A comparison of biological, enzymatic, chemical and hydrothermal pretreatments for producing biomethane from Agave bagasse

Industrial Crops & Products 145 (2020) 112160

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A comparison of biological, enzymatic, chemical and hydrothermal pretreatments for producing biomethane from Agave bagasse

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Idania Valdez-Vazqueza,*, Felipe Alatriste-Mondragónb, Jorge Arreola-Vargasc, Germán Buitróna, Julián Carrillo-Reyesa, Elizabeth León-Becerrild, Hugo O. Mendez-Acostae, Irmene Ortízf, Bernd Weberg a

Unidad Académica Juriquilla, Instituto de Ingeniería, UNAM, Qro., Mexico Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, Mexico c División de Procesos Industriales, Universidad Tecnológica de Jalisco, Guadalajara, Jalisco, Mexico d Centro de Investigación y Asistencia en Tecnología y Diseño del Edo. de Jalisco, AC, Jalisco, Mexico e Dept. de Ingeniería Química, CUCEI-Universidad de Guadalajara, Jalisco, Mexico f Universidad Autónoma Metropolitana, Unidad Cuajimalpa, Cd. de México, Mexico g Facultad de Ingeniería, Universidad Autónoma del Estado de México, Edo. Mex., Mexico b

ARTICLE INFO

ABSTRACT

Keywords: Combined severity factor Diluted acid Native microbiota Ozone Rumen Steam explosion

Agave bagasse is a lignocellulosic agroindustrial waste suitable for biofuel production. This study aimed to compare biological, enzymatic, chemical, and hydrothermal pretreatments applied to Agave bagasse in terms of solubilization of carbohydrates (CHO) and chemical oxygen demand (COD), as well as the biochemical methane potential (BMP) from hydrolyzates. Most pretreatments behave similarly, with an average yield of 0.16 ± 0.02 gCOD/g, only the chemical pretreatment overcame this yield by a factor of 2.6 with a CHO/COD ratio of 0.95. The concentrations of inhibitors – furfural, hydroxymethylfurfural, and phenols – were higher in the chemical hydrolyzates than in the biological and enzymatic hydrolyzates. BMP from most hydrolyzates was the same, on average 219 ± 15 mL/gCODin, hydrolyzates differentiate only in terms of lag phase and the methane production rates.

1. Introduction Human activity has caused soil degradation to fulfill the growing demands for food, feed, and recently biofuels. Depending on the source, between 22 %–66 % of the global land area is considered as degraded soils whilst about 33 % is classified as arid and semi-arid lands (Gibbs and Salmon, 2015). Under this scenario of fertile land scarcity, the great challenge of our society is to continue satisfying agricultural commodities, mainly in a world where new mandates require agricultural feedstocks for biofuel production. Agave plants have a crassulacean acid metabolism that allows them to have improved water use efficiency in semiarid regions. Agave has biomass productivity estimated from < 1–34 Mg/ha-year without irrigation, therefore they constitute an opportunity for expansion in arid and degraded lands for producing biofuel feedstocks (Davis et al., 2011). Mexico has the largest industry of Agave processing for producing tequila which generates from 0.4 to 0.9 million tons of Agave bagasse

(Pérez-Pimienta et al., 2017). Agave bagasse is a lignocellulosic material whose chemical composition is largely determined by the tequila production process where it comes from. The lignocellulosic fiber in Agave bagasse differs from 42 % to 69 % according to its origin, where the cellulose content may range from 38 % to 53 % and the hemicellulose content from 22 % to 54 % (Hernández et al., 2019). In comparison with other lignocellulosic biomasses, Agave bagasse is one of the most recalcitrant lignocellulosic biomasses (Buitrón et al., 2019; Hernandez et al., 2019). In consequence, researchers have applied different types of pretreatments to the Agave bagasse for improving its biodegradability and potential of methane production. In the category of chemical pretreatments, sulfuric acid (H2SO4) was reported as an effective catalyst, obtaining sugar concentrations of 27 g/L from Agave bagasse (Saucedo-Luna et al., 2010), however, the released sulfate ions promoted the growth of sulfate-reducing bacteria that abated the performance. Alternatively, Breton-Deval et al. (2018) proposed the use of hydrochloric acid (HCl) as an attractive catalyst for

⁎ Corresponding author at: Unidad Académica Juriquilla, Instituto de Ingeniería, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, 76230, Querétaro, Mexico. E-mail address: [email protected] (I. Valdez-Vazquez).

https://doi.org/10.1016/j.indcrop.2020.112160 Received 31 October 2019; Received in revised form 16 January 2020; Accepted 20 January 2020 0926-6690/ © 2020 Elsevier B.V. All rights reserved.

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sugar recovery from Agave bagasse, obtaining sugar yields 50 % higher compared to the use of H2SO4 and a four-fold increase during methane production (170 versus 40 mL of methane per gram of chemical oxygen demand, COD). On the other hand, alkaline pretreatment is a selective method due to its potential to remove lignin and consequently to increase the cellulose accessibility. Ávila-Lara et al. (2015) pretreated Agave bagasse with NaOH followed enzymatic hydrolysis obtaining sugar yields above 500 mg/g under the optimum condition of 1.9 % w/ w NaOH, 50 min and 13 % w/w solids loading. Galindo-Hernández et al. (2018) pretreated Agave bagasse with alkaline hydrogen peroxide followed enzymatic hydrolysis. They removed 97 % of lignin and 88 % of holocellulose (cellulose and hemicellulose) by pretreating 5 % w/v of Agave bagasse with 2 % w/v of alkaline hydrogen peroxide at 50 °C, pH 11.5 and 1.5 h. Then, the hydrolyzates obtained were subjected to methanization with a yield of 200 mL/gCOD. In the category of thermal pretreatments, steam explosion pretreatment to Agave bagasse by applying a vapor pressure of 0.98 MPa during 22 min followed one pressure cycle reached maximum solubilization of 0.14 gCOD/g and biochemical methane potential of 290 mL/gCOD (Weber et al., 2019). Differences in furfural content in steam-exploded hydrolyzates are responsible for different degrees of inhibition of the anaerobic digestion. Furfural concentrations ranging from 100 to 500 mg/L inhibited the methane production, but a concentration of 1000 g/L had a stimulating effect on anaerobic digestion (Buitrón et al., 2019). Since Agave bagasse is produced after a thermal process in the tequila factories, the use of enzymatic hydrolysis without pretreatment has been tested. For example, Contreras-Dávila et al. (2017) and Corona-González et al. (2016) using industrial grade cellulolytic enzymes (Celluclast 1.5 L and Macerex PM, respectively) produced enzymatic hydrolyzates with sugars concentrations between 11 and 19 g/L. There is now a substantial body of evidence demonstrating that the pretreatments improve the productivity and yield of methane from Agave bagasse (Arreola-Vargas et al., 2015, 2016; Breton-Deval et al., 2018; Galindo-Hernández et al., 2018; Montiel-Corona and Razo-Flores, 2018; Toledo-Cervantes et al., 2018; Tapia-Rodríguez et al., 2019). However, these results are unclear about which pretreatment is more effective in terms of methane production since most studies reported methane yields < 50 % of the theoretical value of 350 mL/gCODrem. The comparison among studies is difficult to make due to two main facts: a) the Agave bagasse composition varied depending on factors such as the age of plant at which was harvested and the tequila production process that in turn largely influences on the holocellulose content (Hernandez et al., 2019); and b) the conditions for producing methane differed such as bioreactor configuration, substrate concentration, inoculum type, among others. Thus, the aim of this study was to compare biological (ruminal fluids and native microbiota), enzymatic, chemical (diluted hydrochloric acid and ozone), and hydrothermal (two configurations of steam explosion reactors) pretreatments in terms of solubilization of organic matter and BMP.

2.2. Pretreatments 2.2.1. Enzymatic pretreatment Cellulase 50XL was purchased from the Enmex industry (Tlalnepantla, Mexico) with a cellulase activity of 98.39 FPU/mL (Ghose, 1987). Agave bagasse with a particle size of < 1.7 mm was subjected to enzymatic hydrolysis in 125-mL Erlenmeyer flasks with 8.75 % (w/v) of TS, citrate buffer 0.5 M (pH 6), enzyme dose of 10.67 mgprotein/gTS at 42.7 °C, 120 rpm for 24 h. This supernatant is referred to as enzymatic hydrolyzate. 2.2.2. Biological pretreatment with rumen fluid Rumen fluids served as a biocatalyst to solubilize untreated Agave bagasse, according to Barragán-Trinidad et al. (2017). Treatment was performed in triplicate in 1-L anaerobic bottles with a working volume of 0.66 L containing 16.5 g of Agave bagasse and ruminal fluids at an S0/ X0 ratio of 0.33 (gVS/gVS). The medium composition was (in mg/L, McDougall, 1948): (NH4)2SO4, 1300; K2HPO4, 2040; NaHCO3, 400; NaCl, 80; MgSO4·7H2O, 19.2; FeSO4·7H2O, 1.1; CaCl2, 8; KH2PO4, 40. The anaerobic bottles were incubated at 37 °C, 100 rpm, initial pH of 7.0 for 15 days. This supernatant is referred to as rumen bacterial hydrolyzate. 2.2.3. Biological pretreatment with native microbiota The native microbiota present in untreated Agave bagasse was acclimated for 80 days in a semi-continuous bioreactor operated at an organic loading rate of 15 gTS/kg·d, a residence time of 6.6 d and 37 °C. Treatment was performed in triplicate in 1-L anaerobic bottles with a working volume of 0.6 L containing 24.3 g of Agave bagasse and the acclimated microbiota at an S0/X0 ratio of 0.7 (gVS/gVS). The medium composition was (in g/L): urea 0.3, KH2PO4 2.4, K2HPO4 0.5. The anaerobic bottles were statically incubated at 37 °C, initial pH of 6.5 for 4 days. This supernatant is referred to as the native bacterial hydrolyzate. 2.2.4. Steam explosion pretreatments (Reactors A and B) Two steam explosion reactors were used. Volumes of reactor and expansion tanks for reactors A and B were 4.0 and 1.6 L, and 20 and 25 L, respectively. The relation of biomass (dry base) to reactor volume was 13.75 kg/m and 50 kg/m, respectively. Vapor was feed from a steam generator (3 HP) for reactor A while for reactor B from a solar concentrator (collector area 7.2 m2, concentration factor 19). Reactor A operation conditions were total solid content 10 %, an absolute pressure of 0.67 MPa (158 °C) and 20 min of treatment. On the other hand, reactor B was operated with 5 % of total solid content at an absolute pressure of 0.98 MPa (178 °C) for a duration of 24 min (5 cycles of 2 min hold/expansion) (Weber et al., 2019). These supernatants are referred to as steam-exploded hydrolyzates ‘reactor A’ and ‘reactor B’. 2.2.5. Dilute acid pretreatment Agave bagasse was milled in a hammer mill to pass through a 0.84 mm screen. Milled Agave bagasse was pretreated with 1.9 % HCl at 130 °C for 3.22 h as reported by Breton-Deval et al. (2018). This supernatant is referred to as dilute acid hydrolyzate.

2. Materials and experimental methods 2.1. Substrate Agave bagasse (Agave tequilana Weber; var. azul) was collected in 2017 from an industrial facility that produces tequila located in Jalisco, Mexico (latitude 20°50′25″ North, longitude 103°43′30″ West and elevation 1250 m). The facility uses a traditional process where the Agave piña was milled and then brick ovens use moderate heat to steam the Agave piñas. After this stage, Agave bagasse was collected and sun-dried over 10 d and stored indoors in opaque plastic containers at room temperature. Components of Agave bagasse were characterized on a per kilogram basis according to previous methods (APHA, 1999; Sluiter et al., 2012): 934 g of total solids (TS), 899 g of volatile solids (VS), 564 g of cellulose, 109 g of hemicellulose, 152 g of lignin, and 35 g of ash.

2.2.6. Ozone pretreatment and saccharification Agave bagasse with a particle size between 0.60 and 0.70 mm and moisture content of 45 % (w/w) was placed inside a fixed bed reactor consisting of a glass column (0.30 m in height and 0.07 m in diameter) with a concentric bubble-diffuser placed in the center of the reactor. Ozonation was conducted with an ozone dose of 90 mg O3/gTS using a G11 ozone generator (Pacific Ozone Technology, CA, USA) during 60 min at room temperature (27 ± 2 °C) and atmospheric pressure. Ozone-treated bagasse was dried in an oven at 35 ± 5 °C for 48 h and subsequently subjected to enzymatic hydrolysis with 5 % (w/w) of dried ozonated bagasse, 0.05 M citrate buffer (pH 4.8), at 50 °C, 2

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Table 1 Composition of Agave bagasse hydrolyzates. Parameter

Rumen fluid

Native microbiota

Enzymatic hydrolysis

Ozone + Enzymatic hydrolysis

Steam explosion (reactor A)

Steam explosion (reactor B)

Dilute acid

Total carbohydrates (g/L) Chemical oxygen demand (gCOD/ L) Final pH Phenols (mg/L) Furfural (mg/L) Hydroxymethylfurfural (mg/L) Acetic acid (g/L) Other organic acids (g/L)

0.97 ± 0.2 19.9 ± 1.0

0.0 15.1 ± 1.2

11.8 ± 0.02 29.0 ± 1.0

11.46 ± 0.5 29.0 ± 1.2

5.9 ± 0.1 12.8 ± 0.7

21.5 ± 4.5 25.9 ± 0.6

19.5 ± 1.0 20.6 ± 1.4

5.5 ± 0.2 ND ND ND 6.06 ± 1.0 7.47 ± 0.8

5.5 ± 0.1 ND ND ND 6.30 ± 1.3 8.0 ± 0.9

5.5 ± 0.1 ND ND ND ND ND

5.0 ± 0.1 531 ± 23 < 0.005 < 0.005 1.03 ± 0.001 –

4.3 ± 0.2 0.6 ± 0.06 258 ± 9 231 ± 8 ND –

3.7 ± 0.2 308 ± 20 135 ± 13 112 ± 18 6.30 ± 0.7 –

0.6 ± 0.01 190 ± 10 500 ± 50 96 ± 22 1.48 ± 0.14 –

Note: ND, not detected.

120 rpm during 48 h. Celluclast 1.5 L® (Sigma, Louis, MO, USA) with an activity of 90.9 IU/mL was added at an enzyme dose of 4.72 IU/gTS. This supernatant is hereinafter referred to as ozonated-enzymatic hydrolyzate.

CSF = logR 0

(2)

pH

where CSF is the combined severity factor, R0 is the severity factor, and pH is the acidity of the aqueous solution (Lloyd and Wyman, 2005). The experimental data of the biochemical methane assay were fitted to the modified Gompertz equation to obtain the model parameters.

2.3. Characterization of hydrolyzates

BMP = Pmax * exp

All Agave bagasse hydrolyzates were characterized using standard methods. Total carbohydrates were quantified as glucose by the phenolsulfur method (Dubois et al., 1956). COD was quantified using Hach vials (Method 8000, 0–1,500 mgCOD/L) (Hach Company, Loveland, CO). Total phenol content was quantified by the 4-aminoantipyrine spectrophotometric method (APHA, 1992). Furfural and hydroxymethylfurfural were quantified by HPLC while volatile fatty acids were measured using a gas chromatograph-flame ionization detector, as previously was reported (Muñoz-Páez et al., 2020).

exp

2.71828 * Rmax ( Pmax

t)

+1

(3)

where BMP (mL/gCODin) is the cumulative methane production, Pmax is the maximum methane yield (mL/gCODin), Rmax is the methane production rate (mL/d), λ is the lag phase-time (d). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey test. Comparisons with adjusted p values less than 0.05 were considered statistically significant. 3. Results and discussion

2.4. Biochemical methane potential assay

3.1. Effect of pretreatments on solubilization

2.4.1. Inoculum Inoculum consisted of granular sludge from an UASB reactor treating brewery effluents as previously reported by Arreola-Vargas et al. (2018). The contents of total suspended solids and volatile suspended solids (VSS) were of 0.12 g/L and 0.09 g/L, respectively.

Firstly, the pretreatments were compared in terms of solubilization measured as total carbohydrates (CHO) and COD (Table 1). Except for the biological methods, pretreatments released from 10 % to 56 % of the carbohydrates available in the Agave bagasse. Enzymatic hydrolysis (alone or combined with ozone) and steam explosion behave poorly with an average yield of 0.14 ± 0.06 gCHO/gTS (Fig. 1). Opposite, dilute acid gave the highest yield of 0.38 ± 0.04 gCHO/gTS. The two biological pretreatments did not release sugars, instead, these pretreatments produced a mixture of volatile fatty acids (VFA). In consequence, the measured sugars in the biological hydrolyzates were as low as zero to 0.01 gCHO/gTS (1.5 % of the available carbohydrates). Because hydrolyzates had a different composition, the solubilization of Agave bagasse was also compared in terms of COD. In such comparison, most pretreatments behaved similarly, with an average yield of 0.16 ± 0.02 gCOD/gTS, ozonated-enzymatic and dilute acid

2.4.2. Experimental procedure All Agave bagasse hydrolyzates were subjected to anaerobic digestion to determine its BMP using an automatic methane potential test system AMPTS II (Bioprocess Control, Sweden), according to previously reported protocols (Owen et al., 1979; Guwy, 2004). Bottles with a working volume of 360 mL were loaded with 3.6 g-SSV of inoculum, 5 gCOD/L of each hydrolyzate, and basal medium supplemented with 3 g/L of NaHCO3 with an initial pH of 7.5 (Angelidaki and Sanders, 2004). Two control treatments were considered: positive control with inoculum and glucose at 5 gCOD/L, and an endogenous treatment (methane production that was subtracted from the total methane production in all treatments). All assays were performed in triplicate. Results of methane production were expressed at STP conditions. 2.5. Calculations and statistical analysis A combined severity factor (CSF) was calculated for each pretreatment using Eqs. 1 and 2 considering temperature, pH, and contact time as shown in Table S1.

R 0 = t exp

(T

100) 14.75

(1) Fig. 1. Effect of pretreatments on solubilization of total carbohydrates and COD from Agave bagasse. Different letters indicate significant differences at p ≤ 0.05 level.

where R0 is the severity factor, t is the time in minutes, and T is the temperature in °C (Overend and Chornet, 1987). Then, the CSF is defined as follows: 3

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Fig. 2. Effect of pretreatment severity on formation of furfural and hydroxymethylfurfural (HMF) from Agave bagasse.

hydrolyzates overcame the average yield by a factor of 2.0 and 2.6, respectively. The results presented in this study allowed identifying some main patterns that agree with the existing literature on pretreatment of lignocellulosic biomasses. The ratio of total carbohydrates to COD averaged 0.75 ± 0.2 for most hydrolyzates (except for biological hydrolyzates). This ratio is higher than those previously reported for enzymatic hydrolyzates of Agave bagasse ranging from 0.2 to 0.4 (Arreola-Vargas et al., 2016; Galindo-Hernández et al., 2018; MontielCorona and Razo-Flores, 2018), but similar to ratios of acid hydrolyzates ranging from 0.6-0.95 and steam-exploded hydrolyzates whose ratio averaged 0.8 ± 0.1 (Arreola-Vargas et al., 2016; Breton-Deval et al., 2018; Weber et al., 2019). Thus, in this study dilute acid pretreatment overcame the average solubilization yield of 0.16 gCOD/gTS reached by steam explosion, enzymatic and biological pretreatments. In literature, most authors have compared chemical pretreatments by using two (Wang et al., 2015), three (Saha and Cotta, 2010), or four types of catalysts (Park and Kim, 2012). But, despite the relevance of pretreatment, very few studies have contrasted different types of pretreatments. By comparing chemical versus enzymatic pretreatments, Arreola-Vargas et al. (2016) used 2.7 % HCl to pretreat Agave bagasse releasing 1.5 times more sugars than using the enzymatic pretreatment with Celluclast® 1.5 L. This trend was confirmed in this study since acid dilute pretreatment released 2.5 times more sugars than the enzymatic pretreatment with a cocktail of two enzymes. By comparing chemical versus thermal pretreatments, Sambusiti et al. (2013) used 10 % NaOH to pretreat wheat straw and sorghum forage recovering 1.5 times more sugars than applying a thermal pretreatment at 160 °C. Bernier-Oviedo et al. (2018) also proved that a chemical pretreatment with 10 % H2SO4 solubilized most of the hemicellulose from sugarcane bagasse instead of the steam explosion pretreatment at 0.7 MPa for 30 min. In this study, dilute acid solubilized 2.8 times more COD than the steam explosion pretreatments. By comparing more than two pretreatment technologies, Schultz-Jensen et al. (2013) applied chemical (ozone), thermochemical (hydrothermal, wet oxidation and steam explosion), and physical (ball milling) pretreatments to the macroalgae Chaetomorpha linum. These authors demonstrated that thermochemical pretreatments (mainly wet oxidation) overcame the xylan solubilization reached by ozone and physical pretreatments. In this study, four types of pretreatments were contrasted in terms of COD and CHO solubilization. The combined pretreatment of ozonation followed by enzymatic saccharification improved by a factor of 1.5 the CHO recovery in comparison with enzymatic hydrolysis alone. Ozonation degrades lignin making more available the carbohydrates for saccharification (Herrera Adarme et al., 2017). Previous studies demonstrated that alkaline delignification of Agave bagasse before enzymatic hydrolysis improves by a factor of 6–10 the recovery of sugars (Hernández-Salas et al., 2009; Galindo-

Hernández et al., 2018). As individual pretreatment, dilute acid performed best in terms of COD and CHO solubilization. Thermochemical, enzymatic and biological pretreatments solubilized the same amount of COD. Note that, the two biological pretreatments released the same amount of COD from Agave bagasse. Rumen microorganisms were tested for solubilizing rice straw (Zhang et al., 2016), microalgal biomass (Barragán-Trinidad et al., 2017), and corn stover (Jin et al., 2018), but not Agave bagasse. In such studies, rumen microorganisms reached maximum solubilization of 0.4 gCOD/gTS and produced until 8.5 g/L of VFA mainly from the xylan fraction. On the other hand, native microbiotas of lignocellulosic substrates such as grass silage and wheat straw also produced a mixture of VFA, up 12 g/L, when they degraded the lignocellulosic substrate (Li et al., 2012; Pérez-Rangel et al., 2015). In this study, rumen fluids and native microbiota produced on average 12 ± 2 g/L of VFA. 3.2. Effect of pretreatments on formation of inhibitors The use of a CSF that considers time, temperature, pH made possible to compare among pretreatments in terms of inhibitor formation such as furfural and hydroxymethylfurfural (HMF). In this study, the formation of furfural increased linearly with CSF (Fig. 2). The CSF was proposed for comparison among chemical and thermal pretreatments (Lloyd and Wyman, 2005). However, this concept made possible to determine that biological and enzymatic pretreatments had a contribution to the CSF due mainly to its residence times. Thus, at one extreme, the biological and enzymatic pretreatments had the lower values of CSF, on average 2.3 ± 0.3, where the residence time made the highest contribution to CSF ranging from 1,440 min to 21,600 min. Even so, these four pretreatments did not release furfural. At the other extreme, the acid dilute pretreatment gave the highest CSF of 9.6, where the acidic pH contributed 67 % to this CSF. The steam explosion pretreatments (reactors A and B) had a middle-CSF of 6.3 ± 0.9, where pH and temperature contributed equally to CSF. Thus, for 2.3 ≤ CSF ≥ 9.6, the concentration of furfural ranged from 0.1 to 1.1 g/100 g VS (corresponding to 135 mg/L to 500 mg/L in Fig. 2), increasing 0.12 g/ 100 gVS for every increase in CSF. Other authors also have reported that an increase in severity factor resulted in a linear increase in the furfural concentration (Batista et al., 2019). The formation of HMF did not correlate with CSF as furfural did (Fig. 2). Only steam explosion and dilute acid pretreatments released from 0.2 to 0.3 g/100 gVS of HMF. In terms of final concentration, the steam-exploded hydrolyzates contained higher concentrations of HMF than the dilute acid hydrolyzate. This behavior is explained by the higher temperatures applied during the hydrothermal pretreatments which promote the formation of HMF (Zhou et al., 2010). Since 4

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cellulose is the precursor of HMF (Borrero-López et al., 2018), it is stated that the hydrothermal and dilute acid pretreatments solubilized part of the cellulose in the Agave bagasse. The degradation compounds formed during the pretreatments also include phenols due to the lignin degradation and organic acids such as acetic acid due to cleavage of the acetyl linkages in xylan (GalindoHernández et al., 2018). In this study, phenols were detected only in the chemical and hydrothermal hydrolyzates (Table 1). The ozone/enzymatic pretreatment produced the highest concentration of phenols, a phenomenon attributed to the lignin degradation where ozonation has proved to remove until 31 % of insoluble lignin from sugarcane bagasse (Herrera Adarme et al., 2017). There was no pattern in the formation of acetic acid. Firstly, the biological pretreatments yielded the highest concentrations of organic acids where acetic acid represented from 45 % to 60 % of the total VFA. Microorganisms that acted on the Agave bagasse hydrolyzed and fermented the sugar fractions into organic acids being an expected result concentration of acids up to 5 g/L (Li et al., 2012; Jin et al., 2018). For these specific biological pretreatments, the accumulation of these organic acids is a good indicator of performance. Then, the steam explosion ‘reactor B’ produced the highest concentration of acetic acid in comparison with the steam explosion ‘reactor A’ and dilute acid pretreatment. The formation of acetic acid increases with temperature (Batista et al., 2019), a phenomenon that explains the observed trend in this study since ‘reactor B’ was operated at the highest temperature.

untreated Agave bagasse was comparable with previous studies using untreated lignocellulosic biomasses with an average BMP of 190 ± 13 mL/gCODin (Valdez-Vazquez et al., 2016). All pretreatments were effective for increasing the BMP, reaching a 1.3- to 1.9-fold improvement in comparison with untreated Agave bagasse (Fig. 3). The time at which each hydrolyzate produced > 200 mL/ gCODin differed notably. The enzymatic hydrolyzates took the shortest time of three days to reach this BMP, the dilute acid hydrolyzates took five days, the native bacterial hydrolyzates took seven days, and with the longest time, the steam-exploded hydrolyzates took 14 days. In terms of methane production rate (Rmax), the highest Rmax were observed for the enzymatic and dilute acid hydrolyzates (74 and 38 mL CH4/d, respectively), while that the steam-exploded and biological hydrolyzates reached the lowest Rmax (on average, 28 and 20 mL CH4/ d, respectively). At the end of the BMP assay, the different hydrolyzates reached BMP values ranging from 168 to 241 mL/gCODin (Figure S1). BMP from most hydrolyzates was the same (p > 0.05), on average 219 ± 15 mL/ gCODin, which represents 63 % of the theoretical value. Only the steamexploded hydrolyzate ‘reactor A’ and rumen bacterial hydrolyzate produced 20 % lower methane than the average (p < 0.05). It means that different pretreatment methods differed mainly in the time needed to reach the maximum BMP and Rmax, but most hydrolyzates reached the same BMP. These results were surprising due to two facts. First, it means that in spite of different hydrolyzate compositions in terms of CHO/COD ratios, and type and concentrations of inhibitors, all these hydrolyzates reached very close BMP values. In literature, different types of hydrolyzates differed in their BMP. In terms of methane production from soluble COD, Arreola-Vargas et al. (2016) reported that HCl hydrolyzates produced four times more methane than H2SO4 hydrolyzates due to the presence of sulfate-reducing bacteria. Galindo-Hernández et al. (2018) produced two times more methane by using hydrolyzates derived from an enzyme cocktail of two enzymes than by using hydrolyzates derived from an individual enzyme. In contrast, other

3.3. Biochemical methane potential from Agave bagasse hydrolyzates After obtaining different types of Agave bagasse hydrolyzates, BMP assays were carried out using a standard protocol based on recommendations published elsewhere (Hollinger et al., 2016). First, the control consisting of glucose produced 330 ± 22 mL/gCODrem, 95 % of the theoretical value, a result that validated the BMP assay (Angelidaki and Sanders, 2004). Then, the control consisting of untreated Agave bagasse had a BMP of 128 ± 4 mL/gCODin (Fig. 3). The BMP for

Fig. 3. Time course of methane production from different Agave bagasse hydrolyzates and controls (untreated Agave bagasse and glucose). Values correspond to mean ± standard deviation of measurement performed in triplicate. 5

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authors have tested pretreated lignocellulosic substrates, not only the soluble fraction. Sambusiti et al. (2013) produced methane from pretreated wheat straw and sorghum forage (NaOH versus thermal pretreatment), reporting that a combined NaOH/hydrothermal pretreatment reached the theoretical BMP. Mustafa et al. (2018) also demonstrated that the combined Ca(OH)2/hydrothermal pretreatment overcame individual pretreatments in producing methane from sugarcane. In this study, almost all pretreatment methods yielded hydrolyzates with similar BMP. Thus, the standard BMP protocol differentiated hydrolyzates only in terms of lag time and Rmax, but not the BMP. A possible explanation is that all these pretreatment methods reached considerable maturity, that when using the same inoculum and inlet COD concentration, all hydrolyzates gave the same BMP. However, Agave bagasse hydrolyzates could perform differently in continuous reactors where the inoculum is acclimated and the methane productivity increases as the organic loading rate increase. In consequence, is necessary to gain experience in the performance of continuous reactors for methane production fed with different types of Agave bagasse hydrolyzates. Also, differences among hydrolyzates could be observed by comparing methane yields expressed as mL/gVS, as well as by determining the investment and operating costs including productivities, raw materials, and energy utilities needed for each pretreatment. The second fact to highlight is that the maximum BMP was 27 % lower than the theoretical value. This suggests that the Agave bagasse hydrolyzates contain inhibitors that have not been yet characterized. For example, lignin decomposition releases phenolic compounds that in turn form fulvic and humic acids which are detrimental to the anaerobic digestion (Li et al., 2019). A further assumption is that the occurrence of oxalic acid and its salts, terpenoids or steroidal saponins in Agave plants (Monterrosas-Brisson et al., 2013), could be responsible for inhibition of anaerobic digestion. Oxalic acid is biodegradable by anaerobic digestion (Teer et al., 1997). Some oxalateutilizing bacteria include sulfate-reducing bacteria such as Desulfovibrio and lactic acid bacteria such as Enterococcus faecalis which decarboxylate oxalate to formate (Sahin, 2003). The presence of oxalic acid in Agave bagasse hydrolyzates (not confirmed yet), could be the reason for the enrichment of lactic acid bacteria in H2-producing bioreactors (Muñoz-Páez et al., 2020), possibly detrimental to methanization. On the other hand, saponins at concentrations of 50, 100 and 150 ppm inhibit the methane production of food waste (Prabhudessai et al., 2009). So, there could be a negative correlation between the saponin concentration in Agave hydrolyzates with methane production, needing further investigation. Finally, an important factor to consider is the practicality of conducting each pretreatment in full-scale facilities. Based on the BMP results, the biological methods seem the more adequate alternative to produce only biogas. However, for those sugar-based biorefineries dedicated to obtaining high-value chemicals, those pretreatment methods that yield a high CHO/COD ratio should be preferred. In this regard, note that most pretreatments left an important fraction of sugars in the remaining Agave bagasse after pretreatment (see Fig. 1). Therefore, the methane production system could be incorporated into a biorefinery scheme processing the exhausted lignocellulosic solids.

hydrolysis pretreatment released the highest concentration of phenols. When the different hydrolyzates were benchmarked in a BMP assay, most hydrolyzates behave similarly. Individual author contributions I Valdez-Vazquez made the conceptualization, supervised biological pretreatment, analyzed data, and write the final draft. F Alatriste-Mondragón supervised enzymatic pretreatment, analyzed data, and write the first draft. J Arreola-Vargas supervised dilute acid pretreatment, analyzed data, and write the first draft. G Buitrón made the conceptualization, supervised rumen pretreatment, and analyzed data. J Carrillo-Reyes supervised rumen pretreatment, analyzed data, and write the first draft. E León-Becerril supervised ozonation-enzymatic pretreatment, analyzed data, and write the first draft. H O. Mendez-Acosta supervised dilute acid pretreatment and analyzed data. I Ortíz supervised steam explosion pretreatment, analyzed data, and write the first draft. B Weber supervised steam explosion pretreatment, analyzed data, and write the first draft. Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article. Acknowledgements This study was financially supported by “Fondo de Sustentabilidad Energética SENER – CONACYT (Mexico)”, through the project 247006 Gaseous Biofuels Cluster. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2020.112160. References Angelidaki, I., Sanders, W., 2004. Assessment of the anaerobic biodegradability of macropollutants. Rev. Environ. Sci. Biotechnol. 3, 117. https://doi.org/10.1007/s11157004-2502-3. APHA, 1992. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association (APHA), American Water Works Association (AWWA) and Water Pollution Control Federation (WPCF), Washington DC, pp. 5–30. APHA, 1999. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington DC. Arreola-Vargas, J., Ojeda-Castillo, V., Snell-Castro, R., Corona-González, R.I., AlatristeMondragón, F., Méndez-Acosta, H.O., 2015. Methane production from acid hydrolysates of Agave tequilana bagasse: evaluation of hydrolysis conditions and methane yield. Bioresour. Technol. 181, 191–199. https://doi.org/10.1016/j.biortech.2015. 01.036. Arreola-Vargas, J., Flores-Larios, A., González-Álvarez, V., Corona-González, R.S., Méndez-Acosta, H.O., 2016. Single and two-stage anaerobic digestion for hydrogen and methane production from acid and enzymatic hydrolysates of Agave tequilana bagasse. Int. J. Hydrogen Energy 41 (2), 897–904. https://doi.org/10.1016/j. ijhydene.2015.11.016. Arreola-Vargas, J., Snell-Castro, R., Rojo-Liera, N.M., González-Álvarez, V., MéndezAcosta, H.O., 2018. Effect of the organic loading rate on the performance and microbial populations during the anaerobic treatment of tequila vinasses in a pilot-scale packed bed reactor. J. Chem. Technol. Biotechnol. 93, 591–599. https://doi.org/10. 1002/jctb.5413. Ávila-Lara, A.I., Camberos-Flores, J.N., Mendoza-Pérez, J.A., Messina-Fernández, S.R., Saldaña-Duran, C.E., Jimenez-Ruiz, E.I., Sánchez-Herrera, L.M., Pérez-Pimienta, J.A., 2015. Optimization of alkaline and dilute acid pretreatment of agave bagasse by response surface methodology. Front. Bioeng. Biotechnol. 3, 146. https://doi.org/10. 3389/fbioe.2015.00146. Barragán-Trinidad, M., Carrillo-Reyes, J., Buitrón, G., 2017. Hydrolysis of microalgal

4. Conclusions This study compared among five pretreatment methods applied to the Agave bagasse in terms of solubilization of COD and total CHO, the formation of inhibitors, and finally biochemical methane potential from hydrolyzates. In terms of organic matter solubilization, the dilute acid pretreatment outperformed the hydrothermal, biological, and enzymatic pretreatments. The formation of inhibitors was mainly observed for chemical and hydrothermal treatments. The furfural concentration was directly proportional to the CSF increase for 2.3 ≤ CSF ≥ 9.6. Dilute acid and steam explosion pretreatments released the highest concentrations of HMF, while the combined ozone/enzymatic 6

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