J . Chem. Tech. Biotechnol.

1994, 59, 387-393

Continuous Ethanol Production from Carob

Pod Extract by Immobilized Saccharomyces
cerevisiae in a Packed-Bed Reactor
Triantafyllos Roukas
Department of Food Science and Technology, Aristotelian University of Thessaloniki,
Box 250, 540 06 Thessaloniki, Greece
(Received I5 April 1993; revised version received 16 July 1993; accepted 1 1 October 1993)

Abstract: The continuous production of ethanol from carob pod extract by

immobilized Saccharomyces cerevisiae in a packed-bed reactor has been investigated.
At a substrate concentration of 150 g dm-3, maximum ethanol productivity of
16 g d m - 3 h - ' was obtained at D = 0.4 h - ' with 62.3% of theoretical yield and
83.6%sugars' utilization. At a dilution rate ofO.1 h - I, optimal ethanol productivity
was achieved in the pH range 3.5-5.5, temperature range 30-35°C and initial
'.
sugar concentration of 200 g dm- Maximum ethanol productivity of 24.5 g dm-
h - ' was obtained at D = 0 5 h - ' with 58.8% of theoretical yield and 85% sugars'
utilization when non-sterilized carob pod extract containing 200 g d m - 3 total
sugars was used as feed material. The bioreactor system was operated at a constant
dilution rate of 0.5 h - ' for 30 days without loss of the original immobilized
yeast activity. In this case, the average ethanol productivity, ethanol yield
(% of theoretical) and sugars' utilization were 25 g dm-3 h - ', 58.8% and 85.5%,
respectively.
Key words: ethanol, carob pod extract, Saccharomyces cerevisiae, Ca-alginate
beads, continuous fermentation, packed-bed reactor.

1 INTRODUCTION still being used in animal feeds and several applications

The carob pod is the fruit of the carob tree (Ceratonia
siliqua) which is mainly cultivated in the Mediterranean
countries and in many areas of North America. The
annual production is about 340-400 thousand metric
tons.' Greece is a main producer with an annual harvest
of 21 thousand tons in 1985.2 From the utilization
viewpoint, two parts can be distinguished in the pod: the
kibble or 'locust bean' and the seeds or 'locust kernel
gum', a galactomannan highly valued in the food, textile
and cosmetic i n d ~ s t r i e s .Carob
~ kibble contains the
following, expressed as g 100 g-' of kibble: moisture,
10- 15; total sugars (glucose, fructose, sucrose and
maltose), 40-50; protein, 3-4; pectin, 1-2; cellulose, 7;
hemicellulose, 5; phenolic compounds, 20; fat, 0.5-1.0 and
ash, 2-3."6 Before the 20th century, carob pods were
exclusively used as animal fodder and for human
consumption. In more recent years, most carob pods are
387
J . Chem. Tech. Biotechnol. 0268-2515/94/%09.000 1994 SCI. Printed in Great Britain
of the kibble occur, e.g. in the preparation of antiarrheic
and antiemetic products, pastry baking and as cocoa
substitute.' Because of the high concentration of sugars
in the carob kibble it is important to develop new and
more appropriate uses of these sugars. The simplest way
would be the manufacture of a carob sugar syrup.
Previous work in this area is very scarce.'
As demand for the limited global supply of non-
renewable energy resources increases, the prices of oil
and natural gas keep increasing. As a result, production
of ethanol from renewable carbohydrate materials for use
as an alternative liquid fuel has been attracting worldwide
interest. Recently, research has been directed to the
production of ethanol by immobilized microorganisms
using continuous culture. This immobilization technology,
when compared with the other continuous processes, has
several advantages including prevention of organism
washout, high biomass density, high yield, easy control,
388
low risk of contamination and operation with high
dilution rate.' Continuous ethanol production from
chemically-defined media by Saccharomyces cerevisiae
entrapped in microcarriers and ceramic matrices has been
described The production of ethanol from
cheese whey permeate by co-immobilized B-galactosidase
and S . cereuisiae in Ca-alginate beads using batch and
continuous culture has also been reported.'-' ' Ghose
and Bandyopadhyay,' Linko and Linko' and Bravo
and G o n z a l e ~ 'studied
~ the continuous production of
ethanol from molasses by immobilized S. cerevisiae in
Ca-alginate beads.
Recently, considerable interest has been shown in using
agricultural crops and their products such as corn, barley,
sweet potato, sweet sorghum, apples, oranges, dates,
banana pulp, raisins and Jerusalem artichoke tubers for
fuel ethanol production by immobilized S . cerevisiae,
S . uvarum, Zymomonas mobilis and Kluyveromyces
marxianus using continuous culture.' 5--25 Carob has
many distinct advantages over traditional crops, such as
high carbohydrate yield, good growth in poor soil under
favourable dry farming conditions and high tolerance to
various plant disease^.^ However, very little published
information is available on the utilization of carob pod
as a carbohydrate raw material to produce fuel ethanol.'
The production of ethanol from carob pod extract by
immobilized S . cerevisiae in continuous culture has not
been investigated.
The aim of this investigation was to examine the
potential of carob pod as a feed source for ethanol
production by S . cerevisiae entrapped in Ca-alginate
gel using continuous culture. The effects of various
fermentation parameters such as pH, temperature and
initial sugars' concentration on ethanol production were
also studied.
2 MATERIALS AND METHODS
2.1 Microorganism and substrate
Compressed bakers' yeast, Saccharomyces cerevisiae
(ZANAE Co., Thessaloniki, Greece), was used throughout
this investigation.
Carob pods (cultivar Tylliria) were obtained from the
local produce market. After removing the seeds, kibble
was chopped into small particles ranging from 0.3 to
0.6 cm. The particles (45 g) were mixed with 180 cm3
distilled water (solid/liquid ratio 1:4) and the mixture
shaken on a rotary shaker/incubator (Lab-Line Orbit-
Environ shaker, Lab-Line Instr., Inc.) at 250 rev min-'
for 2 h at 70°C in order to extract the sugars. The extract
was then centrifuged at 4000 x g for 15 min and the pH
of the supernatant adjusted to 4.5 with 1 mol dm-3 HCI.
The solution containing 15% (w/v) sugars was sterilized
at 121°C for 15 min.
T. Roukas
2.2 Immobilization of S. cerevisiue
Compressed bakers' yeast (1 35 g) was suspended in
450 cm3 sterile distilled water and the suspension mixed
with 300cm3 of a 5% (w/v) sterile alginic acid sodium
salt solution (Sigma, A-2033). The mixture was extruded
drop by drop with a peristaltic pump into a sterile 2%
CaCl, solution at room temperature while stirring con-
tinuously. The beads (2-3 mm diameter) were hardened
in the above CaCI, solution for 2 h then washed with
sterile physiological saline to remove excess calcium ions
and untrapped yeast.
2.3 Fermentation conditions
Fermentations were carried out in a glass bioreactor of
4.5 cm diameter and 40 cm height. A shallow layer of
glass beads was placed at the bottom of the column. The
bioreactor was sterilized at 121°C for 15 min. After
cooling, the column was filled with 450 g of Ca-alginate
beads with entrapped yeast (2.5 x lo8 cells per gram of
beads). The top of the reactor was fitted with a plastic
lid with one opening to allow removal of fermentation
broth and to prevent washout of the Ca-alginate beads.
The production medium was continuously pumped in to
the column via a tube passing through the lid to the base
of the reactor. Another tube from the medium surface
was used for channelling of the fermentation broth out
of the reactor. The bioreactor was incubated at 30°C
in a thermostatically-controlled chamber. Continuous
operation was initiated at a dilution rate of 0.1 h-' with
medium of 150 g dm-3 initial sugars' concentration,
pH 4.5.
2.4 Analytical techniques
The yeast concentration entrapped in Ca-alginate beads
was determined by dissolving six beads in 10cm3 of
0.3 rnol dm-3 sodium citrate solution (adjusted to pH 5.0
with 1 rnol dm-3 citric acid) for 20 min with continuous
stirring. The number of yeast cells liberated from the gels
were determined by plate counting on MYGP medium
(concentration (w/v): glucose, 2%; malt extract, 0.5%;
yeast extract, 0 5 % ; peptone, 0.5% and agar, 2%) at 30°C
for 48 h.
When steady-state conditions were reached, ethanol
and residual sugars' concentration were determined. A
100cm3 volume of the fermentation broth was mixed
with 150 cm3 of distilled water and the mixture distilled
at atmospheric presure until 170 cm3 distillate was
collected. Ethanol content was measured with an alcohol
meter (Dujardin-Salleron, France) at 15°C. Residual
sugars as glucose were determined by the 3,5-dinitro-
salicylic acid (DNS) method26 after hydrolysis of sugars
in 1 rnol dm-3 HCl for 30 min at 90°C and neutralization
with 1 mol dm-3 NaOH. The dilution rate (D) was
calculated by dividing the flow rate of the medium by
Continuous ethanol production from carob pod extract
the volume of the reactor. Ethanol productivity was
calculated using the equation: R = D P where D is the
dilution rate (h-') and P is the ethanol concentration
(g dm-3).
3 RESULTS AND DISCUSSION
3.1 Effect of dilution rate
The production medium containing 150 g dm-3 sugars,
pH 4.5, was pumped from the bottom of the reactor at
different dilution rates (01,0.2, 0.3, 0.4 and 0.5 h-') in
order to investigate the influence of dilution rate on the
fermentation kinetic parameters. The dilution rate was
increased stepwise from 0.1 to 0.5 h-' until steady-state
operation was achieved at each rate. Temperature was
maintained at 30°C. The ethanol concentration, ethanol
productivity, ethanol yield (% of theoretical) and sugars'
utilization as a function of dilution rate are presented in
Fig. 1.
The ethanol concentration remained constant as the
dilution rate increased from 0.1 to 0.2 h-', but decreased
as the dilution rate increased further from 0.2 to 0.5 h-'.
A maximum ethanol concentration of 46.4 g dm-3 was
obtained at a dilution rate between 0.1 and 0.2 h-',
while the lowest ethanol concentration (32 g dm-3) was
observed at D = 0.5 h-'. The ethanol productivity
increased significantly with the increase of dilution
rate from 0.1 to 0.4 h - ' and remained constant as the
dilution rate was increased beyond 0 4 h-I. A maximum
ethanol productivity of 16 g dm-3 h-' was achieved
at D = 0.4 h - ' and 83.6% sugars' utilization. At D =
0.1 h ' and 94.60/, sugars' utilization, an ethanol produc-
tivity of 4.64 g d m - j h - ' was obtained. Therefore, the
greatest ethanol productivity was achieved at the highest
dilution rate tested, but the most complete utilization of
sugars occured at the lowest dilution rate. This finding is
0.1 0.2 0.3 0.4 0.5
Dilution rate Ch-'l
Fig. 1. Effect of dilution rate on kinetic parameters of carob
pod extract fermentation by immobilized S . cerevisiae in
continuous culture (150 g d m - 3 initial sugars' concentration;
pH 4.5; 30°C).
389
in agreement with earlier studies.8,21.22The lower
ethanol concentration and hence lower ethanol inhibition
of yeast is an adequate explanation of the increased
volumetric productivity at the higher dilution rates.
The ethanol yield (% of theoretical) increased slightly
with increase of dilution rate up to 0.3 h - ' but decreased
beyond this value. A maximum ethanol yield (68.2% of
theoretical) was achieved at D = 0.3 h-', while at the
highest dilution rate tested (D = 0.5 h - ') the ethanol
yield decreased significantly. Sugars' utilization remained
almost unaffected by dilution rate up to 0.2 h - ', but
decreased significantly as the dilution rate was increased
from 0.2 to 0.5 h-'. The highest sugars' utilization
(94.6%) was achieved at D = 0.1 h-', while the lowest
sugars' utilization (80.8%) was obtained at D = 0.5 h-'.
Dallmann and co-workers'6 who studied the production
of ethanol from apple juice by S . cerevisiae entrapped in
Ca-alginate beads in a packed-bed reactor found that an
ethanol productivity of 6.3 g dm-3 h-' was obtained at
D = 0.1 1 h-', while Rosario and Pamatong" found an
ethanol productivity 15 g dm-3 h-', ethanol yield of 89%
of theoretical and 90% sugars' utilization were obtained
'
at D = 0.28 h - when immobilized S . cerevisiae in
K--carrageenan in a packed-bed reactor was grown in
banana extract containing 126 g d m - 3 total sugars.
Koutinas and KanellakiZ2reported an ethanol produc-
tivity of 8.5 g d m - 3 h-', ethanol yield of 68.5% of
theoretical and sugars' utilization of 90.8% were obtained
when Z . mobilis, immobilized on y-alumina pellets in a
fixed-bed reactor, was grown in raisin extract containing
159 g dm-3 total sugars at D = 0.17 h-'. Margaritis and
B a j ~ a iinvestigated
~~ continuous ethanol production
from Jerusalem artichoke extract (100 g dm-3 total
sugars) using immobilized K . marxianus in Ca-alginate
beads in a packed-bed reactor and found an ethanol
productivity of 22.5 g d m - j h-' at D = 0.5 h-' and 92%
sugars' utilization. At D = 2.9 h - ' and 80% sugars'
utilization, an ethanol productivity of 104 g dm-3 h-'
was achieved. Kim and Rhee,25 who studied the con-
tinuous ethanol production from Jerusalem artichoke
extract (100 g dm-3 total sugars) by co-immobilized
inulinase and 2. mobilis in Ca-alginate beads, found an
'
ethanol productivity of 12.6 g d m P 3h - at D = 0 2 6 h -'
using a packed-bed reactor. Also, maximum ethanol
productivity of 55.1 g d m - 3 h - ' was achieved at D =
1.96 h-' with 55% sugars' utilization. There are several
possible reasons for these differences, including the strain
of organism used, chemical composition of the substrate,
reactor design, the immobilization matrix and, generally,
the conditions under which the fermentation takes place
(pH, temperature, dilution rate, etc.).
3.2 Effect of initial pH
Carob pod extract containing 150 g dm-3 total sugars
at different initial pH values (3.5, 4.5, 5.5 and 6.5) was
continuously added to the reactor at a fixed dilution rate
390
-1
-
100 50 II
-
0
2 80
k! Voi-
-
560
L ?30
a-"
cI 40
0
.-
z
ij 10
T 2o
2
W
0 0 35 4.5 5.5 6.5 . L 25
Initial PH
Fig. 2. Effect of initial pH on kinetic parameters of carob
pod extract fermentation by immobilized S. cereuisiae in
continuous culture (1 50 g dm- initial sugars' concentration;
D = 0.1 h-'; 30°C).
of 0 1 h-'. Each pH level was maintained until steady-
state operation was achieved. Temperature was controlled
at 30°C. Figure 2 shows the ethanol concentration,
ethanol productivity, ethanol yield (% of theoretical) and
the sugars' utilization as a function of initial pH. Ethanol
concentration and productivity remained relatively
constant at pH 3.5-5.5 and differed slightly between
pH 4.5 and 6.5. The highest value of ethanol concentra-
tion (46.4 g dm-3) was obtained at pH 4.5. The ethanol
yield (% of theoretical) and the sugars' utilization were
almost constant at pH values between 3.5 and 6.5.
Maximum ethanol yield (64% of theoretical) and the
sugars' utilization (94.6%) were achieved at pH 4.5. These
results agree with those of Bajpai and Margaritisz7 who
studied the effect of pH on kinetic parameters of
Jerusalem artichoke extract fermentation by K . marxianus
entrapped in Ca-alginate beads in static culture. Buzas
and co-workersZ8 reported that the sugars' utilization
remained practically constant over the pH range 2.5-6.2
when S . cerevisiae entrapped in Ca-alginate beads was
grown in a chemically-defined medium in shake flask
culture.
Generally, the results showed that the optimum pH
range for ethanol production and ethanol productivity
was 3.5-5.5, while the optimum ethanol yield (% of
theoretical) and sugars' utilization were obtained at pH
values between 3.5 and 6.5. This was due to the good
yeast growth over the pH range 3.5-6.5.*'
3.3 Effect of temperature
The bioreactor was maintained at different temperatures
(25, 30, 35 and 40°C) to investigate the effect of
temperature on the fermentation kinetic parameters. The
production medium (pH 4.5) contained 150 g dm-3 total
sugars. The reactor was operated at a constant dilution
rate of 0 1 h-I. Each temperature was maintained until
T. Roukas
30 35 40 0
Temperature ("C)
Fig. 3. Effect of temperature on kinetic parameters of carob
pod extract fermentation by immobilized S. cereuisiae in
continuous culture (150 g dm- initial sugars' concentration;
r. A I-, - x * "
1
u = V'I n -;p n WJ).
I,
steady-state operation was achieved. The results are
presented in Fig. 3.
Ethanol concentration, ethanol productivity and
ethanol yield (% of theoretical) increased from 43.5 g
dm-3, 4.35 g d m - 3 h-' and 60.2% to 46.4 g dm-3,
4.64 g d m P 3h-' and 64%, respectively, as the fermenta-
tion temperature increased from 25 to 30°C. The above
parameters remained constant in the temperature range
30-35°C and decreased as the temperature increased
beyond 35°C. This was probably due to the decrease in
the number of viable yeast cells at high temperatures.
Rosa and co-workers" reported that yeast death in
the presence of ethanol at high temperatures was caused
by the enhancement by ethanol of the thermosensitivity
of membranes associated with the thermal death sites,
while Bajpai and Margaritisz7 suggested that high
temperatures caused denaturation of the enzyme system
of K . marxianus. The above results are in agreement with
an earlier study conducted by Rousseau and co-workers3'
who studied the effect of temperature on fermentation
kinetics of waste sulphite liquor by S . cerevisiae in
submerged culture. However, Bajpai and Margaritisz7
found that the ethanol concentration, the ethanol
yield and the sugars' utilization remained constant
over the temperature range 25545°C. The maximum
ethanol concentration (46.4 g dm-3), ethanol produc-
tivity (4.64 g dm-3 h- ') and ethanol yield (64.3% of
theoretical) were obtained at the temperature range
between 30 and 35°C. Finally, the sugars' utilization was
found to be constant (94.2%) at all temperatures.
3.4 Effect of initial sugars
The carob pod extract was diluted with distilled water
or concentrated at 50°C under vacuum in order to
contain 10, 15, 20 and 25% initial sugars. The pH of the
extract was adjusted to 4-5 with 1 moldm-3 HCl.
Continuous ethanol production from carob pod extract 39 1

I 1
4 100 60 - increased with the increase of initial lactose concentration
from 50 to 150 g dm-3 and decreased above 150 g dm-3.
1
p 4
L
c1
0
45 -
There are several hypotheses for these differences,
including the strain used, the composition of the
‘E
v
substrate, the fermentation system and the conditions
50 rr30
v
- under which the fermentation takes place.
9 -0 As shown in Fig. 4, sugars’ utilization remained
.-
25 6
f
15 - unaffected by initial concentration up to 200 g dmP3,but
0 W decreased significantly as the concentration increased
6 0 L-lkJ---- from 200 to 250 g dm-3. The decreased sugars’ utilization
0 100 150 200 250 0
encountered with the highest concentration was probably
Initial sugars (gdm-3)
due to osmotic effects. Above a critical substrate
Fig. 4. Zffect of initial sugars’ concentration on kinetic concentration, decreased water activity and the onset of
parameters of carob pod extract fermentation by immobilized plasmolysis have been reported to combine to cause a
’;
S. cereuisiae in continuous culture (D = 0.1 h - pH 4.5; 30°C). decrease in the rates of fermentation and ethanol
prod~ction.’~Cultures grown at initial sugars’ con-
centrations of 100, 150, 200 and 250gdm-3 utilized
Samples of the prepared carob pod extract (production 93.5, 94.6, 94.6 and 85% of the sugars, respectively.
medium) were used to investigate the effect of initial
sugars’ concentration on the kinetic parameters. Con-
3.5 Ethanol production from non-sterilized carob pod
tinuous operation with 100 g dm-3 sugars medium was
extract
initiated at D = 0.1 h-’. When steady-state conditions
were reached at this dilution rate, the sugar concentration
Non-sterilized carob pod extract (pH 4.5) containing
of the feed input was increased from 100 to 150, 200 and
200gdm-3 total sugars was also used as substrate for
250 g dm-3, respectively. Each sugars’ concentration
the production of ethanol. The bioreactor was operated
level was maintained until steady-state conditions were
at various dilution rates of 0-1, 0.2, 03, 0 4 and 0-5 h-’
achieved. Temperature was maintained at 30°C.
at 30°C. Each dilution rate was maintained until
Figure 4 shows ethanol concentration, ethanol produc-
steady-state conditions were achieved.
tivity, ethanol yield (% of theoretical) and sugars’
Ethanol concentration, ethanol productivity, ethanol
utilization as a function of initial sugars’ concentration.
yield (% of theoretical) and the sugars’ utilization
Ethanol concentration and ethanol productivity increased
are shown in Fig. 5 as a function of the dilution
significantly with the increase in initial sugars’ concentra-
rate. Ethanol concentration decreased as dilution rate
tion up to 200 g d m - j but decreased beyond this value.
increased from 0.1 to 0 5 h-’, while ethanol produc-
The highest values of ethanol concentration (60 g dm-3)
tivity increased rapidly with the same increase of dilution
and ethanol productivity (6 g dm-3 h-’) were obtained
rate. This finding is in agreement with earlier studies
at an initial sugars’ concentration of 200 g dm-3 and
reported by Nguyen and Shieh’ and Gil and co-
94.6% sugars’ utilization. Bajpai and Margaritis3’ found
workers.8 Ethanol concentration (65 g dm-3) and sugars’
that maximum ethanol productivity (5-6 g dm-3 h-’)
was achieved when K . marxianus entrapped in Ca-
alginate beads was grown in Jerusalem artichoke extract
containing 250 g dm-3 initial sugars in static culture. 75 25
Roukas and c o - w o r k e r ~ ,who
~ ~ studied the effect of
initial sugars’ concentration on kinetic parameters of
I +
pre-hydrolysed whey fermentation by immobilized S .
cereuisiae in Ca-alginate beads, found that a maximum
ethanol productivity (5 g d m T 3h-’) was obtained at an
initial lactose concentration of 150 g dm-3 in shake flask
culture.
The ethanol yield (% of theoretical) decreased with the
increase of initial sugars’ concentration from 100 to
250 g dm-3, initially 67% at an initial sugars’ concentra-
tion of 100 g dm-3 and decreased to 51% as the sugars’
0 0.1 0.2 0.3 0.4 05 0
concentration was increased to 250 g drnp3. In contrast,
Diluhon rate (h-’l
Bajpai and Margaritis31 found that the ethanol yield
Fig. 5. Effect of dilution rate on kinetic parameters of non-
remained almost unaffected by initial sugars’ concentra- sterilized carob pod extract fermentation by immobilized S.
tion up to 250 g dm-3 and declined beyond that, while cereuisiae in continuous culture (200 g dm- initial sugars’
Roukas and co-workers3* reported that ethanol yield concentration; pH 4.5; 30°C).
392 T. Roukas

utilization (95.2%)were optimal at the lowest dilution

rate tested (D = 0.1 h-').
As the dilution rate was increased to 0 5 h-', sugars'
utilization decreased significantly. At a dilution rate of
0.5 h- ', ethanol concentration and sugars' utilization
were 49 g dm-3 and 85%, respectively. Ethanol produc-
tivity values varied from 6.5 g dm-3 h-' at D = 0.1 h - '
to 245 g dm-3 h-' at D = 0.5 h-'. The ethanol yield (%
of theoretical) increased slightly as the dilution rate
increased from 0.1 to 0.3 h-' and decreased beyond this L I I I I 1 1 1 I
value. The maximum ethanol yield (68.6% of theoretical) 0 4 8 12 16 M 24 28 32
was achieved at D = 0.3 h-I. Bravo and G o n ~ a l e z ' ~ Time ( d )
found a maximal ethanol productivity of 7 g dm-3 h-' Fig. 6. Long-term continuous ethanol production from non-
sterilized carob pod extract by immobilized S. cereuisiae
at D = 0.15 h-' when S . cerevisiae entrapped in Ca- (200 g dm-3 initial sugars' concentration; D = 0.5 h - '; pH 4.5;
alginate beads was grown in non-sterilized molasses in 30°C).
a fluidized-bed reactor, while Koutinas and co-workers,*'
who studied the production of ethanol from non-
sterilized raisin extract by S. cereuisiae immobilized on
the general performance of the system was stable and
mineral kissiris in a packed-bed reactor, found a
efficient over the period examined. Moreover, other
maximum ethanol productivity of 3.8 g dm-3 h-' at
factors such as high productivity, ease of maintenance
D = 0.52 h-'. Yamade and F u k u ~ h i m a 'used
~ starchy
and absence of sterilization requirements established the
materials to produce ethanol with a novel immobilized
packed-bed reactor as a potentially useful technology for
glucoamylase/S. cerevisiae bioreactor. Ethanol produc-
industrial production of ethanol.
tivies of 3 g dm-3 h-', 3.7 g d m - j h-' and 2.8 g dm-3
The ethanol concentration remained at 48-52 g dm-3
h - ' at D = 0.05 h-' were found when liquified corn,
during the 30 days' operation of the reactor. Average
barley and sweet potato containing 200 g dm-3 sugars
ethanol productivity, ethanol yield (% of theoretical) and
were used as feed materials, respectively. Mehaia and
sugars'utilization were 25 g dm-3 h-', 58.8% and 85.5%,
Cheryan,' who studied the production of ethanol from
respectively (data not shown). The ability of immobilized
date extract containing 150 g dm-3 total sugars by S .
yeast to produce ethanol for a long time has not been
cerevisiae immobilized in a membrane reactor, found an
explained yet. But may be due to the protection of the
ethanol productivity of 28.8 g d m - j h - ' w'Ith 94%
yeast by the immobilization matrix. Rychtera and
sugars' utilization at D = 0.48 h-'.
c o - ~ o r k e r reported
s~~ that immobilized yeast can retain
Results of the plate counting showed no contamination
enzymes activities for a long time due to the different
of the substrate by other microorganisms. This could be
composition of cells (proteins, lipids, RNA, DNA and
due to the large number of microorganisms destroyed
inorganic substances) compared with free yeast.
during the extraction of sugars from carob pod at 70°C
During the continuous fermentation, the structure
for 2 h, the ethanol produced which inhibited growth of
of the Ca-alginate beads was not destroyed. Data
contaminating microorganisms and the high concentra-
obtained during fermentation showed that the leakage
tion of the inoculum which dominated the existing
of yeast outside the beads was low. The high productivity
microflora. The above results showed that the culture
shows that the carob pod extract contained the necessary
grown in non-sterilized medium gave higher ethanol
nutrients to maintain the growth of yeast and the
concentration, ethanol productivity, ethanol yield and
fermentation process without any supplementation.
sugars' utilization than those grown in sterilized medium
In conclusion, our results showed that continuous
under the same fermentation conditions. Moreover, the
production of ethanol from carob pod extract by
production of ethanol from non-sterilized carob pod
immobilized S. cereuisiae is very promising in view of the
extract has the advantages of savings in equipment and
high ethanol productivity obtained at relatively high
energy cost.
sugars' utilization combined with excellent bioreactor
stability.
3.6 Long-term continuous ethanol production

In order to study the operational stability of the

immobilized bioreactor, the system was run at a constant
dilution rate of 0.5 h-' continuously for 30 days.
Non-sterilized carob pod extract (pH 4.5) containing
200 g dm-3 total sugars was used as production medium.
Temperature was maintained at 30°C. Figure 6 shows
the ethanol concentration as a function of time. As shown,
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Wiss. u.-Techno[., 21 (1988) 108-12.
2. Statistical Yearbook of Greece. National Statistical Service
of Greece, Athens, 1988.
Continuous ethanol production from carob pod extract
3. Davies, W. N., Orphanos, P. I. & Papaconstantinou,
J., Chemical composition of developing carob pod. J .
Sci. Food Agric., 22 (1971) 83-6.
4. Binder, R. J., Coit, J. E., Williams, K. T. & Brekke, J. E.,
Carob varieties and composition. Food Technol., 13 (1959)
2 13-6.
5. Calixto, F. S. & Canellas, J., Components of nutritional
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33 (1982) 1319-23.
6. Canellas, J., Pou, J. & Mulet, A., Protein enrichment of
carob pod kibbles after sugar extraction. Lebensm.- Wiss.
u.-Techno!., 22 (1989) 73-7.
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