Comparison of the literature data on biohydrogen production efficiency using pretreated agave bagasse as feedstock.
Nowadays, the use of agro-industrial by-products as alternative sustainable resources to generate bioenergy and high-value bioproducts is one of the most important research topics to tackle environmental concerns related to the excessive consumption of fossil-based fuels and rapid urbanization and industrialization. This chapter provides a broad overview of the potential of the main tequila industry by-products, agave bagasse and tequila vinasse, for biohydrogen (bioH2) and biomethane (bioCH4) production via dark fermentation and anaerobic digestion, respectively. First, pretreatment or conditioning steps commonly applied to tequila by-product streams before downstream biological processes are highlighted. The operational performance of bioH2- and bioCH4-producing reactors is subsequently reviewed, with a focus on reactor configuration and performance, microbial metabolic pathways, and the characterization of microbial communities. Additionally, the development of multi-stage anaerobic digestion processes is comprehensively discussed from a practical point of view. Finally, limitations and potential improvements in the field of bioH2 and bioCH4 production are presented.
- agave bagasse
- tequila vinasse
- dark fermentation
- anaerobic digestion
1. Tequila production process and its main by-products: agave bagasse and tequila vinasse
Tequila is a Mexican alcoholic beverage obtained from the distillation of fermented juice of the mature stems of
At this point, it must be noted that enormous quantities of solid (
Regarding the management and final disposition of AB and TV, it must be highlighted that only a small part of the whole AB generated is used in the manufacturing of different products such as animal feeds, fertilizers, bricks, mattresses, furniture, and packing materials [12, 13]. Therefore, most of AB is treated as waste and returned to the fields in the form of piles that are directly exposed to outdoor conditions, where they may cause leachates, odor generation, and atmospheric pollution [12, 14]. In the case of TV, it has been reported that approximately 80% of the total volume of TV generated is discharged without receiving adequate treatment into receiving water bodies (e.g. rivers, lakes, and sewer system) or directly onto soil, which in turn can result in adverse environmental and human health impacts . To valorize AB and TV and to face such disposal problems, nowadays, engineers and scientists are focusing on using them as potential substrates for the production of biofuels and value-added products in a tequila biorefinery framework. However, there are still several challenges that must be overcome before full-scale facilities could be implemented. This chapter provides an extended insight on (i) the pretreatment or conditioning steps of tequila by-product streams; (ii) the use of AB and TV to produce biogenic hydrogen (bioH2) and methane (bioCH4) via anaerobic fermentation processes, with a special emphasis on reactor configuration and operation, producing/competing metabolic pathways and the characterization of microbial communities; (iii) the development of multi-stage anaerobic digestion (AD) processes; and (iv) limitations and avenues for future research toward improving bioH2 and bioCH4 production.
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) . 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 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).
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.  pretreated cooked and uncooked AB through a dilute acid hydrolysis at 5% (w/v), 56.4–123.6°C, 1.2
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 . Comparing thermochemical, electrochemical, and biological ways of producing H2, the latter is considered the most sustainable because it is eco-friendlier 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 utilizing several different types of carbohydrate-rich wastes as substrate . 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.
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) , 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%
|Pretreatment||Feeding||T (°C)||pH||YH2 (NL/kg AB)||VHPR (NL/L-d)||H2 (% ||Ref.|
|Individual enzymatic hydrolysis||Batch||37||7a||140, 3.4b||2.4||NR|||
|Individual enzymatic hydrolysis||Continuous||37||5.5||67||3.45||26–52|||
|Individual enzymatic hydrolysis||Continuous||35||5.5||105||6||55|||
|Alkaline hydrogen peroxide + binary enzymatic hydrolysis||Batch||37||7.5a||215||0.93||NR|||
|Individual enzymatic hydrolysis||Semi-continuous||37||4.8||1.6c||0.6||49.3d|||
|Acid hydrolysis + detoxification||Batch||37||8.2a||56.2||1.51||NR|||
|Binary enzymatic hydrolysis||Continuous||37||5.5||117.8||13||51–60|||
In another work, Contreras-Dávila et al.  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% (
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.  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  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% (
In another study, Toledo-Cervantes et al.  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.  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.  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 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.
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 ; (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 , fixed bed reactor (FBR) , and CSTR ; (iv) evaluating the feasibility of co-fermentation [11, 36]; and (v) exploring the microbial ecology of the process [32, 36, 37].
|Pretreatment/conditioning||Feeding||T (°C)||pH||YH2 *||VHPR (NL/L-d)||H2 (% ||Ref.|
|Dilution, nutrient supplementation||Semi-continuous||35||5.5||NR||2.2||29.2|||
|Dilution, nutrient supplementation||Semi-continuous||35||5.5||0.12d||1.4||NR|||
|Nutrient supplementation||Batch||35||6.5–5.8||4.8c, 0.12e||3.8||70|||
|Solid removal (centrifugation)||Batch||35||6.5–5.8||4.3b, 0.11e||5.4||71|||
|Solid removal (centrifugation), nutrient supplementation||Continuous||35||5.8||3.4c||12.3||90|||
More particularly, Espinoza-Escalante et al.  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.  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 semi-continuous production of bioH2 from TV. The experiments were performed in 1-L glass vessels inoculated with 10% (
In a similar study, Buitrón and Carvajal  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% (
Later, Buitrón et al.  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 .
Another interesting advance was made by García-Depraect et al. , 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 (
In this field of progressive research, the effect of pH on the bioH2 production efficiency was subsequently studied by García-Depraect et al.  through macro- and 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 . 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 . 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 . 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% (
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 . 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 . 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 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 . 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 . 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 . 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 . In addition, ethanol-type fermentation (reaction 16) generates ethanol, HAc, bioH2, and carbon dioxide. According to Ren et al. , 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 heat-shock 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.
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 . 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 . 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 . 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
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
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 field was conducted by Arreola-Vargas et al. , 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 full-scale 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% (
|Pretreatment||Feeding||Stage||T (°C)||pH||YCH4 *||VMPR (NL/L-d)||CH4 (% ||Ref.|
|Individual enzymatic hydrolysis||Batch||Single||37||8a||0.09b||0.6d||NR|||
|Individual enzymatic hydrolysis||Batch||Two||37||8a||0.24b||0.96||NR|||
|Individual enzymatic hydrolysis||Semi-continuous||Two||37||7||NR||0.41||NR|||
|Acid hydrolysis||Semi-continuous||Single||35||7||0.28b, 130c||NR||NR|||
|Alkaline hydrogen peroxide + binary enzymatic hydrolysis||Batch||Single||37||7.5a||0.2b, 393c||0.67||NR|||
|Individual enzymatic hydrolysis||Continuous||Two||22–25||7.5||0.32b, 225c||6.4||70–76|||
In a later study, Arreola-Vargas et al. , assessed the use of AB hydrolysates (20, 40, 60, 80, and 100%
In another study, Galindo-Hernández et al.  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 (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  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% (
Regarding the use of TV for bioCH4 production (Table 5), Méndez-Acosta et al.  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% (
|Pretreatment/conditioning||Feeding||Stage||T (°C)||pH||YCH4 (NL/g-CODremoved)||VMPR (NL/L-d)||CH4 (% ||Ref.|
|Dilution, nutrient supplementation||Semi-continuous||Two||35||6.8–7.5||0.26||0.29||68|||
|Dilution, solid removal (centrifugation)||Continuous||Single||35||∼7||0.33||NR||60–65|||
With the aim of enhancing the stability of the AD of TV, López-López et al.  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 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.
In another study conducted by Jáuregui-Jáuregui et al. , 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% (
In a further study, Arreola-Vargas et al.  achieved YCH4 ranging from 0.25 to 0.29 NL-CH4/g-CODremoved with 75–90% (
More recently, in two-stage PBRs operated over 335 d, Toledo-Cervantes et al.  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% (
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), and from the syntrophic degradation of HBu (reaction 20) and HPr (reaction 21) . 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 . 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 . 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 advantages [50, 51, 52].
4.3 Microbial communities
AD reactors contain mixed microbial populations . BioCH4 formation from AB and TV has been related with the coexistence of syntrophic bacteria (
5. Multi-stage anaerobic digestion
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 . Indeed, according to Lindner et al. , 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 environmental conditions for the different groups of microorganisms which have differences in terms of physiology, nutrient intake, nutritional requirements, growth rate, optimum growth conditions such as pH, and adaptation to environmental stress conditions . The acidogenesis and methanogenesis separated in space may also produce bioH2 via DF process [17, 24, 35]. However, it is not necessarily desirable to produce bioH2 in all cases . In the latter case, a stream rich in HLac can be obtained through the HLac-type fermentation which can be further fed to the methanogenic stage [36, 37], where hydrogenotrophic may be benefited for the conversion of HLac to HAc by consuming the intermediate bioH2 gas immediately . The possibility of operating at higher organic loading capacity (in the methanogenic stage), reducing alkali addition, and increasing COD removal efficiency are additional advantages of the two-stage AD as compared to single-stage AD [7, 21, 24]. A small number of reactor configurations devoted to bioH2/bioCH4 production from AB/TV can be found in the literature (Figure 3). Among them, for both AB and TV, the CSTR and UASB configurations have shown the highest performance to date for producing bioH2 and bioCH4, respectively, that is, 13 NL-H2/L-d from AB  and 12.3 NL-H2/L-d from TV  and 6.4 NL-CH4/L-d from AB  and 3.5 NL-CH4/L-d from TV .
6. Current limitations and potential improvements
Notwithstanding the enormous efforts made to achieve a better understanding of the DF/AD process of AB/TV, it is still necessary to improve not only bioH2 or bioCH4 productivities and yields but also the (long-term) stability of processes for commercialization purposes. TV is a highly complex wastewater that besides high COD and negligible alkalinity, harbors recalcitrant compounds such as phenols, which may act as inhibitors in DF/AD. While the main limitation to use AB as the feedstock is its recalcitrant structure. As mentioned earlier, some of the pretreatment/conditioning steps used in AB have been optimized not only in terms of hydrolysis yield, reaction time, the generation/release and effect of putative fermentation inhibitory compounds, cost-effectiveness but also in terms of bioH2/bioCH4 production efficiency. However, there is still a need to explore other pretreatments that have not been yet embraced in the field of DF/AD of AB but they have been ascertained as potentially useful in releasing sugars for other applications like the production of bioethanol, such as ammonia fiber explosion (AFEX), autohydrolysis, organosolv, high-energy radiation, ozonolysis, alkaline, ionic liquids, or any combination of those pretreatments. It could be also interesting to explore consolidated processes (direct fermentation) which combine into a single operation the enzymatic hydrolysis of (pretreated) biomass and biological conversion to the desired by-product (in this case bioH2/bioCH4) by mixed consortia.
Besides the features described before, from practical purposes, the highly variable composition of AB/TV constitutes another constraint to produce bioH2 since DF systems are commonly unable to overcome perturbations in feedstock composition. One of the most significant challenges is to assure consistency in the prevailing metabolic pathways during the DF process and favor bioH2-producing pathways over other unwanted routes, for example, homoacetogenesis and methanogenesis. Very little is known about the microbial community structure of DF/AD processes treating AB/TV. In this regard, it is not clear the role of microorganisms and their association with operational parameters (e.g. pH, HRT, and OLR) and process indicators (e.g. VHPR, VMPR, and metabolic composition). Also, much less is known about how microbial assemblage may change through time, and what factors (operating parameters) govern its dynamics. It is worth noticing that HLac monitoring has been disregarded limiting the understanding of integrated DF-AD processes since it, as an intermediate, has a vital role in the carbon flux.
Another concern worth to mention is that most of the previous studies were carried out in batch or semi-continuous reactors. Thus, it is vital to transfer the kinetic knowledge gained from such studies to the expansion of continuous systems. In this context, the development of integrated DF-AD schemes for the continuous production of bioH2 and bioCH4 using AB/TV as feed stocks requires intensive research on interlinking side streams for producing high added-value bioproducts in a biorefinery framework (e.g. HLac-bioH2-bioCH4) for better sustainability of the existing tequila industries.
Tequila industry generates huge amounts of AB and TV, which could be subjected to integrated DF-AD processes to produce bioH2 and bioCH4 while reducing their pollution potential. This chapter focused on the state-of-the-art of configurations and process parameters, metabolic pathways, and microbial ecology of bioH2- and bioCH4-producing reactors. The pretreatment/conditioning steps applied to enhance the valorization of AB/TV were also reviewed. It has been suggested that the HLac-type fermentation coupled to DF and AD can boost the development of cascading design in multi-stage AD processes. This multiproduct approach using AB/TV as resources in the biorefinery scheme may facilitate sustainability to the tequila industry.
This work was financially supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) through the Project-PN-2015-2101-1024. Osuna-Laveaga D.R. acknowledges CONACYT for the Ph.D. scholarship: 267499.
Conflict of interest
The authors declare no conflict of interest.
Acronyms and abbreviations
acetic acid bacteria
alkaline hydrogen peroxide
anaerobic sequencing batch reactor
automatic methane potential test system
chemical oxygen demand
continuously stirred tank reactor
filter paper units
fixed bed reactor
hydraulic retention time
lactic acid bacteria
organic loading rate
packed bed reactor
volatile fatty acids
volatile suspended solids
volumetric biohydrogen production rate
volumetric biomethane production rate
trickling bed reactor
up-flow anaerobic sludge blanket reactor