bioconversion sugar to ethanol

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The Chemical Engineering Journal, 4.3 (1990) Bl - B12 Bl 
Bioconversion of Sugars to Ethanol in an Immobilized Cell Packed Bed 
Bioreactor - Dynamic Response to Perturbations in Process Parameters 
SHARAD K. GUPTA and SUBHASH CHAND* 
Biochemical Engineering Research Centre, Indian Institute of Technology, Delhi Hauz Khas, 
New Delhi - 110 016 (India) 
(Received September 5,1988) 
ABSTRACT 
Saccharomyces cerevisiae cells ionically 
adsorbed onto hexamethylene-diamine- 
treated bagasse (maximum cell loading 
0.410 g cells per gram of carrier, dry basis) 
were used in a packed bed bioreactor at 30 f 
1 “C with sucrose medium (pH 5.0). The 
dynamic behaviour resulting from perturba- 
tions in feed sugar concentration and feed 
flow rate was studied during the continuous 
bioconversion of sugar to ethanol. The time- 
dependent partial differential equations were 
solved numerically by the Crank-Nicolson 
method and the experimental data fit well 
with those computed from the model. 
The RNA and total protein levels and 
alcohol dehydrogenase enzyme activity during 
the transient phase were monitored in a 
continuous stirred tank bioreactor (free cells) 
and their levels provide a reasonable explana- 
tion of the experimentally observed be- 
haviour. 
1. INTRODUCTION 
Most studies on the continuous bioconver- 
sion of carbohydrate feed stocks to ethanol 
using immobilized cells have largely been 
confined to their steady state behaviour [ 1 - 
71. Since actual industrial processes are never 
free from disturbances in the operational 
process parameters, unsteady state behaviour 
assumes significance as the steady state 
analysis is inadequate in furnishing informa- 
tion on the control strategies required for the 
attainment and maintenance of the desired 
operating conditions. In addition, transition 
*Author to whom correspondence should be 
addressed. 
0300-9467/90/$3.50 
from one steady state to another involves 
physiological, metabolic and morphological 
changes that cannot be understood fully from 
the steady state behaviour of the system. 
Only two reports are available on the 
dynamic behaviour of immobilized cell 
systems producing ethanol. Wada et al. [8] 
achieved high ethanol concentration by step- 
wise increase in feed sugar concentration and 
Bajpai and Margaritis [9] in their studies on 
the effect of temperature and pH observed 
that immobilized cells responded faster to 
change in temperature than pH. However, 
both the reports lack theoretical analysis 
and contain only experimental data. The 
present studies were aimed at delineating the 
dynamic response of the immobilized yeast 
cells, both theoretically and experimentally, 
in a packed bed bioreactor following step 
change in operating process parameters, i.e. 
sugar concentration and feed rate, one at a 
time. Similar perturbations were carried out 
using a chemostat (free cells) to provide an 
explanation of the observed dynamic be- 
haviour of the immobilized cell system with 
regard to changes in the physiological state of 
the cells. 
2. MATERIALS AND METHODS 
2.1. Organism and cultivation conditions 
Saccharomyces cerevisiae CBS-5816 was 
obtained from the CBS culture collection, 
Baarni, The Netherlands. The cells were 
cultivated in a 6 1 fermenter (Bioengineering 
AG, Switzerland) for 10 - 12 h at 30 “C! and 
pH 5.0 in a medium with the following 
composition (in g 1-i): sucrose, 20; yeast 
extract, 3; malt extract, 3; peptone, 5. 
@ Elsevier Sequoia/Printed in The Netherlands 
B2 
2.2. Bioconversion medium 
A sucrose medium of the following 
composition was used: sucrose, varied; yeast 
extract, 3.0 g 1-l; MgS04- 7H20, 0.5 g 1-l; 
orthophosphoric acid, 0.3 ml 1-l; pH 5.0. 
2.3. Carrier preparation 
The cells were ionically adsorbed onto the 
activated bagasse (pith portion, chipped to a 
size of 1 - 1.5 cm), which was positively 
charged with an aminohexyl group following 
the method described by Porath and 
Fornstedt [lo]. 
2.4. Immobilization 
After harvesting, the yeast cells were 
centrifuged and resuspended in a fresh sterile 
medium to prepare a suspension with a con- 
centration of the order of 50 g 1-l. Aliquots 
(100 ml) of this suspension were transferred 
into Earlenmeyer flasks (500 ml) containing 
2.0 g dried and autoclaved carrier. The flasks 
were then placed in an orbital shaker main- 
tained at 30 “C for 80 min. The cell suspen- 
sion was separated from the carrier by de- 
cantation and the latter was washed with 
sterile citrate buffer (0.05 mM, pH 5.0) to 
remove loosely bound cells. 
2.5. Analytical techniques 
2.5.1. E than01 and sugar concentration 
Formaldehyde solution (32%) (0.1 ml) 
was added to experimental samples followed 
by centrifugation at 3000 rev min-’ for 15 
min. The supernatant was used subsequently 
in the measurements. The ethanol concentra- 
tion was determined in a dual column and 
dual detector (Porapack, 120 mesh size; 
outer diameter, 0.65 cm; length, 2 m) gas 
liquid chromatograph (AIMIL - 5700, Nucon 
Engineers Pvt. Ltd., India). The total sugars 
(after hydrolysing with 2 N HCl solution in a 
boiling water bath for 10 min and neutralizing 
with 1 N NaOH solution) were measured 
using the dinitrosalicylic acid (DNS) method 
[111. 
2.5.2. Total proteins 
Total proteins were estimated by the 
Lowry method [ 121. 
2.5.3. Alcohol dehydrogenase activity 
A known quantity of cells was disrupted 
by sonication (Sonirep 150, MSE) and 
centrifuged after washing twice with 
phosphate buffer (0.01 M, pH 7.5). The 
supernatant was used for the measurement of 
enzyme activity, with the reaction velocity 
determined by the method of Vallee and 
Hoch [13]. 
2.5.4. RNA content 
The RNA content was measured in super- 
natant samples of the culture filtrate of 
disrupted centrifuged cells by the method of 
Webb [14]. 
2.6. Experimental se t-up 
A glass jacketed column (500 ml, 6.2 cm X 
16.5 cm) was used, with the temperature 
maintained at 30 “C by circulating water 
through the jacket; carbon dioxide was 
released through a vent at the top. A 
schematic diagram of the experimental set-up 
is shown in Fig. 1. The steady state system 
was perturbed by introducing disturbances, 
i.e. a change in feed sugar concentration or 
flow rate, one at a time. Media of different 
feed concentrations were prepared and 
connected to the system via an on/off valve. 
Fermented 
mash 
collector 
Fig. 1. Immobilized cell packed bed bioreactor. 
B3 
When the system was running at the steady 
state with one feed sugar concentration it 
was switched over to another feed by opening 
the valve, while at the same time the previous 
feed was turned off. The change in feed flow 
rate was achieved by adjusting the peristaltic 
pump (previously calibrated) and the actual 
flow rate obtained was monitored. 
3. THEORETICAL ANALYSIS 
The following assumptions were made to 
describe the system’s dynamic response: 
(i) there was no influence of carrier packing 
on the reaction except in its contribution to 
the axial mixing; (ii) no temperature gradients 
(radial or longitudinal) existed; (iii) a dynamic 
equilibrium existed between growing and out- 
going cells, i.e. the concentration of active 
cells remained constant. 
A steady state mass balance on the 
substrate over a differential element (Fig. 2) 
in a packed bed system can be written as 
(1) 
In the case of a packed bed bioreactor 
employing immobilized cells producing 
ethanol, a large fraction of the column 
operates under low ethanol concentrations 
and therefore experiences little product 
inhibition. In addition, under the experi- 
mental range of sugar concentrations used, 
substrate inhibition of the bioconversion 
process can be assumed to be insignificant. 
Hence first-order kinetics offer a good 
approximation for the rate of reaction and 
r,’ = kS 
where the kinetic parameter k accounts for 
the complexity of the bioconversion process 
and assumes different values depending on 
the experimental conditions.Clearly, k is 
a ‘lumped’ parameter and its values were 
obtained experimentally by collecting samples 
Fig. 2. An element of the immobilized cell bioreactor. 
at different bed lengths under various oper- 
ating conditions. 
On substituting the reaction rate in eqn. (1) 
(2) 
Equation (2) is subject to the following condi- 
tions. 
Initial conditions 
s = SO(z) att=OSzO (4) 
X3 
-= 
a.2 
0 atz=L, t>O (5) 
So(z) represents the initial substrate con- 
centration profile in the bioreactor. 
Equation (2) can be made dimensionless by 
using the following dimensionless variables 
4 = SISF 
q =zlL 
ut (6) 
.T= - 
L 
K,= kL 
u 
Thus eqn. (2) becomes 
Dc a2@ a# - - -- - -K&z $ 
UL a7j2 a7j 
Defining the Peclet number Pe as uL/D, 
leads to 
I a24 a4 --__-_ 
Pe aq2 a7 
-K,@= 2 
(7) 
(8) 
The Peclet number was obtained experi- 
mentally at different flow rates using a tracer 
input technique [ 161. 
The initial and boundary conditions given 
by eqns. (3) - (5) become 
7 = 0, 4 = GO(n) for 0 0 
WV 
rl = 1, a4 0 
‘arl= 
for 7 > 0 (11) 
B4 
The most common method for the numerical solution of partial differential equations uses finite 
difference procedures. However, due to numerical instability problems, finite difference methods 
are not straightforward. This is especially true for the explicit difference method where very 
small steps in the time direction must be used to reduce errors and to increase stability. In order 
to avoid these difficulties, the implicit method given by Crank and Nicolson (see ref. 17) was 
used. 
The dimensionless bed length q and time 7 are divided into i and j equal increments respec- 
tively, and the partial derivatives in eqn. (8) are replaced by the following Crank- Nicolson 
differential operators 
a24 J_ 
i 
@i - + _ 1. j 24i, j + 4i - 1, j + 4i + 1, j I- + 1 2&, j + 1 + $i - 1, j + 1 
a77* 2 Ar1* 1 
34 @i+l,j-$i-1,j 
_=- 
ar, 2b 
a4 4i,j+l-4i,j 
.._ _ = - 
a7 AT 
(12) 
(13) 
(14) 
Substituting eqns. (12) - (14) into eqn. (8), the following algebraic equation is obtained on 
rearrangement 
@i + 1, j + Oh, j + T$- 1. j + h@i + 1. j + 1 + Oh, j + 1 + 6th - 1. j + 1 = 0 (15) 
where 
a=&-- 1 
2A7) 
/3=6-K,+; 
1 
y=6+ - 
2A77 
&= 1 
2PeAv* 
1 
a=-2F-z 
The initial and boundary conditions 
represented by eqns. (9) - (11) become 
@i, 0 = @L O(V) for 04). After this 
decrease, the productivity began to rise and 
the exit sugar concentration declined. Similar 
behaviour was obtained on disturbing the 
system from SF = 165 to 197 g 1-l and then 
197 to 232 g 1-l (Fig. 5). The final produc- 
tivity obtained was 21.4 g 1-l h-’ at SF = 
232 g 1-l. This productivity is about 26% 
higher than the productivity obtained when 
the system was directly operated with SF = 
232 g 1-l (Fig. 4). This indicates that the same 
magnitude of increase in sugar feed concen- 
tration (up to 232 g 1-l) does not yield the 
same level of productivity as stepwise in- 
creases. This phenomenon is attributed to 
the slow adaptation of immobilized cells to 
increasing levels of sugar concentration. A 
similar increase in ethanol concentration by 
stepwise increase in feed sugar concentration 
has also been observed by Wada et al. [ 81. 
B7 
60- 
I 
- 70- 
SF=232 &,I-' r------------- ----------- 
; 
G 
I 
I 
; I 
z 60 I 25 
2 I 
VI 
e SF =197gr' 
~5O-___------------ 23f 
AT 
01 I I I I I I I I I I I 113 
88 96 1OL 112 120 126 136 144 152 160 168 176 184 
Time I h) 
Fig. 5. Transient response to stepwise increase in feed sugar concentration from 197 to 232 g 1-r at flow rate of 
98 ml h-l: 0, sugar concentration (experimental); -, model; A, productivity; - - -, step change in SF. 
4.2. Feed flow rate (step change) 
A change in feed flow rate imposes a 
change in growth rate on the organism due to 
a change in substrate concentration. By 
changing the feed flow rate (space velocity), 
varying magnitudes of disturbance were 
introduced and the responses were followed 
during the transient phase until the attain- 
ment of the steady state. The original feed 
condition was reintroduced after reaching 
the steady state. At the steady state at a 
feed flow rate of 98 ml h-’ (space velocity = 
0.376 h-i, calculated on the basis of void 
volume excluding carbon dioxide holdup 
volume) with SF = 160 g I-‘, the system was 
disturbed by raising the feed flow rate to 112 
ml h-l (space velocity = 0.469 h-l). The same 
magnitude of perturbation in space velocity 
(0.093 h-l) was imposed on the system when 
operated at a higher feed flow rate (147 - 
171 ml h-l). In the first case, the behaviour 
of the system remained almost unaltered 
(Fig. 6), whereas in the second case the 
system showed a gradual increase in exit sugar 
concentration following a lag period (Fig. 7), 
resulting in a corresponding decrease in 
ethanol concentration. On reintroducing the 
original feed flow rate, an undershoot in exit 
sugar concentration was noticed and produc- 
llZmlh-' :__...__-_‘__-_______~ 
12 
0 6 * 10 12 IL 16 
Time I h I 
Fig. 6. Dynamic response to step change in feed flow 
rate (98 to 112 ml h-r) (SF= 160 g 1-l): 0, sugar 
concentration (experimental); - theoretical; 0, 
ethanol concentration; A, productivky; - - -, step 
change in SF. 
tivity increased slightly. When returned to the 
original feed flow rate, the steady state was 
restored after passing nearly seven working 
volumes through the bioreactor. 
0 L 12 16 20 32 
ime , h 1 
Fig. 7. Dynamic response to step change in feed flow 
rate (147 to 171 ml h-l) (SF = 160 g 1-l): symbols as 
in Fig. 6. 
Further experiments were conducted 
employing a feed sugar concentration of 
150 g 1-r and larger increments in feed flow 
rate were introduced. The system was 
disturbed by raising the feed flow rate from 
52 to 98 and 152 ml h-l (thus increments of 
46 and 100 ml h-l respectively were 
introduced). Following perturbation an over- 
shoot in exit feed sugar concentration was 
observed in both cases before a new steady 
state was attained (Figs. 8 and 9). The 
observed overshoot indicates that the cells 
could not adapt to an increased metabolic 
activity immediately following a sudden 
increase in flow rate. For AF = 46 ml h-l the 
overshoot was maintained for about 14 h 
before exhibiting a gradual decrease to the 
steady state sugar concentration (Fig. 8). In 
the other case (Fig. 9), the exit sugar con- 
centration remained at the increased level for 
about 24 h before decreasing rapidly. 
It can be inferred from the above studies 
that the response time increases with a larger 
increment in the space velocity. The new 
steady state was achieved in 48 h for AF = 
46 ml h-’ (Fig. 8) and 80 h for AF = 100 
ml h-’ (Fig. 9). Table 1 gives the values of the 
parameters used in predicting the time course 
of the.perturbations theoretically. 
4.3. Dynamics of the cell physiological 
behaviour 
To explain the results obtained following 
perturbation, the dynamics of the physiol- 
6 
4 
0 a 16 2L 32 LO LB 56 
Time I hi 
Fig. 8. Dynamic response to step change in feed flow 
rate (52 to 98 ml h-l) (SF = 150 g 1-l): symbols as in 
Fig. 6. 
ogical behaviour of the cells were studied. 
Due to the practical problems associated with 
the measurement of the biochemical content 
of the immobilized cells during the transition 
phase, the physiological studies were carried 
out in a continuous stirred tank bioreactor 
(CSTB) (free cells) with similar perturbations. 
The changes which occur at the cellular level 
in the immobilized cells can be expected to be 
qualitatively similar to those of free cells. 
However, the response times could be dif- 
ferent due to different micro-environmental 
conditions. The CSTB (free cells) running at 
steady state and at a dilution rate D of 0.05 
h-’ was disturbed by a step increase (75 to 
160 g 1-i) in SF. RNA, protein and alcohol 
dehydrogenase levels were measured following 
the perturbation. A sudden increase in sugar 
level causes synthesis of more RNA, protein 
and enzymes to enhance the capability of the 
organism for growth and ethanol formation. 
Following the perturbation time lags of about 
12 h and 2’7 h were observed in stimulating 
RNA and protein synthesis respectively 
(Fig. 10). This time lag is due to the fact that 
when a culture is shifted to an environment 
which will allow an increase in the specific 
growth rate, its immediate response will be 
limited by the RNA level attained during 
B9 
68 
Time ( h I 
Fig. 9. Dynamic response to step change in feed flow rate (52 to 152 ml h-l) (SF = 150 g 1-l): symbols as in 
Fig. 6. 
Fig. 10. Dynamics of cell physiology following step 
change in feed sugar concentration in CSTB (free 
cells) (D = 0.05 h-l). Step increase in SF (75 to 
160 g 1-l): 0, RNA; A, protein; 0, alcohol dehy- 
drogenase. Step decrease in SF (160 to 75 g 1-l): 
0, RNA; A, protein; m, alcohol dehydrogenase. 
growth in the previous environment. With 
time, however, the RNA level of the culture 
will change and approach a value charac- 
teristic of growth in the new environment, 
thereby allowing the specific growth rate and 
product formation rate of the culture to 
change as well. These observations are the 
basis of RNA-limiting theory [18] which is 
supported by Waldron and Lacroute [19] and 
Koplove and Cooney [20]. However, over- 
shoots in RNA, protein and alcohol de- 
hydrogenase were noticed at various times. 
This suggests that the organism generates 
more active material than is needed for the 
final steady state because of the high level of 
available sugar. This explains the overshoots 
and undershoots observed following a step 
change in the feed sugar concentration (Figs. 
3, 4 and 5). The shift-down response from 
SF = 160 to 75 g 1-l was observed to be faster 
(Fig. 10). No lag time was noticed as the cells 
were not required to synthesize additional 
amounts of the various macromolecules. 
The RNA and protein levels responded soon 
after the perturbation and reached the new 
steady state in 42 h as compared with 54 h in 
the shift-up experiment. Excessive synthesis 
of proteins and enzyme(s) at high sugar 
concentration explains the undershoots 
observed following shift-down in feed sugar 
concentration in Figs. 3, 4 and 5. 
The change at the molecular level following 
a step change in dilution rate was studied 
BlO 
01 0: 
01 2 3 G 
NRV 
Fig. 11. Dynamics of cell physiology for the same increase in dilution rate (II = 0.02 h-l) at different dilution 
rates in CSTB (free cells) (SF = 140 g 1-l). D = 0.02 to 0.04 h-‘: 0, RNA; *, protein; 0, alcohol dehydrogenase; 
D = 0.08 to 0.10 h-l: 0, RNA; *, protein; n , alcohol dehydrogenase. 
NRV 
Fig. 12. Effect of a large increase in dilution rate on cell physiology in CSTB (free cells) (SF = 140 g 1-l). D = 
0.02 to 0.06 h-r: 0, RNA; A, protein; 0, alcohol dehydrogenase; D = 0.08 to 0.12 h-l: 0, RNA; A, protein; n , 
alcohol dehydrogenase. 
to substantiate the observed phenomena 
further and the results are presented in Figs. 
11,12 and 13. As can be seen in Fig. 11 there 
was no significant change in the cellular 
components when D was increased from 0.02 
to 0.04 h-l (AD = 0.02 h-l). However, a 6% 
increase in alcohol dehydrogenase activity was 
observed for the same magnitude of shift 
(AD = 0.02 h-l) but at a higher level of dilu- 
tion rate (D = 0.08 h-l). A slight elevation 
in RNA and protein contents was also 
observed. It can be inferred that the molecu- 
lar composition of yeast cells is not affected 
at the lower dilution rate; therefore the 
steady state is achieved faster at the lower 
dilution rate than at the higher dilution rate 
for the same magnitude of change. This 
explains the results of Figs. 6 and ‘7. Larger 
increments in dilution rate caused a greater 
change in intracellular contents (Fig. 12), 
Bll 
0 I 
O 0 
I I I I 
1 2 3 4 
NW 
Fig. 13. Effect of a decrease in dilution rate from D = 0.12 to 0.08 h-l on cell physiology in CSTB (free cells) 
(SF’ 140 g 1-l): 0, RNA; A, protein; 0, alcohol dehydrogenase. 
with cells showing a prolonged lag period in 
responding to the change. On changing the 
dilution rate from D = 0.02 to 0.06 h-l 
and D = 0.08 to 0.12 h-l, the levels of RNA, 
protein and alcohol dehydrogenase increased 
following a lag period. The prolonged lag 
period, the longer time required to reach the 
steady state and the increased productivity 
(Figs. 8 and 9) are substantiated by these 
results. While observing the change in cellular 
components following a reduction in dilution 
rate (Fig. 13), no lag period was found in 
RNA, protein or alcohol dehydrogenase 
levels, but substantial time was taken for the 
cells to reach the steady state level. This 
confirms the observations made in Figs. 6 - 9. 
5. CONCLUSIONS 
Step changes in the feed sugar concentra- 
tion or the feed flow rate of a continuous 
immobilized cell reactor showed overshoots 
and undershoots in exit sugar concentration 
and productivity. The system behaviour was 
observed to be dependent on the magnitude 
of the change and on the operating conditions 
at which the perturbation was introduced. 
The shift-down response was noted to be 
faster than the shift-up response. A stepwise 
increase in feed sugar concentration (120 to 
232 g 1-l) resulted in a 26% increase in 
productivity compared with the system used 
directly at the high feed sugar concentration. 
Moreover, increased productivity was ob- 
served during the transient phase and this 
suggests that if the system was operated 
permanently in a disturbed mode, it could 
yield better productivity. However, overall 
the system was stable as the disturbance 
disappeared after a finite time period. 
The present studies indicate that the 
levels of the molecular components show an 
overshoot (following a step increase in feed 
sugar concentration) and then assume a 
physiological state appropriate to the new 
operating condition; RNA limiting theory is 
confirmed. 
The model proposed adequately describes 
the dynamic response and the results obtained 
could be used as a basis for controlling the 
reactor operation at a desired level. 
ACKNOWLEDGMENTS 
One of the authors (S.K.G.) is grateful to 
the Council of Scientific and Industrial 
Research for the grant of an associateship 
during this investigation. Thanks are also due 
B12 
to the Head, Biochemical Engineering 
Research Centre, Indian Institute of Tech- 
nology, Delhi for providing the facilities for 
the work. 
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APPENDIX A: NOMENCLATURE 
DC 
F 
AF 
k 
& 
L 
NRV 
P 
rs 
se 
SF 
AS 
t 
U 
Y P/s 
z 
dispersion coefficient (cm2 h-l) 
volumetric flow rate (ml h-l) 
magnitude of perturbation in feed flow 
rate (ml h-l) 
a ‘lumped’ parameter (h-l) 
dimensionless kinetic parameter 
total length of the bioreactor (cm) 
number of reactor volumes passed 
through in a continuous stirred tank 
bioreactor 
product (ethanol) concentration (g 1-i) 
rate of substrate consumption (g 1-l) 
h-l) 
exit feed sugar concentration (g 1-l) 
feed sugar concentration (g 1-l) 
magnitude of perturbation in feed 
sugar concentration (g 1-l) 
time 
linear velocity of the medium (cm h-i) 
product yield coefficient (g g-l) 
differential length of the immobilized 
cell bioreactor (cm) 
Greek symbols 
1) dimensionless length variable (z/L) 
dimensionless time variable (u t/L) 
dimensionless substrate variable (S/SF)