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Temperature and pH Optimization for the

growth of lactobacillus delbrueckii ssp
bulcaricus on whey -based medium

Conference Paper · January 2011

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The 7th International Chemical Engineering Congress & Exhibition (IChEC 2011)
Kish, Iran, 21-24 November, 2011

Temperature and pH Optimization for the growth of lactobacillus

delbrueckii ssp bulcaricus on whey -based medium using response
surface methodology

Marziyeh Aghababaie, Morteza Khanahmadi, Masood Beheshti, Maryam Mirlohi

Biotechnology Department, New Science and Technology Faculty, University of Isfahan
maz_babaie@yahoo.com

Abstract
Industrial production of starter cultures is a note worth field of biotechnology which is strongly
required in food industry especially in developing countries. Lactobacillus delbrueckii ssp
bulgaricus is one of the yogurt starters. To determine the effect of temperature and pH for the
production of a native strain of L. bulgaricus this study was designed using response surface
methodology in 12 batch pH-controlled culture of lactobacillus delbrueckii subssp bulgaricus.
Growth-associated and nongrowth-associated produce formation parameters a and b was estimated
with Luedeking-Piret equation. Effect of pH and temperature on maximum biomass concentration
(Xmax), maximum specific growth rate (μmax), Total lactic acid concentration (P), a and b
parameters has been identified. Second order model for Xmax, μmax and P was significant but a and
b was almost constant. Parameter a was about 0.6 g lactic acid.gbiomass-1 and parameter b was
about 1 g lactic acid.gbiomass-1.h-1. Optimum temperature and pH for maximum biomass and
specific growth rate and acid production were 44°C and 5.7. This study provided the data required
to develop a model for kinetic analysis of a native strain of L. bulgaricus growth and lactic acid
production.

Keywords: Response surface methodology; Lactobacillus bulgaricus, Whey, Lactic acid

production

Introduction

Lactobacillus delbrueckii subsp bulgaricus (L. bulgaricus) is a themophilic bacteria

commonly used as a starter culture in the manufacture of yogurt. Production of native L.
bulgaricus strains develops starters which can be easily used by processors to produce high
quality and more acceptable products. Whey, a byproduct of cheese manufacturing plants is
a nutritious component, containing lactose and milk peptides which make it favorable for the
growth of Lactic acid bacteria. Various aspects of metabolism and growth of L. bulgaricus in
pure culture have been considered in previous researches. The optimal temperatures were
Temperature and pH Optimization for the growth… 

proposed between 42 and 47 ° C, however it was stated that the exact optimal growth
temperature is a strain dependent characteristic[1]. Little work has been done on the optimum
pH for the growth of this bacteria, although some researchers recommended pH values of 5.5
[2, 3] and Temperature of 44° C for L.bulgaricus[3-5]. Growth assosiated and non- growth
associated paramaters for l.bulgaricus has determined in different pH values by Gadgil et
al[6].
Response surface methodology offers a practical way of studying multiple variables, since it
allows for detection of main and quadratic factor effects and first order interactions with a
much smaller number of experiments than in a full factorial design[7]. RSM has been widely
used in predictive microbiology for modeling growth of bacteria with different cultures and
processing conditions. Schepers et al [8] optimized culture conditions for Lactobacillus
helveticus growth using RSM with three factors. Also, RSM was used by Beal et al[3, 5] to
optimize pH and temperature for the growth of a L. bulgaricus and a streptococcus
thermophilus stains.
In this study RSM used to obtain a model of L. bulgaricus growth and lactic acid production
in pH-controlled batch cultures in media containing whey and yeast extract at different pH
and temperature set points. The kinetic model is described in a separate paper. Experiments
were conducted according to a central composite design with five descriptive culture
parameters: maximum specific growth rate (μmax), maximum biomass concentration (Xmax),
amount of lactic acid produced after 5.5 hour of fermentation and growth- and non-growth
associated production rates, which were analyzed statistically with response surfaces.

Material and methods

Inoculums preparation and microorganism used
L.bulgaricus was isolated from a traditional yogurt. After several purification steps, single
colony was identified as L. bulgaricus strain by colony and cell morphology , gram staining,
catalase reaction and sugar fermentation pattern .The pure culture was prepared in de Man,
Rogosa and Sharpe (MRS) medium ( Merck). The cultures were kept in deep-frozen stock (-
80°C). Working cultures was prepared from frozen cultures 1% (v/v), incubated for18h at
40°C. Twenty ml of fresh working culture with the concentration of 108 cell ml-1 was used as
inoculums for biofermentation process.

Medium
Recombined whey solution from whey powder 10 %( W/V) were autoclaved (110°C, 20 min)
and filtered. Eight hundred ml of the filtrate with 1ml Tween 80(Merck) in 1 lit handmade
bioreactor was autoclaved (110°C, 20 min). Solutions of 10g yeast extract (Merck) in 100
milliliter water were prepared and autoclaved (110°C, 20 min) separately, and added to the
sterile whey mixture just before the fermentation.
1M MgSO4 * 7H20 and 1M CaCO3 were autoclaved (110°C, 20 min) and 1ml of each added
to bioreactor just before the fermentation. Mg2+ and Ca2+ has been shown in dechaining and
stimulating the growth of the l.bulgaricus[9].
The 7th International Chemical Engineering Congress & Exhibition (IChEC 2011)
Kish, Iran, 21-24 November, 2011

Analysis
The pH controlled batch cultures were carried out in 1 L bioreactor. Temperature and pH
were controlled depending on the experiment. The pH was controlled by adding 5 N sodium
hydroxide, which was continuously weighted in order to measure produced lactic acid.
Peristaltic pump was used to control pH.
Samples were taken every half an hour, and placed in refrigerator. Growth was observed by
microscopic counting with Thoma with 0.02 mm depth.

Experimental design
The experimental design used here was the Central Composite: Uniform Precision with 12
runs which had 4 factorial, 4 axial and 4 center points. The values of each parameter assigned
to the centre points were chosen from reported optima[3]: T = 44°C and pH = 5.8 for
L.bulgadcus at center points.
The quadratic model for predicting the relationship of response and two controllable factors
is expressed according to equation 1[10].
2 2 2
y = B0 + ∑ Bi x i + ∑ Bii x i2 + ∑ ∑ Bij x i x j + ε 1
i=1 i =1 i > j=1

Modeling
Growth rate data were smoothed and interpolated with fitting to re-parameterized Richards
model using least square method in matlab 2008b [11]:

X( t ) = A(1 + ((X 0 / A) (1−d) − 1) exp(−Kt / d d /(1−d) ))1/(1−d)

2
Where X(t) converted to mass concentration (Xm) and used in Luedeking-piret equation. A is
upper bound of X and X0 indicate the active population of l.bularicus. Cell mass
concentration used to obtain lactic acid production parameters with Luedeking-Piret equation
[12] using least square method with matlab:
dP dX m
rp = =b + aX m 3
dt dt

Results and Discussion

Maximum biomass concentration (Xmax), maximum specific growth rate (μmax), total lactic
acid concentration (P), growth-associated and nongrowth-associated production parameters a
and b were determined for pH-controlled batch cultures using descriptive growth and lactic
acid production models. The results have been brought in table 2.
Temperature and pH Optimization for the growth… 

Table 2. design of experiments based on RSM, 3 basic responses is maximum biomass

concentration (Xmax), maximum specific growth rate (μmax) and lactic acid produced at the end
of fermentation (P)
RUN T PH Xmax (cell/ml) μmax (h-1) P(g/l)
1 40 4.9 1.45E+08 0.891 3.69
2 40 6.5 1.25E+08 0.609 2.385
3 48 4.9 1.3E+08 0.801 3.645
4 48 6.5 0.7E+08 0.5 2.295
5 38.34 5.7 1.8E+08 0.8 5.375
6 49.65 5.7 1.25E+08 0.486 2.7
7 44 4.56 2E+08 0.67 5.9
8 44 6.83 1.1E+08 0.586 2.97
9 44 5.7 1.95E+08 0.92 5.94
10 44 5.7 2.2E+08 0.919 5.265
11 44 5.7 2.16E+08 0.921 6.255
12 44 5.7 1.8E+08 0.88 6.75

Maximum biomass concentration (Xmax)

Fitted model for maximum biomass concentration was significant (Pr<0.05). pH2 and T2 had
the maximum effect on maximum biomass concentration and were significant (Pr<0.05).
Also, first order effect of pH was significant.

Table 2. ANOVA table for maximum biomass concentration (Xmax)

Source DF SS MS F Pr > F
T 1 2.73E+15 2.73E+15 4.13 0.0884
PH 1 5.37E+15 5.37E+15 8.13 0.0292
T*T 1 7.48E+15 7.48E+15 11.32 0.0152
T*PH 1 4.00E+14 4.00E+14 0.61 0.4662
PH*PH 1 6.94E+15 6.94E+15 10.50 0.0177
Model 5 2.05E+16 4.10E+15 6.21 0.0230
(Linear) 2 8.10E+15 4.05E+15 6.13 0.0355
(Quadratic) 2 1.20E+16 6.01E+15 9.09 0.0153
(Lack of 3 2.92E+15 9.72E+14 2.77 0.2121
fit)
(Pure 3 1.05E+15 3.50E+14
Error)
Total 11 2.45E+16
The 7th International Chemical Engineering Congress & Exhibition (IChEC 2011)
Kish, Iran, 21-24 November, 2011

figure1. Response surface for maximum biomass concentration (Xmax)

Maximum specific growth rate (μmax)

x
Maximum specific growth rate was calculated from exponential model (ln = μt ) .second
x0
order model was significant for maximum specific growth rate (Pr<0.05).As same as what
was observed for maximum biomass concentration, second order of pH and temperature had
the maximum effect on maximum specific growth rate and was significant (Pr<0.05). First
order effect of these factors were significant, too (Pr<0.05).

Table 3. ANOVA for maximum specific growth rate (μmax)

Source DF SS MS F Pr > F
T 1 5.17E-02 5.17E-02 7.04 0.0378
PH 1 6.16E-02 6.16E-02 8.39 0.0275
T*T 1 8.81E-02 8.81E-02 12.00 0.0134
T*PH 1 9.00E-05 9.00E-05 0.01 0.9153
PH*PH 1 9.97E-02 9.97E-02 13.59 0.0103
Model 5 2.70E-01 5.40E-02 7.36 0.0153
(Linear) 2 1.13E-01 5.66E-02 7.72 0.0219
(Quadratic) 2 1.57E-01 7.83E-02 10.67 0.0106
(Lack of fit) 3 4.28E-02 1.43E-02 35.63 0.0076
(Pure Error) 3 1.20E-03 4.01E-04
Total 11 3.14E-01
Temperature and pH Optimization for the growth… 

figure2. Response surface for maximum specific growth rate (μmax)

Produced lactic acid

After 5th hour of fermentation all of the batches were in the stationary phase .Therefore lactic
acid concentration which was measured with added sodium hydroxide along 5.5 hours
fermentation picked as a result and analyzed with RSM. Second order model was significant
for prediction of lactic acid produced at the end of fermentation (Pr<0.05). In the order of
efficiency significant effect of T2, pH2 were observed, respectively.

table4. ANOVA for produced lactic acid (P)

Source DF SS MS F Pr > F
T 1 1.9188 1.9188 1.88 0.2199
PH 1 5.7777 5.7777 5.65 0.0550
T*T 1 11.0776 11.0776 10.83 0.0166
T*PH 1 0.0005 0.0005 0.00 0.9830
PH*PH 1 7.9834 7.9834 7.80 0.0314
Model 5 23.6339 4.7268 4.62 0.0448
(Linear) 2 7.6966 3.8483 3.76 0.0874
(Quadratic) 2 15.9369 7.9684 7.79 0.0215
(Lack of fit) 3 4.9786 1.6595 4.29 0.1313
(Pure Error) 3 1.1603 0.3868
Total 11 29.7728
The 7th International Chemical Engineering Congress & Exhibition (IChEC 2011)
Kish, Iran, 21-24 November, 2011

figure3.pH and temperature effect on total lactic acid production

Growth-associated and non-growth associated production parameters a and b

Growth-associated and nongrowth-associated parameters in equation 3 have determined.
None of the factors had the significant effect on these parameters. This result was previously
seen for parameter a [8], but in some reports it was a pH dependant term [12, 13]. Parameter
a was measured by about 0.6 g lactic acid g.biomass-1 and parameter b was measured about 1
g lactic acid gbiomass-1.h-1.

Conclusion
As it was reported by Beal et al[3],Second order model is significant for Xmax, μmax and P.
coefficients of equation 1 are in table4 for maximum biomass concentration, maximum
specific growth rate and produced lactic acid.

table 4. coefficients of models for Xmax, μmax and P according to equation 1

X μ p
B0 -6.00E+09 -18.484 -199.28
T 2.01E+08 0.6336 7.13356
pH 6.92E+08 2.178878 18.9868
T*T -2.14E+06 -0.00733 -0.08223
pH*pH -5.15E+07 -0.19502 -1.7451
T*pH -3.13E+06 -0.00148 -0.00352
R2 83.80% 85.97% 79.38%

Optimum temperature and pH for maximum biomass and specific growth rate and acid
production were 44°C and 5.7. Unless the difference in the mediums, optimum conditions
didn't differs from that of reported by Beal [3]. But the maximum biomass concentration was
lower in this study .This may be due to the strain of the bacteria or medium composition.
Growth associated parameter for acid production is lower from that of reported by other
researchers [6, 8, 14].
Temperature and pH Optimization for the growth… 

Acknowledgments
This study was supported by Isfahan Center for Research on Agricultural Science and Natural
Resources and university of isfahan.

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 The 7th International Chemical Engineering Congress & Exhibition (IChEC 2011)
Kish, Iran, 21-24 November, 2011

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