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Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production
Bioresource Technology 146 (2013) 267–273
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Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production Jose-Axel Flores, Anne Gschaedler, Lorena Amaya-Delgado, Enrique J. Herrera-López, Melchor Arellano, Javier Arrizon ⇑ Unidad de Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Avenida Normalistas 800, Col. Colinas de la Normal, C.P. 44270 Guadalajara, Jal., Mexico
h i g h l i g h t s Fructanase screening by Kluyveromyces marxianus with Agave tequilana as substrate. Simultaneous saccharification/fermentation of A. tequilana fructans in bioethanol production. Correlation between fructanase activity and ethanol production under fermentation. Volatile compounds production from A. tequilana fructans.
a r t i c l e
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Article history: Received 1 April 2013 Received in revised form 16 July 2013 Accepted 18 July 2013 Available online 25 July 2013 Keywords: Kluyveromyces marxianus Agave tequilana fructans Saccharification and fermentation Bioethanol Tequila
a b s t r a c t Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Currently, the Agave tequilana fructans (ATF) are used principally as raw material for the tequila elaboration process (Pinal et al., 2009). These molecules are a complex mixture of short and long branched structures formed by b(2 ? 1) and b(2 ? 6) fructosyl linkages with one internal or external glucosyl residue (Lopez et al., 2003). Due to the complex structure of ATF, complete hydrolysis may require the use of specific fructanase enzymes with high exo-fructanhydrolase activity (Muñoz-Gutierrez et al., 2009; Arrizon et al., 2011). Kluyveromyces marxianus has been reported as an exo-fructanase producer (Singh and Gill, 2006). Some of these non-Saccharomyces yeasts have been isolated from traditional
⇑ Corresponding author. Tel.: +52 (33) 33455200; fax: +52 (33) 33455245. E-mail address: [email protected] (J. Arrizon). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.078
fermentation processes involved during the fermentation of Agave beverages (Cruz-Guerrero et al., 2006). These yeasts produce specific exo-fructanhydrolases for the hydrolysis of ATF (Arrizon et al., 2011, 2012). The fructanases produced by K. marxianus yeasts isolated from these processes have been effective for fructose syrup production from inulin (Barranco-Florido et al., 2001) and ATF (García-Aguirre et al., 2009). In the case of the tequila process, it has been applied fructanases for the hydrolysis of ATF in order to optimize the process with commercial (Avila-Fernandez et al., 2009) and native enzymes (Huitron et al., 2013). Due to its composition, bioethanol production can be achieved from ATF by the saccharification and fermentation, as has been reported for other fructans like inulin from Helianthus tuberosus L (Nakamura et al., 1996; Szambelan et al., 2004; Negro et al., 2006; Hu et al., 2012). Since K. marxianus is capable to produce ethanol at laboratory scale from the whole plant biomass of H. tuberosus L (Kim et al., 2013), and at industrial scale with similar yields to Saccharomyces
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cerevisiae (Lopez-Alvarez et al., 2012; Amaya-Delgado et al., 2013), it can be considered for ATF saccharification and for the conversion of hydrolyzed fructans to ethanol by fermentation with this yeast. Therefore, the objective of the present study was to evaluate the simultaneous saccharification and fermentation of ATF by K. marxianus yeasts isolated from the fermentation process of mezcal for bioethanol and tequila production. To date this is the first work reported for the simultaneous saccharification and fermentation with K. marxianus yeasts using a branched fructan like ATF for bioethanol and tequila production.
2.6. Simultaneous saccharification and fermentation experiments For each K. marxianus yeast strain, 2 106 cells/ml grew in YPD medium were inoculated to the fructan induction medium to produce the inoculums at different induction times. Then, 20% v/v of inoculum was added to the fructan fermentation medium (50 ml in 250 ml Erlen-Meyer flasks at 30 °C without stirring). Samples were taken at 0, 6, 12, 24, 36, 48, 72 and 96 h of fermentation. Cell growth, biomass dry weight, fructanase activity and sugar consumption was determined during fermentation, ethanol and volatile compounds were determined at the beginning and at the end of fermentation.
2. Methods 2.7. Fructanases activity 2.1. Strains and maintenance Fifteen K. marxianus yeast strains were used in this work (DK4, DV, DU3, DI4, SLP1, OFF1, DH4, DV4, DX5, MO6, DF1, DH, DZ5, DA5). They belong to the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) collection. These strains were stored at 80 °C in glycerol (50% v/v) until they were required.
2.2. Agave tequilana fructans A. tequilana fructans were provided by the company ‘‘Bustar Alimentos’’, the fructans were extracted by lixiviation with hot water 70 °C, then they were filtered and lyophilized according to Arrizon et al. (2010). The composition was determined by HPLC and it contained 5% w/w of free sugars (0.5% sucrose, 1% glucose and 3.5% fructose).
The enzymatic activity was measured according to Arrizon et al. (2011). Samples were centrifuged (8000g, 10 min, 4 °C), then 50 ll of supernatant was added on different substrates solutions (sucrose, inulin or ATF at 1% w/v in 100 mM of acetate buffer, pH 5). Incubation was carried out during 15 min at 50 °C. The reaction was stopped by the addition of 100 ll of dinitrosalicylic acid (DNS) and boiling for 5 min at 100 °C, then the mixture was placed on ice. The blank was obtained by inactivation of 50 ll of supernatant with 100 ll of DNS during 15 min, then 50 ll of substrate solution was added (1% w/v). The absorbance was measured with a microplate reader at 540 nm. One unit of enzyme activity was defined as the amount of enzyme liberating 1 lmol of reducing sugars per minute. In order to measure fructanase activity during simultaneous saccharification and fermentation, 500 ll of sample was centrifuged in 10 kDa filters in order to discard soluble fructans and free sugars. 2.8. Sugar consumption
2.3. Pre-inoculum 6
Yeast cells (2 10 cells/ml), were grown in 250 ml Erlenmeyer flask containing 50 ml of YPD medium (yeast extract 1% w/v, bacteriologic peptone 2% w/v, glucose 2% w/v). pH was adjusted to 4.5, and sterilized (121 °C), during 15 min. The flasks were incubated on a rotary shaker at 30 °C, 250 rpm from 12 to 24 h depending of the yeast strain.
In order to measure the consumption of ATF, total sugar content was determined according to Arrizon et al. (2010) using anthrone: 100 ll of sample (supernatant of the same sample used for enzymatic activity) was mixed with 200 ll of anthrone (200 mg of anthrone in 100 ml of H2SO4), and the mix was shaken and heated for 10 min at 100 °C. The reaction was stopped by immersion in ice for 5 min. The sugar content was obtained by comparing the absorbance of the sample at 625 nm against a standard curve of sucrose (0–100 lg/ml).
2.4. Fructanase induction and fructan fermentation media 2.9. Cell growth and biomass production For fructanase production, the following medium was used; a salt solution (urea 8 g/l, K2HPO4 3 g/l and MgSO4 2 g/l) was adjusted at pH 4.5 and sterilized (121 °C, 15 min). Later an ATF solution (20 g/l) used as a carbon source was sterilized by filtration (0.45 lm) and added to the salt solution to reach a final concentration of 10 g/l of fructans. For ethanol and tequila fermentation, the salt solution was added with ATF solution (200 g/l) to reach a final concentration of 125 g/l of fructans.
Cell growth was determined by cell counting in a Neubauer chamber. Yeast biomass was determined by measuring dry weight. The cellular dry weight was obtained by harvesting the cells from 1 ml of the centrifuged sample used for enzymatic activity, rinsed with the same amount of distilled water, and desiccated at 60 °C until constant weight was obtained. 2.10. Ethanol and volatile compounds production
2.5. Fructanase induction experiments Yeasts were grown on pre-inoculum medium. 2 106 cells/ml were inoculated on 50 ml of fructanase induction medium in 250 ml Erlen-Meyer flasks at 30 °C and 250 rpm (rotary shaker). Samples were taken at 0, 12, 24, 36, 48, 72 and 96 h, to measure the fructanase activity on A. tequilana fructans in order to determine the induction time at which the highest enzymatic activity was detected. The induction time was considered as the propagation time for the inoculums used in the saccharification and fermentation experiments.
Analysis of ethanol and volatile compounds was carried out in a Hewlett–Packard 6890 gas chromatograph (Palo Alto, CA, USA) with a flame ionization detector (FID) equipped with an HP-Innowax PEG column (60 m 0.320 lm). The initial column temperature was 45 °C for 7 min, and was then ramped at 10 °C/min to 160 °C, followed by a 20 °C/min ramp to 220 °C and maintained during 4 min. Injector and detector temperatures were maintained at 250 °C. The injection system consisted in a head-space Hewlett– Packard 7694E. The preparation program and injection sample started with the following conditions: vial temperature at 80 °C, loop temperature at 110 °C, transfer line temperature at 115 °C.
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The cycle time of head space and gas chromatograph 40 min, the vial equilibrium time 5 min, the pressurization time 0.2 min, the filling loop time 0.2 min, the loop equilibrium time 0.5 min, injection time 1 min and finally the agitation time 1 min. The volatile compounds measured in this study were acetaldehyde, ethyl acetate, 1-propanol, isobutyl alcohol 1-butanol, amyl alcohols, ethyl caprate, ethyl lactate, furfural and ethyl caproate (all volatile compounds were Sigma–Aldrich). Quantification was performed according to an external standard method. Ethanol yield was calculated considering the ethanol produced (final ethanol concentration initial ethanol concentration) and the sugar consumed (initial sugar concentration final sugar concentration). The alcoholic efficiency was calculated considering the ethanol yield over the maximum theoretical ethanol yield.
3. Results and discussion 3.1. Induction time of inoculums To perform the simultaneous saccharification and fermentation of A. tequilana fructans (ATF), induction experiments for fructanase production were carried out with different K. marxianus yeasts, in order to find the induction time at which the inoculums have the maximum extracellular fructanase activity, as well as yeast cells for the simultaneous ATF hydrolysis and ethanol production during fermentation. In the induction experiments, it was observed that the induction time where the highest fructanase activity was reached depended on the yeast strain. Half of them (DK4, DV, DU3, DI4, SLP1, OFF1 and DH4) reached the maximum enzymatic activity within (24–48 h), while the strains DV4, DX5, MO6, DF1, DH, DZ5, DA5 and DL of K. marxianus found the maximum activity at 96 h of induction (Fig. 1). The strain DV4 showed the highest fructanase activity, followed by DK4, SLP1 and DU3, while the rest of the yeasts exhibited values lower than the mean value (0.396 U/ ml). The differences observed in the induction time and fructanase activity, could be associated to variations in the capacity of synthesis and secretion of enzymes between the K. marxianus yeasts, these variations depended also on the yeast strain, carbon source (inducer substrate), nitrogen source, oxygenation, mineral composition and fermentation system (Singh and Gill, 2006; Chi et al., 2009; Silva-Santisteban et al., 2009), as well as the catalytic capacity of the secreted fructanases (Zhang et al., 2009; Singh et al., 2007; Treitchel et al., 2009; Arrizon et al., 2011). During induction experiments, cell growth and biomass were determined (data not shown), a great variation was observed in the initial cell population of the different inoculums (100–600 106 cells/ml, representing 1–4.5 g/l of biomass respectively).
3.2. Fructanase activity of inoculums on different substrates Once the induction time was set to develop the inoculums of each K. marxianus strain, the fructanase activity was determined again before they were added to the ATF medium for the simultaneous saccharification and fermentation process. The activity was measured on inulin and sucrose, in order to elucidate the preference of the fructanases for short and large fructans in comparison to the substrate of interest (ATF). Fig. 2 shows the maximum fructanase activity on different substrates for these inoculums. The hydrolytic capacity and the enzymatic preferences on different substrates varied in function of the K. marxianus yeast tested, in general DV4 showed the highest enzymatic activity in all the substrates, followed by SLP1, DH4, DU3 and DH, the rest of the yeast strains showed lower enzymatic activity (Fig. 2). For all the yeast strains, the highest enzymatic activity was observed on sucrose (2–12 U/ml, Fig. 2a), while the lowest was observed on inulin (0.02–0.2 U/ml, Fig. 2b), which is normal for K. marxianus yeasts as they are recognized as an exo-fructanhydrolase producers with a high ratio of the enzymatic activity on sucrose/inulin (Pandey et al., 1999; Singh and Gill, 2006; Arrizon et al., 2011). In the case of fructanase activity on ATF the yeast strain DV4 showed the highest enzymatic activity, followed by SLP1 and DU3 (Fig. 2c). It can be also observed that for most of the yeasts the fructanase activity of the inoculums on ATF was higher (0.05–1.2 U/ml) than inulin (0.02–0.2) (Fig. 2b and c respectively). Therefore, these enzymes showed a substrate preference for ATF, which was previously observed with a purified exo-fructanhydrolase from a K. marxianus strain isolated from fermenting musts of mezcal (Arrizon et al., 2011).
3.3. Sugar consumption and fructanase activity, biomass and ethanol production Regarding ATF consumption during the saccharification and fermentation, even though the low fructanase activity of some inoculums of the K. marxianus strains evaluated (Fig. 2), all of them consumed more than 90% of the initial ATF concentration with different rates. Therefore, the hydrolytic capacity of the extracellular enzymes, as well as the fructose uptake of the different inoculums depends of the yeast strain. As an example, Fig. 3 represents the ATF consumption and fructanase activity only for the five inoculums with the highest sugar consumption and fructanase activity (DV4, DH4, SLP1, DU3 and DH). It can be seen that DV4 and DH4 showed the faster consumption of ATF followed by DH, SLP1 and DU3 (Fig. 3a), while for fructanase activity, the highest activity was observed for the yeast strain DV4, which was increased during
Enzymatic activity (U ml-1)
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Fig. 1. Maximum fructanase activity on Agave tequilana fructans for the extracellular enzymes of the Kluyveromyces marxianus inoculums induced at 24 h (DK4, DV, DU3, DI4), 48 h (SLP1, OFF1, DH4) and 96 h (DV4, DX5, MO6, DF1, DH, DZ5, DA5, DL) of culture.
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Enzymatic activity ( U ml -1)
14
(a)
12 10 8 6 4 2 0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Enzymatic activity (U ml-1)
0.25
(b) (b) 0.20
0.15
0.10
0.05
0.00 DK4
DV
DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1
DH
DZ5 DA5
DL
Enzymatic activity (U ml -1)
1.4
(c)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Fig. 2. Maximum fructanase activity on sucrose (a), inulin (b) and Agave tequilana fructans (c) for the extracellular enzymes of Kluyveromyces marxianus inoculums induced at 24 h (DK4, DV, DU3, DI4), 48 h (SLP1, OFF1, DH4) and 96 h (DV4, DX5, MO6, DF1, DH, DZ5, DA5, DL) of culture.
the saccharification and fermentation (Fig. 3b). For the yeast strains DH4 and SLP1 a slighter increase was observed, while the fructanase activity for DH and DU3 remained constant (Fig. 3b). Even though the similarity of the sugar consumption rate between the K. marxianus strains DV4 and DH4, a higher fructanase activity was observed for the strain DV4, thus, it could be possible that the strain DH4 has a higher fructose uptake capacity than DV4. According with the sugar consumption yield for all the K. marxianus strains, the inoculums composed of cells and extracellular enzymes were effective for the simultaneous hydrolysis and sugar consumption during the fermentation process. Biomass production varied for all the yeast from 1 to 5 g/l, as an example Fig. 4 shows the biomass production of the five yeast with the highest sugar consumption rate during the saccharification and fermentation. Regarding ethanol production, the three K. marxianus yeasts (DA5, OFF1 and
DZ5) were the highest producers (Table 1), which exhibited low fructanase activity on the three substrates in their respective inoculums (Figs. 1 and 2). On the contrary, a low ethanol production level was observed for yeast strains with high fructanase activity in the inoculums, such as the DV4 and DH4 yeast strains (Table 1). Recently, it has been found that K. marxianus is an industrial ethanol producer (Lopez-Alvarez et al., 2012; Amaya-Delgado et al., 2013), and as other ethanol producer yeasts, the generated alcohol might modify the composition and rigidity of the membrane (Ramirez-Cordova et al., 2012), which could have effect on the enzyme secretion capacity of yeasts (Singh and Bhermi, 2008). On the other hand, ethanol could have effect also on the catalytic capacity of the extracellular fructanases, as has been observed in intracellular enzymes of S. cerevisiae during alcoholic fermentation (Ogawa et al., 2000; Alexandre et al., 2001; Chandler
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Agave tequilana fructans (g l-1 )
100
(a) 80
60
40
20
0 0
20
40
60
80
Time (h)
Table 1 Ethanol produced, ethanol yield and alcoholic efficiency at the end of the saccharification and fermentation process of Agave tequilana fructans. Yeast
Yp/s (g g1)
Efficiency (%)
Ethanol produced (g/l)
DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5 DL
0.323 ± 0.02 0.263 ± 0.01 0.505 ± 0.02 0.420 ± 0.02 0.491 ± 0.01 0.497 ± 0.02 0.396 ± 0.05 0.324 ± 0.00 0.467 ± 0.01 0.447 ± 0.03 0.418 ± 0.08 0.491 ± 0.03 0.506 ± 0.03 0.499 ± 0.01 0.483 ± 0.05
63.3 ± 4.4 51.6 ± 3.2 99.1 ± 4.8 82.3 ± 4.5 96.3 ± 2.3 97.6 ± 3.4 77.6 ± 9.8 63.5 ± 0.2 91.6 ± 1.4 87.8 ± 6.3 82.0 ± 15.3 96.3 ± 6.0 99.3 ± 6.4 98.0 ± 1.8 94.7 ± 11.1
24.5 ± 1.21 21.5 ± 0.94 47.5 ± 1.64 32.4 ± 1.27 44.9 ± 0.77 49.7 ± 1.22 34.8 ± 3.12 28.7 ± 0.06 37.5 ± 0.42 37.4 ± 1.91 34.5 ± 4.58 46.0 ± 2.04 47.1 ± 2.14 49.9 ± 0.65 45.9 ± 3.81
Enzymatic activity (U ml-1)
30
(b)
25 20 15 10 5 0 0
20
40
60
80
Time (h) Fig. 3. Agave tequilana fructans consumption (a) and fructanase activity (b) during saccharification and fermentation with five Kluyveromyces marxianus yeasts strains; DV4 (d), DH4 (s), SLP1 (.), DU3 (4) and DH (j).
5
Biomass (gl-1)
4 3 2 1 0
0
20
40
60
80
Time (h) Fig. 4. Biomass production during saccharification and fermentation with five Kluyveromyces marxianus yeasts strains; DV4 (d), DH4 (s), SLP1 (.), DU3 (4) and DH (j).
et al., 2004). As the fructanase activity of the K. marxianus strain DV4 was increased during the saccharification and fermentation (Fig. 3b), it could be possible that this K. marxianus strain produce enzymes more tolerant to ethanol than the other strains. Since most of the K. marxianus yeasts strains produce extracellular fructanases (Singh and Gill, 2006; Chi et al., 2009; Arrizon et al., 2012), the role of ethanol on the enzyme secretion capacity and on the hydrolytic activity of the fructanases has to be elucidated for a better understanding of the simultaneous saccharification and
fermentation of fructans by K. marxianus yeasts. The yeast strains SLP1, DZ5, DA5, DU3 and DH, showed high fructanase activity on ATF of their respective inoculums before they were inoculated (Fig. 2), as well as high ethanol concentration at the end of fermentation (Table 1). Therefore, these yeast strains are suitable for simultaneous saccharification and fermentation for bioethanol production from ATF. Comparing the quantity of ethanol obtained in this study (20–50 g/l, Table 1) with other works, it corresponded to 2.5–6.3% v/v respectively, which was higher than the production observed in the simultaneous saccharification and fermentation of inulin from H. tuberosus L. using a K. marxianus yeast (4.1% v/v of ethanol, Negro et al., 2006), but lower than the concentrations reached with a mixture of Kluyveromyces fragilis, S. cerevisiae and Zymomonas mobilis (12% v/v) and with a mixture of Aspergillus niger and S. cerevisiae (20.1% v/v) with this substrate (Szambelan et al., 2004; Nakamura et al., 1996, respectively). Hu et al. (2012) evaluated the simultaneous saccharification and fermentation capacity in an H. tuberosus inulin medium (200 g/l), using a S. cerevisiae and a K. marxianus yeast strains, a higher ethanol concentration was also observed (9.3 and 8.2% v/v respectively). Thus, the yield of simultaneous saccharification and fermentation depends also of the type and concentration of substrate; as well as, the type of microorganism used. Recently, Kim et al. (2013) showed that K. marxianus ferment the mixture of glucose, xylose and fructose to ethanol from whole H. tuberosus biomass hydrolyzates, this is possible because K. marxianus has also the capacity to assimilate xylose as a carbon source (Lane et al., 2011). During the tequila elaboration process a high bagasse and vinasse quantities are generated as byproducts (Espinoza-Escalante et al., 2009). Therefore, it could be also possible the bioethanol production from whole biomass hydrolyzates of A. tequilana using K. marxianus strains. The simultaneous saccharification and fermentation of ATF not only can be used in the production of bioethanol, it is also useful for the industrial tequila process, as it can reduce the energy consumption in the cooking step for fructan hydrolysis with simultaneous ethanol production (Avila-Fernandez et al., 2009; Huitron et al., 2013). Additionally, nowadays the number of industries involved in the extraction of fructans from A. tequilana has been increased, principally to produce ingredients for the food industries (García-Aguirre et al., 2009), thus it could be available in the future for other biotechnological applications. As tequila is an alcoholic beverage, the production of volatile compounds during fermentation is important for the quality of the beverage (Arrizon et al., 2006; Arrizon and Gschaedler, 2007; Arellano et al., 2008; Pinal et al., 2009). Therefore, the volatile compounds were determined at the end of the saccharification and fermentation for all the K. marxianus yeast strains evaluated.
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Table 2 Volatile compounds production at the end of the saccharification/fermentation process for the Kluyveromyces marxianus yeasts tested. Yeast strain
DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5 DL
Volatile compounds (mg l1) Acetaldehyde
Ethyl acetate
Methanol
1-Propanol
Isoamyl acetate
Isobutyl alcohol
1-Butanol
Amyl alcohols
6.8 ± 2.5 12.2 ± 4.1 25.8 ± 4.2 27.1 ± 6.9 27.3 ± 0.4 25.6 ± 0.4 29.6 ± 3.3 37.1 ± 7.8 22.8 ± 6.2 33.0 ± 0.9 25.3 ± 4.9 23.7 ± 1.8 12.6 ± 6.1 33.3 ± 5.5 17.7 ± 3.6
19.1 ± 0.8 38.4 ± 23.5 32.8 ± 5.1 51.9 ± 16.3 58.7 ± 5.2 39.5 ± 9.4 56.1 ± 12.1 21.9 ± 11.2 43.1 ± 10.0 60.5 ± 11.7 58.7 ± 9.4 37.6 ± 15.7 34.7 ± 1.7 49.0 ± 8.1 49.6 ± 21.8
5.3 ± 2.4 3.7 ± 1.2 3.8 ± 1.5 2.6 3.6 ± 1.3 4.3 ± 0.3 3.4 4.3 ± 0.2 4.6 ± 0.8 2.6 ± 0.6 2.4 ± 0.6 3.8 ± 1.1 3.7 ± 0.2 3.8 ± 0.2 4.1 ± 0.4
0 0 6.18 0.38 ± 0.1 0.68 ± 0.4 0.31 ± 0.01 0.45 ± 0.01 0 0.41 ± 0.01 0.5 ± 0.05 0.7 ± 0.3 0.41 0.33 ± 0.01 0.7 ± 0.01 0.4 ± 0.01
37.8 ± 1.4 33.9 ± 1.8 52.1 ± 0.3 49.1 ± 2.8 54 ± 0.3 81.2 ± 2.8 64 ± 1.7 25.7 ± 0.3 48.7 ± 9.7 56.1 ± 1.6 53.4 ± 2.7 64.3 ± 19 70.6 ± 3.7 57.3 ± 2.0 68.4 ± 0.4
49 ± 1.7 27.4 ± 1.4 75 ± 11.3 74.8 ± 12 59.4 ± 8.5 98.8 ± 2 81.4 ± 5.2 21.2 ± 4.5 81 ± 0.6 90.6 ± 2.6 94.3 ± 2.9 65.3 ± 14.9 79.1 ± 13.1 72.7 ± 13.8 73.4 ± 13.5
0 0 1.9 0 0.1 0 0 0 0 0.1 0.1 0 0 0.2 0
2.8 ± 0.02 4 ± 0.7 1.6 ± 0.2 1.7 ± 0.1 1.9 ± 0.1 2.5 ± 0.2 2.2 ± 0.1 2.8 ± 0.2 2 ± 0.15 1.5 ± 0.3 1.2 ± 0.3 2 ± 0.5 2.1 ± 0.2 1.6 ± 0.6 1.6 ± 0.07
3.4. Volatile compounds production From 16 volatile compounds measured only 8 were detected in all the fermentation musts (Table 2). In general, isobutyl alcohol was the highest volatile compound produced by the yeasts, followed by isoamyl acetate, ethyl acetate and acetaldehyde, while lower levels of 1-butanol, 1-propanol and amyl alcohols were observed. Previously Lopez-Alvarez et al. (2012), compared at laboratory and industrial scale a K. marxianus yeast (isolated from the tequila process) against a bakery S. cerevisiae yeast and a higher production of ethanol, amyl alcohols, isobutyl alcohol, phenylethyl alcohol and propanol was observed with respect to the bakery S. cerevisiae yeast strain. Thus, with the exception of isobutyl alcohol, the production of volatile compounds for this K. marxianus yeast was different regarding to the behavior observed for the K. marxianus of the present work. Recently, Amaya-Delgado et al. (2013) evaluated at industrial tequila conditions a K. marxianus and a Pichia kluyveri yeast strains (both isolated from mezcal fermentation) and a S. cerevisiae yeast strain isolated from tequila fermentation, a lower C/N ratio was used than the work of Lopez-Alvarez et al. (2012). The K. marxianus yeast produced less ethanol than the P. kluyveri and S. cerevisiae yeast strains, however it produced more isobutyl alcohol and ethyl acetate than the P. kluyveri and S. cerevisiae yeasts, which is similar to the results of the present work. Therefore, as it can be seen, the origin of the K. marxianus yeast and the C/N ration influenced the volatile compounds production capacity. It can be noticed that a common characteristic observed for all the K. marxianus yeast strains in the tequila process is the production of isobutyl alcohol. It is well known that the higher alcohols are derived from precursors of the degradation or synthesis of amino acids according with the Ehrlich pathway. In particular, isobutyl alcohol is biochemically derived from valine (Hazelwood et al., 2008). Therefore, the biochemical conversion of valine to isobutyl alcohol has to be studied in the future for K. marxianus yeasts. Regarding the capacity of volatile compounds production between the different K. marxianus strains, it can be observed that DH4, followed by MO6, DF1 and OFF1 were in general the highest producers (Table 2). With the exception of OFF1, all of them produced low ethanol (Table 1) but a high biomass level (3– 4.5 g/l). Therefore, the conversion of the carbon source to bioethanol is less effective for these K. marxianus yeasts except the OFF1 strain. On the contrary, DA5, DU3, DZ5, DH and SLP1 produced high yields of ethanol (Table 1), but less volatile compounds (Table 2), as well as biomass (2–2.5 g/l), thus they have a high yield to convert the carbon source in bioethanol (Table 1). Even though OFF1 K. marxianus strain showed low fructanase activity, it has a high capacity to transform the carbon source in ethanol and volatile
compounds with low biomass production, thus this yeast strain is suitable for tequila fermentation. For the next volatile compounds; acetaldehyde, ethyl acetate, 1-propanol, isoamyl acetate, isobutyl alcohol, 1-butanol and amyl alcohols, the yeast strains DV4, MO6, DU3, OFF1, OFF1, DU3 and DV were the higher producers respectively (Table 2). Thus, a high metabolic diversity between the K. marxianus yeasts was observed. 4. Conclusions In spite of the complex structure of ATF, they can be used as a substrate for simultaneous saccharification/fermentation process with K. marxianus for bioethanol production. Most of the K. marxianus inoculums can perform the ATF hydrolysis during the fermentation, nevertheless few reached an alcoholic efficiency higher than 95% and some of them were more suitable only for the saccharification process. Volatile compounds such as isobutyl alcohol was the highest volatile compound produced for most of the yeasts, thus the regulation of isobutyl alcohol production has to be elucidated. The yeast strain OFF1 showed a great potential for tequila fermentation. Acknowledgement We would like to thank the Consejo Nacional de Ciencia y Tecnología CONACYT for the economical support received on the project 181766. References Alexandre, H., Ansanay-Galeote, V., Blonding, S., Dequin, S., 2001. Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae. FEBS Lett. 498, 98–103. Amaya-Delgado, L., Herrera-Lopez, E., Arrizon, J., Arellano-Plaza, M., 2013. Performance evaluation of Pichia kluyveri,Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation. World J. Microbiol. Biotechnol.. http://dx.doi.org/10.1007/s11274-012-1242-8. Arellano, M., Pelayo, C., Ramirez, J., Rodriguez, I., 2008. Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice. J. Ind. Microbiol. Biotechnol. 35, 835–841. Arrizon, J., Fiore, C., Acosta, G., Romano, P., Gschaedler, A., 2006. Fermentation behaviour and volatile compounds production by agave and grape must yeasts in high sugar Agave tequilana and grape must fermentations. Antonie van Leeuwenhoek 89, 181–189. Arrizon, J., Gschaedler, A., 2007. Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration. J. Appl. Microbiol. 102, 1123–1131. Arrizon, J., Morel, S., Gschaedler, A., Monsan, P., 2010. Comparison of the water soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages. Food Chem. 122, 123–130.
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agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker yeast used in tequila production. J. Biosci. Bioeng. 113, 614–618. López, M.G., Mancilla-Margalli, N.A., Mendoza-Díaz, G., 2003. Molecular structures of fructans from Agave tequilana Weber var. azul. J. Agric. Food Chem. 51, 7835–7840. Muñoz-Gutierrez, I., Rodríguez-Alegría, M.E., López-Munguía, A., 2009. Kinetic behaviour and specificity of b-fructosidases in the hydrolysis of plant and microbial fructans. Process Biochem. 44, 891–898. Nakamura, T., Ogata, Y., Hamada, S., Ohta, K., 1996. Ethanol production from Jerusalem artichoke tubers by Aspergillus niger and Saccharomyces cerevisiae. J. Ferment. Bioeng. 81, 564–566. Negro, M.J., Ballesteros, I., Manzanares, P., Oliva, J.M., Saez, F., Ballesteros, M., 2006. Inulin-containing biomass for etanol production. Appl. Biochem. Biotechnol. 129–132, 922–932. Ogawa, Y., Nitta, A., Uchiyama, H., Imamura, T., Shiomoi, H., 2000. Tolerance mechanism of the etanol-tolerant mutant of sake yeast. J. Biosci. Bioeng. 90, 313–320. Pandey, A., Soccol, C.R., Selvakumar, P., Soccol, V.T., Krieger, N., Fontana, J.D., 1999. Recent developments in microbial inulinases: its production, properties and industrial applications. Appl. Biochem. Biotechnol. 81, 35–52. Pinal, L., Cornejo, E., Arellano, M., Herrera, E., Nuñez, L., Arrizon, J., Gschaedler, A., 2009. Effect of Agave tequilana age, cultivation field location and yeast strain on the tequila fermentation process. J. Ind. Microbiol. Biotechnol. 36, 655–661. Ramirez-Cordova, J., Drnevich, J., Madrigal-Pulido, J.A., Arrizon, J., Allen, K., Martinez-Velazquez, M., Alvarez-Maya, I., 2012. Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice. Antonie van Leeuwenhoek 102, 247–255. Silva-Santisteban, Y.B.O., Converti, A., Maugeri Filho, F., 2009. Effects of carbon and nitrogen sources and oxygenation on the production of inulinase by Kluyveromyces marxianus. Appl. Biochem. Biotechnol. 152, 249–261. Singh, R.S., Bhermi, H.K., 2008. Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis. Bioresour. Technol. 99, 7418–7423. Singh, P., Gill, P.K., 2006. Production of inulinases: recent advances. Food Technol. Biotechnol. 44 (2), 151–162. Singh, R.S., Rajesh, D., Puri, M., 2007. Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of highfructose syrup. J. Microbiol. Biotechnol. 17 (5), 733–738. Szambelan, K., Nowak, J., Czarnecki, Z., 2004. Use of Zymomonas mobilis and Saccharomyces cerevisiae mixed with Kluyveromyces fragilis for improved ethanol production from Jerusalem artichoke tubers. Biotechnol. Lett. 26, 845–848. Treitchel, H., Mazutti, M.A., Filho, F.M., Rodrigues, M.I., 2009. Technical viability of the production, partial purification and characterization of inulinase using pretreated agroindustrial residues. Bioprocess Biosyst. Eng. 39, 425–433. Zhang, T., Gong, F., Chi, Z., Liu, G., Chi, Z., Sheng, J., Li, J., Wang, X., 2009. Cloning and characterization of the inulinase gene from a marine yeast Pichia guilliermondii and its expression in Pichia pastoris. Antonie van Leeuwenhoek 95, 13–22.
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Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production Jose-Axel Flores, Anne Gschaedler, Lorena Amaya-Delgado, Enrique J. Herrera-López, Melchor Arellano, Javier Arrizon ⇑ Unidad de Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Avenida Normalistas 800, Col. Colinas de la Normal, C.P. 44270 Guadalajara, Jal., Mexico
h i g h l i g h t s Fructanase screening by Kluyveromyces marxianus with Agave tequilana as substrate. Simultaneous saccharification/fermentation of A. tequilana fructans in bioethanol production. Correlation between fructanase activity and ethanol production under fermentation. Volatile compounds production from A. tequilana fructans.
a r t i c l e
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Article history: Received 1 April 2013 Received in revised form 16 July 2013 Accepted 18 July 2013 Available online 25 July 2013 Keywords: Kluyveromyces marxianus Agave tequilana fructans Saccharification and fermentation Bioethanol Tequila
a b s t r a c t Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Currently, the Agave tequilana fructans (ATF) are used principally as raw material for the tequila elaboration process (Pinal et al., 2009). These molecules are a complex mixture of short and long branched structures formed by b(2 ? 1) and b(2 ? 6) fructosyl linkages with one internal or external glucosyl residue (Lopez et al., 2003). Due to the complex structure of ATF, complete hydrolysis may require the use of specific fructanase enzymes with high exo-fructanhydrolase activity (Muñoz-Gutierrez et al., 2009; Arrizon et al., 2011). Kluyveromyces marxianus has been reported as an exo-fructanase producer (Singh and Gill, 2006). Some of these non-Saccharomyces yeasts have been isolated from traditional
⇑ Corresponding author. Tel.: +52 (33) 33455200; fax: +52 (33) 33455245. E-mail address: [email protected] (J. Arrizon). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.078
fermentation processes involved during the fermentation of Agave beverages (Cruz-Guerrero et al., 2006). These yeasts produce specific exo-fructanhydrolases for the hydrolysis of ATF (Arrizon et al., 2011, 2012). The fructanases produced by K. marxianus yeasts isolated from these processes have been effective for fructose syrup production from inulin (Barranco-Florido et al., 2001) and ATF (García-Aguirre et al., 2009). In the case of the tequila process, it has been applied fructanases for the hydrolysis of ATF in order to optimize the process with commercial (Avila-Fernandez et al., 2009) and native enzymes (Huitron et al., 2013). Due to its composition, bioethanol production can be achieved from ATF by the saccharification and fermentation, as has been reported for other fructans like inulin from Helianthus tuberosus L (Nakamura et al., 1996; Szambelan et al., 2004; Negro et al., 2006; Hu et al., 2012). Since K. marxianus is capable to produce ethanol at laboratory scale from the whole plant biomass of H. tuberosus L (Kim et al., 2013), and at industrial scale with similar yields to Saccharomyces
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cerevisiae (Lopez-Alvarez et al., 2012; Amaya-Delgado et al., 2013), it can be considered for ATF saccharification and for the conversion of hydrolyzed fructans to ethanol by fermentation with this yeast. Therefore, the objective of the present study was to evaluate the simultaneous saccharification and fermentation of ATF by K. marxianus yeasts isolated from the fermentation process of mezcal for bioethanol and tequila production. To date this is the first work reported for the simultaneous saccharification and fermentation with K. marxianus yeasts using a branched fructan like ATF for bioethanol and tequila production.
2.6. Simultaneous saccharification and fermentation experiments For each K. marxianus yeast strain, 2 106 cells/ml grew in YPD medium were inoculated to the fructan induction medium to produce the inoculums at different induction times. Then, 20% v/v of inoculum was added to the fructan fermentation medium (50 ml in 250 ml Erlen-Meyer flasks at 30 °C without stirring). Samples were taken at 0, 6, 12, 24, 36, 48, 72 and 96 h of fermentation. Cell growth, biomass dry weight, fructanase activity and sugar consumption was determined during fermentation, ethanol and volatile compounds were determined at the beginning and at the end of fermentation.
2. Methods 2.7. Fructanases activity 2.1. Strains and maintenance Fifteen K. marxianus yeast strains were used in this work (DK4, DV, DU3, DI4, SLP1, OFF1, DH4, DV4, DX5, MO6, DF1, DH, DZ5, DA5). They belong to the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ) collection. These strains were stored at 80 °C in glycerol (50% v/v) until they were required.
2.2. Agave tequilana fructans A. tequilana fructans were provided by the company ‘‘Bustar Alimentos’’, the fructans were extracted by lixiviation with hot water 70 °C, then they were filtered and lyophilized according to Arrizon et al. (2010). The composition was determined by HPLC and it contained 5% w/w of free sugars (0.5% sucrose, 1% glucose and 3.5% fructose).
The enzymatic activity was measured according to Arrizon et al. (2011). Samples were centrifuged (8000g, 10 min, 4 °C), then 50 ll of supernatant was added on different substrates solutions (sucrose, inulin or ATF at 1% w/v in 100 mM of acetate buffer, pH 5). Incubation was carried out during 15 min at 50 °C. The reaction was stopped by the addition of 100 ll of dinitrosalicylic acid (DNS) and boiling for 5 min at 100 °C, then the mixture was placed on ice. The blank was obtained by inactivation of 50 ll of supernatant with 100 ll of DNS during 15 min, then 50 ll of substrate solution was added (1% w/v). The absorbance was measured with a microplate reader at 540 nm. One unit of enzyme activity was defined as the amount of enzyme liberating 1 lmol of reducing sugars per minute. In order to measure fructanase activity during simultaneous saccharification and fermentation, 500 ll of sample was centrifuged in 10 kDa filters in order to discard soluble fructans and free sugars. 2.8. Sugar consumption
2.3. Pre-inoculum 6
Yeast cells (2 10 cells/ml), were grown in 250 ml Erlenmeyer flask containing 50 ml of YPD medium (yeast extract 1% w/v, bacteriologic peptone 2% w/v, glucose 2% w/v). pH was adjusted to 4.5, and sterilized (121 °C), during 15 min. The flasks were incubated on a rotary shaker at 30 °C, 250 rpm from 12 to 24 h depending of the yeast strain.
In order to measure the consumption of ATF, total sugar content was determined according to Arrizon et al. (2010) using anthrone: 100 ll of sample (supernatant of the same sample used for enzymatic activity) was mixed with 200 ll of anthrone (200 mg of anthrone in 100 ml of H2SO4), and the mix was shaken and heated for 10 min at 100 °C. The reaction was stopped by immersion in ice for 5 min. The sugar content was obtained by comparing the absorbance of the sample at 625 nm against a standard curve of sucrose (0–100 lg/ml).
2.4. Fructanase induction and fructan fermentation media 2.9. Cell growth and biomass production For fructanase production, the following medium was used; a salt solution (urea 8 g/l, K2HPO4 3 g/l and MgSO4 2 g/l) was adjusted at pH 4.5 and sterilized (121 °C, 15 min). Later an ATF solution (20 g/l) used as a carbon source was sterilized by filtration (0.45 lm) and added to the salt solution to reach a final concentration of 10 g/l of fructans. For ethanol and tequila fermentation, the salt solution was added with ATF solution (200 g/l) to reach a final concentration of 125 g/l of fructans.
Cell growth was determined by cell counting in a Neubauer chamber. Yeast biomass was determined by measuring dry weight. The cellular dry weight was obtained by harvesting the cells from 1 ml of the centrifuged sample used for enzymatic activity, rinsed with the same amount of distilled water, and desiccated at 60 °C until constant weight was obtained. 2.10. Ethanol and volatile compounds production
2.5. Fructanase induction experiments Yeasts were grown on pre-inoculum medium. 2 106 cells/ml were inoculated on 50 ml of fructanase induction medium in 250 ml Erlen-Meyer flasks at 30 °C and 250 rpm (rotary shaker). Samples were taken at 0, 12, 24, 36, 48, 72 and 96 h, to measure the fructanase activity on A. tequilana fructans in order to determine the induction time at which the highest enzymatic activity was detected. The induction time was considered as the propagation time for the inoculums used in the saccharification and fermentation experiments.
Analysis of ethanol and volatile compounds was carried out in a Hewlett–Packard 6890 gas chromatograph (Palo Alto, CA, USA) with a flame ionization detector (FID) equipped with an HP-Innowax PEG column (60 m 0.320 lm). The initial column temperature was 45 °C for 7 min, and was then ramped at 10 °C/min to 160 °C, followed by a 20 °C/min ramp to 220 °C and maintained during 4 min. Injector and detector temperatures were maintained at 250 °C. The injection system consisted in a head-space Hewlett– Packard 7694E. The preparation program and injection sample started with the following conditions: vial temperature at 80 °C, loop temperature at 110 °C, transfer line temperature at 115 °C.
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The cycle time of head space and gas chromatograph 40 min, the vial equilibrium time 5 min, the pressurization time 0.2 min, the filling loop time 0.2 min, the loop equilibrium time 0.5 min, injection time 1 min and finally the agitation time 1 min. The volatile compounds measured in this study were acetaldehyde, ethyl acetate, 1-propanol, isobutyl alcohol 1-butanol, amyl alcohols, ethyl caprate, ethyl lactate, furfural and ethyl caproate (all volatile compounds were Sigma–Aldrich). Quantification was performed according to an external standard method. Ethanol yield was calculated considering the ethanol produced (final ethanol concentration initial ethanol concentration) and the sugar consumed (initial sugar concentration final sugar concentration). The alcoholic efficiency was calculated considering the ethanol yield over the maximum theoretical ethanol yield.
3. Results and discussion 3.1. Induction time of inoculums To perform the simultaneous saccharification and fermentation of A. tequilana fructans (ATF), induction experiments for fructanase production were carried out with different K. marxianus yeasts, in order to find the induction time at which the inoculums have the maximum extracellular fructanase activity, as well as yeast cells for the simultaneous ATF hydrolysis and ethanol production during fermentation. In the induction experiments, it was observed that the induction time where the highest fructanase activity was reached depended on the yeast strain. Half of them (DK4, DV, DU3, DI4, SLP1, OFF1 and DH4) reached the maximum enzymatic activity within (24–48 h), while the strains DV4, DX5, MO6, DF1, DH, DZ5, DA5 and DL of K. marxianus found the maximum activity at 96 h of induction (Fig. 1). The strain DV4 showed the highest fructanase activity, followed by DK4, SLP1 and DU3, while the rest of the yeasts exhibited values lower than the mean value (0.396 U/ ml). The differences observed in the induction time and fructanase activity, could be associated to variations in the capacity of synthesis and secretion of enzymes between the K. marxianus yeasts, these variations depended also on the yeast strain, carbon source (inducer substrate), nitrogen source, oxygenation, mineral composition and fermentation system (Singh and Gill, 2006; Chi et al., 2009; Silva-Santisteban et al., 2009), as well as the catalytic capacity of the secreted fructanases (Zhang et al., 2009; Singh et al., 2007; Treitchel et al., 2009; Arrizon et al., 2011). During induction experiments, cell growth and biomass were determined (data not shown), a great variation was observed in the initial cell population of the different inoculums (100–600 106 cells/ml, representing 1–4.5 g/l of biomass respectively).
3.2. Fructanase activity of inoculums on different substrates Once the induction time was set to develop the inoculums of each K. marxianus strain, the fructanase activity was determined again before they were added to the ATF medium for the simultaneous saccharification and fermentation process. The activity was measured on inulin and sucrose, in order to elucidate the preference of the fructanases for short and large fructans in comparison to the substrate of interest (ATF). Fig. 2 shows the maximum fructanase activity on different substrates for these inoculums. The hydrolytic capacity and the enzymatic preferences on different substrates varied in function of the K. marxianus yeast tested, in general DV4 showed the highest enzymatic activity in all the substrates, followed by SLP1, DH4, DU3 and DH, the rest of the yeast strains showed lower enzymatic activity (Fig. 2). For all the yeast strains, the highest enzymatic activity was observed on sucrose (2–12 U/ml, Fig. 2a), while the lowest was observed on inulin (0.02–0.2 U/ml, Fig. 2b), which is normal for K. marxianus yeasts as they are recognized as an exo-fructanhydrolase producers with a high ratio of the enzymatic activity on sucrose/inulin (Pandey et al., 1999; Singh and Gill, 2006; Arrizon et al., 2011). In the case of fructanase activity on ATF the yeast strain DV4 showed the highest enzymatic activity, followed by SLP1 and DU3 (Fig. 2c). It can be also observed that for most of the yeasts the fructanase activity of the inoculums on ATF was higher (0.05–1.2 U/ml) than inulin (0.02–0.2) (Fig. 2b and c respectively). Therefore, these enzymes showed a substrate preference for ATF, which was previously observed with a purified exo-fructanhydrolase from a K. marxianus strain isolated from fermenting musts of mezcal (Arrizon et al., 2011).
3.3. Sugar consumption and fructanase activity, biomass and ethanol production Regarding ATF consumption during the saccharification and fermentation, even though the low fructanase activity of some inoculums of the K. marxianus strains evaluated (Fig. 2), all of them consumed more than 90% of the initial ATF concentration with different rates. Therefore, the hydrolytic capacity of the extracellular enzymes, as well as the fructose uptake of the different inoculums depends of the yeast strain. As an example, Fig. 3 represents the ATF consumption and fructanase activity only for the five inoculums with the highest sugar consumption and fructanase activity (DV4, DH4, SLP1, DU3 and DH). It can be seen that DV4 and DH4 showed the faster consumption of ATF followed by DH, SLP1 and DU3 (Fig. 3a), while for fructanase activity, the highest activity was observed for the yeast strain DV4, which was increased during
Enzymatic activity (U ml-1)
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Fig. 1. Maximum fructanase activity on Agave tequilana fructans for the extracellular enzymes of the Kluyveromyces marxianus inoculums induced at 24 h (DK4, DV, DU3, DI4), 48 h (SLP1, OFF1, DH4) and 96 h (DV4, DX5, MO6, DF1, DH, DZ5, DA5, DL) of culture.
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Enzymatic activity ( U ml -1)
14
(a)
12 10 8 6 4 2 0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Enzymatic activity (U ml-1)
0.25
(b) (b) 0.20
0.15
0.10
0.05
0.00 DK4
DV
DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1
DH
DZ5 DA5
DL
Enzymatic activity (U ml -1)
1.4
(c)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5
DL
Fig. 2. Maximum fructanase activity on sucrose (a), inulin (b) and Agave tequilana fructans (c) for the extracellular enzymes of Kluyveromyces marxianus inoculums induced at 24 h (DK4, DV, DU3, DI4), 48 h (SLP1, OFF1, DH4) and 96 h (DV4, DX5, MO6, DF1, DH, DZ5, DA5, DL) of culture.
the saccharification and fermentation (Fig. 3b). For the yeast strains DH4 and SLP1 a slighter increase was observed, while the fructanase activity for DH and DU3 remained constant (Fig. 3b). Even though the similarity of the sugar consumption rate between the K. marxianus strains DV4 and DH4, a higher fructanase activity was observed for the strain DV4, thus, it could be possible that the strain DH4 has a higher fructose uptake capacity than DV4. According with the sugar consumption yield for all the K. marxianus strains, the inoculums composed of cells and extracellular enzymes were effective for the simultaneous hydrolysis and sugar consumption during the fermentation process. Biomass production varied for all the yeast from 1 to 5 g/l, as an example Fig. 4 shows the biomass production of the five yeast with the highest sugar consumption rate during the saccharification and fermentation. Regarding ethanol production, the three K. marxianus yeasts (DA5, OFF1 and
DZ5) were the highest producers (Table 1), which exhibited low fructanase activity on the three substrates in their respective inoculums (Figs. 1 and 2). On the contrary, a low ethanol production level was observed for yeast strains with high fructanase activity in the inoculums, such as the DV4 and DH4 yeast strains (Table 1). Recently, it has been found that K. marxianus is an industrial ethanol producer (Lopez-Alvarez et al., 2012; Amaya-Delgado et al., 2013), and as other ethanol producer yeasts, the generated alcohol might modify the composition and rigidity of the membrane (Ramirez-Cordova et al., 2012), which could have effect on the enzyme secretion capacity of yeasts (Singh and Bhermi, 2008). On the other hand, ethanol could have effect also on the catalytic capacity of the extracellular fructanases, as has been observed in intracellular enzymes of S. cerevisiae during alcoholic fermentation (Ogawa et al., 2000; Alexandre et al., 2001; Chandler
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Agave tequilana fructans (g l-1 )
100
(a) 80
60
40
20
0 0
20
40
60
80
Time (h)
Table 1 Ethanol produced, ethanol yield and alcoholic efficiency at the end of the saccharification and fermentation process of Agave tequilana fructans. Yeast
Yp/s (g g1)
Efficiency (%)
Ethanol produced (g/l)
DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5 DL
0.323 ± 0.02 0.263 ± 0.01 0.505 ± 0.02 0.420 ± 0.02 0.491 ± 0.01 0.497 ± 0.02 0.396 ± 0.05 0.324 ± 0.00 0.467 ± 0.01 0.447 ± 0.03 0.418 ± 0.08 0.491 ± 0.03 0.506 ± 0.03 0.499 ± 0.01 0.483 ± 0.05
63.3 ± 4.4 51.6 ± 3.2 99.1 ± 4.8 82.3 ± 4.5 96.3 ± 2.3 97.6 ± 3.4 77.6 ± 9.8 63.5 ± 0.2 91.6 ± 1.4 87.8 ± 6.3 82.0 ± 15.3 96.3 ± 6.0 99.3 ± 6.4 98.0 ± 1.8 94.7 ± 11.1
24.5 ± 1.21 21.5 ± 0.94 47.5 ± 1.64 32.4 ± 1.27 44.9 ± 0.77 49.7 ± 1.22 34.8 ± 3.12 28.7 ± 0.06 37.5 ± 0.42 37.4 ± 1.91 34.5 ± 4.58 46.0 ± 2.04 47.1 ± 2.14 49.9 ± 0.65 45.9 ± 3.81
Enzymatic activity (U ml-1)
30
(b)
25 20 15 10 5 0 0
20
40
60
80
Time (h) Fig. 3. Agave tequilana fructans consumption (a) and fructanase activity (b) during saccharification and fermentation with five Kluyveromyces marxianus yeasts strains; DV4 (d), DH4 (s), SLP1 (.), DU3 (4) and DH (j).
5
Biomass (gl-1)
4 3 2 1 0
0
20
40
60
80
Time (h) Fig. 4. Biomass production during saccharification and fermentation with five Kluyveromyces marxianus yeasts strains; DV4 (d), DH4 (s), SLP1 (.), DU3 (4) and DH (j).
et al., 2004). As the fructanase activity of the K. marxianus strain DV4 was increased during the saccharification and fermentation (Fig. 3b), it could be possible that this K. marxianus strain produce enzymes more tolerant to ethanol than the other strains. Since most of the K. marxianus yeasts strains produce extracellular fructanases (Singh and Gill, 2006; Chi et al., 2009; Arrizon et al., 2012), the role of ethanol on the enzyme secretion capacity and on the hydrolytic activity of the fructanases has to be elucidated for a better understanding of the simultaneous saccharification and
fermentation of fructans by K. marxianus yeasts. The yeast strains SLP1, DZ5, DA5, DU3 and DH, showed high fructanase activity on ATF of their respective inoculums before they were inoculated (Fig. 2), as well as high ethanol concentration at the end of fermentation (Table 1). Therefore, these yeast strains are suitable for simultaneous saccharification and fermentation for bioethanol production from ATF. Comparing the quantity of ethanol obtained in this study (20–50 g/l, Table 1) with other works, it corresponded to 2.5–6.3% v/v respectively, which was higher than the production observed in the simultaneous saccharification and fermentation of inulin from H. tuberosus L. using a K. marxianus yeast (4.1% v/v of ethanol, Negro et al., 2006), but lower than the concentrations reached with a mixture of Kluyveromyces fragilis, S. cerevisiae and Zymomonas mobilis (12% v/v) and with a mixture of Aspergillus niger and S. cerevisiae (20.1% v/v) with this substrate (Szambelan et al., 2004; Nakamura et al., 1996, respectively). Hu et al. (2012) evaluated the simultaneous saccharification and fermentation capacity in an H. tuberosus inulin medium (200 g/l), using a S. cerevisiae and a K. marxianus yeast strains, a higher ethanol concentration was also observed (9.3 and 8.2% v/v respectively). Thus, the yield of simultaneous saccharification and fermentation depends also of the type and concentration of substrate; as well as, the type of microorganism used. Recently, Kim et al. (2013) showed that K. marxianus ferment the mixture of glucose, xylose and fructose to ethanol from whole H. tuberosus biomass hydrolyzates, this is possible because K. marxianus has also the capacity to assimilate xylose as a carbon source (Lane et al., 2011). During the tequila elaboration process a high bagasse and vinasse quantities are generated as byproducts (Espinoza-Escalante et al., 2009). Therefore, it could be also possible the bioethanol production from whole biomass hydrolyzates of A. tequilana using K. marxianus strains. The simultaneous saccharification and fermentation of ATF not only can be used in the production of bioethanol, it is also useful for the industrial tequila process, as it can reduce the energy consumption in the cooking step for fructan hydrolysis with simultaneous ethanol production (Avila-Fernandez et al., 2009; Huitron et al., 2013). Additionally, nowadays the number of industries involved in the extraction of fructans from A. tequilana has been increased, principally to produce ingredients for the food industries (García-Aguirre et al., 2009), thus it could be available in the future for other biotechnological applications. As tequila is an alcoholic beverage, the production of volatile compounds during fermentation is important for the quality of the beverage (Arrizon et al., 2006; Arrizon and Gschaedler, 2007; Arellano et al., 2008; Pinal et al., 2009). Therefore, the volatile compounds were determined at the end of the saccharification and fermentation for all the K. marxianus yeast strains evaluated.
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Table 2 Volatile compounds production at the end of the saccharification/fermentation process for the Kluyveromyces marxianus yeasts tested. Yeast strain
DK4 DV DU3 DI4 SLP1 OFF1 DH4 DV4 DX5 MO6 DF1 DH DZ5 DA5 DL
Volatile compounds (mg l1) Acetaldehyde
Ethyl acetate
Methanol
1-Propanol
Isoamyl acetate
Isobutyl alcohol
1-Butanol
Amyl alcohols
6.8 ± 2.5 12.2 ± 4.1 25.8 ± 4.2 27.1 ± 6.9 27.3 ± 0.4 25.6 ± 0.4 29.6 ± 3.3 37.1 ± 7.8 22.8 ± 6.2 33.0 ± 0.9 25.3 ± 4.9 23.7 ± 1.8 12.6 ± 6.1 33.3 ± 5.5 17.7 ± 3.6
19.1 ± 0.8 38.4 ± 23.5 32.8 ± 5.1 51.9 ± 16.3 58.7 ± 5.2 39.5 ± 9.4 56.1 ± 12.1 21.9 ± 11.2 43.1 ± 10.0 60.5 ± 11.7 58.7 ± 9.4 37.6 ± 15.7 34.7 ± 1.7 49.0 ± 8.1 49.6 ± 21.8
5.3 ± 2.4 3.7 ± 1.2 3.8 ± 1.5 2.6 3.6 ± 1.3 4.3 ± 0.3 3.4 4.3 ± 0.2 4.6 ± 0.8 2.6 ± 0.6 2.4 ± 0.6 3.8 ± 1.1 3.7 ± 0.2 3.8 ± 0.2 4.1 ± 0.4
0 0 6.18 0.38 ± 0.1 0.68 ± 0.4 0.31 ± 0.01 0.45 ± 0.01 0 0.41 ± 0.01 0.5 ± 0.05 0.7 ± 0.3 0.41 0.33 ± 0.01 0.7 ± 0.01 0.4 ± 0.01
37.8 ± 1.4 33.9 ± 1.8 52.1 ± 0.3 49.1 ± 2.8 54 ± 0.3 81.2 ± 2.8 64 ± 1.7 25.7 ± 0.3 48.7 ± 9.7 56.1 ± 1.6 53.4 ± 2.7 64.3 ± 19 70.6 ± 3.7 57.3 ± 2.0 68.4 ± 0.4
49 ± 1.7 27.4 ± 1.4 75 ± 11.3 74.8 ± 12 59.4 ± 8.5 98.8 ± 2 81.4 ± 5.2 21.2 ± 4.5 81 ± 0.6 90.6 ± 2.6 94.3 ± 2.9 65.3 ± 14.9 79.1 ± 13.1 72.7 ± 13.8 73.4 ± 13.5
0 0 1.9 0 0.1 0 0 0 0 0.1 0.1 0 0 0.2 0
2.8 ± 0.02 4 ± 0.7 1.6 ± 0.2 1.7 ± 0.1 1.9 ± 0.1 2.5 ± 0.2 2.2 ± 0.1 2.8 ± 0.2 2 ± 0.15 1.5 ± 0.3 1.2 ± 0.3 2 ± 0.5 2.1 ± 0.2 1.6 ± 0.6 1.6 ± 0.07
3.4. Volatile compounds production From 16 volatile compounds measured only 8 were detected in all the fermentation musts (Table 2). In general, isobutyl alcohol was the highest volatile compound produced by the yeasts, followed by isoamyl acetate, ethyl acetate and acetaldehyde, while lower levels of 1-butanol, 1-propanol and amyl alcohols were observed. Previously Lopez-Alvarez et al. (2012), compared at laboratory and industrial scale a K. marxianus yeast (isolated from the tequila process) against a bakery S. cerevisiae yeast and a higher production of ethanol, amyl alcohols, isobutyl alcohol, phenylethyl alcohol and propanol was observed with respect to the bakery S. cerevisiae yeast strain. Thus, with the exception of isobutyl alcohol, the production of volatile compounds for this K. marxianus yeast was different regarding to the behavior observed for the K. marxianus of the present work. Recently, Amaya-Delgado et al. (2013) evaluated at industrial tequila conditions a K. marxianus and a Pichia kluyveri yeast strains (both isolated from mezcal fermentation) and a S. cerevisiae yeast strain isolated from tequila fermentation, a lower C/N ratio was used than the work of Lopez-Alvarez et al. (2012). The K. marxianus yeast produced less ethanol than the P. kluyveri and S. cerevisiae yeast strains, however it produced more isobutyl alcohol and ethyl acetate than the P. kluyveri and S. cerevisiae yeasts, which is similar to the results of the present work. Therefore, as it can be seen, the origin of the K. marxianus yeast and the C/N ration influenced the volatile compounds production capacity. It can be noticed that a common characteristic observed for all the K. marxianus yeast strains in the tequila process is the production of isobutyl alcohol. It is well known that the higher alcohols are derived from precursors of the degradation or synthesis of amino acids according with the Ehrlich pathway. In particular, isobutyl alcohol is biochemically derived from valine (Hazelwood et al., 2008). Therefore, the biochemical conversion of valine to isobutyl alcohol has to be studied in the future for K. marxianus yeasts. Regarding the capacity of volatile compounds production between the different K. marxianus strains, it can be observed that DH4, followed by MO6, DF1 and OFF1 were in general the highest producers (Table 2). With the exception of OFF1, all of them produced low ethanol (Table 1) but a high biomass level (3– 4.5 g/l). Therefore, the conversion of the carbon source to bioethanol is less effective for these K. marxianus yeasts except the OFF1 strain. On the contrary, DA5, DU3, DZ5, DH and SLP1 produced high yields of ethanol (Table 1), but less volatile compounds (Table 2), as well as biomass (2–2.5 g/l), thus they have a high yield to convert the carbon source in bioethanol (Table 1). Even though OFF1 K. marxianus strain showed low fructanase activity, it has a high capacity to transform the carbon source in ethanol and volatile
compounds with low biomass production, thus this yeast strain is suitable for tequila fermentation. For the next volatile compounds; acetaldehyde, ethyl acetate, 1-propanol, isoamyl acetate, isobutyl alcohol, 1-butanol and amyl alcohols, the yeast strains DV4, MO6, DU3, OFF1, OFF1, DU3 and DV were the higher producers respectively (Table 2). Thus, a high metabolic diversity between the K. marxianus yeasts was observed. 4. Conclusions In spite of the complex structure of ATF, they can be used as a substrate for simultaneous saccharification/fermentation process with K. marxianus for bioethanol production. Most of the K. marxianus inoculums can perform the ATF hydrolysis during the fermentation, nevertheless few reached an alcoholic efficiency higher than 95% and some of them were more suitable only for the saccharification process. Volatile compounds such as isobutyl alcohol was the highest volatile compound produced for most of the yeasts, thus the regulation of isobutyl alcohol production has to be elucidated. The yeast strain OFF1 showed a great potential for tequila fermentation. Acknowledgement We would like to thank the Consejo Nacional de Ciencia y Tecnología CONACYT for the economical support received on the project 181766. References Alexandre, H., Ansanay-Galeote, V., Blonding, S., Dequin, S., 2001. Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae. FEBS Lett. 498, 98–103. Amaya-Delgado, L., Herrera-Lopez, E., Arrizon, J., Arellano-Plaza, M., 2013. Performance evaluation of Pichia kluyveri,Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation. World J. Microbiol. Biotechnol.. http://dx.doi.org/10.1007/s11274-012-1242-8. Arellano, M., Pelayo, C., Ramirez, J., Rodriguez, I., 2008. Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice. J. Ind. Microbiol. Biotechnol. 35, 835–841. Arrizon, J., Fiore, C., Acosta, G., Romano, P., Gschaedler, A., 2006. Fermentation behaviour and volatile compounds production by agave and grape must yeasts in high sugar Agave tequilana and grape must fermentations. Antonie van Leeuwenhoek 89, 181–189. Arrizon, J., Gschaedler, A., 2007. Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration. J. Appl. Microbiol. 102, 1123–1131. Arrizon, J., Morel, S., Gschaedler, A., Monsan, P., 2010. Comparison of the water soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages. Food Chem. 122, 123–130.
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