J. Chem. T ech. Biotechnol.

1997, 68, 271276

Kinetics of Lactic Acid Fermentation by

Lactobacillus delbrueckii Grown on Beet Molasses
Jose M. Monteagudo,* Lourdes Rodr guez, Jesusa Rinco n & Juan Fuertes
Department of Chemical Engineering, University of Castilla-La Mancha, Campus Universitario, s/n, 13004
Ciudad Real, Spain
(Received 13 February 1996 ; revised version received 29 July 1996 ; accepted 11 September 1996)

Abstract : The fermentation kinetics for the conversion of beet molasses, a valu-
able and economical fermentation substrate, to lactic acid by the homo-
fermentative organism L actobacillus delbrueckii C.E.C.T. 286 have been studied
at controlled pH and temperature under anaerobic batch conditions. An inhibi-
tory eect of lactic acid on fermentation of beet molasses has been found. The
bacterium was able to produce lactic acid even after growth ceased. A kinetic
model for the fermentation is proposed. From this model, the maximum allow-
able lactic acid concentration above which growth stops and the lactic acid level
above which bacteria stop producing lactic acid were found to be 45 g dm~3 and
57 g dm~3, respectively.

Key words : Lactic acid, fermentation kinetics, beet molasses, L actobacillus del-
brueckii

NOTATION k Specic growth rate (h~1)

k Maximum specic growth rate (h~1)
max
A Rate constant (g lactic acid g~1
biomass) 1 INTRODUCTION
B Rate constant (g lactic acid h~1)
K Monod constant (g dm~3) Lactic acid is an industrially important product with a
s
m Coefficient of maintenance (g large market due to its attractive properties. For
sucrose h~1 g~1 biomass) example, the acid and salts are preferred to other acids
P Lactic acid concentration (g dm~3) in the food industry because they do not dominate
P Lactic acid concentration above other avors and also act as preservers. Furthermore,
max
which bacteria do not grow the possibility of directly converting lactic acid to
(g dm~3) acrylic acid1 has also turned lactic acid into an impor-
P@ Lactic acid concentration above tant raw material for the chemical industry.
max
which bacteria cease lactic acid pro- Rened sucrose, although expensive, is the substrate
duction (g dm~3) most commonly used for producing lactic acid by fer-
S Substrate concentration (g dm~3) mentation.2 However, production costs could be
t Time (h) reduced if sucrose from beet molasses was used instead,
X Biomass concentration (g dm~3) especially if the microorganism could produce lactic
Y Product yield on the utilized sub- acid directly from molasses, a valuable and economical
P@S
strate (g lactic acid g~1 sucrose) fermentation substrate as a by-product of sugar manu-
Y Product yield on the formed facture. As reported previously,3,4 L actobacillus del-
P@X
biomass (g lactic acid g~1 biomass) brueckii C.E.C.T 286 is one of the organisms which
Y Biomass yield on the utilized sub- carries out the fermentation of molasses into lactic acid.
X@S
strate (g biomass g~1 sucrose) Although the production of lactic acid from beet
molasses has been studied by several groups,3h7 the
* To whom correspondence should be addressed. development of kinetic models for the design and
271
J. Chem. T ech. Biotechnol. 0268-2575/97/$09.00 ( 1997 SCI. Printed in Great Britain
272
Fig. 1. Schematic diagram of fermentation system used for the
production of lactic acid by L actobacillus delbrueckii from
beet molasses.
control of biochemical reactors has only been treated in
one previous work,4 probably due to the complexity of
the process. Nevertheless, since the study of fermenta-
tion rates can be very useful for the design and control
of both continuous and batch systems, in this paper the
dynamic state of the batch fermentation of beet molas-
ses has been expressed by three kinetic equations rep-
resenting the concentrations of biomass, lactic acid and
substrate in the batch culture of L . delbrueckii on beet
molasses. To formulate these equations the major ele-
mentary mechanisms of the process were taken into
account.
In order to obtain the kinetic parameters of the
model equations, batch fermentation experiments were
performed at controlled pH and temperature under
anaerobic batch conditions. Then, the experimental
data were numerically analyzed and the kinetic param-
eters of the proposed equations evaluated. Although the
mathematical model developed simplies the true
process, an adequate description of the fermentation
kinetics of this system was provided.
2 MATERIALS AND METHODS
2.1 Microorganism
L actobacillus delbrueckii C.E.C.T. 286, obtained from
the Spanish Type Culture Collection, was used in all the
fermentation experiments. The bacteria were kept
frozen in a 20% (w/v) glycerol solution until ready for
use.
2.2 Media
Beet molasses (50% sucrose w/w) were diluted to obtain
sucrose concentrations between 20 and 120 g dm~3.
The medium was adapted from an optimum nutrient
composition described previously.3
J. M. Monteagudo, L . Rodr guez, J. Rinco n, J. Fuertes
2.3 Inoculum preparation
The preparation of inoculum started with the transfer of
the frozen organisms to a 500 cm3 Erlenmeyer ask
containing 250 cm3 of liquid MRS medium8 containing
(g dm~3) peptone, 10 ; yeast extract, 4 ; dextrose, 20 ;
K HPO , 2 ; CH COONa . 3H O, 5 ; C H O (NH ) H,
2 4 3 2 6 5 7 42
2 ; MgSO . 7H O, 02 ; MnSO . 4H O, 005 and Tween
4 2 4 2
80, 10~3 dm3. The ask was subsequently incubated at
50C for 15 h, the time needed for the microorganism to
reach the exponential growth phase. Then, 150 cm3 of
the growing biomass suspension was injected into the
fermentor containing molasses medium to initiate the
fermentation process. The organism concentration in
the inoculum was 311% (v/v), determined by centrifu-
gation at 3200 rev min~1.
2.4 Batch equipment and procedures
All fermentations were performed under anaerobic con-
ditions in a 5 dm3 stirred jar fermentor, shown sche-
matically in Fig. 1. During the experiments, temperature
and agitation rate were controlled at 49C and
100 rev min~1, respectively. The pH was maintained
constant by automatic addition of 2 mol dm~3 NaOH
solution. The total time of fermentation was approx-
imately 26 h. The bacterial, sucrose and lactic acid con-
centrations were followed during the course of the
fermentation. Samples were withdrawn at approx-
imately 30 min intervals and analyzed immediately.
2.5 Analytical methods
Biomass was measured by constructing a calibration
curve of optical density as a function of dry mass. Dry
mass was determined by ltration of a suitable volume
(410 cm3, depending on the optical density) through a
045 km pore size membrane lter (Millipore, Bedford,
MA, USA), washing with distilled water, drying at
100C for 24 h and weighing. The optical density was
measured on a Spectronic 20 D spectrophotometer at
620 nm. At the wavelength used the medium was found
to have 100% transmission so changes in concentration
did not aect optical density readings in the late stages
of the fermentation.
Sucrose concentrations were measured using a Gilson
isocratic High Performance Liquid Chromatography
System provided with an HPLC column for sugars
(Spheri-5 Amino, Phase Sep, Connecticut, USA) and
using CH CNH O (77 : 23) as eluent.
3 2
Lactic acid concentration was measured using the
same HPLC System but with a column for organic
acids (Polipore H, Phase Sep, Conneticut, USA) using
H SO (015 mol dm~3) as eluent. Acid production was
2 4
also measured by the amount of base (NaOH) added on
demand to maintain the pH constant.
Kinetics of lactic acid fermentation by L. delbrueckii
3 MODEL DEVELOPMENT
Unstructured Batch Growth Models9 describe that the
rate of increase in biomass is a function of the biomass
only. Thus,
dX/dt \ f (X) (1)
One of the simpler models belonging to the general
form given by eqn (1) is Malthus law,9 which uses
f (X) \ kX (2)
where k is a constant. Thus,
dX/dt \ kX (3)
The specic growth rate, k, is usually expressed as a
function of the limiting substrate concentration, S, by a
Monod-type relationship :9
k \ k [S/(K ] S)] (4)
max S max
The Monod equation only applies to the growth
phase and in the present case production of lactic acid
diverts substrate and inhibition restricts growth. There-
fore, eqn (3) must be extended to include the lactic acid
concentration P, i.e.
dX/dt \ kX(1 [ P/P ) (5)
max
where k is a function of both substrate and product
concentrations.
Equation (5) predicts a continuous decrease of the
growth rate as the product concentration rises. Further-
more, growth ceases at nite product concentration,
P .
max
In relation to the kinetics of product formation, the
simplest kinetics arise when there is a simple stoichio-
metric connection between product formation and sub-
strate utilization or growth. In this last case, the
product formation rate, dP/dt, can be written as :
dP/dt \ Y dX/dt (6)
P@X
The alcohol fermentation is an example of this class.
Such product formation kinetics are sometimes called
growth-associated.
In many fermentations, especially those involving sec-
ondary metabolites, signicant product formation does
not occur until relatively late in a batch cultivation,
perhaps approaching or into the stationary phase. The
penicillin fermentation exemplies such behaviour.
Occasionally, a simple non growth-associated model
suffices for product formation kinetics in such cases. In
273
these models, production rate is proportional to
biomass concentration rather than growth rate.
The classic study of Luedeking and Piret10 on the
lactic acid fermentation by L . delbrueckii indicated that
the product formation kinetics combined growth-
associated and non growth-associated contributions :
dP/dt \ A dX/dt ] BX (7)
This two-parameter kinetic expression, often termed
LuedekingPiret kinetics, has proved extremely useful
and versatile in tting product formation data for many
dierent fermentations. However, since previous studies
has demonstrated the inhibitory eect of lactate, by
removal from the culture medium using dialysis,11 this
model may be improved by the addition of a term indi-
cating dependence of the rate of acid production on
inhibitor concentration, the lactic acid itself :
dP/dt \ (A dX/dt ] BX)(1 [ P/P@ ) (8)
max
According to this equation, dP/dt will become zero
when P approaches P@ , the concentration greater than
P above which bacteria do not produce lactic acid.
max
Finally, substrate utilization kinetics may be
expressed by an equation12,13 which considers both
substrate consumption for maintenance and substrate
conversion to biomass and product. The rate of sub-
strate utilization is related stoichiometrically to the
rates of formation of biomass and lactic acid. The sub-
strate requirement to provide energy for maintenance is
usually assumed to be rst-order with respect to
biomass concentration, mX.
dS/dt \ [1/Y dX/dt [ 1/Y dP/dt [ mX (9)
X@S P@S
4 RESULTS AND DISCUSSION
To obtain the parameters of the equations that rep-
resent the concentrations of biomass, lactic acid and
substrate in the batch culture of L . delbrueckii on beet
molasses, batch runs (triplicate experiment) were per-
formed under optimal conditions,3,7 as shown in Table
1.
The data obtained from these experiments are shown
in Fig. 2. They were tted to eqns (5), (8) and (9) using a
computer program that compared the batch fermenta-
tion data with the proposed rate expressions in such a
way that the dierence between the model predictions
and the actual experimental values were minimized. To
obtain the best tting rate equations, a nonlinear regres-
sion analysis based on Marquardt Algorithm14 com-
bined with a RungeKutta method for dierential
equations was used. The estimated parameters are listed
in Table 2.
274 J. M. Monteagudo, L . Rodr guez, J. Rinco n, J. Fuertes

TABLE 1 tion of glucoseyeast extract medium using L . del-

Experimental Conditions for Batch Runs of L . delbrueckii
Grown on Beet Molasses
Yeast extract concentration : 531 g dm~3
Peptone concentration : 508 g dm~3
Sucrose concentration : 65 g dm~3
Temperature : 49C
pH : 590
Agitation rate : 100 rev min~1
Using data from the table, the ratio A/B equals 270.
Compared with the value for A/B of 40 obtained by
Luedeking and Piret10 in the lactic acid fermentation of
glucose by L . delbrueckii, there is less growth-associated
product formation in the present work. Further, the
small value of the ratio A/B obtained here indicated the
almost total independence of the. lactic acid production
rate from the growth rate. Bacteria produce lactic acid
proportionally to the concentration, not depending on
their growth phase. A low maintenance coefficient
would thus be expected, 009.
The maximum specic growth rate, k , was
max
0831 h~1. This result compared favorably with results
obtained by Tyree et al.15 who found a maximum spe-
cic growth rate of 0722 h~1 for L actobacillus xylosus
grown on glucose. The overall bacterial yield, Y , had
X@S
a maximum value, 0270, at pH 590, a value higher
than obtained by Hanson and Tsao16 in the fermenta-
brueckii.
The maximum yield of lactic acid from sucrose (on
beet molasses), Y , was 091 g g~1, close to the stabil-
P@S
ized value of 09 for all homofermentative lactobacilli17
at a pH value of 590. In Table 3, this parameter is com-
pared with the yields obtained by other workers.16,18,19
The results from Hanson and Tsao16 and Luedeking18
agree with those obtained here, i.e. the yield increased
up to pH values close to 590. Finns19 results, however,
indicated that the highest yield occurred at lower pH
values.
The degree of reproducibility of the experimental
system is shown in Fig. 2. The symbols represent the
experimental data and the solid line the model predic-
tions. The fermentations were characterized by a short
lag phase followed by exponential growth and a simul-
taneous biosynthesis of lactic acid with growth.
4.1 Biomass
Biomass production is presented in Fig. 2. The data
show a marked decline in biomass concentration from
about 15 h. This trend can be explained by including
the possibility of endogeneous metabolism in the model.
In endogeneous metabolism, reactions in bacteria
consume bacterial material. An assumption often made
for the appearance of the phase of decline (and some-
times also for the stationary phase) is that inhibitory
products of metabolism accumulate during growth and
their subsequent interaction with the viable organisms
results in death. For the data obtained in the present
study, eqn (5), which accounts for this, was found to
accurately express the relationship between the biomass
concentration and lactic acid concentration. From eqn
(5) the maximum allowable lactic acid concentration
above which bacteria do not grow, P , was predicted
max
to be 45 g dm~3.

4.2 Lactic acid

Fig. 2. Mathematical simulation of batch fermentations. (), Similar to the analysis of biomass production, the
Theoretical model ; (L sucrose, K lactic acid, = biomass) dependence of the rate of lactic acid production on con-
experimental data. centration was studied. Typical lactic acid concentra-

TABLE 2 TABLE 3
Fermentation Parameters for L . delbrueckii Grown on Beet Comparison of Lactic Acid Yields from Batch Fermentations
Molasses
pH Y Y Y Y
P@S P@S P@S P@S
(this work) (Ref. 16) (Ref. 18)a (Ref. 19)a
A (g lactic acid g~1 biomass) 0235
B (g lactic acid h~1) 0087 495 079 074 083 091
m (g sucrose h~1 g~1 biomass) 0090 533 083 086 087 088
k (h~1) 0831 585 088 090 090 087
max 590 091
Y (g cells g~1 sucrose) 0270
X@S
Y (g lactic acid g~1 sucrose) 0910
P@S a Interpolated.
Kinetics of lactic acid fermentation by L. delbrueckii
tion curves are also presented in Fig. 2. The
experimental data show that the lactic acid concentra-
tion at the end of the log phase (about 22 h) is approx-
imately 57 g dm~3. The lactic acid had a noticeable
eect on the growth rate at concentrations about
20 g dm~3.
An expression for modelling inhibition due to lactic
acid production is that derived from the Luedeking and
Piret equation and given in the form of eqn (8). Accord-
ing to eqn (8), the lactic acid-producing capability of the
bacteria was completely inhibited at a lactic acid con-
centration of 57 g dm~3 (P@ ). Thus, the model equa-
max
tion was able to predict that bacteria were capable of
producing lactic acid even after growth ceases.
Lactic acid is an inhibitor of both the growth of bac-
teria and its own biosynthesis. Figure 3 shows the eect
of lactic acid on fermentation rate during batch fermen-
tation with dierent initial lactic acid concentrations. As
the initial amount of lactic acid in the medium was
increased, sucrose in the medium was consumed more
slowly. When the initial concentration of lactic acid was
60 g dm~3, total inhibition occurred.
4.3 Substrate
Substrate concentration curves are also illustrated in
Fig. 2. Sugar was not completely utilized in the fermen-
tations. The fermentations were considered complete
when the sugar concentration dropped below 5 g dm~3.
After this point, no biomass growth or lactic acid pro-
duction was observed.
For the sucrose concentration data obtained in the
present study, eqn (9) was found to accurately express
the relationship between the concentrations of sub-
strate, biomass and lactic acid. From this equation, the
sugar concentration at which bacteria did not grow and
stopped producing lactic acid was found to be
485 g dm~3. As indicated above, eqn (9) is the sum of
three terms representing the growth of bacteria, the bio-
Fig. 3. Eect of lactic acid concentration on growth rate and
fermentation of L . delbrueckii grown on beet molasses (= :
lactic acid, 20 g dm~3 ; K : lactic acid, 40 g dm~3 ; L : lactic
acid, 60 g dm~3).
275
synthesis of product and maintenance. A similar model
was used to express the kinetics of substrate utilization
in the lactic acid fermentation by S. cremoris12 and S.
cerevisiae.13
5 CONCLUSIONS
Lactic acid, a product of industrial importance, can be
produced by fermentation of the sucrose contained in
beet molasses, a by-product of sugar manufacture. The
experimental results obtained using beet molasses were
similar to those obtained using synthetic sucrose solu-
tions. Thus, lactic acid ready to use in the food industry
may be obtained by fermentation of beet molasses, an
economical fermentation substrate.
The batch fermentation kinetics of this fermentation
process were analyzed and two main conclusions
derived. Firstly, lactic acid was an inhibitor of beet
molasses fermentation. Secondly, bacteria were able to
produce lactic acid even after growth ceased.
To model the dynamic state of the batch fermentation
three rate equations that represent growth, lactic acid
production and sugar utilization have been proposed.
The model has been found to provide an adequate
description of the fermentation kinetics.
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