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The effect of repeated temperature shock on baker's. W. J. Groot, R. H. Waldram, R. G. J. M. van der Lans, and K. Ch. A. M. Luyben. Department of Biochemical ...
Appl Microbiol Biotechnol (1992) 37:396-398

App/ A Microb/o/ogy B/otechno/ogy © Springer-Verlag 1992

The effect of repeated temperature shock on baker's yeast W. J. Groot, R. H. Waldram, R. G. J. M. van der Lans, and K. Ch. A. M. Luyben Department of Biochemical Engineering, Julianalaan 67, NL-2628 BC Delft, The Netherlands Received 24 Septemer 1991/Accepted 13 February 1992

Summary. In a recycle system in which evaporation is used for ethanol recovery during fermentation, temperature changes of the broth in the loop will occur. These repeated temperature shocks may have an effect on the microbial ethanol production rate. In this study such repeated temperatue changes were simulated in a recycle system with ethanol production by baker's yeast. The magnitude of the temperature change, as well as the time of exposure to this change were found to have an effect on the ethanol production rate. A temperature increase from 30°C in the fermentor to 35°C or more in the recycle loop led to a significantly lower ethanol concentration in the broth. This effect became negligible at a short exposure time of 18 s of the yeast to the higher temperature.

function of a constant temperature is well documented: see Slapack et al. (1987) for a review on this subject. Also the effect of a non-recurrent heat shock has been extensively investigated, e.g. for pasteurization processes. However it is not known whether a repeated temperature shock will influence the ethanol production rate. In this paper the effect of a repeated heat shock on ethanol production by baker's yeast in a recycle system is described. A continuous culture with a recycle loop is used, comparable to an integrated fermentation/pervaporation process with respect to the overall dilution rate, residence time in the recycle loop and volume of the recycle loop. In the experiments notably a shift-up in temperature in the recycle loop was considered, since higher temperatures are desirable in evaporation methods in order to obtain high mass transfer rates.

Introduction In fermentation equipment, microorganisms can be exposed to several gradients, notably in systems with recycling. A recycle fermentor consists of a reactor and a unit operation in which separation or addition takes place. In ethanol fermentations cell recycling can be found in cell retention systems (e.g. centrifugation, microfiltration, settling) or systems with in-situ ethanol recovery. These recovery methods include evaporation methods, for example the flashferm process (Maiorella et al. 1981), the recycle variant of the well-known vacuferm process. Recently pervaporation has been studied for ethanol recovery (Mulder and Smolders 1986; Gudernatsch et al. 1988; Groot et al. 1991). The use of ethanol-selective membranes offer prospects of cost reduction in the downstream processing of ethanol. In a recycle system with stripping or pervaporation the microorganisms will be exposed to temperature gradients, since the evaporation of ethanol and water withdraws heat from the broth. The physiology of yeast as a

Correspondence to: R. G. J. M. van der Lans

Materials and methods Fermentation. Ethanol fermentations were carried out with the baker's yeast strain Saccharomyces eerevisiae CBS 8066. The fer-

mentation temperature was 30° C. The yeast was kept on slants of GPY (glucose/peptone/yeast extract) agar. The inocula for the fermentations were prepared in a shake flask with a medium of 10 kg m-3 of glucose and 10 kg m-3 of yeast extract paste, and grown overnight in an incubator. The fermentation media contained 150kgm -3 of glucose, 7.5 kg m -3 of yeast extract paste, 5 kg m -3 of NH4C1, 1.0 kg m -3 of KHzPO4, 0.25 kgm -3 of MgSO4"7H20 and 0.20kgm -3 of CaC12.2H20 in demineralized water. The solution containing glucose and CaClz'2H20, and the solution containing the rest of the ingredients of the medium were sterilized apart at 110° C. Fermentation equipment. Figure 1 gives a schematic representa-

tion of the experimental set-up. A fermentor (Applikon, Schiedam, The Netherlands), thermostatted at 30° C, was coupled to a stirred thermostatted flask with a working volume of 0.121. Before entering the flask the broth was warmed up in a heating coil, and after the flask the broth was cooled down to 30° C in a cooling coil. The broth was returned from the flask to the fermentor by overpressure. The effluent was withdrawn from the principal fermentor by suction at the liquid surface. The total fermentation volume was 1.00 1. The pH was controlled at pH 5.0, by adding

397 1 M NaOH. Polypropylene glycol (P2000) was used as antifoam. Air was sparged into the broth at a rate of 0.1 m 3 m -3 min -1. The fermentation equipment was sterilized by autoclaving at ll0°C.

8O kg/m 3

Analytical methods. Biomass concentrations were determined as dry mass by centrifuging a 5 ml sample, washing the pellet with demineralized water and drying overnight at 110 ° C. Glucose concentrations were determined in the supernatant with a Skalar autoanalyser type 5100 (Skalar, Breda, The Netherlands). First the sample was dialysed against a solution containing phenol, then glucose oxidase/peroxidase (Boehringer, Mannheim, FRG) and 4aminophenazone (Merck, Amsterdam, The Netherlands) was added to the dialysate, yielding a coloured solution, the absorption of which was measured at 520 nm. Ethanol concentrations were determined in the supernatant by gas chromatography (Chrompack 437A gas chromatograph, Chrompack, Middelburg, The Netherlands; HaySep Q column, 80-100 mesh; flame ionisation detection; column temperature, 170° C, temperature of the injection port and detector, 200 ° C).

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First the influence of the temperature was investigated for a continuous culture without recycle at 30, 35 and 40 ° C, at a dilution rate of 0.030 h -~. The ethanol and biomass concentrations were 73 and 6.0 kg m - 3 respectively at 30 ° C, and decreased to 57 and 4.5 kg m - 3 , respectively, at 35 ° C. The biomass and product yield, calculated with the help of the glucose conversion, were slightly lower at 35°C compared to 30 ° C. The specific ethanol production rate was slightly higher at 35 ° C. At 40°C the yeast was able to grow in continuous culture; however, a large fluctuation in the biomass and ethanol concentration was observed, with ethanol levels ranging f r o m 15 to 45 kg m - 3. In the experiments with the recycle system the broth was circulated over a loop with a stirred vessel at a different temperature (Fig. 1). Experiments were carried out with an increasing temperature difference at a fixed recirculation flow, and with a decreasing recirculation flow at a fixed temperature difference. The dilution rate based on total volume was 0.050 h - ~ . For a recirculation rate of 6 1 h - a the measured ethanol and biomass concentrations are shown in Fig. 2. At a temperature of 33°C in the loop the ethanol and biomass concentrations were increased slightly. When the temperature difference was increased the ethanol and biomass levels

Fig. 2. Effect of a heat shock on ethanol (Q) and biomass ( + ) production. Fermentation temperature 30 ° C. Recirculation rate, 61 h - 1; overall dilution rate (D) = 0.050 h -

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were significantly lower, and again large fluctuations were found. The concentration range observed is depicted in the figure with an error bar. Figure 3 shows that at low recirculation rates a heat shock f r o m 30 to 35°C yields low ethanol and biomass concentrations and large fluctuations. At a high recirculation rate of 24 1 h - ~ however, the data become comparable to those of a normal continuous culture at 30 ° C

398 Table 1. The effect of temperature shocks in the range 25-30°C

on ethanol production by baker's yeast Temperature shock a

Dilution rate (h-1)

Ethanol concentration (kg m-3)

Biomass concentration (kg m-3)

30--*30° C 30~25 ° C 25~30 ° C

0.061 0.061 0.059

39-42 38-51 28-51

5.6- 6.0 6.0- 7.1 9.6-10.6

Recirculation rate 6 1h - 1 The first value represents the temperature in the fermentor; the second value represents the temperature in the loop

(for the reference culture see Fig. 2). In general the ethanol and biomass yield was lower in the heat-shock experiments compared with a normal continuous culture. It m a y also be of interest that in some heat-shock experiments flocculation of the yeast was induced. Some experiments were carried out with the recycle system with a temperature in the loop or fermentor of 25°C (see Table 1)~ A temperature shock of 5°C at a recirculation rate of 6 1 h - 1 leads to unstable cultures. Nevertheless the average ethanol concentrations in both experiments with a temperature shock of 25 to 30 ° C and 30 to 25°C were comparable to a normal continuous culture at 30°C. The average biomass concentrations, however, were higher.

Discussion

The ethanol concentration in a continuous fermentation of sugar by yeast is determined by several factors: yeast strain, medium and operating conditions. The growth rate and the ethanol production rate of the yeast are limited by product inhibition by ethanol. The combination of both rates results in a certain concentration of biomass and ethanol in the broth. The temperature at which the fermentation is carried out has an effect on both the growth rate and ethanol tolerance of the yeast. For most yeasts the o p t i m u m temperature for growth is about 30 ° C. This was also found in the experiments with and without recycle: compared with a temperature of 30°C the biomass (and ethanol) concentration was lower at 35°C and higher. In general the optimum temperature for ethanol production is slightly higher than that for growth (Rose and Harrison 1987). This difference was observed in most experiments, for example when the fermentor was maintained at 25°C (Table 1).

In the recycling experiments the yeast was repeatedly exposed to a different temperature. This repeated temperature shock resulted in an additional negative effect on the ethanol production rate, compared with simply maintaining the fermentor at a different temperature. In the system without recycling the ethanol production rate was about 20% lower when increasing the temperature f r o m 30 to 35 ° C. In the system with recycling and a temperature shock f r o m 30 to 35 °C, the ethanol production rate was about 50% lower than in the fermentation without recycling at 30 ° C (Fig. 2). For the last case it must be noted that in addition to the decrease in ethanol production, a relatively unstable culture was obtained. This instability as such is important and needs further investigation. A refractory period exists for the yeast to react to a temperature shock. At a recirculation rate of 6 1 h - 1 implying a residence time in the loop of 72 s, marked effects were found. These effects diminished at a residence time in the loop of 18 s. With respect to integrated fermentation/recovery processes with evaporation of ethanol/water mixtures in a recycle loop (Groot et al. 1991), it can be concluded that a repeated temperature shock of more than 3°C should be avoided. On the other hand the phenomenon of the optimum temperature for ethanol production being larger than for growth m a y also profitably be used in a recycle system. More experimental data however are needed for such optimization.

Acknowledgement. This work was financially supported by the IOPm (Innovative Research Programme on membranes issued by the Dutch Ministry of Economic Affairs).

References

Groot W J, Lans RGJM van der, Luyben KChAM (1991) The design of a membrane-based integrated ethanol production process. Appl Biochem Biotechnol 28/29:539-547 Gudernatsch W, Kimmerle K, Stroh N, Chmiel H (1988) Recovery and concentration of high vapour pressure bioproducts by means of controlled membrane separation. J Membr Sci 36 : 331-342 Maiorella B, Wilke CR, Blanch HW (1981) Alcohol production and recovery. Adv Biochem Eng 20:43-92 Mulder MHV, Smolders CA (1986) Continuous ethanol production controlled by membrane processes. Process Biochem 21 : 35-38 Rose AH, Harrison JS (1987) The yeasts, vol 2, chapter 3. Academic Press, New York Slapack GE, Russell I, Stewart GG (1987) Thermophilic microbes in ethanol production. CRC Press, Boca Raton, Fla.