The Influence of Cultivation Conditions on Mycelial Growth and

International Journal of Medicinal Mushrooms, 10(3):279–292 (2008)

The Influence of Cultivation Conditions on Mycelial Growth and Exopolysaccharide Production of CulinaryMedicinal Mushroom Pleurotus citrinopileatus Singer (Agaricomycetideae) Chiu-Yeh Wu,1 Jeng-Leun Mau,2 & Zeng-Chin Liang 3,* Department of Biotechnology, Chungchou Institute of Technology, Yuanlin, Changhua 51003, Taiwan, ROC; Department of Food Science and Biotechnology, National Chung-Hsing University, Taichung 40227, Taiwan, ROC; 3Department of Bioresources, Da-Yeh University, Datsuen, Changhua 51591, Taiwan, ROC 1 2

* Address all correspondence to Zeng-Chin Liang, Department of Bioresources, Da-Yeh University, 112, Shanchiao Road, Da-Tusen, Changhua, Taiwan 51591, ROC; [email protected]

ABSTRACT: The effects of submerged culture conditions on the mycelial growth and exopolysaccharide (EPS) production by Pleurotus citrinopileatus in submerged culture were studied. Using the one-factor-at-a-time method, the suitable rotation speed and inoculation density for the mycelial biomass and EPS production were found to be 100 rpm and 10%, respectively. The medium volume, carbon and nitrogen sources were 100 mL fructose and soy peptone for the mycelial growth and 50 mL glucose and peptone for the EPS production. To study the interactions between glucose (1.32–4.68 g/100 mL), peptone (0.32–3.68 g/100 mL), and initial pH (3.32–6.68), the central composite rotatable design and response surface methodologies were used. The trials were performed in 250-mL flasks containing 50 mL of medium under the condition of 25°C and 100 rpm for 14 days. The components were found to be 3.50 g glucose/100 mL, 3.68 g peptone/100 mL, and initial pH 5.0, and the mycelial biomass of 1.34 g dry cell weight/100 mL and EPS of 64.20 mg/100 mL were obtained in submerged cultures, which were higher than those obtained in the basal medium, respectively. In a 5-L stirred-tank bioreactor, maximum mycelial biomass of 1.10 g dry cell weight/100 mL and EPS of 90 mg/100 mL were achieved, and the fermentation time was shortened from 14 days to 10 days under these cultivation conditions. KEY WORDS: culinary-medicinal mushrooms, exopolysaccharide, mycelial biomass, submerged culture, Pleurotus citrinopileatus, response surface methodology

I. INTRODUCTION During the last decades, various biological and pharmacological activities of polysaccharides produced by mushrooms have been reported.1–9 Polysaccharides can be obtained from fruiting bodies cultivated on solid substrates, mycelial biomass, and culture broth by submerged fermentation.10–14

Submerged culture gives rise to many potential advantages, including higher yields of mycelial biomass or other biopolymers in a compact space and within a shorter time frame, with fewer chances of contamination.12,15 It is known that mycelial biomass or exopolysaccharide (EPS) production in submerged culture is influenced by environmental factors, such as initial pH values, rotation speeds,

ABBREVIATIONS CCRD: central composite rotatable design; EPS: exopolysaccharide; PDA: potato dextrose agar; PDB: potato dextrose broth; RSM: response surface methodology. 1521-9437/08/$35.00 © 2008 by Begell House, Inc.

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medium volumes, temperatures, inoculation densities, and medium compositions (e.g., carbon and nitrogen sources).12,16–23 The one-factor-at-a-time method is usually used to obtain the optimal submerged culture condition for the production of mycelial biomass and EPS.24,25 This method varies one factor while keeping all others constant and thus ignores the interactions between different variables and involves a relatively large number of experiments. Therefore, statistically based experimental designs were applied for the screening of influential factors for mycelial biomass and EPS production in submerged culture, such as the orthogonal layout for Cordyceps pruinosa14 and Agrocybe cylindracea23 and the Plackett-Burman design for Cordyceps militaris NG326 and Boletus spp.27 In addition, the central composite rotatable design (CCRD)17 and response surface methodology (RSM)27 were used to study the interactions between medium compositions and obtain the optimal culture requirements. Pleurotus citrinopileatus Singer (Pleurotaceae) is a yellow oyster mushroom, also known as Yu-huang-mo in Chinese and Nireohma in Japanese. Many reports indicate that this mushroom possesses biological and pharmacological activities, such as antitumor activity,28,29 an antihyperglycemic effect,30 fatigue resistance, immunoenhancing, the ability to delay aging,31 and antihyperlipidemic and antioxidant effects.32 Apparently, this mushroom has shown great potential for application in medicinal usage. In this research, the effects of the cultivation conditions on mycelial biomass and EPS production were studied using the one-factor-at-a-time method. In addition, the CCRD was used to explore the interactions between medium compositions, and RSM was applied to adequate medium composition for the enhancement of mycelial biomass and EPS productivity.

II. MATERIALS AND METHODS

ET AL.

the Archer Daniels Midland Company (Rotterdam, Netherlands). NH4NO3 and (NH4)2SO4 were from BASF Aktiengesellschaft (Ludwigshafen, Germany). Sucrose and monosodium L-glutamate were food-grade commercial products from Tai-Sugar Corp. (Taipei, Taiwan) and Ve-Wang Corporation (Taipei, Taiwan), respectively. Soypeptone and tryptone were purchased from American Laboratories Inc. (Omaha, NE, USA). Peptone was purchased from Kyokuto Pharmaceutical Industrial Co. (Tokyo, Japan). Yeast extract was purchased from Merck Co. (Darmstadt, Germany). KH2PO4, K2HPO4, and MgSO4•7H2O were purchased from Kanto Chemical Co. (Tokyo, Japan). B. Fungus Origin and Growth Conditions Pleurotus citrinopileatus, obtained from You-Hao Mushroom Research Institute (You-Hao City, Heilungkiang, China), was grown on PDA slants for 14 days at 25°C, maintained at 4°C, and subcultured every 3 months. To prepare the inoculum, the mycelium of P. citrinopileatus was transferred to a Petri dish containing PDA medium at 25°C for 14 days. Mycelial agar discs (Ø 2 cm) were obtained by a sterilized knife and were transferred to the inoculum medium, which contained 50 mL of PDB in 250-mL flasks, and incubated on an orbital shaking incubator at 100 rpm and 25°C for 1 day. A shaken flask culture was performed in a 250-mL flask, which contained 100 mL of the basal medium. The basal medium (100 mL) consisted of 4 g glucose, 2 g peptone, 100 mg yeast extract, 10 mg MgSO4•7H2O, 20 mg KH2PO4, and 40 mg K2HPO4. The pH value was adjusted to the desired value by the addition of either 6 N HCl or NaOH. The medium was sterilized at 121°C for 20 minutes and cooled to room temperature; then, the inoculum was inoculated at a density of 5% (v/v). The flasks were incubated on a shaking incubator at 100 rpm and 25°C for 14 days.

A. Media and Chemicals Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Himedia Laboratories (Mumbai, India). Fructose, galactose, glucose, lactose, maltose, and mannose were obtained from

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C. One-Factor-at-a-Time Method To seek optimum environmental factors, different levels of rotation rates (50, 100, and 150 rpm),

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medium volumes (50, 100, 150, and 200 mL), inoculation densities (1, 3, 5, and 10%), and initial pH values (4, 5, 6, and 7) were used to substitute for the original culture conditions, respectively. Furthermore, to study medium compositions, the original medium was also replaced with different types: 4 g/100 mL carbon (glucose, fructose, mannose, galactose, maltose, lactose, and sucrose) and 2 g/100 mL nitrogen sources [NH4NO3, (NH4)2SO4, monosodium L-glutamate, peptone, soypeptone and tryptone]. The flasks were incubated on a shaking incubator at 25°C for 14 days. Each trial was performed in triplicate, and the values obtained were the means of three calculations. D. Central Composite Rotatable Design The variables for CCRD experiments were selected according to the results of the one-factor-at-a-time strategy. The independent variables and their levels are presented in Table 1. To investigate the effect of carbon and nitrogen sources and pH values on mycelial growth and EPS production, a CCRD consisting of a 23 full design to provide for the estimation of curvature of a model with a star configuration (6 axial points) and 6 central points for the estimation of a pure error mean square, totaling 20 experiments (Table 2), was carried out to obtain a second-order model. The distance of the axial points was ±1.682, calculated from d = (2)N/4, where d is the distance of the axial points and N is the number of independent variables.33 Experiments were randomized in order to maximize the

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effects of unexplained variability in the observed responses due to extraneous factors. The trials were performed in triplicate in 250-mL flasks containing 50 mL of medium under the condition of 25°C and 100 rpm for 14 days. The CCRD experimental results were fitted with a second-order polynomial equation by a multiple regression technique. The equation proposed for the response (Y1 and Y2) was as follows: Yi = a0 + a1X1 + a2X2 + a3X3 + a11X12 + a22X22 + a33X32 + a12X1X2 + a13X1X3 + a23X2X3 where X is the coded independent variable; Yi (i = 1 and 2) is the predicated response for mycelial growth and EPS production, respectively; a0 is the value of the fitted response at the center point of the design; and ai, aii, and aij are the linear, quadratic, and cross-product terms, respectively. Furthermore, to deduce optimum workable conditions, a graphical technique was used by one fixed variable at a predetermined optimal condition. Responses were monitored and results compared with model predictions. The fitness of the second-order model was expressed by the coefficient of determination (R2), and its statistical significance was determined by an F test. The significance levels of regression and validation were determined by a t test. The SAS software (version 8.0, SAS Institute Inc., Cary, NC, USA) and SigmaPlot 2001 software (version 7.0, SPSS, Inc., Chicago, IL, USA) were used for regression and graphical analyses of the data, respectively. The suitability of the polynomial models for predicting

TABLE 1 Independent Variables and Experimental Levels for the Central Composite Rotatable Design Coded level Variable Glucose (g/100 mL) Peptone (g/100 mL) pH

Coded symbol

+d *

+1

0

–1

–d

X1 X2 X3

4.68 3.68 6.68

4.00 3.00 6.00

3.00 2.00 5.00

2.00 1.00 4.00

1.32 0.32 3.32

* d = (2)N/4 = 1.682, where d is the distance of the axial points and N is the number of independent variables.

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TABLE 2 Central Composite Rotatable Design and Responses in the Mycelial Biomass and Exopolysaccharide Production of Pleurotus citrinopileatus

Trial no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Variable level

Response*

X1

X2

X3

Mycelial biomass (g/100 mL)

Exopolysaccharide (mg/100 mL)

+1 +1 +1 +1 –1 –1 –1 –1 +d** –d 0 0 0 0 0 0 0 0 0 0

+1 +1 –1 –1 +1 +1 –1 –1 0 0 +d –d 0 0 0 0 0 0 0 0

+1 –1 +1 –1 +1 –1 +1 –1 0 0 0 0 +d –d 0 0 0 0 0 0

1.42 1.12 1.07 0.85 1.14 1.19 0.70 0.80 1.03 0.96 1.26 0.72 1.20 0.50 1.14 1.12 1.13 1.15 0.94 0.99

43.22 53.20 14.58 14.33 40.72 44.17 14.60 5.03 29.67 27.68 54.86 12.99 27.54 39.56 31.67 28.27 32.76 34.46 32.76 28.50

* Incubated at 25°C and 100 rpm for 14 days. ** d = (2)N/4 = 1.682, where d is the distance of the axial points and N is the number of independent variables.

the optimum response values was tested under the recommended optimal conditions. Both experimental response data were separately analyzed by the SPSS software (version 7.0, SPSS).

transferred to the fermentation medium and was cultivated for 14 days. F. Analytical Methods

E. Bioreactor Fermentation After previous experiments were completed, the fermentation medium was inoculated with 10% (v/v) of the inoculum and then cultivated at 25°C in a 5-L stirred-tank bioreactor (Model FMT-5L, Yuh Chuen Chiou Ind. Co., Ltd., Kaohsiung, Taiwan). Fermentations were conducted under the following conditions: temperature, 25°C; aeration rate, 1 vvm; agitation speed, 100 rpm; initial pH, 5.0; and working volume, 3-L. The inoculum was

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The mycelial growth of P. citrinopileatus was expressed as dry cell weight. The mycelia were harvested at the end of the incubation, filtered through preweighed filter paper (Whatman No. 1), and dried to constant weight at 50°C. To measure EPS, all supernatants were collected and then crude EPS was precipitated with the addition of two volumes of 95% ethanol, stirred vigorously, and left overnight at 4°C. The precipitated EPS was recovered by centrifugation at 3000 rpm for 20 minutes and then by discarding the supernatant. The precipitate



was washed twice with 75% ethanol and then lyophilized. The EPS content was measured using a phenol-sulfuric acid method according to Dubois et al.34 The pH was measured using a digital pH meter (Model SP 2200, Suntex Instruments Co., Hsi-Chih City, Taipei County, Taiwan).

III. RESULTS AND DISCUSSION A. Effect of Environmental Factors

50 40 30 20 10 0

EPS (mg/100 mL)

60 50 40 30 20 10 0

Mycelial biomass (g/100mL)

EPS (mg/100 mL)

60


Three rotation rates on mycelial growth and EPS production were studied and maximal mycelial biomass and EPS concentrations were 1.07 g/100 mL and 48.74 mg/100 mL at 100 rpm (Fig. 1A). The lower biomass yields found at the rotation rate above 100 rpm might be a result of increased shear stress. The shear stress increased concurrently at higher rotation rates was a detrimental factor to mycelial

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growth.20 Probably, high rotation speeds resulted in shearing inactivation of some key necessary for synthesizing the polysaccharides or breaking up of mycelial pellets or both.22 In addition, in calculating the specific EPS productions, the values were 16.20, 45.53, and 45.80 mg EPS/g dry cell weight at 50, 100, and 150 rpm, respectively. There was no significant difference in the values between 100 and 150 rpm. For further experiments, a rotation rate of 100 rpm was used. To find out a suitable medium volume for the production of mycelial biomass and EPS, the mycelium was cultivated in shaken flask culture at various medium volumes, ranging from 50 to 200 mL, with 100 rpm at 25°C for 14 days. The adequate medium volume for mycelial growth was 100 mL, whereas the optimal medium volume for EPS production was 50 mL (Fig. 1B). The highest value of specific EPS production was 54.50 mg EPS/g dry cell weight for the medium volume of

A

1.3

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7.50

C

7.00

1.1

6.50

0.9

6.00 pH Mycelial biomass EPS

0.7 0.5

1.5 1.3

50

100 150 Rotation rate (rpm)

B

0

2 4 6 8 10 Inoculation density (%)

5.50 5.00

7.50

D

7.00

1.1

6.50

0.9

6.00

0.7

5.50

0.5

50 100 150 200 Medium volume (mL)

4

Final pH

OF

5 6 Initial pH

7

Final pH

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5.00

FIGURE 1. Effect of rotation rate (A), medium volume (B), inoculation density (C), and initial pH (D) on mycelial biomass and exopolysaccharide production of Pleurotus citrinopileatus.


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50 mL. The highest specific EPS production for a medium volume of 50 mL was in accordance with the EPS production. The fermentation in the flasks was subjected to oxygen-limiting conditions. Furthermore, the accumulation of EPS resulted in the increase in the viscosity of broth, thereby limiting the transference of oxygen.12 This revealed that a high oxygen transfer rate favored mycelial biomass and EPS formation. For further experiments, a medium volume of 50 mL was used. Previous reports22,24 revealed that inoculation density was one of the important factors affecting mycelial growth and EPS production in submerged culture. To find out a suitable inoculation density, the mycelia were inoculated into the flasks with various inoculation densities, ranging from 1% to 10%. The yields of mycelial biomass and EPS increased with increased inoculation density from 1% to 10% (Fig. 1C). The suitable inoculation density, 10%, for both mycelial growth and EPS production was used in the following experiments. The value of specific EPS production was 45.04 mg EPS/g dry cell weight with an inoculation density of 10%. The initial pH value may affect cell membrane function, cell morphology and structure, the uptake of various nutrients, and metabolic biosynthesis.18,35 The initial pH values for mycelial growth and EPS production were determined in the basal medium over a pH range of 4.0 to 7.0. The suitable initial pH for both mycelial growth and EPS production was around pH 6.0, and the mycelial biomass and EPS content reached 1.07 g/100 mL and 48.74 mg/100 mL, respectively (Fig. 1D). Specific EPS productions were 61.90, 55.15, 45.53, and 40.24 mg EPS/g dry cell weight under initial pH values of 4.0, 5.0, 6.0, and 7.0, respectively. But the highest value of specific EPS production was obtained at initial pH 4.0 due to the lower growth yield. The amounts of mycelial biomass and EPS production were higher at initial pH 6.0 than 7.0. Therefore, the initial pH of 6.0 mL was used for further experiments.

concentration of 4 g/100 mL for 14 days. Among carbon sources tested, lactose and sucrose were not effective in mycelial growth as evidenced by their lower biomass contents of 0.83 and 0.56 g/100 mL, respectively (Fig. 2A). The medium containing glucose was the most efficient carbon source for EPS production (48.70 mg/100 mL). It seems that the favored carbon sources of P. citrinopileatus were similar to that of P. florida.36 The specific EPS productions for glucose, fructose, mannose, galactose, maltose, lactose, and sucrose were 45.49, 21.15, 27.40, 22.83, 26.50, 36.78, and 52.77 mg EPS/g dry cell weight, respectively. Although the highest specific EPS production (mg EPS/g dry cell weight) of P. citrinopileatus was obtained under the medium supplied with sucrose, the maximum amount (mg EPS/100 mL) of EPS production was observed with glucose as the carbon source. For further experiments, glucose was used as the carbon source. One of six nitrogen sources at 2 g/100 mL was used in the basal media for submerged cultures of P. citrinopileatus. Soypeptone gave rise to the highest mycelial growth (3.01 g/100 mL), whereas the highest EPS production (48.70 mg/100 mL) was found in the medium containing peptone (Fig. 2B). The specific EPS productions for NH4NO3, (NH4)2SO4, monosodium L-glutamate, peptone, soypeptone, and tryptone were 21.14, 35.66, 10.87, 45.49, 8.07, and 17.55 mg EPS/g dry cell weight, respectively. It seems that peptone can provide the mycelium with amino acids essential for enzyme synthesis in EPS production by P. citrinopileatus. Obviously, inorganic nitrogen sources gave rise to relatively lower mycelial growth and EPS production than organic nitrogen sources. A similar phenomenon has also been reported in the submerged cultivation of many kinds of mushrooms.15,19,20,22,23 It was suggested that certain crucial amino acids could scarcely be synthesized from inorganic nitrogen sources in the fermentation of higher fungi.13 For further experiments, peptone was used as the nitrogen source.

B. Effect of Carbon and Nitrogen Sources

C. Optimization of Medium Compositions for Mycelial Biomass and EPS Production

To find out a suitable carbon source, the mycelium of P. citrinopileatus was incubated in basal media containing one of seven carbon sources at a

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After a series of preliminary experiments using the one-factor-at-a-time method, the results


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A

50 40

1.2

30 0.8

20

0.4 0.0

10 e e e e e e e cos uctos annos lactos altos actos ucros u l r L S M G F M Ga

EPS (mg/100 mL)


EFFECT

0

3.5 3.0 2.5 2.0

60

B

50 40

Mycelial biomass EPS

30

1.5

20

1.0

10

0.5 0.0

e e e e O3 on epton ypton SO 4 amat t N p ) 2 t Pe yp Tr N H 4 ( N H 4 L - gl u So Na

EPS (mg/100 mL)


Carbon source

0

Nitrogen source

FIGURE 2. Effect of carbon (A) and nitrogen sources (B) on the mycelial biomass and exopolysaccharide production of Pleurotus citrinopileatus.

indicated that different requirements for medium volume, carbon, and nitrogen sources were observed for mycelial biomass and EPS produced by P. citrinopileatus. Since EPS demonstrated several physiological activities,4 the selection of suitable conditions mainly depended on the requirements for EPS production. To optimize both the production of mycelial biomass and EPS, the combined effect of carbon source (glucose), nitrogen source (peptone), and initial pH was investigated using the statistical approach of CCRD, which could identify and quantify the interactions between variables.


Considerable variations were observed in the results of mycelial biomass and EPS (Table 2). The mycelial biomass ranged from 0.50 g/100 mL to 1.42 g/100 mL, with trail numbers 14 and 1 having the minimum and maximum biomass values, respectively. EPS production ranged from 5.03 mg/100 mL to 54.86 mg/100 mL, with trail numbers 8 and 11 having the minimum and maximum biomass values, respectively. The mycelial biomass and EPS production at the center of the design (trial numbers 15–20) were 0.94–1.15 g/100 mL and 28.27–34.46 mg/100 mL, respectively.

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An analysis of variance (ANOVA) was conducted to assess the significant effects of independent variables on the responses, which were notably affected by the various treatment combinations. In submerged culture by P. citrinopileatus, the effect of peptone concentration was significant for mycelial growth at the 1% level and for EPS production at the 0.1% level (Table 3). The initial pH of the medium had a significant effect on mycelial growth at the 5% level. However, glucose concentration did not affect the mycelial growth and EPS yield at the 5% level. In other words, a small change of peptone concentration could cause a large variation in mycelial biomass and EPS production. It is possible that peptone supplementation was used previously for synthesizing enough enzymes to metabolize glucose for mycelial biomass and EPS production by P. citrinopileatus during fermentation. ANOVA was performed to determine the lackof-fit and the significance of the linear, quadratic, and cross-product effects of independent variables on the quality attributes. The lack-of-fit test was a measure of failure of a model to represent data in the experimental domain at which points were not included in the regression.37 ANOVA values of the polynomial models for mycelial biomass and EPS production and the coefficients of determination (R2) are presented in Table 4. As can be seen in Table 4, the models for mycelial biomass and EPS production were significant at the 5% and 0.1% levels, respectively. More specifically, the linear effects for mycelial biomass and EPS production were significant at the 1% and 0.1% levels, respec-

ET AL.

tively. However, no significant effect was found for the quadratic and cross-product effects. The R2 values for mycelial growth and EPS production were 0.81 and 0.95, respectively. In other words, a high correlation coefficient (r = 0.90) was obtained for the significant model of mycelial biomass production with an insignificant lack-offit variation. Also, an extremely high correlation coefficient (r = 0.97) was obtained for the response surface models of the EPS yield in submerged culture by P. citrinopileatus. The coefficients of variation for both models were within acceptable ranges (< 13.55%). Therefore, the response surface models developed were desirable. The estimated coefficients of the polynomial models for the mycelial biomass and EPS production are presented in Table 5. The intercepts of polynomial equations for mycelial biomass and EPS production were significant at the 0.1% level. They showed that peptone concentration (X2) had a significant effect on both mycelial biomass and EPS production at the 0.1% level. Positive coefficients of X1, X2, and X3 indicated a linear effect to increase the yield of mycelial biomass. Therefore, the negative coefficients of quadratic terms (X12, X22, and X32) and the cross-product term (X1X2) had slightly negative effects on the yield of mycelial biomass. However, negative coefficients of the linear term (X3), the quadratic term (X12), and the cross-product terms (X1X3 and X2X3) also showed slightly negative effects for EPS production by P. citrinopileatus. The contour plots (Figs. 3A and 3B) were superimposed, and the shaded overlapping area

TABLE 3 Analysis of Variance (ANOVA) Table Mean square Source of variation

df

Mycelial biomass

Exopolysaccharide

Glucose Peptone Initial pH

4 4 4

0.026 0.104** 0.071*

28.7 772.9*** 35.1

* Significant at 5% level. ** Significant at 1% level. *** Significant at 0.1% level.

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TABLE 4 Significance of ANOVA Values of Independent Variables as Linear, Quadratic, or Cross-Products on Mycelial Biomass and Exopolysaccharide Production of Pleurotus citrinopileatus Mean square Source of variation

df

Mycelial biomass

Exopolysaccharide

9 3 3 3

0.082* 0.208** 0.017 0.021

361.05*** 1035.75*** 14.20 33.20

Model Linear Quadratic Cross-product Total error Lack of fit Pure error

10 5 5

R2 Coefficient of variation (%)

0.017 0.026 0.008

17.11 27.95 6.27

0.81

0.95

12.81

13.55


was selected as the optimum condition (Fig. 3C) at which the mycelial growth and EPS production were over 1.30 g/100 mL and 56.50 mg/100 mL,

respectively. A point in this area at 3.68 g/100 mL peptone, 3.50 g/100 mL glucose, and initial pH 5.0 could be recommended as the practical optimum for

TABLE 5 Estimated Coefficients for the Fitted Second-Order Polynomials Representing the Relationship between the Responses of Mycelial Biomass and Exopolysaccharide Production of Pleurotus citrinopileatus Estimated coefficient Independent variable

df

Mycelial biomass

Exopolysaccharide

Intercept X1 X2 X3 X12 X22 X32 X1X2 X1X3 X2X3

1 1 1 1 1 1 1 1 1 1

1.08*** 0.05 0.17*** 0.11** –0.01 –0.01 –0.06 –0.03 0.08 0.02

31.51*** 1.77 14.88*** –1.74 –1.67 0.18 0.05 0.28 –1.98 –2.91



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6.68

A

1.30

1.50 1.40

5.00 1.30 1.20

4.16

1.10

3.32 6.68

40.10 44.20

5.84 Initial pH

1.60

1.40

5.84 Initial pH

ET AL.

1.00

0.90

0.80

40.10 44.20 48.30 52.40

B

48.30

56.50

5.00

52.40

60.60

56.50

4.16

64.70

60.60

3.32 6.68 5.84 Initial pH

C

1.30

EPS 56.50

5.00

1.30 56.50

4.16 3.32

Mycelial biomass

0.32

1.16

2.00

2.84

3.68

Glucose concentration (g/100 mL) FIGURE 3. Contour plots of mycelial growth (A) and exopolysaccharide production (B) as functions of glucose concentration versus initial pH at 3.68 g peptone/100 mL, respectively, and optimum condition (C) showing the shaded overlapping area for both mycelial growth and exopolysaccharide production by Pleurotus citrinopileatus after superimposition of contour plots (A) and (B).

the range of variables studied. Under this condition, the predicted yields of mycelial biomass and EPS by P. citrinopileatus in submerged culture would be raised to 1.34 g/100 mL and 57.76 mg/100 mL, respectively (Table 6). The maximal mycelial biomass and EPS concentration using the one-factor-at-a-time

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method were 1.07 g/100 mL and 48.74 mg/100 mL, respectively. It seems that the predicted productivities of mycelial biomass and EPS showed increments of 25.2% and 18.6%, respectively. The suitability of the polynomial models for predicting the response values was tested under the


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recommended combination of the above cultivation conditions. The mycelial biomass was as expected, whereas the experimental value of the EPS production (64.20 mg/100 mL) was even higher than that predicted. However, no significant difference between the predicted and experimental values was found at the 95% confidence level. It is obvious that the optimization of the mycelial biomass and EPS production by P. citrinopileatus in submerged culture was established. D. Fermentation Results in a 5-L StirredTank Bioreactor To investigate the mycelial biomass and EPS production by P. citrinopileatus in a 5-L stirred-tank bioreactor, the microorganism was cultivated under the above resultant medium (100 mL) consisted of 3.50 g glucose, 3.68 g peptone, 100 mg yeast extract, 10 mg MgSO4•7H2O, 20 mg KH2PO4, 40 mg K2HPO4, and initial pH 5.0. First, a significant variance was observed on the morphology of cells grown in the shaken flask culture and stirred-tank bioreactor: 1- to 2-mm diameter compact pellets were developed in the shaken flask culture, and feather-like mycelial clumps were developed in the stirred-tank bioreactor (figures not shown). A comparison between the mycelial biomass and EPS yields in the shaken flask culture and the stirred-tank bioreactor indicated that the higher

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mycelial biomass production existed in the shaken flask culture, whereas the higher EPS production was in the stirred-tank bioreactor. It seems that the most productive mycelial morphology of P. citrinopileatus for maximum mycelial biomass and EPS could be compact pellet and feather-like mycelial clumps, respectively. Lee et al.22 pointed out that feather-like morphology was more favorable than pellets in the production of mycelial biomass and EPS from Grifola frondosa. In contrast, results from other mushroom fermentation indicate that the most desirable morphology was often the compact pellet.15,16 At an agitation speed of 100 rpm (Fig. 4A), the maximum yields of mycelial biomass and EPS were 1.14 g/100 mL at the 8th day and 51 mg/100 mL at the 10th day, respectively. At 150 rpm (Fig. 4B), the maximum yields of mycelial biomass and EPS were 0.86 g/100 mL at the 9th day and 94 mg/100 mL at the 10 the day, respectively. These results revealed that higher shear rates caused a decrease in cell growth but favored EPS production. The mechanical shear that was raised by agitation in the stirred-tank bioreactor and caused decreased yields of mycelial biomass was observed in some fungal fermentation.22,38,39 Under higher agitation conditions, the high EPS yield may be due to efficient mixing and may lead to the formation of the desired morphology (feather-like mycelial clumps). According to the above observations, the initial fermentation condition was adjusted to aeration

TABLE 6 Predicted and Experimental Values for the Responses at the Recommended Cultivation Condition of Pleurotus citrinopileatus Treatment variable

Coded level

Actual level

Glucose Peptone Initial pH

0.50 1.68 0.00

3.50 g/100 mL 3.68 g/100 mL 5.00

Response

Predicted

Experimental*

1.34 57.76

1.34 ± 0.15 64.20 ± 0.93

Mycelial biomass (g/100 mL) Exopolysaccharide (mg/100 mL)

* Not significant in t test at 95% level confidence (n = 5).


289

ET AL.

8

0.8

4 0.4

2

B

A

0

0.0

4

6

8

10

12

14

0

2

4

Incubated time (days)

6

8

1.2

6

0.8 4

0.4

C

2

0.0

0 0

2

12

60 40 20 0

14


8

pH

10

80

4

6

8

10

12

100 80 60 40

EPS (mg/ 100 mL)

2

Mycelial biomass (g /100 mL)

0


6 pH

100

1.2

pH Mycelial biomass EPS

EPS (mg/ 100 mL)

CHIU-YEH WU

20 0

14


FIGURE 4. The time profiles of mycelial biomass and exopolysaccharide production of Pleurotus citrinopileatus in a 5-L stirred-tank fermenter. The stirred-tank fermenter was operated under the following conditions: aeration rate 1 vvm and agitation speed 100 rpm (A), aeration rate 1vvm and agitation speed 150 rpm (B), and 0–3 days, aeration rate 1 vvm without agitation; 4–14 days, aeration rate 1.5vvm and agitation speed 100 rpm (C).

rate 1 vvm without agitation from day 0 to day 3 to avoid the damage of shear rates on mycelia; then the aeration rate and agitation were increased to 1.5 vvm and 100 rpm from the 4th day to the 14th day, respectively, to change the mycelia to the desired morphology (feather-like mycelial clump) and enhance the mixing of the production medium. The results (Fig. 4C) showed that the maximum mycelial biomass and EPS yields were 1.10 g/100 mL at the 8th day and 90 mg/100 mL at the 10th day, respectively. There was no marked difference in maximum mycelial biomass yield between Figures 4A and 4C or in EPS yield between Figures 4B and 4C. This indicates that higher aeration was an efficient strategy for enhanced EPS production. The change in morphology during the fermentation period showed that

290

pellets were formed initially. After agitation, only a minority of the pellets (approximately 0.05-mm diameter) and most of the feather-like mycelial clumps remained in the broth.

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