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Eect of temperature on lactic acid production from cheese
whey using Lactobacillus helveticus under batch conditions
M.S.A. Tango, A.E. Ghaly *
Biological Engineering Department, Dalhousie University, P.O. Box 1000, Halifax, Nova Scotia, Canada, B3J 2X4
Received 29 December 1997; received in revised form 9 July 1998; accepted 17 July 1998
Abstract
A 5 L continuous mix, batch bioreactor was used to investigate the eect of temperature on the growth of
Lactobacillus helveticus and production of lactic acid from lactose. The temperature levels used were 238C (no
control), 378C (no control), 378C (controlled) and 428C (controlled). No pH control was provided. The temperature
and pH were monitored during the fermentation process. The pH steadily decreased from the initial value of 4.4 to
less than 3.0, due to the lactic acid formation. Increasing the fermentation temperature from 238C to 428C (with no
pH control), enhanced the lactose utilization and lactic acid production by 26.6 % and 6.2 g L1, respectively.
Maximum specic growth rate, lactose utilization and lactic acid production were 0.25 h1, 60.6 % (of initial
concentration), and 10.0 g L1 respectively, for fermentation with temperature control at 428C. The results showed
the need for controling both temperature and pH during batch lactic acid fermentation from cheese whey to avoid
yield losses. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Batch fermentation; Cheese whey; Lactose; Lactic acid; Lactobacillus helveticus; PH; Temperature; Cell growth
1. Introduction
Lactic acid is a natural organic acid which has
many applications in pharmaceutical, food and
chemical industries. These include: uses as an
acidulant, preservative and as a substrate for the
production of biodegradable plastics (polylactidepolymers, polyhydroxybutryate) and some other
organic acids [1]. Lactic acid can be produced by
fermentation from a variety of sugar containing
substrates. Such a substrate is cheese whey.
Cheese whey is a by-product of the cheese-
making process. It contains about 93% water,
5% lactose, 0.9% nitrogen compounds, 0.6%
minerals and vitamins, 0.3% fat and 0.2% lactic
acid. There is continued interest in utilizing lac-
tose from cheese whey for the production of
value added end products. Several researcheshave utilized anaerobes or facultative anaerobes
to ferment lactose to single cell protein, ethanol,
biogas, lactic acid and acetate [210].
Lactobacillus helveticus has been chosen for the
production of lactic acid from cheese whey
because it appears to be among the most pro-
ductive bacteria for lactic acid production from
Biomass and Bioenergy 16 (1999) 6178
0961-9534/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
P I I : S 0 9 6 1 -9 5 3 4 (9 8 )0 0 0 6 2 -2
PERGAMON
* Corresponding author.
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lactose [11, 12]. It is a thermophilic and acido-
philic bacterium, that will grow under conditions
inhibitory for most contaminant microorganisms
[13, 14]. Temperature and pH are the key en-
vironmental parameters that aect the lactic acid
fermentation process. It is, therefore, important
to determine the temperature regimes at which
optimum microbial growth is achieved. Better
understanding of the temperature eects on lac-
tose fermentation will facilitate improvement of
the process. In addition, the rates of substrate
utilization and lactic acid production during the
lag, growth, stationary and death phases of
Lactobacillus helveticus under batch culture oper-
ation need to be quantied.
2. Objective
The main aim of this study was to investigate
the eect of temperature on the production of
lactic acid from acid cheese whey using
Lactobacillus helveticus in a continuous mix
batch bioreactor and to evaluate the performance
characteristics of the fermentation process as
measured by microbial growth rate, lactic acid
production and lactose conversion eciency.
3. Materials and methods
3.1. Substrate collection and preparation
Cheese whey was obtained from Farmers
Cooperative Dairy Plant in Truro, Nova Scotia,
Canada in 40 L plastic bags. The cheese whey
bags were kept in a storage facility (Associate
freezers of Canada, Dartmouth, Nova Scotia) at
258C to minimize microbial and enzymatic
degradation. The amount required for the exper-
iments was pasteurized in 4 L glass bottles
according to the procedure described by Ghalyand El-Taweel [5]. The bottles were immersed in
water bath (Fisher Scientic, Model No. 4391,
Montreal, Canada,) for 45 min at 708C. They
were then cooled suddenly to 08C in an ice bath
for 30 min and then kept at room temperature
(208C) for 24 h. This process of alternating heat-
ing, cooling and warming up was repeated three
times. The pasteurized cheese whey stock was
then stored in refrigerator at 48C. Some of the
characteristics of cheese whey used in this study
are shown in Table 1.
3.2. Inoculum preparation
Lactobacillus helveticus (ATCC 15009) was
obtained from the American Type Culture
Collection (Rockville, Maryland). The bacteria
was revived and maintained in tomato juice-yeast
extract broth (ATCC medium 17) which con-
tained skim milk (100 g/L), tomato juice (100
mL/L) and yeast extract (5 g/L). The medium
was sterilized at 1218C and 103.4 kPa for 15 min
in an autoclave (Market Forge Sterilmatic,Model No. STM-E, New York). The rehydated
bacterial culture was grown in the incubator
(Precision Scientic Co., Model 815, Chicago,
Illinois) in Petri dishes with fresh agar containing
skim milk (100 g/L), tomato juice (100 mL/L),
yeast extract (5 g/L) and agar (15 g/L) at 378C
for 3 days. The pasteurized cheese whey was
transferred to several 250 mL sterilized
Erlenmeyer asks (150 mL/ask). Inoculum
(10% v/v) for batch fermentation came from a
culture grown at 378C for 48 h in the pasteurized
cheese whey. The Lactobacillus helveticus were
transferred from the stock culture to the 150 mL
of pasteurized cheese whey in each sterilized
Erlenmeyer ask (from two Petri dishes of pure
culture). The asks were capped with cotton
plugs and mounted on a controlled environment
reciprocating shaker (Incubator Shaker, Series
25, New Brunswick Scientic Co. Inc., New
Jersey). The temperature in the shaker chamber
was maintained at 378C and the shaker was oper-
ated at 200 rpm for 48 h. The contents of the
asks were transferred to one large sterilized con-
tainer and thoroughly mixed, then refrigerated at
48C until ready for use.
3.3. Experimental apparatus
The experimental apparatus (Fig. 1) consisted
of (a) four bioreactors each with a mixing system
(b) a temperature control system and (d) a data
acquisition system.
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A 5 L batch bioreactor was used in this study.
The fermenter was constructed from a plexiglas
cylinder of 5 mm thickness. Its dimensions are as
shown in Fig. 2. Four vertical baes (positioned
at 908 apart) made from plexiglas were used in
the fermenter to improve the top to bottom turn-
over and to reduce vortex eect. Provisions were
made on the cover for mounting the temperature
probe, pH probe, dissolved oxygen probe and
mixing shaft. In addition, two ports for sample
collection and pressure release were incorporated.
Agitation was facilitated by a mixing system
which consists of an electric motor (DaytonElectric MFG Co., model 4Z142, Chicago,
Illinois) with a controller and a mixing shaft. The
motor was mounted onto the top of the reactor
cover and was connected to the motor with a
exible coupling collar. The mixing shaft had
two at-bladed impellers of 75 mm diameter,
mounted at 148 mm apart with the bottom
impeller being 30 mm from the fermenter oor.
The mixing speed was maintained at 150 rpm.
The fermentation temperature was controlled
using a 1080 260 370 mm well insulated
water bath. The water bath was constructed from
a double wall stainless steel sheet (1 mm thick-
ness) with 20 mm thick styrofoam sheet placed in
between. Water ow rate within the water bath
was controlled by a submersible pump
(Tecumseh Products Co., Model 1-MAT, Cat.
No. 521286, Oklahoma City, Oklahoma) inserted
in the water bath and discharges water to the
heater unit. Uniform distribution of water fromthe heater unit was facilitated by holes around a
steel tube containing a heater element (2.0 kW).
Temperature sensor was used to detect the water
temperature and the control unit regulated the
temperature at a preset value.
The data acquisition system consisted of a
data logger, pH probes, dissolved oxygen probes,
Table 1
Some characteristics of cheese whey
Characteristics Measured value Units
Total solids 68 298.00 mg L1
Fixed solids 6748.00 mg L1
Volatile solids 61 550.00 mg L1
Percent volatile solids 90.12 %
Percent xed solids 9.88 %
Suspended solids 25 160.00 mg L1
Fixed solids 225.00 mg L1
Volatile solids 24 935.00 mg L1
Percent volatile solids 99.11 %
Percent xed solids 0.89 %
Total Kjeldahl nitrogen 1560.00 mg L1
Ammonium nitrogen 263.00 mg L1
Organic nitrogen 1297.00 mg L1
Percent organic nitrogen 83.14 %Percent ammonium nitrogen 13.92 %
Total chemical oxygen demand 81 050.00 mg L1
Soluble chemical oxygen demand 68 050.00 mg L1
Insoluble chemical oxygen demand 13 000.00 mg L1
Percent soluble chemical oxygen demand 84.96 %
Percent insoluble chemical oxygen demand 16.04 %
Lactose 4.82 %
Lactic acid 0.22 %
Potassium 1670.00 mg L1
Sulfur 154.00 mg L1
Phosphorus 483.00 mg L1
pH 4.90
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Fig.
1.
Schematicdiagram
ofexperimentalapparatusforbatchoperation.
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Fig. 2. Diagram of batch bioreactor.
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thermocouples, signal conditioning unit and a
personal computer. The data logger (Syscon
International Inc., Model 525 S.SYSCON, South
Bend, Indiana) was connected to the signal con-
ditioning unit and to IBM PS2 personal compu-
ter through a serial communication port. Four
pH probes (Fisher Scientic, Model No. 13-620-
104, Montreal, Canada) and four dissolved oxy-
gen probes (Cole-Parmer, Model No. 25643-04,
Chicago, Illinois) were connected to the data
data logger through the signal conditioning unit
whereas four type-T thermocouple sensors (Cole
Parmer, Cat. No. L-08530-74, Chicago, Illinois)
were directly connected to the data logger. All
probes were calibrated prior to each experimental
run. The temperature of the medium was moni-tored (for this study only) using four ther-
mometers (Fisher Scientic, Cat. No. 14-983-10B,
Montreal, Canada, ) to an accuracy of20.58C
while pH measurements were recorded using pH
probes (Fisher Scientic, Cat. No. 13-620-104,
Montreal, Canada,) attached to a microprocessor
based pH tester BNC (OAKTON, Cat. No. WD-
35624-10). A Quick Basic environment was used
to develop the software and operate the data ac-
quisition system.
3.4. Experimental procedure
In this study, four temperature levels (ambient
temperature of 238C, initial temperature of 378C,
continuous temperature control at 378C and con-
tinuous temperature control at 428C) were used
to investigate the growth of Lactobacillus helveti-
cus, lactose utilization and lactic acid production.
The bioreactor and its components were chemi-
cally sterilized using 2% potassium metabisul-
phite, then thoroughly cleaned by hot distilled
water. The fermenter was lled with 4.32 L pas-
teurized cheese whey and immediately 480 mL
inoculum was added. At this moment the mixingmotor for each fermenter was turned on (150
rpm), the data acquisition system and the compu-
ter program were activated. The ambient tem-
perature, medium temperature and pH were
monitored throughout the fermentation process.
For the runs at ambient temperature and no con-
trol (238C), the fermenter with pasteurized cheese
whey was placed at room temperature. For the
runs at initial temperature of 378C and no con-
trol, the fermenter with pasteurized cheese whey
was rst immersed in water bath until steady
temperature of medium was 378C then water was
emptied from the bath. For the runs at tempera-
tures of 37 and 428C and continuous temperature
control, the fermenters with pasteurized cheese
whey were maintained in the water bath at the
preset temperatures. In all of the experimental
runs, inoculum was separately brought to the in-
dividual initial temperature conditions prior to
addition to the respective fermenter.
3.5. Samples and analyses
Samples of about 5 mL each were collected
throughout the experiment for cell number, lac-
tose and lactic acid analyses. The samples were
collected at zero hour, initially every after 2 h
until 12 h, and thereafter at intervals of 4 h
(between 12 to 24 h), 6 h (between 24 to 48 h)
and 8 h (between 48 to 64 h). The cell concen-
tration was determined using plate count pro-
cedure and dehydrogenase activity according to
the procedure described by Ghaly and Ben
Hassan [15]. Lactose concentrations were deter-
mined using sugar analyzer (YSI Model 27,
Yellow Springs, Ohio), while lactic acid concen-
trations were determined using glucose/L-lactate
analyzer (YSI Model 2000, Yellow Springs,
Ohio).
4. Results and discussions
4.1. pH
The results of pH measurements are shown in
Fig. 3. The initial pH of the fermenter medium
(after the addition of inoculum) was 4.4. Therewas a steady decrease in the pH of the medium
during the fermentation of cheese whey with no
temperature control (room temperature) until it
reached a value of 3.4 at the end of the fermenta-
tion process. For the experiment with an initial
temperature of 378C (no temperature control),
the pH of the medium decreased from 4.4 to 2.9
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within the rst 20 h and then decreased slowly
reaching 2.8 at the end of the experiment. In the
case of continuously controlled temperatures at
both 37 and 428C, the respective pH of the med-
ium dropped at a fast rate to 2.7 and 3.6 within
the rst 10 h, and then decreased gradually
reaching a steady values of 2.7 and 3.0 at the end
of the experiments.
The correlation between the pH versus operat-
ing temperature tted the following parabolic
function (R 2=0.998):
pH 15X31 0X74 T 0X011 T
2
1where T is the fermentation temperature (8C).
Similarly, the correlation between the pH ver-
sus cell number tted the following equation
(R 2=0.980):
pH 11X01 4X91N
N0 20X4 ln
N
N0
2
where N0 is the initial cell number (106 cells
mL1); N is the cell number at time t (106 cells
mL1).
Since the temperature has a direct eect on the
cell number, which in turn has a direct eect on
the pH of the medium due to cell activities, the
eect of both temperature and cell number on
pH can be deduced in the following general ex-
pression:
pH fTxfNyX 3
Substituting the expressions for f(T) and f(N)from Eqs. (1) and (2) into Eq. (3), the following
equation was obtained (R 2=0.985):
pH 15X31 0X74 T 0X011 T2h i1X11
11X01 4X91N
N0 20X4 ln
N
N0
!0X15X 4
Fig. 3. pH changes during batch fermentation.
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The above expression was tested at various tem-
perature levels (20, 30 and 408C) and cell survival
ratios (N/N0) of 2, 5 and 10. The pH of the med-
ium decreased as the N/N0 and/or the tempera-
ture increased. The model was more sensitive to
changes in the temperature than those in the cell
survival ratios (N/N0) within the ranges tested.
The pH values at which maximum specic
growth rate occurred were 4.2, 3.5, 3.4, and 4.0
for the experiments with room temperature (no
control), initial temperature of 378C (no control),
continuously controlled at 37 and 428C, respect-
ively. Ghaly and El-Taweel [5] mentioned that
the decrease in pH could be attributed to the for-
mation of lactic acid, conversion of carbon diox-
ide to carbonic acid and the buer capacity ofthe inorganic salts present in the cheese whey. A
reduced pH is usually desirable as microbial cul-
tures are less susceptible to microbial contami-
nation. Another point of view is that, if better
fermentation performance (high specic growth
rate, high lactic acid yield and ecient lactose
utilization) was obtained at such low pH levels,
then the task of pH control would be unnecess-
ary, thus, avoiding the related costs. Apparently,
the results of this study showed that bacterial ac-
tivity was signicantly reduced at such low pH
values caused by acid formation and, thus, the
need for pH control is justied.
4.2. Temperature
The temperature results are shown in Fig. 4. The
rst experiment was conducted at a controlled
room temperature and, thus, the initial temperature
of the medium remained relatively constant
(23.020.58C) until the end of the experiment.
During the experiment in which the initial tempera-
ture of the medium was raised to 378C, a fastdecrease in the medium temperature from the initial
value of 378Ctoavalueof258C occurred within the
rst 8 h. Then, a slow decrease of temperature (from
25 to 238C) was observed during the next 12 h.
Thereafter, no change in temperatures was observed
until the end of the fermentation process. For the
continuous temperature controlled experiments,
Fig. 4. Room and fermenter temperatures measured during batch fermentation.
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the medium temperatures were successfully main-
tainedat 3720.58Cand4220.58C, respectively.
In industrial fermentation processes, the oper-
ating temperature of the fermenter is often raised
to optimum level to increase microbial activity.
There is a maximum temperature at which the
growth rate is highest and that depends on the
characteristics of the microorganism used as well
as on the environmental conditions. However,
when the temperature of the medium is above or
below that required for optimum growth, the mi-
crobial activity is substanstially reduced and the
organisms may eventually die [16, 17]. Roy et
al. [11] reported that for Lactobacillus helveticus,
the optimum range for growth is 42458C. Roy et
al. [8] observed high specic growth rate (m) of0.639 h1 for Lactobacillus helveticus at a tem-
perature of 428C. Simulated studies by Peleg [18]
have demonstrated that the bacterial growth rate
improved by about two-fold when the operating
temperature was increased from 15 to 458C. In
this study, higher cell growth, higher lactose util-
ization and higher lactic acid production were
obtained at continuously controlled temperature
experiments (37 and 428C). In biological systems,
the temperature aects the rate of biochemical
reactions, the activity of extracellular enzymes,
the generation time, and the activity of the micro-
organisms involved. Tchobanoglous [19] reported
that the rate of reaction for microorganisms
increases with increasing temperature (doubling
with every 108C rise in temperature) until a limit-
ing maximum temperature is reached, after which
the growth rate decreases very rapidly. It is, there-
fore, important that the fermentation temperature
be maintained as constant as possible since bac-
teria grow optimally within a narrow temperature
range and are adversely aected by sudden tem-
perature unctuations.
4.3. Cell growth
The eect of temperature on the microbial
growth during batch fermentation is shown in
Fig. 5. The four distinct growth phases (lag
phase, during which the cell growth rate was
zero; exponential phase, during which the cell
growth rate was maximum; stationary phase,
during which the cell growth rate was zero; and
death phase, during which the cell death rate was
maximum) were observed. These four phases
were interconnected by transition intervals where
the growth rate changed continuously. The cell
number obtained from each of the experiment
were plotted on a semilogarithmic ordinate scale
to linearize the exponential portions of the
growth curves to ascertain the specic growth
rate and length of lag period.
Table 2 shows some of the kinetic parameters
of the batch culture obtained from this study.
The shortest lag period was 1.9 h and the longest
one was 4.7 h for continuously controlled tem-
perature at 428C and room temperature (238C),
respectively. The results showed that the lagperiod decreased (from 4.7 to 1.9 h), the expo-
nential phase decreased (from 11.6 to 5.9 h), no
remarkable change in the stationary phases, and
slight increase in death phase (7.011.2 h) as the
temperature of the medium was increased from
room temperature (238C with no control) to con-
tinuously controlled temperature at 428C. The
lowest and maximum ratios of nal cell number
to maximum cell number of 0.24 and 0.61 were
obtained during continuously controlled tempera-
ture at 378C and room temperature (238C and no
control), respectively.The specic growth rates (m) of the
Lactobacillus were found to be 0.090, 0.010,
0.210 and 0.250 h1 for fermentations at room
temperature (238C with no control), initial tem-
perature of 378C (no control), and continuously
controlled temperatures of 378C and 428C, re-
spectively (Fig. 6). These values were low com-
pared to those obtained by Roy et al. [8] which
ranged from 0.146 h1 (at 348C) to 0.492 h1 (at
428C). Mercier et al. [20] obtained a maximum
specic growth rate of 0.310 h1 (at pH 5.4) and
0.400 h1
(at pH 6.5). Similarly, Venkatesh etal. [13] and Siebold et al. [21], using initial sub-
strate concentrations of 50 and 30 gL1, reported
specic growth rates of 0.310 h1 (at pH 5.6) and
0.490 h1 (at pH 6.0), respectively. These results
suggest that the specic growth rate of the micro-
organisms is signicantly improved for pH con-
trolled experiments in the range of 5.4 to 6.5.
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Ghaly et al. [22] reported that the length of lag
phase usually depends on the extent to which the
new medium and environmental factors (pH, and
temperature) are dierent from those at which
inoculum was prepared. In this study, the pH
level was the highest (4.04.5) at the initial stage
Fig. 5. Eect of temperature on cell number during batch fermentation.
Table 2
Some kinetic parameters of the batch culture
Temperature
No control Control
Parameter 238C 378C 378C 428C
Lag phase (h) 4.7 2.8 2.4 1.9Exponential phase (h) 11.6 12.2 7.6 5.9
Specic growth rate, m (h1) 0.09 0.10 0.21 0.25
Stationary phase (h) 6.7 7.5 6.3 5.0
Specic cell death rate, Kd (h1) 0.040 0.060 0.080 0.090
Death phase (h) 7.0 9.5 11.7 11.2
Initial cell number (106 cells mL1) 13.0 14.0 13.5 14.0
Maximum cell number (106 cells mL1) 41.0 60.0 82.0 83.0
Final cell number (106 cells mL1) 25.0 30.0 20.0 24.0
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controlled experiment at 428C, while the lowest
Kd value of 0.040 h1 was deduced for exper-
iment at room temperature with no temperature
control (Fig. 6). The specic growth and death
rates at dierent temperature conditions during
fermentation appear to increase as fermentation
temperature was increased from room tempera-
ture with no control to continuously controlled
temperature at 428C. Roy et al. [11] mentioned
that after the exponential growth phase (8 h), cell
death rate increased at higher pH values (4.7
6.3). This death phase of organisms occur when
lactic acid concentration in the broth is greater
than 20 g L1.
4.4. Lactose utilization
Fig. 7 displays the lactose concentration in the
medium with the initial value of 48.0 g L1. The
residual lactose concentrations were found to be
31.8, 29.5 25.0 and 19.0 g L1 indicating that
34.0, 37.9, 47.9 and 60.6% of the initial lactose
concentrations were utilized during the fermenta-
tion process at room temperature (at 238C and
no control), an initial temperature of 378C (no
control), a continuous temperature control at 37and at 428C, respectively. Table 3 shows the per-
cent of lactose utilization and specic lactose
uptake rate during the four growth phases.
About 2.7 and 29.7% of the initial lactose con-
centration were utilized during lag and exponen-
tial phases during the fermentation with
temperature controlled at 428C (corresponding to
specic lactose uptake rates of 0.047 109 and
0.076 109 g lactose cell1 h1 during lag and
exponential phases), respectively. During the
growth phases, the lactose utilization rate
increased from 29.3 to 53.2% as the fermentationtemperature was increased from room tempera-
ture (with no control) to that continuously con-
trolled one at 428C.
General correlations between lactose utilization
and each of the pH, cell number, and tempera-
ture tted the following equations (R 2=0.985,
0.960, and 0.995):
Fig. 7. Lactose concentration during batch fermentation.
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Lu 251X55 166X05pH
36X45pH2 2X66pH3Y 9
Lu 5X16 5X13N
N0 0X59 ln
N
N0Y 10
Lu 99X8
1 exp11X78 0X14 T 1a4X64 X 11
where: Lu is the cumulative lactose utilization (g
L1).
Lactose utilization as a function of pH, tem-
perature and cell number can be determined by
the following general expression:
Lu fpHp1 fNp2 fTp3 X 12
Substituting the expressions for f(pH), f(N) and
f(T) from Eqs. (9)(11) into Eq. (12), the follow-
ing equation is obtained (R 2=0.995):
Lu
251X55 166X05pH 36X45pH2
2X66pH30X337
5X16 5X13N
N0 0X59 ln
N
N0
0X003
99X8
1 exp11X78 0X14 T 1a4X64
Hd Ie1X700X 13Evaluating Eq. (13) revealed that the lactose util-
ization increased when the cell survival ratio (N/
No) increased from 2 to 10, at the optimum pH
of 6 and the optimum temperature of 408C. The
lactose utilization decreased when the pH varied
from 6 to 5 or 7, at the cell survival ratio (N/No)of 5 and the temperature of 408C. However, the
lactose utilization increased by three-fold (7.3
20.3 g L1) when the temperature increased from
20 to 408C, at the pH of 6 and the cell survival
ratio (N/No) of 5, thus indicating that the model
was sensitive to changes in temperature than
those in pH and cell ratio.
Higher values of lactose utilization ranging
from 4493% when nutrient supplements were
added have been reported by other
investigators [11]. Similar work by Chiriani et
al. [23] on whey ultraltrate containing 30 g L1
initial lactose concentration using Lactobacillus
helveticus at temperature of 428C and pH of 5.7
gave a sugar conversion of 94.07% after 48 h.
For continuous pH control (at pH = 5.6), about
89.7% of lactose was converted to lactic acid
after 95 h of fermentation; whereas, under no pH
control conditions, using Lactobacillus bulgaricus,
46.2% of the initial lactose concentration (30 g
L1) was converted to lactic acid during 40 h of
fermentation [24]. According to the authors, the
low initial lactose concentration and optimum
pH level (pH 5.65.7) at which the fermentation
were conducted, have contributed to such highsugar conversion levels. The results of this study
showed that a maximum lactose utilization of
53.2% was achieved at continuously controlled
temperature of 428C. The lactose conversion was
low compared to other studies, due to the fact
that the fermentation experiments were con-
ducted at pH in the low range of 2.74.0.
Table 3
The lactose utilization rate and specic lactose uptake rate during the growth phases
TemperatureNo control Control
238C 378C 378C 428C
Phase (%) (g cell1h1) 109 (%) (g cell1h1) 109 (%) (g cell1h1) 109 (%) (g cell1h1) 109
Lag phase 3.1 0.023 2.5 0.029 3.5 0.051 2.7 0.047
Growth phase 13.5 0.022 18.9 0.020 21.0 0.038 29.7 0.076
Stationary phase 7.9 0.014 5.7 0.006 7.7 0.007 8.7 0.009
Death phase 4.8 0.010 9.0 0.010 10.8 0.010 13.1 0.010
Total 29.3 36.1 43.0 53.2
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4.5. Lactic acid
The chemical reactions involved during conver-
sion of lactose to lactic acid (using bacteria)
under anaerobic conditions are as follows:
(a) Product formation and respiration:
90 C12H22O11 360 H2O 4bacteria 270 C3H6O3
270 CO2 1080 H EnergyX 14
(b) Growth (Synthesis):
10 C12H22O11 24 NH4 4
bacteria24 C5H7NO2
62 H2O 24 HX 15
The net chemical reaction of the above two
equations can be written as follows:
100 C12H22O11 24 NH4 360 H2O 4
bacteria
270 C3H6O3 24 C5H7NO2 270 CO2
61 H2O 1104 H EnergyX 16
From Eq. (16), the stoichiometric lactic acid
yield (YP/S) was estimated to be 0.71 g lactic
acid/g lactose and the cell yield (YX/S) was found
to be 0.08 g cell/g lactose.
The results (Fig. 8) revealed that the initial
lactic acid concentration in the cheese whey
was about 2.2 g L1 whereas the nal lactic acid
Fig. 8. Eect of temperature on lactic acid production.
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Table4
Lacticacidproductionandyieldduringtheduringthegrowthphasesasafunctionoffourtemperaturelevels
Temperature
Nocontrol
Control
238C
378C
378C
428C
Phase
P
(g/L)YP/N
(gLA/cell)
109
YP/S
(gLA/glac)
P
(g/L)YP/N
(gLA/cell)
109
YP/S
(gLA/glac)
P
(g/L)YP/N
(gLA/cell)1
09
YP/S
(gLA/glac)
P
(g/L)YP/N
(gLA/cell)
109
YP/S
(gLA/glac)
Lag
0.2
0
0.0
14
0.1
3
0.1
7
0.0
11
0.1
5
0.2
2
0.0
15
0.1
3
0.2
2
0.0
15
0.1
7
Growth
1.5
1
0.0
60
0.2
3
2.5
0
0.0
67
0.2
8
3.5
2
0.1
00
0.3
4
3.7
4
0.1
15
0.2
6
Stationary
0.5
7
0.0
14
0.1
5
1.3
3
0.0
23
0.4
9
2.2
8
0.0
28
0.6
2
2.8
4
0.0
35
0.6
8
Death
0.6
5
0.0
19
0.2
8
1.1
0
0.0
24
0.2
6
2.4
7
0.0
54
0.4
7
2.5
0
0.0
45
0.4
0
Total
2.9
3
0.2
0
5.1
0
0.2
9
8.4
9
0.4
0
9.3
0
0.3
6
P=
thelacticacidconcen
tration(g/L);YP/N=theproductyieldcoe
cient(glacticacid/cell);YP/S=theproductyieldcoecient(glacticacid/glactose)
concentration was in the range of 6.012.3 g L1.
Thus, the lactic acid production varied from 3.8
to 10.1 g L1.
Table 4 shows the summary of some kinetic
parameters of lactic acid production and yield
during the various phases of growth. The maxi-
mum lactic acid yield (lactic acid production/lac-
tose utilization) was 36.0%, which was obtained
during the fermentation with continuous tem-
perature control (at 428C). Thus, the maximum
lactose conversion eciency (lactic acid yield/
stoichiometric lactic acid yield) of the batch fer-
mentation process was found to be 50.7%. Fig. 9
summarizes the eect of fermentation tempera-
ture on lactic acid production during the various
growth phases of Lactobacillus helveticus. The
minimum and maximum lactic acid productions
were eected during the lag and exponential
phases respectively. Comparable amounts of
lactic acid were produced during stationary and
death phases for each temperature level. These
results indicate that at pH in the range of
4.53.5, the lactic acid produced was mainly by
growth associated mechanism, whereas, at low
pH it was due to cell maintenance. For no
pH control conditions, about 30% of the lactic
acid produced was due to growth associated
Eq. (13).The experimental data for the lactic acid con-
centration and the transient pH of the medium
was curve tted and found to conform to
Weibull distribution. The general correlation
obtained was of the form shown below
(R 2=0.985):
P 9X364X58 103 exp 359X3pH2X88
17
where P is the lactic acid concentration (g L1);
pH is the pH of the medium ().
In the same manner, the correlation betweenthe lactic acid concentration and the fermenta-
tion temperature tted the following sigmoid
equation (R 2=0.950):
P 9X54
1 exp11X45 0X33 T 1a3X29 X 18
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The correlation between cell number and the lac-
tic acid concentration tted the following ex-
pression (R 2=0.998):
P 24X53 24X31 exp 0X01N
N0
2X074 519
where N/N0 is the cell survival ratio ().
Similarly, the correlation between the lactic
acid concentration and the lactose utilization can
be described by the following expression
(R 2=0.997):
P 9X50 9X0exp 0X001Lu2X41
h iX 20
The lactic acid production as a function of the
temperature, cell survival ratio and lactose utiliz-
ation can be expressed as follows (R 2=0.991):
p 9X54
1 exp11X45 0X33 T 1a3X29
VX
WaY
0X950
24X53 24X31exp
40X01
N
N0
2X075VX
WaY
0X002
9X50 9X0exp
h0X001 Lu
2X41
i& '0X049
21
Eq. (21) showed that the lactic acid production
increased when the cell survival ratio (N/N0) was
increased from 2 to 10 (at the temperature and
lactose utilization of 408C and 30 g L1, respect-
ively) or when the lactose utilization was
increased from 10 to 30 g L1 (at the temperature
and the cell survival ratio of 408C and 5, respect-
ively) or when the temperature was increased
from 20 to 408C (at the cell survival ratio and
the lactose utilization of 5 and 30 g L1, respect-
ively). However, the increase in lactic acid pro-
duction as a result of temperature increase was
almost three-fold indicating that the model wasmore sensitive to changes in the temperature
than those in cell survival ratio and the lactose
utilization.
Early inhibition conditions were noted during
the fermentation process. Since the lactate con-
centration was low in the initial period of fer-
mentation, this inhibition was mainly due to
lactic acid produced and the lactose concen-
tration level at the prevailing pH of the medium.
Roy et al . [11] observed death phases of
Lactobacillus helveticus at lactic acid concen-
tration higher than 20 g L1. Higher lactic acid
concentration have been reported (3560 g L1)
for initial lactose concentration varying from
37.2 to 50 g L1 [19, 11]. Chiriani et al . [23]
obtained a lactic acid yield of 26.0 g L1 using
whey containing 30 g L1 lactose and
Lactobacillus helveticus at a temperature of 428C
and a pH of 5.7 after 48 h. Under a controlled
pH of 5.9 and a temperature of 428C, Roy et
al. [8] obtained high lactic acid production of
about 30 g L1. The maximum lactic acid pro-
duced in this study was only 10 g L1
. This lowconcentration of lactic acid is due to the fact that
the pH of the medium decreased as the fermenta-
tion process proceeded reaching conditions un-
favourable for the growth activity of the
fermenting bacteria.
5. Conclusions
The eect of broth temperature on the per-
formance characteristics of lactic acid fermenta-
tion from cheese whey lactose was investigated in
the absence of pH control. In all the four fermen-
tation conditions studied, the pH value of the
broth decreased from 4.4 to values less than 3.0
as fermentation proceeded reaching conditions
unfavourable for cell growth activity. The maxi-
mum specic growth rate (m) obtained was
0.25 h1 for fermentation conducted at the con-
trolled temperature of 428C.
Although considerable growth activity was
noted during the fermentation process, this
appears inadequate for better lactose utilization
since only about 34.060.6% of the initial lactose
concentration was utilized. For the four fermen-tation conditions studied, the lactic acid concen-
tration was only about 6.012.3 g L1. The
maximum lactic acid yield was 37.9%, which was
obtained during fermentation with continuously
controlled temperature at 428C. The correspond-
ing lactose conversion eciency was 53.3%.
Cell number, lactose consumption and lactic acid
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