Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.     

Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice

The production of aroma compounds during tequila fermentation using four native yeast strains isolated from agave juice was quantified at controlled (35 degrees C) and uncontrolled temperatures (room temperature) by gas chromatography (FID). Three of the four strains were identified as Saccharomyces cerevisiae (MTLI 1, MALI 1 and MGLI 1) and one as Kloeckera apiculata (MALI 2). Among the aroma compounds produced, acetaldehyde has the highest accumulation at the controlled temperature and before 50% of sugar was consumed. The S. cerevisiae strains produced ethyl acetate in almost the same quantity at a concentration of 5 mg/L and the K. apiculata produced six-times more (30 mg/L) than the S. cerevisiae strains, independent of the fermentation temperature. The rate and amount of 1-propanol, amyl alcohols and isobutanol production were affected by the type of yeast used. The K. apiculate strain produced 50% less of the higher alcohols than the Saccharomyces strains. The results obtained showed that indigenous isolated yeasts play an important role in the tequila flavor and suggest that mixtures of these yeasts may be used to produce tequila with a unique and desirable aroma.     

A new agenda for blue agave landraces: food,energy and tequila

Agave tequilana Weber (Rigidae, Agavaceae), blue agave, is a native Mexican plant that has been associated with tequila since the 17th century. The tequila industry has matured over time and now has a geographical indication (Denominación de Origen; DOT). The tequila industry has grown substantially in the last 15 years (19.82% annual increase between 1995 and 2008), resulting in an increase in agave production and associated residue (leaves) and bagasse that can be used for second‐generation biofuels. At a time when the biofuel industry is undergoing unprecedented changes, with diversified demand and predictions of increased competitiveness, this paper presents a review of agave landraces that have been affected by tequila production but may be beneficial for a biofuel industry. Conventional botanical studies have revealed domestication syndromes in races related to blue agave (‘azul listado’, ‘sigüín’ and ‘pata de mula’) specifically for production of fructans in the plant core as would be expected in mezcal agaves (including those used for tequila). Some others, such as the ‘moraleño’ and ‘bermejo’ cultivars (Sisalanae) show domestication syndrome only in the fibers, while others, such as ‘chato,’A. americana L. subtilis (Americanae) show domestication syndrome in fructans and fibers and ‘zopilote,’A. rhodacantha (Rigidae) a relatively low domestication syndrome. No specimens of the cultivars named ‘mano larga’, ‘mano anchaque’ and ‘cucharo’ were found in the Tequila Region of Origin (Western Mexico). The genetic resources from landraces ignored by the tequila industry may be valuable for both ethanol production and conservation.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> age,cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.     

Volatile compounds flavoring obtained from Brazilian and Mexican spirit wastes by yeasts

Vinasse is a waste obtained from the production of beverages, such as tequila and cachaça. The presence of acids, alcohols, sugars, minerals, amino acids, peptides, and nitrogen salts make vinasse a hazardous liquid waste to the environment, affecting the fauna, flora, and microbiota of rivers and lagoons. This study used biological treatment concomitant to volatile compound production. The yeasts used in the study were Saccharomyces cerevisiae (CCMA 0187 and CCMA 0188), Candida parapsilosis (CCMA 0544), and Pichia anomala (CCMA 0193). A higher percentage reduction in chemical and biochemical oxygen demand was observed in the tequila vinasse than in the cachaça vinasse. However, a higher production of volatile compounds was observed in the cachaça vinasse. C. parapsilosis CCMA 0544 produced the highest concentration of 2-phenylethanol (162 mg L^?1). These results indicated that the environmental damage of vinasse can be reduced by treating vinasse with yeasts, and this treatment produces aroma compounds. This biological treatment has high economic potential, especially for the tequila industry.     

Beyond the point of no return? A comparison of genetic diversity in captive and wild populations of two nearly extinct species of Goodeid fish reveals that one is inbred in the wild

The relative importance of genetic and non-genetic factors in extinction liability has been extensively debated. Here, we examine the levels of genetic variability at 13 (seven informative) loci in wild and captive populations of two endangered species of Mexican Goodeid fish, Ameca splendens and Zoogoneticus tequila. Allelic diversity was higher in the wild populations, and F(IS) lower. Values of theta (=4Nemu) were estimated using a coalescent approach. These implied that the effective population size of all captive populations of A. splendens were smaller than that of the wild population; qualitatively similar results were obtained using an analytical method based on within-population gene identity disequilibrium. However, the wild population of Z. tequila did not show a significantly greater estimate of theta. We used the Beaumont approach to infer population declines, and found that both species showed clear evidence of a decline in effective population size, although this was stronger and probably occurred over a longer period of time in Z. tequila than in A. splendens. The decline in Z. tequila probably occurred before captive populations were established. We discuss implications for the conservation of critically endangered populations.     

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Mouse bone marrow cytogenetic damage produced by residues of tequila

Five concentrations (50-860 mg/kg) of residues obtained after distillation and lyophilization of commercial tequila were injected into mice for evaluation of chromosome aberrations, sister-chromatid exchanges, and proliferation kinetics in mouse bone marrow cells. Appropriate positive and negative controls were included. Our results showed significant dose-related increases of chromosomal aberrations starting at 50 mg/kg and for sister-chromatid exchanges at 430 mg/kg. Cellular proliferation kinetics showed no alterations. With these data we demonstrated that the residues of tequila are genotoxic in vivo.     

Agave for tequila and biofuels: an economic assessment and potential opportunities

This paper explores the economic viability of producing biofuels from Agave in Mexico and the potential for it to complement the production of tequila or mescal. We focus on Agave varieties currently being used by the tequila industry to produce two beverages, tequila and mescal, and explore the potential for biofuel production from these plants. Without competing directly with beverage production, we discuss the economic costs and benefits of converting Agave by‐products to liquid fuel as an additional value‐added product and expanding cultivation of Agave on available land. We find that the feedstock cost for biofuel from the Agave piña alone could be more than US$3 L^?1 on average. This is considerably higher than the feedstock costs of corn ethanol and sugarcane ethanol. However, there may be potential to reduce these costs with higher conversion efficiencies or by using sugar present in other parts of the plant. The costs of cellulosic biofuels using the biomass from the entire plant could be lower depending on the conversion efficiency of biomass to fuel and the additional costs of harvesting, collecting and transporting that biomass.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

The effect of tequila in the synaptonemal complex structure of mouse spermatocytes.

The effect of tequila in the synaptonemal complex (SC) of mouse spermatocytes was determined. We tested 3 dosages (2.1, 4.2 and 8.4 g/kg) administered in a single intraperitoneal inoculation. The frequency of SC alterations was established in pachytenic nuclei 5 days after the administration using a silver impregnation technique. Three types of alterations were observed (desynapses, breaks and multiaxials) and the rate of each alteration was compared with that obtained with appropriate controls, including cyclophosphamide (CP) (150 mg/kg). The results showed a significant increase induced by tequila only in the frequency of desynapses. This damage began at the second highest dose (4.2 g/kg). The other SC alterations were in the control range. CP, however, induced a significant increase in all 3 types of SC alterations.     

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from <Emphasis Type="Italic">Agave tequilana</Emphasis> bagasse to produce ethanol by <Emphasis Type="Italic">Pichia caribbica</Emphasis>

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147°C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40°C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R ² = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.     

Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration

AIMS: To study the effect of the addition of different nitrogen sources at high sugar concentration in the tequila fermentation process. METHODS AND RESULTS: Fermentations were performed at high sugar concentration (170 g l(-1)) using Agave tequilana Weber blue variety with and without added nitrogen from different sources (ammonium sulfate; glutamic acid; a mixture of ammonium sulfate and amino acids) during the exponential phase of growth. All the additions increased the fermentation rate and alcohol efficiency. The level of synthesis of volatile compounds depended on the source added. The concentration of amyl alcohols and isobutanol were decreased while propanol and acetaldehyde concentration increased. CONCLUSIONS: The most efficient nitrogen sources for fermentation rate were ammonium sulfate and the mixture of ammonium sulfate and amino acids. The level of volatile compounds produced depended upon types of nitrogen. The synthesis of some volatile compounds increased while others decreased with nitrogen addition. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of nitrogen could be a strategy for improving the fermentation rate and efficiency in the tequila fermentation process at high sugar Agave tequilana concentration. Furthermore, the sensory quality of the final product may change because the synthesis of the volatile compounds is modified.     

Utilization of by-products from the tequila industry. Part 2: Potential value of Agave tequilana Weber azul leaves

The leaves of the agave plant are left in the field after harvesting the heads for tequila production. Different types of agave leaves were isolated, classified, and their content in the total plant determined. The usable fractions were collected and their properties determined. Of the total wet weight of the agave plant, 54% corresponds to the agave head, 32% corresponds to materials which could be usable for sugar and fiber production which leaves 14% of the wet plant without apparent utility. The fractions with higher total reducing sugars (TRS) content were the fresh fraction of partially dry leaves stuck to the head and the leaf bases with a TRS content of 16.1% and 13.1%, respectively. The highest TRS concentration (16-28%) is in the agave head which is used for tequila production. The leaves are 90-120 cm long and 8-12 cm wide and contain fiber bundles that are 23-52 cm long and 0.6-13 mm wide. The ultimate fiber length is approximately 1.6 mm with an average width of 25 microns. There are several types of leaf fibers that can be utilized depending on what part of the plant they come from and what product is desired. Agave leaf fibers were pulped using a soda pulping process and the pulp was hand formed into test sheets. Test sheets made from pulped agave leaf fibers had a breaking length comparable to paper made from both pine and eucalyptus fibers, but the tear index and burst index were lower than the other two papers.     

Yeast communities in a natural tequila fermentation

Fresh and cooked agave,Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated byClavispora lusitaniae and an endemic species,Metschnikowia agaveae. (2)Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particularHanseniaspora spp.,Pichia kluyveri, andCandida krusei. (3)Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but includedSaccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity.Torulaspora delbrueckii, Kluyveromyces marxianus, andHanseniaspora spp. progressively ceded the way toS. cerevisiae, Zygosaccharomyces bailii, Candida milleri, andBrettanomyces spp. With the exception ofPichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process.Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantialDrosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.     

Parametric and genetic analysis of Drosophila appetitive long‐term memory and sugar motivation

Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aversive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long‐term memory (LTM), a long‐lasting memory dependent on de novo protein synthesis. In contrast, one session of odour–sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative conditioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short‐term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the unconditioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addition, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247‐P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.     

Regulation of the organic pollution level in anaerobic digesters by using off-line COD measurements

A sampled delayed scheme is proposed to regulate the organic pollution level in anaerobic digestion processes by using off-line COD measurements. The proposed scheme is obtained by combining an error feedback control with a steady state estimator to track constant references and attenuate process load disturbances. The controller performance is tested experimentally for the treatment of tequila vinasses over a period of 68 days under different set-point values and several uncertain scenarios which include badly known kinetic parameters and load disturbances. Experimental results show that the COD concentration can be effectively regulated under the influence of set-point changes and high load disturbances by using only a daily off-line COD measurement, which makes the industrial application of the proposed control scheme feasible.     

Increasing fermentation efficiency at high sugar concentrations by supplementing an additional source of nitrogen during the exponential phase of the tequila fermentation process

In the tequila industry, fermentation is traditionally achieved at sugar concentrations ranging from 50 to 100 g x L(-1). In this work, the behaviour of the Saccharomyces cerevisiae yeast (isolated from the juices of the Agave tequilana Weber blue variety) during the agave juice fermentation is compared at different sugar concentrations to determine if it is feasible for the industry to run fermentation at higher sugar concentrations. Fermentation efficiency is shown to be higher (above 90%) at a high concentration of initial sugar (170 g x L(-1)) when an additional source of nitrogen (a mixture of amino acids and ammonium sulphate, different than a grape must nitrogen composition) is added during the exponential growth phase.     

Production of Fermentable Sugars and Hydrogen-Rich Gas from <Emphasis Type="Italic">Agave tequilana</Emphasis> Biomass

The Mexican tequila industry annually processes approximately 1 × 10⁶ Agave tequilana plants, generating approximately 1.78 × 10⁸ kg of bagasse per year. This biomass is considered an attractive alternative to fossil fuels as an energy source and to produce biofuels and/or chemical products because it is produced and used without adversely affecting the environment. The first aim of the present work was to determine the effect of temperature, the concentration of H₂SO_4, and reaction time on the hydrolysis of agave bagasse to maximize the fermentable sugars using a steam explosion. This step process generated 71.11 g/L of reducible sugars in the supernatant (59.29 % glucose, 29.05 % xylose, and 11.66 % fructose) and unconverted organic matter of enzymatic hydrolysis bagasse (35.4 % α-cellulose, 7.33 % hemicellulose, 49.91 % lignin, and 7.31 % ashes). A mathematical surface response analysis of the hydrolysis was used for process optimization. The second aim involves the study of the thermodynamics of the reforming of unconverted organic matter from enzymatic hydrolysis of Agave tequilana bagasse (ATB) evaluated by the Gibbs free energy minimization method for hydrogen production. The effect of the parameters on the system performance measures, such as reaction temperature (T), Water/Biomass ratio (WBR), and pressure (P), were also investigated. The maximum H₂ production obtained was 23.2 mol of H₂/271.5 g ATB with a WBR ≥ 11 and a temperature of 740 °C. These findings indicate that the temperature and WBR are essential factors in the production of H₂, which was reflected in the efficiency of the process.