Appl Biochem Biotechnol (2011) 164:1160–1171

DOI 10.1007/s12010-011-9202-6

D(−)-LacticAcid Production by Leuconostoc

mesenteroides B512 Using Different Carbon
and Nitrogen Sources

Luciana Fontes Coelho & Cristian J. Bolner de Lima &

Marcela Piassi Bernardo & Jonas Contiero

Received: 8 September 2010 / Accepted: 8 February 2011 /

Published online: 1 March 2011
# Springer Science+Business Media, LLC 2011

Abstract Sugar concentration from sugarcane juice and yeast autolysate increased lactic
acid production more than the other agro-industrial substrates tested. The concentrations of
these two components were further optimized using the Plackett–Burman design and
response surface method. A second-order polynomial regression model estimated that a
maximal lactic acid production of 66.11 g/L would be obtained when the optimal values of
sugar and yeast autolysate were 116.9 and 44.25 g/L, respectively. To validate the
optimization of the medium composition, studies were carried out using the optimized
conditions to confirm the result of the response surface analysis. After 48 h, lactic acid
production using the shake-flask method was at 60.2 g/L.

Keywords Response surface method . Medium optimization . D(−)-Lactic acid . Residues

Introduction

Lactic acid has been used in biodegradable plastics, such as polylactic acid (PLA), and can
be used to improve the physical properties in the production of garbage bags, agricultural
plastic sheeting, and food packaging [1]. It can also be used in sutures and surgical implants
due to its biocompatible and bioabsorbable characteristics [2]. Lactic acid is industrially
produced either through chemical synthesis or microbial fermentation. The advantage of the
biological method is that an optically pure lactic acid can be obtained by choosing a strain
of lactic acid bacteria, whereas chemical synthesis always results in a racemic mixture of
lactic acid [3]. The optical purity of lactic acid is very important to the physical properties
of PLA and obtaining a more stable crystalline polymer than that achieved with a racemic
lactic acid [4, 5]. Polymers made with L(+)-lactic acid have a melting point of 175 °C.
However, this melting point can be increased by adding D(−)-lactic acid, thereby producing
a stereocomplex with a melting point of around 230 °C [6, 7].

L. F. Coelho : C. J. B. de Lima : M. P. Bernardo : J. Contiero (*)

Department of Biochemistry and Microbiology, Univ. Estadual Paulista—UNESP,
Biological Sciences Institute, CEP 13506-900 Rio Claro, SP, Brazil
e-mail: jconti@rc.unesp.br
Appl Biochem Biotechnol (2011) 164:1160–1171 1161

Leuconostoc mesenteroides B512F is a lactic bacteria that produces optically pure D(−)-
lactic acid. This species as well as all lactic bacteria has complex nutritional requirements
due to their limited ability to biosynthesize B vitamins and amino acids [8]. Refined sugars
such as glicose, fructose, sucrose, and lactose are fermented by Leuconostoc. Furthermore,
a considerable amount of an expensive complex nitrogen source, such as yeast extract, must
be added to the medium to produce lactic acid within a reasonable timeframe. However, this
is economically unfavorable, as pure sugars and pure complex nitrogen sources are
expensive. Therefore, raw materials for the industrial production of lactic acid need to have
characteristics such as low cost, low levels of contaminants, rapid fermentation, and year-
round availability [3]. According to Tejayadi and Cheryan [9], the cost of raw materials
represents 68% of the total cost of lactic acid production. A number of industrial bio-
products or wastes have been evaluated as substrates for lactic acid production with the aim
of decreasing the cost of the process, such as sugarcane [10], molasses [11], cassava
wastewater [12], and whey [13] as carbon sources and corn steep liquor (CSL) [14] and
yeast autolysate [15] as nitrogen sources. However, there are a few studies on D(−)-lactic
acid fermentation using these substrates in comparison with L(+)-lactic acid.
Sugarcane juice, sugarcane molasses, and whey are renewable, abundant, and cheap
sources of carbon. They were also proven to be an economically feasible raw material
for industrial production of lactic acid once they have enough nutrients necessary for
the growth of lactic acid bacteria. Furthermore, sugarcane molasses and sugarcane juice
do not require treatment to convert starch to fermentable sugars as the sugar content is
almost all in the form of sucrose [10] or in the form of lactose in the case of whey. This
way, it is possible to reduce the cost of culture medium by using these substrates in order
to increase economic viability. These substrates are renewable, abundant, and cheap
sources of carbon and they have enough nutrients necessary for the growth of lactic acid
bacteria.
The aim of the present study was to evaluate the potential use of different types of cheap
agro-industrial substrates as well as investigate the effects of different medium components
for the optimization of the production of D(−)-lactic acid by L. mesenteroides B512.

Materials and Methods

Microorganism

L. mesenteroides NRRL B 512 was provided by Department of Food Engineering,

Unicamp, Brazil. The strain was stored in a Man, Rogosa, and Sharpe (MRS) medium with
20% (v/v) glycerol at −20 °C.

Culturing

The inoculum was prepared through the transference of 1 mL of stock culture to

Erlenmeyer flasks containing 100 mL of growth medium (MRS). The following was the
composition of the medium (g/L): peptone (10.0), yeast extract (5.0), meat extract (10.0),
glucose (20.0), sodium acetate (5.0), ammonium citrate (2.0), K2HPO4 (5.0), MgSO4·7H2O
(0.1), and MnSO4·4H2O (0.05). All experiments and inoculum preparations were carried
out in flask cultures in orbital shakers at 200 rpm and 30 °C. The initial pH was adjusted to
6.2. 10% (v/v) of the inoculum and 100 g/L of calcium carbonate was added to 250-mL
Erlenmeyer flasks with 20 mL of the experimental media.
1162 Appl Biochem Biotechnol (2011) 164:1160–1171

Effect of Different Carbon Sources

Fermentations were carried out with MRS medium without glucose and supplemented
with different carbon sources at an initial concentration of 120 g/L of reducing sugar.
Three carbon sources were studied: cheese whey, sugarcane juice, and sugarcane
molasses. The sugarcane molasses and sugarcane juice were supplied by the Santa
Lucia sugar processing plant and the cheese whey powder was provided by Tavolaro
Dairy. Both factories are located in the state of São Paulo, Brazil.
The cheese whey powder containing 72% lactose was dissolved in water and the
resulting solution was heated to boiling point for 5 min in order to coagulate the
proteins. The solution was then cooled naturally to room temperature and filtered
through a coffee filter to remove the proteins; the supernatant contained 200 g/L of
lactose. The sugarcane molasses and sugarcane juice contained 1,000 and 240 g/L
of reducing sugar, respectively. These three carbon sources (whey, sugarcane
molasses, and sugarcane juice) were diluted to reach the desired sugar concentration
(120 g/L).

Effect of Different Nitrogen Sources

The fermentation medium was composed of sugarcane juice (120 g/L of sugar),
5 g/L of sodium acetate, 0.1 g/L of magnesium sulfate, 0.05 g/L of sulfate
manganese, 2 g/L of ammonium citrate, and 1 mL/L of Tween 80, complemented
with four nitrogen sources: yeast autolysate, yeast extract, corn steep liquor, and
pro-flo (cottonseed protein).
The corn steep liquor and pro-flo were supplied by Corn Products and the US company
Traders Protein, respectively. Both factories are located in the state of São Paulo, Brazil.
Table 1 displays the concentrations of nitrogen sources.

Plackett–Burman Experimental Design

The purpose of this first optimization step was to identify the medium components that
have a significant effect on lactic acid production. Twelve experiments were generated
using eight factors: sugar from sugarcane juice, sodium acetate, ammonium citrate,
magnesium sulfate (MgSO4), manganese sulfate (MnSO4), potassium phosphate (K2HPO4),
Tween 80, and yeast autolysate. Variables with a confidence level greater than 95% were
considered to have a significant influence over lactic acid production. The Plackett–Burman
experimental design was based on the first-order model with no interaction among the
factors. Table 2 displays the concentrations used for each variable. A central composite
design (CCD) was performed with the variables that significantly increased the production
of lactic acid.

Table 1 Concentration of each

nitrogen source Nitrogen sourcea Amount (g/L)

a Yeast extract 30
Concentration of each nitrogen
source determined based on the Yeast autolysate 25.56
concentration of nitrogen CSL 73.5
contained in 30 g/L of yeast Pró-floo 30.94
extract
Appl Biochem Biotechnol (2011) 164:1160–1171 1163

Table 2 Variables and levels used in design

Variables Codes Range and levels

−1 +1

Sugar from sugarcane juice (g/L) X1 50 150

Sodium acetate (g/L) X2 0 10
Amonium citrate (g/L) X3 0 4
MgSO4 (g/L) X4 0 0.4
MnSO4 (g/L) X5 0 0.2
K2HPO4 (g/L) X6 0 4
Tween 80 (mL/L) X7 0 2
Yeast autolysate (g/L) X8 0 40

Central Composite Design

A CCD for two independent variables, each at five levels with four star points (α=1.41)
and four replicates at the center points, was used to develop a second-order polynomial
model, which determined the optimal values of variables for lactic acid production.
Screened through the previous work, sugar from sugarcane juice and yeast autolysate was
taken as the variable for investigation.
The variables of the experiments were coded based on the following equation:
ðXi Xcp Þ ð1Þ
xi ¼ ΔXi i ¼ 1; 2; :::::; K

in which xi is the coded value of an independent variable, Xi is the real value of an

independent variable, Xcp is the real value of an independent variable at the center point,
and ΔXi is the step chance value.
The behavior of the system was explained by the following quadratic equation:
X X X
Y ¼ b0þ bixiþ biixi2þ bijxixj ð2Þ

in which Y is the predicted response, i.e., lactic acid concentration; b0 is the offset term; bi is
the linear effect; bii is the squared effect; bij is the interaction effect; and xi is the independent
variable.
The Statistica 7.0 software package (Stat Soft, USA) was used for the experimental
design and regression analysis of the experimental data. The response surface was
generated to determine interactions among the variables. The optimal points for the
variables were obtained from Maple 9.5 (Waterloo Maple Inc., Ontario, Canada).
Using the CCD method, a total of 12 experiments was conducted with various
combinations of sugar from sugarcane juice and yeast autolysate. Table 3 displays the range
and concentrations of the variables investigated in these experiments.
To validate the optimization of the medium composition, tests were carried out using the
optimized conditions in order to confirm the results of the response surface analysis.

Analysis

Lactic acid concentrations were determined using a high-performance liquid chromatography

system (HPLC) equipped with a UV detector at 210 nm. A Rezex ROA (300×7.8 mm,
1164 Appl Biochem Biotechnol (2011) 164:1160–1171

Table 3 Real values of variables used in central composite design

Variables (g/L) Codes Range and levels

−1.41 −1 0 +1 +1.41

Sugar from sugarcane juice X1 29.5 50 100 150 170.5

Yeast autolysate X2 8.85 15 30 45 51.15

The independent variables used in the central composite design were identified as significant to increasing
lactic acid production using a Plackett–Burman two-level factorial design

phenomenex) column was eluted with 5 mM H2SO4 as a mobile phase at a flow rate of
0.4 mL/min and the column temperature was maintained at 60 °C. Optical purity of D(−)-
lactic acid was determined with HPLC using a chirex 3126 phenomenex (150×4.6 mm)
column with 1 mM of CuSO4 as the mobile phase at 1 mL/min (30 °C). Reducing sugars
were measured using the 3,5-dinitro salicylic acid method [16].

Results and Discussion

Effect of Different Carbon Sources

The results of the influence of three carbon sources (cheese whey, sugar cane juice, and
molasses) over the production and productivity of D (−)-lactic acid are displayed in Table 4.
Among the three carbon sources investigated, L. mesenteroides achieved the highest
production and productivity of D(−)-lactic acid with sugarcane juice. Sugarcane juice contains
13% to 16% (w/v) of sucrose and has the advantage of being a renewable, abundant, cheap
carbon source. Lower production was observed with molasses, probably due to the presence
of inhibitory compounds. Furthermore, when molasses is used in fermentation, it may cause
serious problems in the purification and treatment of the substrate [17].

Effect of Different Nitrogen Sources

The results of the influence of four nitrogen sources added to sugarcane juice, yeast autolysate,
yeast extract, corn steep liquor, and pro-flo (cottonseed protein) over the production and
productivity of D(−)-lactic acid after 48 h of fermentation are displayed in Table 5.
Among the four nitrogen sources evaluated, yeast extract and yeast autolysate achieved the
highest D(−)-lactic acid production (59.2 and 44.48 g/L, respectively) and productivity (2.3
and 1.61 g/L h, respectively). Vahvaselka and Linko [18] investigated the effect of yeast
extract, hydrolyzed casein, and hydrolyzed protein from whey cheese, corn steep liquor, and

Table 4 Effect of different carbon sources on the production and productivity of D(−)-lactic acid after 48 h
of fermentation with 120 g/L of initial sugar

Carbon source Production (g/L) Productivity (g/L h)

Cheese whey 41.16 0.85

Molasses 38.4 0.80
Sugarcane juice 43.8 0.91
Appl Biochem Biotechnol (2011) 164:1160–1171 1165

Table 5 Effect of different nitrogen sources added to sugarcane juice (120 g/L of initial sugar) on lactic acid
production and productivity

Nitrogen sourcea (g/L or mL/L) Productionb (g/L) Productivityc (g/L h)

Yeast extract 59.2 2.3

Yeast autolysate 44.48 1.61
CSL 36 1.25
Pro-flo 26.4 1.31
a
Initial amount of each nitrogen source determined based on the concentration of nitrogen contained in 30 g/
L of yeast extract, i.e., 2.94 g/L of nitrogen
b
Lactic acid production in 48 h of fermentation
c
Lactic acid productivity in 24 h of fermentation

malt extract on lactic acid production by Lactobacillus helveticus in ultra-filtered milk. Yeast
extract (5 g/L) achieved the best result, followed by the hydrolysates, whereas corn steep
liquor and malt extract had little effect on production. Cox and MacBean [19] report a
significantly better effect with yeast extract in comparison to corn steep liquor for a strain of
Lactobacillus bulgaricus. However, the high cost of yeast extract is unfavorable to industrial
production [20]. Thus, autolyzed yeast may be a viable alternative.

Plackett–Burman Experimental Design

The Plackett–Burman design matrix (real and coded values) of 12 experiments with eight
variables (X1=sugar from sugarcane juice, X2=acetate, X3=citrate, X4=MgSO4, X5=MnSO4,
X6=K2HPO4, X7=Tween80, and X8=yeast autolysate) and respective results (D-lactic acid

Table 6 Plackett–Burman design (real and coded values) with respective results regarding lactic acid
production

Run Independent variables (g/L or mL/L) Result

X1 X2 X3 X4 X5 X6 X7 X8 Lactic acid (g/L)

1 150 (1) 0 (−1) 4 (1) 0 (−1) 0 (−1) 0 (−1) 2 (1) 40 (1) 48

2 150 (1) 10 (1) 0 (−1) 0.4(1) 0 (−1) 0 (−1) 0 (−1) 40 (1) 45.2
3 50 (−1) 10 (1) 4 (1) 0 (−1) 0.2(1) 0 (−1) 0 (−1) 0 (−1) 12
4 150 (1) 0 (−1) 4 (1) 0.4(1) 0 (−1) 4 (1) 0 (−1) 0 (−1) 8
5 150 (1) 10 (1) 0 (−1) 0.4(1) 0.2(1) 0 (−1) 2 (1) 0 (−1) 10.2
6 150 (1) 10 (1) 4 (1) 0 (−1) 0.2(1) 4 (1) 0 (−1) 40 (1) 17.5
7 50 (−1) 10 (1) 4 (1) 0.4(1) 0 (−1) 4 (1) 2 (1) 0 (−1) 2.1
8 50 (−1) 0 (−1) 4 (1) 0.4(1) 0.2(1) 0 (−1) 2 (1) 40 (1) 27
9 50 (−1) 0 (−1) 0 (−1) 0.4(1) 0.2(1) 4 (1) 0 (−1) 40 (1) 16
10 150 (1) 0 (−1) 0 (−1) 0 (−1) 0.2(1) 4 (1) 2 (1) 0 (−1) 1.15
11 50 (−1) 10 (1) 0 (−1) 0 (−1) 0 (−1) 4 (1) 2 (1) 40 (1) 24
12 50 (−1) 0 (−1) 0 (−1) 0 (−1) 0 (−1) 0 (−1) 0 (−1) 0 (−1) 26

(−1) and (1) are coded levels

X1 sugar from sugarcane juice, X2 acetate, X3 citrate, X4 MgSO4, X5 MnSO4, X6 K2HPO4, X7 Tween80, X8
yeast autolysate
1166 Appl Biochem Biotechnol (2011) 164:1160–1171

production) are displayed in Table 6. It was also observed in Table 6 that the decrease in
nitrogen source results in a decreased production of lactic acid, indicating nutrient
limitation.
Yeast autolysate proved as the most influential variable in the production of lactic acid,
followed by K2HPO4, MnSO4, and sugar from sugarcane juice. Among these variables,
only sugar and yeast autolysate had a significant positive effect on lactic acid production
with a 95% confidence level and were therefore used to optimize the production of lactic
acid. The effects of the variables are illustrated in Fig. 1 (Pareto chart).
Using the Plackett–Burman design to study solid-state fermentation with Lactobacillus
amylophilus GV6, Naveena et al. [21] report that MnSO4·H2O achieved a negative
coefficient, whereas MgSO4, sodium acetate, and corn steep liquor were found to be
insignificant. However, ammonium citrate and Tween 80 improved the production of lactic
acid.
According to Honorato et al. [22], the addition of phosphate to the culture medium
increases microorganism growth and lactic acid production as this component maintains
the pH near the optimal value for growth and allows the conduction of fermentation for a
longer period of time. However, K2HPO4 had a negative effect on lactic acid production
(Fig. 1). This may be explained by the excess of this component in the medium. Thus, the
addition of K2HPO4 and MnSO4 to sugarcane juice is not necessary and should be
avoided.

Response Surface Method

Sugar and yeast autolysate were further optimized using response surface optimization. The
design matrix of the variables in coded units and real values and the respective
experimental results are displayed in Table 7.
By comparing tests 9, 10, 11, and 12 with test 7 where the concentration of X2 was
reduced by threefold (maintaining X1 constant), the lactic acid production was reduced,

Fig. 1 Pareto chart for lactic acid production

Appl Biochem Biotechnol (2011) 164:1160–1171 1167

Table 7 Central composite design for optimization of two variables (each at five levels) and experimental
values for production of lactic acid

Run Independent variables (g/L) Results (g/L)

Sugar from sugarcane juice (X1) Yeast autolysate (X2) Lactic acid

1 50 (−1) 15 (−1) 16
2 50 (−1) 45 (1) 28.56
3 150 (1) 15 (−1) 6.4
4 150 (1) 45 (1) 58.8
5 29.5 (−1.41) 30 (0) 6.8
6 170.5 (1.41) 30 (0) 18.4
7 100 (0) 8.85 (−1.41) 4.72
8 100 (0) 51.15 (1.41) 59.4
9 100 (0) 30 (0) 56.8
10 100 (0) 30 (0) 56.5
11 100 (0) 30 (0) 57.12
12 100 (0) 30 (0) 57

(−1.41), (−1), (0), (1), and (1.41) are coded levels

indicating nutrient limitation. In the same way, by reducing X1 by half (test 2), lactic acid
decreases by the same amount.
These results indicate that the increased availability of nitrogen assimilated by L.
mesenteroides B512 probably influences the increased production of lactic acid during the
fermentation process. Since lactic acid bacteria are nutritionally fastidious, amino acids and
vitamins are required for growth. Choosing the right type of nitrogen source appears to be
very important because of the rise in biomass concentration.
The application of multiple regression analysis methods yielded the following regression
equation (Eq. 3) for the experimental data:
Y ¼ 56:85 þ 4:63X1 þ 17:78X2  20:85X1 X1 þ 9:96X1 X2  11:12X2 X2 ð3Þ
in which Y is the predicted response (lactic acid production) and X1 and X2 are,
respectively, the coded values of the test variables sugar from sugarcane juice and yeast
autolysate.
The highest production of lactic acid was 59.4 g/L, obtained from 100 mL/L of
sugarcane juice and 51.15 g/L of yeast autolysate (Table 7). The response surface quadratic
model was performed in the form of analysis of variance (ANOVA) and the results are
summarized in Table 8. Fisher’s F-test was used to check the statistical significance of
Eq. 3.
ANOVA of the quadratic regression model demonstrates that the model is highly
significant [as evident from Fisher’s test (Fcalc (5,6)=100.8525>Ft (5,6)=4.39)] and has a
very low probability value [(Pmodel>F)=0.000011]. The fit of the model was checked by the
coefficient of determination (R2) and multiple correlation coefficient (R). The R2 value
(0.988) for Eq. 3 indicates that the sample variation of 98.8% for lactic acid was attributed
to the independent variables and only 1.2% of the total variation cannot be explained by the
model. The adjusted R2 value (0.978) is also high, which indicates the high significance of
the model. The high R value (0.994) demonstrates a strong agreement between observed
and predicted values.
1168 Appl Biochem Biotechnol (2011) 164:1160–1171

Table 8 Analysis of variance for the quadratic model

Source Sum of squares Degree of freedom Mean square F-value P>F

Model 6,203.364 5 1,240.673 100.8525 0.000011

Error 73.811 6 12.302
Lack of fit 73.591 3 24.530 334.05
Pure error 0.220 3 0.073
Total 6,277.176

R2 =0.988; adjusted R2 =0.978; R=0.994

Table 9 displays the Student’s t-distribution and probability (P) values. These values
serve as a tool to check the significance of each coefficient, which represents the interaction
pattern between the test variables. A smaller P-value denotes a more significant
corresponding coefficient. All independent variables had a significant effect (small P-
values). The parameter estimate indicate that X2 (yeast autolysate) is the parameter that
contributes more strongly to the response (lactic acid). Moreover the variables X1 and X2
and their interaction (X1X2) had a positive effect, with an increase in their concentration
leading to an increase in the response (lactic acid production).
Figure 2 displays the 3D response surface and contour plots, a graphical representation
of the regression equation plotted for one to understand the interaction of the variables and
locate the optimal level of each variable for maximal response.
There was a strong interaction between sugar and yeast autolysate in the production of
lactic acid (Fig. 2). The area of greatest lactic acid production is located between 102 and
132 g/L of sugar from sugarcane sugar and between 37 and 52 g/L of yeast autolysate.
However, both variables may inhibit lactic acid production at higher concentrations due to
significant carbon and nitrogen repression. As lactic acid bacteria are nutritionally
fastidious and require various amino acids and vitamins for growth, choosing the right
nitrogen and carbon sources is very important. Nitrogen is necessary to the synthesis of
amino acids, lipids, enzyme cofactors, some carbohydrates, and other substances. The
nitrogen source is a major factor of influence over the growth of Lactobacillus [23]. As the
synthesis of lactic acid by fermentation is associated with cell growth, there is no product
formation if the medium does not have an adequate concentration of nitrogen [24].
However, high concentrations of nitrogen can lead to cell death [15].
Using an MRS medium containing 20 g/L of yeast extract and 120 g/L of sugarcane
juice, Calabia and Tokiwa [10] obtained 107 g/L of lactic acid. The authors attribute this
high production to the high nutritional value of sugarcane juice, which contains natural

Table 9 Least-squares fit and parameter estimates

Term Estimate Standard error t Pr>−t|

Intercept 56.855 1.75 32.42 0.000

X1 4.63 1.24 3.73 0.0096
X2 17.786 1.24 14.34 0.000007
X1X2 9.96 1.753 5.678 0.001284
X12 −20.85 1.386 −15.038 0.000005
X22 −11.12 1.386 −8.02 0.000201
Appl Biochem Biotechnol (2011) 164:1160–1171 1169

Fig. 2 Response surface and contour plots of D(−)-lactic acid production by L. mesenteroides showing the
interaction between sugar from sugarcane juice and yeast autolysate

sugars, proteins, amino acids, and vitamins essential to the growth of lactic acid bacteria.
Sule et al. [25] tested different concentrations of sucrose in a culture medium (5, 30, 50, and
100 g/L) and achieved maximal lactic acid production (21 g/L) using 50 g/L of sucrose,
whereas the use of 100 g/L of sucrose resulted in a decrease in the production of lactic acid.
The authors attribute this to the increased viscosity of the medium due to the high
concentration of sucrose.
The point of maximal lactic acid production was determined through a canonical
analysis of the adjusted model. A study was carried out to identify the nature of the
stationary point (maximal point, low response, or even a saddle point). An algorithm carried
out on the Maple 9.5 program (Waterloo Maple, Inc., Canada) was used to calculate the
stationary point (P0) for the synthesis of lactic acid. These values are displayed in Table 10.
The l values for sugar and yeast autolysate indicate that these responses have a maximal
point, as they have equal and negative signs (Table 10). A second-order polynomial
regression model estimated that a maximal lactic acid production of 66.11 g/L would be
obtained when optimal sugar concentration from sugar and yeast autolysate values were
116.9 and 44.25 g/L, respectively. All optimal points were located within the experimental
region and varied around their center points to different extents. Three additional experiments
in a shaker were performed with this optimal medium composition in which mean lactic acid
production was 60.2 g/L, which was a bit smaller than the predicted value (66.11 g/L).

Table 10 Stationary point for lactic acid production and codified values of variables X1 and X2 on the
optimization point

P0 Lactic acid Coordinates Lactic acid

l1 −22.947 X1 0.338
l2 −9.023 X2 0.951
1170 Appl Biochem Biotechnol (2011) 164:1160–1171

Sugarcane juice is a renewable, abundant, and cheap source of carbon. Furthermore, it is

an economically feasible raw material for the industrial production of lactic acid once it has
enough nutrients necessary for the growth of lactic acid bacteria. Therefore, optically pure
D-lactic acid can be obtained using L. mesenteroides B512F, and the cost of culture medium
could be reduced by using this cheap substrate.

Conclusions

Based on the optimization of the responses, the best result for D(−)-lactic acid production
(60.2 g/L) was obtained with 116.9 g/L of sugar and 44.25 g/L of yeast autolysate. Thus,
the use of these substances with L. mesenteroides is feasible as there was considerable
production of D(−)-lactic acid, requiring only the completion of a cheap source of nitrogen
(yeast autolysate). The Plackett–Burman design, central composite design, and response
surface method, including regression analysis and model generation, were effective
methods regarding medium optimization for the production of D(−)-lactic acid.

Acknowledgments The authors are grateful to Tavolaro Dairy and Corn Products for kindly supplying the
whey and corn steep liquor, respectively, the Sta. Lucia plant for supplying sugarcane molasses and
sugarcane juice, the Brazilian fostering agency São Paulo Research Foundation—FAPESP, and the National
Council for Scientific and Technological Development (CNPq) for the fellowships and financial support.

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