Ethanol production potential of Saccharomyces fragilis IZ 275 using cheese whey powder solution
Potencial de producción de etanol de Saccharomyces fragilis IZ 275 usando solución de polvo de suero de queso
Geyci de Oliveira Colognesi1, Leandro Freire dos Santos1*, Raul J. H. Castro Gomez2, Salvador Masseguer Roig1, Hélio Hiroshi Suguimoto1
1 North Paraná University, Centre for Postgraduate Studies and Research, Science and Technology of Dairy Products, Londrina – PR, Brazil. *Author for correspondence. (leandrofreire@onda.com.br).
]]> 2 State University of Londrina, Department of Animal Husbandry, Londrina – PR, Brazil.
Received: February, 2014.
Approved: March, 2015.
Abstract
The use of residues from dairy industry is interesting because of the ease of acquisition and a relatively low cost. The objective of this study was to analyze ethanol production by the Saccharomyces fragilis varying the concentration of cheese whey powder (CWP) solution, initial pH and inoculum concentration using a factorial design technique; besides, semi-batch operations to add lactose was investigated. Statistical analysis was performed by ANOVA (p≤0.05). The concentrations of CWP solution and initial pH were significant in the fermentation medium for ethanol production. The optimum conditions were CWP solution 15 %, pH 5.0, inoculum concentration 5 % and a fermentation time of 18 h, and ethanol production reached 7.6 % (v/v). Additionaly, semi-batch operations performed to add lactose also modified the ethanol yield (10.67 % v/v). This would be the first time that a high ethanol production rate was obtained from S. fragilis using cheese whey powder solution and a factorial design technique.
Key words: Ethanol, Saccharomyces fragilis, cheese whey powder solution.
Resumen
]]> El uso de residuos de la industria lechera es interesante por la facilidad de adquisición y costo relativamente bajo. El objetivo de este estudio fue analizar la producción de etanol por Saccharomyces fragilis variando la concentración de solución de suero en polvo de queso (CWP), pH inicial y concentración del inóculo, con una técnica de diseño factorial; además, se investigaron operaciones de semi lotes para agregar la lactosa. El análisis estadístico realizado fue ANOVA (p≤0.05). Las concentraciones de la solución CWP y el pH inicial fueron significativas en la fermentación del medio para la producción de etanol. Las condiciones óptimas fueron 15 % de solución CWP, pH 5.0, concentración de inóculo de 5 % y tiempo de fermentación de 18 h, y la producción de etanol alcanzó 7.6 % (v/v). Además, por operaciones de semi lotes realizadas para agregar lactosa también se modificó la producción de etanol (10.67 % v/v). Esta sería la primera vez que se obtiene una tasa alta de producción de etanol de S. fragilis usando solución de polvo de suero de queso y una técnica de diseño factorial.Palabras clave: etanol, Saccharomyces fragilis, solución de polvo de suero de queso.
Introduction
Dairy production is unique because it is produced every day of the year (Douphrate et al., 2013). The modern dairy industry produces high amounts of residues (wastewater) due to scale-up approaches and inherent manufacturing processes (Krzeminska et al., 2013). Cheese whey (CW), an important residue from dairy industry, is a significant environmental problem because of its high organic matter content (Carvalho et al., 2013). The world production of CW is 108 t year-1 but only a small amount is used by industries for producing value-added compounds such as organic acids, oligonucleotides and biodegradable plastics (Hungaro et al., 2013; Madureira et al., 2013; Sharma and Luzinov, 2012). Thus, using CW for producing green energy or ethanol and CW powder (CWP) would give better results due to its high lactose content (Guo et al. 2010; Koushki et al. 2012). So far, there are only a few reports available about CWP fermentation.
Saccharomyces cerevisiae, the most used yeast for ethanol production and the common distiller's yeast, cannot ferment lactose because it lacks both lactose permease and β-galactosidase, which transport lactose into the cytoplasm and hydrolyze it into glucose and galactose (Zou et al., 2013). Cheese whey contains lactose as a carbon source which would prevent its fermentation by S. cerevisae (Pisponen et al., 2013), although this yeast might use whey/lactose by means of hybrid recombination and co-immobilization strategies (Guo et al., 2012; Kisielewska, 2012; Tahoun et al., 2002). Besides, there are metabolic differences, such as those in the glucose and fructose consumption, in various Saccharomyces wine species (Tronchoni et al., 2009). Thus, the question is whether other Saccharomyces spp. would possess lactose permease and β-galactosidase levels. Singh et al. (2009) isolated and purificated β-galactosidase from S. fragilis, suggesting that this yeast could ferment cheese whey and produce ethanol. Therefore, the objective of the present study was to determine a factorial design to produce ethanol by S. fragilis using CWP solution, and besides to investigate other metabolic aspects.
Materials and Methods
Cheese whey powder was obtained from Central Cooperative of Agro-industrial (Confepar®). For the preparation of CWP solution, an appropriate CWP amount was dissolved in 0.5 - 1 L of distilled water and autoclaved – continuous flow (100 °C, 30 min) and the precipitate (mostly proteins) was removed by filtration. The lactose content in the cleared solution was determined and the solution was diluted with sterile water in order to adjust the initial lactose concentration (8.58 g L-1; pH 4.8) (Silva et al., 2010). When required, glucose and lactose levels were measured with a spectrophotometer using glucose oxidase and methylamine reaction methods (Ohizumi et al., 1989).
Saccharomyces fragilis IZ 275 (SF), obtained from the Centre for Postgraduate Studies and Research – UNOPAR, Brazil, was first cultured on PDA; the colonies were inoculated into 100 mL of CWP solution (250 mL Erlenmeyer flasks) supplemented with yeast extract (12 g L-1), KH2PO4 (5 g L-1), (NH4)2SO4 (6 g L-1) and MgSO4 (0.6 g L-1), pH 5.5, and incubated 24 h at 35 °C by shaking them at 3 g. After incubation growth was suspended in sterile peptone (1 %) and the number of viable cells in suspension was evaluated by colony-forming units (CFU) methodology (Pereira et al., 2013). To carry out the growth kinetic experiments, ethanol, glucose and lactose content were evaluated (Silva et al., 2010). Ethanol levels were measured with the potassium dichromate method (Nair and Zuhara, 2008).
]]> Factorial planning was used during the optimization stage to assess concentration of CWP solution, initial pH and inoculums concentration and, according to Santos et al. (2013), a factorial design can be a strategy to increase the productivity of the desired product. Thus, a complete factorial design 33 with three replications at the central point was used (Table 1) to determine the optimal concentration of CWP solution, initial pH and inoculum concentration for ethanol production by S. fragilis. The temperature and fermentation time was fixed at 35 °C and 18 h (Yong et al., 2013). The generated model was validated using the best conditions obtained. The Statistica 5.0 software by Statsoft was used for planning and analyzing the data (Santos et al., 2013).
Results and Discussion
The ANOVA shows that the ethanol production was influenced (p≤0.05) by CWP solution (Table 1). This result would give a better understanding about the improvement in fermentation performance (ethanol production) and lactose content, which is linearly related to the concentration of CWP solution (Guo et al., 2010; Koushki et al., 2012). Ethanol production was modeled by the equation z=15.26-0.26y-0.15y2-8.92x+0.99x2+1.05xy-0.13yx2+0.2y2x-0.28*2.86y+0.01*2.86y2-0.02*2.86x+3.4. The value of the adjusted R2 was acceptable (0.81) and the lack of fit of the model was not significant (p>0.05). The mathematical model may explain 97 % of the responses because R2 was 0.97.
The ANOVA shows that the ethanol production was influenced (p≤0.05) by CWP solution (Table 1). This result would give a better understanding about the improvement in fermentation performance (ethanol production) and lactose content, which is linearly related to the concentration of CWP solution (Guo et al., 2010; Koushki et al., 2012). Ethanol production was modeled by the equation z=15.26-0.26y-0.15y2-8.92x+0.99x2+1.05xy-0.13yx2+0.2y2x-0.28*2.86y+0.01*2.86y2-0.02*2.86x+3.4. The value of the adjusted R2 was acceptable (0.81) and the lack of fit of the model was not significant (p>0.05). The mathematical model may explain 97 % of the responses because R2 was 0.97.
The optimum conditions for ethanol production (7.6 % v/v) were CWP solution 15 %, pH 5.0, inoculum concentration 5 % and 18 h of fermentation (Figure 1A and Table 1). To confirm the validity of the statistical experimental model, two runs of additional confirmation experiments were carried out. Guo et al. (2010) show that high ethanol production (5.3% v/v) from lactose fermentation was possible using mixed cultures of immobilized cells of Kluyveromyces marxianus and S. cerevisiae. Thus, the monocultures of S. fragilis are very desirable in ethanol fermentation using whey as medium. This result also agrees with those published by Guo et al. (2012), who used CWP and intergeneric fusion technique, and obtained 3.8 % (v/v).
The predictive ability of the model (Figure 1B) shows a good correlation between the observed and the predicted values. The promising results of ethanol production from S. fragilis IZ 275 using cheese whey powder solution were possible only because of the lactose permease and β-galactosidase expression, which facilitates the passage of lactose into the cytoplasm and hydrolysis of lactose into glucose and galactose (Zou et al., 2013); thus, glucose and galactose may follow to glycolytic pathway. In conclusion, this result agrees with those published by Singh et al. (2009) which isolated and purified β-galactosidase from S. fragilis.
Figure 2A shows the kinetic parameters (ethanol, glucose, lactose and CFU) at optimum conditions (CWP 15 %, pH 5.0 and inoculum concentration 5 %) for ethanol production, which increased linearly between 6 and 16 h at a rate of 0.67 % h-1 (R2=0.991). This linear production behaviour is observed using other experimental designs; thus, Xiao et al. (2010) report a linear production of acetoin while increasing the inoculum biomass.
Ethanol production was 8.21 % at 16 h of fermentation, but lactose depletion was detected at 14 h (Figure 2A). These results lead to a key question: Could ethanol production increase with semi-batch process (recharge with lactose)? The results of own study indicate that the ethanol production significantly increased (10.67 % v/v) (Figures 2B, C), when lactose was added (Figure 2E) and, certainly, β-galactosidase activity was increased when the degradation product of lactose was analysed (Figure 2D). Potential benefits of semi-batch operations for ethanol production were also reported by Davis et al., 2006). Besides, this observation agrees with that of Maria and Cocuz (2011), who point out that semi-batch reactors may be advantageous considering the feeding policy.
]]> Conclusion
To our knowledge, this is the first time that a high ethanol production rate was obtained from Saccharomyces fragilis using cheese whey powder solution and a design factorial technique. Besides, semi-batch operations to add lactose modified the ethanol yield. Probably, ethanol production from S. fragilis using cheese whey powder solution cannot replace the available strategies for ethanol production from sugar cane bagasse (traditional Brazilian perspective), but it might complement them.
Acknowledgements
The authors gratefully acknowledge the Coordination of Personnel Improvement – Superior Level (CAPES), North Paraná University, Fundação André Tosello, Confepar and Brazil's National Council for Scientific and Technologic Development (CNPq), Brazil, for financial support and technical assistance.
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