Materials and Methods

Raw material

The buds are made up by a conical structure formed by the more tender leaves grouped in the center of the plant and it is regenerated after cutting, so it can be harvested from the same plant several times. The specimens of Agave lechuguilla (10 buds) were collected in Ramos Arizpe municipality, Coah.

For its conservation and storage in the laboratory of Environmental Biotechnology laboratory of the Faculty of Chemical Sciences of the Autonomous University of Coahuila (Biotecnología Ambiental de la Facultad de Ciencias Químicas de la Universidad Autónoma de Coahuila), the buds were dried in a dehydrator of Trays Koleff Mod. KL10, at 45 °C for 24 h and subsequently were discarded, milled and sieved in a Retsch SM100 cutting mill, until reaching 2 mm average particle size. Finally, the material was mixed and stored at room temperature in 28 °C in plastic containers (30 × 15 × 15 cm) until its pretreatment.

Chemical characterization of the biomass of Agave lechuguilla

The moisture content was specified with a moisture analyzer (OHAUS BL-MB23). The extractive and ash were separated by the analytical methods of the National Renewable Energy Laboratory of the United States of America (NREL): NREL / TP-510-42619 (^{Sluiter et al., 2005}) and NREL/TP-510- 42622, respectively (^{Sluiter et al., 2008}). The determination of cellulose (glucan), hemicellulose (xylan) and lignin was carried out according to the Laboratory Analytical Procedure (LAP): NREL /TP-510-42618) modified by ^{Mussatto et al. (2011)}. 500 mg of the material was hydrolyzed with 72 % H₂SO4 (w / w) for 7 min at 50 °C. Subsequently, the obtained hydrolyzate was diluted up to 4 % with distilled water.

A second hydrolysis of the reaction mixture was made by autoclaving at 121 °C for 1 h. The solution subjected to the autoclave was passed through 0.2 μm PVDF filters for analysis by high performance liquid chromatography (HPLC). The solid residues derived from the filtration were used to calculate the insoluble lignin in acid (Klason lignin). The protein content was determined by the Kjeldahl method (^{Nielsen, 1994}).

Obtaining spores of Phanerochaete chrysosporium H-298

The culture of the strain P. chrysosporium H-298 was obtained from the Collection of Microbial Cultures (CDBB) of Center for Research and Advanced Studies of the National Polytechnic Institute (Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional) (CINVESTAV/IPN), Mexico. Its propagation was done on potato dextrose agar (PDA) for 2 days at 30 °C. The spores were collected and resuspended in sterile distilled water and stored at 4 °C until use.

Pretreatment of A. lechuguilla with P. chrysosporium H-298

For this purpose, nine Erlenmeyer flasks of 125 mL with 5 g of dried and milled core of A. lechuguilla previously sterilized with a volume of modified Kirk's medium solution (in g L^-1: 2.0 de KH₂PO₄, 0.5 g L^-1 MgSO₄(5H₂O, 0.1 de CaCl₂(H₂O, 0.03 de MnSO₄(7H₂O, 0.012 of yeast extract, 0.2 of ammonium tartrate and 1 mg L^-1 of thiamin) en, until obtaining an initial humidity of 60 %. The medium was inoculated with 5 μl of the spore solution of P. chrysosporium H-298, under sterile conditions using a laminar flow hood. The flasks were placed in a New Brunswick ™ 124 / 24R orbital incubator at 30 °C for an incubation time of 10, 15 and 20 days. After each test, the biomass of A. lechuguilla was washed with distilled water and dried in an oven at 45 °C for 24 h. From each sample the cellulose, hemicellulose and lignin content was determined according to the protocols described.

Optimization of the biomass delignification process of A. lechuguilla with P. chrysosporium H-298

The experiments were carried out in 21 Erlenmeyer flasks of 250 mL, in which 10 g of Agave lechuguilla bud previously sterilized, 50 mL of Kirk medium solution and 10 μL of spore inoculum of P. chrysosporium H-298 were incorporated. The flasks were placed in an orbital shaker (New Brunswick™ 124/24R) at 30 °C and 150 rpm.

The optimization was done through a factorial design 3², with the statistical package Minitab™ version 17 (^{Minitab Inc., 2010}), in which the factors of interest were 1) the incubation time (20, 40 and 60 days) and 2 ) the concentration of nitrogen (ammonium tartrate) in medium (1M, 2M and 3M) (tables 1 and 2). After each test, the biomass of A. lechuguilla was washed with distilled water and dried in an oven (Koleff Mod. KL10) at 45 °C for 24 h. At the conclusion of the procedure, each sample was determined its cellulose, hemicellulose and lignin content according to the protocol described.

Enzymatic hydrolysis of the biomass of Agave lechuguilla pretreated with P. chrysosporium H-298

As in the previous tests, three Erlenmeyer flasks of 125 mL were used, in which 10 g (dry basis) of biomass of A. lechuguilla pretreated under optimal conditions with P. chrysosporium H-298 were placed. Subsequently, 40 g of a citrate buffer at a pH of 4.8 was added, which corresponds to a solids loading of 25 % (w/w). The commercial enzymatic complex Cellic™ CTec3 was incorporated into the flasks at an enzyme load of 25 Units of Filter Paper (UPF) g^-1 of glucans. The hydrolysis was carried out at 50 °C and a stirring speed of 200 rpm for 92 h. At hydrolysis times of 12, 24, 48, 72 and 92 h, samples were taken from the hydrolyzate and centrifuged at 10 000 rpm for 10 min (Haraus^TM Megafuge^TM 16 R). The liquid fraction was filtered through 0.22 μm PVDF filters. Samples were analyzed on an Agilent 1260 Infinity liquid chromatograph (HPLC) equipped with a refractive index detector at 45 °C using an Agilent Hi-Plex H column at 35 °C (7.7 × 300 mm), 5 mM H2SO4 cone as phase mobile, at a flow rate of 5.0 mL min^-1. The yield of enzymatic hydrolysis (saccharification) was expressed as g of glucose released g^-1 from initial glucans in the pretreated material.

Fermentation of enzymatic hydrolyzate of biomass of A. lechuguilla

The fermentation of the hydrolysates was carried out in three Erlenmeyer flasks of 125 mL, with 30 mL of enzymatic hydrolyzate. The hydrolysates were supplemented with the following nutrients (in g L^-1): yeast extract 10, KH₂PO₄ 1.17, CaCl₂ 0.09, MgSO₄(7H₂O 0.36, (NH₄)₂SO₄ 4.14. The medium was supplemented with 15 mL L^-1 of a saline solution (stock saline solution) containing NaCl 1.26, CuSO₄(5H₂O 0.26, FeSO₄(5H₂O 0.22, MnCl₂(4H₂O 0.12 y ZnCl₂(7H₂O 0.32.The fermentation conditions were: 5.5 of medium pH, 30 °C, 100 rpm agitation speed, 10 % (v/v) inoculum (Saccharomyces cerevisiae ATCC 4126), during 10 h of incubation (Table 3). took a sample of the fermentation broth at 0, 2, 4, 6, 8 and 10 h, which were centrifuged at 10 000 rpm for 10 min, and the liquid fraction was filtered through PVDF filters for the quantification of ethanol and glucose by HPLC.

Analysis

Glucose and ethanol were determined by HPLC (Agilent 1260) equipped with a refractive index detector at 45 °C, using an Agilent Hi-Plex H column at 35 °C (7.7 × 300 mm) and as a mobile phase as a mobile solution. of 5 mM H₂SO₄ at a flow of 0.5 mL min^-1. All experiments were done in triplicate and the average values were recorded. The analysis of variance (ANOVA) applied together with a Fisher F test with a value of p <0.05 with the Minitab^® version 17 statistical package (^{Minitab Inc., 2010}).

Pretreatment of A. lechuguilla with P. chrysosporium H-298

The kinetics of degradation of lignin during exploratory studies of the biomass pre-treatment of A. lechuguilla with P. chrysosporium H-298 is illustrated in Figure 1. The results obtained indicate a maximum degradation of lignin (15.9 %) after 15 days of treatment at a temperature of 30 °C, which remains without significant changes after 20 days. Cellulose and hemicellulose remain unchanged through this process.

Lignina = Lignin; Tiempo (días)= Time (days)

Figure 1 Kinetic degradation of lignin of Agave lechuguilla Torr. with P. chrysosporium H-298 at 30 °C. 

^{Zeng et al. (2013)} performed a purification and biochemical characterization study of two extracellular enzymes of P. chrysosporium (lignin peroxidase-LiP and manganese peroxidase-MnP) responsible for the degradation of lignin, which resulted in both peroxidases showing a maximum activity at 30 °C.

Optimization of the biomass delignification process of A. lechuguilla with P. chrysosporium H-298

Table 1 describes the degradation of biomass lignin from A. lechuguilla with P. chrysosporium H-298 at different treatment times and at different concentrations of ammonium tartrate as a source of nitrogen. A maximum removal of lignin (36.15 %) can be observed after 60 days of treatment at a concentration of 1M of ammonium tartrate; those that exceeded 1M caused a low degradation of lignin (maximum 6 % in the 2M and 3M tests), behavior that was also observed in the study carried out by ^{Haddadin et al. (2009)} who concluded that high concentrations of nitrogen manage to inhibit the production of lignin peroxidases and lacquers, as well as low yields of lignin removal; however, small concentrations of nitrogen are necessary for the optimal growth of P. chrysosporium.

It was observed that the cellulose and hemicellulose content remains without major changes during pretreatment, with a loss of only 2 % of cellulose, which demonstrates the efficiency of the pretreatment, which, in addition to increasing the digestibility through the degradation of lignin, allows to preserve the structural sugars for its hydrolysis in the saccharification stage. Some studies by ^{Zhang et al. (2012)} proved that the pretreatment time is the most important factor for the degradation of lignin with P. chrysosporium in rice straw.

Figure 2 illustrates the increase in lignin removal from 16 % to 36.15 % in treatments at 20 and 60 days respectively, which corroborates that the treatment time was the most important factor for this process (^{Zhang et al., 2012}).

Degradación de lignina = Delignification; Tiempo = Time; Concentración de nitrógeno = Concentration of nitrogen.

Figure 2 Response surface graph of the optimization study of the biomass delignification process of Agave lechuguilla Torr. with Phanerochaete chrysosporium H-298. 

Enzymatic hydrolysis of biomass of A. lechuguilla pretreated with P. chrysosporium H-298

Table 2 shows the data indicating a maximum glucose release of 44.90 g L^-1 corresponding to 93.09 % hydrolysis yield of the pretreated material at 92 h. This yield was more than 2 times higher than that obtained with the biomass without pretreatment (37.92 %, with a glucose release of 18.72 g L^-1). This increase was not significant after 48 h of incubation for both cases. It is likely that in this period, the cellulases began to be inhibited. This result can be attributed to an increase in the diffusional limitation of cellulases due to a high load of solids, or lignin affected the action of enzymes, blocking access to cellulose (Ríos et al., 2017).

When comparing the maximum yield of hydrolysis obtained with previous results by Ortíz et al. (2017), using biomass of A. lechuguilla pretreated by autohydrolysis and the Celluclast 1.5 L enzyme complex (60.85 %, with a glucose release of 59 g L^-1), it can be proved that the effectiveness of biological pretreatment with P. chrysosporium H -298. However, contrasting this same parameter with the same enzyme complex (Cellic^® CTec3), the previous study by ^{Morales et al. (2017)} achieved a similar performance (96.5 %, with a glucose release of 108.0 g L^-1). In both cases the material was pretreated by autohydrolysis, the release of glucose was higher than that resulted in the present work. This is due to the fact that during the hydrothermal pretreatment, all the extractives are removed and more than 90 % of the hemicellulose is hydrolyzed, which allows the material to be enriched in cellulose and duplicates its composition with respect to the non-raw material (> 40 %).

Fermentation of enzymatic hydrolyzate of biomass of A. lechuguilla pretreated with P. chrysosporium H-298

Table 3 shows the results of the fermentation process of the enzymatic hydrolyzate obtained by Saccharomyces cerevisiae ATCC 4126. The maximum ethanol production was 16.53 g L^-1, in a fermentation time of 10 h, equivalent to an ethanol concentration of 2 % v/v. The maximum conversion efficiency according to the theoretical value (EC) was 90.03 % at 6 h of incubation; after this period, the EC decreased to 82.69 % at 10 o'clock. This behavior is normal due to the reduction of the concentration of glucose in the fermentation medium.

The concentration of ethanol obtained is attributed to the fact that it may be higher to increase the fermentation time, since a constant consumption of glucose was noticed until 10 h. However, this period was not sufficient to detect a total glucose consumption (glucose remaining of 5.7 g L^-1). In the fermentation of enzymatic hydrolysates of biomass from Agave lechuguilla treated by autohydrolysis, ^{Ortiz et al. (2017)} recorded a maximum conversion efficiency of 91 %, lower than that obtained in this study. This difference in CD can be attributed to the fact that biological pretreatment with P. chrysosporium H-298 does not generate inhibitory byproducts of the fermentation stage, unlike the autohydrolysis process, in which inhibitors such as acetic acid, furfural and hydroxymethylfurfural are produced (HMF ) during pretreatment.