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Ethanol production from two varieties of henequen (Agave fourcroydes Lem)



    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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    1. Instituto de Biología, Universidad Nacional Autónoma de México, 3er. Circuito S/N, Ciudad Universitaria, 04510, México, D.F.
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    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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    1. Centro de Investigación Científica de Yucatán, A.C., Calle 43 no. 130, Col. Chuburná de Hidalgo, Mérida, 97200, Yucatán, México
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A. Larqué Saavedra, tel. +52 999 942 8370, fax 52 999 981 3900, e-mail: larque@cicy.mx


This work demonstrates the significant differences in ethanol production of two henequen (Agave fourcroydes Lem) varieties. Yaax ki, or green henequen, surpasses Sac ki, or white henequen, in weight, sugar accumulation capability and ethanol production. The study was carried out with the ‘piña’ (stem and basal part of the leaves that remain attached to it after harvesting the leaves) of 5- and 9-year-old plants, cultivated in two localities of Yucatán, México. At 5 years of age, Yaax ki piñas are 33% larger than those of Sac ki, and at 9 years this difference can increase to 59%. Juice from 5-year-old piñas of Yaax ki contains 15.6% more reducing sugars, whereas in the must it can exceed 67%. Values obtained in the 9-year-old plants indicate that the Yaax ki variety accumulates 30.6% more reducing sugars than the Sac ki, whereas in the must the difference is in favor of Yaax ki is 21.7%. To produce 1 L of ethanol at 40% concentration by volume from 5-year-old plants, 48 kg of Sac ki piñas and 29 kg of Yaax ki are required, whereas with the 9-year-old plants, only 23 and 19 kg are needed, respectively.


Agave species, all of which are native to the American continent, are now being considered as potential bioenergy feedstocks for two main reasons, they are known to be good producers of ethanol and the incorporation of such water-use efficient plants on dry lands where no food crops can grow could result in potentially large benefits.

Henequen (Agave fourcroydes Lem.) is a well-known fiber producing plant that grows in shallow infertile soil. It was domesticated by the Mayans in pre-Hispanic times, through the selection of wild populations of its ancestor, A. angustifolia Haw. The Mayans succeeded in differentiating at least seven landraces with distinctive biological and productive characteristics, adapted to different ecological conditions of the Yucatan Peninsula. It has been used primarily for fiber, but traditionally henequen had many uses and was also selected for its high carbohydrate content. At the beginning of the 20th century this diversity was almost lost as a consequence of the establishment of extensive plantations of Sac ki, the variety preferred for the cordage industry. Today, only three of the seven original varieties can be found: the predominant Sac ki, the less common Yaax ki, which is very similar to Sac ki but more robust, and the almost extinct Kitam ki, with a smaller size and softer fiber making it more adequate for textile uses (Colunga-GarcíaMarín & May-Pat, 1993; Colunga-GarcíaMarín, 2003). The evaluation and comparison of Sac ki and Yaax ki capability to accumulate carbohydrates will contribute to the possible use of its germplasm for ethanol production, and could show a possible fingerprint of its differentiated selection by humans historically.

Henequen is a crassulacean acid metabolism (CAM) plant (Nobel, 1985). A great proportion of the carbohydrates produced by its leaves are allocated to fiber formation following a metabolic pathway not fully described as yet, although some of the carbohydrates are stored in the piña. Metabolism, translocation and accumulation of these carbohydrates have not been widely studied to date.

A process for obtaining ethanol from Sac ki piñas, left in the field as byproducts of the fiber industry, has been described using a commercial wine strain of Saccharomyces cerevisiae as fermenting yeast (Larqué-Saavedra et al., 2004). The piña of agaves is an important reservoir of carbohydrates of nonstructural sugars such as oligofructans (inulin, neoinulin, levan and highly branched fructans) (Aspinall & Gupta, 1959; Wang & Nobel, 1998; Cedeño & Alvarez-Jacobs, 2000; López et al., 2003; Gómez-Ayala et al., 2004; García-Albornoz, 2006). When hydrolyzed, these polymers yield mainly fructose, glucose and sucrose which represent between 24% and 27% of the total carbohydrate content.

Microbiological studies were performed to identify the autochthonous yeasts present in this plant variety and during the spontaneous fermentation of agave must, which could be used to produce ethanol (Lappe et al., 2004; Tapia-Tussell et al., 2006; Pérez-Brito et al., 2007). Among these yeasts, one strain of Kluyveromyces marxianus (denominated CICY-Ki) was selected due to its biochemical characteristics, rapid fermentation of fructose, glucose, sucrose, and high temperature tolerance (>52 °C) (Gough et al., 1996; Kourkoutas et al., 2002; Lappe et al., 2004).

A series of experiments with henequen carried out by our group demonstrated that the amount of sugars present in fresh henequen piña used for ethanol production is affected by climatic conditions and plant age (Rendón-Salcido et al., 2009). Total soluble solids (°Brix) concentration varied from 11.7 in 8-year-old plants to 15.2 in 19-year-old plants and from 9.7 in the autumn rainy season to 17.4 during the spring dry period. The results indicate that depending on the age of the plant and the time of year, between 1.6 and 3.9 kg of piña are required to produce 1 L of must which, once fermented, will produce 114 mL of ethanol.

The present study was carried out to evaluate and compare the capability of Sac ki and Yaax ki varieties of different ages to accumulate carbohydrates for ethanol production, and to determine the yield in ethanol production when autochthonous fermenting yeast is used.

Material and methods

Biological material

Two varieties of henequen (A. fourcroydes Lem.) were selected for the present study: Sac ki (white henequen) and Yaax ki (green henequen). Three 5-year-old plants and three 9-year-old plants of each variety were collected from henequen plantations located in the neighboring towns of Chicxulub Pueblo and Mocochá in Yucatán, México. These towns are located 8 m asl where the plantations have a slightly alkaline soil with a pH of 7.3 and 8.0, respectively (Duch, 1988, 1991). The plants were collected in two sites because it was impossible to find a single plantation with both varieties at different ages. Yaax ki plants are very rare and difficult to find.

The study was carried out with piñas (stem and basal part of the leaves that remain attached to it after removal of the leaves for fiber extraction) collected during the rainy season. The piñas were harvested and weighed in situ and thereafter taken to the laboratory for further processing.

Extraction of henequen juice

Henequen juice was extracted from 500 g piña samples (cut into pieces) with a hydraulic press (at/kg cm−2) and the amount of soluble solids was then measured for a fast gross sugar level content. Crude extract was filtered to eliminate the suspended solids and reducing sugars were measured.

To obtain the agave must, 500 g piña samples were cut into pieces and sterilized in an autoclave (Model GE3, FAMSA, Mexico) at 121 °C for 4 h at 1 atm pressure to hydrolyze the oligofructans.

Analytical methods

Concentration of total soluble solids (°Brix) was determined with a portable refractometer (RHB32 Cole Parmer, Vernon Hills, IL, USA). Reducing sugars were estimated in the crude extract and must using the 3,4-dinitrosalicylic acid (DNS) method (Miller, 1959).

Sample absorbance was read in a spectrophotometer at 550 nm and sugar concentrations were calculated by comparison with a glucose calibration curve (intervals of 0.01–0.1 g L−1).

Ethanol concentration was determined by the dichromate method (Williams & Reese, 1950). Sample absorbance was read at 490 nm and ethanol concentrations were determined by comparison with an ethanol calibration curve (intervals 2–20g L−1).

Fermentation conditions and yeast strain

For the fermentation process, agave must with pH 4.4 was adjusted to 9°Brix with water; 1.5 g L−1 of ammonium sulfate were added as nitrogen source; must was maintained at 30±2 °C in darkness, according to Larqué-Saavedra et al. (2004). Once the temperature was reached, 50 mL L−1 (30 × 106 yeasts m L−1) of K. marxianus CICY KI strain were added to ferment the juice without stirring and in darkness.

Samples were collected every 12 h to monitor the fermentation process evolution, soluble solids concentrations were measured and reducing sugars were determined at the beginning and end of fermentation.

K. marxianus CICY-Ki strain was isolated from henequen leaf bases following classical microbiological procedures. It was identified by morpho-physiological tests following the methodology of Yarrow (1998) and the keys of Kurtzman & Fell (1998), Barnett et al., (2001) and the Central Bureau voor Schimmelculture (CBS Database). The identity was confirmed by sequence analysis of the D1/D2 domain of 26S rDNA; sequence was aligned and edited with bioedit v5 program (Hall, 2005) and compared with GenBank sequence database. Sequence obtained was deposited in the GenBank with accession number AY894821 (Lappe et al., 2004; Pérez-Brito et al., 2007).

Ethanol distillation

After 60 h fermentation, ethanol distillation was performed at normal pressure in a Vigreux column at temperatures ranging from 65 to 98 °C. Ethanol volume was recorded and adjusted to 40%, as commonly performed for alcoholic beverages.

Statistical analyses

All of the results are expressed as mean±SE. A comparison of average values between varieties was performed with a two-way analysis of variance (anova), statistica 7 program (Statsoft, Tulsa, OK, USA). Values with the same letter are not significantly different (Tukey's test).


Plant weight

Five-year-old plants were found in Chicxulub Pueblo and 9-year-old plants in Mocochá. Sac ki and Yaax ki 5- and 9-year-old piñas presented significant differences in weight, regardless of the collection site. Five-year-old Yaax ki piñas were 33% heavier than the Sac ki piñas, and in 9-year-old piñas the difference increased to 59% (Fig. 1).

Figure 1.

 Weight of piñas of Sac ki and Yaax.ki agave varieties at different ages. Each bar is the mean value of three independent samples ± SE. Values with the same letters are not significantly different (Tukey's test P≤0.01).

Soluble solids and sugar determinations

°Brix found in juice and must at 5 years in both varieties showed values that varied from 9.8 to 10.0, indicating a similar value for sugar content that can potentially be transformed to ethanol (Table 1). In the case of juices from the 9-year-old piñas, a greater difference was registered in favor of the Yaax ki variety, with 16% in crude extracts and 13.6% in the must.

Table 1.   Soluble solids (°Brix) and reducing sugars (RS) present in juice and must in two henequen varieties at two different ages
Site of harvest
Chicxulub PuebloJuiceMust
Sac kiYaax kiSac kiYaax ki
  1. Data is the mean value of three independent plants ± SE. Values with the same letters indicate no significant differences present Tukey's test at P≤0.0001.

Five-year-old piñas
°Brix10.00 ± 1.05A9.80 ± 1.18A10.00 ± 1.06A9.80 ± 1.18A
RS (g kg−1 piña)31.96 ± 1.71a36.96 ± 1.23a−b31.47 ± 2.78a52.58 ± 3.48c
Nine-year-old piñas
°Brix9.80 ± 0.96A11.40 ± 0.63 A10.80 ± 1.42A12.27 ± 1.43A
RS (g kg−1 piña)43.60 ± 2.52b56.96 ± 2.69c55.78 ± 4.28c67.93 ± 3.78d

Table 1 also shows the results of reducing sugars. The values obtained in 5- and 9-year-old plants, regardless of the collection site, were highest in the Yaax ki variety. In the 5-year-old crude extract, Yaax ki had 15.6% more reducing sugars and in the must up to 67% more in comparison with Sac ki.

Values obtained with 9-year-old plants indicate that the piñas of both varieties have more reducing sugars than those of 5-year-old plants. Yaax ki accumulated 30.6% more sugars in the juice and 21.7% in the must. The concentration of reducing sugars was higher in Yaax ki must at both ages.

Figure 2 represents total sugar content per average weight of Sac ki and Yaax ki piñas. The capability to accumulate carbohydrates is significantly higher in Yaax ki than in Sac ki: 136% more in 5-year-old piñas and 103% in 9-year-old piñas.

Figure 2.

 Sugar content in the must of piñas of Sac ki and Yaax ki at different ages. Each bar is the mean value of three independent samples ± SE. Values with the same letters are not significantly different (Tukey's test P≤0.01).

Fermentation and ethanol results

Once the musts from both varieties were obtained, the fermentation carried out with the native yeast K. marxianus followed a fairly similar pattern. Figure 3 shows the remaining concentration of total soluble solids (6° Brix) after 48 h fermentation when yeast activity stopped. Similar data have been reported by Tzec Gamboa (2006), Rendón-Salcido et al. (2009) and Segura-García (2010) in A. fourcroydes and A. angustifolia musts indicating that, in all probability, the remnant soluble solids are non-fermentable sugars or other compounds that are not able to be metabolized by the yeast to generate ethanol.

Figure 3.

 Pattern of fermentation of must by Kluyveromyces marxianus of two varieties of henequen. Each point is the mean value of three independent samples ± SE.

Ethanol was obtained by distillation of must. Figure 4 shows the volumes of ethanol at 40%, obtained by distillation in both Sac ki and Yaax ki musts obtained from 5- and 9 year-old cooked piñas. At both ages, a greater quantity of ethanol per piña was obtained from Yaax ki than from Sac ki. At 5 and 9 years, Yaax ki produced 110% and 72% more ethanol than Sac ki, respectively. In other words, in order to produce 1 L of ethanol using 5-year-old plants, 48.23 kg of Sac ki piña or 29.98 kg of Yaax ki piña are required; if 9-year-old plants are used, 23.12 and 19.9 kg are required.

Figure 4.

 Volume of ethanol at 40% from piñas of two varieties of henequen of different ages. Each bar is the mean value of three independent samples ± standard error. Values with the same letters are not significantly different (Tukey's test P≤0.01).


These data confirm that henequen piñas are excellent reservoirs of sugars that can be metabolized into ethanol. The most relevant aspect of this work is the confirmation of significant differences between the two varieties of henequen evaluated. The Yaax ki variety surpasses the Sac ki variety in weight, sugar accumulation capability and ethanol production. These differences may be the result of differential human selection in pre-Hispanic times, as Sac ki is a variety selected for its coarser and longer fiber, good for cordage, and Yaax ki is a double purpose variety, with shorter fiber but greater carbohydrates. In the context of biodiversity conservation, the results of this study are of particular importance due to the fact that preference has been given to the Sac ki variety in most plantations because its leaves are longer and the market demands longer fibers. The Yaax ki variety, on the other hand, is in danger of extinction. From the data obtained in this study, it is clear that Yaax ki should be recommended for commercial plantations aimed at the production of ethanol.

Yaax ki's capability to accumulate greater quantities of sugars in the first 9 years is probably due to its shorter life cycle in comparison with the Sac ki variety. No previous experimental work has measured its potential for alcohol production and this aspect requires further investigation since precocity is an important value if the objective is to produce ethanol from this agave in the shortest period of time possible.

In the year 2002, the use of henequen was proposed for two purposes, fiber and alcohol. Data published by Rendón-Salcido et al. (2009) together with the data from this study confirm the potential of this double proposal. However, it is important to note that the alcohol targeted in these studies was for the production of an alcoholic beverage from henequen, such as mezcal, tequila or bacanora distilled from different Agave species. This aspect notwithstanding, the data are valid in that they provide an important antecedent for the consideration of henequen and agaves in general as potentially useful in the production of biofuels. In this context, ethanol that can be produced from both the leaves and the piñas must be considered. In this respect, it is noteworthy that a recent study by Cáceres-Farfán et al. (2008) indicated that 15 L of juice from henequen leaves are required to produce 1 L of alcohol. Therefore, the current annual production of 75 million liters of juice from the defibering plants in the Yucatán could produce 5 million liters of ethanol per year. Potential ethanol production from leaves and the piña make henequen a promising plant for biofuel feedstock.

Based on the data presented in this study, 48 kg of piña from 5-year-old Sac ki are required to produce 1 L of ethanol at 40%, whereas at 9 years only 23 kg are needed. From 5- and 9-year-old Yaax ki piñas 29 or 19 kg are required to produce 1 L of ethanol at 40%, respectively. These data show that larger quantities of henequen are needed to produce 1 L of ethanol than from Agave tequilana which has been selected for many years to produce alcoholic beverages that only require about 8–10 kg of piña, whereas Agave angustifolia in the mezcal industry requires about 12–14 kg of piña tissue (Colunga-GarcíaMarín, et. al., 2007).

Agaves are also known for their high biomass, which could be converted into ethanol. However, the most expensive part of making ethanol from lignocelluloses is pretreating the biomass to make it accessible to the enzymes which will degrade the polymers into sugars that can be fermented into ethanol. Further work is required to determine the optimum enzymatic and microbial processing to convert henequen biomass into ethanol before the agaves can be selected for biofuel production. More comprehensive studies are required to determine the true potential of these CAM plants in the generation of biofuels.


To CONACYT (Grant 173442) for the economic support to carry out the present research work and the master studies formation of the first author Jesús Martínez Torres; the assistance of Mirbella Cáceres is acknowledged and to Olivia Hernández-González for her support in the statistical analysis.