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Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous
Bioresource Technology 97 (2006) 26–31
Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous Wenjun Wang, Longjiang Yu *, Pengpeng Zhou School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China Received 23 August 2004; received in revised form 31 January 2005; accepted 18 February 2005 Available online 19 April 2005
Abstract Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina M-23 were examined to determine their effects on the growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. The results showed that different fungal elicitor could cause diversely stimulating effects. Among the fungal elicitors tested, the M. mucedo elicitor concentration of 30 mg l1 promoted the biomass and total carotenoids yield most remarkably, resulting in 69.81 ± 6.00% and 78.87 ± 4.15% higher than the control, respectively. At the concentration of 30 mg l1, R. glutinis elicitor stimulated the highest astaxanthin yield with a 90.60 ± 5.98% increase compared to the control. The R. rubra elicitor concentration of 30 mg l1 resulted in the optimal total carotenoids and astaxanthin content to be 42.24 ± 0.49% and 69.02 ± 0.72% higher than the control, respectively. At the concentration of 30 mg l1, R. rubra elicitor gave the highest increase in the ratio of astaxanthin in total carotenoids by 18.85 ± 0.11% of the control. 2005 Published by Elsevier Ltd. Keywords: Fungal elicitor; Astaxanthin; Total carotenoids; Xanthophyllomyces dendrorhous/Phaffia rhodozyma
1. Introduction Carotenoids are used as additives in the food and feed industries. Astaxanthin (3,3 0 -dihydroxy-b,b 0 -carotene-4,4 0 -dione) is an interesting carotenoid owing to its high market price and the growing demand. There has been growing interest in the use of astaxanthin as a pigment for aquaculture, especially as feed supplement for farmed trout, salmon and prawns. Since these animals cannot synthesize astaxanthin, it must be included in the feed to obtain a color appealing to consumers (Fang and Wang, 2002; Ramı´rez et al., 2001). Moreover, due to its special structure, astaxanthin is a more powerful scavenger of singlet oxygen (1O2) and peroxyl * Corresponding author. Tel.: +86 27 8754 3633; fax: +86 27 8754 0184. E-mail addresses: [email protected] (W. Wang), yulongjiang@ mail.hust.edu.cn (L. Yu).
0960-8524/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.biortech.2005.02.012
radicals (H2O2) than b-carotene, cantaxanthin, and zeaxanthin (3,3 0 -dihydroxyl-b-carotene). Its antioxidant activity is much stronger than all other carotenoids. Furthermore, astaxanthin may exert antitumor activities through the enhancement of immune responses (Lai et al., 2004; Persike et al., 2002). Traditionally astaxanthin production has been achieved by chemical synthesis (Britton et al., 1996). However, consumer and governmental concerns regarding chemical additives in foods have stimulated research in biological systems to produce astaxanthin by biotechnology (Carlos and Johnson, 2004). These systems are mainly focused on microorganisms such as algae, bacteria or yeast. The red yeast Xanthophyllomyces dendrorhous (formerly known as Phaffia rhodozyma) is one of the most promising microorganisms for the commercial production of astaxanthin (Cruz and Parajo´, 1998). A number of research works on this subject have been reported over the last few years. Some of them deal with
W. Wang et al. / Bioresource Technology 97 (2006) 26–31
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the optimization of fermentation methodologies (FloresCotera et al., 2001; Ramı´rez et al., 2001; Vazquez and Martin, 1998), mutagenesis (An et al., 1991), chemical stimulants such as mevalonic acid, ethanol, lycopene and acetic acid (Calo et al., 1995; Gu et al., 1997; Johnson and Lewis, 1979; Meyer and du Preez, 1993), and genetic and metabolic engineering (Norihiko and Hiroshi, 1998; Visser et al., 2003), but the production levels in these hosts are still low. Up to now, employment of pathogenic and non-pathogenic fungal preparations and chemicals, termed as elicitors, thus becomes one of the most important strategies to improve secondary metabolite production in intro cell cultures (Yu et al., 2002). Previously it has been reported that the fungus Epicoccum nigrum stimulated astaxanthin formation by the yeast X. dendrorhous (Carlos and Johnson, 2004). In this study, the effects of fungal elicitors prepared from six fungus were investigated in an attempt to increase the growth, total carotenoids and astaxanthin production of X. dendrorhous.
atus, C. versicolor, M. mucedo and M. alpina M-23 were grown in potato dextrose broth for 7 days at 30 C in the 150 rpm shaking incubators. After suspension cultures had been filtered, mycelial walls were resuspended in water, and filtered again. A total of 40 g (fresh weight) mycelial walls were homogenized in 0.1 M sodium acetate buffer (pH 5.6) in a Waring blender at room temperature for 10 min, and then were blended with 120 ml ethyl acetate in a mixture at room temperature for 24 h. Mixture was filtered with decompress filter and the filtrate containing lipids was discarded. The remains were collected, and 100 ml deionized water was added, then the pH value was adjusted to 2 with 1 mol l1 HCl, and then it was autoclaved at 121 C for 1 h. The suspension was filtered and filtrate was adjusted to pH 5.8 with 0.5 mol l1 NaOH and used as elicitors (Yu et al., 2001). Their carbohydrate concentrations were determined by the orcinol– sulfuric acid method (Francois et al., 1962).
2. Methods
Each fungal elicitor was added into the proliferation media at the concentrations of 5, 10, 20, 30 and 40 mg l1 carbohydrate equivalent. Then, all media were sterilized by autoclaving for 20 min at 121 C. Proliferation experiments were carried out for 4 days at 22 C in orbital shakers (agitation speed, 200 rpm) with 500 ml flasks containing 50 ml autoclaved proliferation media. Each experiment was performed twice at the same time to check the reproducibility.
2.1. Yeast and fungal strains P. rhodozyma AS 2.1557 (now X. dendrorhous) was obtained from China General Microbiological Culture Collection Center (CGMCCC, Beijing, China), maintained on slants of yeast malt (YM) agar at 4 C and transferred monthly. Rhodotorula rubra AS 2.670, Rhodotorula glutinis AS 2.703, Panus conchatus AS 5.154, Coriolus versicolor AS 5.48, Mucor mucedo AS 3.2531 were obtained from CGMCCC, and Mortieralla alpina M-23 was isolated from the campus of Huazhong University of Science and Technology (Wuhan, China) and identified by Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands) (Zhu et al., 2004). Six fungal strains were mainly maintained on slants of potato dextrose agar (PDA) at 4 C in our laboratory. 2.2. Media and chemicals The components of YM media were 10 g glucose, 5 g bactopeptone, 3 g yeast extract, 3 g malt extract, and 20 g agar (for plates) in 1 l distilled water. The components of proliferation media (designed for this study) were 30 g glucose, 10 g ammonium chloride, 2 g magnesium sulfate, 4 g potassium dihydrogen phosphate and 1 g disodium hydrogen phosphate in 1 l tap water (pH 6.0). Astaxanthin standard was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.3. Preparation of fungal elicitors R. rubra and R. glutinis were grown in YM media for 4 days at 22 C in 200 rpm shaking incubators. P. conch-
2.4. Culture condition
2.5. Analytical procedures Biomass was measured by DCW (dry cell weight). The total carotenoids yield was determined by the DMSO method (Sedmark et al., 1990) and astaxanthin yield was measured by HPLC. Chromatographic separations were performed on HPLC (Waters 496, USA), carbanate 5 lm, 250 · 4.6 mm column Alltech with a 5-lm 45 · 4.6 mm guard column. The eluting solvent was methanol:methyl cyanide (90:10 v/v) and flow rate was 1 ml min1. The astaxanthin HPLC peak was monitored at 480 nm. Synthetic astaxanthin standard was used as external standard.
3. Results 3.1. Effects of fungal elicitors on growth of X. dendrorhous The effects of the six fungal elicitors on the growth of X. dendrorhous were shown in Fig. 1. All fungal elicitors derived from the different strains tested showed diverse effects on the growth. M. mucedo and P. conchatus elicitors showed more notably stimulative effects on the
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W. Wang et al. / Bioresource Technology 97 (2006) 26–31
growth of X. dendrorhous than the other four fungal elicitors. Among all the elicitors tested, the M. mucedo elicitor concentration of 30 mg l1 resulted in the maximal biomass, causing 69.81 ± 6.00% higher than the control (5.31 ± 0.11 g l1). At the concentration of 30 mg l1, P. conchatus elicitor could improve the growth of X. dendrorhous markedly with an increase of 68.87 ± 5.66% compared to the control. However, R. rubra elicitor could not influence the growth of X. dendrorhous as prominently as the other five elicitors, and it would even restrain the cell proliferation at high addition. 3.2. Effects of fungal elicitors on total carotenoids yield and content of X. dendrorhous The effects of different fungal elicitors on the total carotenoids yield and content of X. dendrorhous were prominent (Fig. 2(a) and (b)). Among six elicitors used, each elicitor showed a clear positive influence on the
Biomass of X. dendrorhous (g l-1)
9.5
total carotenoids yield, and there was a steady increase of the total carotenoids yield along with the concentrations, reaching the maximum at certain level. The optimal dosages of obtaining the highest total carotenoids yields for elicitor treatments were high, but low concentrations of elicitors resulted in the maximal total carotenoids content (Table 1). M. mucedo and R. glutinis elicitors at low levels could increase the total carotenoids yields more evidently than the other four elicitors. The M. mucedo elicitor concentration of 30 mg l1 increased the total carotenoids yield most significantly, resulting in 78.87 ± 4.15% higher than the control. The total carotenoids content was determined by the biomass and total carotenoids yield of X. dendrorhous. R. rubra elicitor effectively promoted the total carotenoids content at concentration of 30 mg l1 to gain its maximum, giving an increase of 42.24 ± 0.49% compared to the control. Though the other five elicitors could enhance the total carotenoids content as well, but their effects were not very palpable. 3.3. Effects of fungal elicitors on astaxanthin yield and content of X. dendrorhous
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1) Fig. 1. Effects of different elicitors at various concentrations on growth of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
To improve higher astaxanthin production, the particular attention was paid to the fungal elicitors stimulating more astaxanthin biosynthesis. As depicted in Fig. 3(a) and (b), R. rubra and R. glutinis elicitors showed more significantly stimulating activities in the astaxanthin accumulation of X. dendrorhous. The optimal dosages of the highest astaxanthin yield and content of X. dendrorhous with different fungal elicitors were shown in Table 2. The R. rubra concentrations of 20 mg l1 and 30 mg l1 increased in the astaxanthin yield and content of X. dendrorhous significantly, resulting in 88.29 ± 6.15% and 69.02 ± 0.72% higher than the control, respectively. At the concentrations of 20 mg l1 and 10 mg l1, R. glutinis elicitor gave the increases in
3.8
0.54
Total carotenoids yield of X. dendrorhous (mg l-1)
3.6
Total carotenoids content of X. dendrorhous (mg g-1 DCW)
(a)
3.4 3.2 3.0 2.8 2.6 2.4 2.2
(b)
0.52 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36
2.0
0.34 0
5
10
15
20
25
30
35
40 -1
Concentration of elicitors (mg l )
45
0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1)
Fig. 2. Effects of different elicitors at various concentrations on total carotenoids yield (a) and content (b) of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
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Table 1 Optimal dosages of the highest total carotenoids yield and content of X. dendrorhous with different elicitors Elicitors
Concentration (mg l1)
Total carotenoids yield (mg l1)
Concentration (mg l1)
Total carotenoids content (mg g 1 DCW)
R. rubra R. glutinis P. conchatus C. versicolor M. alpina M-23 M. mucedo Control
20 30 40 10 30 30 0
3.217 ± 0.115 3.337 ± 0.102 3.176 ± 0.092 2.971 ± 0.079 3.094 ± 0.076 3.488 ± 0.081 1.950 ± 0.072
30 5 5 5 20 5 0
0.5233 ± 0.0018 0.4256 ± 0.0053 0.4191 ± 0.0029 0.4333 ± 0.0003 0.4529 ± 0.0099 0.4427 ± 0.0061 0.3679 ± 0.0006
2.4
0.40
Astaxanthin content of X. dendrorhous (mg g-1 DCW)
Astaxanthin yield of X. dendrorhous (mg l-1)
(a) 2.2 2.0 1.8 1.6 1.4
(b)
0.38 0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20
1.2
0.18 0
5
10
15
20
25
30
35
40
45
0
5
Concentration of elicitors (mg l-1)
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1)
Fig. 3. Effects of different elicitors at various concentrations on astaxanthin yield (a) and content (b) of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
the astaxanthin yield and content of X. dendrorhous markedly by 90.60 ± 5.98% and 31.57 ± 0.41% of the control, respectively. Among six fungal elicitors tested, M. mucedo elicitor mainly showed the negative effect on astaxanthin content.
They could improve both the total carotenoids and astaxanthin yields of X. dendrorhous, but not increase the ratios of astaxanthin in total carotenoids.
4. Discussion 3.4. Effects of fungal elicitors on ratio of astaxanthin in total carotenoids As shown in Fig. 4, R. rubra and R. glutinis elicitors showed the more effectively stimulative activities in the ratios of astaxanthin in total carotenoids than the other four fungal elicitors, resulting in 18.85 ± 0.11% (at 30 mg l1) and 17.13 ± 0.46% (at 40 mg l1) higher than the control (0.599 ± 0.004), respectively. In comparison with them, the other four fungal elicitors mainly decreased the ratios of astaxanthin in total carotenoids.
Six fungal elicitors tested could enhance the growth, total carotenoids and astaxanthin production of X. dendrorhous at different degrees as mentioned above, but the mechanism by which fungal elicitors stimulated carotenoid biosynthesis of X. dendrorhous is unknown. Previous studies have shown that carotenoid precursors and chemical stimulants such as mevalonic acid, ethanol, lycopene and acetic acid could enhance astaxanthin yields in P. rhodozyma (Calo et al., 1995; Gu et al., 1997; Johnson and Lewis, 1979; Meyer and du Preez, 1993).
Table 2 Optimal dosages of the highest astaxanthin yield and content of X. dendrorhous with different elicitors Elicitors
Concentration (mg l1)
Astaxanthin yield (mg l1)
Concentration (mg l1)
Astaxanthin content (mg g 1 DCW)
R. rubra R. glutinis P. conchatus C. versicolor M. alpina M-23 M. mucedo Control
20 30 40 20 30 30 0
2.203 ± 0.072 2.230 ± 0.071 1.910 ± 0.001 1.526 ± 0.065 1.622 ± 0.059 1.680 ± 0.072 1.170 ± 0.051
30 10 5 5 20 5 0
0.3732 ± 0.0016 0.2905 ± 0.0009 0.2470 ± 0.0013 0.2412 ± 0.0039 0.2406 ± 0.0038 0.2241 ± 0.0017 0.2208 ± 0.0012
W. Wang et al. / Bioresource Technology 97 (2006) 26–31
Ratio of astaxanthin in total carotenoids
30 0.74 0.72 0.70 0.68 0.66 0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1) Fig. 4. Effects of different elicitors at various concentrations on ratio of astaxanthin in total carotenoids of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
R. rubra and R. glutinis could produce carotenoid because both of them owned the biosynthetic pathway of carotenoid and were believed to contain the structural genes of carotenoid biosynthesis (An et al., 1991). This is understandable that the fungal elicitors prepared from R. rubra and R. glutinis could probably promote the biosynthesis of astaxanthin by elicitation or providing the precursors. It was found that the contaminant of fungus (which was identified Epicoccum nigrum later) on a plate of X. dendrorhous markedly affected carotenogenesis in various strains of the yeast, and the astaxanthin and total carotenoids yields were increased by nearly 40% in the wild-type X. dendrorhous UCD 67-385 (ATCC 24230) (Carlos and Johnson, 2004). The E. nigrum was a plant pathogen and known to synthesize secondary metabolites including isoprenoids and certain compounds or intermediates which could affect carotenoid formation (Ekah, 1970; Foppen and Grivanovski-Sassu, 1968, 1969). The plant pathogens or wood-rotting fungi produced oxidative enzymes which could lead plant cell walls degraded, resulting in generating reactive oxygen species (ROS) including peroxyl radicals and singlet oxygen which could enhance astaxanthin yield of P. rhodozyma (Schroeder and Johnson, 1995a,b). It was conceivable that oxidizing agents produced from the metabolic activities of E. nigrum and some wood-rotting fungus could stimulate astaxanthin production of X. dendrorhous (Carlos and Johnson, 2004). P. conchatus and C. versicolor were the wood-rotting fungus which could allow access to plant nutrients such as cellulose and lignin, so it was probable that the elicitors prepared from them stimulated the growth and carotenoid formation by X. dendrorhous. The red yeast X. dendrorhous was originally isolated by Phaff and his collaborators in the late 1960Õs from fluxes of deciduous trees rich in sugar in mountainous regions of Japan and Alaska
and could ferment sugars such as glucose and saccharose to produce carotenoid, primary astaxanthin (Phaff et al., 1972). It was presumed that during the long-term process of yeast–pathogen interaction the red yeast X. dendrorhous has gradually formed a series of complex and effectively protective mechanism to defend against the incursion of pathogens and white-rotting fungus. Therefore the red yeast X. dendrorhous co-existing with the wood-rotting fungus or plant pathogens were induced to stimulate the pigment formation. The components of elicitors prepared from fungal cell walls were very complicated, including proteins, polysacchrides, glycoproteins, peptides, oligosaccharides and lipoids (Ebel and Scheel, 1992). It was possible that they could stimulate the growth or carotenogenesis of X. dendrorhous. The components of cell walls of six fungus in this study, including yeast, white-rotting fungus and mucor, were different. It was clear that there were chitinglucan in the cell walls of the yeast (such as R. glutinis and R. rubra) and white-rotting fungus while peptidoglycan in those of the mucor. It was probable the later were more abundant in ingredients which were advantageous to the growth of X. dendrorhous. It was interesting that there was a novel approach to increase the carotenoid production of X. dendrorhous by additives such as the fungal elicitors in the industrial fermentations. It has been proved that some kinds of microbial elicitors stimulated the biomass and carotenoid yield of Penicillium sp. PT95 (Han et al., 2002). In conclusion, stimulation by fungus may be a more common regulatory mechanism for carotenogenesis than has previously been recognized.
5. Conclusion The present work suggested that six fungal elicitors which were prepared from R. rubra, R. glutinis, P. conchatus, C. versicolor, M. mucedo, M. alpina M-23 could influence the growth, total carotenoids and astaxanthin formation by X. dendrorhous at different concentrations. It offered a new approach to achieve higher biomass and carotenoid production by X. dendrorhous. The M. mucedo elicitor concentration of 30 mg l1 promoted the biomass and total carotenoids yield most significantly with a 69.81 ± 6.00% and 78.87 ± 4.15% increase compared to the control, respectively. At the concentration of 30 mg l1, R. rubra elicitor stimulated the highest total carotenoids and astaxanthin content, causing 42.24 ± 0.49% and 69.02 ± 0.72% higher than the control, respectively. R. glutinis elicitor (at 30 mg l1) resulted in the maximal astaxanthin yield with 90.60 ± 5.98% higher than the control. The R. rubra elicitor concentration of 30 mg l1 gave the maximal increase in the ratio of astaxanthin in total carotenoids by 18.85 ± 0.11% of the control. It
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could be inferred that the fungal elicitors might stimulate the growth and carotenoid biosynthesis of X. dendrorhous by the active ingredients in the fungal extracts.
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Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous Wenjun Wang, Longjiang Yu *, Pengpeng Zhou School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China Received 23 August 2004; received in revised form 31 January 2005; accepted 18 February 2005 Available online 19 April 2005
Abstract Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina M-23 were examined to determine their effects on the growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. The results showed that different fungal elicitor could cause diversely stimulating effects. Among the fungal elicitors tested, the M. mucedo elicitor concentration of 30 mg l1 promoted the biomass and total carotenoids yield most remarkably, resulting in 69.81 ± 6.00% and 78.87 ± 4.15% higher than the control, respectively. At the concentration of 30 mg l1, R. glutinis elicitor stimulated the highest astaxanthin yield with a 90.60 ± 5.98% increase compared to the control. The R. rubra elicitor concentration of 30 mg l1 resulted in the optimal total carotenoids and astaxanthin content to be 42.24 ± 0.49% and 69.02 ± 0.72% higher than the control, respectively. At the concentration of 30 mg l1, R. rubra elicitor gave the highest increase in the ratio of astaxanthin in total carotenoids by 18.85 ± 0.11% of the control. 2005 Published by Elsevier Ltd. Keywords: Fungal elicitor; Astaxanthin; Total carotenoids; Xanthophyllomyces dendrorhous/Phaffia rhodozyma
1. Introduction Carotenoids are used as additives in the food and feed industries. Astaxanthin (3,3 0 -dihydroxy-b,b 0 -carotene-4,4 0 -dione) is an interesting carotenoid owing to its high market price and the growing demand. There has been growing interest in the use of astaxanthin as a pigment for aquaculture, especially as feed supplement for farmed trout, salmon and prawns. Since these animals cannot synthesize astaxanthin, it must be included in the feed to obtain a color appealing to consumers (Fang and Wang, 2002; Ramı´rez et al., 2001). Moreover, due to its special structure, astaxanthin is a more powerful scavenger of singlet oxygen (1O2) and peroxyl * Corresponding author. Tel.: +86 27 8754 3633; fax: +86 27 8754 0184. E-mail addresses: [email protected] (W. Wang), yulongjiang@ mail.hust.edu.cn (L. Yu).
0960-8524/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.biortech.2005.02.012
radicals (H2O2) than b-carotene, cantaxanthin, and zeaxanthin (3,3 0 -dihydroxyl-b-carotene). Its antioxidant activity is much stronger than all other carotenoids. Furthermore, astaxanthin may exert antitumor activities through the enhancement of immune responses (Lai et al., 2004; Persike et al., 2002). Traditionally astaxanthin production has been achieved by chemical synthesis (Britton et al., 1996). However, consumer and governmental concerns regarding chemical additives in foods have stimulated research in biological systems to produce astaxanthin by biotechnology (Carlos and Johnson, 2004). These systems are mainly focused on microorganisms such as algae, bacteria or yeast. The red yeast Xanthophyllomyces dendrorhous (formerly known as Phaffia rhodozyma) is one of the most promising microorganisms for the commercial production of astaxanthin (Cruz and Parajo´, 1998). A number of research works on this subject have been reported over the last few years. Some of them deal with
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the optimization of fermentation methodologies (FloresCotera et al., 2001; Ramı´rez et al., 2001; Vazquez and Martin, 1998), mutagenesis (An et al., 1991), chemical stimulants such as mevalonic acid, ethanol, lycopene and acetic acid (Calo et al., 1995; Gu et al., 1997; Johnson and Lewis, 1979; Meyer and du Preez, 1993), and genetic and metabolic engineering (Norihiko and Hiroshi, 1998; Visser et al., 2003), but the production levels in these hosts are still low. Up to now, employment of pathogenic and non-pathogenic fungal preparations and chemicals, termed as elicitors, thus becomes one of the most important strategies to improve secondary metabolite production in intro cell cultures (Yu et al., 2002). Previously it has been reported that the fungus Epicoccum nigrum stimulated astaxanthin formation by the yeast X. dendrorhous (Carlos and Johnson, 2004). In this study, the effects of fungal elicitors prepared from six fungus were investigated in an attempt to increase the growth, total carotenoids and astaxanthin production of X. dendrorhous.
atus, C. versicolor, M. mucedo and M. alpina M-23 were grown in potato dextrose broth for 7 days at 30 C in the 150 rpm shaking incubators. After suspension cultures had been filtered, mycelial walls were resuspended in water, and filtered again. A total of 40 g (fresh weight) mycelial walls were homogenized in 0.1 M sodium acetate buffer (pH 5.6) in a Waring blender at room temperature for 10 min, and then were blended with 120 ml ethyl acetate in a mixture at room temperature for 24 h. Mixture was filtered with decompress filter and the filtrate containing lipids was discarded. The remains were collected, and 100 ml deionized water was added, then the pH value was adjusted to 2 with 1 mol l1 HCl, and then it was autoclaved at 121 C for 1 h. The suspension was filtered and filtrate was adjusted to pH 5.8 with 0.5 mol l1 NaOH and used as elicitors (Yu et al., 2001). Their carbohydrate concentrations were determined by the orcinol– sulfuric acid method (Francois et al., 1962).
2. Methods
Each fungal elicitor was added into the proliferation media at the concentrations of 5, 10, 20, 30 and 40 mg l1 carbohydrate equivalent. Then, all media were sterilized by autoclaving for 20 min at 121 C. Proliferation experiments were carried out for 4 days at 22 C in orbital shakers (agitation speed, 200 rpm) with 500 ml flasks containing 50 ml autoclaved proliferation media. Each experiment was performed twice at the same time to check the reproducibility.
2.1. Yeast and fungal strains P. rhodozyma AS 2.1557 (now X. dendrorhous) was obtained from China General Microbiological Culture Collection Center (CGMCCC, Beijing, China), maintained on slants of yeast malt (YM) agar at 4 C and transferred monthly. Rhodotorula rubra AS 2.670, Rhodotorula glutinis AS 2.703, Panus conchatus AS 5.154, Coriolus versicolor AS 5.48, Mucor mucedo AS 3.2531 were obtained from CGMCCC, and Mortieralla alpina M-23 was isolated from the campus of Huazhong University of Science and Technology (Wuhan, China) and identified by Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands) (Zhu et al., 2004). Six fungal strains were mainly maintained on slants of potato dextrose agar (PDA) at 4 C in our laboratory. 2.2. Media and chemicals The components of YM media were 10 g glucose, 5 g bactopeptone, 3 g yeast extract, 3 g malt extract, and 20 g agar (for plates) in 1 l distilled water. The components of proliferation media (designed for this study) were 30 g glucose, 10 g ammonium chloride, 2 g magnesium sulfate, 4 g potassium dihydrogen phosphate and 1 g disodium hydrogen phosphate in 1 l tap water (pH 6.0). Astaxanthin standard was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.3. Preparation of fungal elicitors R. rubra and R. glutinis were grown in YM media for 4 days at 22 C in 200 rpm shaking incubators. P. conch-
2.4. Culture condition
2.5. Analytical procedures Biomass was measured by DCW (dry cell weight). The total carotenoids yield was determined by the DMSO method (Sedmark et al., 1990) and astaxanthin yield was measured by HPLC. Chromatographic separations were performed on HPLC (Waters 496, USA), carbanate 5 lm, 250 · 4.6 mm column Alltech with a 5-lm 45 · 4.6 mm guard column. The eluting solvent was methanol:methyl cyanide (90:10 v/v) and flow rate was 1 ml min1. The astaxanthin HPLC peak was monitored at 480 nm. Synthetic astaxanthin standard was used as external standard.
3. Results 3.1. Effects of fungal elicitors on growth of X. dendrorhous The effects of the six fungal elicitors on the growth of X. dendrorhous were shown in Fig. 1. All fungal elicitors derived from the different strains tested showed diverse effects on the growth. M. mucedo and P. conchatus elicitors showed more notably stimulative effects on the
28
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growth of X. dendrorhous than the other four fungal elicitors. Among all the elicitors tested, the M. mucedo elicitor concentration of 30 mg l1 resulted in the maximal biomass, causing 69.81 ± 6.00% higher than the control (5.31 ± 0.11 g l1). At the concentration of 30 mg l1, P. conchatus elicitor could improve the growth of X. dendrorhous markedly with an increase of 68.87 ± 5.66% compared to the control. However, R. rubra elicitor could not influence the growth of X. dendrorhous as prominently as the other five elicitors, and it would even restrain the cell proliferation at high addition. 3.2. Effects of fungal elicitors on total carotenoids yield and content of X. dendrorhous The effects of different fungal elicitors on the total carotenoids yield and content of X. dendrorhous were prominent (Fig. 2(a) and (b)). Among six elicitors used, each elicitor showed a clear positive influence on the
Biomass of X. dendrorhous (g l-1)
9.5
total carotenoids yield, and there was a steady increase of the total carotenoids yield along with the concentrations, reaching the maximum at certain level. The optimal dosages of obtaining the highest total carotenoids yields for elicitor treatments were high, but low concentrations of elicitors resulted in the maximal total carotenoids content (Table 1). M. mucedo and R. glutinis elicitors at low levels could increase the total carotenoids yields more evidently than the other four elicitors. The M. mucedo elicitor concentration of 30 mg l1 increased the total carotenoids yield most significantly, resulting in 78.87 ± 4.15% higher than the control. The total carotenoids content was determined by the biomass and total carotenoids yield of X. dendrorhous. R. rubra elicitor effectively promoted the total carotenoids content at concentration of 30 mg l1 to gain its maximum, giving an increase of 42.24 ± 0.49% compared to the control. Though the other five elicitors could enhance the total carotenoids content as well, but their effects were not very palpable. 3.3. Effects of fungal elicitors on astaxanthin yield and content of X. dendrorhous
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1) Fig. 1. Effects of different elicitors at various concentrations on growth of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
To improve higher astaxanthin production, the particular attention was paid to the fungal elicitors stimulating more astaxanthin biosynthesis. As depicted in Fig. 3(a) and (b), R. rubra and R. glutinis elicitors showed more significantly stimulating activities in the astaxanthin accumulation of X. dendrorhous. The optimal dosages of the highest astaxanthin yield and content of X. dendrorhous with different fungal elicitors were shown in Table 2. The R. rubra concentrations of 20 mg l1 and 30 mg l1 increased in the astaxanthin yield and content of X. dendrorhous significantly, resulting in 88.29 ± 6.15% and 69.02 ± 0.72% higher than the control, respectively. At the concentrations of 20 mg l1 and 10 mg l1, R. glutinis elicitor gave the increases in
3.8
0.54
Total carotenoids yield of X. dendrorhous (mg l-1)
3.6
Total carotenoids content of X. dendrorhous (mg g-1 DCW)
(a)
3.4 3.2 3.0 2.8 2.6 2.4 2.2
(b)
0.52 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36
2.0
0.34 0
5
10
15
20
25
30
35
40 -1
Concentration of elicitors (mg l )
45
0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1)
Fig. 2. Effects of different elicitors at various concentrations on total carotenoids yield (a) and content (b) of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
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Table 1 Optimal dosages of the highest total carotenoids yield and content of X. dendrorhous with different elicitors Elicitors
Concentration (mg l1)
Total carotenoids yield (mg l1)
Concentration (mg l1)
Total carotenoids content (mg g 1 DCW)
R. rubra R. glutinis P. conchatus C. versicolor M. alpina M-23 M. mucedo Control
20 30 40 10 30 30 0
3.217 ± 0.115 3.337 ± 0.102 3.176 ± 0.092 2.971 ± 0.079 3.094 ± 0.076 3.488 ± 0.081 1.950 ± 0.072
30 5 5 5 20 5 0
0.5233 ± 0.0018 0.4256 ± 0.0053 0.4191 ± 0.0029 0.4333 ± 0.0003 0.4529 ± 0.0099 0.4427 ± 0.0061 0.3679 ± 0.0006
2.4
0.40
Astaxanthin content of X. dendrorhous (mg g-1 DCW)
Astaxanthin yield of X. dendrorhous (mg l-1)
(a) 2.2 2.0 1.8 1.6 1.4
(b)
0.38 0.36 0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20
1.2
0.18 0
5
10
15
20
25
30
35
40
45
0
5
Concentration of elicitors (mg l-1)
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1)
Fig. 3. Effects of different elicitors at various concentrations on astaxanthin yield (a) and content (b) of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
the astaxanthin yield and content of X. dendrorhous markedly by 90.60 ± 5.98% and 31.57 ± 0.41% of the control, respectively. Among six fungal elicitors tested, M. mucedo elicitor mainly showed the negative effect on astaxanthin content.
They could improve both the total carotenoids and astaxanthin yields of X. dendrorhous, but not increase the ratios of astaxanthin in total carotenoids.
4. Discussion 3.4. Effects of fungal elicitors on ratio of astaxanthin in total carotenoids As shown in Fig. 4, R. rubra and R. glutinis elicitors showed the more effectively stimulative activities in the ratios of astaxanthin in total carotenoids than the other four fungal elicitors, resulting in 18.85 ± 0.11% (at 30 mg l1) and 17.13 ± 0.46% (at 40 mg l1) higher than the control (0.599 ± 0.004), respectively. In comparison with them, the other four fungal elicitors mainly decreased the ratios of astaxanthin in total carotenoids.
Six fungal elicitors tested could enhance the growth, total carotenoids and astaxanthin production of X. dendrorhous at different degrees as mentioned above, but the mechanism by which fungal elicitors stimulated carotenoid biosynthesis of X. dendrorhous is unknown. Previous studies have shown that carotenoid precursors and chemical stimulants such as mevalonic acid, ethanol, lycopene and acetic acid could enhance astaxanthin yields in P. rhodozyma (Calo et al., 1995; Gu et al., 1997; Johnson and Lewis, 1979; Meyer and du Preez, 1993).
Table 2 Optimal dosages of the highest astaxanthin yield and content of X. dendrorhous with different elicitors Elicitors
Concentration (mg l1)
Astaxanthin yield (mg l1)
Concentration (mg l1)
Astaxanthin content (mg g 1 DCW)
R. rubra R. glutinis P. conchatus C. versicolor M. alpina M-23 M. mucedo Control
20 30 40 20 30 30 0
2.203 ± 0.072 2.230 ± 0.071 1.910 ± 0.001 1.526 ± 0.065 1.622 ± 0.059 1.680 ± 0.072 1.170 ± 0.051
30 10 5 5 20 5 0
0.3732 ± 0.0016 0.2905 ± 0.0009 0.2470 ± 0.0013 0.2412 ± 0.0039 0.2406 ± 0.0038 0.2241 ± 0.0017 0.2208 ± 0.0012
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Ratio of astaxanthin in total carotenoids
30 0.74 0.72 0.70 0.68 0.66 0.64 0.62 0.60 0.58 0.56 0.54 0.52 0.50 0.48 0.46 0
5
10
15
20
25
30
35
40
45
Concentration of elicitors (mg l-1) Fig. 4. Effects of different elicitors at various concentrations on ratio of astaxanthin in total carotenoids of X. dendrorhous. Symbols: R. rubra (–j–), R. glutinis (–d–), P. conchatus (–m–), C. versicolor (–.–), M. alpina M-23 (–r–), M. mucedo (–%–).
R. rubra and R. glutinis could produce carotenoid because both of them owned the biosynthetic pathway of carotenoid and were believed to contain the structural genes of carotenoid biosynthesis (An et al., 1991). This is understandable that the fungal elicitors prepared from R. rubra and R. glutinis could probably promote the biosynthesis of astaxanthin by elicitation or providing the precursors. It was found that the contaminant of fungus (which was identified Epicoccum nigrum later) on a plate of X. dendrorhous markedly affected carotenogenesis in various strains of the yeast, and the astaxanthin and total carotenoids yields were increased by nearly 40% in the wild-type X. dendrorhous UCD 67-385 (ATCC 24230) (Carlos and Johnson, 2004). The E. nigrum was a plant pathogen and known to synthesize secondary metabolites including isoprenoids and certain compounds or intermediates which could affect carotenoid formation (Ekah, 1970; Foppen and Grivanovski-Sassu, 1968, 1969). The plant pathogens or wood-rotting fungi produced oxidative enzymes which could lead plant cell walls degraded, resulting in generating reactive oxygen species (ROS) including peroxyl radicals and singlet oxygen which could enhance astaxanthin yield of P. rhodozyma (Schroeder and Johnson, 1995a,b). It was conceivable that oxidizing agents produced from the metabolic activities of E. nigrum and some wood-rotting fungus could stimulate astaxanthin production of X. dendrorhous (Carlos and Johnson, 2004). P. conchatus and C. versicolor were the wood-rotting fungus which could allow access to plant nutrients such as cellulose and lignin, so it was probable that the elicitors prepared from them stimulated the growth and carotenoid formation by X. dendrorhous. The red yeast X. dendrorhous was originally isolated by Phaff and his collaborators in the late 1960Õs from fluxes of deciduous trees rich in sugar in mountainous regions of Japan and Alaska
and could ferment sugars such as glucose and saccharose to produce carotenoid, primary astaxanthin (Phaff et al., 1972). It was presumed that during the long-term process of yeast–pathogen interaction the red yeast X. dendrorhous has gradually formed a series of complex and effectively protective mechanism to defend against the incursion of pathogens and white-rotting fungus. Therefore the red yeast X. dendrorhous co-existing with the wood-rotting fungus or plant pathogens were induced to stimulate the pigment formation. The components of elicitors prepared from fungal cell walls were very complicated, including proteins, polysacchrides, glycoproteins, peptides, oligosaccharides and lipoids (Ebel and Scheel, 1992). It was possible that they could stimulate the growth or carotenogenesis of X. dendrorhous. The components of cell walls of six fungus in this study, including yeast, white-rotting fungus and mucor, were different. It was clear that there were chitinglucan in the cell walls of the yeast (such as R. glutinis and R. rubra) and white-rotting fungus while peptidoglycan in those of the mucor. It was probable the later were more abundant in ingredients which were advantageous to the growth of X. dendrorhous. It was interesting that there was a novel approach to increase the carotenoid production of X. dendrorhous by additives such as the fungal elicitors in the industrial fermentations. It has been proved that some kinds of microbial elicitors stimulated the biomass and carotenoid yield of Penicillium sp. PT95 (Han et al., 2002). In conclusion, stimulation by fungus may be a more common regulatory mechanism for carotenogenesis than has previously been recognized.
5. Conclusion The present work suggested that six fungal elicitors which were prepared from R. rubra, R. glutinis, P. conchatus, C. versicolor, M. mucedo, M. alpina M-23 could influence the growth, total carotenoids and astaxanthin formation by X. dendrorhous at different concentrations. It offered a new approach to achieve higher biomass and carotenoid production by X. dendrorhous. The M. mucedo elicitor concentration of 30 mg l1 promoted the biomass and total carotenoids yield most significantly with a 69.81 ± 6.00% and 78.87 ± 4.15% increase compared to the control, respectively. At the concentration of 30 mg l1, R. rubra elicitor stimulated the highest total carotenoids and astaxanthin content, causing 42.24 ± 0.49% and 69.02 ± 0.72% higher than the control, respectively. R. glutinis elicitor (at 30 mg l1) resulted in the maximal astaxanthin yield with 90.60 ± 5.98% higher than the control. The R. rubra elicitor concentration of 30 mg l1 gave the maximal increase in the ratio of astaxanthin in total carotenoids by 18.85 ± 0.11% of the control. It
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could be inferred that the fungal elicitors might stimulate the growth and carotenoid biosynthesis of X. dendrorhous by the active ingredients in the fungal extracts.
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