Biofuels produced from lignocellulosic biomass are an environmentally friendly source of renewable energy. Lignocellulosic biomass is an abundant source and is composed mainly by cellulose, hemicellulose, and lignin. However, for it use a pretreatment is needed to deconstruct lignocellulosic matrix. Acid pretreatment hydrolyzes hemicellulose to monosaccharides and reduces the crystallinity of cellulose (Arreola-Vargas et al., 2015). While alkaline pretreatments primarily remove lignin (Xu and Huang, 2014), this process also increases the enzymatic digestibility of biomass. Sequential pretreatments have been proposed to improve the enzymatic hydrolysis of the remaining solid fraction (Pedersen et al., 2010; Rezende et al., 2011). The hydrolysates generated can be used by various microorganisms for the generation of liquid and gaseous biofuels such as bioethanol and methane (Arreola-Vargas et al., 2015; Xu and Huang, 2014).
One source of lignocellulosic biomass with a high potential for biofuels are plants of the genus Agave. Mexico is the center of origin for 76% of all agave species. Agave tequilana is the most commercially exploited plant due to its use in the preparation of tequila, it is also the taxon on which most of the works has been done on. Other species such as A. salmiana have received less attention. Then the information about the susceptibility of its structural carbohydrates to sugar release is scarce (Li et al., 2015).
In the present investigation, we applied an acid-alkali pretreatment to A. salmiana biomass. Following that, the resulting solid fraction was subject to enzymatic hydrolysis. We used acid and enzymatic hydrolysates as a substrate for bioethanol and methane production.
For this objective, we obtained A. salmiana lignocellulosic biomass from the Perote Valley, in the state of Veracruz, Mexico. We cut, dried, water-washed, and stored the dried biomass for later use. We performed an acid pretreatment of agave biomass with sulfuric acid in an autoclave at a solid/liquid ratio of 1/10 w/v. After that, pretreated biomass was separated through vacuum filtration. The solid fraction was washed with abundant distilled water to remove the acid remains, while the liquid phase (filtrate) was stored. The procedure for alkali pretreatment was similar to acid pretreatment. The solid fraction obtained from acid-alkali pretreatment was submitted to enzymatic hydrolysis. The experiments were performed in 50 mM citrate buffer solution (pH 4.8). Reactions were carried out in an incubator at 50 °C with shaking at 150 rpm for 72 h. We used the commercial enzyme Celluclast 1.5L. Alcoholic fermentation process was done with using the acid (H-ACD) and enzymatic (H-ENZ) hydrolysates. The tests were performed in conic tubes at 30 °C without agitation. Saccharomyces cerevisiae "Ethanol Red" was used at a concentration of 107 cells/mL. The pH of the H-ACD was adjusted to pH 4.8 with NH4OH. The hydrolysates were sterilized by filtration (0.2 μm membrane) and supplemented with NH4H2PO4 and MgCl2 at a concentration of 1 and 0.02 g/L, respectively. Methane production was performed in a test system for automatic methane potential (AMPTS II). A reaction volume of 350 mL containing 10 g COD/L of the H-ACD and H-ENZ which were adjusted to a pH of 7.5. We used an inoculum granular sludge from a wastewater treatment plant. We filled the headspace of bottles with helium gas to ensure anaerobic conditions. The equipment operated at 37ºC with a shaking of 120 rpm.
In the results, it was found that application of a pretreatment on agave biomass changes the proportion of the main components. After acid pretreatment, both percentages of lignin as cellulose increased due to removal of hemicellulose. The sulfuric acid was effective to hydrolyze the hemicellulose yielding a hydrolysate rich in sugars, mainly xylose. The concentration of reducing sugars of H-ACD was 19.85±0.11 g/L. After, acid and subsequent alkali pretreatment to agave biomass, the 91% of lignin were removed. The enzymatic hydrolysis applied to solid fraction obtained from acid-alkali subsequent pretreatment yielded of 83.24±3.11% and 45.74±2.03 g/L of reducing sugars.
During alcohol fermentation, the consumption of sugars from H-ACD was 7.23±0.85 g/L and produced 1.73 ± 0.07 g/L of ethanol. The fermentation yield of these experiments was 47.41±6.82%. However, the consumption of sugars from H-ENZ was 24.52±0.79 and the ethanol production was 9.75±0.28 g/L. This corresponds to a fermentation yield of 77.84 ± 4.28%. Fermentation yields of H-ENZ were higher than H-ACD.
On the other hand, we used H-ACD and H-ENZ as substrate for methane production. H-ACD produced a total volume of 148.1 ± 3.68 mL after of 43 h. When the reactors had the H-ENZ as substrate they produced a total volume of 260 ±9.19 mL of biogas. The yields of methane, using H-ACD and H-ENZ were 12.09 ± 0.30% and 21.22 ± 0.75%, respectively.
Our work on the biomass of A. salmiana is a significant contribution to the knowledge of this plant as a source for biofuel feedstock. Further studies are needed to optimize each step of the process for biofuels production.
This work was carried out thanks to the economic support of the 2016 Mobility Program of the Red Temática Mexicana Aprovechamiento Integral Sustentable y Biotecnología de los Agaves (AGARED) and the SAGARPA-CONACYT project (2011-15-174696). Láinez, M. thanks CONACyT for the scholarship granted (275210).