Statistical Evaluation of Medium Components by Plackett-Burman method for Laccase from Pseudomonas aeruginosa ADN04 using Submerged Fermentation

 

Arunkumar.T^1*, Narendrakumar.G², Alex Anand.D³

¹Department of Bioinformatics, Sathyabama University, Chennai – 600119

²Department of Biotechnology, Sathyabama University, Chennai – 600119

³Department of Biomedical Engineering, Sathyabama University, Chennai – 600119

 

ABSTRACT:

The effect of medium components on laccase production from using Pseudomonas aeruginosa ADN04 was studied. Placket Burman design was used to evaluate the significant parameters that have large effect on the fermentation and with the experimental design a successful result was obtained. K₂HPO₄, NaNO_3, CaCl_2, NH₄Cl_, CuSO_4, NH₄H₂PO_4, ZnSO_4, Glucose          , Sucrose_, Yeast Extract_,MgSO_4, KH₂PO₄ was screened for the better media composition. Sucrose, K₂HPO₄, NH₄Cl, Yeast Extract, CaCl₂, were standardized according to the F values.

 

KEYWORDS: Plackett-Burman Design (PBD), Laccase, submerged fermentation

 

 

INTRODUCTION:

PBD, an efficient way to identify the important factors among a large number of variables, was used in the present study to screen the important variables that significantly influenced laccase production. In this study, a 12-run Plackett-Burman design was applied to asses eleven factors (including two variables that were kept as dummy) ^1-4. Each variable was observed at two levels: –1 for the low level and +1 for the high level. It illustrated the variables and their corresponding levels used in the experimental design. The values of two levels were set according to our preceding initial experimental results.

 

Plackett-Burman designs are substitute to fractional factorials for selection. One beneficial characteristic is that the sample size is a multiple of four rather than power of two. There are no two-level fractional factorial designs with sample sizes between 16 and 32 runs. However, there are twenty run, twenty-fourrun, and twenty-eightrun Plackett-Burman designs^5-8.The main effects are orthogonal and two-factor interactions are puzzled with main effects. This is dissimilar from resolution of three fractional where two factor interactions are indistinguishable from main effects.

 

MATERIALS AND METHOD:

Bacterial Culture:

Pseudomonas aeruginosa ADN04 (bacterial culture) was isolated, identified biochemically and 16S rRNA sequencing⁹. The isolated pure culture was preserved in nutrient agar slants at 4°C in Department of Biotechnology, Sathyabama University. All the experimental analysis was executed using these strains.

 

Plackett-Burman design:

Differenet variables were identified to have influence on the production of laccase. But the selection of the variable are perform statisically using Plackett-Burman design using JMP 10 –version 17. The difference between the average of H ( high ) and L ( low ) responses for each independent and dummy variable.

 

 Different chemical componets were assessed using this method, K₂HPO₄, NaNO₃, CaCl₂, NH₄Cl, CuSo_4, NH₄H₂PO_4, ZnSO_4, Glucose, Sucrose,Yeast Extract, MgSO₄, KH₂PO₄ (Table-1).

 

ZnSO₄ and CuSO₄ were used as dummy variable and other components were used to analyse the effect on the enzyme production. The design of experiment was proposed using JMP-10 and the analysis was performed accordingly. On the basis of the response (enzyme activity) the Fisher value (F-value) was calculated.(Fisher value determines the selection of variables, higher the value more the interaction).

 

RESULT AND DISCUSSION:

The organism was isolated from the soil sample (Harur forest, Tamilnadu), was preserved as pure culture in the Department of Biotechnology, Sathyabama University, Chennai. The media which was optimized by Arunkumar et al., 2014 was used to as Fermentation media. After incubation, the culture was centrifuged and the supernatant was used for purification.

Table 1:PB Design Screening of media using Plackett Burman (B Design) (Plackett RL, Burman JP, 1946) (JMP10)

L – Low , H- High

 

Table 2: Statistical analysis of Plackett Burman design for the production of laccase using Ps.aeruginosa ADN04

 

Table-2  express the interaction of variables represented by F-value. Higher the value more the influence. Sucrose, K₂HPO₄, NH₄Cl, Yeast Extract, CaCl₂On the basis of PB design, the high influencing components were selected for further analysis.

 

Fig. 1: Curve fitting between the actual and predicted value

 

The curve fitting between the Actual and the predicted values shows the R2 value to be 97.283 that shows the significance of the reaction performed (Figure 1).

 

 

Table 3:Summary of Fit

 

Table-3 and Table-4express the fit of the model as R2 value is 97.2% corresponding to the Adj. R2 value of 94.9%. The values justify that the results given by the experimental analysis and the report generated by the software was relevant.

 

Table 4: Analysis of Variance

 

Table 5: Parameter Estimates

 

Table:6 Sorted Parameter Estimates

Term	Estimate	Std Error	t Ratio	t Ratio	Prob>|t|
Sucrose [5]	-5.178	1.570429	-3.30		0.0132*
K2HPO4[1]	-4.843	1.570429	-3.08		0.0177*
NH4Cl[1]	-2.192	1.570429	-1.40		0.0054*
Yeast Extract [1]	2.157	1.570429	1.37		0.0120*
CaCl2[1]	1.482	1.570429	0.94		0.0037*
Glucose[5]	-1.352	1.570429	-0.86		0.4178
NH4H2PO4[0.1]	1.317	1.570429	0.84		0.4294
NaNO3[2]	-1.138	1.570429	-0.72		0.4922
MgSO4[0.2]	0.938	1.570429	0.60		0.5691
CuSO4[0.001]	0.428	1.570429	0.27		0.7931
KH2PO4[1]	-0.182	1.570429	-0.12		0.9110
ZnSO4[0.01]	0.057	1.570429	0.04		0.9721

 

Fig.2: Prediction Profiler

 

Figure 2 summaries the structural prediction of the compound in various composition.

Table 7: Screening for YContrasts

Term	Contrast	Plot of t-Ratio	Length t-Ratio	Individual p-Value	Simultaneous p-Value
Sucrose	5.17800		2.62	0.0257*	0.2620
K2HPO4	4.84300		2.45	0.0307*	0.2967
NH4Cl	2.19200		1.11	0.2524	0.9926
YE	-2.15700		-1.09	0.2599	0.9937
CaCl2	-1.48200		-0.75	0.4317	1.0000
Glucose	1.35200		0.68	0.4720	1.0000
NH4H2PO4	-1.31700		-0.67	0.5299	1.0000
NaNO3	1.13800		0.58	0.5899	1.0000
MgSO4	-0.93800		-0.47	0.6543	1.0000
CuSO4	-0.42800		-0.22	0.8337	1.0000
KH2PO4	0.18200		0.09	0.9291	1.0000
ZnSO4	-0.05700		-0.03	0.9775	1.0000
Sucrose*K2HPO4	-2.75952		-1.40	0.1622	0.9207
Sucrose*NH4Cl	-0.04048		-0.02	0.9841	1.0000
K2HPO4*NH4Cl	-1.10475		-0.56	0.6003	1.0000
Sucrose*YE	1.90019		0.96	0.3160	0.9993
K2HPO4*YE	-1.61220		-0.82	0.3932	1.0000
NH4Cl*YE	-1.32031		-0.67	0.4830	1.0000
Sucrose*CaCl2	0.68814		0.35	0.7368	1.0000

 

 

Fig.3:  Half Normal Plot

 

Length PSE=1.97798Asterisked terms were forced orthogonal. Analysis is order dependent.p-Values derived from a simulation of 10000 Lenth t ratios.

 

Figure -2 and Figure -3 shows the use a normal and half normal probability plot of the effects to evaluate the significance and statistical importance of main and interaction effects from a 2-factorial design. The fitted line indicates where you would expect the points to fall if the effects were zero. Significant effects have a label and fall toward the left or right side of the graph.

 

Table 5, table 6, table 7 states the effective prediction of the interaction of compounds present in the medium by statistical approach. Identifying appropriate components for the medium was a time-consuming and arduous process involving large number of experiments. The PBD is the basic groundwork technique for fast screening of the effects of different medium constituents. Initial various carbon, nitrogen and salts have been analysed to choose best for the maximum laccase production. PBD was used to assess theimportance of various medium components and to enhance the laccase production in submergefermentation. The variables represent the nutrient components, the independent variables andtheir respective high and low concentration was used in PB optimization study. The maximum laccaseproduction was 232.11 mg/L was obtained in PB optimization study using Pseudomonas aeruginosa. This technique evidenced tobe appreciated in screening enormous number of constituents in production media effectively.

 

CONCLUSION:

K₂HPO₄, NaNO₃, CaCl₂, NH₄Cl, CuSO₄, NH₄H₂PO₄, ZnSO₄, Glucose, Sucrose, Yeast Extract, MgSO₄, KH₂PO₄ were selected as the variables and by using Plackett Burman method Sucrose, K₂HPO_4, KH₂PO₄, NH₄Cl, , CuSO₄, NaNO₃, MgSO₄ found to have maximum impact on the production of laccase enzyme.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 21.03.2017           © RJPT All right reserved