ÁREA: Bioquímica e Biotecnologia
TÍTULO: PROCESS OPTIMIZATION AND THE EFFECTS OF LACTIC ACID ON THE ETHANOL PRODUCED BY SACCHAROMYCES CEREVISIAE
AUTORES: OLIVEIRA, K. F. (ICB-USP) ; SOUZA, C. S. (ICB-USP) ; CAPELA, M. V. (IQ-UNESP) ; TOGNOLLI, J. O. (IQ-UNESP) ; LALUCE C (IQ-UNESP)
RESUMO: Effects of increasing lactic acid concentrations on viability and ethanol formation by strain 63M at 34°C were studied using a 2^3 full central composite design. Aiming at retaining a maximal viability and a maximal ethanol produced at the end of the fermentation, the MINITAB 14.0 gave the following optimized values: 0.65 g/L lactic acid, 200 g/L initial sucrose and inoculum of 40 g/L. Under the given condition, the viability was 86.4% and the ethanol produced was 72.1 g/L. Minimization of the effects of lactic acid was studied at 34°C in pulse fed-batch cultures in the medium having 40g/L inoculum, 200g/L sucrose and 0 - 4.0g/L lactic acid, with initial pH restored to 4.5. The yeast cells were able to tolerate 3.0g/L lactic acid as shown by 87.0% viability and 95.6g/L ethanol.
PALAVRAS CHAVES: lactic acid, fermentation, s. cerevisiae
INTRODUÇÃO: Lactic acid is produced by lactic-acid bacteria and this organism is almost an inevitable contamination in industrial fermentations. Increases in ethanol productivity (BAYROCK & INGLEDEW, 2004) and reduced fermentation capacity (NARENDRANATH et al, 2001) have been reported for yeast cells resistant to lactic acid. Recently, it was shown that lactic acid does not completely inhibit ethanol production by yeasts in corn mashes but decreases in ethanol productivity were observed (GRAVES et al, 2006). Viability of the yeast population cells can not be significantly changed in order to allow a long lasting succession of fermentation cycles with cell reuse. The optimization of the fermentation process in the presence of lactic acid has not yet been tried with the aim of avoiding losses in viability with gains in ethanol yield. Optimizing an industrial process with respect to ethanol concentration, productivity and yield in the presence of lactic acid require the quantification of the dynamic behaviour of the yeast population at very high ethanol concentrations and this is not a simple task under continuous culture conditions (ALFENORE et al, 2004). It is easier to define optimal monitoring strategies for a fermentation process when the extreme limits of the yeast’s tolerance to ethanol and lactic acid are previously determined. In the present work, the determination of extreme limits of the yeast’s tolerance to lactic acid, sugar concentrations and high inoculum showed that it is possible to obtain high ethanol yields in short fermentation periods at 34°C and in the presence of lactic acid. A comparison between simple batch and pulse-fed batch culture was also made to minimize the effects of added lactic acid (0-4g/L) on ethanol production while retaining high viability.
MATERIAL E MÉTODOS: - Yeast strain: strain 63M (LALUCE et al., 2002).
- Synthetic medium: The medium used was that described by THOMAS et al. (1998) containing sucrose as carbon source. Variations were made: a) added lactic acid=0-4.0 g/L, c) sucrose = 100-200 g/L.
- Inoculum propagation: overnight growth on the same synthetic medium described above containing 10% sucrose and 2% yeast extract.
- Batch fermentation: carried out for optimization studies in agitated (100 rpm) Erlenmeyer flasks containing 100 mL of synthetic medium at pH 4.5 adjusted before inoculation and lactic acid addition.
-Fed-batch fermentation: A 200 mL mini-reactor was operated as follows: a) a 2-fold concentrated synthetic medium without sucrose (50 mL) at pH 4.5 and a concentrated yeast cream (4 g/25mL, dry weight); b) Addition of decreasing pulses (first two-hour period) of sucrose (800g/L) every hour: 10mL, 8mL and 7mL; c) addition of a volume of lactic acid solution (85%, v/v) after the addition of the first sucrose pulse; d) restoring of the pH to 4.5 by adding 3 mol/L NaOH solution; d) final working volume of 100 mL at 34°C.
- Analytical methods: a) cell viability was determined by using the metyhylene blue method (LEE et al., 1981); b) residual sugar using the 3.5-dinitrosalicylic acid method (MILLER, 1959); c) ethanol using a gas chromatograph; c) cell biomass was washed and dried at 105°C until constant weight.
- Experimental design method and statistical analysis involving the following: a) Response surface design: 2^3 full central composite design; b) Independent variables: temperature, initial sucrose and inoculum concentrations; c) Responses: ethanol and viability; d) Software: MINITAB 14 and STATISTICA 6.0.
RESULTADOS E DISCUSSÃO: Coefficient of determination (R2) for ethanol was 0.968 and for viability was 0.845. This indicates that the models can explain 96.8% of the variability for ethanol production response and 84.5% for viability. The quadratic model for ethanol showed strong quadratic effects of inoculum and sucrose on ethanol production. Nevertheless, the linear model for viability showed a strong linear effect of lactic acid and inoculum. Based on these statistical models, the optimal conditions to obtain 80.0g/L ethanol production (maximal response) and 80.4% viability after 4h fermentation at 34°C were: 0.65g/L added lactic acid, 200g/L sucrose and 40g/L inoculum.
In order to minimize the effects of high concentrations of lactic acid, studies were carried out in batch and fed-batch cultures. Without pH adjustment of the batch cultures, activation of the fermentation was observed at concentrations of lactic acid ≤ 0.2g/L. A beneficial effect of lactic acid on ethanol production was mentioned by THOMSSOM and LARSSON (2006). The lowest effects of added lactic acid on ethanol production (g/L) were observed for the control experiment in pulse-fed cultures and simple batch cultures when the initial pH was adjusted to 4.5. Nevertheless, addition of lactic acid led to decreases in ethanol yields, particularly in simple batch cultures when the pH was readjusted.
Taking into account the control experiment without lactic acid, the lowest drops in pH during fermentation were observed in the simple batch culture rather than in the fed-batch culture. Nevertheless, the increases in pH in the presence of lactic acid, due to its buffering capacity at increasing concentrations, were greater in fed- batch cultures.
CONCLUSÕES: In presence of 0.65g/L lactic acid, the experimental validation carried out at 34°C gave the following results: 72.1g/L ethanol, 86.4% viability and constant values of residual sugar and ethanol yields reached after 6h fermentation. The buffering capacity of the increasing concentrations of lactic acid was observed in both batch and fed-batch cultures at initial pH readjusted to 4.5. The readjustment of the initial pH to 4.5 at the beginning of the feeding is essential to minimize the negative effects of pH and lactic acid (tolerance up to 2-3g/L) on levels of ethanol and viability retention.
AGRADECIMENTOS: The authors are grateful by FAPESP (process no. 2005/03681-2).
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