 (NL/g-CODremoved)

Continuous Single 35 7 0.33 NR 60–65 [44]

Single 32 8 0.28 2.3<sup>a</sup> 90 [46]

VMPR (NL/L-d)

0.26 0.29 68 [35]

Calculated from provided information; NR: not

CH4 (% v/v) Ref.

Pretreatment/ conditioning

Dilution, nutrient supplementation

reported;

Table 5.

117

Dilution, solid removal (centrifugation)

Dilution Semi-

In a later study, Arreola-Vargas et al. [17], assessed the use of AB hydrolysates (20, 40, 60, 80, and 100% v/v) obtained either from acid or enzymatic pretreatment for bioCH4 production in single- and two-stage AD processes. The experiments were conducted in the AMPTS II system at 37°C, 120 rpm, initial pH of 8, and using 10 g-VSS/L of anaerobic granular sludge collected from a full-scale UASB reactor treating TV as inoculum. The highest VMPR for single- (0.84 NL-CH4/L-d) and two-stage (0.96 NL-CH4/L-d) processes were achieved in the assays with enzymatic hydrolysates at 100% and 20%, respectively. Regarding YCH4 results, the highest value with the single-stage process of 0.16 NL-CH4/g-CODremoved was obtained in the assays with 20% hydrolysate from enzymatic pretreatment, while the two-stage process attained up to 0.24 NL-CH4/g-CODremoved, also at 20% hydrolysate regardless of the type of pretreatment used. Although both hydrolysates harbor potential fermentation inhibitors (i.e. organic acids, furan derivatives, and polyphenols) in different concentrations, results showed no negative effects in the AD performance. Toledo-Cervantes et al. [7] also evaluated the bioCH4 production from the spent medium of DF of enzymatic hydrolysate of AB. The authors found that bioCH4 production in an AnSBR was severely inhibited likely because the remaining catalytic activity of the enzyme used may have contributed to the degradation of CH4 biocatalyst. In the same year, Breton-Deval et al. [18] contrasted the bioCH4 production from acid AB hydrolysates previously obtained using two different acid catalysts, that is, HCl and H2SO4. The experiments were carried out in the AMPTS II at 35°C, 120 rpm, initial pH of 7.5, an organic load of 8 g-COD/L, and using 10 g-VSS/L of anaerobic granular sludge collected from a full-scale UASB reactor treating TV as inoculum. The results showed that HCl hydrolysate outperformed the H2SO4 one by obtaining a four-fold increase on YCH4, that is, 0.17 versus 0.04 NL-CH4/g-CODremoved, respectively. The impairment of the methanogenic activity was attributed to the fact that the addition of sulfate ions favored the activity of sulfate-reducing bacteria (SRB). However, when using optimized HCl hydrolysates based on bioCH4 production (1.8% HCl, 119°C, and 103 min) rather than sugar recovery (1.9% HCl, 130°C, and 133 min), the highest YCH4 of 0.19 NL-CH4/g-CODremoved (0.09 NL-CH4/g-VS of AB) was obtained indicating that other components of the hydrolysates besides sugars may influence bioCH4 production, for example, extractives, potential microbial inhibitors.

In another study, Galindo-Hernández et al. [22] evaluated the bioCH4 production potential from AB previously pretreated with AHP followed by enzymatic saccharification with hemicellulases and cellulases. The experiments were performed in the AMPTS II system at 37°C, 150 rpm, initial pH of 7.0, and using an organic load of 5 g-COD/L, 10 g-VS/L of inoculum (anaerobic granular sludge from a mesophilic full-scale TV treatment plant) and a defined mineral solution. Under such conditions, the YCH4 and VMPR were found as 0.2 NL-CH4/g-CODremoved

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

(0.39 NL-CH4/g of AB) and 0.67 NL-CH4/L-d, respectively, indicating the potential advantage of integrating a delignification pretreatment and the use of synergistic enzymatic mixtures before the AD process.

Regarding continuous processes, Montiel and Razo-Flores [21] studied the effect of OLR on the VMPR using a mesophilic (23–25°C) 1.5-L UASB reactor (with a working volume of 1.25 L) feeding with diluted (and supplemented with nutrients) acidogenic effluent generated during the DF of enzymatic hydrolysates of AB. The reactor was inoculated with 20 g-VS/L of anaerobic granular sludge from a full-scale UASB reactor treating TV and operated for 80 d to achieve OLRs between 1.35 and 24 g-COD/L-d by increasing the COD concentration of the influent and then by decreasing the HRT from 21 to 10 h. The highest VMPR and YCH4 of 6.4 NL-CH4/L-d and 0.32 NL-CH4/g-CODfed (225 NL-CH4/kg of AB) were achieved at an OLR of 20 g-COD/L-d (14 h HRT). Under such conditions, the COD removal efficiency was above 90% and the CH4 content in the gas phase was of 73% (v/v).

Regarding the use of TV for bioCH4 production (Table 5), Méndez-Acosta et al. [43] assessed the mesophilic AD of TV in a lab-scale CSTR reactor for 250 d at HRTs of 14–5 d corresponding to increments in the OLR from 0.7 to 6 g-COD/L-d (influent COD concentrations of 10–33 g/L). The highest YCH4 of 0.32 L-CH4/g-CODremoved and VMPR of 2.8 L-CH4/L-d with bioCH4 concentrations in the biogas greater than 65% (v/v) and COD removal efficiencies over 90% were obtained, even with an unbalanced COD/N/P ratio, at 6 g-COD/L-d OLR. However, a relatively long start-up of 50 d and continuous supplementation of external alkalinity were needed in order to provide stability to the process.

With the aim of enhancing the stability of the AD of TV, López-López et al. [44] investigated the influence of alkalinity and volatile fatty acids (VFAs) on the performance of a 2-L UASB reactor. The UASB reactor was inoculated with anaerobic granular sludge and operated under mesophilic conditions during 235 d at OLRs from 2.5 to 20 g-COD/L-d with recirculation of the treated effluent at recycling flow rate to influent flow rate ratios of 1:1 to 10:1 in one-unit increments. In that study, it was found that, by maintaining a VFAs to alkalinity ratio ≤ 0.5 with recirculation 1:10, the recirculation of the effluent could induce stable performances by reducing the impact of VFAs and organic matter concentration present in the effluent, attaining a COD removal efficiency higher than 75% with a YCH4 of 0.33


Notes: All studies were conducted using anaerobic granular sludge; <sup>a</sup> Calculated from provided information; NR: not reported;

#### Table 5.

Comparison of the literature data on biomethane production efficiency using tequila vinasse as feedstock.

field was conducted by Arreola-Vargas et al. [8], who evaluated the feasibility of producing bioCH4 from acid uncooked AB hydrolysates under two conditions, that is, with and without nutrient addition. The experiments were conducted in a mesophilic (32°C) AnSBR (with recirculation) at an OLR of 1.3 g-COD/L-d (influent concentration of 5 g-COD/L). The reactor had a working volume of 3.6 L and was inoculated with 5.8 g-VSS/L of anaerobic granular sludge collected from a fullscale UASB reactor treating brewery wastewater. The total cycle time was 72 h with a reaction time of 71 h and an exchange ratio of 80% (v/v). Unexpectedly, the best performance was obtained without additional supplementation of nutrients, achieving a volumetric bioCH4 production rate (VMPR) of 0.3 NL-CH4/L-d and a bioCH4 yield (YCH4) of 0.26 NL-CH4/g-CODremoved with a CH4 content in the

In a later study, Arreola-Vargas et al. [17], assessed the use of AB hydrolysates

In another study, Galindo-Hernández et al. [22] evaluated the bioCH4 production potential from AB previously pretreated with AHP followed by enzymatic saccharification with hemicellulases and cellulases. The experiments were

performed in the AMPTS II system at 37°C, 150 rpm, initial pH of 7.0, and using an organic load of 5 g-COD/L, 10 g-VS/L of inoculum (anaerobic granular sludge from a mesophilic full-scale TV treatment plant) and a defined mineral solution. Under such conditions, the YCH4 and VMPR were found as 0.2 NL-CH4/g-CODremoved

pretreatment for bioCH4 production in single- and two-stage AD processes. The experiments were conducted in the AMPTS II system at 37°C, 120 rpm, initial pH of 8, and using 10 g-VSS/L of anaerobic granular sludge collected from a full-scale UASB reactor treating TV as inoculum. The highest VMPR for single- (0.84 NL-CH4/L-d) and two-stage (0.96 NL-CH4/L-d) processes were achieved in the assays with enzymatic hydrolysates at 100% and 20%, respectively. Regarding YCH4 results, the highest value with the single-stage process of 0.16 NL-CH4/g-CODremoved was obtained in the assays with 20% hydrolysate from enzymatic pretreatment, while the two-stage process attained up to 0.24 NL-CH4/g-CODremoved, also at 20% hydrolysate regardless of the type of pretreatment used. Although both hydrolysates harbor potential fermentation inhibitors (i.e. organic acids, furan derivatives, and polyphenols) in different concentrations, results showed no negative effects in the AD performance. Toledo-Cervantes et al. [7] also evaluated the bioCH4 production from the spent medium of DF of enzymatic hydrolysate of AB. The authors found that bioCH4 production in an AnSBR was severely inhibited likely because the remaining catalytic activity of the enzyme used may have contributed to the degradation of CH4 biocatalyst. In the same year, Breton-Deval et al. [18] contrasted the bioCH4 production from acid AB hydrolysates previously obtained using two different acid catalysts, that is, HCl and H2SO4. The experiments were carried out in the AMPTS II at 35°C, 120 rpm, initial pH of 7.5, an organic load of 8 g-COD/L, and using 10 g-VSS/L of anaerobic granular sludge collected from a full-scale UASB reactor treating TV as inoculum. The results showed that HCl hydrolysate outperformed the H2SO4 one by obtaining a four-fold increase on YCH4, that is, 0.17 versus 0.04 NL-CH4/g-CODremoved, respectively. The impairment of the methanogenic activity was attributed to the fact that the addition of sulfate ions favored the activity of sulfate-reducing bacteria (SRB). However, when using optimized HCl hydrolysates based on bioCH4 production (1.8% HCl, 119°C, and 103 min) rather than sugar recovery (1.9% HCl, 130°C, and 133 min), the highest YCH4 of 0.19 NL-CH4/g-CODremoved (0.09 NL-CH4/g-VS of AB) was obtained indicating that other components of the hydrolysates besides sugars may influence bioCH4 production, for example, extractives, potential

(20, 40, 60, 80, and 100% v/v) obtained either from acid or enzymatic

biogas of 70–74% (v/v).

New Advances on Fermentation Processes

microbial inhibitors.

116

NL-CH4/g-CODremoved. However, even though the high recirculation ratio led to the recovery of alkalinity without any addition of external alkalinity, the granular sludge tended to become flocculent with a reduction in the average size from 2.5 to 1.5 mm.

and from the syntrophic degradation of HBu (reaction 20) and HPr (reaction 21) [48]. Thus, an even production and consumption rate of organic acids is a sign of healthy single-stage AD processes. Contrarily, excessive accumulation of organic acids in the effluent has been related to reactor upset and failure, causing a drop in biogas production and COD removal efficiency. For instance, the presence of HPr in a HPr/HAc ratio ≥ 1 is usually matched with operational instability [43]. The alkalinity ratio, α = intermediate alkalinity (pH = 5.75)/partial alkalinity (pH = 4.3), roughly relates the amounts of VFAs and bicarbonate alkalinity in anaerobic reactors, measuring the buffer potential of the systems [49]. Values ≤0.3 are reported as adequate for achieving stable operation; however, in the case of TV-fed anaerobic reactors, stable processes have been achieved at slightly higher range of α between 0.2 and 0.5 [44, 47]. Moreover, bioCH4 production can be disrupted by the formation of certain by-products such as long chain fatty acids or solvents, which may jeopardize the suitable availability of bioCH4 precursors. In this regard, in the case of integrated DF-AD schemes, special attention must be also paid to the concentration and composition of organic acids coming from the DF stage. At this point, it should be mentioned that the redirection of carbon through HLac has been reported as a strategy to enhanced AD processes due to its thermodynamic

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

AD reactors contain mixed microbial populations [15]. BioCH4 formation from

(Anaerolineaceae, Candidatus, Cloacamonas, Syntrophobacter, Syntrophomonas, and Syntrophus), hydrogenotrophic (Methanobacterium and Methanocorpusculum) and acetoclastic (Methanosaeta and Methanosarcina) methanogens [7, 18, 47]. It has been previously observed that the two-stage AD of TV at low concentrations of VFAs (low OLRs) favored the acetoclastic pathway, in contrast, hydrogenotrophic methanogens enriched at high concentrations (high OLRs) [7]. This change in diversity has been also observed in an AnSBR digester fed with acid AB hydrolysates [53]. However, the opposite trend was observed during the single stage AD of TV using a pilot-scale PBR [47]. Regardless of the tequila by-product used, loss of syntrophic relationships for interspecies H2/HFor transfer and interspecies HAc transfer has been associated with microbial imbalance, which subsequently affects negatively bioCH4 production [8, 53]. However, in the case of multi-stage AD processes, unsuitable concentrations of hydrolytic/acidogenic bacteria in DF effluent may be quite detrimental for the granular methanogenic sludge [15]. In addi-

AB and TV has been related with the coexistence of syntrophic bacteria

tion, other bacteria which can compete with the methanogens for bioCH4 precursors may also be present in AD reactors, for example, SRB [15, 18].

Since TV has negligible levels of alkalinity and high concentrations of components with a tendency to suffer very rapid acidification [43, 44], two-stage AD processes have emerged as important operational strategies to provide enhanced stability of the CH4-producing stage [7, 24]. However, the multi-stage AD approach seems to be also applicable for pretreated AB [17, 21]. In fact, a two-stage AD process fed with AB hydrolysates showed up to 3.3-fold higher energy recovery than a single-stage process [17]. Indeed, according to Lindner et al. [16], two-stage systems seem to be only recommendable for digesting sugar-rich feed stocks, which undergo a quick hydrolysis/acidogenesis. This approach allows to provide optimal

advantages [50–52].

4.3 Microbial communities

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

5. Multi-stage anaerobic digestion

119

In another study conducted by Jáuregui-Jáuregui et al. [45], after a start-up period of 28 d, a mesophilic up-flow FBR inoculated with anaerobic granular sludge withdrawn from a full-scale UASB reactor treating brewery wastewater exhibited a YCH4 of 0.27 NL-CH4/g-CODremoved with a CH4 content of 75% (v/v) and COD removal efficiencies of up to 90% under an OLR of 8 g-COD/L-d and an HRT of 4 d. However, the authors also reported the inhibition of biogas production due to digester clogging, which led to an excessive VFAs accumulation. In the same year, Buitrón et al. [35] reported the performance of a UASB reactor treating the resulting effluent of a DF stage at three different COD concentrations, that is, 0.4, 1.08, and 1.6 g/L, and two HRTs, that is, 24 and 18 h. The maximal content of CH4 in the gas phase (68% v/v) and COD removal (67%) were achieved at the concentration of 1.6 g-COD/L with an HRT of 24 h. A further decrease in HRT resulted in lower efficiencies, that is, 40% CH4 content and 52% removal efficiency.

In a further study, Arreola-Vargas et al. [46] achieved YCH4 ranging from 0.25 to 0.29 NL-CH4/g-CODremoved with 75–90% (v/v) CH4 content and 85% COD removal using a bench scale AnSBR inoculated with anaerobic granular sludge and fed with diluted TV (8 g-COD/L), the reaction time varied within 3–9 d. Interestingly, later, the same research group performed a pilot scale study for the mesophilic AD treatment of TV using a 445-L packed bed reactor (PBR) which was operated for 231 d under increasing OLRs, from 4 to 12.5 g-COD/L-d [47]. The PBR showed a stable performance exhibiting COD removals and YCH4 in the range of 86–89% and 0.24–0.28 NL-CH4/g-CODremoved, respectively. Meanwhile, the highest VMPR of 3.03 NL-CH4/L-d was reached at the highest OLR of 12.5 g-COD/L-d [47].

More recently, in two-stage PBRs operated over 335 d, Toledo-Cervantes et al. [7] achieved the highest YCH4 of 0.29 NL-CH4/g-CODremoved at OLRs in the range of 2.7–6.8 g-COD/L-d (6–2.4 d HRT) with COD removal efficiencies between 81 and 95%, and with average CH4 contents around 80% (v/v). However, further increasing the OLR to 12 g-COD/L-d (2.2-d HRT) decreased the removal efficiency of COD (from 81 to 74%) accompanied with HAc and HPr accumulation.

### 4.2 Metabolic pathways

As shown in Table 6, the majority of bioCH4 produced in AD systems occurs from the use of HAc and bioH2 via acetoclastic (reaction 17) and hydrogenotrophic (reaction 4) pathways, respectively. However, bioCH4 can also be evolved from HFor (reaction 18), compounds with the methyl group like methanol (reaction 19),

