World Journal of Microbiology & Biotechnology 20: 767–773, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Optimization of submerged culture conditions for exopolysaccharide production in Sarcodon aspratus (Berk) S.lto TG-3 Ji Hoon Joo1, Jong Min Lim1, Hyun Oh Kim1, Sang Woo Kim1, Hye Jin Hwang1, Jang Won Choi2 and Jong Won Yun1,* 1 Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 712-714, Korea 2 Department of Natural Resource, Daegu University, Kyungsan, Kyungbuk 712-714, Korea *Author for correspondence: Tel.: +82-53-850-6556, Fax: +82-53-850-6559, E-mail:
[email protected] Received 1 December 2003; accepted 2 April 2004
Keywords: Exopolysaccharides, optimization, orthogonal matrix method, Sarcodon aspratus, submerged culture
Summary The effect of medium components (carbon, nitrogen, and mineral sources) and environmental factors (initial pH and temperature) for mycelial growth and exopolysaccharide (EPS) production in Sarcodon aspratus (Berk) S.lto TG-3 was investigated. The optimal temperature (25 C) and initial pH (5.0) for the EPS production in shake flask cultures of S. aspratus were determined using the two-dimensional contour plot. The most suitable carbon, nitrogen, and mineral sources for EPS production were glucose, yeast extract, CaCl2 and KH2PO4, respectively. Notably, the EPS production was significantly enhanced by supplementation of calcium ion. Subsequently, the optimum concentration of glucose (30 g l)1), yeast extract (15 g l)1), CaCl2 (1.1 g l)1), and KH2PO4 (1.2 g l)1) were determined using the orthogonal matrix method. The effects of nutritional requirement on the mycelial growth of S. aspratus were in regular sequence of glucose > KH2PO4 > yeast extract > CaCl2, and those on EPS production were in the order of glucose > yeast extract > CaCl2 > KH2PO4. Under the optimal culture conditions, the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68 g l)1 after 4 days of fermentation, which was 6fold higher than that at a basal medium. The two-dimensional contour plot and orthogonal matrix method allowed us to find the relationship between environmental factors and nutritional requirement by determining optimal operating conditions for maximum EPS production in S. aspratus. The statistical experiments used in this work can be useful strategies for optimization of submerged culture processes for other mushrooms.
Introduction The polysaccharides of higher fungi show various biological activities such as immuno-stimulating activity and antitumour activity (Mizuno et al. 2000; Moon et al. 2002; Cui & Chisti 2003). Sarcodon aspratus (Berk) S.lto TG-3 is a Basidiomycete fungus belonging to the order Aphyllopherales, and the family Telephoraceae. In recent years, the polysaccharides with biological functions originated from this mushroom have been reported by several investigators (Mizuno et al. 2000; Moon et al. 2002). It has been used as an item traditional haute cuisine and as a remedy for digestive upsets after eating meat food in Korea. However, it takes several months to obtain the polysaccharides from the fruiting body of S. aspratus. To overcome this problem, a submerged fermentation process for the production of exopolysaccharides (EPS) is strongly desirable due to its low cost, high productivity and the feasibility of mass production in a compact space. In fact, many investigators have
attempted to optimize the submerged cultures for EPS production from higher fungi (Peiris et al. 1998; Fang et al. 2002; Berovic et al. 2003; Hwang et al. 2003). In the present study, the contour plot method and the orthogonal matrix method were used to identify the nutritional requirements and environmental conditions in liquid culture of S. aspratus. The contour plot showing the influence of multi-parameters by a threedimensional diagram has been used together with other statistical experiments (Hung et al. 2002; Marcos et al. 2002). The orthogonal matrix method to determine the optimal setting has been applied to study the relations between the experimental variables and their effects on mycelial growth and EPS production (Escamilla et al. 2000; Xu et al. 2003). The objective of the present study was to investigate the design combination of nutrients and environment factors, thereby optimizing EPS production by S. aspratus. To the best of our knowledge, this is the first report regarding submerged culture of S. aspratus.
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Materials and methods
Culture broth Centrifugation (9000 x g, 20 min)
Microorganism and media Sarcodon aspratus (Berk) S.lto TG-3 (S. aspratus) from the culture collection of our laboratory was originally isolated from a mountainous district in Korea. The stock culture was maintained on potato dextrose agar (PDA) slants in 25% glycerol solution at )20 C for about 2 months and subcultured every 4 weeks. Slants were incubated at 25 C for 4 days and then stored at 4 C. The seed cultures were grown in 250 ml flasks containing 50 ml of MCM medium (Mushroom complete medium; 20 g l)1 glucose, 2 g l)1 meat peptone, 2 g l)1 yeast extract, 0.46 g l)1 KH2PO4, 1 g l)1 K2HPO4, 0.5 g l)1 MgSO4 Æ 7H2O) at 25 C on a rotary shaker incubator at 150 rev min)1 for 3 days (Kim et al. 2002).
Mycelium
Supernatant
Constant weight (60°C, 24 h)
Filtration (Whatman No. 2) Filtrate
Dried mycelium
Ethanol precipitation (Supernatant:Ethanol=1:4)
Supernatant
Precipitate Lyophilization
Inoculum preparation and flask cultures S. aspratus was initially grown on PDA medium in a petri dish, then transferred into the seed culture medium by punching out 5 mm of the agar plate culture with a self-designed cutter (Park et al. 2001). Shake flask cultures were carried out in 250 ml flasks containing 50 ml of the MCM medium at 25 C for 4 days, using 4% (v/v) inocula. All experiments were performed at least in duplicate to ensure reproducibility. Fermentation in a 5-l stirred-tank bioreactor MCM medium was used as the basal medium. The fermentation medium was inoculated with 4% (v/v) of the seed culture and then cultivated for 132 h at 25 C in a 5-l batch bioreactor (Ko-BioTech Co., Seoul, Korea) with a working volume of 3-l. Unless otherwise specified, the initial pH, temperature, aeration rate, and agitation rate were controlled at 5.0, 25 C, 2 v/v/m, and 150 rev min)1, respectively. Comparative fermentation experiments were conducted between the basal and the optimum medium.
Exopolysaccharides
Figure 1. Recovery process of exopolysaccharides produced by submerged culture of Sarcodon aspratus (Berk) S.lto TG-3.
tion (9000 · g for 20 min) and then dried at 60 C for 24 h, to a constant weight. The supernatant was filtered through a membrane filter (Whatman no. 2) and then the resulting culture filtrate was mixed with four volumes of absolute ethanol, stirred vigorously and left overnight at 4 C. The precipitated EPS were centrifuged at 9000 · g for 20 min and the supernatant was discarded. The precipitate of crude EPS was dried in an oven and the EPS weight was estimated. The filtrate from membrane filtration (Whatman no. 2) was analysed quantitatively for residual sugar concentration by HPLC (Shimadzu Co., Kyoto, Japan), using an Aminex HPX-42C column (0.78 · 30 cm; Bio–rad, USA) equipped with a refractive index detector (Bae et al. 2000).
Results and discussion Optimization of nutrients using the orthogonal matrix method The orthogonal matrix method was used to optimize the concentrations of four effective nutrients (glucose, yeast extract, CaCl2, and KH2PO4), which resulted from each factor investigated in flask experiments. The notation La(bc) is used to represent the orthogonal array where ‘a’ is the number of experimental runs, ‘b’ the number of levels for each factor or variable and ‘c’ the number of factors investigated (Escamilla et al. 2000). Hence L9(34) is the orthogonal array of the present study. Analytical methods The procedure for isolation of EPS is shown in Figure 1. Dry weight of mycelium was measured after centrifuga-
Effect of initial pH and temperature A significant effect of environmental factors such as initial pH and temperature in submerged cultures of higher fungi has been documented in literatures (Yang & Liau 1998; Fang & Zhong 2002a). In order to investigate the effect of initial pH and temperature on mycelial growth and EPS production, S. aspratus was cultivated with different initial pH values and temperatures in shake culture conditions. The maximum mycelial growth (10.22 g l)1) and EPS (0.51 g l)1) production were obtained at an initial pH of 5.0 and at 25 C as shown in the two-dimensional contour plots (Figure 2). The previous models were used to calculate the contours of constant response for the parameters. This two-dimensional contour plot has been used for
Exopolysaccharide production in Sarcodon aspratus 6
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Figure 2. Two-dimensional contour plots showing the effect of initial pH and temperature on mycelial growth and exopolysaccharide production by Sarcodon aspratus (Berk) S.lto TG-3 in shake flask cultures. A: mycelial growth (g l)1) and B: exopolysaccharide concentration (g l)1).
system optimization by many researchers (Hung et al. 2002; Marcos et al. 2002). The horizontal rhombic shape in the maximum area suggests that simultaneous optimization of the initial pH and temperature is considered important to achieve maximum growth and EPS production in submerged culture of S. aspratus. These optimal culture conditions are quite similar to those of other mushrooms in their submerged cultures (Lee et al. 2003; Yang et al. 2003).
Effect of carbon and nitrogen source To investigate the requirement of major nutrients for mycelial growth and EPS production, various carbon
sources (2%, w/v) and nitrogen sources (0.4%, w/v) were supplemented to the basal medium. Among the carbon sources tested, the maximum mycelial growth (10.62 g l)1) and maximum EPS production (0.46 g l)1) were observed in the glucose medium (Table 1). Among the 12 nitrogen sources examined, meat peptone was relatively favourable to mycelial growth of S. aspratus. However, the maximum EPS production was achieved in the yeast extract medium. Inorganic nitrogen sources yielded a relatively lower mycelial growth and EPS production compared to the organic nitrogen sources. This result is in accordance with nutritional requirement for EPS production in several mushrooms (Sudhakaran & Shewale 1988; Fang & Zhong 2002b).
Table 1. Effect of carbon and nitrogen sources on mycelial growth and exopolysaccharide (EPS) production in shake flask cultures of Sarcodon aspratus (Berk) S.lto TG-3a. Mycelial growth (g l)1)
EPS (g l
Carbon source (2%) Sucrose Glucose Fructose Maltose Lactose Xylose
2.08b ± 0.08 10.62 ± 0.32 9.44 ± 0.42 2.44 ± 0.36 7.71 ± 0.61 7.08 ± 0.84
0.24 0.46 0.25 0.13 0.41 0.17
± ± ± ± ± ±
0.02 0.00 0.04 0.06 0.01 0.01
7.43 4.36 4.42 7.27 4.90 3.87
± ± ± ± ± ±
0.12 0.09 0.03 0.08 0.08 0.04
Nitrogen source (0.4%) Poly peptone Casein peptone Tryptone Meat peptone Soy peptone Martone A-1 Yeast extract NaNO3 NH4NO3 (NH4)2HPO4 NH4Cl (NH4)2HC6H5O7
7.89 8.07 7.15 11.48 5.60 9.37 10.70 1.48 0.93 1.09 0.84 0.67
0.46 0.30 0.34 0.45 0.44 0.53 0.54 0.32 0.23 0.40 0.19 0.28
± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.00 0.02 0.04 0.04 0.01 0.00 0.00 0.01 0.00 0.01 0.02
3.82 3.53 3.70 4.01 4.53 3.94 3.89 4.50 1.75 2.62 1.76 4.56
± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.09 0.01 0.11 0.05 0.01 0.09 0.00 0.03 0.02 0.01 0.04
a b
± ± ± ± ± ± ± ± ± ± ± ±
0.51 0.37 0.33 0.06 0.04 0.37 0.04 0.04 0.03 0.05 0.04 0.05
Fermentations were carried out for 4 days at 25 C with initial pH 5. Values are mean of triple determinations with standard deviation (±).
)1
)
Final pH
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Table 2. Effect of mineral sources on mycelial growth and exopolysaccharide (EPS) production in shake flask culture of Sarcodon aspratus (Berk) S.lto TG-3a.
Table 3. Factors and levels of instrumental factors matching the Latin square. Levels
Mineral (7 mM)
KH2PO4 MgSO4 Æ 7H2O MgCl2 Æ 6H2O K2HPO4 MnCl2 Æ 4H2O KCl CaCl2 CuCl2 Æ 2H2O Na2HPO4 Æ 12H2O
Mycelial growth (g l)1)
EPS (g l )1)
7.09 b ± 0.27 11.01 ± 0.03 10.12 ± 0.00 6.84 ± 0.36 6.73 ± 0.47 9.83 ± 0.67 10.02 ± 0.70 5.08 ± 0.22 6.76 ± 0.80
0.55 0.45 0.39 0.50 0.54 0.37 0.98 0.23 0.51
± ± ± ± ± ± ± ± ±
Factors
Final pH
0.03 0.01 0.03 0.02 0.08 0.01 0.01 0.03 0.05
3.76 4.32 3.99 3.76 4.55 4.25 4.27 4.03 3.73
± ± ± ± ± ± ± ± ±
0.04 0.12 0.03 0.04 0.09 0.05 0.15 0.04 0.15
a
Fermentations were carried out for 4 days at 25 C with initial pH 5. b Values are mean of triple determinations with standard deviation (±).
Effect of mineral source Table 2 shows the influence of mineral sources on mycelial growth and EPS production in S. aspratus. Among the various mineral sources examined, both Mg2+ and Ca2+ had a beneficial effect on mycelial growth, whereas the maximum EPS production was achieved in the media containing calcium ion. This result pointed out that addition of mineral sources is effective for EPS accumulation in liquid culture of S. aspratus. Supplementation of calcium ion to the medium has been frequently regarded as one of effective ways to accelerate mycelial growth and to induce some metabolites from many microorganisms in submerged cultures (Chardonnet et al. 1999; MacArtain et al. 2003; Taherzadeh et al. 2003). Although it is still in doubt how calcium ions effect the enhanced EPS production, it is presumably linked to the increase in cell membrane permeability, as usually found in the production of some secondary metabolites (Chardonnet et al. 1999; Maccio et al. 2002).
1 2 3
Glucose A (%)
Yeast extract B (%)
CaCl2 C (%)
KH2PO4 D (%)
2 3 4
1.0 1.5 2.0
0.110 0.132 0.154
0.081 0.120 0.136
Each factor is denoted by symbols A, B, C, and D. The optimum level for each factor is obtained by concentration experiment of each factor.
The three levels of the factors associated with the orthogonal matrix are shown in Table 3. They are set based on the concentration levels of each factor. The smallest orthogonal array is selected with minimum experiments that can assign all the design variables to their columns. The chosen four factors (e.g. glucose, yeast extract, CaCl2, and KH2PO4) are assigned to columns A–D; that is, the columns are mutually orthogonal without overlapping. The orthogonal matrix method was obviously a serviceable experimental design to simultaneously investigate the relationship between the effect of medium components and their optimal concentrations. The synergistic effects on the mycelial growth and EPS production can be examined in medium supplemented with the optimal concentrations of glucose, yeast extract, CaCl2, and KH2PO4. In addition, the orthogonal matrix can simultaneously investigate many more factors than for instance central composite design, and thus facilitates economical benefit, experimental convenience, while being competitively rapid and precise. It means that the orthogonal matrix method can be a useful tool for optimizing the determination conditions of EPS production in a submerged fermentation process. Some researchers have successfully applied it to optimization of culture media for the production of primary and secondary metabolites in fermentation processes (Escamilla et al. 2000; Xu et al. 2003; Fan et al. 2004).
Experimental results of L9(34) orthogonal matrix method Sequence of effects of factors The orthogonal experimental design technique is a mathematical method that enables one to study the relationships among various factors (Escamilla et al. 2000; Li et al. 2001; Xu et al. 2003). The Latin square used to optimize the nutrimental factors was L9(34) (Table 3), where L9(34) indicates a Latin square with nine combinations of variables and (34) denotes four factors with three levels. Appropriately selecting the factor can improve the quality of the product, especially when at least two quality characteristics are to be simultaneously considered. The selection of the factor should account for the objectives of the study and feasibility and ease of handling the optimization process (Houng et al., 2003). In our case, four factors are the carbon source (glucose), the nitrogen source (yeast extract), and two mineral sources (CaCl2 and KH2PO4).
The fermentation results of mycelial growth and EPS production by the orthogonal matrix method are given in Table 4. The variance in factor/level combination shows the range over which the maximum mycelial growth and EPS production changed with levels for each factor. The effects of these control factors on mycelial growth and EPS production were determined by the analysis of variance technique to determine which factors were statistically significant. Intuitive analyses and statistical calculations are shown in Table 5 based on historical data analysis. According to the relative magnitude order of R value, the sequence of effect of all factors or mycelial growth and EPS production could be determined. The reference sequence of effects of factors on mycelial growth was in the order of
Exopolysaccharide production in Sarcodon aspratus
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Table 4. Arrangements of the L9(34) Latin square. Exp. no.
Factors
1 2 3 4 5 6 7 8 9
Responded results
A
B
C
D
Mycelial growth (g l)1)
EPS (g l)1)
1 1 1 2 2 2 3 3 3
1 2 3 1 2 3 1 2 3
1 2 3 2 3 1 3 1 2
1 2 3 3 1 2 2 3 1
11.58 12.36 12.95 13.96 13.28 13.54 12.44 14.36 13.53
2.10 2.77 2.15 2.52 3.10 3.63 2.55 3.32 3.30
± ± ± ± ± ± ± ± ±
0.02 0.59 0.38 0.42 0.57 0.94 0.81 1.01 1.01
± ± ± ± ± ± ± ± ±
0.24 0.19 0.05 0.07 0.08 0.33 0.01 0.19 0.38
The assignments of column A, B, C, and D were performed by orthogonal array consisted of nine experiments corresponding to the nine rows and four columns. Values are mean of triple determinations with standard deviation (±).
glucose > KH2PO4 > yeast extract > CaCl2. On the other hand, that on EPS production was glucose > yeast extract > CaCl2 > KH2PO4. Therefore, glucose was determined as a more influential factor than the other nutrients. This result indicated that glucose gave good mycelial growth and EPS production. The relative magnitude of factors were determined to simultaneously optimize the multiple quality characteristics via the conventional orthogonal matrix method. Xu et al. (2003) also have reported that glucose was determined as an effective factor in submerged culture of P. tenuipes C240.
Fermentation results in shake flask experiments Figure 3 shows the optimal levels and relation of each combination. If the experimental result given in Figure 3 is studied carefully, it can be seen that this ratio of factor affected the yield in EPS production. The results were found to be as follows: (1) to obtain maximum mycelial growth, the optimum composition was 3% (w/v) glucose, 2% (w/v) yeast extract, 0.132% (w/v) CaCl2, and 0.136% (w/v) KH2PO4; (2) to obtain maximum EPS production, the optimum composition was 3% glucose, 1.5% yeast extract, 0.11% CaCl2, and 0.12% KH2PO4.
In this study, optimum productivity of EPS was achieved by the combined levels of each factor. Fermentation results in a 5-l stirred-tank bioreactor Figure 4 shows the typical time profiles of mycelial growth and EPS production in a 5-l stirred-tank reactor at the basal and the optimum culture medium. In a basal medium (Figure 4A), EPS concentration reached a maximum level of 0.48 g l)1 at 108 h, while maximum mycelial growth concentration was 8.56 g l)1 at 72 h. The initial pH value of the fermentation broth slowly decreased from 5.0 to 4.3, and reached 6.0 at the end of fermentation. Under the optimized culture condition (Figure 4B), the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68 g l)1 after 4 days of fermentation, which was 6-fold higher than that with the basal medium. Also, the maximum mycelial concentration was 9.44 g l)1 at 72 h. Kim et al. (2002) have reported that maximum EPS production of S. aspratus was found to be 0.137 g l)1 in MCM medium at 5 days. The EPS yield was enhanced 4-fold after medium optimization. Hence, it is suggested that the orthogonal matrix method used in this work can be widely applied to other processes for optimization of submerged culture conditions for the mushrooms. To the best of our
Table 5. Analysis of experiments according to the orthogonal matrix method. Mycelial growth (g l A K1a K2 K3 k1b k2 k3 Rc Optimal level
36.89 40.78 40.33 12.30 13.59 13.44 1.30 2
)1
Exopolysaccharides (g l)1)
)
B ± ± ± ± ± ± ±
0.99 1.93 2.83 0.33 0.64 0.94 0.97
37.98 40.00 40.02 12.66 13.33 13.34 0.68 3
C ± ± ± ± ± ± ±
1.25 2.17 2.33 0.42 0.72 0.78 1.20
39.48 39.85 38.67 13.16 13.28 12.89 0.39 2
D ± ± ± ± ± ± ±
1.97 2.02 1.76 0.66 0.67 0.59 1.26
38.39 38.34 41.27 12.80 12.78 13.76 0.98 3
A ± ± ± ± ± ± ±
1.97 2.54 1.81 0.66 0.85 0.60 1.45
7.02 9.25 9.17 2.34 3.08 3.06 0.74 2
B ± ± ± ± ± ± ±
0.48 0.48 0.58 0.16 0.16 0.19 0.32
7.17 9.19 9.08 2.39 3.06 3.03 0.67 2
C ± ± ± ± ± ± ±
0.32 0.46 0.76 0.10 0.15 0.25 0.25
9.05 8.59 7.80 3.02 2.86 2.60 0.42 1
D ± ± ± ± ± ± ±
0.76 0.64 0.14 0.25 0.21 0.05 0.30
P ki = k of all experiment at the same factor level. Values are mean of triple determinations with standard deviation (±). Average of ki. Values are mean of triple determinations with standard deviation (±). c R = max{average of ki})min{average of ki}. Values are mean of triple determinations with standard deviation (±). a
b
8.50 8.95 7.99 2.83 2.98 2.66 0.32 2
± ± ± ± ± ± ±
0.70 0.53 0.31 0.23 0.18 0.10 0.28
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3
1
4
Yeast extract (g l-1) 1.5
2
12 10
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8 6
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14
1 0.11
0.132
0.154
CaCl2 (g l-1)
0.081
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KH2PO4 (g l-1)
Figure 3. Analysis of the relationship on media between mycelial growth (d) and exo-polysaccharide (s) production by Sarcodon aspratus (Berk) S.lto TG-3 in shake flask cultures.
10
20
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Figure 4. Typical time profiles of mycelial growth (d) and exopolysaccharide (s) production in basal medium (A) and optimum medium (B) in a 5-l stirred-tank reactor.
knowledge, this is the first report on optimizing EPS production in submerged culture of S. aspratus.
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