Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures

Process Biochemistry 39 (2004) 931–937

Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures Chin-Hang Shu∗ , Ming-Yeou Lung Department of Chemical and Materials Engineering, National Central University, Chung-Li, Taoyuan 320, Taiwan, ROC Received 20 November 2002; received in revised form 10 April 2003; accepted 7 June 2003

Abstract The effects of culture pH ranging from pH 3.0 to 6.0 on cell growth, exopolysaccharide biosynthesis and molecular weight distribution of exopolysaccharides of Antrodia camphorata were examined both in shake flask culture and in a stirred tank fermenter. In a controlled pH stirred tank fermentation, the optimum pH for cell growth was 4.0 with a cell yield at 0.3 g/g while that for exopolysaccharide formation was 5.0 with a product yield at 5.05 mg/g. A relatively high molecular weight exopolysaccharide with a lower yield was obtained at low pH values while a relatively low molecular weight exopolysaccharide with a high yield was obtained at higher pH values. The average molecular weight of the exopolysaccharide in the flask culture was higher than that in the stirred tank fermenter. A two stage pH process that maximized product formation was demonstrated with a high product yield of 148 mg/l with the relatively high average molecular weight of 2.18 × 105 . © 2003 Elsevier Ltd. All rights reserved. Keywords: Antrodia camphorata; Exopolysaccharide; Submerged fermentation; Two-stage pH process; Molecular weight; pH

1. Introduction A number of polysaccharides isolated from fungi or yeasts have been reported with some potential pharmaceutical applications such as anti-tumour, mitogenic acitivity, and activation of alternative pathway complement and plasma clotting activity [1–6]. Antrodia camphorata is used in traditional medicine in Taiwan has been identified as a fungus of the family Basidiomycetes [7]. Some bioactive compounds of A. camphorata have been isolated and characterized including sesquiterpene lactone, steroids and triterpenoids [8–12]. Recently, polysaccharides extracted from fruiting bodies and mycelial cultures of A. camphorata have been shown to have anti-hepatitis B virus activity [13]. In spite of these potential pharmaceutical applications, relatively rare information regarding the process aspects of producing these bioactive compounds has been published. This might be partially due to the fastidious nature of A. camphorata that grows specifically in the inner wall of the rotting trunk of Cinnamomum kanehirai native only in Taiwan [14]. Little success has been achieved by solid-state culture of ∗ Corresponding author. Tel.: +886-3-422-7151x4227; fax: +886-3-426-3749. E-mail address: [email protected] (C.-H. Shu).

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00220-6

A. camphorata and submerged culture might be the major route of production of valuable metabolites including exopolysaccharide. The pH of culture broth is one of the most critical environmental parameters affecting growth and biosynthesis of exopolysaccharides in submerged cultures. However, the effect of pH on the biosynthesis of exopolysaccharides and cell growth varies with different microorganisms [15–24], operational conditions [15–17] and medium composition [18,19]. In general, the optimal medium pH for cell growth is around the lower range from 2.0 to 4.0 but the optimal medium pH for exopolysaccharide formation is around the high range from 5.0 to 7.0. Thus, a two-stage control of pH for improving exopolysaccharide production has been demonstrated [21]. pH effects are often investigated using the same microorganism in flask experiments with different initial pH values. In this system an inconsistent result was obtained. Lower pH values favoured the production of exopolysaccharide of Ganoderma lucidum [22] as compared to that of a previous study [17]. Exopolysaccharides with antitumour activity differ greatly in their chemical composition and configuration. Although it is difficult to correlate the structure and antitumour activity of complex exopolysaccharides, differences in activity can be correlated with the size of the molecules and the

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branching pattern. It is generally believed that ␤-(1→3) linkages in the main chain of the glucan and additional ␤-(1→6) branch are needed for antitumour activity [25]. High molecular weight glucans seem to be more effective than those of low molecular weight [26–28]. Thus, it is essential to correlate the process parameters such as pH on the exopolysaccharide with its molecular weight for the development of a fermentation process with high quality exopolysaccharides from A. camphorata. The aim of this study was to determine the effects of pH on exopolysaccharide formation and their molecular weight distribution by culturing A. camphorata both in shake flasks and in stirred tanks bioreactors. A two-stage pH batch fermentation strategy was proposed to optimize the production of exopolysaccharide.

2. Materials and methods 2.1. Microorganism A. camphorata CCRC 35396, was purchased from the Culture Collection and Research Center (Hsinchu, Taiwan). The culture was maintained on malt extract agar of Blakeslee’s formula and transferred to a fresh agar plate every month, grown at 28 ◦ C, then stored at 4 ◦ C for about 2 weeks [7]. 2.2. pH control and culture conditions The effect of initial pH on the fungus culture was studied using shake flask cultures at different initial pH values. 250 ml Erlenmeyer flasks contained 100 ml medium consisting of the following components (g/l): glucose 25, peptone 5, malt exact 3, yeast extract 3, KH2 PO4 ·H2 O 1, MgSO4 ·7H2 O 1, vitamin B1 1. The medium pH was adjusted to 3 to 6 in steps of 1.0 pH unit by adding 1 N NaOH or 1 N HCl prior to sterilization. The culture was incubated on a rotary shaker at 28 ◦ C and 150 rpm for 2 weeks. The effect of pH on the fungus culture was also studied by batch fermentation in a 5-l bioreactor (B. Braun, Germany) with pH control. A range of culture pH was examined from 3.0 to 6.0 in steps of 1.0 pH unit. The medium used in this study was the same as that of the shake flask culture. The inoculum of around 300 ml was prepared by flask culture at 28 ◦ C and 150 rpm for 5 days. The fermentation with 3.3 l of medium was operated at temperature 28 ◦ C, 1 vvm aeration and agitation 300 rpm for about 14 days. A pH shift experiment was demonstrated by controlling the culture pH at 4.0 for cell growth in the first stage then at 5.0 for product formation. 2.3. Analytical methods Biomass concentration was determined by dry weight measurement involving filtration of broth samples through

pre-weighed filter discs (Whatman Ltd., Maidstone, UK). The filtrate was collected and stored at −20 ◦ C for the measurement of pH, residual glucose and exopolysaccharide. Biomass was washed and dried in the vacuum oven at 60 ◦ C until measurement of cell dry weight was not changed with time. The off-line pH value was measured using a pH meter (Model TS2, SUNTEX, Taiwan). The residual glucose of the culture was assayed by a DNS method [29]. The exopolysaccharide in the filtrate was determined by phenol-sulphuric acid assay [30]. The relative amount of ␤-(1→3)-d-glucans in polysaccharide were estimated by a fluorescence method [31]. The molecular weights of exopolysaccharide were determined by a gel permeation chromatography (GPC) system equipped with a GPC column (Shodex OHPak SB-804HQ) and a RI detector (SFD, RI 2000). The polyethylene glycol (PEG) standards (Polymer Laboratories, UK) with molecular weights ranged from 1.9 × 103 to 1.26 × 106 were used to construct a calibration curve. All of the sample solutions were dialyzed against deionized water using a dialysis tubular membrane with a molecular weight cut-off of 6000–8000 for 2 days before injection. The flow rate of the mobile phase was 1 ml/min deionized water.

3. Results and discussion 3.1. Fermentation kinetics Time-course data on cell, exopolysaccharide and glucose concentration of one batch culture with initial pH 4.0 and four pH controlled batch cultures with pH set point ranging from 3.0 to 6.0 are shown in Figs. 1 and 2. While cell growth showed a distinct exponential phase and a stationary phase, exopolysaccharide biosynthesis occurred throughout the culture and continued even when all the residual sugars were consumed (Fig. 1). As the set point of pH in the pH controlled batch cultures was adjusted from 3.0 to 6.0, cell growth rate decreased and fermentation time was extended from 16 to 21 days (Fig. 2). 3.2. Effect of pH control on fermentation kinetics The importance of pH control on fermentation processes of A. camphorata is illustrated in Fig. 1 by the contrast between the controlled pH process at 4.0 and an uncontrolled pH process with initial pH 4.0. While culture pH was uncontrolled it fell gradually from 4.0 to 2.0 at the end of fermentation. As a result, the fermentation time was shortened from 17 days of the controlled process to 13 days of the uncontrolled process. The yield of exopolysaccharide (YP/S ) was enhanced from 1.42 of the uncontrolled process to 3.45 mg/g of the controlled process. However, there was little effect on the yield of biomass (YX/S ) and both the cell yields of the fermentation processes in Fig. 1 were around 0.3 g/g. In other words, the influence of culture pH on the biosynthesis of exopolysaccharides is significant and it is essential

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Fig. 1. Time-course data of batch fermentation in a stirred tank fermenter with initial pH at 4.0 (a) uncontrolled pH; (b) controlled pH, respectively. Exopolysaccharide (䊊); glucose (䊏); biomass (䊐); pH (䊉).

to elucidate the pH effects on exopolysaccharide production using the pH controlled processes rather than the pH uncontrolled processes. Nevertheless, with the uncontrolled system, the flask experiment of different initial pH values might provide the preliminary information of the pH effects on exopolysaccharide fermentation. 3.3. Effect of initial pH on exopolysaccharide formation by flask experiments Results of the flask experiments after 14 days of fermentation with various initial pH ranging from 3.0 to 6.0 are shown in Fig. 3. An optimal initial pH for cell growth occurred at 4.0 with cell density around 6.4 g/l, while an optimal initial pH for exopolysaccharide formation occurred at 5.0 with exopolysaccharide yield at 24 mg/l (Fig. 3). The influence of pH on cell growth was not significant as compared to that of exopolysaccharide formation. In spite of the uncontrolled pH nature of flask cultures, these results indicate that cell growth might favour a lower culture pH range and exopolysaccharide formation a higher culture pH range. 3.4. Effect of pH on the cell growth of pH controlled fermentation Results of all experiments in batch fermentations were listed in Table 1. While the culture pH was controlled,

Fig. 2. Time-course data of pH controlled batch fermentation in a stirred tank fermenter with set point at (a) pH 3.0; (b) pH 5.0; (c) pH 6.0, respectively. Exopolysaccharide (䊊); glucose (䊏); biomass (䊐).

the influence of pH on cell growth became significant as compared to that of the flask cultures. While the set point of culture pH of fermentation was controlled from 3.0 to 6.0, the specific growth rate (␮) decreased monotonically from 0.49 to 0.20 day−1 , and the fermentation time increased from 16 to 21 days. The maximum cell density (Xmax ) of each controlled pH fermentation showed an optimal value at 8.87 g/l at pH 4.0, and decreased by 47% at pH at 6.0 and decreased by 25% at pH at 3.0. In other words, the optimal cell yield (YX/S ) and the optimal cell production rate (QX ) occurred at pH 4.0 were 0.3 g/g and 0.6 g/l day, respectively. Higher or lower culture pH than 4.0 would inhibit cell formation.

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Fig. 3. Effects of initial pH on biomass (䊐) and expolysaccharide (䊊) in the flask cultures after 14 days of cultivation with various initial pH from 3.0 to 6.0.

3.5. Effect of pH on exopolysaccharide formation of pH controlled fermentation The influence of pH on exopolysaccharide formation of A. camphorata in the pH controlled fermentation was significant as indicated in Table 1. The maximum exopolysaccharide concentration (Pmax ) of each pH controlled fermentation showed an optimal value 118 mg/l at pH 5.0, and decreased by 62% at pH 6.0 and decreased by 49% at pH 3.0. Likewise, the optimal specific product yield (YP/X ) was 18.05 mg/g obtained at pH 5.0. The optimal product yield (YP/S ) and the optimal production formation rate (QP ) occurred at pH 5.0 were 5.0 mg/g and 6.6 mg/l day, respectively. Higher or lower culture pH than 5.0 inhibited exopolysaccharide formation. 3.6. Effect of pH on the molecular weight of exopolysaccharide Results of the distribution of molecular weight of exopolysaccharide produced under different initial pH in flask cultures and those of pH-controlled fermentation are listed in Table 2. The effect of culture pH on the distribution of molecular weights of exopolysaccharide was significant as indicated in Table 2. In order to elucidate the pH ef-

fects on the molecular weight of exopolysaccharide, the molecular weight of exopolysaccharide of A. camphorata on the chromatograms of gel permeation chromatography was characterized by the fraction of molecular weight dis¯ n ). The tribution and number-average molecular weight (M molecular weight of exopolysaccharides of A. camphorata was divided into three fractions: low molecular weight (less than 5.0 × 104 ), medium molecular weight (less than 5.0 × 104 –4.0 × 105 ) and high molecular weight (greater than 4.0 × 105 ). From the change of the fraction of molecular weight distribution obtained under different pH controlled processes, the pH effect was revealed qualitatively. ¯ n might be a quantitative and useful index for However, M taking the relative amount of different fraction of molecular weight distribution of exopolysaccharide into account. As the initial pH of flask cultures was adjusted from 3.0 to 6.0, the distribution of the molecular weight of exopolysaccharide shifted from higher molecular weights toward lower ¯ n decreased monotonically from molecular weights and M ¯ n of the 7.98 × 105 to 2.18 × 105 (Table 2). Likewise, M controlled-pH fermentation with set point ranged from 3.0 to 6.0 in the stirred tank fermenters decreased from 2.43 × 105 to 9.15 × 104 after 14 days of fermentation. This observation was consistent with that of pullulan fermentation by Madi et al. [31]. However, an opposite conclusion was reported in that a high molecular weight portion of pullulan fermentation was obtained at a higher pH 6.5 [32]. Under similar culture pH conditions, the exopolysaccharides produced by the flask experiments displayed higher molecular weights than those of stirred tank fermenters as ¯ n of the uncontrolled pH shown in Fig. 4. For example, M batch fermentation with initial pH 4.0 in the flask culture ¯n was 6.8 × 105 , which was larger than 1.78 × 105 , the M of a stirred tank fermenter. This might be due to the difference of environmental conditions between a flask and a fermenter; for instance, auto-shifting of culture pH, oxygen transfer rate, and shear rate. However, further experiments are required to substantiate the arguments. While using the same stirred tank fermenter with the same oxygen transfer ¯ n of an uncontrolled pH experiment and shear rate, the M with initial pH 4.0 was 1.78 × 105 , which was slightly larger ¯ n of the controlled pH fermentation than 1.22 × 105 , the M at pH 4.0. This might be explained by that the culture pH of the uncontrolled fermentation decreasing towards an acidic

Table 1 Fermentation parameters of the batch experiments under various pH controlled processes in a stirred tank fermenter Different pH controlled processes

µ (day−1 )

QX (g/l day)

QP (mg/l day)

Xmax (g/l)

Pmax (mg/l)

YP/X (mg/g)

YX/S (g/g)

YP/S (mg/g)

t (day)

Controlled at pH 3.0 Controlled at pH 4.0 Controlled at pH 5.0 Controlled at pH 6.0 Initial pH 4.0 (Uncontrolled) Two-stage: pH 4.0 → pH 5.0a

0.49 0.34 0.29 0.20 0.51 0.33

0.33 0.60 0.46 0.36 0.41 0.17

3.74 5.11 6.55 2.14 2.69 8.71

6.64 8.87 7.85 4.60 8.72 8.32

60 87 118 45 36 148

11.03 11.41 18.05 12.64 4.82 22.32

0.23 0.30 0.28 0.14 0.29 0.26

2.51 3.45 5.05 1.79 1.42 5.77

16 17 18 21 13 17

a

Culture pH was controlled at pH 4.0 within the first 0–8 day and then shifted to pH 5.0 until the end of the fermentation.

0.61 0.38 0.28 0.37 0.29 0.13 b

Uncontrolled pH during the fermentation process. Culture pH was controlled at pH 4.0 within the first 0–8 day and then shifted to pH 5.0 until the end of the fermentation.

Fig. 4. Effects of culture pH on the number-average molecular weight ¯ n ) of exopolysaccharide in the flask cultures (䉱) and in the stirred (M tank fermenters ( ).

a

10 68 90 100 95 98 2.43 1.22 1.03 0.91 1.78 1.08 42 20 10 0 25 18 28 42 47 53 55 49 30 38 43 47 20 33 2.8 3.8 3.9 3.8 – – 7.98 6.80 4.69 2.18 – – 58 52 28 0 – – 42 48 52 69 – – 0 0 20 31 – – pH 3.0 pH 4.0 pH 5.0 pH 6.0 Initial pH 4.0a Two-stageb

High Mw (%) Medium Mw (%) Low Mw (%)

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90 32 10 0 5 2

¯ n (Da) × M 105 Medium Mw (%) Low Mw (%) High Mw (%) Final pH

Low Mw (%)

Medium Mw (%)

¯ wn (Da) × M 105

Culture in stirred tanks Stirred tank fermentation

¯ n (Da) × M 105 Shake flask fermentation

After 14 days of cultivation

Initial pH or pH set point

Table 2 Effect of pH on the molecular weight distributions of exopolysaccharides from the batch fermentations at different pH controlled processes

At the end of the fermentation

C.-H. Shu, M.-Y. Lung / Process Biochemistry 39 (2004) 931–937

environment during the course of fermentation. This favoured the formation of high-molecular-weight exopolysaccharide. In addition to culture pH, fermentation time was another factor affecting the distribution of molecular weight of exopolysaccharide as indicated in Table 2. The portion of high molecular weight of exopolysaccharide declined with fermentation time, and this was consistent with pullulan fermentations [31,32]. In fact, there was no higher molecular weight exopolysaccharide present at the end of fermentation. It is speculated that the changes of molecular weight of exopolysaccharide with fermentation time are due to the presence of an endo-amylase type enzyme capable of partially hydrolyzing the exopolysaccharides [33]. Thus, if higher molecular weight exopolysaccharide was desired, the fermentation time should be minimized. 3.7. Two-stage batch fermentation process for optimal exopolysaccharide production The fermentation process in a stirred tank with two-stage pH operation was performed to optimize exopolysaccharide production as demonstrated in Fig. 5. In the first stage, the culture pH was controlled at pH 4.0 around 8 days for cell growth. It was then shifted to pH 5.0 for exopolysaccharide production in the secondary stage. The results of fermentation parameters and the molecular weight distribution of exopolysaccharides are listed in Tables 1 and 2, respectively. As expected, exopolysaccharide production of the two-stage batch fermentation process further enhanced by 25% as compared to that of the fermentation at pH 5.0, reaching 148 mg/l. The cell yield of the two-stage process was 0.26 g/g, which was slightly less than the YX/S , 0.30 g/g, of the fermentation at pH 4.0. However, the highest specific product yield (YP/X ) and productivity of the two-stage fermentation process were achieved at 22.32 mg/g and 8.71 mg/l. day,

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[2]

[3]

[4]

[5]

[6] Fig. 5. Two-stage batch fermentation with culture pH shifted from pH 4.0 to 5.0 after 8 days of cultivation. Exopolysaccharide (䊊); glucose (䊏); biomass (䊐); pH (䊉).

¯ n of the two-stage batch process after respectively. The M 14 days of fermentation was 1.08 × 105 , which was slightly larger than that of pH controlled fermentation at pH 5.0. In ¯ n subjected to the culture pH other words, the change of M was also demonstrated by the two-stage process.

[7]

[8]

[9]

[10]

4. Conclusion

[11]

The culture pH in shake flask cultures and in stirred tank fermentation of A. camphorata has a critical influence on cell growth, exopolysaccharide formation and biopolymer molecular weight distribution. The optimal pH for biomass formation was around 4.0, whereas that for exopolysaccharide was around 5.0. High-molecular-weight exopolysaccharide was obtained at lower pH values with lower yields but the low-molecular-weight exopolysaccharide was obtained at higher pH values with higher yields. In addition, the average molecular weight of exopolysaccharide in the shake flask was larger than that of the stirred tank fermentation. A two-stage pH process that maximizes product formation has been successfully demonstrated. This two-stage pH fermentation process offers certain advantages including high product yields with relatively higher molecular weights.

[12]

Acknowledgements

[19]

The authors would like to thank the National Science Council of the Republic of China, Taiwan for financially supporting this research under contract no. NSC 91-2622-E-008-003-CC3.

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