Biotechnology Letters Vol 6 No 7 465-470 (1984)

HORIZONTAL REACTOR FOR THE CONTINUOUS PRODUCTION OF ETHANOL BY YEASTS IMMOBILIZED IN PECTIN

Antonio Navarre , Hugo Marangoni, Ignacio MagaSa Plaza I , Danley Callieri 2 C~tedra de Microbiologla Industrial, Facultad de Bioqufmica, Universidad Nacional de Tucum~n

Casilla Correo 90 Suc 2, (4000) Tucum~n, ARGENTINA

SUMMARY. Yeast cells (Saccharomyces cerevX~iae) were immobilized in pectin gel, incubated 12 h at 30°C and then used for the continuous production of ethanol employing a wedge-shaped horizontal reactor and sugar cane molasses as the carbon source. Under steady state conditions the mean residence time was 1.6 h and the volumetric productivity 40 g EtOH/hl. The gas evolved was easily released. Successive batch incubation in a synthetic medium substantially restored the fermentative capacity of the beads already used in the continuous assay.

INTRODUCTION. Much research work, dealing with both continuous and batch production of alcohol (Parisi et al., 1977; Jackson, 1976; Ghose and Tyagi, 1979; Gencer and Mutharasan, 1980) as well as with the influence that different facts, such as the increase in the concentration of microorganisms inside the reactor (Cysewsky and Wilke, 1978; Sitton et al., 1979; Rosario et al., 1979; Garcla and Rolz, 1978; Dawson, 1977; Margaritis and Wilke, 1978; Wick and Popper, 1977; Panchal and Stewart, 1981) exert on production, has already been carried out. In continuous processes, the immobilization of cells in inactive supports has proved useful in maintaining a high cell concentration inside the reactor (Sitton and Gaddy, 1980; Moo-Young et al., 1980; Wada et al., 1980; Williams and Munnecke, 1981; Mc Ghee et al., 1982; Ryu et al., 1982; Tyagy and Ghose, 1982), one of the questions dealt with being an efficient way to cope with the CO 2 gas phase effect (Shiotani and Yaman6, 1981; Navarre, 1978) that occurs during fermentation especially when vertical reactors are used. This paper deals with the continuous production of ethanol from yeasts immobilized in pectin gel in a horizontal reactor designed to reduce the negative influence of gas release. The viability of yeasts, both during and after fermentation, taking into account whether they were immobilized on the surface or in inner gel layers of the beads and using samples taken from different areas of the reactor, was also studied.

MATERIALS AND METHODS. Microorganism: Saccharomyces ce~evisiae CMI-120, from our own collection.

Molasses fermentation medium: Composition in g/l: MgSO4.7H20 , 0.5; yeast extract, 1.5; urea, 1.5; sugar cane molasses with 50% total reducing sugars (including 1.9% non fermentable sugars), 320. The pH was adjusted to 5.0 with H2SO 4 at a 10% concentration.

Synthetic fermentation medium: Composition in g/l: CaCI2, 5.0; MgSO4.7H20 , 0.5; yeast extract, 1.5; urea, 1.5; H2KP04, 1.5; glucose, 160; acetate buffer, 0.2 M; pH 4.5, 300 ml.

TDepartamento de Biotecnologla y Bioingenierfa, Centre de Investigaci6n y de Estudios Avanzados del IPN, M6xico D.F.

2Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Cientificas y T@cnicas (CONICET), Argentina.

4 6 5

Reactor: Horizontal reactors of the same working volume but of different shapes and dimensions relationships were tested. It was thus found, obviously empirically, that the design shown in Fig. 1 seemed to be the most suitable, above all concerning the gas release. It is under run a study aimed to confirm these conclusions through a mathematical analysis.

C

8 ~ B B

T . . ; y ', ',:' ~""- /

U U L ........,,L. ,_~,r..', I ,A ±

, 3 0 c m i

Fig. I. Continuous fermentor design. A, medium inlet; B, gas outlets; C, termometer; D, effluent outlet.

Fermentation assays: Beads of 4 mm diameter, 4% pectin and 20 g/l yeast cells were prepared. 400 g beads and enough molasses fermentation medium to cause the level of the liquid exceed in about 2 cm the upper surface of the bead bed were placed inside the reactor and incubated for about 12 h at 30°C to allow multiplication of the entrapped cells, above all those immobilized near the surface of the beads. Continuous feeding of medium was then started and regulated so as to obtain a mean residence time of 1.6 h. Samples of effluent were collected every 12 h for the determination of ethanol (EtOH), total reducing sugars (RS) and yeast concentration. After 30 days the assay was stopped and samples of beads from the inlet and outlet areas of the reactor were separately collected and allowed to drain dry on filter paper. 5 g of those beads and 50 ml of synthetic medium were placed in a flask and incubated at 30°C. The liquid was removed and replaced by fresh medium every 12 h and both residual glucose and ethanol concentrations, as well as number of cells, were determined in the fermented medium in order to find out if there was a recovering of the fermentative capacity. For the purpose of determining the influence that a complex carbohydrate source such as molasses might have on fermentation, the continuous assay was repeated under the same conditions as above, but using a synthetic medium.

Viability of yeasts according to their position i n the Support: Samples of beads taken from the central area of the reactor were subjected to the following treatment: a) vigorous shaking in a physiological solution in order to shake loose cells located on the surface; b) the outer layer, about 0.5 - 0.8 mm thick, which is clearly visible and easily detachable, was removed by means of dissection instruments; c) the rest of the bead was carefully sliced trying to leave a central cubic portion of about 1.5 mm side. The three fractions obtained from (b) and (c) were dissolved in 5% EDTA solution. The number of living cells in each of the three suspensions was counted with a Neubauer chamber using the methylene blue stain test.

RESULTS. The analysis of samples of liquid taken from different areas along the reactor showed that it possesses characteristics that are similar to those found in plug-flow type reactors, with increasing ethanol concentrations and decreasing sugar concentrations from the medium inlet to the effluent outlet.

4 6 6

z - 8 0 - O z

so . z u o z ~o40"

<-r 30-

I I I I i

5 10 15 20 25 days

~E

m,,,

~_-J

8> m_o

• o • o • o

Fig. 2. The time course of continuous fermentation of sugar cane molasses medium using yeasts immobilized in pectin gel.

Fig. 2 shows that during the first three days EtOH concentration in the effluent increased rapidly until a maximum of 70 g/l. The system then reached the steady state, during which productivity was 40 g EtOH/h liter, estimated on the basis of a liquid working volume of 290 ml. During this period yield was 0.49 g EtOH/g consumed sugars and the fermentable substrate utilization about 90%. After approximately fifteen days without significant variations, the performance of the system began to decrease and 30 days after the beginning of the assay productivity was 40% lower. The variation in the number of yeasts in the effluent followed a similar pattern to that of productivity. The number of immobilized living cells decreased from the surface to the center of the beads and it was highest in the beads located in the inlet area of the reactor. A general substantial decrease of the living population occurred after 22 days of continuous fermentation (Table i).

Continuous o

(da!s)

I0

Percentage of viable yeasts in the beads I

Centre First layer

97

Second layer

9O 88

15 94 82 75

18 90 75 61

22 91 74 59

25 74

45 30

59

42

45

40

Table i. Viabi'lity of the irmnobilized yeasts according to their position in the beads. Beads were taken from the central area of the reactor.

467

Successive batch incubation in a synthetic medium of the beads already

used for the continuous assay ~artially restored the cell population,

and hence the fermentative activity of the beads, even if not in the same degree,depending on the location of the beads in the reactor (Table2)

~ Fermented medium

Batch:N ° % viable

3 J.

72

]lucose (g/liter)

A B

48 95

42 75

43 78

42 75

44 59

Ethanol (g/liter)

A B

41.3 19.3

42.2 26.4

44.6 32.1

44.0 31.4

43.0 36.0

81

yeas ts

49

72

4 ' , - -

5 80 73 Table 2. Effect of successive 12 h-batch incubation on the recovery of the fermentative capacity of beads taken from the inlet (A) and outlet (B) area of the continuous reactor.

When a synthetic medium was used, the evolution of the continuous fermentation process as well as the alcohol production and amount of living yeasts in the effluent during the steady state were only slightly enhanced with respect to the results obtained with molasses. However, loss of activity at the end of the assay was much smaller, reaching only 15%. Because of the plug-flow characteristics of the reactor it was considered useful to see if there was some effect on the performance of the system when the direction of the feeding of medium into the reactor was inverted after 30 days of continuous fermentation. It was then observed that during the following six days the total activity of the system gradually increased until a 7% recovery was reached. After that the performance started to decrease again. It must also be mentioned that during the assays, both with molasses and with a synthetic medium, the CO 2 produced was easily released.

DISCUSSION. The gradual decrease in the performance of the fermentor was in all probability due to the inhibitory effect of ethanol on fermentation as well as to its negative action on the viability of yeasts. These effects were more noticeable in the outlet area, where ethanol concentration was maximal and therefore the low number of living immobilized cells found in the beads taken from this section of the reactor and also the partial recovery of the activity of the system when the medium inlet in the reactor was inverted. The decrease in the amount of living yeasts from the surface to the centre of the beads might have been due to the fact that the diffusion of substrate was not anough to keep them viable. Pirt (1975) uses the name "active layer" for the one formed cells which are placed on the surface and considers that the cells beneath such layer have practically no participation in the fermentation process as a whole. This is why it is desirable to prepare the beads with relatively low concentration on immobilized yeasts and subsequently incubate them to favour the development of the surface layer, as described in this paper. In this way a higher specific productivity is reached than that obtained when working with beads prepared with a high concentration of cells but without incubation. Another factor that may have a bearing on the activity of immobilized yeasts is a high ethanol concentration in the beads. This was not the case in the present assay since the highest alcohol concentration was found in the medium, a fact which implies an easy diffusion of the product from the beads. The greater ethanol concentration in the medium also indicates that there was

468

a poor diffusion of the product towards the inside of the beads. The greater loss of activity observed in the assay with molasses as the carbon source might have been due to the negative influence of osmotic pressure (Panchal and Stewart, 1980 a,b) which in molasses reaches considerable values because of its saline content. In addition to this, there is the more direct and simpler effect of the molasses colloids, which settle on the beads, as was evinced by the progressive colouring that the beads acquired along the assay. The short residence time, the low pH and the considerable ethanol concentration conform an unfavourable environment for most microorganisms, thus indicating the possibility of fermenting unsterilized molasses medium. In order to do this it is necessary to evolve a working method which allows for a continuous incorporation of the required amount of molasses into the fermentor, in order to avoid the development of contaminants, which would certainly be the case if the medium was to remain unsterilized in a separate feeding reservoir. Another important aspect in the lengthening of the working life of yeasts is the incorporation of small amounts of air in the reactor (Nagodawithana et al., 1974; Jones et al., 1981; Nagodawithana and Steinkraus, 1976; Ghose and Tyagi, 1979), which in this case proved to be difficult to do, given the present design of the fermentor used. This is the reason why the modifications that are necessary in order to determine the influence of different air concentrations in the medium on the yield of the process are at present being studied.

ACKNOWLEDGEMENTS. This work was partially supported by grants from Secreta rfa de Ciencia y Tecnologla, Direcci6n Nacional del Az~car and Consejo de Investigaciones de la Universidad Nacional de Tucum~n, Argentina.

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