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09603085.htm Trans IChemE, Vol 81, Part C, September 2003
ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION
BY LACTOBACILLUS DELBRUECKII NRRL B-445
S. J. TE

LLEZ-LUIS
1,2
, A. B. MOLDES
2
, M. VA

ZQUEZ
1,3
and J. L. ALONSO
2
1
Department of Food Science and Technology, UAM Reynosa-Aztlan, Universidad Autonoma de Tamaulipas, Mexico
2
Department of Chemical Engineering, University of Vigo (Facultad de Ourense), Vigo, Spain
3
A

rea de Tecnologa de los Alimentos, Escuela Politecnica Superior, Departamento de Qu mica Anal tica, Universidad
de Santiago de Compostela, Lugo, Spain
L
actic acid bacteria are generally recognized as nutritionally fastidious. The complexity
of the media increases the cost of lactic acid production. In this study a low-cost
nutrient medium based on corn steep liquor (CSL) was developed for lactic acid pro-
duction by Lactobacillus delbrueckii NRRL B-445. Starting from a medium containing
90 g l
1
glucose and 20 g l
1
CSL as a sole nutrient source, 70.7 g l
1
lactic acid was obtained
with an economic efciency of 98g lactic acid per

nutrient. Other media, made with CSL
and each individual component (yeast extract, peptone, sodium acetate, sodium citrate,
K
2
HPO
4
, MgSO
4
7H
2
O, MnSO
4
H
2
O or FeSO
4
7H
2
O) of a general (Mercier) lactobacilli
medium were also assayed. The highest economical efciency (134g lactic acid per

nutrient)
was obtained supplementing 10 g l
1
CSL with 0.05 g l
1
FeSO
4
7H
2
O. Additionally, lactic
acid production and glucose consumption were mathematically modelled and the regression
parameters obtained were correlated with CSL concentration by linear or exponential
equations.
Keywords: Lactobacillus delbrueckii; lactic acid, corn steep liquor, nutrients study;
mathematical modelling.
INTRODUCTION
Many applications in dairy, beverage, confectionery, meat
and poultry industries have been found for lactic acid
(2-hydroxy propionic acid) and its derivates. Lactic acid is
employed in food industry as acidulant, avour and preser-
vative due to its mild taste that does not hide the weaker
aromatic avours of some foods. Additionally, it has a
technological application during cheese and yoghurt produc-
tion, producing the coagulation of the casein fraction.
An other important application of this compound is the
production of polylactic acid (PLA)-based degradable plas-
tics (Chahal, 1991; Ozen and Ozilgen, 1992).
Lactic acid can be obtained by chemical synthesis from
petroleum-based products or by microbial fermentation.
Many lactobacilli strains and some fungus like Rhizopus
oryzae can bioconvert glucose and other sugars to lactic acid
(Zhou et al., 1999; Hofvendahl and Hahn-Hagerdal, 2000).
Owing to its asymmetric carbon, lactic acid can occur in two
optically active enantiomers, L and D (Vick-Roy, 1985), but
an important advantage of microbial fermentation over
chemical synthesis is that it is possible to produce exclu-
sively one of the isomers.
Lactic acid bacteria use sugars via different pathways
resulting in homo-, hetero- or mixed acid fermentations.
Homofermentation gives only lactic acid as the end product
of glucose metabolism by the EmbdenMeyerhofParnas
pathway. In heterofermentation, equimolar amounts of lactic
acid, carbon dioxide and ethanol or acetate are formed from
glucose via the phosphoketolase pathway (Chahal, 1991).
Several factors that affect lactic acid production by micro-
organisms are medium composition (carbohydrate source,
sugar concentration and growth factors), temperature,
presence of oxygen, pH and product concentration
(Burgos-Rubio et al., 2000). Lactobacillus delbrueckii
NRRL B-445, also named as Lactobacillus rhamnosus
ATCC 10863 (Hofvendahl and Hahn-Hagerdal, 2000), is a
homofermentative lactic acid bacterium that produces
mainly L-lactic acid.
In spite of the advantages, fermentations must be cost
competitive with chemical synthesis. Fermentation medium
can represent almost 30% of the cost for a microbial
fermentation (Miller and Churchill, 1986). Lactic acid
bacteria have limited capacity to synthesize B-vitamins
and amino acids (Hofvendahl and Hahn-Hagerdal, 2000).
Yeast extract is used as the main source of nitrogen and
vitamins for lactic acid production by microorganisms, but it
is too expensive for large-scale fermentations. Complex
media commonly employed for growth of lactic acid
bacteria are not economically attractive due to their high
amount of expensive nutrients such as yeast extract, peptone
and salts (Mercier et al., 1992). Various nitrogen sources
were tested for lactic acid production by bacteria but none
of these gave lactic acid concentrations as high as that
obtained with yeast extract (Nancib et al., 2001; Tellez-
Luis et al., 2003). However, new low-cost media for lactic
acid fermentation are desirable in order to decrease the
production cost.
250
Corn steep liquor (CSL) is a low-cost nutritional medium
employed successfully in the production of ethanol by
Zymomonas mobilis (Kadam and Newman, 1997; Silveira
et al., 2001), succinic acid by Anaerobiospirillum succini-
ciproducens (Lee et al., 2000) or arabinanase by Fusarium
oxysporum Cheilas et al., 2000). It could replace some of
the expensive nutrients in the complex medium employed to
grow L. delbrueckii.
The aim of this study was to develop a low-cost nutrient
medium based on CSL for lactic acid production by
L. delbrueckii NRRL B-445. Additionally, lactic acid produc-
tion and glucose consumption were modelled and the regres-
sion parameters obtained were correlated with CSL
concentration.
MATERIALS AND METHODS
Strai ns and Cul ture Condi tions
L. delbrueckii NRRL B-445 was obtained from the
United States Department of Agriculture Northern National
Research Laboratory in Peoria, IL. The strain was grown on
plates using the complete media proposed by Mercier et al.
(1992), which contains 20 g l
1
glucose, 5 g l
1
yeast
extract, 10 g l
1
peptone, 5 g l
1
sodium acetate, 2 g l
1
sodium citrate, 2 g l
1
K
2
HPO
4
, 0.58g l
1
MgSO
4
7H
2
O,
0.12 g l
1
MnSO
4
H
2
O, 0.05 g l
1
FeSO
4
7H
2
O and 10 g l
1
agar at 37

C for 24 h. Inocula were prepared by washing

cells from plates with 5 ml sterile water. Biomass in the
inocula was measured by optical density at 600 nm and
adjusted to equivalent values by dilution with water to
obtain 6 g l
1
dry cells. Inocula were 5 ml. The experiments
were carried out in 250 ml Erlenmeyer asks with a nal
volume of 100 ml using different media. The content of
nitrogen in the yeast extract, peptone and CSL used was
11.9, 12 and 13% in dry basis, respectively. The content of
water in the yeast extract, peptone and CSL used was 3.5, 4
and 50%, respectively.
Calcium carbonate (10 g) was added previously to inocu-
lation. After inoculation, fermentations were carried out in
orbital shakers at 41.5

C and 200 rpm for 9698 h. The pH

was kept constant around 6 due to the lactic acid formed was
neutralized by the present of calcium carbonate. Samples
(2 ml) were taken at random time intervals and centrifuged
at 16,000g for 3 min. The supernatants were used immedi-
ately for various analyses.
Analytical Methods
Glucose, lactic acid and acetic acid were determined by
high performance liquid chromatography (HPLC) using a
Transgenomic ION-300 column and an isocratic elution
with a ow rate of 0.4 ml min
1
. The mobile phase was
0.0025 M H
2
SO
4
. The oven temperature was 65

C and a
refractive index (RI) detector was used.
Stati sti cal Analysi s
All experimental data were obtained in triplicate and
mean values are given. Linear and non-linear regression
analyses of experimental data were performed using
commercial software (Microsoft Excel 2000, Microsoft
Corporation, Redmond, WA, USA).
RESULTS AND DISCUSSION
The cost of nutrients is an important aspect in the
fermentation of glucose to lactic acid by L. delbrueckii.
General lactobacilli media such as Mercier medium and
MRS medium are very complex with many expensive
nutrients. Table 1 shows the components of the Mercier
medium as well as the cost of each nutrient. The price of
CSL is also included in the same table. As it can be noted,
the price of CSL is two to three times lower than the price of
yeast extract and peptone, respectively.
Comparative Study Between Mercier Medi um
and CSL-based Media
Media containing different concentrations of CSL (1, 3, 5,
10 or 20g l
1
) were tested in the fermentation of 90 g l
1
glucose to lactic acid. For comparative purposes a batch
fermentation using the Mercier medium with 90 g l
1
glucose was also performed.
Figure 1 shows the results of the batch experiments
for lactic acid and glucose concentrations. The highest
lactic acid concentration was obtained using the Mercier
medium (76.2 g l
1
). However, a similar pattern was shown
by the fermentation with 20 g l
1
CSL, obtaining 70.7 g l
1
lactic acid at the end of the fermentation. Using lower
concentrations of CSL, lower lactic acid concentrations
and volumetric productivities were obtained. These facts
suggest that CSL at concentration lower than 20 g l
1
did
not supply the required nutrients for the metabolism of
L. delbrueckii. Additionally, acetic acid was quantied
and negligible concentrations were obtained (data not
shown). This was important because it demonstrated that
L. delbrueckii maintains the homofermentative pathway in
the presence of CSL.
In the experiment using the Mercier medium, the glucose
concentration was completely consumed at the end of the
fermentation (Figure 1b). However, a nal glucose concen-
tration of 12.0 g l
1
was observed in the experiments carried
out with medium containing 20g l
1
CSL. Using lower
concentrations of CSL, higher concentrations of residual
glucose were obtained. This suggested that CSL is limited in
some nutrients.
Table 2 shows numerical values of lactic acid concentra-
tion, product yield (Y
p
=
s
), product efciency (E
p
=
s
) and
economic efciency (E
p
=
) after 98h of fermentation.
Product yield was dened as grams lactic acid produced
per gram glucose consumed, product efciency as grams
lactic acid produced per gram initial glucose and economic
Table 1. Prices of nutrients used in
experiments.
Nutrient Cost (

=kg)
Corn steep liquor 36.06
Yeast extract 76.74
Peptone 112.27
Sodium acetate 13.94
Sodium citrate 20.73
K
2
HPO
4
30.29
MgSO
4
10.58
MnSO
4
15.03
FeSO
4
11.54
ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION 251
Trans IChemE, Vol 81, Part C, September 2003
efciency as grams lactic acid produced per cost unit of
nutrients (

). As it can be observed, E
p
=
s
decreased with the
decrease of CSL concentration. However, Y
p
=
s
was main-
tained around 0.9 g g
1
using the Mercier medium or media
containing 20 or 10g l
1
CSL.
It is interesting to select the cheapest media that allow the
highest lactic acid concentration to be obtained. The E
p
=
is
an adequate parameter to compare media from an econom-
ical point of view. The parameter E
p
=
showed that it was
more protable to use a medium with 20 g l
1
CSL than the
Mercier medium because using 20 g l
1
CSL, 98 g of lactic
acid were produced per euro of nutrients while only 50 g
lactic acid were obtained per euro when the Mercier medium
was employed.
Mathemati cal Model l ing of Fermentation wi th
CSL as a Sole Nutriti onal Source
Lactic acid production and glucose consumption were
mathematically modelled and the regression parameters
obtained were correlated with CSL concentration by linear
or exponential equations. A mathematical model was
adopted from another study to describe the fermentative
production of lactic acid (Mercier et al., 1992):
dP
dt
P
r
P 1
P
P
m

(1)
where t is time, P is lactic acid concentration, P
m
is
maximum concentration of lactic acid, and P
r
is the ratio
between the initial volumetric rate of product formation (r
p
)
and the initial product concentration P
0
. Equation (1) can be
directly solved to give the following expression:
P
P
0
P
m
e
P
r
t
P
m
P
0
P
0
e
P
r
t
(2)
From the series of experimental data for lactic acid
concentration during fermentation, the model parameters
P
0
, P
m
and P
r
can be calculated for each fermentation
medium by non-linear regression using the least-squares
method. Table 3 shows the kinetic and statistical parameters.
Figure 1a shows the experimental and predicted data for
these batches. The coefcient r
2
showed a good agreement
between experimental and predicted data. The value of the
F-test probability showed that the model for 1 g l
1
CSL
medium is the least accurate due to the low value of lactic
acid concentration obtained for this medium.
The models predict maximumlactic acid concentrations of
74.9 g l
1
for the Mercier medium, 66.6 g l
1
for 20 g CSL
l
1
medium and 25.9 g l
1
for 10 g CSL l
1
medium at 98 h.
The regression parameters obtainedfor each experiment were
Figure 1. Experimental and calculated dependence of lactic acid and
glucose concentrations on the fermentation time corresponding to fermen-
tations of 90 g l
1
glucose with different concentrations of corn steep liquor
and the Mercier medium.
Table 2. Results for the lactic acid production by Lactobacillus delbrueckii
using different concentrations of CSL and the Mercier medium. All
fermentations were with 90 g l
1
glucose.
Medium
Lactic acid
concentration
(g l
1
)
Y
p=s
(g g
1
)
E
p=s
(g g
1
)
E
p=

(g
1
)
Mercier medium 76.20 0.85 0.85 50
20 g l
1
CSL 70.73 0.91 0.79 98
10 g l
1
CSL 27.13 0.90 0.30 75
5 g l
1
CSL 15.59 0.66 0.17 86
3 g l
1
CSL 8.55 0.42 0.10 79
1 g l
1
CSL 2.57 0.20 0.03 71
Table 3. Results obtained by regression analysis of lactic acid production and glucose consumption by Lactobacillus delbrueckii using different media (all
media include 90 g l
1
glucose).
Lactic acid production Glucose consumption
Medium P
0
(g l
1
) P
m
(g l
1
) P
r
(h
1
) r
2
F-test of
probability Y
p=s
(g g
1
) r
2
F-test of
probability
Mercier medium 2.81 74.97 0.181 0.9982 0.9759 0.91 0.9966 0.9931
20 g l
1
CSL 4.82 66.66 0.091 0.9783 0.9041 0.91 0.9935 0.9933
10 g l
1
CSL 1.69 25.91 0.137 0.9851 0.9311 0.86 0.9834 0.9767
5 g l
1
CSL 0.99 15.38 0.166 0.9941 0.9559 0.63 0.9883 0.9782
3 g l
1
CSL 0.67 8.88 0.199 0.9892 0.9432 0.46 0.9808 0.9648
1 g l
1
CSL 0.28 2.73 0.412 0.9856 0.8505 0.21 0.9536 0.9775
Trans IChemE, Vol 81, Part C, September 2003
252 TE

LLEZ-LUIS et al.
correlated with the CSL concentration by mean of empirical
equations. P
0
, P
m
and P
r
were related to the CSL concentra-
tion given by equations (3)(5), respectively:
P
0
0:2371C
csl
0:1555 (3)
P
m
3:3189C
csl
1:9735 (4)
P
r
0:3788 C
0:4733
csl
(5)
The coefcient r
2
(0.9710) for P
0
, (0.9858) for P
m
and
(0.9749) for P
r
conrmed that the empirical equations t the
data well. By combining equations (3)(5) with the model of
equation (2), it is possible to predict the lactic acid concen-
tration at any time for CSL concentrations and time in the
range studied (098 h and 120 g l
1
CSL). Figure 2 shows
how the generalized model predicts the dependence of lactic
acid concentration on different CSL concentrations and time
using the model parameters. This kind of surface response
allows the selection of different conditions in order to
achieve the same results.
The consumptionof glucose by L. delbrueckii can be given
by the following equation (obtained from the Y
p
=
s
denition):
S S
0

1
Y
p=s
(P P
0
) (6)
where Y
p
=
s
, P and P
0
were dened above, S is the glucose
concentration (g l
1
) and S
0
is the initial glucose concentra-
tion. Using the series of experimental data concerning
glucose concentration
=
time and the regression parameters
of equation (2), the model parameter Y
s
=
p
can be calculated
for each fermentation medium by non-linear regression
using the least-squares method. Table 3 lists the numerical
values of Y
p
=
s
and statistical parameters obtained for the
glucose consumption and Figure 1b shows the experimental
and predicted data for these fermentations. The parameter
Y
p
=
s
varied with C
csl
according to the following equation:
1
Y
p=s
4:163 C
0:5072
csl
(7)
The statistical parameter r
2
for the empirical equation (7)
was signicant (0.9645). Combining equation (5) with
equation (6), a generalized model for predicting glucose
consumption in CSL media was also developed. Figure 3
shows the prediction of the generalized model for the
dependence of glucose concentration with the CSL concen-
tration and time. The model predicts that more than 45 g l
1
glucose remained in the mediumwhen less than 10 g l
1
CSL
is used. Both models would be very useful for optimization.
Fermentation of Suppl emented CSL Medi a
In order to increase the lactic acid production and the
economic efciency, experiments were conducted using
10 g l
1
CSL supplemented with one component of the
Figure 2. Prediction of the generalized model for the dependence of lactic
acid concentration on the corn steep liquor concentration and time.
Figure 3. Prediction of the generalized model for the dependence of glucose
concentration on the corn steep liquor concentration and time.
Table 4. Results for the lactic acid production by Lactobacillus delbrueckii using CSL supplemented with other nutrients from the
Mercier medium.
Medium
Lactic acid
concentration (g l
1
) Y
p=s
(g g
1
) E
p=s
(g g
1
)
E
p=
(g lactic acid
per

nutrients)
10 g l
1
CSL 27.13 0.90 0.30 75
10 g l
1
CSL10 g l
1
peptone 76.71 0.85 0.85 52
10 g l
1
CSL5 g l
1
yeast extract 77.64 0.89 0.86 104
10 g l
1
CSL2 g l
1
citrate 41.30 0.72 0.46 103
10 g l
1
CSL5 g l
1
acetate 40.04 0.77 0.44 111
10 g l
1
CSL2 g l
1
K
2
HPO
4
29.10 0.66 0.32 69
10 g l
1
CSL0.58g l
1
MgSO
4
25.49 0.84 0.27 67
10 g l
1
CSL0.12g l
1
MnSO
4
37.35 0.94 0.41 103
10 g l
1
CSL0.05g l
1
FeSO
4
48.53 0.85 0.54 134
Trans IChemE, Vol 81, Part C, September 2003
ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION 253
Mercier medium at a time, in the same concentrations.
Table 4 lists the medium used and results for lactic acid
concentration obtained and Y
p
=
s
, E
p
=
s
and E
p
=
calculated at
98 h of fermentation for media of supplemented CSL.
Figure 4 shows the experimental and calculated results
from the fermentation of 10 g l
1
CSL alone as control and
10 g l
1
CSL supplemented with peptone or yeast extract
(the main nutritional components of the Mercier medium).
Both exhibited a signicant effect. The highest lactic acid
concentration (77.64g l
1
) was obtained by supplementing
10 g l
1
CSL with 5 g l
1
yeast extract. Similar lactic acid
concentration (76.71g l
1
) was also obtained by supple-
menting with 10 g l
1
peptone. These values compare very
well with those achieved with the Mercier medium and the
20 g l
1
CSL medium. The E
p
=
s
was also higher in the above
two cases. Although the glucose consumed was different,
the Y
p
=
s
was similar or slight lower than that without
supplementation. The values of Y
p
=
s
compare well with
those reported using other microorganisms like Rhizopus
oryzae (Zhou et al., 1999). The E
p
=
was decreased by
30.66% using the medium supplemented with peptone and
increased by 38.66% when using the medium supplemented
with yeast extract (Table 4). The E
p
=
values showed that it
is most economically interesting to supplement CSL with
yeast extract than with peptone. The importance of yeast
extract in the preculture media is stressed (Amrane and
Prigent, 1994). They proposed that the main contributors of
yeast extract are the purine and pyridine bases and B group
Figure 4. Experimental and calculated dependence of lactic acid and
glucose concentrations on the fermentation time corresponding to fermen-
tations of 90 g l
1
glucose with different concentrations of corn steep liquor
alone and supplemented with peptone or yeast extract.
Figure 5. Experimental and calculated dependence of lactic acid and
glucose concentration on the fermentation time corresponding to fermenta-
tions of 90 g l
1
glucose with different concentrations of corn steep liquor
supplemented with citrate, acetate or phosphate.
Figure 6. Experimental and calculated dependence of lactic acid and
glucose concentration on the fermentation time corresponding to fermenta-
tions of 90 g l
1
glucose with different concentrations of corn steep liquor
supplemented with MgSO
4
, MnSO
4
or FeSO
4
.
Trans IChemE, Vol 81, Part C, September 2003
254 TE

LLEZ-LUIS et al.
vitamins. The importance of yeast extract to Lactobacilli has
been reported (Hujanem and Linko, 1996).
Figure 5 shows experimental results for the fermentation
of 10 g l
1
CSL supplemented with the carboxylic salts and
mineral acids (sodium citrate, sodium acetate and sodium
phosphate) of the Mercier medium. Citrate, acetate and phos-
phate decreased signicantly the value of Y
p
=
s
but the E
p
=
s
was increased after supplementing with citrate or acetate.
Using 10 g l
1
CSL alone, lactic acid concentration was
27.13g l
1
. This value slightly increased with supplementa-
tion with acetate or citrate but enhancement was not observed
with phosphate. The involvement of citrate and acetate in the
metabolismcycles could be the explanation. The supplemen-
tation of CSL with citrate and acetate enhanced the economy
of the lactic acid fermentation. E
p
=
was 111 g lactic acid per

nutrient for the fermentation of the 10 g l

1
CSL medium
supplemented with 5 g l
1
acetate. The E
p
=

of media with
citrate was the same as that for yeast extract (Table 4).
Figure 6 shows the experimental results from the fermen-
tation of 10g l
1
CSL supplemented with MgSO
4
, MnSO
4
and FeSO
4
, the mineral sources of the Mercier medium. The
lactic acid concentration obtained by supplementing CSL
with MgSO
4
remained the same as that of medium without
supplementation at 98h. It is reported that magnesium is
a key element in lactic acid fermentation (Thomas and
Ingledew, 1990). In our study, addition of MgSO
4
had no
effect on lactic acid production. This must have occurred
because CSL contains 1.5% Mg
2
on a dry basis (Zabriskie
et al., 1980). Using MgSO
4
as a component of the CSL
medium decreased the E
p
=
of the process.
Better results were obtained by supplementing with
MnSO
4
or FeSO
4
(Table 4). Although the highest lactic
acid concentration was obtained by supplementing with
yeast extract, the E
p
=
showed that the better supplement
is 0.05 g l
1
FeSO
4
because 134 g lactic acid per

of
nutrients was obtained. This value is 75% higher than the
E
p
=
obtained with 10 g l
1
CSL, 36% higher than to
20 g l
1
CSL medium and 168% higher than the E
p
=

value when the Mercier medium was used.

Mathemati cal Modell i ng of Fermentation wi th CSL
Supplemented wi th Nutri ti onal Source
The experimental lactic acid production and glucose
concentration data were examined using equation (2) and
equation (6). The kinetic parameters of P
0
, P
m
and P
r
were
calculated for each fermentation medium by non-linear
regression. The results are shown in Table 5 together with
the statistical parameters. The coefcient r
2
showed that all
the equations obtained were well tted and Figures 46
conrm the good agreement between experimental and
predicted data. The value of F-test probability also showed
that the model was accurate. The values of P
0
obtained are
higher than the values reported by others (Parajo et al.,
1996). This was because the CSL contains a low concentra-
tion of lactic acid (Hull et al., 1996).
Table 5 also shows the parameter Y
p
=
s
and statistical
parameters for the consumption of glucose. The coefcient
showed a good agreement between experimental and
predicted data. Figures 46 also display comparison
between experimental and predicted data. The values of
Y
p
=
s
are in agreement with those reported in the literature
(Parajo et al., 1996).
CONCLUSIONS
Alternative media based on CSL were evaluated in order
to improve the economic efciency of the lactic acid
production by Lactobacillus delbrueckii NRRL B-445. CSL
is a cheaper nutrient source than other complex media like
that proposed by Mercier. In this work, it was demonstrated
that a medium containing 10g l
1
corn steep liquor is more
economically efcient than the Mercier medium but it is not
a balanced nutritional medium for Lactobacillus delbrueckii.
It can be improved by adding other supplements such as
yeast extract or mineral salts. A medium containing CSL
(10 g l
1
) with 0.05 g l
1
FeSO
4
is an economically efcient
medium for lactic acid production by Lactobacillus
delbrueckii NRRL B-445.
REFERENCES
Amrane, A. and Prigent, Y., 1994, Mathematical model for lactic acid
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a
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a
The CSL concentration was 10 g l
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ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION 255
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ACKNOWLEDGEMENTS
The Authors are grateful to Xunta de Galicia for the nancial support of
this work (Project XUGA PGIDT00PXI38301PR). A grant from the
PROMEP program of the Secretar a de Educacion Publica (Mexico) to
author Tellez-Luis is gratefully acknowledged.
ADDRESS
Correspondence concerning this paper should be addressed to
Dr M. Vazquez, A

rea de Tecnolog a de los Alimentos, Escuela Politecnica

Superior, Departamento de Qu mica Anal tica, Universidad de Santiago
de Compostela, Campus de Lugo, 27002 Lugo, Spain.
E-mail: vazquezm@lugo.usc.es
The manuscript was received 7 May 2002 and accepted for publication
after revision 30 April 2003.
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