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Optimization of lactic acid production from

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NCIMB 8130

Article in World Journal of Microbiology and Biotechnology July 2002

DOI: 10.1023/A:1015523126741

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 World Journal of Microbiology & Biotechnology 18: 441448, 2002. 441

2002 Kluwer Academic Publishers. Printed in the Netherlands.

Optimization of lactic acid production from beet molasses

by Lactobacillus delbrueckii NCIMB 8130

Ch. Kotzamanidis1, T. Roukas2,* and G. Skaracis1

1
Hellenic Sugar Industry S.A., Department of Planning-R & D, Laboratory of Industrial Microbiology, 57400
Thessaloniki, Greece
2
Aristotle University of Thessaloniki, Department of Food Science and Technology, Box 250, 54124 Thessaloniki,
Greece
*Author for correspondence: Tel.: +30-31-998776; Fax: +30-31-998789, E-mail: roukas@agro.auth.gr

Received 16 October 2001; accepted 6 March 2002

Keywords: Beet molasses, fermentation, lactic acid, Lactobacillus delbrueckii, mathematical modelling

Summary

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake ask
fermentation was investigated. Shake asks proved to be a better fermentation system for this purpose. Substitution
of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid
concentration was achieved without treatment of molasses. A Central Composite Design was employed to
determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast
extract, CaCO3). A satisfactory t of the model was realized. Lactic acid production was signicantly aected both
by sucroseyeast extract and sucroseCaCO3 interactions, as well as by the negative quadratic eects of these
variables. Sucrose and yeast extract had a linear eect on lactic acid production while the CaCO3 had no signicant
linear eect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast
extract and CaCO3 of 89.93, 45.71 and 59.95 g/l, respectively.

Introduction ranose), nitrogen compounds, organic acids, amino

Lactic acid is used in the pharmaceutical, chemical, and
food industries primarily as an acidulant and preserva-
tive agent (Roukas & Kotzekidou 1998). It is commer-
cially produced by fermentation of corn sugars,
molasses, and whey with homofermentative lactic acid
bacteria.
Production of lactic acid has been reported from a
synthetic medium and whey permeate by free as well as
immobilized lactic acid bacterial cells using batch,
fed-batch and continuous culture (Audet et al. 1989;
Aeschlimann & Stockar 1990; Guoqiang et al. 1991;
Roukas & Kotzekidou 1991, 1996, 1998; Chiarini et al.
1992; Demirci & Pometto 1995; Bruno-Barcena et al.
1999). Hang (1989), Zhang & Cheryan (1991), and
Yumoto & Ikeda (1995) have studied lactic acid
production from starch by Rhizopus oryzae, Lactobacil-
lus amylovorus and L. amylophilus, respectively. How-
ever, utilization of glucose, sucrose or starch as carbon
source is not economical, and the exploitation of a less
expensive carbohydrate source could prove benecial.
Molasses is a byproduct of the sugar industry readily
available at relatively low cost. It contains water,
approximately 50% sugars (sucrose, glucose, fructose,
acids, heavy metals, etc. Monteagudo et al. (1997)
studied the kinetics of lactic acid fermentation by
L. delbrueckii grown on beet molasses. The production
of lactic acid from beet molasses by free and immobi-
lized L. delbrueckii IFO 3202 cells using batch and
continuous culture has been described (Goksungur &
Guvenc 1997, 1999). Monteagudo et al. (1993, 1994)
studied the eect of the fermentation parameter (tem-
perature, pH, inoculum concentration, and initial sugar
concentration) on lactic acid production from beet
molasses by L. delbrueckii CECT 286. Moreover, these
authors used a Central Composite Design to determine
the most suitable nutrient medium for obtaining a
maximum cell concentration.
High heavy metal (iron, zinc, copper, manganese,
magnesium, calcium, etc.) concentrations in the medium
cause a critical problem during fermentation as they
inhibit the growth of microorganisms, inuence sub-
strate pH, and are involved in the inactivation of
enzymes associated with product biosynthesis. During
fermentation of molasses to lactic acid, the medium pH
decreases resulting in a signicant decrease of microor-
ganism biomass. To avoid drop of pH, calcium carbon-
ate is added to the medium thus maintaining the pH
442
between 6.5 and 7.0. The most important factors for the
production of lactic acid are the concentration of sugar,
yeast extract and calcium carbonate. Thus, mathemat-
ical modelling was proposed to determine the most
suitable concentrations of the above parameters that
would contribute to maximum lactic acid production.
Given that yeast extract is quite expensive, other low-
cost nitrogen compounds were tested as replacements.
Also, dierent techniques were used to remove the heavy
metals from molasses in order to increase the production
of lactic acid by L. delbrueckii NCIMB 8130.
Materials and methods
Microorganism and culture conditions
Lactobacillus delbrueckii NCIMB 8130 was used
throughout this study. The microorganism was main-
tained on MRS agar plates at 4 C and subcultured
every 3 weeks. Cells for inoculation of the production
medium at a level of 10% (v/v) were obtained from
cultures grown on MRS broth (pH 7.0) at 37 C for
16 h.
Pretreatment of molasses
Beet molasses, obtained from the sugar plant of Hellenic
Sugar Industry at Platy, Imathias was diluted with
distilled water to obtain 10% (w/v) total sugar concen-
tration. This molasses solution was used for all the
pretreatment methods (cation exchange resin, sulphuric
acid, tricalcium phosphate, potassium ferricyanide and
EDTA treatment) as described in our previous work
(Roukas 1998).
Carbon activated treatment
Fifteen gram activated carbon (Riedel-de Haen, K-
903386) was added to 200 ml of molasses solution
containing 10% (w/v) sugars and the mixture was
heated with continuous stirring at 50 C for 15 min.
Activated carbon was removed by ltration and the
process was repeated four times until the solution was
colourless. The pH of the ltrate was adjusted to 7.0
with 1 N NaOH and the solution sterilized at 121 C for
15 min.
Sources of proteins
Yeast extract from lager tank sediment
Lager tank sediment, brewery yeast, was the sludge
obtained after wort fermentation and lagering of beer.
The lager tank sediment was centrifuged at 5000 g for
15 min and the yeast cells were treated by dierent
methods. The yeast cells were lyophilized and the
sediment used as supplement in the molasses solutions.
The yeast cells were diluted in physiological saline and
Ch. Kotzamanidis et al.
disrupted in a sonicator (Sonics and Materials Inc.,
Danbury, Connecticut, USA, model VC375) for 25 min.
The solution was centrifuged at 5000 g for 15 min and
the supernatant was lyophilized. The powder was used
as source of protein in the molasses solution.
Protein from cheese whey
Cheese whey was obtained from a local feta cheese plant
(Stamoulas, Thessaloniki). It contained 5% (w/v) lac-
tose and had a pH of 5.0. Proteins were precipitated by
heating the whey at 90 C for 20 min. The protein was
removed by centrifugation at 5000 g for 15 min and
dried at 50 C under vacuum overnight, and was used to
supplement the molasses solution.
Study of fermentation parameters
Initial sugar concentration
The molasses was diluted with distilled water to give
concentrations of 8, 10 and 12% (w/v) initial sugar
(0.26, 0.33 and 0.40% (w/v)) total nitrogen compounds
(betaine, amino acids, nitrates, nitrites), respectively. A
series of conical asks containing 200 ml molasses
solution supplemented with 5% (w/v) yeast extract
and 5% (w/v) calcium carbonate (pH 7.0) were inocu-
lated with 20 ml of the inoculum and incubated at 45 C
in a rotary shaker/incubator (orbital shaker, Forma
Scientic, USA) at 150 rev/min.
Yeast extract
A series of fermentation experiments were performed in
conical asks to examine the eect of yeast extract on
lactic acid production. Molasses solution containing
10% (w/v) initial sugar (0.33% total nitrogen com-
pounds) was supplied with dierent concentrations of
yeast extract (Scharlau, 7-079), cheese whey proteins,
and yeast extract from brewery yeasts (produced by
dierent methods as earlier described). The pH of the
medium was adjusted to 7.0 with 1 M NaOH or 1 M
HCl and sterilized at 121 C for 15 min. All experiments
were carried out in 500 ml conical asks containing
200 ml of fermentation medium and incubated as
described above for 24 h.
Calcium carbonate
A set of conical asks containing 200 ml molasses
solution (initial sugar 10%, yeast extract 5%, total
nitrogen compounds 0.33%, pH 7.0) at dierent calcium
carbonate concentrations (1, 3, 5, 7 and 9%) were
incubated at 45 C in a rotary shaker incubator at
150 rev/min for 24 h.
Treated molasses
A series of conical asks containing 200 ml treated
molasses solution (initial sugar 10%, yeast extract 5%,
calcium carbonate 5%, pH 7.0) were inoculated with
20 ml of the inoculum and incubated at 45 C as already
described.
Lactic acid production from molasses 443
Analytical techniques Table 2. Experimental design.
At specic time intervals, the asks were removed and
Runs Sucrose Yeast Calcium Lactic acid
the fermentation broth was analysed. Lactic acid con- (g/l) extract (g/l) carbonate (g/l) concentration (g/l)
centration was measured by HPLC using a PL Hi Plex
H column 300 7.7 mm (Polymer Laboratories Inc, 1 70.000 30.000 30.000 63
USA) and a u.v. detector at 210 nm with 0.005 M 2 120.000 30.000 30.000 53
3 70.000 70.000 30.000 66
H2SO4 as the eluent at a ow rate of 0.6 ml/min at 4 120.000 70.000 30.000 20
55 C. The number of living cells was determined by 5 70.000 30.000 80.000 60
plate counting on MRS agar at 37 C for 48 h. Residual 6 120.000 30.000 80.000 63
sugars (glucose, fructose, sucrose) were determined as 7 70.000 70.000 80.000 66
sucrose by the method of Dubois et al. (1956). 8 120.000 70.000 80.000 27
9 52.955 50.000 55.000 55
Data presented are means of triplicate experiments. 10 137.045 50.000 55.000 22
Variability was also expressed by coecient variation 11 95.000 16.364 55.000 53
(cv) values (cv sd=
x  100). 12 95.000 83.635 55.000 29
13 95.000 50.000 12.955 78
Experimental design and statistical analysis 14 95.000 50.000 97.044 85
15 95.000 50.000 55.000 88
16 95.000 50.000 55.000 88
The statistical analysis of the data was performed using 17 95.000 50.000 55.000 86
the Minitab package. Details of response surface meth- 18 95.000 50.000 55.000 88
odology can be found elsewhere (Neter et al. 1996). The 19 95.000 50.000 55.000 86
levels of factors used in the experimental design are 20 95.000 50.000 55.000 88
listed in Table 1. The concentrations of the factors were
chosen after a series of experiments as shown in Figures
24. In this design there are ve experimental levels: a, ecient estimates with b0 having the role of a scaling
1, 0, +1, +a where a 2n =4, n is the number of constant.
variables and 0 correspond to central point. The actual
level of each factor was calculated using the following
equation (Neter et al. 1996). Results and discussion

Actual value High level

2
Low level
Static vs. shake ask fermentation
Coded value High level  Low level
2
Production of lactic acid from molasses by L. delbrueckii
Twenty experiments were conducted using a central in static and shake ask fermentation is shown in
composite statistical design for the study of three factors Figure 1. The cultures grown in static and shake asks
each at ve levels (Table 2). Response surface model produced the same amount of lactic acid (45 g/l) after 24
was tted to one response variable, namely lactic acid and 18 h, respectively. The shorter fermentation time of
concentration (g/l). The most commonly used empirical shake ask culture probably is due to a better diusion
model, polynomial response surface, was tted to of substrate into the cells by improving the mass transfer
measure the response variable. The second-order poly- characteristics with respect to substrate and product/
nomial model was tted to response giving an equation byproducts of the fermentation broth. As shown in
of the term Figure 1a, b biomass concentration decreased after the
rst 6 h of fermentation, while at the same time lactic
Y b0 b1 A b2 B b3 C b11 A2 b22 B2 b33 C 2 acid concentration increased. This means that there is
not a parallel relationship between biomass and lactic
b12 AB b13 AC b23 BC 1 acid concentration. These results agree with those of
Monteagudo et al. (1997) who studied the production of
where A, B, and C represent the levels of the factors lactic acid from molasses by L. delbrueckii. The mech-
according to Table 1, and b0 ; b1 ; . . . ; b23 represent co- anism for lactic acid inhibition of growth is not fully
understood. The accepted mechanism of inhibition by
weak organic acids is related to the solubility of the non-
dissociated form within the cytoplasm and the insolu-
Table 1. Levels of the factors used in the experimental design.
bility of the ionized acid form. This causes acidication
Factor Name Levels of the cytoplasm and the collapse of the proton motive
force, resulting in inhibition of nutrient transport.
)a )1 0 +1 +a
Finally, the energy gained by lactate production is no
A Sucrose (g/l) 52.955 70.000 95.000 120.000 137.045 longer available for cell growth but is completely used
B Yeast extract (g/l) 16.364 30.000 50.000 70.000 83.635 for maintenance of pH homeostasis (Goncalves et al.
C Calcium 12.955 30.000 55.000 80.000 97.044
1997). Moreover, autolysis of the cell occurs due to the
carbonate (g/l)
high concentration of lactic acid. Thus, the viable cell
444 Ch. Kotzamanidis et al.
culture (fermentation time 18 vs. 24 h). Thus, the shake
ask was selected over static fermentation for all
subsequent studies.

Eect of initial sugars

As shown in Figure 2, lactic acid concentration in-

creased with the increase of initial sugar concentration
up to 100 g/l but then signicantly decreased beyond
this value (Figure 2a). The highest concentration of
lactic acid (90 g/l) was obtained after 24 h with an initial
sugar concentration of 100 g/l. When initial sugar
concentrations were 80 and 120 g/l, maximum lactic
acid concentration was lowered by 22.2 and 55.5%,
respectively. Goksungur & Guvenc (1997) and Monte-
agudo et al. (1997) reported a maximum lactic acid
concentration of 57 and 80 g/l when L. delbrueckii was
grown on beet molasses containing 65 and 107 g/l initial
sugar after 22 and 38 h of incubation, respectively.

Figure 1. Fermentation kinetics of L. delbrueckii during lactic acid

production from beet molasses in static (-s-) and shake ask (-h-)
culture. Data are means of triplicate experiments; cv values did not
exceed 3.5% in all cases (initial sugar concentration 70 g/l).

count is decreased and the liberated enzymes continue to

produce lactic acid. Biomass concentration was higher
in culture grown in static rather than in shake ask
culture. Lactobacillus delbrueckii is an anaerobic micro-
organism. Consequently, the microorganism grows bet-
ter in static culture where the fermentation conditions
are anaerobic. Biosynthesis of lactic acid was carried out
during the growth and death phase of the microorgan-
ism. The maximum viable cell number (0.8 108 c.f.u./
ml) was observed in static culture after 6 h of the
fermentation.
As expected, the concentration of residual sugars
decreased during the fermentation and there was an
increase in lactic acid production. The concentration of
residual sugars fell rapidly during the rst 18 h of
fermentation, after which it slowly decreased owing to
the rapid increase of lactic acid concentration observed
at the same time. When the maximum concentration of
lactic acid was achieved in static and shake ask culture,
97% of sugars had been converted to lactic acid.
Consequently, both static and shake ask culture were
Figure 2. Fermentation kinetics of L. delbrueckii during lactic acid
found to be satisfactory fermentation systems for the
production from beet molasses in shake ask culture at dierent sugar
production of lactic acid from beet molasses by concentrations. (-s-), (-h-), (-n-): Initial sugar concentration 80, 100
L. delbrueckii. However, the shake ask system gave a and 120 g/l, respectively. Data are means of triplicate experiments; cv
higher lactic acid productivity as compared to the static values did not exceed 3.0% in all cases.
Lactic acid production from molasses 445
Roukas & Kotzekidou (1998) reported that a high
concentration of lactic acid (25 g/l) was obtained when a
co-culture of L. casei and Lactococcus lactis was grown
in deproteinized whey, while Yumoto & Ikeda (1995)
found that a maximum lactic acid concentration (53.4
g/l) was obtained when L. amylophilus was grown in
synthetic medium containing 100 g/l starch. In all cases,
the number of viable cells decreased during fermenta-
tion. When the initial sugar concentration was increased
from 100 to 120 g/l, a signicant decrease in viable cells
was observed during the rst 18 h of fermentation, due
to the inhibition of growth by the high sugar concen-
trations (Roukas 1993). As shown in Figure 2, the
residual sugar concentration decreased during fermen-
tation. Increasing the initial sugar concentration from 80
to 120 g/l resulted in a signicant increase of the residual
sugar concentration. This was expected since there was
also a decrease in biomass and lactic acid concentration.
The increase in residual sugars was due to the inability
of the microorganism to metabolize high levels of
sugars. When the maximum concentration of lactic acid
was achieved in cultures grown at an initial sugar
concentration of 80, 100 and 120 g/l, 96.7% of sugar Figure 3. Eect of yeast extract on kinetic parameters of beet molasses
fermentation by L. delbrueckii in shake ask culture. (-s-) lactic acid;
consumed was converted to lactic acid. In this case, the
(-h-) viable cells number; (-d-) lactic acid yield; (-j-) sugar utilization.
total amount of sugar utilized was 87.5, 93 and 37.5% in Data are means of triplicate experiments; cv values did not exceed
fermentations with 80, 100 and 120 g/l initial sugar 2.5% in all cases (fermentation time 24 h).
concentration, respectively. Monteagudo et al. (1994)
reported a maximum lactic acid yield of 87.8% when L.
delbrueckii was grown in beet molasses in submerged grown in whey permeate supplemented with 1.52.5%
fermentation. The decreased sugar utilization encoun- yeast extract. Several possible explanations for such
tered with the highest concentration probably was due dierences include the strain of microorganism, the
to osmotic eects. It has been reported that above a chemical composition of the substrate, the fermentation
critical substrate concentration, the decreased water system, and generally the conditions employed during
activity and the onset of plasmolysis combine to cause a fermentation.
decrease in the rates of fermentation and lactic acid
production (Roukas 1993). Substitution of yeast extract with other protein sources

Eect of yeast extract
As shown in Figure 3, the lactic acid concentration,
number of viable cells, lactic acid yield and sugar
utilization all increased with the increase of yeast extract
concentration from 1 to 5% (w/v), while higher con-
centrations caused a decrease of the fermentation
parameters, indicating that these concentrations of yeast
extract might be toxic. Maximal lactic acid concentra-
tion (90 g/l), number of viable cells (0.8 105 c.f.u./ml),
lactic acid yield (96.7%), and sugar utilization (93%)
were obtained in culture grown in medium supplement-
ed with 5% (w/v) yeast extract. These results are in
contrast with those of Goksungur & Guvenc (1997) who
studied the eect of yeast extract on lactic acid produc-
tion from beet molasses by L. delbrueckii IFO 3202 and
found that the maximum lactic acid concentration (60
g/l) was obtained when the medium was supplemented
with 1% (w/v) yeast extract. On the other hand,
Aeschlimann & Stockar (1990) and Chiarini et al.
(1992) reported that a maximum lactic acid concentra-
tion (4245 g/l) was obtained when L. helveticus was
Yeast extract is the most important medium component
in lactic acid fermentation, being a principal growth
factor for lactic acid bacteria. However, yeast extract is
an expensive material to be used at industrial level
process. For this reason, yeast extract was replaced by
low cost protein sources such as proteins from brewery
yeast and cheese whey. The amount of each protein
source was calculated to give the equivalent concentra-
tion of 5% (w/v) yeast extract. The results are shown in
Table 3. As can be seen, the medium supplemented with
yeast extract gave the highest lactic acid concentration
(90 g/l) compared with the other media supplied with
other protein sources. In all cases, the fermentation
parameters (except lactic acid yield) were very low
compared to those of the medium supplemented with
yeast extract. In conclusion, supplementation of molas-
ses with yeast extract signicantly aects lactic acid
concentration, biomass and sugar utilization. The
importance of yeast extract supplement could be ex-
plained by the fact that it contains critical amounts of
vitamins and trace elements essential for lactic acid
biosynthesis.
446
Table 3. Lactic acid production from molasses by L. delbrueckii using dierent protein sources (fermentation time 24 h).
Substrates Lactic
acid (g/l)
Molasses (10% initial sucrose, 5% yeast extract, 90.0
5% calcium carbonate)
Molasses + 5% (w/v) dried brewery yeast cells 41.0
Molasses + 5% (w/v) protein extracted 14.0
from brewery yeast cells by sonication
Molasses + 5% (w/v) protein precipitated 21.0
from cheese whey by heating
Eect of calcium carbonate
Lactobacillus delbrueckii has an optimum growth pH
between 6.0 actobacillus and 7.0. In static and shake
ask cultures, the pH was controlled at 6.07.0 with the
addition of CaCO3. Various concentrations of CaCO3
were used in order to examine its eect on lactic acid
production. Almost all fermentation parameters (except
lactic acid yield) increased with the increase of CaCO3
concentration up to 5% (w/v), remained constant
between 5 and 7% and then decreased (Figure 4). Lactic
acid yield practically remained constant. The highest
values of lactic acid concentration, lactic acid yield,
number of viable cells and sugar utilization were
obtained at a CaCO3 concentration of 5% (w/v). The
decrease in lactic acid concentration at concentrations of
CaCO3 higher than 7% (w/v) may be due to the
Figure 4. Eect of CaCO3 on kinetic parameters of beet molasses
fermentation by L. delbrueckii in shake ask culture. Symbols as in
Figure 3. Data are means of triplicate experiments; cv values did not
exceed 4.0% in all cases (fermentation time 24 h).
Ch. Kotzamanidis et al.
Viable cells Lactic acid Sugar
number (c.f.u/ml) yield (%) utilization (%)
0.8 105 96.7 93.0
1.3 103 95.3 43.0
5.6 102 93.3 15.0
1.6 102 95.4 22.0
inhibition of the enzyme activities which are responsible
for the biosynthesis of lactic acid. Moreover, high
concentrations of CaCO3 inhibit the growth of micro-
organisms.
Eect of treatment of beet molasses on lactic acid
production
Production of lactic acid from pretreated beet molasses
using dierent chemical methods to precipitate the
heavy metals and remove the coloured substances is
shown in Table 4. As can be seen, untreated molasses
gave the highest concentration of lactic acid compared
to the other methods. This means that beet molasses
contained desirable substances which signicantly af-
fected lactic acid production. Highest concentration of
lactic acid (90 g/l), number of viable cells (0.8
105 c.f.u./ml), lactic acid yield 96.7%, and sugar utili-
zation 93% were obtained in culture grown on untreated
molasses. A general conclusion is that sucrose, yeast
extract and CaCO3, signicantly inuence lactic acid
production from molasses by L. delbrueckii. In turn, a
Central Composite Design was used to determine the
optimum level of these parameters leading to a maxi-
mum lactic acid concentration.
Optimization of lactic acid production
The eect of the three previously mentioned variables,
each at ve levels, and their interactions on lactic acid
concentration has been determined. Analysis of variance
for lactic acid production is presented in Table 5. The
analysis tests the value of the model and determines if a
more complex model could provide a better t. If the F-
test for the model is signicant at the 5% level (i.e.
P < 0:05), then the model can adequately account for
the variation observed. If the F-test for lack of t is
signicant, then a more complicated model is needed. As
shown in Table 5, R2 was 0.998 and the F-test for the
regression was signicant at a level of 5% (P < 0:05).
Moreover, the lack of t was not signicant at the 5%
level (P > 0:05). Consequently, the model chosen can
satisfactorily explain the eects of the three factors
(sucrose, yeast extract, CaCO3) on lactic acid produc-
tion from beet molasses by L. delbrueckii. The following
model was tted for lactic acid concentration:
Lactic acid production from molasses 447
Table 4. Lactic acid production from pretreatment molasses by L. delbrueckii in shake ask culture (fermentation time 24 h).

Pretreatment Lactic Viable cells Lactic acid Sugar

of molasses acid (g/l) number (c.f.u./ml) yield (%) utilization (%)

Sulfuric acid treatment 11.0 8.3 102 91.6 12.0

Tricalcium phosphate treatment 44.0 3.1 104 97.7 45.0
Potassium ferricyanide treatment 83.0 4.1 104 97.6 85.0
EDTA treatment 86.0 5.8 104 96.6 89.0
Cation exchange resin 15.0 3.8 102 93.7 16.0
Carbon activated treatment 12.0 1.9 102 92.3 13.0
Untreated molasses 90.0 0.8 105 96.7 93.0

Table 5. Analysis of variance for lactic acid concentration (R2 = 0.998).

Source DF Seq SS Adj SS Adj MS F P

Regression 9 10517.9 10517.86 1168.65 547.61 0.000

Linear 3 2379.2 5401.00 1800.33 843.61 0.000
Square 3 7328.1 7328.12 2442.71 1000.00 0.000
Interaction 3 810.5 810.50 270.17 126.60 0.000
Residual Error 10 21.3 21.34 2.13
Lack of t 5 16.0 16.01 3.20 3.00 0.126
Pure error 5 5.3 5.33 1.07
Total 19 10539.2

responding to the optimum levels of sucrose, yeast

Y 287:452 5:531A 5:547B  0:027A2
 0:041B2  0:003C 2  0:020AB 0:004AC
2 tting of the experimental data to Equation (2) allowed
where A, B and C are the coded levels ()a, +a) of
factors shown in Table 1. The full second-order model,
Equation (1), was simplied by omitting two terms (C,
BC) that were not statistically signicant. Table 6 shows
that sucrose and yeast extract have a strong positive
linear eect on the response (P  0:05). There were also
signicant negative quadratic eects of sucrose, yeast
extract, and CaCO3, indicating that the lactic acid
concentration increases with the increase of these
parameters, then it reaches a maximum, and nally it
decreases at even higher concentrations of them. Signif-
icant interactions were noted between sucrose and yeast
extract, as well as sucrose and CaCO3. On the other
hand, CaCO3 and the interactions between CaCO3 and
yeast extract appeared to have no signicant eect on
lactic acid production (Table 6, P  0:05). In order to
determine the maximum lactic acid concentration cor-
extract and CaCO3, a second-order polynomial model
was used to calculate the values of these variables. The
the determination of the concentration of sucrose
(A 89:93 g/l), yeast extract (B 45:71 g/l), and
CaCO3 (C 59.95 g/l) giving a maximum lactic acid
concentration (88.0 g/l) and, therefore, the optimization
of lactic acid production from beet molasses by
L. delbrueckii. Finally, a maximum lactic acid concen-
tration of 90.0 g/l was obtained under optimized con-
ditions and this is close to 88.0 g/l predicted using
mathematical Equation (2). Hence, it is concluded that
lactic acid production from beet molasses has been
optimized using L. delbrueckii NCIMB 8130.
Conclusions
The results show some important aspects of lactic acid
production from beet molasses by L. delbrueckii. Shake
ask culture has proven a better fermentation system

Table 6. Estimated regression coecients for lactic acid concentration.

Term Coecient SE coecient T P

Constant )287.452 9.7299 )29.543 0.000

Sucrose 5.531 0.1366 40.484 0.000
Yeast extract 5.547 0.1500 36.979 0.000
Calcium carbonate 0.035 0.1169 0.301 0.769
Sucrose*Sucrose )0.027 0.0006 )44.523 0.000
Yeast extract* Yeast extract )0.041 0.0009 )42.226 0.000
Calcium carbonate* Calcium carbonate )0.003 0.0006 )5.016 0.001
Sucrose*Yeast extract )0.020 0.0010 )18.877 0.000
Sucrose*Calcium carbonate 0.004 0.0008 4.840 0.001
Yeast extract*Calcium carbonate )0.0 0.0010 )0.000 1.000
 448
than static culture. Replacement of yeast extract by
other protein sources was unsuccessful, resulting in very
low concentrations of the product. Pretreatment of beet
molasses with dierent chemical methods did not
improve the production of lactic acid. Sucrose, yeast
extract and CaCO3 were of paramount importance in
lactic acid production.
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