The efficiency and economics of the lactic acid production through microbial
fermentation is still under problematic conditions if considered from
many points of view and among them, media composition plays a vital role
in the improvement of such a process (Stanbury et al., 2003). Fermentation
of lactic acid by microbes such as bacteria using different media plays
an important role in the final product of the process (Lee, 2005). Proper
design of the media does affect the performance of microorganisms in optimizing
the lactic acid production (Krishnan et al., 1998; Telez-Luiz et
Nowadays, research effort is focused on looking not only for new and
effective nutritional sources but also for new progressive fermentation
techniques which enable the achievement of both high substrate conversion
and high production yields. The improvement of lactic acid production
has been studied under the control of various factors and media components
(Arasaratnam et al., 1996; Wenge and Methews, 1999; Hujanen et
al., 2001; Molinier et al., 2004; Rao et al., 2004).
In our previous studies (Ismail et al., 2006), eleven components
of media were screened using Placket Burman Design and the results indicated
that, the main media components that affected the lactic acid production
process by L. rhamnosus fermentation were glucose and peptone.
The correlation between those two variables was analyzed using Response
Surface Methodology (RSM). In this study, the media compositions of the
fermentation process were optimized using the CCD method. The advantage
of CCD method is that the effect of variable at a distance alpha from
the design centre can be studied (Liew et al., 2005).
MATERIALS AND METHODS
Design of experiment (DOE): Experiment was conducted at Bioprocess
Engineering Laboratory of IIUM Malaysia and was designed by Central Composite
Design (CCD) using STATISTICA Software. CCD is a set of technique designed
to find the best value of response.
Microorganism and media: The microorganism used in this study
was Lactobacillus rhamnosus (L. rhamnosus, the homo-fermentative
lactic acid bacteria (Takumi et al., 2001). The culture media used
was the MRS medium containing glucose, peptone, yeast extracts, lactose,
Tween 80, K2HPO4, sodium acetate, (NH4)2SO4,
MnSO4, MgSO4 and distilled water.
Inoculums preparation: The preparation of inoculums started with
transferring the stock culture into a liquid MRS media. After the growth
of culture, the microorganisms were transferred to a plate of solid MRS
medium. The plate was incubated at 37°C for 48 h in order to allow
sufficient growth of colonies.
The grown colonies were either used to initiate a fermentation process
or were stored back at 4°C as stock culture which can be prepared
by culturing the colonies in slant agar followed by adding 30% of sterilized
glycerol. The L. rhamnosus inoculums were prepared by inoculating
a single colony of them into 10 mL broth media which was then incubated
at 37°C for 24 h. One milliliter of inoculums was transferred into
bijou bottle containing 9 mL media. Cultures were incubated for 10 h at
37°C before being transferred into shake flask.
Sampling: Sample in shake flasks were taken by using aseptic technique
for every 2 h by flaming the cap swabbed with 70% ethanol. Twelve milliliter
of sample was transferred into a bijou bottle, which was then being divided
for measuring optical density (OD, A660 nm), product (lactate),
substrate (glucose) and cell dry weight. The flasks then were transferred
back to the thermostat rotary incubator shaker to continue the fermentation
Analytical method: Optical density analysis (OD), total cell number
(TCN) and Cell Dried Weight (CDW) were analyzed as described by Maizirwan
et al. (2006).
Glucose and lactate analysis: One milliliter sample was transferred
into 1.5 mL Eppendorf tube and centrifuged at 3000 rpm for 10 min. The
supernatant was transferred into a cuvette and analyzed using the YSI
2700 Biochemical Analyzer.
Experimental design for media optimization: The experiment for
media optimization was designed using the Central Composite Design (CCD)
method. From the media screening experiment, since peptone and glucose
were found to be the most significant component that affects the production
of lactic acid, they were selected as independent variables to be optimized
in order to increase the productivity of lactic acid.
||Experimental design for media optimization using central
1 shows the actual parameters value used in media optimization experiment
of media using CCD method.
Fermentation in shake flask: In order to find the exact value
of the optimum concentrations of peptone and glucose in producing the
optimum concentration of lactic acid, experiments were carried out to
find the growth kinetic of the bacteria and the productivity of lactic
acid using the formulated media components by CCD method. For each run,
10 shake flasks were used. In each flask, 10 mL inoculums were transferred
into 90 mL formulated media under aseptic condition. The shake flask was
capped with cotton and swabbed with 70% ethanol and then incubated in
a thermostat rotary incubator shaker for 30 h under setting temperature
of 37°C and rotation speed of 150 rpm. The critical value or optimized
value in percentage of peptone and glucose was studied by STATISTICA analysis.
RESULTS AND DISCUSSION
Media optimization: The results showed that the cell growth rates of
L. rhamnosus are slightly different with the different media component
(Fig. 1). At the beginning, the growth was quite similar but
after certain time, the growth becomes slower not only due to the decrease of
substrate concentration but also the resistance of these bacteria with acidic
condition and other inhibitors.
From Fig. 2, it could be concluded that the production
of lactate is different with different concentration of the peptone and
glucose. The graph showed that Run 6 is the best run due to the high amount
of lactate produced. The rate of productivity is increasing steadily.
The substrate conversion (glucose) is decreasing as the fermentation getting
longer (data not hown).
||The growth profile of L. rhamnosus in T-Flask
||The profile of lactic acid production for each run
||The growth profile, glucose reducing and produced lactate
for Run 6
This mean that even though the cell growth is
not much different but the lactate produced is significantly different.
Many factors have been involved in this lactate production such as the
combination of media quantity, pH and others environmental factors. Since L. rhamnosus is a gram positive, it needs longer time to secrete
secondary metabolite compared to gram negative via metabolic pathway.
For run 6 (Fig. 3), the graph was extended to see the
relation among the growth performance of the bacteria, glucose consumption
and the lactate produced. The yield is increased as the substrate is decreasing.
When all the substrates were consumed, the production was stopped. The
Lactobacillus is non-growth associated, as can be viewed from the
graph that even the cell growth had reached the stationary phase after
20 h of fermentation, the lactate still being produced with high rate.
At this condition, the glucose has been converted to lactate via sugar
The total of carbon source plays an important role in the lactate production.
||Interaction pattern between glucose and peptone on Lactate
||Critical value of glucose and peptone
||ANOVA table of quadratic regression model
In many of the fermentation processes, sugars are controlled so that it
will be completely metabolized to produce a targeted product but not the
by-product as it will become the inhibitor for the cell itself. This homo-fermentative
bacterium is hoped to produce lactate as a main product with less by-product
such as ethanol and CO2.
Critical value: From the Table 2, 9.8% of glucose
and 9.98% of peptone was needed by L. rhamnosus in order to increase
the production of acid lactic. This critical value was used for further
optimization process in the bioreactor.
The ANOVA of quadratic regression model demonstrate the model was highly
significant as shown in Table 3 with lower probability
value (p-value). The pattern of interaction between the glucose and peptone
(Fig. 4) was also indicated by this coefficient.
||Productivity and growth kinetic study of L. rhamnosus
using optimized media
on the p-value obtained from the ANOVA table, it shows that the glucose
is the main factor to promote the lactic acid production, followed by
the combination of glucose and peptone.
Growth kinetic study: From the Table 4, it shows
that the cells grow fastest in Run 1 and 3 but with lower productivity.
The highest productivity has been obtained at Run 5 with the value of
0.630 g g-1 h which corresponds to the fifth faster in the
cell growth rate; its about 0.341 h-1. The value of growth
rate is still within the range of value that had been obtained by Zannini
et al. (2005); it ranged from 0.16 to 0.390 h-1. The
highest productivity has been achieved at lower concentration of glucose
and shorter fermentation time. As reported by Serna and Rodríguez
(2006), the productivity of lactic acid production using Lactobacillus
lactis has been obtained at 0.29 g L-1h using 20 g L-1
of glucose and 48 h of fermentation time. In this study, with low amount
of substrate (<10 g L-1 of glucose) and short fermentation
time (<40 h) has contributed to generate the high productivity of lactic
The Central Composite Design (CCD) is practical to be used in the optimization
of fermentation media. The critical concentration of the most significant
variables (glucose and peptone) which critically influence the cell growth
and lactic acid production was 9.80 and 9.98 g, respectively. From ANOVA
analysis, the most influence variable can be determined by p-value. The
smaller p-value indicates the most significant variable toward the lactate
production. The maximum lactic acid production was observed in Run 5 which
produces 0.630 g L-1 of the acid which correspond to specific
growth rate of 0.341 h-1.
The authors would like to thank IIUM Research Centre for funding this
research under Project No. IIUM 504/022/3/LT 27.