ISSN   0974-3618  (Print)                  www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

Optimization of Cellulase Producing Aspergillus flavus SB4 by Solid State Fermentation using Response Surface Methodology (RSM)-CCD

 

N. Utharalakshmi1*, A. Ganesh Kumar2 and G. Narendrakumar1

1Department of Biotechnology, Faculty of Bio and Chemical Engineering, Sathyabama University, Chennai 600119, India.

2National Institute of Ocean Technology, NIOT Campus, Chennai - 600 100, India.

*Corresponding Author E-mail: uthiral@gmail.com

 

 

ABSTRACT:

Solid State fermentation is widely applied to produce many enzymes and metabolites mainly from fungi. Multiple substrate (Paddy straw, Sugarcane bagasse, Rice husk and wheat bran) were mixed in different combination. Aspergillus flavus isolated from rhizosphere was inoculated. After incubation, the cellulase concentration was quantified using FPase. The optimum parameter for the production of cellulase was analysed using RSM –CCD . The results were evaluated on 28th day of incubation. R2 value was 0.989, adjusted R2 was 0.9788 and predicted R2 is 0.9505. The quadratic model gave a satisfactory description of the experimental data. Under optimum conditions, a maximum cellulase concentration of 55.43 IU/g was predicted according to the developed model.

 

KEYWORDS: Aspergillus flavus, SSF, Optimization, RSM-CCD, cellulase.

 


INTRODUCTION:

Solid state fermentation (SSF) is recognized as a fermentation process takes place in the lack or near-absence of free water by using a biological solid substrate. Many microorganisms are capable of growing on solid substrates such as filamentous fungi, which can grow to significant extent in the absence of free       water. (1, 2)

 

Agro-industrial residues are generally considered as suitable substrates for the production of enzymes, especially cellulase, under solid state fermentation3. The agricultural waste has to be converted into useful components as they are a concern of environmental pollution. These products may be a solution to this problem.

 

 

 

 

 

 

 

Received on 18.03.2015          Modified on 26.03.2015

Accepted on 30.03.2015         © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 349-354

DOI: 10.5958/0974-360X.2015.00058.X

 

Lignocellulose compounds contain enormous quantity of cellulose that can be degraded by many microorganisms to convert them into useful products4. A cellulolytic enzyme system is a multi-enzyme system comprising of endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), exo-glucanase (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91) and glucosidase (β-D-glucosideglucanohydrolase, cellobiase, EC 3.2.1.21) that acts synergistically to degrade cellulosic substrate(5, 6).

Response surface methodology (RSM) is a statistical method for modeling and optimization of multiple variables that determine optimum process conditions by combining experimentation data with interpolation following second order polynomial equation in a sequential testing. RSM is successfully applied for the optimization of enzymatic hydrolysis of other bioprocesses. In this present study RSM-CCD was adopted to determine the optimal nutritional conditions for the production of cellulase from Aspergillus flavus SB4 by solid state fermentation using multiple substrate combinations7.

 

 

MATERIALS AND METHODS:

Selection of Agricultural wastes:

Paddy straw, Sugarcane bagasse, Rice husk and wheat bran were selected for cellulase production and these wastes were collected from different parts of Kanchipuram district, Tamilnadu, India.

 

Microorganism and Cultural conditions:

Cellulose hydrolyzing fungi Aspergillus flavus SB4 was isolated from rhizosphere was used in this study. The culture was maintained on CMC agar slants and stored in the refrigerator (4oC) for further analysis. The organism was sequenced and submitted in GENBANK with an accession number of HE8145988.

 

Pre-treatment of substrate:

Raw substrates were sun dried individually to reduce the moisture content to make them more susceptible for crushing9.

 

Estimation of moisture content

The initial moisture content of the solid medium was fixed by soaking the substrate with the appropriate quantity of distilled water. The sample was then dried at 105°C . The dry weight was recorded. The percentage of moisture content was calculated as follows;

 

 

Moisture content (initial)of solid medium (%)

= (wt .of substrate – dry wt.)/(dry wt)×100

 

The crushed substrate was then sieved individually to get powdered form that was used in different combination for Solid state fermentation.

 

Inoculum Preparation

Aspergillus flavus SB4 maintained as stock culture on PDA slants were revived. The growth condition was at room temperature for 4 days by inoculation in 100 ml of minimal media (NH4NO3 – 0.14 g, KH2PO4 – 0.2 g, CaCl2 – 0.03 g, NH4NO3 – 0.14 g, K2HPO4 – 0.2 g, peptone – 0.7 g, FeSO4 – 0.50 g MnSO4 – 0.16 g ZnSO4 – 0.14 g in 100 ml distilled water) at 100 rpm for 48 hours.

 

Solid State Fermentation

Solid state fermentation was carried out in 250 ml Erlenmeyer flasks that contained 30 g of combination of different raw material (obtained from RSM table) and 15 ml of distilled water (Moistening agent). The flask were Sterilized at 121°C for 15 min and cooled to room temperature. About 1 ml of inoculum was added, mixed well and incubated at 27°C in a humidified incubator for 28 days. The flasks were periodically mixed by gentle shaking. After incubation period, enzyme activity was determined by FPase assay method10.

 

RSM

Experimental Design and Statistical Analysis:

RSM was used to optimize the cultural conditions for the cellulase production by Aspergillus flavus SB4. Central composite design was used consisting of four factors at two level pattern. Different combination of shade dried, powdered and sieved Paddy straw, Sugarcane bagasse, Rice husk and wheat bran were used in this study (Table 1). The model was analyzed using Design Expert 7.0 software (Stat-Ease Inc. Minneapolis)

 

The quadratic equation

Y= βo+ β1A + β2B + β3C + β4D + β5A2 + β6B2 + β7C2 + β8D2 + β9AB+ β10BC + β11CD + β12DA + β13AC + β14BD

 

Where Y is the measured response (cellulase activity (IU/g)), A, B, C and D are the coded independent input variables, βo is the intercept term, β1, β2, β3 and β4 are the coefficients showing the linear effects, β5, β6, β7 and β8 arethe quadratic coefficients showing the squared effects and β9, β10, β11, β12, β13, and β14 are the cross product coefficients showing the interaction effects. (11, 12)

 

Filter paper activity (FPase) assay for Cellulases

A modified protocol of IUPAC was followed for measuring the ability of the cellulase to hydrolyze both crystalline and amorphous regions of cellulose. FPase assay is the best measure of the activities of both endo- and exo- type cellulase .

 

To a test tube 1 ml buffer and 0.5 ml enzyme was added. At least two dilutions must be made of each enzyme sample investigated. One dilution should release slightly less than 2 mg of glucose in the reaction conditions. 50 mm Whatmann filter paper strip was inserted into the test tube and incubated at 50°C for 1 hour. The mixture was boiled for 20 min followed by addition 20 ml water. The mixture was filtered with a glass filter paper. The filtrate was measured against reference at 540 nm. A linear glucose standard was constructed using the absolute amounts of glucose (mg/0.5 ml) plotted against 540 nm. Using this standard, the absorbance values of the sample tubes (after subtraction of enzyme blank) was converted into glucose units.

 

Calculation for FPase Unit (FPU):

 

 

The units of FPU is based on the International Unit (IU)

1 IU = 1 μmol min-1 of substrate converted

        = 1 μmol min-1 of glucose formed during the hydrolysis reaction

        = 0.18 mg min-1 when product is glucose

 

Assay of Cellulase

After the incubation time, the media was washed with PBS and the cell free extract was used for analysis. The fungal crude extract was prepared by following method. 10 ml of cell free extract was centrifuged at 5000 rpm for 15 minutes. The activity of Cellulase was assayed using standard DNS method. One unit (U) enzyme activity was defined as the amount of enzyme required to liberate 1 μmol reducing sugar from the appropriate substrate per min under the assay conditions.

 

RESULTS AND DISCUSSION:

In the present work different agricultural waste that are available abundantly in the surrounding were used for the production of cellulase enzyme. The organism used was Aspergillus flavus SB4 which was reported to have higher productivity of cellulase. The cultural conditions were characterized in pervious experiments8. The cellulase activity was evaluated using FPase method.

 

Optimization of media

Central composite Design (CCD) was used to investigate the effects of four independent variable on the cellulase production. The experimental design and the actual response along with the predicted response are given in Table 2.

 

The quadratic equation

R1 = 53.94 -1.59067 A+ 3.198833 B -0.38167 C + 0.703333 D + 0.154 AB+ 2.17675 AC-0.70625 AD - 1.055 BC+0.34 BD+ 0.40975CD - 4.97696A2- 9.99896 B2 -4.83746 C2 -8.32496 D2

 

The quadratic regression equation was analyzed for predicted response. The 30 design conditions the experimental value FPase activity was ranging from 8.56 to 56.17 IU/g of dry substrate. The multiple correlation coefficient R2 was 0.989 adjusted R2 was 0978812 and predicted R2 was 0.95 were in reasonable agreement.

 


 

Table -1 Independent variables and their coded levels for the central composite design used in production of cellulase by Aspergillus flavus SB4

Factor

Name

Units

Type

Low Actual

Mean

High Actual

Variables

-1

0

+1

A

Paddy straw

mg

Numeric

0

7.5

15

B

Sugarcane bagasse

mg

Numeric

0

7.5

15

C

Rice husk

mg

Numeric

0

7.5

15

D

Wheat bran

mg

Numeric

0

7.5

15

 

Table 2: Central composite design for cellulase production by Aspergillus flavus SB4

Std

Factor 1

A:Paddy straw

Factor 2

B:Sugarcane bagasse

Factor 3

C:Rice husk

Factor 4

D:Wheat bran

Actual Value

Predicted

Value

Mg

Mg

Mg

mg

FTP

1

0

0

0

15

21.25

25.19

2

15

0

0

0

20.09

18.76

3

0

15

0

0

34.22

32.71

4

15

15

0

0

26.04

26.90

5

0

0

15

0

22.69

21.36

6

15

0

15

0

23.06

23.64

7

0

15

15

0

23.81

24.66

8

15

15

15

0

27.53

27.56

9

0

0

0

15

27.53

26.51

10

15

0

0

15

16.74

17.26

11

0

15

0

15

34.60

35.39

12

15

15

0

15

26.41

26.75

13

0

0

15

15

23.81

24.32

14

15

0

15

15

23.25

23.78

15

0

15

15

15

28.64

28.98

16

15

15

15

15

31.62

29.05

17

-7.5

7.5

7.5

7.5

38.32

37.21

18

22.5

7.5

7.5

7.5

30.13

30.85

19

7.5

-7.5

7.5

7.5

8.56

7.55

20

7.5

22.5

7.5

7.5

19.72

20.34

21

7.5

7.5

-7.5

7.5

36.46

35.35

22

7.5

7.5

22.5

7.5

33.11

33.83

23

7.5

7.5

7.5

-7.5

20.09

19.23

24

7.5

7.5

7.5

22.5

21.58

22.05

25

7.5

7.5

7.5

7.5

54.68

53.94

26

7.5

7.5

7.5

7.5

55.43

53.94

27

7.5

7.5

7.5

7.5

53.20

53.94

28

7.5

7.5

7.5

7.5

56.17

53.94

29

7.5

7.5

7.5

7.5

51.71

53.94

30

7.5

7.5

7.5

7.5

52.45

53.94

 

 

 

 

 

 

Figure 1 Surface plots illustrating the effect of substrate on cellulase activity measured in FPase/g (dry weight)

 

Table 3 ANOVA for cellulase production

Source

Sum of squares

df

Mean square

F value

p-value

Model

4853.107

14

346.6505

96.69219

< 0.0001

Significant

A-Paddy straw

60.72529

1

60.72529

16.93827

0.0009

B-Sugarcane bagasse

245.5808

1

245.5808

68.50055

< 0.0001

C-Rice husk

3.496067

1

3.496067

0.975168

0.3391

D-Wheat bran

11.87227

1

11.87227

3.311564

0.0888

AB

0.379456

1

0.379456

0.105843

0.7494

AC

75.81185

1

75.81185

21.14641

0.0003

AD

7.980625

1

7.980625

2.226058

0.1564

BC

17.8084

1

17.8084

4.967347

0.0415

BD

1.8496

1

1.8496

0.515914

0.4836

CD

2.686321

1

2.686321

0.749303

0.4003

A2

679.4088

1

679.4088

189.5094

< 0.0001

B2

2742.286

1

2742.286

764.9134

< 0.0001

C2

641.8561

1

641.8561

179.0347

< 0.0001

D2

1900.935

1

1900.935

530.2332

< 0.0001

Residual

53.7764

15

3.585093

Lack of Fit

38.27739

10

3.827739

1.234833

0.4316

Not significant

Pure Error

15.49901

5

3.099802

Cor Total

4906.883

29

Std. Dev.

1.893434

R2

0.989041

Mean

31.42933

Adjusted R2

0.978812

C.V. %

6.024417

Predicted R2

0.950519

PRESS

242.7963

Adequate precision

34.65149

 


Nibedita Sarkar and KaustavAikat13 suggested the similar results when Aspergillus flavus was used as the organism of interest and rice bran in solid state fermentation. About 2.45 FPase was reported when the temperature, pH, incubation time and Basal medium 35, 1.5, 84 and 40 respectively. FPase production was significantly affected by linear terms of the temperature, initial pH, incubation period at (p < 0.0001) whereas medium had small effect (p<0.005).

 

The present study indicates that the strain produced cellulase enzyme which hydrolyzed paddy straw, Sugarcane bagasse, Wheat bran and rice husk all in the combination of 7.5 g each to be converted into fermentable sugars 55.43 IU/g. Wang et al.14 report cellulase activity from Trichoderma reesei using SSF in 72 hours at 28°C at 5.5 pH. Brijwani et al.15 analyzed the productivity of cellulase using mixed culture so that the organism will be in efficiency of producing both endo- and exocellulase.

 

CONCLUSION:

The present study demonstrates the usage of optimization methodology to produce cellulase with significant activity. In this investigation RSM based on CCD was used. The results presented here confirm the possibility of medium optimization and cultivation environment to improve cellulase activity with different combination of raw materials. Optimization of process parameters resulted cellulase activity to 55.43I U/g by Aspergillus flavus.

 

ACKNOWLEDGEMENT:

The facilities provided in the Department of Biotechnology, Faculty of Bio and Chemical Engineering, Sathyabama University to carry out this study are gratefully acknowledged.

 

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