Enhanced methane production and organic matter removal from tequila vinasses by anaerobic digestion assisted via bioelectrochemical power-to-gas

Journal Pre-proofs Enhanced methane production and organic matter removal from tequila vi‐ nasses by anaerobic digestion assisted via bioelectrochemical power-to-gas Estrada-Arriaga Edson Baltazar, Reynoso-Deloya Ma. Guadalupe, GuillénGarcés Rosa Angélica, Falcón-Rojas Axel, García-Sánchez Liliana PII: DOI: Reference:

S0960-8524(20)31618-7 https://doi.org/10.1016/j.biortech.2020.124344 BITE 124344

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

25 September 2020 24 October 2020 26 October 2020

Please cite this article as: Edson Baltazar, E-A., Ma. Guadalupe, R-D., Rosa Angélica, G-G., Axel, F-R., Liliana, G-S., Enhanced methane production and organic matter removal from tequila vinasses by anaerobic digestion assisted via bioelectrochemical power-to-gas, Bioresource Technology (2020), doi: https://doi.org/10.1016/ j.biortech.2020.124344

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Enhanced methane production and orgsanic matter removal from tequila vinasses by anaerobic digestion assisted via bioelectrochemical power-to-gas

Estrada-Arriaga Edson Baltazara*, Reynoso-Deloya Ma. Guadalupeb, Guillén-Garcés Rosa Angélica c, Falcón-Rojas Axeld, García-Sánchez Lilianad

aSubcoordinación

de Tratamiento de Aguas Residuales, Instituto Mexicano de

Tecnología del Agua, Paseo Cuauhnáhuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, México. bFacultad

de Ingeniería, Universidad Nacional Autónoma de México, Paseo

Cuauhnahuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, México. cUniversidad

Politécnica del Estado de Morelos, Paseo Cuauhnáhuac 566, Lomas del Texcal, Jiutepec, Morelos 62550, México

dSubcoordinación

de Tecnologías Apropiadas, Instituto Mexicano de Tecnología del

Agua, Paseo Cuauhnáhuac 8532, Progreso, Jiutepec, Morelos C.P. 62550, México

*Corresponding author. E-mail address: [email protected] (EstradaArriaga E.B.)

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Abstract In this study, showed a strategy to generate methane and remove organic matter removal from tequila vinasses through of anaerobic digestion assisted via bioelectrochemical power to-gas. Specific methanogenic activity (SMA) assays in batch mode were tested and a single-stage bioelectrochemical upflow anaerobic sludge blanket reactor (UASB) was evaluated to generate methane during tequila vinasses treatment. The results showed that the methane production in the bioelectrochemical UASB reactor applied at low voltage of 0.5 V and under HRT of 7 d was higher than the in the conventional UASB reactor. The specific methane production rate in bioelectrochemical UASB reactor was up to 2.9 NL CH4/L d, with a maximum methane yield of 0.32 NL CH4/g CODremoved. Similar COD removals were observed in the bioelectrochemical UASB reactor and conventional reactors (92-93%). High carbon dioxide reduction and hydrogen production were observed in the bioelectrochemical UASB reactor.

Keywords: Anaerobic digestion; Bioelectrochemical power-to-gas; Bioelectrochemical UASB reactor; Methane production; Tequila vinasses.

1. Introduction Recently, the application of bioelectrochemical systems (BES) for wastewater treatment has gained interest from the scientific community because of its efficacy during wastewater treatment and biogas production (Choi and Lee, 2019). In particular, the BES called “microbial electrolysis cells (MECs)” oxidizes the organic matter contained in the wastewater through bioelectrochemical reactions carried out at the anode’s surface (Zhang et al., 2019). An external supplied voltage (0.14-1.0 V) is required to carry out the bioelectrochemical reactions forming electrons and protons which are subsequently

2

used in the redox reaction during fermentation or methanogenesis process (Kim et al., 2018). During these reactions, it is possible to increase the hydrogen and methane production. These concepts of fermentation or methanogenesis through electrochemical reactions is called “electrofermentation” and “electromethanogenesis,” respectively (Geppert et al., 2016). The electromethanogenesis or “bioelectrochemical power to-gas” is a new bioelectrochemical concept by which methane is obtained through the reduction of carbon dioxide and hydrogen by means of electrotrophic methanogens and hydrogenotrophic methanogens, developed at the cathode of the MECs (Zakaria and Dhar, 2019; Gepper et al., 2016). In recent years, several studies have been carried out through technology assisted via bioelectrochemical power to-gas, whether to increase the methane production or to treat wastewaters (Wang et al., 2019; Choi and Lee, 2019; Park et al., 2019; Yin et al., 2019; Luo et al., 2018; Hirano and Matsumoto, 2018; Feng et al., 2017; Gajaraj et al., 2017; Fu et al., 2015). These studies have demonstrated that a methane yield of over 90% was obtained, with regards to the theoretical methane yield (0.35 NL CH4/g CODremoved), and that a better reactor performance over conventional anaerobic digestion was also achieved. Several of these studies have been carried out to treat synthetic wastewaters. Few studies have been carried out using different real wastewater through BES, assisted via bioelectrochemical power to-gas (Hirano and Matsumoto, 2018; Fen et al., 2017; Cerillo et al., 2016; De Vrieze et al., 2014; Cusick et al., 2011). They reported that is necessary to carry more studies to understand the electromethanogenesis phenomena that take place during real wastewater treatment and determine which are the variables, electrode materials and operating conditions that affect the performance of anaerobic digestion assisted via bioelectrochemical power to-gas to generate methane and remove organic matter.

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Currently, the nutrient recovery, biogas production (methane and hydrogen), bioelectricity, and other valuable by-products (ethanol, bioplastic), occurs during highstrength wastewater treatment, especially agroindustrial wastewater, which contains high concentrations of organic matter measured as chemical oxygen demand (COD) > 4,000 mg/L and other pollutants (Xiong et al., 2020, Lim et al., 2020). These agroindustrial wastewaters are preferably treated by anaerobic technology generating a potential for energy generation such as methane and hydrogen (Hamza et al., 2016). Traditionally, the anaerobic digestion has been used to treat this type of industrial effluents. Several anaerobic technologies have been suggested to treat high-strength wastewaters, among them: upflow anaerobic sludge blanket reactor (UASB), anaerobic membrane bioreactor (AnMBR), anaerobic sequencing batch reactor (AnSBR), upflow packed-bed reactor (PBR) and continuously stirred tank reactor (CSTR).

Despite the fact that favorable organic matter removals and relatively high specific methane production rates have been obtained with different anaerobic treatment technologies, further studies are needed to find alternative methods to treat high-strength wastewater and obtain higher organic matter removal efficiencies and higher methane productions (Li et al., 2019). Tequila vinasses is a type of high-strength wastewater that is considered highly polluting because it is released into the environment at temperatures close to 90 ºC, at a pH lower than 4.0 units, and with a high COD (5-150 g/L) and biological oxygen demand (BOD) of 2-60 g/L (García-Depraect et al., 2020; ToledoCervantes, et al., 2018; Arreola-Vargas et al., 2018). Furthermore, tequila vinasses contain

phenolic

compounds,

aldehydes,

sulfides,

methyl

2-furoate,

5-

hydroxymethylfurfural, high amounts of potassium, long and short chain fatty acids that can cause inhibition to methanogens thus generating lower methane yields and low

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efficiencies removal of COD in the anaerobic digestion (Arreola-Vargas et al., 2018; Buitrón et al., 2014; Mendez-Acosta et al., 2010). Tequila is a traditional alcoholic beverage in Mexico that is obtained from the distillation of the fermented agave must from the variety of Agave tequilana var. Azul. The vinasses generated by tequila industries are obtained after distilling fermented agave must. For each liter of tequila obtained, between 10 and 12 liters of vinasses are produced (García-Depraect et al., 2020). Almost 80% of tequila vinasses are discharged directly to the water bodies and land without receiving a previous treatment, thus affecting the environment (ToledoCervantes et al., 2018). Anaerobic digestion is one of the main processes used to treat tequila vinasses. According to the available literature, several anaerobic reactor configurations have been assessed for the treatment of tequila vinasses such as CSTR anaerobic digester (García-Depraect et al., 2020; García-Depraect et al., 2020b; GarcíaDepraect and León-Becerril, 2018), AnSBR (García-Depraect et al., 2020c; ArreolaVargas et al., 2016; Buitrón et al., 2014), UASB reactor (García-Depraect et al., 2020; López-López, et al., 2015; Buitrón, et al., 2014), PBR (Toledo-Cervantes et al., 2018; Arreola-Vargas et al., 2018; Jáuregui-Jáuregui et al., 2014) and two-stage system (GarcíaDepraect et al., 2020; García-Depraect et al., 2020c; Toledo-Cervantes et al., 2018). These authors have reported COD removals in a range of 70-96% and specific methane production rates of 0.1 to 3.2 L CH4/L d. Up to this moment, there have not been studies about the treatment of tequila vinasses using BES assisted via bioelectrochemical power to-gas for methane production and COD removal. The objective of this study was to evaluate the methane production and COD removal through bioelectrochemical power to-gas, during the anaerobic digestion of tequila vinasses. This study was divided into three stages. In the first stage, specific methanogenic activity (SMA) assays were evaluated to select the best substrate/inoculum ratio (S/X) for methane production from

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tequila vinasses. The second stage, SMA assays via bioelectrochemical power-to-gas were performed to determine the voltage input for methane production and COD removal from tequila vinasses. Finally, the last stage consisted on assessing a continuous flow single-stage UASB reactor via bioelectrochemical power-to-gas performance for methane production and COD removal during tequila vinasses treatment, which was compared to a conventional UASB reactor.

2.Materials and methods

2.1.Tequila vinasses The tequila vinasses were obtain from a tequila company located in Jalisco, Mexico, which physical-chemical characteristics are shown in Table 1. For this study, the vinasses were previously filtrated using a fiberglass filter 1.2 µm. Then, the tequila vinasses were stored in a temperature of 4 °C until used.

Table 1 2.2.Specific methanogenic activity assays The specific methanogenic activity (SMA) test has the objective of determining the sludge capacity to produce methane using tequila vinasses as a source carbon. The SMA assays also were used to determine the anaerobic biodegradability of tequila vinasses. For the SMA tests, anaerobic granular sludge was used, which was obtained from an UASB reactor that treats wastewaters from a paper mill company. The granular sludge was previously acclimated to the tequila vinasses with a COD of 3,000 mg/L (10% raw vinasses and 90% tap water) during three months, at a temperature of 35 °C, with a pH of 7. The pH was adjusted with a 10 N NaOH solution. The diluted vinasses were

6

changed every 8 days. Once the granular sludge was acclimated, the SMA tests were carried out.

The SMA were conducted in a batch-mode assay. The granular sludge and the vinasses were added to 250-mL serum bottles. Different S/X ratio were tested (0.020, 0.17, 0.43, 0.54, 0.71, 0.82, and 1.2 g COD/g VTS). The variation of S/X ratio was carried out varying the COD of the vinasses, keeping the same VTS concentration. The COD of the vinasse was adjusted by diluting it (%)/tap water (%) of 2/98 up to 100/0. The pH of the vinasses were adjusted to a pH of 7 using a solution of phosphate buffer of 0.2 M. Afterwards, the serum bottles were sealed with rubber septa and aluminum crimp caps and then they were gassed with N2 gas for 1 min. The serum bottles were placed in an orbital shaker at 100 r.p.m. and they were placed in an incubator and maintained at 35°C. The VTS concentration of acclimated anaerobic granular sludge was 60.7 g/L. At the same time, a negative control bottle was assembled, which consisted of anaerobic granular sludge and a synthetic solution that contained sodium acetate, macro, and micronutrients as carbon source. One additional bottle containing only inoculum (endogenous control) was included for background methane production. The tests were carried out in triplicate.

2.3.Specific methanogenic activity assays assisted via bioelectrochemical power-to-gas The SMA assays via bioelectrochemical power-to-gas were performed in a batchmode assay. The best S/X ratio for methane production was 0.17 g COD/g STV obtained from the SMA from the previous test which was selected to carry out these assays. The bioelectrochemical reactors used for SMA assays via bioelectrochemical power-to-gas were placed in 250-mL serum bottles. See details in the Supporting Material. In the

7

bioelectrochemical reactor, electrodes were introduced to carry out the electrochemical reactions. Carbon felts with a surface area of 3.0 cm2 (3.0 cm x 1.0 cm) were used as anode and cathode. The electrodes were immersed in the anaerobic granular sludge and placed 0.5 cm above the bottom of the reactor vessels. To separate the anode and cathode, glass fiber of 1.0 mm of thickness was used between both electrodes. Later, the bioelectrochemical reactors were sealed with rubber septa and aluminum crimp caps and then were gassed with N2 gas for 1 min. After the serum bottles were placed in an orbital shaker at 100 r.p.m., they were placed in an incubator and maintained at 35 ° C. Voltages of 0.1, 0.3, 0.5 and 1.0 V were tested in the SMA assays via bioelectrochemical powerto-gas. The voltage applied to the reactors was supplied through a UNIT-T UTP3313TFL DC power supply. A titanium wire was used as electrical conductor. Negative control bioelectrochemical reactors were mounted simultaneously during the SMA tests via bioelectrochemical power-to-gas working under the same S/X ratio, voltage, and temperature conditions. These negative control reactors consisted on anaerobic granular sludge and electrodes fed with a synthetic solution that contained sodium acetate as carbon, macro, and micronutrients. One 250-mL serum bottle (control reactor) was fed with vinasses (S/X ratio of 0.17 g COD/g VTS) without applying the voltage. This, in order to compare the biogas production with biogas production via bioelectrochemical power-to-gas. The pH of the vinasses was adjusted to 7 with phosphate buffer solutions of 0.2 M. The COD of the vinasses in all reactors was maintained at 8,910 mg/L. Anaerobic granular sludge used as inoculum was the same utilized in the previous test. One additional bottle containing only inoculum (endogenous control) was included for background methane production. The tests were carried out in triplicate.

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2.4.Experimental set up of the UASB reactor via bioelectrochemical power-to-gas for continuous runs To evaluate methane production and removal of organic matter during the treatment of tequila vinasses, a single-stage UASB reactor via bioelectrochemical powerto-gas (bioelectrochemical UASB reactor) at laboratory-scale was used in this study. The performance of the bioelectrochemical UASB reactor was compared to the conventional UASB reactor’s. Both reactors have 4.25 L in working volume and were inoculated with acclimated anaerobic granular sludge. The volume of granular sludge inoculated in the reactors was 1.0 L. One anode and cathode were completely immersed inside the sludge bed. Graphite felt was used as anode and cathode, which were separated by a 1 mm thick fiberglass cloth. The size of the electrodes was 10 cm high and 5 cm wide (surface area 50 cm2). Titanium wire was used as the electrical conductor. In order to apply the electrical load required for the bioelectrochemical UASB reactor, a UNIT-T UTP3313TFL DC power supply was used. A HRT of 7, 3 and 1 d were tested in both UASB reactors. The voltage applied to the bioelectrochemical UASB reactor was obtained according to the results of the SMA via bioelectrochemical power-to-gas, which was 0.5 V. Both reactors had a recirculation of wastewater with a superficial velocity between 0.5 and 0.7 m/h. The temperature in both UASB reactors was maintained at 35 °C. The biogas production was measured using the volumetric method. The pH of the reactors was kept at 7, which was controlled through a 10 M NaOH solution and a 2 M H2SO4 solution.

2.5.Energy recovery calculations from UASB reactors The electrical energy input (WE) for biogas production (CH4 or H2) into the bioelectrochemical UASB reactor is calculated as:

9

𝑛

𝑊𝐸 = ∑1(𝐼𝐸𝑎𝑝∆𝑡 ― 𝐼2𝑅𝑒𝑥∆𝑡)

Eq. 1

where I is the current (A) calculated from the voltage across the resistor (I = V/Rex ), Eap (V) is the external applied voltage, Δt (min) is the time interval of measurements made during steady state, and Rex = 1 Ω is the external resistor. In this study, the current was constant over time, then Eq. 1 was simplified to WE = I Eap Δt.

The energy efficiency (𝜂E) relative to the electrical input was calculated from the ratio of energy for biogas production to the input electrical energy (Eq 2).

η𝐸 =

𝑊𝐶𝐻4𝑜𝑟 𝐻2 𝑊𝐸

Eq. 2

where WCH4 or H2 = nCH4 or H2 ΔHCH4 or H2, where nCH4 or H2 is the moles of methane or hydrogen produced and ΔHCH4 = 891 kJ/mol and ΔHH2 = 285.83 kJ/mol is the heat of combustion of methane and hydrogen, respectively (upper heating value) (Gajaraj et al., 2017; Call and Logan, 2008). The efficiency relative to the consumed substrate (𝜂S) is

η𝑆 =

𝑊𝐶𝐻4𝑜𝑟 𝐻2 𝑊𝑆

Eq. 3

where WS = nS ΔHS, where nS is the moles of COD removed and ΔHS = -870.28 kJ/mol is the heat of combustion of substrate (in terms of acetate) (Call and Logan 2008). To convert the COD of the tequila vinasses (g-COD/L) to moles of acetate, a conversion factor of 0.78 g-COD/g-sodium acetate was used. 10

The total energy recovery (𝜂S

) for bioelectrochemical UASB reactor was

+ E

calculated based on the relative energy of COD removed, and the electrical energy input according to the Eq 4

𝑊𝐶𝐻4𝑜𝑟 𝐻2

η𝑆 + 𝐸 = 𝑊𝑆 + 𝑊𝐸 𝑥100

Eq. 4

For the conventional UASB reactor, the energy efficiency was determined with Eq 4 where WE is zero value.

2.6.Analytical methods and statistical analysis The determination of BOD, solids, nitrogen forms, phosphorous form, total alkalinity, pH, temperature, apparent color, turbidity, mineral acid, total acid, true color, electrical conductivity, total dissolved solids, chlorides, sulfide, sulfate, phenols and metals were carried out according to the standard methods (APHA, 2005). TCOD and SCOD were measured using the HACH test (HACH Odyssey DR/2500). TOC was measured in a Shimadzu Total Organic Carbon Analyzer TOC-LCSH/CPH series. Biogas composition (H2, CH4, and CO2) was determined with a gas chromatography SRI 8610 C GC equipped with a thermal conductivity. The column used was a Micropacked Colum 2m 1mm/16” OD SILCO (RESTEK). Helium was used as carrier gas at a pressure of 6080 psi. The temperature of column and oven was maintained at 50°C/2 min, then increased to 120°C at a ramp of 20°C/min. The analysis of variance (ANOVA) was applied to compare the means of different groups of data from the study and p values less than 0.05 were considered statistically significant.

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3.Results and discussions

3.1.SMA assays at different S/X ratio Fig. 1 shows the cumulative methane production from tequila vinasses at different S/X ratios from SAM assays. The highest values of methane production were obtained with a S/X ratio of 0.17 g COD/g VTS. It was observed that when the value of the S/X ratio increased, methane production was affected, which was related to the increase in the COD of tequila vinasses. The cumulative methane volume under these conditions was 171 mL. When the COD increased to 71 g/L (S/X = 1.2 g COD/g VTS), the cumulative methane volume dropped to values of 44 mL. The SMA and methane yield were directly related to the organic matter concentration contained in the tequila vinasses observing maximum values of SMA of 0.37 g COD-CH4/g VTS d and a methane yield of 321 NmL CH4/CODremoved for a COD of 10.6 g/L. The methane yield corresponded to 92% of the maximum theoretical methane yield (350 NmL CH4/CODremoved). The negative control shows a SMA value of 0.7 g COD-CH4/g VTS and a methane yield of 349 NmL CH4/CODremoved. The values obtained from negative control showed a good methanogenic activity of anaerobic granular sludge. Methane content was in the range of 13-58% obtaining maximum values for a S/X ratio of 0.17 g COD/g VTS and low values for a S/X ratio of 1.2 g COD/g VTS. The results obtained showed that methane content decreases when the concentration of organic matter is increased. See details in the Supporting Material. The effects of lactate fermentation on the biochemical methane potential of tequila vinasses were evaluated by Díaz-Cruces et al. (2020), who obtained a maximum chemical methane potential of 435 NmL CH4/g VSadded and a maximum methane production rate of 105 NmL CH4/d with the increase of lactate-type fermentation.

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Fig. 1 The results of the SMA assays showed that at a high S/X ratio, the methanogenic activity is inhibited. Increasing COD, the inhibition increased reducing methane production probably because of volatile fatty acids accumulation. In addition, the tequila vinasses contain a high level of inhibitor compounds. The best results of methane production occurred when the tequila vinasses were diluted to a vinasse: water ratio of 1:6 (S/X ratio of 0.17 g COD/g VTS). Barrera et al. (2014) showed that a SO42-/COD ratio above 0.1 causes methanogens inhibition by H2Sfree, HS- and S2-, and propionic acid accumulation during the treatment of very high strength and sulfate rich vinasses. In our study, the maximum SO42-/COD ratio was 0.02, so the inhibition of methane production due to the presence of sulfates was not possible. The vinasses are formed, mostly, by complex organic compounds of great molecular size (sugars and carbohydrates) and by recalcitrant compounds, such as phenols, heavy metals, tannic acids, humic acids, melanoidins, alcohols, organic acids sulphates, and furfurals. The combination of all of these compounds negatively affects the growth of methanogens, thus generating slower microbial activities and a lower methane production (Parsaee et al., 2019; Barrera et al., 2014; Retes-Pruneda et al., 2014). These microbial activities were seen during the SMA assays. The duration of the lag phase (λ) during the methane production from the tequila vinasses treatment were of between 5.4 and 9.4 h, increasing with the increase in the S/X ratio. The lag phase in the SAM assays indicate the time that it takes for the microorganisms to start producing methane from any substrate. The lag phase for negative control reactor was 0.1 h, indicating a fast methane production. Since there are no inhibitory compounds and since there is an organic compound easily biodegradable such as acetate, the activity of methanogenic microorganisms during the anaerobic digestion was almost instantaneous. In this way it was proved that tequilas vinasses contain

13

inhibitory organic and inorganic compounds that affect the methane production during the anaerobic digestion.

3.2.SMA assays assisted via bioelectrochemical power-to-gas In this experimental phase, methane production from tequila vinasses was carried out at a S/X ratio of 0.17 g COD/d VTS (CODadded of 9 g/L), applying different levels of voltage input from 0.1 to 1.0 V. After 120 h of monitoring cumulative methane production, the maximum methane production observed was 216 mL, applying a voltage of 0.5 V (Fig. 2a). It was observed that by applying a voltage during the anaerobic digestion of tequila vinasses, methane production was higher, compared to the anaerobic digestion without applying voltage (30% more, regarding control reactor). A significant difference in the methane volumes produced at applied voltages between 0.1 V and 1.0 V were observed (p-values ≤ 0.001) in the anaerobic digestion of tequila vinasses as well as in the acetate (negative control reactor (Fig. 2a and Fig. 2c). Zhao et al. (2016) indicate that methane production in a bioelectrochemical UASB reactor increase when the anode potential is increased. By applying electricity during the anaerobic digestion of the tequila vinasses, the presence of hydrogen was detected in the biogas, with a content of between 3 and 8%. Most hydrogen production was obtained by applying a voltage of 1.0 V (24 mL) and the lower production of hydrogen was of 8.7 mL with a voltage de 0.3 V (Fig. 2b). Compared to the control reactor, there was a significant enhancement in the methane and hydrogen produced in the SMA assisted via bioelectrochemical power-to-gas (pvalues < 0.001). Fig. 2 The generation of hydrogen occurred because the electrons generated by the oxidation of organic matter are combined with the protons formed at the anode to form

14

hydrogen through the hydrogen evolution reaction: 2H

+

+ 2e- → H2 (g) (Zakaria and

Dhar, 2019). Electroactive bacteria at the anode requires a voltage input of 0.25-0.1.0 V for the hydrogen evolution reaction to occur in a MECs. This voltage is required to overcome thermodynamic barriers in cathodic reactions (cathodic overpotentials). The hydrogen formed during the bioelectrochemical reactions carried out during the anaerobic digestion of the tequila vinasses served as a substrate for the hydrogenotrophic methanogens developed in the cathode. In this way, part of the hydrogen formed was consumed and combined with carbon dioxide to generate methane. Negative control bioelectrochemical reactors fed with a synthetic solution were assembled simultaneously during the SMA tests, via bioelectrochemical power-to-gas from tequila vinasses. Fig. 2c and Fig. 2d shows the cumulative methane and hydrogen productions from negative control reactors. The cumulative methane volumes at voltages of 0.1 to 1.0 V were from 79.2 to 94.8 mL, which were lower than the ones observed during the anaerobic digestion of the tequila vinasses. During these tests, hydrogen in the biogas was detected at between 7 and 16%. The cumulative hydrogen volumes observed were from 8.4 to 18.4 mL, which were also lower during the anaerobic digestion of tequila vinasses. The maximum cumulative methane and hydrogen volumes were obtained by applying lower voltages, compared to the ones obtained in the anaerobic digestion of the tequila vinasses. This is due, mainly, to the fact that since there is no interference such as solids, the internal resistance within the bioelectrochemical reactor is lower, thus needing lower voltage inputs for the biogas production. The high concentration of total solids and other pollutants contained in high-strength wastewater could generate high internal resistance, resulting in overpotential and ohmic loss problems, so in order to break that barrier, it is necessary to apply a higher voltage (Gajaraj et al., 2017).

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The maximum SMA and methane yield obtained during anaerobic digestion from tequila vinasses via bioelectrochemical power-to-gas was 0.75 g CH4-COD/g VTS and 327.1 NmL CH4/CODremoved respectively, at 0.5 V, which represents 93.5% of theoretical methane yield (Table 2). The lower SAM value was obtained by applying a voltage of 0.3 V, having 0.42 g CH4-COD/g VTS as a result. Comparing these results to the control reactor’s (SMA of 0.38 g CH4-COD/g VTS), it was observed that the electric power input stimulated the activity of methanogenic microorganism up to two times more during the methane production from the tequila vinasses treatment. The lowest methane yield was observed at 0.1 V with a value of 286.5 NmL CH4/g CODremoved (82% of theoretical methane yield). Yu et al. (2018) observed that electro-methanogenic bacteria increases methane production in the anaerobic digesters. Once the biogas production was stabilized (120 h of anaerobic digestion), the maximum COD removal achieved was 45% when 1.0 V was applied. There was no significant difference on the COD removal at 0.5 and 1.0 V (p-value 0.2). An increase in the COD removal was observed when the power input increased. The COD removal of control reactor was 24% which was the lower one, compared to the anaerobic digestion by electro-methanogenesis.

As it can be observed in Table 2, the results about the SMA, methane, and hydrogen yield vary according to each voltage. It would be expected that by increasing the electric power input the SMA values, methane and hydrogen yield also increase. Nevertheless, due to the complexity of the tequila vinasses, which are mostly complex organic compounds can be cause internal resistances that generate fluctuations during the production of methane. This generates that the protons and electrons are transported to the cathode at different speeds. This behavior could be proved during the tests with the negative control reactor via electro-methanogenesis. It was observed that by increasing

16

the voltage input in the negative control reactors, methane production, SMA, and methane yield also increased. Likewise, it was observed that the methane produced increased, compared to the control reactor.

Methane content, hydrogen, and carbon dioxide generated via electromethanogenesis during the tequila vinasses treatment were in the range of 72-74%, 6-8%, and 19-22%, respectively (Table 2). For control reactor, methane and carbon dioxide content was 58 and 42% respectively, the presence of hydrogen was not observed. During the anaerobic digestion of tequila vinasses assisted via bioelectrochemical power-to-gas, an increase in the methane content was presented, as well as a reduction of the carbon dioxide. The formation of hydrogen and decrease of carbon dioxide was very important for the increase of methane since part of these gases were reduced and used at the cathode by the electrotrophic methanogens, acetoclatic methanogens and hydrogenotrophic methanogens to form methane (Zakaria and Dhar, 2019; Gajaraj et al., 2017). The cathodic electro-methanogenesis can occur directly via electron transport to electrotrophic methanogens to generate methane by carbon dioxide reduction indirectly via hydrogen formation at the cathode, which is consumed by hydrogenotrophic methanogens to form methane. It can also occur by hydrogenotrophic methanogenesis via where hydrogen turns into acetate by homoacetogens. This acetate can be utilized by acetoclastic methanogens or by electroactive bacteria for anodic oxidation to form methane (Fu et al., 2015). In our experiments, three pathway for the methane formation were present during the anaerobic digestion of tequila vinasses (Zakaria and Dhar, 2019; Ishii et al., 2019; Feng et al., 2017). This same behavior was observed during the tests in negative control reactor via electro-methanogenesis.

17

Table 2 3.3.Bioelectrochemical and conventional UASB reactors performance 3.3.1.COD removal In this experimental phase, the tequila vinasses were treated in a single-stage bioelectrochemical UASB reactor under continuous mode. In order to compare the performance of the biolectrochemical reactor, it was operated in parallel with a conventional UASB reactor. Both reactors were fed with tequila vinasses with an average COD of 9,360 mg/L. In order to obtain these concentrations of organic matter, the tequila vinasses were diluted to a vinasse:water tap ratio of 1:6 since above these concentrations, methane production started to be inhibited. The power input applied to the biolectrochemical UASB reactor was 0.5 V.

COD profiles in the UASB reactors at different HRTs are shown in Fig 3. The organic loading rates (OLR) were varied from 1.3 to 9.7 kg COD/m3d. At a HRT of 7 d, the effluent COD in both reactors was for bioelectrochemical UASB and a conventional reactor of 732 and 688 mg/L, respectively. COD removal in both reactors under an OLR of 1.3 kg COD/m3d was 92-93%. Once the reactors were stabilized under a HRT of 7 d, the organic load increased to 3.2 kg COD/m3d by decreasing the HRT from 7 to 3 d. Similar UASB reactor performances were observed in both reactors during the tequila vinasses treatment at a HRT of 7 and 3 d showed a non-significant difference (p-values 0.4) on the COD effluents and COD removals (Fig. 3a). When the OLR was increased to 9.7 kg COD/m3d (once reaching the steady state), COD removal efficiencies were decreased in both reactors (Fig. 3b). The conventional UASB reactor was more affected by the increased OLR compared to the bioelectrochemical UASB reactor. For the bioelectrochemical UASB reactor, the COD removal was of 60% with a COD effluent of 3,668 mg/L. For the conventional UASB reactor, the COD removal and COD effluent 18

was 48% and 4,839 mg/L, respectively. These decreases of COD removal were related to a decrease in the pH and the alkalinity, possibly due to an accumulation of volatile fatty acids due to an overload on the reactors (García-Depraect et al., 2020; Arreola-Vargas et al., 2018).

The bioelectrochemical UASB reactor has a great potential to treat high-strength industrial wastewaters as acid distillery wastewater or similar wastewater, showing that the removal efficiencies of organic matter are higher, compared to the conventional anaerobic reactors (Park et al., 2019; Yin et al., 2019; Gajaraj et al., 2017). The effect of the OLR was studied by Park et al. (2019) indicating that the increase of OLR during high-strength wastewater treatment affects the conventional anaerobic reactor performance to a greater extent, compared to a bioelectrochemical anaerobic reactor. Feng et al. (2017) investigated the treatment of acidic distillery wastewater by upflow anaerobic bioelectrochemical reactor reporting a COD removal between 82 and 96%.

On the other side, COD removals of 75-95% have been reported during the treatment of tequila vinasses by using an OLR from 0.6 to 20 kg COD/m3 d (HRT of 1 to 6 d) using CSTR anaerobic digester, fixed-bed reactor and anaerobic sequential batch reactor, and conventional UASB reactor (Arreola-Vargas et al., 2016; Lopez-Lopez et al., 2015; Hernandez-Martinez et al., 2014; Jáuregui-Jáuregui et al., 2014). Recently, a twostage system with dominant lactate-type fermentation in acidogenesis was used to treat tequila vinasses (García-Depraect et al., 2020). The effluent from acidogenic CSRT reactor fed the methanogenic UASB reactor with an OLR from 2.5 to 12.5 kg COD/m3d. These authors reported that by increasing the OLR from 2.5 to 10.1 kg COD/m3d, COD removal increased from 91 to 93%. Nevertheless, by operating at an OLR of 12.5 kg

19

COD/m3 d, the methanogenic performance was drastically affected thus inhibiting the methanogenic process due to an overloading caused by the accumulation of volatile fatty acids. This same behavior was observed by Toledo-Cervantes et al. (2018). They employed a two-stage anaerobic system to treat tequila vinasses. The methanogenic reactor they utilized was a PBR, which was operated at an OLR from 2.7 to 12 kg COD/m3d (HRT of 6 to 2.2 d). Under these conditions, COD removals were of 74 to 95%, showing a decrease of the COD removal and methane content at a high OLR. ArreolaVargas et al. (2018) assessed a PBR operated at different OLRs (between 4 and 12.5 kg COD/m3d) for the treatment of tequila vinasses, showing a stable performance at different OLRs (COD removal of 86-89%). Fig. 3 3.3.2.Biogas production Fig. 4 shows the biogas production in bioelectrochemical and conventional UASB reactors at different HRTs during the treatment of tequila vinasses. Throughout the whole study, the biogas production was higher in the bioelectrochemical UASB reactor, compared to the conventional UASB reactor. The maximum specific methane production rate in the bioelectrochemical and conventional UASB reactors was 2.9 and 2.1 NL CH4/L d, respectively. During the first two operating stages, methane production was up to 42% higher, compared to the conventional treatment. An increase in methane production was observed in both reactors when the OLR was changed from 1.3 to 3.2 kg COD/m3d (Table 3). Nevertheless, by incrementing the OLR to up to 9.7 kg COD/m3 d, methane production decreased by 35% in both reactors, because of the pH drop of the reactors due to the overload. The low methane production was related to the decrease in the COD removal efficiencies. An increase in the production of hydrogen was observed during last stage in the bioelectrochemical UASB reactor, which was related to the increase of protons at the

20

cathode, which also caused a higher development of the hydrogen-producing bacteria than electrotrophic methanogens and acetoclastic methanogens. One part of the hydrogen is indirectly formed by the consumption of hydrogenotrophic methanogens (Fig. 4c). In the conventional UASB reactor hydrogen production was not observed. In the bioelectrochemical UASB reactor the specific hydrogen production rate was from 0.27 to 0.48 NL H2/L d, which increased when the OLR increased. The amount of carbon dioxide produced in the bioelectrochemical UASB reactor was 50% less compared to the conventional UASB reactor during all the experimental stages (Fig. 4d). This behavior was due to the fact that during the cathodic electro-methanogenesis processes, the electrons formed at the anode are transported directly to the electrotrophic methanogens developed at the cathode, which utilizes carbon dioxide for its reduction and forms methane. Both combinations of electro-methanogenesis and hydrogenotrophic methanogenesis increase the carbon dioxide reduction (thus generating less content of carbon dioxide in the biogas) which increases the methane production according to Eq. 5 and Eq. 6. As it is shown in Fig. 5 and Table 3, methane content was higher in the bioelectrochemical UASB reactor with concentrations of between 55 and 74%, while for the conventional UASB reactor, methane content was between 54 and 62%. By increasing the OLR, the methane content was reduced in both reactors. Carbon dioxide content in the conventional UASB reactor remained constant during the experiment with concentrations of 38-46%, while in the bioelectrochemical UASB reactor, carbon dioxide was between 18 and 32%, showing higher concentrations of carbon dioxide as the OLR increased. The high content of carbon dioxide in the bioelectrochemical UASB reactor at

21

an OLR of 9.7 kg COD/m3 was related to the inhibition of the electrotrophic methanogens which were not capable of reducing carbon dioxide to methane.

4H2 + CO2

CH4 + 2H2O

CO2 + 8 H+ + 8e-

CH4 + 2H2O

Eq. 5 Eq. 6

Fig. 4 Fig. 5 On the other hand, the maximum methane yield obtained was 0.32 NL CH4/g CODremoved which corresponds to the 91% of the theoretical methane yield and was obtained in the bioelectrochemical UASB reactor at a HRT of 3 d. Under three operational conditions, the methane yield was higher compared to a conventional UASB reactor’s. The hydrogen yield obtained from the bioelectrochemical UASB reactor ranged from 0.03 to 0.09 NL H2/g CODremoved, which represents from 2 to 6% of the theoretical hydrogen yield (1.4 NL H2/g CODremoved) (Table 3). Feng et al. (2017) reported that methane production and methane content were higher in an upflow anaerobic bioelectrochemical reactor compared to a conventional UASB reactor treating acidic distillery wastewater. A methane yield of 0.33 NL CH4/g CODremoved (biogas methane content of 83%) and specific methane production rate of 3.2 L CH4/L d at an OLR of 10.1 kg COD/m3d were obtained from a two-stage anaerobic digestion to continuously treat tequila vinasses (García-Depraect, et al., 2020). Toledo-Cervantes et al. (2018) used upflow continuous PBRs in two-stage anaerobic digestion for tequila vinasses treatment at OLRs from 7.7 to 29 kg COD/m3 d, which showed a 87% of the theoretical methane yield under an OLR of 19.4 kg COD/m3 d. Buitrón et al. (2014) achieved a methane yield of 0.26 NmL CH4/g CODremoved and a methane content of 68% using a conventional UASB

22

reactor during the treatment of a tequila vinasses acidogenic effluent derived from a hydrogen-producing under a HRT of 1.0 d. Arreola-Vargas et al. (2018) reported a maximum methane yield of 0.24-0.28 NL CH4/g CODadded and a specific methane production rate of 3.03 L CH4/L d during a tequila vinasses treatment using a pilot-scale PBR at an OLR of 12.5 kg COD/m3 d (HRT of 2 d). The influence of alkalinity and volatile fatty acids ratio on tequila vinasses treatment in an UASB reactor was investigated by Lopez-Lopez et al. (2015) obtaining a methane yield of 0.33 NL CH4/g CODremoved at a ratio of VFAs/Alk ≤ 0.5. In an anaerobic sequential batch reactor, a methane yield of up to 0.29 NL CH4/g CODadded and methane content of 92% were obtained during tequila vinasses treatment at an OLR of 0.67-1-01 kg COD/m3 d (Arreola-Vargas et al., 2016).

3.3.3.Energy recovery Energy recovery ηS + E for the bioelectrochemical UASB reactor ranged from 77 to 80% for methane production and 2.5 to 7% for hydrogen production (Table 3). For the UASB conventional reactor, the energy recoveries for methane production ranged from 46 to 75% and were based only on the efficiency relative to the consumed substrate ηS. The energy recoveries were higher in the bioelectrochemical UASB reactor than in the conventional UASB reactor. The increase in the OLR, based on the variation of HRT, improved the biogas production and energy recoveries in both reactors. However, it was observed that with an OLR of 9.7 kg COD/m3 d, the energy recovery for methane production in the bioelectrochemical UASB reactor was affected by a decrease in the electro-methanogenesis and hydrogenotrophic methanogenesis processes. According to Zakaria and Dhar et al. (2019), Fen et al. (2017), and Zhao et al. (2016) the energy efficiencies of a BES for methane production is favored by the presence and enriching of

23

the electroactive bacteria and hydrogenotrophic methanogens into the reactor, compared to conventional anaerobic digesters. Table 3

4.Conclusions The tequila vinasses treatment through of anaerobic digestion assisted via bioelectrochemical power-to-gas enhanced methane production, and COD removal, compared to the conventional anaerobic digestion. The strategy developed in this study through the SMA assays allowed to select the best starting and operating conditions of a single-stage bioelectrochemical UASB reactor for the treatment of tequila vinasses. The low power input applied to granular anaerobic sludge allowed to enrich the electrotrophic methanogens and hydrogenotrophic methanogens during tequila vinasses treatment. The low content of carbon dioxide in the biogas is a parameter that can indicate the good performance of a bioelectrochemical reactor.

Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online

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Figure captions

Fig. 1 Cumulative methane production at different S/X ratio from tequila vinasses.

Fig. 2 SMA assays assisted via bioelectrochemical power-to-gas for biogas production via bioelectrochemical power-to-gas. a) cumulative methane production, b) cumulative hydrogen production, c) cumulative methane production at different voltage input from negative control reactor (acetate), and d) cumulative hydrogen production at different voltage input from negative control reactor (acetate).

Fig. 3 COD profiles in the UASB reactors at different HRT. a) OLR, and b) COD removals.

Fig. 4 Biogas production during tequila vinasses treatment in UASB reactors at different HRT. a) overall biogas production, b) methane production, c) hydrogen production and d) carbon dioxide production.

Fig. 5 Biogas content during tequila vinasses treatment in UASB reactors at different HRT.

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Table captions

Table 1. Composition of the tequila vinasses used in this work.

Table 2. Overall results of SMA assays from tequila vinasses assisted via bioelectrochemical power-to-gas.

Table 3. Performance comparison of conventional and bioelectrochemical UASB reactors treating tequila vinasses at stabilized operational conditions.

CRediT authorship contribution statement Estrada-Arriaga Edson Baltazar: Supervision, Investigation, Conceptualization, Methodology, Writing - Original Draft, Resources, Project administration. Reynoso-Deloya Ma. Guadalupe: Investigation, Methodology, Validation, Formal analysis Guillén-Garcés Rosa Angélica: Methodology. Falcón-Rojas Axel: Methodology, Conceptualization. García-Sánchez Liliana: Methodology, Investigation, Formal analysis

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Cumulative CH4 production@STP (mL)

180 160 140 120 100 80 60 40 20 0 0

20

40

60 Time (h)

80

S/X=0.02

S/X=0.17

S/X=0.43

S/X=0.71

S/X=0.82

S/X=1.2

100

120

S/X=0.54

240

a)

210 180 150 120 90 60 30 0 0 28 24

20

40

60

80

b)

20 16 12 8 4 0

33

100

120

umulative H2 production@STP Cumulative CH4 production@STP (mL) (mL)

umulative H2 production@STP Cumalative CH4production@STP (mL) (mL)

Fig. 1

100 90 80 70 60 50 40 30 20 10 0

c)

0 28 24 20 16 12 8 4 0

20 d)

40

Fig. 2

34

Influent Effluent conventional UASB reactor Effluent bioelectrochemical UASB reactor OLR

COD (mg/L)

HRT 1 d

HRT 3 d

HRT 7 d

12,000

25

10,000

20

8,000

15

6,000 10

4,000 2,000

5

0

0 0

10

20

30

40 Time (d)

50

60

70

0

10

20

30

40 Time (d)

50

60

70

80

COD removal (%)

b) 100 80 60 40 20 0

conventional UASB reactor Fig. 3

35

80

bioelectrochemical UASB reactor

OLR (kg COD/m3 d)

a)

Biogas production (L/d) CH4 production (L/d)

HRT 7 d

20

HRT 3 d

HRT 1 d

a)

16 12 8 4 0 15

0

10

20

30

40

50

60

70

80

10

20

30

40

50

60

70

80

10

20

30

40

50

60

70

80

10

20

30

b)

10 5 0

H2 production (L/d)

3

0 c)

2 1

CO2 production (L/d)

0 8

0 d)

6 4 2 0 0

40 50 60 70 80 Time (d) conventional UASB reactor bioelectrochemical UASB reactor Fig. 4

36

HRT 7 d

HRT 3 d

HRT 1 d

Biogas content (%)

100 80 60 40 20 0 0

10

20

30

40 Time (d)

CH4 CO2 bioelectrochemical UASB reactor H2

50

60

CH4 CO2 H2

70

80

conventional UASB reactor

Fig. 5

Table 1. Parameter Total chemical oxygen demand (TCOD) Soluble chemical oxygen demand (SCOD) Biochemical oxygen demand (BOD) Total organic carbón (TOC) Total solids (TS) Fixed total solids (FTS) Volatile total solids (VTS) Total suspended solids (TSS) Volatile suspended solids (VSS) Total nitrogen Ammonia nitrogen Nitrite nitrogen Nitrate nitrogen

Value (mg/L) 105,000 71,000 43,200 26,000 35,624 2,579 33,045 6,600 5,650 813 575 0.3 0.1 37

Parameter Calcium Copper Iron Manganese Magnesium Lead Sodium Potassium Zinc Chlorides Temperature (°C) Electrical conductivity (mS/cm) Total dissolved solids

Value (mg/L) 564 0.3 24 0.7 178 0.1 22.5 322 0.4 0.5 90 3.7 1,530

Total phosphorus Phosphate Sulfide Sulfate Phenols pH (unit)

325 108 9 1,275 0.5 3.5

Apparent color (Pt-Co) True color (Pt-Co) Turbidity (NTU) Mineral acid Total acid

51,250 20,600 2,750 192 584

Table 2. Voltage applied (V)

0.0 (control reactor)

0.1

0.3

S/X (g COD/g VTS) Biogas λ (h) Pmax (mL) Rmax (mL/h) Biogas content (%) SMA (g COD/g VTSadded d) Methane yield (NmL CH4/g CODremoved) Hydrogen yield (NmL H2/g CODremoved) COD removal (%)

0.5

1.0

0.17 CH4 6.4 156.2 5.9 58

H2 0 0 0 0

CO2 CH4 H2 CO2 CH4 H2 CO2 CH4 H2 CO2 CH4 H2 CO2 4.3 4.3 3.1 4.1 7.9 8.0 7.2 7.2 174.7 17.3 162.4 7.2 175.5 13.7 167.4 19.7 7.5 0.7 6.6 0.3 11.7 1.0 9.8 1.1 42 72 7 19 72 3 25 74 6 22 71 8 21

0.38

0.48

0.42

0.75

0.62

321.9

286.5

273.9

327.1

296.6

0

28.2

11.8

25.4

34.9

24

37

38

44

45

Table 3. Conventional UASB reactor 7 3 1 9,110 9,629 9,336 1.3 3.2 9.7 732 688 4,839 92 93 48

HRT (d) Influent (mg/L) OLR (kg COD/m3.d) Effluent COD (mg/L) COD removal (%)

38

Bioelectrochemical UASB reactor 7 3 1 9,110 9,629 9,336 1.3 3.2 9.7 633 632 3,668 93 93 62

Biogas content (%) CH4 H2 CO2

62 38

58 42

54 46

74 8 18

68 8 24

55 15 32

Specific methane production rate (NL/L.d) Specific hydrogen production rate (NL/L.d) Methane yield (NL CH4/g CODremoved) Hydrogen yield (NL H2/g CODremoved) ηS methane (%) ηS + E methane (%) ηS + E hydrogen (%)

1.5 0.18 46 -

2.1 0.24 60 -

1.4 0.30 75 -

2.6 0.27 0.30 0.03 80 77 2.5

2.9 0.35 0.32 0.04 83 80 3

1.8 0.48 0.31 0.09 82 77 7

S/X ratio

HIGHLIGHTS

 

Voltage applied

Bioelectrochemical UASB reactor is developed to treat tequila vinasses High methane production from tequila vinasses was achieved by electroAnode methanogenesis  Methane production and organic removal wasMethane improved at a voltage input of 0.5 V fiber  Glass High carbon dioxide reduction was observed in bioelectrochemical UASBMethane reactor Cathode separator  Bioelectrochemical reactions improved the conventional anaerobic digestion

Bioelectrochemical UASB reactor Conventional UASB reactor

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Tequila vinasses