Hydrolysis of Agave fourcroydes Lemaire (henequen) leaf juice and fermentation with Kluyveromyces marxianusfor ethanol production
© Villegas-Silva et al.; licensee BioMed Central Ltd. 2014
Received: 5 July 2013
Accepted: 11 February 2014
Published: 14 February 2014
Carbon sources for biofuel production are wide-ranging and their availability depends on the climate and soil conditions of the land where the production chain is located. Henequen (Agave fourcroydes Lem.) is cultivated in Yucatán, Mexico to produce natural fibers from the leaves, and a juice containing fructans is produced during this process. Fructans can be hydrolyzed to fructose and glucose and metabolized into ethanol by appropriate yeasts. In Mexico, different Agave species provide the carbon source for (distilled and non-distilled) alcoholic beverage production using the stem of the plant, whilst the leaves are discarded. In this work, we investigated the effect of thermal acid and enzymatic hydrolysis of the juice on the amount of reducing sugars released. Growth curves were generated with the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus and fermentations were then carried out with Kluyveromyces marxianus to determine alcohol yields.
With thermal acid hydrolysis, the greatest increase in reducing sugars (82.6%) was obtained using 5% H2SO4 at 100°C with a 30 min reaction time. Statistically similar results can be obtained using the same acid concentration at a lower temperature and with a shorter reaction time (60°C, 15 min), or by using 1% H2SO4 at 100°C with a 30 min reaction time. In the case of enzymatic hydrolysis, the use of 5.75, 11.47 and 22.82 U of enzyme did not produce significant differences in the increase in reducing sugars. Although both hydrolysis processes obtained similar results, the difference was observed after fermentation. Ethanol yields were 50.3 ± 4 and 80.04 ± 5.29% of the theoretical yield respectively.
Final reducing sugars concentrations obtained with both thermal acid and enzymatic hydrolysis were similar. Saccharomyces cerevisiae, a good ethanol producer, did not grow in the hydrolysates. Only Kluyveromyces marxianus was able to grow in them, giving a higher ethanol yield with the enzymatic hydrolysate. The leaves account for a non-negligible weight of the total agave plant biomass, so this work complements the knowledge already developed on agave fermentations by making it possible to produce ethanol from almost the entire plant (stem and leaves).
Comparison of Agave fourcroydes L. biomass productivity with other energy crops
Dry biomass productivity (t∙ha-1∙year-1)
Although producing ethanol from the juice (stem and/or leaves) of agaves is not a direct fermentation of simple sugars (as with sugar cane or sweet sorghum), it is not as difficult as producing it from lignocellulosic materials. There is no need for solid material pretreatments, but a hydrolysis step is needed because fermentable sugars are not directly available. Unlike fructans from chicory, which are mainly composed of fructose molecules joined by β(2-1) linkages, agave fructans contain an important amount of β(2-6) linkages that result in branched molecules [7, 9, 10]. This can affect, for example, the choice of enzyme for the hydrolysis of the fructan chains. Commercial enzyme preparations that contain endo- and exo-inulinases can be used to achieve good yields in agave fructans hydrolysis. Inulinases can mainly be produced by microorganisms such as yeast strains (Candida sp., Sporotrichum sp., Pichia sp., and Kluyveromyces sp.) or fungi (Aspergillus and Penicillium species) . Thermal acid hydrolysis can also be used, but this process is associated with the production of unwanted by-products (hydroxymethyl furfural and fructose dianhydride) that can affect further biological steps or the purity of final products .
The juice from the leaves of the henequen plant has previously been studied for ethanol production . In that work, the juice was diluted to simulate the concentrations obtained from the mills. The low sugar content was compensated for by the addition of molasses, another industrial residue. However, technical proposals exist to obtain pure juice from the mills, meaning that sugar concentrations could be higher and there would be no need to add other carbon sources. The use of a mixture of yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus, helped to improve ethanol yields, although they were still low. The aim of this work was to investigate the effect of thermal acid and enzymatic hydrolysis of the juice on the amount of reducing sugars released from these processes. Temperature, time and sulfuric acid concentrations were varied for thermal acid hydrolysis. Temperature and enzyme concentration were varied for enzymatic hydrolysis. The treatment that gave the highest reducing sugars yields was used to continue the study. Growth curves were generated with the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus and fermentations were then carried out with Kluyveromyces marxianus to determine alcohol yields.
Results and discussion
Thermal acid hydrolysis
Increase in reducing sugars after thermal acid hydrolysis of henequen juice
Heating time (min)
Reducing sugars2(g L-1)
Reducing sugars increase (%)
40.25 ± 0.64a
48.84 ± 2.58b
74.59 ± 1.05c
53.55 ± 1.83d
73.36 ± 1.7c
64.38 ± 2.3e
74.01 ± 1.49c
74.4 ± 3.29c
74.95 ± 1.41c
The selection of one yeast over the other depends on the nature of the must and fermentation conditions. The use of a mixture of both yeasts can even be envisaged .
With the higher yields obtained in this work, it is possible to produce 0.038 L of ethanol per liter of leaf juice. Taking into account that a henequen leaf contains 0.78-0.89 L of juice and approximately 250 million leaves are processed per year, 7.125 million liters of ethanol could therefore be produced with this residue. As for production costs, they will be higher than for processes employing direct fermentation of simple sugars, such as sugar cane or sweet sorghum juice, due to the inclusion of the enzymatic hydrolysis step. The amount of enzyme used, however, is minimal. Production costs will be lower than for solid lignocellulosic residues, because there is no pre-treatment step before saccharification. It is easier and cheaper to process liquid materials.
To our knowledge, no data has previously been reported on hydrolyzed agave leaf juice fermentations to produce ethanol.
Final reducing sugars concentrations obtained with both thermal acid and enzymatic hydrolysis were similar, but enzymatic hydrolysis gave better ethanol yields after fermentation. Saccharomyces cerevisiae, a good ethanol producer and one used in agave stem must fermentations, could not grow in either hydrolysate. The development of strains resistant to inhibitory compounds present in agave leaves is important for commercial exploitation of this part of the plant. Fermentation of thermal acid and enzymatic hydrolysates with Kluyveromyces marxianus resulted in theoretical ethanol yields of 50.3 ± 4 and 80.04 ± 5.29% respectively. This yeast is capable of producing inulinase, but attempts to ferment the leaf juice without hydrolysis failed, meaning that preliminary hydrolysis of the fructans present in the juice appears to be necessary. K. marxianus can also be manipulated in order to attain higher ethanol yields. These studies, together with the isolation of new strains from henequen leaf juice, are being carried out by our group. This work complements the knowledge already developed on agave juice fermentation. Agave leaves (fourcroydes or tequilana) account for a non-negligible weight of total plant biomass and comprise an important carbohydrate source that can be used to produce ethanol as biofuel.
All reagents were analytical grade. Inulinase enzyme (Sigma-Aldrich) was obtained from Aspergillus niger with a declared activity of 2000 U · mL-1 and a density of 1.13 g · cm-3. One enzymatic unit is defined as the amount of enzyme necessary to release one g of reducing sugars per minute at 50°C.
Henequen leaves were obtained from healthy plants grown in the gardens of Centro de Investigación Científica de Yucatán AC. They were rinsed with tap water and passed through a three-roller mill to extract the juice, which was filtered to eliminate solid leaf residues and stored at -20°C until use.
Thermal acid hydrolysis
The variables considered important in this process were temperature, heating time and H2SO4 concentration. A 32 full factorial design leading to eight sets of experiments was used to determine the effect of the above mentioned variables on the release of reducing sugars. The levels for each variable were determined on the basis of previous experience with henequen juice hydrolysis and were decided as follows: temperature = 60 and 100°C, heating time = 15 and 30 min, H2SO4 concentration = 1 and 5% (v/v). These levels were decided not only based on reaction system characteristics, but also based on economic and practical factors. Thermal acid hydrolysis experiments were carried out in 250 mL Erlenmeyer flasks with 80 mL of henequen leaf juice. Fresh juice was used as a reference for the initial reducing sugars concentration.
To study enzymatic hydrolysis, a more classical (one variable at a time) approach was used due to the lack of previous data. Conditions of preliminary assays were based on enzyme data provided by the manufacturer. They were carried out in 25 mL Erlenmeyer flasks with 5 mL of raw henequen juice at pH 4.5. Two temperatures, 55 and 60°C, were tested using 22.82 U of enzyme and 90 min incubation time. Subsequently, three enzyme concentrations were tested at 60°C: 5.75, 11.47 and 22.82 U of enzyme and 40 min incubation time.
For fermentation assays, enzymatic hydrolysis was carried out in 250 mL Erlenmeyer flasks with 180 mL of raw henequen juice. An enzyme concentration of 5.75 U was used. Incubation was carried out at 60°C for 30 min and pH 4.5. All enzymatic hydrolysis incubations were performed at 150 rpm.
Kluyveromyces marxianus was isolated from the base of henequen leaves following classical isolation procedures. It was characterized by phenotypical and molecular tests . Commercial Saccharomyces cerevisiae was obtained from Safmex S.A. de C.V. (Mexico). Both yeasts were maintained in Petri dishes on YPGA medium containing glucose (20 g∙L-1), yeast extract (5 g∙L-1), peptone (10 g∙L-1) and agar (20 g∙L-1), and incubated at 30 ± 2°C in darkness. The cultures were collected in sterile water and kept at 4°C until use. Viability of the cell suspensions was measured by staining with methylene blue. Cell suspension viabilities ≥ 95% of both strains were used in all experiments.
Growth curves were generated in 250 mL Erlenmeyer flasks with 100 mL of thermal acid or enzyme hydrolyzed henequen juice adjusted to pH 4.5. Prior to hydrolysis, soluble solids of the raw henequen juice were adjusted to 6°Bx. Ammonium sulfate (1.5 g∙L-1) was used as the nitrogen source  and a cell concentration of 3×107 cell∙mL-1 was employed in all cases. The flasks were incubated at 30 ± 2°C with 150 rpm agitation for 33 h. Samples were harvested every 3 h. Non-hydrolyzed henequen juice was used as a reference. In this case, to avoid contamination, the juice was heated to 80°C and immediately cooled to room temperature after adjusting soluble solids to 6°Bx.
Inocula for fermentations were prepared with thermal acid or enzymatically hydrolyzed henequen juice with a soluble solids concentration of 6°Bx and an initial cell concentration of 3×107 cell∙mL-1. pH was adjusted to 4.5 and 1.5 g∙L-1 ammonium sulfate was added. Growth was maintained at 30 ± 2°C for 18 hours. The amount of the inoculum was 10% of total fermentation volume. Fermentations were carried out in 250 mL Erlenmeyer flasks with 180 mL of non-diluted thermal acid or enzymatically hydrolyzed henequen juice. pH was adjusted to 4.5 and 1.5 g∙L-1 ammonium sulfate was added. After inoculation, the flasks were kept at 30 ± 2°C for 48 hours without agitation.
Soluble solids concentration was measured with a portable refractometer (Cole-Parmer FG103/113) and expressed as °Bx. Reducing sugars concentrations were determined in the hydrolysates and during fermentations by the 3, 5-dinitrosalicilic acid method . The absorbance of the samples was read at 550 nm. For ethanol quantification, 25 mL of fermented sample was diluted with 25 mL of distilled water and distilled at 100°C until 25 mL of distillate was recovered. Ethanol concentration was determined by the dichromate method . The absorbance of the samples was read at 585 nm.
All experiments were performed in triplicate (n = 3). For statistical analysis, the Statgraphics Centurion XV (Statpoint technologies Inc., Warrenton, VA, USA) software was employed. Significant differences among treatments were determined using the Tukey HSD test with p ≤ 0.05. For response surface analysis, the MINITAB 16 (Minitab Inc., State College, PA, USA) software was used.
PAVS received his M.Sc. in Renewable Energy working on this project. TTT is a laboratory technician at CICY working in the Renewable Energy Department. BBCC is head of the Biotechnology Department at CICY. Her research interests are on enzymatic hydrolysis. ALS is a researcher in the Natural Resources Department at CICY. His main area of expertise is Biomass and Productivity. LFBP is head of the Renewable Energy Department. His research area involves biofuel production.
The authors would like to thank Gerardo Rivera for his advice and María Guadalupe Serrano for her support during the experimental work.
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