Research J. Pharm. and Tech.

9(4): April 2016

ISSN

0974-3618 (Print)
0974-360X (Online)

www.rjptonline.org

RESEARCH ARTICLE

Application of Response Surface Methodology (RSM) to optimize culture

media for the production of rhamnolipids by Pseudomonas aeruginosa
Athira Gopakumar1, Jacyntha Thomas1, G.Narendrakumar1, Preethi. T.V2
1

Department of Biotechnology, Sathyabama University, Chennai 600 119.

Research Scholar, Department of Microbiology, School of Life Sciences, Vels University, Pallavaram, Chennai
600 117.
*Corresponding Author E-mail:

ABSTRACT:
Rhamnolipids, naturally existent biosurfactantsassembledbyrhamnose sugar molecules and hydroxyalkanoicacids that has wide range of promising profitable applications. In the study Pseudomonas
aeruginosabacterial isolates was identified and screened for rhamnolipid production. P. aeruginosa achieved the
production of rhamnolipid at room temperature in static cultures of a mineral salts medium containing olive oil.
The pure culture was enhanced and optimized at pH- 7, temperature-37oC and olive oil as effective carbon
source. The cultural media was optimized using response surface methodology - Box-Behnken with 30 ml of
olive oil, 5 g/l NaNO3, 4 g/l KH2PO4 and 7.5 g/l FeSO4 mixture contributes maximum production of
rhamnolipid. The R2 value was 99.8 that correlated with adjusted R2 value of 99.7 and predicted R2 value of 99.5
shows the model is significant.

KEYWORDS: Rhamnolipids, RSM-BB, olive oil, optimization

INTRODUCTION:
The regular method of media optimization changing one
factor at one time consume a lot of chemicals and time
hence optimization can be performed using response
surface methodology (RSM) that involves factorial
design and regression analysis with multifactorial
experiments(12,13).

A surfactant is a molecule that has both a hydrophilic

end, which attracts water and a hydrophobic end, which
repels water and attracts non-polar chemicals. A biosurfactant are produced naturally including rhamnolipid
that are produced by Pseudomonas aeruginosa and a
number of other bacteria. Surfactants are chemicals that
reduce the surface tension of water(1-6).

The aim of this work is to optimize the media

components for higher production of rhamnolipid.

Bio-surfactants have been applied in many fields, such

as microbial enhanced oil recovery, bioremediation,
cosmetics, rhamnolipidrmaceuticals, detergents, food
and other industries.The rhamnose-containing glycolipid
biosurfactants produced by Pseudomonas aeruginosa
were first described in 1949. Rhamnolipids are class of
compound that contain one or more 3-hydroxy fatty
acids of various chain length (C7 C21) esters lined to a
mono or di rhamnose and produced as a complex
molecule(7-11).
Received on 16.03.2016
Accepted on 25.04.2016

MATERIALS AND METHODS:

P. aeruginosa was isolated from oil contaminated soil
using serial dilution technique. Isolated organisms were
confirmed by biochemical methods.
Preparation of minimal salt medium:14
Minimal Salt Medium (MSM) for 1000 ml for
Rhamnolipid
production.
MgSO4.7H2O
-0.5g,
FeSO4.7H2O -0.01g, KH2PO4- 1.0g, K2HPO4- 0.5g,
NaNO3- 2.0 g, CaCl2- 0.01 g, KCl - 0.01 g. maintained
at pH 7.

Modified on 04.04.2016
RJPT All right reserved

Research J. Pharm. and Tech. 9(4): April, 2016; Page 1-3

DOI:

Research J. Pharm. and Tech. 9(4): April 2016

Seed Culture:15
Pseudomonas aeruginosa was grown on plate count agar
was transferred to the seed culture medium. Seed culture
grown in 250 ml flask contain Mini mum Salt Medium at
30oC at rotary shaker at 180 rpm for 14 hrs.

Extraction of Rhamnolipids
Each flask received 100 ml of distilled water. The flasks
were agitated for 1 h at 200 rpm at 30C on an orbital
shaker. The suspension was filtered through cheese
cloth; the excess liquid being squeezed out manually.
This procedure was done three times. The extract was
centrifuged for 10 min at 12,500g, and the supernatant
was extracted at least three times with chloroform
methanol (3:1, v/v), with 15 ml of this solvent mixture
being used for each extraction. The organic
rhamnolipidse was concentrated at reduced pressure at
40C, giving rise to a crude extract containing the
rhamnolipids26.

Preparation of Inoculation medium (g/L) :16

Carbon source (glucose, glycerol, liquid paraffin,
Dextrose, lactose) were sterilized separately.NaNO3 2.125, (NH4)2SO2-1.65, NH4NO3-1.0, MgSO4-7H2O-0.5,
K2HPO4 -2.0, KH2PO4 -2.0, Yeast extract - 1.0, NaCl1.0, CaCl 2-2H2O -0.01, MnSO4-5H2O -0.01, FeSO42H2O - 0.01. From the seed culture, the inoculum was
transferred to Inoculation medium.

Analytical method:
Optimization
of
Rhamnolipid
at
different Rhamnolipid concentration was quantified by
temperature:17
spectrophotometer method as rhamnose content using a
The production of rhamnolipid was optimized by using standard. Rhamnolipid27.
different temperature 27 C, 37C, 42C.

RESULT AND DISCUSSION:

Optimization of Rhamnolipid at different pH:18

Seed culture grown in 250ml flask contain Minimal salt
medium (MSM). The sample pH is adjusted to 4 to 10
respectively by adding 1N HCl.

15

Rhamnolipids (g/dm3)

Optimization of Rhamnolipid (by different carbon

source) :19
To optimize the production of rhamnolipid different
carbon source like fish oil, sugarcane syrup, glycerol,
olive oil, liquid paraffin

Rhamnolipids (g/dm3)

15

10

Response Surface Methodology:

Statistical analysis Response Surface Methodology
5
(RSM) was used to investigate the main effects of
dependable variables on the production of Rhamnolipid
by the organism. Olive oil (A), NaNO3 (B), KH2PO4(C),
FeSO4 (D), were selected as independent variables. BoxCarbon source
Behnken was used for the experimental data and data
Figure -1 (a) Effect of carbon source
were fitted to a second order polynomial model and
regression coefficients obtained. The software used for
15
this analysis was Design expert version - 7.0(20-22).
Fish oil

Oliv e oil

Glycer ol

Sugarcane syr up

10

liquid paraffin

Oliv e oil

Glycer ol

Sugarcane syr up

Rhamnolipids (g/dm3)

Rhamnolipids (g/dm3)

Fish oil

Rhamnolipids (g/dm3)

15

Regression analysis for different models indicated that

the fitted quadratic models accounted for more than 95%
of the variations in the experimental data, which were
significant. The ANOVA and F ratio test have been
10
performed to justify the goodness of fit of the developed
mathematical models. The calculated values of F ratios
for lack-of-fit have been compared to standard values of
F ratios corresponding to their degrees of freedom to
5
find the adequacy
of the developed mathematical
(23-25)
models
.

10

pH

Figure -1(b) Effect of pH

liquid paraffin

Carbon source

12

10

20

Research J. Pharm. and Tech. 9(4): April 2016

Rhamnolipids (g/dm3)

Table -1 The design summary used in RSM

Study
Response Surface
Runs
Type
Factor
Name
Units
Type
A
Olive oil
v/v
Numeric
B
NaNO3
g/l
Numeric
C
KH2PO4
g/l
Numeric
D
FeSO4
g/l
Numeric

29
Low Actual
10
1
2.5
5

Initial
Design
High Actual
50
9
5.5
10

Low Coded
-1
-1
-1
-1

Design
Model
High Coded
1
1
1
1

Quadratic
Mean
30
5
4
7.5

Std. Dev.
12.86535
2.57307
0.964901
1.608169

important parameters such as carbon sources,

temperature and pH were varied. From the estimated
rhamnolipid yield under these varying conditions, it was
determined that the highest yield by Pseudomonas
aeruginosa was at pH 7, temperature at 35C and olive
oil containing medium. A similar study on optimization
of rhamnolipid was done by Maier and Chavez21 in
which the Pseudomonas sp showed the maximum
rhamnolipid accumulation at pH- 7, temperature 35C

12

Response surface methodology:

The quadratic equation
Response = o+ 1 X1 + 2 X2 + 3 X3 + 4 X4 +5
X12 + 6 X22 + 7 X32 + 8 X42 +9 X1 X2+ 10 X2
X3 + 11 X1 X3+ 12 X1 X4+ 13 X2 X4+ 1 X3 X4
Where Response is the measured response, X1, X2, X3
and X4 are the coded independent input variables, o is
the intercept term, 1, 2, 3 and 4 are the coefficients
showing the linear effects, 5, 6, 7 and 8, are the
quadratic coefficients showing the squared effects and
9, 10, 11, 12, 13 and 14 are the cross product
coefficients showing the interaction effects24.

0
10

Box-Behnken

20

30

40

50

Temperature

Figure -1(c) Effect of Temperature on the production of

Rhamnolipid

Optimization of rhamnolipid Production

The organism was found to accumulate highest
concentration of rhamnolipid at 35oC in olive oil
containing medium (Figure 1a). To determine the ideal
growth condition for highest rhamnolipid yield, the most
Table -2 Design of Experiment and its response
Std
Factor 1
Factor 2
A:Olive oil
B:NaNO3
v/v
g/l
1
10
1
2
50
1
3
10
9
4
50
9
5
30
5
6
30
5
7
30
5
8
30
5
9
10
5
10
50
5
11
10
5
12
50
5
13
30
1
14
30
9
15
30
1
16
30
9
17
10
5
18
50
5
19
10
5
20
50
5
21
30
1
22
30
9
23
30
1
24
30
9

Factor 3
C:KH2PO4

Factor 4
D:FeSO4

4
4
4
4
2.5
5.5
2.5
5.5
4
4
4
4
2.5
2.5
5.5
5.5
2.5
2.5
5.5
5.5
4
4
4
4

7.5
7.5
7.5
7.5
5
5
10
10
5
5
10
10
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
5
5
10
10

Response 1
Actual
g/dm3
4.1
4
5.5
5.4
5.4
4.2
2.7
7.2
5.6
4.9
5.1
5.4
1.5
4.9
5.1
4.8
2.5
5.1
7.1
4.2
3.8
5.6
4.7
5.1

Predicted
4.1
3.9
5.4
5.3
5.3
4.2
2.6
7.2
5.6
5.0
5.2
5.6
1.7
4.9
5.3
4.8
2.5
5.1
7.0
4.1
3.7
5.8
4.5
5.2

Research J. Pharm. and Tech. 9(4): April 2016

30
30
30
30
30

5
5
5
5
5

4
4
4
4
4

Table -3 ANOVA Table

Source
Sum of
Squares
Model
427.8906
A-Olive oil
0.0675
B-NaNO3
5.4675
C-KH2PO4
9.1875
D-FeSO4
0.040833
AB
0
AC
7.5625
AD
0.25
BC
3.4225
BD
0.49
CD
8.1225
A^2
138.0509
B^2
172.9298
C^2
171.2593
D^2
132.1309
Residual
0.497
Lack of Fit
0.245
Pure Error
0.252
Cor Total
428.3876
Std. Dev.
Mean
C.V. %
PRESS

df

7.5
7.5
7.5
7.5
7.5

Mean
Square
30.56361
0.0675
5.4675
9.1875
0.040833
0
7.5625
0.25
3.4225
0.49
8.1225
138.0509
172.9298
171.2593
132.1309
0.0355
0.0245
0.063

14
1
1
1
1
1
1
1
1
1
1
1
1
1
1
14
10
4
28
0.188414
6.42069
2.934489
1.80495

14.2
14.5
14.7
14.7
14.2

p-value
Prob > F
< 0.0001
0.1896
< 0.0001
< 0.0001
0.3016
1.0000
< 0.0001
0.0189
< 0.0001
0.0023
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001

0.388889

0.8971

7.44917

Rhamnolipid
Design Points
14.7

Design-Expert Software

15

1.5

X1 = A: Olive oil
X2 = C: KH2PO4

Actual Factors
B: NaNO3 = 5.00
D: FeSO4 = 7.50

15

11.75
4.75

C: KH2PO4

Rhamnolipid

Actual Factors
C: KH2PO4 = 4.00
D: FeSO4 = 7.50
5

5.00

6.52093
8.51473

X1 = A: Olive oil
X2 = C: KH2PO4

1.5

Actual Factors
B: NaNO3 = 5.00
D: FeSO4 = 7.50

12

4.00

8.5

5.25

3.00

5.69125

9.20709

20.00

3.00

B: NaNO3

1.00
30.00

40.00

8.51473

4.52713

A: Olive oil

20.00

3.25

C: KH2PO4

6.52093

A: Olive oil

2.50 10.00

2.50

1.00 10.00
20.00

50.00

30.00

4.00

10.5085

6.52093

30.00

5.00

40.00

4.75

8.51473

40.00

7.00
7.44917
5.69125

5.50

50.00

9.00
10.965
7.44917

10.00

12.5023

3.25

12.7229

10.00

20.00

30.00

40.00

50.00

50.00

A: Olive oil

A: Olive oil

(a)

(b)

Des ign-Expert Software

Des ign-Expert Software

Rham nolipid
14.7

Rham nolipid

Rhamnolipid
14.7

Rhamnolipid

Des ign-Expert Software

10.00

5.50

1.5

8.12
9.705

1.5

14.7

9.705

7.50

9.8

X1 = B: NaNO3
X2 = C: KH2PO4
Actual Factors
A: Olive oil = 30.00
D: FeSO4 = 7.50

12.875

20.00

30.00

40.00

12.3742

30.00

7.50

9.00
7.00

4.75

A: Olive oil

5.00

4.00

10.236

5.95967

20.00

6.25

D: FeSO4

10.00

5.50
3.25

8.09783

8.12
6.535

5.00

4.5

40.00

8.75
11.29

8.12
6.535

4.00

50.00

10.00
9.705

9.705

Actual Factors
A: Olive oil = 30.00
D: FeSO4 = 7.50

4.75

7.35

4.9

6.25

15

11.5

Rhamnolipid

Actual Factors
B: NaNO3 = 5.00
C: KH2PO4 = 4.00

1.5

X1 = B: NaNO3 8.09783
X2 = C: KH2PO4

8.09783

1.5

12.25

Actual Factors
B: NaNO3 = 5.00
C: KH2PO4 = 4.00

D: FeSO4

X1 = A: Olive oil
X2 = D: FeSO4

Rham nolipid
Des ign Points
14.7

8.12

X1 = A: Olive oil
X2 = D: FeSO4

8.75

C: KH2PO4

Rham nolipid
Des ign Points
14.7

Rhamnolipid

Des ign-Expert Software

C: KH2PO4

8.09783

3.00

3.25

3.8215

B: NaNO3

2.50 1.00

5.95967

5.00 10.00

2.50

50.00

1.00

3.00

5.00

A: Olive oil

7.00

9.00

(c)

B: NaNO3

(d)
Des ign-Expert Software

Des ign-Expert Software

Rham nolipid

10.00

10.00

9.09042

X1 = B: NaNO3
X2 = D: FeSO4

15

11.75

Actual Factors
A: Olive oil = 30.00
C: KH2PO4 = 4.00
5

7.50

8.53139

1.5

8.75

12

8.75

X1 = C: KH2PO4
X2 = D: FeSO4

8.53139

1.5

Rhamnolipid

Actual Factors
A: Olive oil = 30.00
C: KH2PO4 = 4.00

6.54315

9.09042

X1 = C: KH2PO4
X2 = D: FeSO4

D: FeSO4

1.5

X1 = B: NaNO3
X2 = D: FeSO4

15

14.7

4.55491

Rham nolipid
Des ign Points
14.7

7.29361

1.5

7.29361

Actual Factors
A: Olive oil = 30.00
B: NaNO3 = 5.00

Actual Factors
A: Olive oil = 30.00
B: NaNO3 = 5.00

Rhamnolipid

Rham nolipid
Des ign Points
14.7

Rhamnolipid
Rham nolipid

Des ign-Expert Software

Rhamnolipid
14.7

Des ign-Expert Software

D: FeSO4

Actual Factors
C: KH2PO4 = 4.00
D: FeSO4 = 7.50

9.20709

7.00

B: NaNO3

X1 = A: Olive oil
X2 = B: NaNO3

Rhamnolipid

8.51473

Rhamnolipid
Design Points
14.7

7.44917

X1 = A: Olive oil
X2 = B: NaNO3

1.5

0.99884
0.99768
0.995787
94.28807

5.50

1.5

9.20709

not significant

Rhamnolipid14.7

Design-Expert Software

Rhamnolipid

9.00

Significant

R-Squared
Adj R-Squared
Pred R-Squared
Adeq Precision

Design-Expert Software

Rhamnolipid
14.7

Design-Expert Software

14.5
14.5
14.5
14.5
14.5

F
Value
860.9469
1.901408
154.0141
258.8028
1.150235
0
213.0282
7.042254
96.40845
13.80282
228.8028
3888.757
4871.262
4824.205
3721.997

Rhamnolipid

25
26
27
28
29

7.50

8.5

5.25

2
6.25

6.25

12.684

9.00

10.00

7.29361
5.49681

D: FeSO4
1.00

3.00

5.00

7.00

3.00

6.25
5.00

B: NaNO3

1.00

3.25

4.00

4.75

D: FeSO4

5.50

C: KH2PO4

B: NaNO3

(e)

(f)

Figure -2 Contour Plot and Surface graph showing the interaction between the components
(a) A and B (b) A and C (c) A and D (d) B and C (e) B and D (f) C and D

5.50

4.00

7.50

6.54315
5.00
2.50

4.75

8.75

6.54315

10.5196

9.00

10.00

8.53139

5.00

7.50

7.29361

5.00

12.5079
8.53139

7.00

8.75

9.09042
10.8872

3.25

6.25
5.00 2.50

C: KH2PO4

Research J. Pharm. and Tech. 9(4): April 2016

15.

CONCLUSION:
The application of RSM was used to optimize culture
media for the production of rhamnolipids by
Pseudomonas aeruginosa isolated from oil contaminated
soil. The pure culture was enhanced and optimized at
pH- 7, temperature-37oC and olive oil as effective
carbon source. Further the cultural media was optimized
using RSM -BB perceiving with 30 olive oil, 5 NaNO3, 4
KH2PO4 and 7.5 FeSO4 mixture contributes maximum
production of rhamnolipid. The R2 value was 99.8 that
correlated with Adjusted R2 value of 99.7 and Predicted
R2 value of 99.5 shows the model is significant.

16.
17.

18.

19.

20.

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