Table 6.

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

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 advantages [50–52].

#### 4.3 Microbial communities

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

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 [45], after a start-up period of 28 d, a mesophilic up-flow FBR inoculated with anaerobic granular sludge withdrawn from a full-scale UASB reactor treating brewery wastewater exhibited a YCH4 of 0.27 NL-CH4/g-CODremoved with a CH4 content of 75% (v/v) and COD removal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

In a further study, Arreola-Vargas et al. [46] achieved YCH4 ranging from 0.25

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-

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

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),

4H2 þ CO2 ! CH4 þ 2H2O (4) HAc ! CH4 þ CO2 (17) 4HFor ! CH4 þ 3CO2 þ 2H2O (18) 3CH3OH þ H2 ! CH4 þ H2O (19) 4HPr þ 2H2O ! 4HAc þ CO2 þ 3CH4 (syntrophic conversion) (20) HBu þ 2H2O ! 4HAc þ CO2 þ CH4 (syntrophic conversion) (21)

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. Interest-

ingly, later, the same research group performed a pilot scale study for the

of COD (from 81 to 74%) accompanied with HAc and HPr accumulation.

efficiencies, that is, 40% CH4 content and 52% removal efficiency.

1.5 mm.

New Advances on Fermentation Processes

COD/L-d [47].

Table 6.

118

Biomethane-producing reactions.

4.2 Metabolic pathways

AD reactors contain mixed microbial populations [15]. BioCH4 formation from AB and TV has been related with the coexistence of syntrophic bacteria (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 addition, other bacteria which can compete with the methanogens for bioCH4 precursors may also be present in AD reactors, for example, SRB [15, 18].

## 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 [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

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 [16]. 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 [7]. 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 [52]. 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 singlestage 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 [23] and 12.3 NL-H2/L-d from TV [38] and 6.4 NL-CH4/L-d

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

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

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

feedstock is its recalcitrant structure. As mentioned earlier, some of the

processes since it, as an intermediate, has a vital role in the carbon flux.

tainability of the existing tequila industries.

7. Conclusions

121

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 sus-

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

from AB [21] and 3.5 NL-CH4/L-d from TV [54].

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

6. Current limitations and potential improvements

#### Figure 3.

Types of reactor configurations used for biohydrogen and biomethane production from tequila processing byproducts. (a) Batch reactor, (b) continuously stirred tank reactor (CSTR) with recirculation, (c) CSTR, (d) anaerobic sequencing batch reactor (AnSBR), (e) trickling bed reactor with recirculation, (f) packed bed reactor, (g) up-flow anaerobic sludge blanket (UASB) reactor. AnSBR can integrate mechanical or hydraulic mixing. UASB can operate with effluent recycle.

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

13 NL-H2/L-d from AB [23] and 12.3 NL-H2/L-d from TV [38] and 6.4 NL-CH4/L-d from AB [21] and 3.5 NL-CH4/L-d from TV [54].
