Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran

New Biotechnology  Volume 28, Number 6  October 2011

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Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran Yun-Tao Liu1,2, Ze-Yu Luo1, Chuan-Nan Long1, Hai-Dong Wang1, Min-Nan Long3 and Zhong Hu1, 1

Department of Biology, Shantou University, Shantou, China School of Food Science and Technology, Jiangnan University, Wuxi, China 3 School of Energy Research, Xiamen University, Xiamen, China 2

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box–Behnken’s design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 308C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g1, increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g1) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects. Introduction As the most abundant renewable natural biological energy resource [1], lignocellulose is continually replenished, with the help of sunlight, by photosynthetic reduction of carbon dioxide. Accumulation of this biomass in large quantities every year results in not only deterioration of the environment but also loss of potentially valuable materials that can be processed to yield energy, food, and chemicals [2]. Therefore, the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings [3]. The chemical composition of cellulose is very simple, consisting of only glucose residues connected by b-1,4-glycosidic bonds. However, no single enzyme is able to degrade crystalline cellulose. To degrade crystalline cellulose to glucose, at least three enzymes are needed for cooperation: endoglucanase (EC 3.2.1.4), exoglucanase (cellobiohydrolase, EC 3.2.1.91) and b-glucosidase (EC Corresponding author: Hu, Z. ([email protected]), ([email protected]) 1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.12.003

3.2.1.21) [4]. Cellulolytic enzymes from fungi have been extensively studied in some model organisms such as Trichoderma reesei [5]. But their cellulase system is, in general, deficient in b-glucosidases, causing accumulation of cellobiose, which results in repression and end product inhibition of the enzymes [1]. We have reported that a cellulolytic fungus Penicillium decumbensL-06 [6,7] can produce not only endoglucanase and exoglucanase but also high-level b-glucosidases (4.61 IU g1). Therefore, the strain is suitable for preparing new mutants. During the past four decades, a lot of efforts have been devoted to preparing mutagenesis and genetic modifications and obtaining improved strains capable of producing high levels of cellulases [8,9]. Although these efforts were made to produce high-level cellulase for the purpose of degrading waste cellulose, no commercially efficient enzyme complex has been produced yet [1]. Optimization of the substrate and culture techniques is one of the most important ways to increase activity and reduce cost in industrial production of cellulase complex, although strain is of great importance [10]. Submerged fermentation (SmF) is used for industrial production www.elsevier.com/locate/nbt

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of cellulase, but its high cost and low yield are the major factors that restrict its industrial applications [11]. However, the solid-state fermentation (SSF), as reported, can be used to produce cellulases because of its low investment and operating expenses. To our knowledge, the use of fungus P. decumbens to produce cellulase with rice bran (broken husks of the seeds of rice grains which are separated from the flour by sifting) as the substrate by SSF has not been reported. Because of different processing methods and machineries, rice bran ingredients are different, and generally it is about 5–5.5% (in weight) of the ordinary rice. Of rice bran, the oil is normally 14–24%; protein 12–18%; carbohydrate 33–53% (about 20% crude fiber); water 7–14%; and ash 8–12%. Besides, it is also rich in vitamins E and B, protein and other nutrients [12]. This study describes the effects of different conditions on promoting the production of the cellulase enzyme by mutant ML-017 in SSF; and uses the statistical techniques (response surface methodology, RSM) to analyze the optimal parameter so as to maximize the filter paper activity (FPA).

Materials and methods Microorganism and culture media P. decumbens L-06 (GenBank accession number EU273880) from Microorganism Laboratory of Shantou University, Shantou, PR China was maintained on potato dextrose agar (PDA) and subcultured once every three months. Rice bran was obtained from Shantou, PR China and was milled and utilized as the substrate of SSF. The size of rice bran particle milled was 100 meshes in average. Cellulose-Congo red medium [13] and filter paper medium [14] were prepared for the isolation of mutants; SSF medium: the substrate (washed to neutral) was mixed thoroughly with Mandel’s solution [15], and the modulation of the initial pH value was adjusted with Mandel’s solution. The contents were sterilized at 1218C, 1.1 kg/cm2 for 30 min.

Isolation and selection of mutants P. decumbens L-06 (wild strain) was grown on PDA for 15 days at 308C. Conidia that generated on slant were transferred into pH 5.8, 10 mM phosphate buffer with 0.1% Tween-80, and count was adjusted to 108 spores/ml. EMS (50 mg) was added to 20 ml suspension at 108 spores/ml. This suspension was kept at room temperature for 12–48 h followed by UV-irradiation for 2– 10 min (10 cm to lamp, 25 W). 99% of the spores were killed in the irradiation. The treated suspension (500 ml) was inoculated in filter paper medium. The flasks were incubated at 150 rpm and 308C for 3 days. 100 ml of the solution from the filter paper medium where the strains grew fastest and the paper festered the most was spread on cellulose-Congo red medium. In the Congo red medium, the colonies with the bigger hydrolyzed circle were isolated as possible mutants.

Enzyme extraction and activity determination Cellulases were extracted by suspending the fermented substrate (fermented for 72 h) in 10 times distilled water and mixing it for one hour at 200 rpm and 508C. The suspended material and the fungal biomass were separated by 2-layer gauze filtering and then by centrifugation (10,000  g for 15 min). The clarified supernatant was used as the crude enzyme. 734

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Cellulase activity assay with filter paper as the substrate is considered a widely accepted and typical method [16,17]. FPA was determined by the methodology of Ghose [18]. After 30 min incubation at 508C, the reducing sugar liberated in the reaction mixture was measured by the dinitrosalicylic (DNS) acid method [19]. The FPA was expressed as IU g1 of dry substrate. One unit (IU) of enzyme activity is defined as the amount of enzyme required to liberate 1 mmol of product per min at 508C.

Evaluation and optimization of solid-state fermentation conditions Based on our previous reports [6,7], three major factors, that affect FPA production, were further studied. A three-level and threevariable Box–Behnken’s design (BBD) (Minitab software 13.0) was applied to determine the best condition combination for cellulase production. The variables considered were the initial pH, culture temperature and moisture content in the experimental design. Both the independent and dependent factors used in this design are listed in Table 1. Table 2 shows the definitions and coded levels of the three dependent variables. Each experiment was performed in triplicates (it means that the same steps were all repeated for three times in all optimization experiments, and the TABLE 1

Definition and coded levels for Box–Behnken’s design matrix Independent variables

Symbol

Coded levels 0

1 Temperature

X1

Initial pH

X2

Moisture content

X3

258C

+1

308C

4.5

358C

5.5

65%

70%

6.5 75%

TABLE 2

Box–Behnken’s design matrix and the response of the FPA activity*,y Experiment order

FPA (IU g1)

Independent variables X1

X2

X3

1

+1

0

+1

4.17

2

1

0

1

3.41

3

0

1

+1

4.66

4

+1

1

0

3.93

5

0

+1

+1

4.82

6

1

1

0

3.29

7

0

+1

1

4.52

8

0

0

0

5.06

9

+1

0

1

4.15

10

1

0

+1

3.49

11

0

0

0

5.17

12

+1

+1

0

4.19

13

1

+1

0

3.49

14

0

1

1

4.11

15

0

0

0

5.14

* y

Experimental FPA activity was averages of triplicates. Coded levels in Table 1.

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Y¼

X

A0 þ

3 3 2 X 3 X X X Ai X i þ Aii X2i þ Ai j Xi X j i¼1

i¼1

i¼1 j¼iþ1

where Y was the response variable; A0, Ai, Aii and Aij were the regression coefficients of variables for intercept, linear, quadratic and interaction terms, respectively; and Xi and Xj were independent variables. The responses obtained from each set of experimental design (Table 2) were subjected to multiple non-linear regression using the software Minitab, Version 13.1 to obtain the coefficients of the second-order polynomial model. The quality of the fit of the polynomial model equation was expressed by the coefficient of determination R2, and the statistical and regression coefficient significance were analyzed by Fisher’s F-test.

Results and discussion Mutation of Penicillium decumbens P. decumbens L-06 was subjected to successive mutagenic treatment with EMS followed by UV-irradiation. The screening media gave fairly reliable indication of increased cellulolytic activities. 265 colonies, which grew fast on filter paper medium, were isolated and were cultured in the Congo red medium. Finally, ten mutants (ML-01, ML-017, ML-039, ML-047, ML-093, ML-132, ML-144, ML145, ML-181 and ML-203) could grow in Congo red medium and produce hydrolyzed circle (diameter  2 cm) surrounding each colonies. One of the mutants, ML-017 (GenBank accession number FJ458446) has shown the largest hydrolyzed circle(diameter = 3.7 cm) on cellulose-Congo red medium. Actually, Congo red medium is widely used in isolating and screening the cellulose-decomposing strain. Recently, the adoption of similar method with our study was reported [20–22]. Indeed, Congo red medium mainly reflects the change in endoglucanase activity, but in our study filter paper medium could be assisted in isolating and screening the cellulose-decomposing strains. Many reports have shown that strain mutation is a traditional method to successfully enhance the expression level of enzymes. Several strains are mutated to obtain improved strains such as JUA10 [23]. Some reports show that subjecting the spores to UVirradiation cannot obtain obvious results [8]. Therefore, we adopted the mutation procedure involving treatment of spores with EMS for 12–48 h, followed by UV-irradiation for 2–10 min.After successive mutagenic treatments, a mutant ML-017 was obtained. The strain exhibits larger hydrolyzed circle surrounding the colony on the cellulose-Congo red medium. Therefore, ML-017 was used in investigating the conditions of cellulase production.

TABLE 3

Estimated regression coefficients for Y (FPA activity)* Independent variables

ML-017 Coefficient

t-Value

P-value

Constant

5.123

70.556

0.000

X1

0.345

7.759

0.001

X2

0.129

2.895

0.034

X3

0.119

2.671

0.044

X1X1

1.060

16.201

0.000

X2X2

0.338

5.163

0.004

X3X3

0.258

3.940

0.011

X1X2

0.015

0.239

0.821

X1X3

0.015

0.239

0.821

X2X3

0.062

0.994

0.366

*

Statistically significant at 95% of confidence level.

as research object. The experiment design is shown in Table 1, with the actual levels of variables for each of the experiments in the design matrix calculated, and the results are outlined in Table 2. Multiple regression analysis was performed on the experimental data, and the coefficients are presented in Table 3. The analysis of variance (ANOVA) for the BBD is shown in Table 4.The mathematical model that represented FPA within the region being as the effects of independent variables was expressed in the form of equation: Y ML-017 ¼ 5:123 þ 0:345X1 þ 0:12875X2 þ 0:11875X3  1:06042X21  0:33792X22  0:25792X23 where Y was the FPA, and X1, X2 and X3 were the coded variables for the culture temperature, initial pH and moisture content respectively. As shown in Tables 3 and 4, the R2 value of this model was determined to be 0.962, which showed that the regression model defined well the true behavior of the system. The Pvalue of the model was 0.000 (P < 0.05) (Table 4), which indicated that the model fitness was significant. According to the P-value (Table 3) (the value, in case of below 0.05, indicated significance level) X1, X2, X3, X12, X22 and X33 were significant, but X1 and X2, X1 and X3, as well as X2 and X3 interactions were not significant. Three dimension surface plots were constructed. And the effects of TABLE 4

Analysis of variance for Y (filter paper activity) Source

Degree of freedom

Adj SS

Adj MS

F-value

P-value

Regression

9

5.70431

0.63381

40.07

0.000

Linear

3

1.19763

0.39921

25.24

0.002

Square

3

4.48926

1.49642

94.60

0.000

Interaction

3

0.01742

0.00581

0.37

0.780

Optimization of culture conditions with RSM

Residual error

5

0.07909

0.01582

FPA is a standard of comprehensively measuring cellulase activity. The degradation of natural cellulose is not the role of a single enzyme, but the cooperation result of many kinds of cellulases. The purpose of this work is to establish a rapid assessment on the application value of the mutant strains. Therefore, we used the FPA

Lack-of-fit

3

0.07262

0.02421

7.49

0.120

Pure error

2

0.00647

0.00323

Total

14

S = 0.1258 R2 = 98.6%; R2(adj) = 96.2%

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data in this study are the average values of the three experiments repeated) and the average yield of cellulase was taken as the response variable, Y. Regression analysis was performed, based on the experimental data, and was fitted into an empirical second-order polynomial model as shown below in the following equation:

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the culture temperature, initial pH and moisture content, as well as their interactions, are shown in Fig. 1. To further validate the optimal culture condition, the impact on the FPA by the optimal values of the variables was calculated in the equation through the software, and the results are as follows:

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X1 = 30.1856; X2 = 5.66961; X3 = 71.80885. Therefore, the optimal combination of the variables was concluded as follows: the culture temperature at 308C, initial pH 5.7, and moisture content 72%. And under these conditions, the predicted value of Y (FPA) from the model was 5.15 IU g1. When P. decumbens ML-017 was culti-

Research Paper FIGURE 1

Response surface (3D) and contour plots showing interaction effects added on the response Y (filter paper activity). (a1) and (a2) were the effects of the temperature (X1) and initial pH (X2); (b1) and (b2) were the effects of the culture temperature (X1) and moisture content (X3); (c1) and (c2) were the effects of initial pH (X2) and moisture content (X3). 736

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vated under the optimal combination achieved by RSM method, the FPA was found to be 5.76 IU g1 (increased by 44.12% when compared with the original strain). And the difference between the predicted value and actual value was 11.8%, within the error allowed (less than 20%). The maximum value was very close to the theoretical one predicted that indicated the validity of RSM method, and the effectiveness of the experimental design matrix for the cellulase yield in this study. The RSM allowed the calculation of maximum yield based on the data from few experiments in which all the factors varied within chosen ranges. This method had been successfully applied in the optimization of medium compositions [24], conditions of enzymatic hydrolysis [25] and enzyme production [26]. In our work, RSM was applied to optimize the SSF medium for cellulase production. The cellulase activity reported by other studies which adopted the similar substrate and methods [1] is lower than our result. Certainly, the cellulase activity reported by some research is higher than that in our study [27,28], but we need only 3 days for the fermentation. In addition, because rice is the local main crop, rice bran can be easily acquired is of and low-cost. This process of fermentation is simpler and demands less on the equipment required. Finally, the substrate in this study does not need to be pre-treated.

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It has been reported that P. decumbensL-06 used bagasse as the substrate for cellulase production in SSF, and the maximum cellulase (FPA) production was 3.89 IU g1 [7], but ML-017 was 3.65 IU g1 only. Thus, compared with bagasse, rice bran is more suitable to be used as the substrate for ML-017. According to the results X2 (initial pH) of P-value in this study, initial pH significantly affected the FPA production (Table 3). However, other researches [23,29] and our earlier report [7] indicated that the FPA did not vary significantly within the pH value of 5–6.5. It is possible that the mutagenesis caused changes in the physiological characteristics of strain or the substrate was the untreated bagasse of other studies [7]. However, what we used was rice bran which was milled to 100 mesh powder. Moreover, rice bran contains more abundant nutrients than bagasse. So pH value may be affected by the solubility of these nutrients, which will affect the fermentation.

Acknowledgements This work was supported by the Program for Key International S&T Cooperation Projects of China (No. 2009DFA60930), the Chinese National Programs for High Technology Research and Development (No. 2006AA05Z111), and National Science Foundation of China (No. 41076106).

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