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Optimization of lactic acid production by Escherichia coli harboring ldhA gene
from Rhizopus oryzae
Thanawan Watthanaphorna,b*, Ruethairat Boonsombata
aInstitute
of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand
in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
bProgram
*
Author for correspondence; email: Thanawan.W@Student.chula.ac.th
Abstract
Lactic acid is widely used for many industries such as food, pharmaceutical, cosmetic and biodegradable plastic. Lactic
acid can be produced by fermentation of many microorganisms such as Rhizopus oryzae. Although this fungus has many
advantages such as optically pure lactic acid production and simple nutrient requirement, one of the obstacles for lactic
production is its morphology. The filamentous form of R. oryzae results in an increase in broth viscosity, but a decrease in
oxygen transfer. The aim of my research is to apply genetic engineering techniques to overcome the problem of R. oryzae
morphology. Escherichia coli is used for this study due to its rapid growth and well-studied genetic information. The genetically
modified E. coli strain RB24 generated by transforming the plasmid containing R. oryzae ldhA gene into E. coli background with
knocked out chromosomal ldhA and pta is utilized for L-lactic acid production. Previous study found that when E. coli strain
RB24 was fermented in anaerobic condition with 100 g/L initial glucose in shake-flask level, lactic acid was obtain at the
concentration of 5.03±4.149 g/L. Due to the different characteristics between R. oryzae and E. coli, fermentation conditions and
glucose concentrations are required to optimized for lactic acid production when the strain RB24 is used.
1. Introduction
Lactic acid is an organic acid, which was
discovered in 1780 by Scheele and used in many
industry such as food industry, pharmaceutical
industry, cosmetic industry, chemical industry and
textile industry [1]. Recently, the consumption of
lactic acid trends to be increased rapidly. Lactic acid
can be used as a monomer for poly(lactic acid) that
can be applied in the production of biodegradable
plastic which is a worldwide concern due to the global
warming [2,3].
Lactic acid has two optical isomers: L(+)-lactic
acid and D(-)-lactic acid. It can be produced by
fermentation of many microorganisms or chemical
synthesis [4]. The advantages of microbial
fermentation include low substrate cost and energy
consumption for lactic acid production [5]. The purity
of lactic acid is important for industry application [6].
The isomers of lactic acid produced from
microorganisms depend on the enzyme lactate
dehydrogenase. Lactate dehydrogenase catalyzes
pyruvate to lactate in glycolysis [7]. The efficiency of
poly(lactic acid) production also depends on purity of
the isomers which L(+)-lactic acid is more favorable
for industry than D(-)-lactic acid. It has been many
studies about biological fermentation for lactic acid
production which lactic acid bacteria is commonly
used in various industries due to the high productivity.
However, it still has some problems such as complex
nutrient requirement [8]. Other microorganisms such
as a fungus, Rhizopus oryzae, can produce pure L(+)lactic acid when starch or glucose is used as a carbon
source. However, lactic acid production from R.
oryzae has some limitations, for example, a high
concentration of oxygen requirement, low yield lactic
acid production, and the contaminations of fumaric
and ethanol produced during lactic acid fermentation
[1]. Moreover, its filamentous morphology is not
suitable for fermentation in bioreactors [8]. Therefore,
we are interested in the expression of ldhA gene from
R. oryzae in other microorganisms that requires simple
nutrients for growth and produces pure and high L(+)lactic acid.
The aim of this research is to apply genetic
engineering techniques to overcome the problem of R.
oryzae morphology. The genetically modified
Escherichia coli strain RB24 used in this study is
generated by transforming the plasmid containing R.
oryzae ldhA gene into E. coli background with
knocked out chromosomal ldhA and pta to allow the
expression of R. oryzae ldhA on the plasmid and
reduce the contamination of other substances
produced during fermentation, respectively [9]. This
E. coli strain RB24 is expected to combined the
advantages of E. coli, which are rapid growth, simple
nutritional requirements and well-studied genetic
information, and R. oryzae, which are simple medium
requirement and production of optically pure L(+)lactic acid, in one particular organisms.
The previous study suggests that L(+)-lactic acid
production from E. coli RB24 in shake flask level
under anaerobic condition gives a low yield of L(+)lactic acid when fermented with 100 g/L glucose
(5.03±4.149 g/L). Furthermore, the high residual
glucose
concentration
may
inhibit
lactic
acid production. Therefore, the optimization of
fermentation conditions and glucose concentrations is
necessary for lactic acid production from this E.
coli harboring R. oryzae ldhA gene.
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slightly faster than RB7 and RB24 (at 6 hr for
JC13509 comparing to at 9 hr for RB7 and RB24).
2. Materials and Methods
2.1 Microorganisms and media
All strains used in this study are JC13509
derivatives. E. coli K-12 strain JC13509 from Dr.
Steven Sandelr’s lab, University of Massachusetts,
Amherst, USA, is used as the wild-type strain. The
strain which chromosomal ldhA and pta are knocked
out is named as RB7. The strain RB24 is the strain
RB7 that harbors the plasmid with ldhA from R.
oryzae NRRL395. All strains were grown in LB
medium at 37 °C for 24 hour. but 100 µg/ml
Ampicillin was added in RB24 culture to maintain the
plasmid.
The 1 L of growth medium (LB medium)
consists of 10 g of bactro-peptone, 5 g of yeast
extract, 10 g of NaCl and 2% bactro-agar (for plates).
The 1 L of fermentation broth consists of 5 g of yeast
extract, 5 g of peptone, 0.25 g of KH2PO4, 0.25 g of
K2HPO4 and salt solution 5 mL (10 ml salt solution
consist of 400 mg MgSO4.7H2O, 20 mg MnSO4.5H2O,
20 mg FeSO4.7H2O) with pH 6.8. The concentration
of glucose in fermentation broth was varied as 10, 20,
30, 50, 70 and 100 g/L. All media was sterilized at
121 °C for 15 min.
2.2 Inoculum preparation
Cells from LB agar medium were transferred into
5 ml of fermentation broth and incubated at 37 °C,
200 rpm for 10 hour in a rotary shaker.
2.3 Fermentation
The inoculum was transferred into 50 ml
fermentation broth with varied glucose concentration.
Fermentation conditions as in Table 1 were used in
this experiment. The 1 ml of sample was harvested at
different points of time.
Table 1: Fermentation conditions for lactic acid
production.
Condition
1
2
37 °C for 48 hour no shaking
37 °C for 48 hour, anaerobic
Figure 1: Growth curve of E. coli strain JC13509
(squares), RB7 (triangles) and RB24 (circles) for 48
hour.
The results of L(+)-lactic production with various
glucose concentrations under limited oxygen
condition (37 °C for 48 hour no shaking) are shown in
Table 2 and those under anaerobic condition are
shown in Table 3.
Table 2: L(+)-Lactic acid (LA) production and
residual glucose under limited oxygen condition (37
°C no shaking) for 24 and 48 hr.
Initial
glucose
(g/l)
10
20
30
50
2.4 Analytical methods
Cell growth was determined by Spectrophotometer
at 600 nm. The samples harvested at different points
of time were centrifuged at 10,000 rpm for 7 minute.
Then, the concentrations of L(+)-lactic acid and
residual glucose in the supernatant were measured by
YSI selector and high performance liquid
chromatography (HPLC).
70
100
E. coli
strains
24 hr
48 hr
JC13509
LA
(g/l)
0.20
Glucose
(g/l)
0.00
LA
(g/l)
0.19
Glucose
(g/l)
0.00
RB7
0.19
0.04
0.21
0.04
RB24
JC13509
0.24
0.19
0.50
9.56
0.24
0.24
0.00
0.45
RB7
0.04
8.93
0.14
0.61
RB24
JC13509
0.14
0.18
4.75
1.43
0.26
0.20
0.05
8.45
RB7
0.17
1.66
0.14
9.59
RB24
JC13509
0.18
0.20
1.61
38.0
0.22
0.21
13.6
29.6
RB7
0.12
40.6
0.15
28.7
RB24
JC13509
0.32
0.22
40.7
58.8
0.34
0.21
32.3
48.9
RB7
ND*
ND
ND
ND
RB24
JC13509
0.37
0.21
62.3
93.9
0.71
0.23
54.5
82.6
RB7
ND
ND
ND
ND
RB24
0.33
96.1
0.40
89.9
3. Results and Discussion
*ND = Not determined
The growth curve of E. coli JC13509, RB7 and
RB24 for 48 hour is represented as in Figure 1. The
strain JC13509 seemed to stay in the log phase
slightly less than RB7 and RB24 (at 3 to 6 hr for
JC13509 comparing to at 3-9 hr for RB7 and RB24).
The JC13509 also seemed to enter to stationary phase
From this experiment, the highest lactic acid
concentration of 0.71 g/l was measured when E. coli
RB24 was fermented with the initial glucose of 70 g/l
for 48 hr under limited oxygen condition (Table 2).
This may results from the gene ldhA from R. oryzae
which is an organism that produce glucose under
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aerobic condition while the mechanism for lactic acid
production in E. coli may be triggered when oxygen is
diminished. The plasmid in RB24 contains not only
the coding region of ldhA gene, but also the promoter
which may be regulated by oxygen. Therefore, L(+)lactic acid production from this E. coli RB24, which
ldhA gene from R. oryzae is expressed E. coli, may
require small amount of oxygen to induce the
expression of lactate dehydrogenase. Thus, the
production of L(+)-lactic acid from E. coli RB24
seems to be better under limited oxygen condition
than anaerobic condition (Table 2 Vs Table 3).
Table 3: L(+)-Lactic acid (LA) production and
residual glucose at 37 °C under anaerobic
condition for 24 and 48 hr.
Initial
glucose
(g/l)
E. coli
strains
24 hour
48 hour
JC13509
LA
(g/l)
0.22
Glucose
(g/l)
0.05
LA
(g/l)
0.24
Glucose
(g/l)
0.03
RB7
0.18
1.26
0.17
0.04
RB24
JC13509
0.14
0.20
0.24
4.80
0.25
0.23
0.00
1.30
RB7
0.14
9.69
0.21
4.86
RB24
JC13509
0.21
0.22
9.12
13.8
0.26
0.20
5.98
7.99
RB7
0.15
18.2
0.19
13.1
RB24
JC13509
0.21
0.19
17.2
36.6
0.25
0.23
14.7
28.2
RB7
0.17
39.8
0.14
32.6
RB24
JC13509
0.28
0.22
42.4
56.2
0.30
0.21
35.2
47.2
RB7
ND*
ND
ND
ND
RB24
JC13509
0.34
0.24
61.7
92.2
0.33
0.24
54.5
85.0
RB7
ND
ND
ND
ND
RB24
0.35
90.5
0.38
84.3
Therefore, the future work is to optimize other
conditions and also glucose concentrations to improve
L(+)-lactic production from the E. coli RB24 strain
which is useful for scaling up the L(+)-lactic acid
production.
Acknowledgements
This research is granted by Thai Research Fund
(TRF) and National Budget 2012-2013. It is also
supported by The Institute of Biotechnology and
Genetic Engineering, program in Biotechnology,
Faculty of Science, Chulalongkorn University,
Bangkok, Thailand.
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10
20
30
50
70
100
*ND = Not determined
[3]
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4. Conclusions
E. coli RB24 was a genetically modified strain that
harbors ldhA from R. oryzae. In this study, the lactic
acid concentration was still obtained with low yield in
all conditions and glucose concentration was still high.
The other factors such as pH will be monitored
because the low pH may inhibit lactic acid production.
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