Tango Ghaly 1999 Effect of Temperature L Helve Tic Us

8/3/2019 Tango Ghaly 1999 Effect of Temperature L Helve Tic Us

1/18

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.


2/18

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.

M.S.A. Tango, A.E. Ghaly / Biomass and Bioenergy 16 (1999) 617862


3/18

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

M.S.A. Tango, A.E. Ghaly / Biomass and Bioenergy 16 (1999) 6178 63


4/18

Fig.

1.

Schematicdiagram

ofexperimentalapparatusforbatchoperation.



5/18

Fig. 2. Diagram of batch bioreactor.



6/18

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



7/18

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.



8/18

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.



9/18

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.



10/18

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



11/18


12/18

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.



13/18

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



14/18

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.



15/18

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



16/18

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



17/18


18/18

[6] Ghaly AE, El-Taweel AA. Kinetics of batch production

of ethanol from cheese whey. Biomass and Bioenergy

1994;6(6):46578.

[7] Ghaly AE, Ben-Hassan RM, Mansour MH. Eect of pHcontrol on the growth and survival of Kluyveromyces fra-

gilis in cheese whey under aerobic condition. Applied

Biochemistry and Biotechnology 1992;23(3):21935.

[8] Roy D, LeDuy A, Goulet J. Kinetic of growth and lactic

acid production from whey permeate by Lactobacillus

helveticus. The Canadian Journal of Chemical

Engineering 1987;68(8):597603.

[9] Reddy CA, Henderson HE, Erdman MD. Bacterial fer-

mentation of cheese whey for production of a ruminant

feed supplement rich in crude protein. Applied and

Environmental Microbiology 1976;32:76976.

[10] Luedeking R, Piret EL. A kinetic study of lactic acid fer-

mentation. Batch process at controlled pH. Journal of

Biochemical and Microbiological Technology andEngineering 1959;1(4):393412.

[11] Roy D, Goulet J, LeDuy A. Batch fermentation of whey

ultraltrate by Lactobacillus helveticus for lactic acid pro-

duction. Applied and Microbiology Biotechnology

1986;24:20613.

[12] Cox GC, MacBean RD. Lactic acid production by

Lactobacillus bulgaricus in supplemented whey ultraltra-

tion. The Australian Journal of Dairy Technology

1977;32:1922.

[13] Venkatesh KV, Okos MR, Wankat PC. Kinetic model of

growth and lactic acid production from lactose by

Lactobacillus bulgaricus. Process Biochemistry

1993;28:23141.

[14] Norton S, Lacroix C, Vuillemard J-C. Kinetic study ofcontinuous whey permeate fermentation by immobilized

Lactobacillus helveticus for lactic acid production.

Enzyme and Microbial Technology 1994;16(6):45766.

[15] Ghaly AE, Ben-Hassan RM. Dehydrogenase activity

measurement in yeast fermentation. Applied

Biochemistry and Biotechnology 1993;43(1):7792.

[16] Rosso L, Lobry JR, Bajard S, Flandrois JP. Convenientmodel to describe the combined eects of temperature

and pH on microbial growth. Applied and

Environmental Microbiology 1995;61(2):6106.

[17] Peleg M. A model of temperature eects on the mi-

crobial populations from growth to lethality. Journal of

the Science of Food and Agriculture 1995;68:839.

[18] Peleg M. A model of microbial growth and decay in a

closed habitat based on combined Fermi's and the

Logistic equations. Journal of the Science of Food and

Agriculture 1996;71:22530.

[19] Tchobanoglous, G, Wastewater Engineering: Treatment,

Disposal, Reuse. 2nd ed. New York: McGraw-Hill, 1979.

[20] Mercier P, Yerushalmi L, Rouleau D, Dochain D.

Kinetics of lactic acid fermentation on glucose and corn

by Lactobacillus amylophilus. Journal of ChemicalTechnology and Biotechnology 1992;55:11121.

[21] Siebold M, Frieling Pv, Joppien R, Rindeisch D,

Schugerl K, Roper H. Comparison of the production of

lactic acid by three dierent Lactobacilli and its recovery

by extraction and electrodialysis. Process Biochemistry

1995;30(1):8195.

[22] Ghaly AE, Kok R, Ingrahm JM. Growth rate determi-

nation of heterogeneous microbial population in swine

manure. Applied Biochemistry and Biotechnology

1989;22:5978.

[23] Chiriani L, Mara L, Tabacchioni S. Inuence of growth

supplements on lactic acid production in whey ultral-

trate by Lactobacillus helveticus. Applied Microbiology

and Biotechnology 1992;36:4614.

[24] Shahbazi A, Du M, Kumar JV, Tan KH, 1996, Lacticacid production by Lactobacillus helveticus. Liquid fuels

for renewable resources. In Proceedings of an alternative

energy conference. 277.


Date post:	06-Apr-2018
Category:	Documents
Upload:	antonija-trontel
View:	222 times
Download:	0 times

Tango Ghaly 1999 Effect of Temperature L Helve Tic Us

Documents