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

¹Department of Biotechnology, Sathyabama University, Chennai – 600 119.

²Research 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 β-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-37^oC 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:

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 bio-surfactant 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).

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).

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).

The aim of this work is to optimize the media components for higher production of rhamnolipid.

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.

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 30^oC at rotary shaker at 180 rpm for 14 hrs.

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, NaCl-1.0, CaCl ₂-2H₂O -0.01, MnSO₄-5H₂O -0.01, FeSO₄-2H₂O - 0.01. From the seed culture, the inoculum was transferred to Inoculation medium.

Optimization of Rhamnolipid at different temperature:¹⁷

The production of rhamnolipid was optimized by using different temperature 27 °C, 37°C, 42°C.

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.

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

Response Surface Methodology:

Statistical analysis Response Surface Methodology (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. Box-Behnken was used for the experimental data and data were fitted to a second order polynomial model and regression coefficients obtained. The software used for this analysis was Design expert – version - 7.0^(20-22).

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 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 find the adequacy of the developed mathematical models^(23-25).

Extraction of Rhamnolipids

Each flask received 100 ml of distilled water. The flasks were agitated for 1 h at 200 rpm at 30°C 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 12,500×g, 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 40°C, giving rise to a crude extract containing the rhamnolipids²⁶.

Analytical method:

Rhamnolipid concentration was quantified by spectrophotometer method as rhamnose content using a standard. Rhamnolipid²⁷.

RESULT AND DISCUSSION:

Figure -1 (a) Effect of carbon source

Figure -1(b) Effect of pH

Table -1 The design summary used in RSM

Study Type	Response Surface		Runs	29	Initial Design	Box-Behnken	Design Model	Quadratic
Factor	Name	Units	Type	Low Actual	High Actual	Low Coded	High Coded	Mean	Std. Dev.
A	Olive oil	v/v	Numeric	10	50	-1	1	30	12.86535
B	NaNO3	g/l	Numeric	1	9	-1	1	5	2.57307
C	KH2PO4	g/l	Numeric	2.5	5.5	-1	1	4	0.964901
D	FeSO4	g/l	Numeric	5	10	-1	1	7.5	1.608169

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 35^oC in olive oil containing medium (Figure – 1a). To determine the ideal growth condition for highest rhamnolipid yield, the most 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 35°C 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 35°C

Response surface methodology:

The quadratic equation

Response = ɛo+ ɛ1 X1 + ɛ2 X2 + ɛ3 X3 + ɛ4 X4 +ɛ5 X12 + ɛ6 X22 + ɛ7 X32 + ɛ8 X42 +ɛ9 X1 X2+ ɛ10 X2 X3 + ɛ11 X1 X3+ ɛ12 X1 X4+ ɛ13 X2 X4+ ɛ1 X3 X4

Where Response is the measured response, X1, X2, X3 and X4 are the coded independent input variables, ɛo is the intercept term, ɛ1, ɛ2, ɛ3 and ɛ4 are the coefficients showing the linear effects, ɛ5, ɛ6, ɛ7 and ɛ8, are the quadratic coefficients showing the squared effects and ɛ9, ɛ10, ɛ11, ɛ12, ɛ13 and ɛ14 are the cross product coefficients showing the interaction effects²⁴.

Table -2 Design of Experiment and its response

Std	Factor 1 A:Olive oil	Factor 2 B:NaNO3	Factor 3 C:KH2PO4	Factor 4 D:FeSO4	Response 1
					Actual	Predicted
	v/v	g/l			g/dm^3
1	10	1	4	7.5	4.1	4.1
2	50	1	4	7.5	4	3.9
3	10	9	4	7.5	5.5	5.4
4	50	9	4	7.5	5.4	5.3
5	30	5	2.5	5	5.4	5.3
6	30	5	5.5	5	4.2	4.2
7	30	5	2.5	10	2.7	2.6
8	30	5	5.5	10	7.2	7.2
9	10	5	4	5	5.6	5.6
10	50	5	4	5	4.9	5.0
11	10	5	4	10	5.1	5.2
12	50	5	4	10	5.4	5.6
13	30	1	2.5	7.5	1.5	1.7
14	30	9	2.5	7.5	4.9	4.9
15	30	1	5.5	7.5	5.1	5.3
16	30	9	5.5	7.5	4.8	4.8
17	10	5	2.5	7.5	2.5	2.5
18	50	5	2.5	7.5	5.1	5.1
19	10	5	5.5	7.5	7.1	7.0
20	50	5	5.5	7.5	4.2	4.1
21	30	1	4	5	3.8	3.7
22	30	9	4	5	5.6	5.8
23	30	1	4	10	4.7	4.5
24	30	9	4	10	5.1	5.2
25	30	5	4	7.5	14.2	14.5
26	30	5	4	7.5	14.5	14.5
27	30	5	4	7.5	14.7	14.5
28	30	5	4	7.5	14.7	14.5
29	30	5	4	7.5	14.2	14.5

Table -3 ANOVA Table

Source	Sum of Squares	df	Mean Square	F Value	p-value Prob > F
Model	427.8906	14	30.56361	860.9469	< 0.0001	Significant
A-Olive oil	0.0675	1	0.0675	1.901408	0.1896
B-NaNO3	5.4675	1	5.4675	154.0141	< 0.0001
C-KH2PO4	9.1875	1	9.1875	258.8028	< 0.0001
D-FeSO4	0.040833	1	0.040833	1.150235	0.3016
AB	0	1	0	0	1.0000
AC	7.5625	1	7.5625	213.0282	< 0.0001
AD	0.25	1	0.25	7.042254	0.0189
BC	3.4225	1	3.4225	96.40845	< 0.0001
BD	0.49	1	0.49	13.80282	0.0023
CD	8.1225	1	8.1225	228.8028	< 0.0001
A^2	138.0509	1	138.0509	3888.757	< 0.0001
B^2	172.9298	1	172.9298	4871.262	< 0.0001
C^2	171.2593	1	171.2593	4824.205	< 0.0001
D^2	132.1309	1	132.1309	3721.997	< 0.0001
Residual	0.497	14	0.0355
Lack of Fit	0.245	10	0.0245	0.388889	0.8971	not significant
Pure Error	0.252	4	0.063
Cor Total	428.3876	28
Std. Dev.		0.188414		R-Squared		0.99884
Mean		6.42069		Adj R-Squared		0.99768
C.V. %		2.934489		Pred R-Squared		0.995787
PRESS		1.80495		Adeq Precision		94.28807

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

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-37^oC 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.

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Received on 16.03.2016 Modified on 04.04.2016

Accepted on 25.04.2016 © RJPT All right reserved

Research J. Pharm. and Tech. 9(4): April, 2016; Page 335-339

DOI: 10.5958/0974-360X.2016.00059.7