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.

465
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 UL ........,,L. ,_~,r..', I ,A

, 30cm 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.

466
 z-80-
Oz
~E

m,,,
so.
zu
oz
~o40" ~_-J
<-r 30-
8>
m_o
• o
• o
• o
I I I I i
5 10 15 20 25 days
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 Percentage of viable yeasts in the beads I

o

(da!s) First layer Second layer Centre

I0 97 9O 88
15 94 82 75
18 90 75 61
22 91 74 59
25 74 59 45
30 45 42 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 ° ]lucose (g/liter) Ethanol (g/liter) % viable yeas ts
A B A B

48 95 41.3 19.3 72 49
42 75 42.2 26.4
3 J.
43 78 44.6 32.1 81 72

4 ' 42 75 44.0 31.4 , - -

5 44 59 43.0 36.0 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.

REFERENCES
Cysewsky, R.G., Wilke, C.R. (1978) Process design and economic studies of
alternative fermentation methods for the production of ethanol. Biotechnol
Bioeng 20: 1421-1444.
Dawson, P.S.S. (1977) Continuous fermentation. Annu Rep Ferment Process i,
Perlman, D. (ed), Academic Press, New York, pp 73-93
Garcla, R., Rolz, C. (1978) Desarrollo de tecnologfa para producir etanol
para carburante a partir de la ca~a de az~car. I. Evaluaci~n de la tecno-
logla tradicional. ICAITI, Divisi6n Investigaciones Aplicadas, Guatemala
Gencer, M.A., Mutharasan, R. (1980) Ethanol fermentation in a yeast immobilized
Column fermentor. Adv Biotechnol, vol I. Moo-Young,M., Robinson, C.W.,
Vezina, C. (eds) Pergamon Press, London, pp 627-633
Ghose, T.K., Tyagi R.D. (1979) Rapid ethanol fermentation of cellulose
hydrolysate. I. Batch versus continuous systems. Biotechnol Bioeng 21:
1387-1400
Jackson, E.A. (1976) Brazil's national alcohol program. Process Biochem Ii
(5): 29-30
Jones, R.P., Pamment, N, Greenfield, P.F. (1981) Alcohol fermentation by yeasts
The effect of environmental and other variables. Process Biochem 42-49
Margaritis, A., Wilke, C.R. (1978) ~le rotor fermentor. II. Application to
ethanol fermentation. Biotechnol Bioeng 20:727-753
Mc Ghee, J.E., Julian, G.S.T., Detroy, R.W., Bothast, R.J. (1982) Ethanol
production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum
and Zymomonas mobilis. Biotechnol Bioeng 24: 1155-1163.
Moo-Young, M., Lamptey, J., Robinson, C.W.(1980) Immobilization of yeasts
cells on various supports for ethanol production. Biotechnol Lett 2:541-548
Nagodawithana, T.W., Castellano, C., Steinkraus, K.H. (1974) Effect of
dissolved oxygen, temperature, initial cell count and sugar concentration
on the viability of Saccharomyces cere~isiae in rapid fermentations.
Appl Microbiol 28 (3): 383-391.

469
Nagodawithana, T.W., Steinkraus, K.H. (1976) Influence of ethanol production
and accumulation on the viability of Saccharomyces cerevisiae in "rapid
fermentation". Appl and Environ Microbiol 31 (2): 158-162
Navarro, A.R., Rubio, M.C., Callieri, D~A.S. (1983) Production of ethanol
by yeasts immobilized in pectin. Eur J Appl Microbiol Biotechnol 17:
148-151
Navarro, J.M. (1978) Production d'ethanol par cellules adsorbees. SMT
Coloque. Tolouse, France, 211-217.
Panchal, C.J., Stewart, G.G. (1980a) The effect of osmotic pressure on the
production and excretion of ethanol and glycerol by a brewing yeast
strain. J Inst Brew 86:20]-210
Panchal, C.J., Stewart, G.G. (1980b) Regulatory factors in alcohols
fermentations. Adv in Biotechnol. In: Stewart, G.G., Russell (eds) Fifth
international yeast symposium: 3-8
Panchal, C.J., Stewart, G.G. (1981) Ethanol production by a highly
flocculent brewing yeast strain. Dev Ind Microbiol 22, Underkofler LA
Wulf ML (eds) 711-717
Parisi, F., del Borghi, M, Ferraiolo, G. (1977) Kinetic considerations and
diluting factor for alcoholic fermentation of beet molasses. Alcohol
Ind Res 163-169
Pirt, S.J. (1975) Principles of microbe and cell cultivation. Blackwell
Scientific Publications, Halsted Press, New York, p 223-233.
Rosario, E.J., Lee, K.J., Rogers, P.L. (1979) Kinetics of alcohol fermenta-
tion at high yeast levels. Biotechnol Bioeng 21:1477-1482
Ryu, Y.W., Navarro, J.M., Durand, G. (1982) Comparative study of ethanol
production by an immobilized yeast in a tubular reactor and in a
multistage reactor. Eur J Appl Microbiol Biotechnol 15: 1-8
Shiotani, T, Yamaha, T. (1981) A horizontal packed-bed bioreactor to
reduce CO 2 gas holdup in the continuous production of ethanol by
immobilized yeast cells. Eur J Appl Microbiol Biotechnol 13:96-101
Sitton, O.C., Foutch, G.L., Book, N.L., Gaddy, J.L. (1979) Ethanol from
agricultural residues. Process Biochem 14(9): 7-i0
Sitton, O.C., Gaddy, J.L. (1980) Ethanol production in an immobilized cell
reactor. Biotechnol Bioeng 22: 1735-1748
Tyagi, R.D., Ghose, T.K. (1982) Studies on immobilized Saccharomyces
cerevisiae. Analysis of continuous rapid ethanol fermentation in
immobilized cell reactor. Biotechnol Bioeng 24:781-795
Wada, M., Kato, J, Chibata, I. (1980) Continuous production of ethanol
using immobilized growing yeast cells. Eur J Appl Microbiol Biotechnol
i0:275-287
Wick, E., Popper, K. (1977) Continuous fermentation in slant tubes.
Biotechnol Bioeng 19:235-246
Williams, D., Munnercke, D.M. (1981) The production of ethanol by
immobilized yeast cells. Biotechnol Bioeng 23: 1813-1825

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