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MATHEMATICAL MODEL OF L-LACTIC ACID

FERMENTATION IN A RDC COUPLED WITH PRODUCT
SEPARATION BY ION EXCHANGE
a a a
J. P. LIN , S. D. RUAN & P. L. CEN
a
Department of Chemical Engineering , Zhejiang University , Hangzhou, 310027, People's
Republic of China
Published online: 30 Mar 2007.

To cite this article: J. P. LIN , S. D. RUAN & P. L. CEN (1998) MATHEMATICAL MODEL OF L-LACTIC ACID FERMENTATION IN A
RDC COUPLED WITH PRODUCT SEPARATION BY ION EXCHANGE, Chemical Engineering Communications, 168:1, 59-79, DOI:
10.1080/00986449808912707

To link to this article: http://dx.doi.org/10.1080/00986449808912707

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 Chem. Eng. Comm..1998. V o l 168. pp. 59-79 0 1998 OPA (Ovcrrcas Publirhcn Association) N.V.
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MATHEMATICAL MODEL OF L-LACTIC

ACID FERMENTATION IN A RDC COUPLED
WITH PRODUCT SEPARATION
BY ION EXCHANGE
J. P. LIN, S. D. RUAN and P. L. CEN*
Department of Chemical Engineering, Zhejiang University,
Hangzhou 310027, People's Republic of China

(Received I July 1997; In final form I0 December 1998)

A Rotating Disk Contactor (RDC) was designed to perform the L-lactic acid fermentation with
a filamentous fungus, Rhizopus oryzae, which was immobilized on the surface of rotating disks.
The bioreactor was operated in repeated batch or continuous modes. The growth rate of the
fungi was about I mm/day perpendicular to the disks' surface. A weak-base anionic resin, D354,
was selected which was high in selectivity for lactic acid separation. Even at low concentration,
the ion exchange capability was about 0.5 g Laclg dry resin. A coupled process of L-lactic acid
fermentation and ion-exchange separation was evaluated experimentally. The results indicated
that the pH value of the fermentation broth could be maintained at pH 3.5 without any addition
of alkali. The conversion ratio of glucose to L-lactic acid was about 0.7glg and the fermentation
rate was able to reach as high as 62.5g glucose per hour per square meter of the disk surface
area. A mathematical model was proposed to describe the simultaneous process of L-lactic acid
fermentation and separation by ion exchange, in which the thickness increase of mycelia as well
as the substrate and product inhibitions were included. The model simulation was in good
agreement with the experimental data.

Keywords: L-lactic acid; rotating-disk contactor; ion-exchange resin; coupled fermentation and
separation process; mathematical model

Lactic acid is one of the most important organic acids and is widely applied
in food and pharmaceutical industries (Buchta, 1983; Penning er al., 1992).
It can be produced by either chemical o r biochemical processes (Buchta,
1983; Motosugi et al,, 1984). Because lactic acid can be produced from
renewable resources with high yield, the fermentation method is more

'Corresponding author
 60 J. P. LIN er a/.

attractive (Aksu et a/., 1986; Chiarini et al., 1992; Aschlimann et a/., 1991;
Abe et a/., 1991). The optically active L-lactic acid is the form which can be
metabolized by human body, which makes L-lactic acid more suitable for
food additive. The biodegradable polymer made of L-lactic acid has special
properties and applications. Therefore interest in L-lactic acid fermentation
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has increased recently (Mattey, 1992; Hang et al., 1990; Yu et al., 1989;
Hamamci el a[., 1994).
Many strains have been identified which have the ability to accumulate L-
lactic acid (Shusan, 1985; lshizaki et al., 1992). Among them, a filamentous
fungus, Rhizopus oryzae, is more attractive because of its high conversion
ratio and production of almost pure optically active L-lactic acid. Rhizopus
oryzae growths aerobically with long mycelia which tend to form large sized
pellets or even mats and causes a dramatic increase in oxygen mass transfer
resistance. In conventional stirred tank bioreactors, the fungi will anchor on
the internal elements of the fermentor, such as heat exchanger, bafflers and
stirrers. The fermentation period is long (72-96 hoursj and the yield is low
(60-70%) because of local anaerobic condition. The immobilization of
Rhizopus oryzae can reduce the size of the pellets and enhance oxygen mass
transfer. The cell immobilization by gel entrapment method have been
widely investigated for various microorganisms including Rhizopus oryzae
(Audet et al., 1989; Hang et a/., 1989). The disadvantage of gel
immobilization method for industrial application is the difficulty to make
small immobilized particles in large amounts in sterilized conditions. The
rotating disk contactor (RDC) has been widely applied in waste water
treatment processes (Benefield et al., 1980). The microbes are immobilized
on the surface of disks by physical adsorption which is easy to operate and
to scale up. The spores of Rhizopus oryzae can be adsorbed on the rough
surface, and then mycelia can cover whole surface of the disk. The mycelia
are directly in contact with sterilized air in the head space of the RDC,
which will enhance oxygen mass transfer.
L-lactic acid is toxic to strain growth. In the traditional lactic acid
fermentation processes, alkali o r alkali salt must be added to the
fermentation broth to neutralize the produced lactic acid and to regulate
the pH value. The most commonly used neutralizer in industry is solid
calcium carbonate, which is difficult to sterilize and to feed into the
fermentation broth. Furthermore the solubility of calcium lactate limits the
increase of initial substrate concentration. The simultaneous fermentation
and product separation process can remove the accumulated product to
eliminate product inhibition (Cen et a / . , 1993). The separation methods,
such as adsorption, ion-exchange, solvent extraction, membrane separation
 FERMENTATION WITH PRODUCT SEPARATION 61

etc., can be coupled into the process. The separation can be carried out
in-silu or with an external separator. The other advantages of the coupled
fermentation and separation processes include the increase in both
fermentation rate and substrate concentration, the simplification in the
product recovery and purification processes.
Ion-exchange resins are applied in organic acid separation (De Corte el ol.,
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1973; Srivastava et al., 1992). When it is applied in the coupled fermentation

and separation process, it should be biocompatible with the particular
strain. In addition, the resin should be high in separation selectivity and
capability. Because the product concentration in the fermentation broth is
generally very low (it can be even lower in the coupled process), it is desired
that the ion-exchange capability at low concentrations should not be
reduced very much.
The mathematical model of the coupled fermentation and separation
process is important for process analysis and scale-up. There are a lot of
models which have proposed to describe the fermentation and separation
alone. How to simulate the coupled process is a new task we are facing.
The objectives of this work are: to examine the feasibility of L-lactic acid
fermentation in a RDC bioreactor, to screen a resin which is biocompatible
with R. oryiae and with high selectivity and capability for L-lactic acid
separation, to evaluate the possibility of simultaneous L-lactic acid
fermentation in R D C and product separation by ion-exchange method,
and to establish a mathematical model for the coupled process.

MATERIALS AND METHODS

Microorganism and Medium

The strain used in this work is Rhizopus oryzae N R R L 395. A potato-
glucose agar was used for solid media to obtain spores. Liquid media
consists of (per liter): 2 g urea, 0.6 g KH2P04, 0.25 g MgS04.7H20, 0.044 g
Z u S 0 4 . 7 H 2 0 , and glucose is added as experiments required.

lmmobilization of R. oryzae
The spores of R. oryiae were washed down from the solid media with
sterilized physiological saline. The spore suspension was then added to RDC
and mixed with sterilized liquid medium. The rotating rate of disk was in the
range of 20-50rpm and sterilized air was passed through the head space of
 62 J. P. LIN er al.

RDC. The broth became clear after about 18 hours, it indicated that the
spores had been adsorbed on the surface of the disks. Then white spots
appeared on the disks because of the germination of spores. After about
30 hours, which depends on the inoculation volume, the mycelia of R. oryzae
covered whole surface of the disks. The immobilization procedure was
finished.
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Experimental Apparatus

The experimental apparatus for the simultaneous L-lactic acid fermentation

and separation is shown in Figure 1. It consists of one RDC and four fixed-
bed ion-exchange columns. The dimensions of the RDC were 185mm in
diameter and 320mm in length with a total volume of 8.5liters. Eight disks
were installed on the axis with equal distance. The ion-exchange columns
were 90 mm in diameter and 800mm in height and 1.5 kg of D354 resin were
added to each column. A peristaltic pump was used to recycle fermentation
broth between the RDC and one of the columns. The other columns were in
different stages of regeneration by acid or alkali solution. After one of the
columns was saturated with L-lactic acid, which was determined by change
in color and volume of the resin, another regenerated column will be shifted
in to replace the saturated one. In case of fermentation without coupled ion-

i;;
14
r
FIGURE I Experimental apparatus of coupled fermentation and ion-exchange separation.
1 . Air system; 2. Motor (variable speed); 3. Dosing bottle; 4. Dosing pump; 5,8. Temperature
control system; 6. Thermometer; 7. Rotating disc contactor ($185 x 320; seven disks, $165 x 5,
dislance between two discs: 40mm); 9. Exhaust cooler; 10, 1 1 . Sampler; 12, 14. Peristaltic pump;
13. Ion-exchange column ($90 x 800, 1.5kg resin in each column); 15. Alkali reservoir; 16.
Sterile deionic water reservoir.
 FERMENTATION WITH PRODUCT SEPARATION 63

exchange column, the sterilized calcium carbonate must be added to

neutralize L-lactic acid.

Resin, Ion-exchange lsotherm and Resin Regeneration

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The resin evaluated in this study are all commercial products in China,
among them, a weak-base anionic resin, D354, which is made in Linan
Resin Factory, is nontoxic to R. oryzae and is used in the simultaneous L-
lactic acid fermentation and separation process. The ion-exchange isotherms
were measured by shake flask method. The regeneration of the resin can be
carried out by two procedures. First, acid such as sulfuric acid can be used
to replace L-lactate from the resin to obtain L-lactic acid, then the resin has
to be regenerated by alkali into alkali-type resin for reuse. Second, the resin
is directly regenerated by alkali solution, such as sodium hydroxide, so the
final product is sodium L-lactate. When the resin changed from -OH and to
-lactate form, the volume of resin will increase by about 50%.

RESULTS AND DISCUSSIONS

Screening of Ion-exchange Resins

A screening procedure was designed to select the most favorable resin or
adsorbent for lactic acid separation. Among various strong-base, weak-
base, macroporous adsorbent resins as well as activated carbon, a weak-base
anionic resin, D354,is the best. The results are shown in Table I.

TABLE I Capability of various ion-exchange resins and adsorbents to separate lactic acid
(Po = 53.4 g/L, V = IOOml, T = 25°C)

resinlodsorbenr dry weight, g p, g / L q p . g Lac/g dry resin

2.3313 46.5 0.333
2.6914 46.5 0.269
D296
D354
D301 R
AB-8
D4006
X-5
PVP
Activated
Carbon
nd: not detectable.
 64 J . P. LIN er a1

The ion-exchange isotherms of lactic acid on D354 resin were shown

in Figure 2. The main advantage of D354 resin is the high ion-exchange
capability even a t low concentration. For example, at I% of lactic acid,
which is equivalent to about p H 3 3 - a limitation for R. oryzae growth, the
ion-exchanged amount has reached more than 80% of that at saturation.
This is a very important feature because of low lactic acid concentration in
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fermentation broth, especially in simultaneous fermentation and separation

process (SFS).
The erect of nutrient salts, that exist in the medium, on the ion exchange
of lactic acid is negligible. The results are listed in Table 11. It indicates that

+ + + + + 35OC
x x x x x 25
model correlation

FIGURE 2 The ion-exchange isotherms of lactic acid on D354 resin.

TABLE I I EKect of inoreanic salts on ion exchange of lactate with D354 resin
I n o r ~ a n i csalt salt concenrrarion, g / L qp, g La& resin

MgS04
ZnS04
KH2P04
Urea
M$+ + zn2+ +
K +Urea
without salt
 FERMENTATION WITH PRODUCT SEPARATION 65

the inorganic salts in the medium will not interfere the ion-exchange
capibility of lactate on D354 resin.
The another feature of ion-exchange isotherms of lactic acid on D354 resin
is the extra-equivalent adsorption. The ion-exchange amount of D354 resin is
4.7mmol/g dry resin by standard hydrogen chloride method as shown in
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Figure 2 (dashed line). The possible reasons include the non-specific physical
adsorption, multi-layer adsorption and self-polymerization of lactic acid at
high concentration. Therefore, the total ion-exchanged amount can be
assumed to be contributed by two factors: ion exchange and non-specific
adsorption, which can be described by two independent Langmuir equations
as following:

The parameter q,, represent the maximum ion-exchange amount, which

should equal to 4.7 mmol/g (or 0.423 g Lac/g resin) for D354 resin measured
by standard method. The other parameters in Eq. (I) can be correlated from
experimental data and are listed in Table 111. The comparisons between
model calculation and experimental data are also shown in Figure 2.
Glucose can be physically adsorbed by D354 resin. The adsorption
isotherms are shown in Figure 3 and can be described by linear isotherm
equation:

The slope, ks, of the straight lines in Figure 3 are 0.00276L/g, 0.00167 L/g
and 0.00143 L/g at 20,27 and 30"C, respectively.

Repeated Fed-batch and Continuous L-lactic Acid

Fermentation in RDC
After immobilization procedure of R. oryzae was finished, the RDC was
ready to perform the L-lactic acid fermentation either in repeated fed-batch
or in continuous operation mode. The repeated fed-batch operation can be

TABLE Ill The parameters of the ion-exchange isotherms of lactic acid on D354 resin

value at 3 0 ~ ~
value at 35°C
 J. P. LIN er a/.
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FIGURE 3 The adsorption isotherms o f glucose on D354 resin (0 - 20"C, A -

30°C).

carried out as follows: after last batch fermentation has finished, withdraw
all the broth from the RDC and add fresh sterilized medium to start the next
batch fermentation. In this stage, ion-exchange column was not coupled
to the processes, therefore, sterilized calcium carbonate was added to
neutralize the produced L-lactic acid during fermentation.

I . Conrinuous Fermentation

During operation period of the RDC after immobilization of R. oryzae, the

length of mycelia will grow continuously. The relationship between
mycelium thickness on the disks' surface and time is shown in Figure 4.
The fermentation rate is not only a function of total surface area of disks
but also that of the thickness of mycelia. Because fermentation rate is much
faster than growth rate of mycelia, quasi-steady-state can be reached in
continuous operation. The results at different operation conditions are
summarized in Table IV.
Assuming the density of mycelia is a function of the thickness of mycelia
on the disks' surface, i.e.,
 FERMENTATION WITH PRODUCT SEPARATION
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FIGURE 4 The relationship between mycelium thickness and time (two batches with difterent
inoculation ratio).

TABLE IV Quasi-steady-state results in continuous L-lactic acid fermentation in the RDC

p,ocHa (a>0) (3)

Then the total biomass on total surface area of the disks is:

M =k p + ' (4)
 68 J. P. LIN er a/.

where, k is a constant. Because of mass transfer limitation, the interior

mycelium grows slower than the mycelia at surface. The overall specific
growth rate of the mycelia should be a function of the thickness of mycelia.
Assuming:
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From the independent experimental data of free-cell fermentation, the

specific growth rate of R. oryzae is inhibited by both substrate and product
(Lin, J. P., 1997), the specific growth rate of R. oryzae on the disks' surface
should be:

L-lactic acid is a product of energy metabolism, so, the growth-coupled

assumption can be adopted:

in which,

and L is the maximum thickness of mycelia, which equals half of the

distance between two disks. For continuous fermentation at quasi-steady-
state, the material balance equations are:

The parameters in above equations can be correlated from experimental

data as followings:

A = 33.59, n = 0.3044, K,,, = 0.6367g/L,

Ks = 71 1.0 g/L, K p = 653.3 g/L.

The comparisons of calculated and experimental volumetric L-lactic acid

formation rate were shown in Table IV and Figure 5 with good agreement.
 FERMENTATION WITH PRODUCT SEPARATION
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FIGURE 5 Comparison of fermentation rate between the experimental data and data
calculated from mathematical model.

From Eq. (7) following equations can be obtained:

Functions f, (4, f 2 ( S ) and f3(P) represents the contribution of mycelia

thickness, substrate concentration and product concentration to the L-lactic
acid formation rate, respectively. The relationships between 6 andf, (4,S a n d
fi(S), as well as P and f3(P) were shown in Figures 6-8. The results indi-
cated that the thickness of mycelia was an important factor to the fermenta-
tion rate; the substrate concentration had little effect on the fermentation
rate after S greater than 10g/L; and lactate inhibition was negligible.

2. Repeated Fed-batch Fermentation

Typical experimental results were shown in Figures 9- 10. The substrate
comsumption and product formation curves were almost straight lines. For
 J . P. LIN el a/,
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FIGURE 6 The relationship between/, (6) and mycelium thickness.

FIGURE 7 The relationship betweenf2(S) and concentration of glucose.

 FERMENTATION WITH PRODUCT SEPARATION
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FIGURE 8 The relationship b e t w e e n h ( ~ )and concentration of L-lactic acid.

FIGURE 9 Time course of repeated-batch fermentation ( I ) (H: 1 -2.5mm, V,: 5.0L).

 1. P. LIN er a/.

' O 0 1
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FIGURE 10 Time course of repeated-batch fermentation (2) (H: 4.5-5.0mm. V,: 5.5L).

the repeated fed-batch process, the mathematical models can be written as:

The thickness of mycelia during each batch fermentation can be assumed

growth linearly:
Hf - Hi
H ( t ) = H, +-[total
(Hf 2 H ( t ) 2 Hi)

where, ttot,l is the period time of one batch fermentation. Then substrate
consumption and product formation curves can be predicted by the model
with the parameters correlated from continuous fermentation. T h e
comparisons between predicted curves and experimental data were also
shown in Figures 9- 10.

Simultaneous L-lactic Acid Fermentation in RDC and

L-lactic Acid Separation by Ion Exchange
The experiments were carried out with the apparatus shown in Figure 1. The
typical repeated fed-batch results were shown in Figure 11. Initially, both
 FERMENTATION WITH PRODUCT SEPARATION
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FIGURE 1 1 Time courses of a repeated-batch operation coupled with ion-exchange

separation. ( 0 - S in RDC; rn - P in RDC; A-Sch in column exit; A - p H value in column
exit, F=4.3 Ljhr, T=5.0-6.0mm.

RDC and ion-exchange column were filled with same medium. Because of
physical adsorption of glucose on the resin, the glucose concentration in the
column was indeed lower than that in the RDC. After recycle pump was
switched on, sharp drop in glucose concentration was observed because of
mixing of mediums between RDC and column, as well as the consumption
by biomass. After initial stage, the glucose concentration was almost
reduced linearly with time. The glucose concentration in the outlet of the
column was always higher than that in the RDC because of the residence
time in the column without glucose consumption. The p H value in the outlet
stream was kept around pH6.0, which would guarantee the pH value in
RDC at pH3.5 without any addition of alkali or alkali salt. The lactic acid
concentration in RDC was lower than 8g/L, and zero in the effluent of
column because of ion-exchange separation.
The coupled fermentation and separation system can be simplified as
shown in Figure 12.
The material balance equations of RDC can be expressed as:
 J. P. LIN er a1
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Bioreactor

Ion -Exchange Column

FIGURE 12 Simplified schematic diagram for the simulation of fermentation processcoupled
with ion-exchange.

where, k p H is a constant considering the effect of pH value difference

between calcium carbonate neutralization process and SFS process on
the fermentation rate. According to the study of breakthrough curves of
ion-exchange column previously, the ion-exchange dynamics of the column
can be described by a continuous-flow plate model (Lin, J. P., 1997). The
column consists of ten tanks-in-series and a head space. For the ith tank,
the material balance equations are:

dSi 1 - c d G F
--
dt
-
E dt
+-
VT&
(Si-1 - Si)

where ks is the mass transfer rate constant of substrate and is assumed

independent of concentration.
 FERMENTATION WITH PRODUCT SEPARATION 75

dPi 1 - E dqpai F
dt
-
E d?
+-(Pi-1 - Pi)
VTE
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where k p is the mass transfer rate of constant of product and is also assumed
independent of concentration. The head space of the column can be
considered as a perfect mixing tank. Then,

The initial and boundary conditions are:

at

From breakthrough curves of lactic acid on D354 resin, the mass transfer
coefficients ks and k p at flow rate of 4.3 L/hr were correlated as: k p = 0.5 I/
hr, ks = 35.0 I/hr (Lin, J. P., 1997). The correction coefficient k p Hrepresent
the effect of pH value on the L-lactic acid production rate r~~~ Because the
pH value in the coupled process (pH3.5) was lower than that in the process
with calcium carbonate as neutralizer @H4.8), kpHmust be correlated from
the experimental data of the SFS processes.
The comparisons of experimental data and model predictions were shown
in Figures 13 and 14. The predicted glucose and L-lactic acid concentrations
either in the RDC or in the effluent of the ion-exchange column were in good
agreement with the experimental data.

Comparisons Among Various Operation Methods

Free-cell culture of R. oryzae in a B. Braun fermentor was carried out.
The results were shown in Table V. Also results from repeated fed batch
 J. P. LIN er a1

- calculated curves
in RDC
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*. ---calculated curves
n ,A!;\- 1 in outlet of column

FIGURE 13 Comoarisons between exoerimental data and model simulation curves ( H : 5.0-

- calculat8_.
ed curves
.. ._

in RDC
---calculated curves
in outlet of column
0 I
I

10 - \

FIGURE 14 Comparisons between experimental data and model simulation curves ( H : 6.0-
6.5mm, F = 4 . 3 L/hr, kpll=0.88).
 FERMENTATION WITH PRODUCT SEPARATION 77

TABLE V Comparisons of L-lactic acid fermentation in stirred tank, RDC with or without
coupled separation
Method pH value Production Rate Yield
in broth g / ( L . hr) gk
Free cells with CaCO, 6-7 0.65-1.5 0.65-0.7
RDC with CaCO, 6-7 3-4 0.7-0.8
RDC with coupled separation 3.5 3-4 0.7-0.75
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operation in RDC with or without coupled separation process were listed in

Table V.

CONCLUSION

1. RDC bioreactor is suitable one to be used to immobilize R. oryzae and to

perform L-lactic acid fermentation with high fermentation rate and high
L-lactic acid yield.
2. Weak-base anionic resin D354 is high in ion-exchange capability and
selectivity for L-lactic acid separation.
3. L-lactic acid fermentation and coupled product separation by ion-
exchange method can eliminate product inhibition and simplify fermen-
tation and product separation processes.
4. The proposed mathematical model is applicable to L-lactic acid
fermentation in RDC bioreactor with or without coupled separation
processes.

Acknowledgement

This work was supported by National Natural Science Foundation of

China.

NOMENCLATURES

A constant in fermentation kinetic model

C concentration, g/L
d diameter of ion-exchange column, m
F recycle flow rate between reactor and column, L/hr
H thickness of mycelium film, mm
k~~ inhibition coefficient of low pH to fermentation
kp transfer coefficient for lactic acid, hr-'
 78 J. P. LIN et al.

adsorption equilibrium constant for glucose, Llg

coefficient in ion-exchange isotherm, giL
coefficient in ion-exchange isotherm, giL
limiting substrate constant, giL
product inhibition constant, g/L
substrate inhibition constant, g/L
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length of ion-exchange: column, m

n coefficient in fermentation kinetic
p L-Iactic acid concentration, giL
q the ion-exchange or adsorption amount, glg (dry)
maximum chemical exchange amount oflactic acid, gig (dry)
maximum extra-equivalent adsorption amount of lactic acid,
gig (dry)
q adsorption amount on unit resin, giL-resin
q* equilibrium exchange amount, g/Lcresin
rF fed rate, g-glucosc/hr
rLa production rate of Ldactic acid, g/hr
S glucose concentration, giL
t time, hr
YP j S product yield, gig

Greek

zero dimensional thickness of mycelium film

CT total porosity of fixed-fed
Pr real density of resin, giL

Subscripts

a lactic acid
c calculated data
ch head space of ion-exchange column
e experimental data
g glucose
ith cell of column
p product (Ldacticacid)
S substrate (glucose)
X biomass
o initial value
 FERMENTATION WITH PRODUCT SEPARATION 79

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Abe, S. and Takagi, M. (1991) Simultaneous Saccharification and Fermentation of Cellulose to

Lactic Acid, Biotechnol. Bioeng., 37, 93.
Aksu, Z. and Kutsal, T. (1986) Lactic Acid Production from Molasses Utilizing Lactobacillus
delbrueckii and Invertase together, Biotechnol. Letters, 8, 157.
Aschlimann, A. and von Stockar, U. (1991) Continuous Production of Lactic Acid from Whey
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Permeate by Lactobacillus helveticus in a Cell-Recycle Reactor, Enzyme Microb. Technol.,

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