Influence of exogenous stress factors on production of carotenoids by some strains of carotenogenic yeasts

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1 Annals of Microbiology, 54 (1), (2004) Influence of exogenous stress factors on production of carotenoids by some strains of carotenogenic yeasts I. MAROVA 1 *, E. BREIEROVA 2, R. KOCI 1, Z. FRIEDL 1, B. SLOVAK 1, J. POKORNA 1 1 Faculty of Chemistry, Technical University of Brno, Purkynova 118, Brno, Czech Republic; 2 Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovak Republic Abstract The aim of this study was to comparise composition and content of carotenoids produced by some yeasts strains in optimal growth conditions and in the presence of exogenous stress factors. Nine strains of carotenogenic yeasts were grown aerobically on glucose medium. As the stress factors 10 mmol/l H 2 O 2 and 5-10% NaCl were used, which were added into media i) at the beginning of growth and ii) to the exponentially growing cells. Changes of growth parameters as well as carotenoid production (lycopene, α-carotene and β-carotene) were followed. Ergosterol production was followed as additional parameter of biomass quality. Analyzed strains partially differed in the spectrum of produced carotenoids; the highest content of β-carotene was detected in Sporidiobolus salmonicolor CCY Stress factors added to yeast cultures resulted in different responses. As good producents of enriched biomas could serve above all strains Rhodotorula glutinis and S. salmonicolor grown under salt stress. Carotenoids act as lipid-soluble membrane antioxidants whose production is considered as an adaptive mechanism against adverse stress effects. Ability of red yeasts to adapt by means of overproduction of industrially significant metabolites could be of increasing interest for potential biotechnological applications. Key words: yeasts, carotenoids, osmotic stress, oxidative stress. INTRODUCTION Carotenoids are isoprenoid membrane-protective antioxidant pigments that efficiently scavenge 1 O 2 and peroxyl radicals; the antioxidative efficiency of which is apparently related to their structure. To date, almost 600 carotenoids have been identified. There are two major carotenoid families, the class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls) (Goodwin and Brit- * Corresponding Author. Phone: ; Fax: ; marova@fch.vutbr.cz 73

2 ton, 1988). Carotenoids are lipophilic compounds and can be stored in a lipophilic environment. The most significant part in the molecule is the conjugated double bond system that determines their color and biological action (Sandmann, 2001). These pigments occur universally in photosynthetic systems of higher plants, algae and phototrophic bacteria. In plants and algae, the most important function of carotenoids is to protect against photosensitized oxidation by quenching the excitation energy of the chlorophyll triplet state or singlet oxygen. Carotenoids also appear to be involved in the response of plants to environmental stress through the zeaxanthin-violaxanthin xanthophyll cycle. In nonphotosynthetic organisms carotenoids are important in protecting against photooxidative damage by quenching photosensitizers, interacting with singlet oxygen and scavenging peroxyl radicals. Thus, many non-phototrophic bacteria and fungi rely on carotenoids for protection when growing in light and air. Protective effect of carotenoids is dependent on their chemical stuctures, which can differ in the length of the polyene chromophore, the nature of the end groups and the various substituents that they contain (Britton et al., 1995). Carotenoid formation in microorganisms is dependent on the strain, the culture medium and the conditions of cultivation (Sandmann et al., 1999). During growth different types of environmental and physiological stress conditions constantly challenge all microorganisms. To cope with the deleterious effects of stress, cells have developed rapid molecular responses to repair the damage and protect against further exposure to the same and other forms of stress. In microorganisms, the environment of which is highly variable, where conditions such as temperature, osmolarity or nutrient availability are far from constant, stress responses are of particular importance. During stress different classes of substances are overproduced, e.g. heat shock proteins, antioxidative enzymes and low-molecular weight antioxidants including carotenoids, glycerol and other lipidic substances, some carbohydrates etc. Yeast cells exposed to mild stress can develop tolerance not only to higher doses of the same stress, but also to stress caused by other agents. This phenomenon is known as cross-protection and suggests the existence of an integrating stress response mechanism (Sigler et al., 1999). In recent years evidence is accumulating that carotenoids play an important role in human health by preventing degenerative diseases (Krinsky, 1989). The industrial use of carotenoids involves their application in nutrient supplementation, for pharmaceutical purposes, as food colorants and in animal feeds (Sandmann, 2001). As a result, the production of carotenoids by biotechnology is of increasing interest (Misawa and Shimada, 1997). Carotenoids are the most widespread natural lipid-soluble pigments with many important biological activities and industrial applications. The aim of this study is to evaluate the composition and content of carotenoids produced by some strains of carotenogenic yeasts under optimal growth conditions as compared to those cultivated in the presence of exogenous stress factors. MATERIAL AND METHODS Strains. Nine strains of carotenogenic yeasts from different environments were studied: five strains of the genus Rhodotorula (R. glutinis CCY river 74 I. MAROVA et al.

3 Donau, signed R. glutinis A; R. glutinis CCY soil, grass, signed R. glutinis B; R. mucilaginosa CCY river Little Donau, R. aurantiaca CCY soil, wood, and R. minuta CCY Jacob s Lake), two strains of Sporidiobolus salmonicolor (CCY river Little Donau, signed S. salmonicolor A; CCY soil, wood, signed S. salmonicolor B) and two strains of Cystofilobasidium capitatum (CCY river Donau, signed C. capitatum A; CCY soil, wood, signed C. capitatum B). All strains were obtained from the Czechoslovak Collection of Yeasts (CCY), Bratislava, Slovakia. Cultivation of microorganisms. All strains were grown on a medium containing (g): yeast autolysate 4, (NH 4 ) 2 SO 4 10, glucose 20, KH 2 PO 4 1, K 2 HPO 4 0.1, NaCl 0.1, CaCl 2 0.1, MgSO 4 0.5, and solution of microelements 0.25 ml (components per liter: H 3 BO g, CuSO 4 5 H 2 O 0.1 g, KI 0.25 g, MnSO 4 5 H 2 O 1 g, FeCl 3 6 H 2 O 0.5 g, (NH 4 ) 6 Mo 7 O 24 4 H 2 O 0.5 g, ZnSO 4 7 H 2 O 1 g). As exogenous stress factors: 10 mmol/l H 2 O 2 (induction of oxidative stress) and 10% NaCl (induction of osmotic stress), which were added into growth media. In addition, strains S. salmonicolor and C. capitatum were exposed to 5% NaCl. Stress factors were added: i) at the beginning of growth, and ii) to the exponentially growing cells. Extraction of carotenoids from yeast cells. Cells were collected by centrifugation (3000 rpm; 30 min). For subsequent isolation of carotenoids the whole biomass obtained from 250 ml of medium was used. Yeast cells were disintegrated using a mechanical disruption by shaking with glass beads ( U.S sieve). A mixture of pigments and other organic compounds was extracted from the cell homogenate using 50 ml of acetone. After saponification of the extract by ethanolic KOH carotenoids were extracted twice with 50 ml of diethyl ether. The diethyl ether extracts were collected and dried under vacuum. After evaporation, the residue was dissolved in 1-2 ml of absolute ethanol and further purified using solid phase extaction (Aparatus SPEC Manifold, Ansys Diagnostics USA, vacuum pump, Barnat Co., USA). Discs (SPEC.PLUS 3 ml; sorbent C18 AR) were conditioned by 5 ml of methanol. Sample (ethanolic cell extract; see above) was rinsed with 5 ml of ethanol. Carotenoids bound to the sorbent were then eluted twice with 5 ml of cyclohexane. Eluates were collected and evaporated under vacuum to dryness. The residue was dissolved in 0.5 ml of methanol (gradient grade) and used for HPLC chromatographic analysis. Analysis of carotenoids. Carotenoid pigments extracted from yeast cells were individually identified and quantified by RP-HPLC using a chromatographic system described previously (Marova et al., 1999). Samples (10 microliter valve) were filtered through PTFE filters and injected onto Nucleosil 100 C18 column, 7 mm, 150 x 4.6 mm with guard column 30 x 4.6 mm, 7 mm that had been equilibrated with a mobile phase (methanol/water; 95:5). Isocratic elution was carried out at 45 C by a flow rate of 1.0 ml/min. with expection of the R. glutinis strains. Visible detection of lycopene, a-carotene and b-carotene was achieved at 450 nm, while UV detection at 285 nm was used for detection of ergosterol. Data processing (integration) of analyses was assessed using a Ann. Microbiol., 54 (1), (2004) 75

4 CSW Integrator v.1.7. (Chromatographic Station Software, DataApex Co., Prague). Individual carotenoids as well as ergosterol were quantified using external standards. Statistical analysis. A dataset of experimental results was processed by correlation and principal component analysis (PCA) using software STATGRAPH- ICS ver (Manugistics, Rockwille 1992). The eigenvalues of the correlation matrix, corresponding component weights and principal components were used to describe mutual relationship among yeast strains, metabolites and/or stress factors studied. Relevant PCA data are available on request. RESULTS Production of biomass Since carotenoids are produced as secondary metabolites mainly during the stationary phase of growth, yeast cells were collected after 80 hours of cultivation. In optimal conditions all strains were grown at similar growth rate (see Table 1, control). Addition of stress factors into cultivation medium led to different changes of growth according to the yeast species, type of stress factor or growth phase, in which stress factors were added. Addition of H 2 O 2 caused in most strains substantially greater biomass production decrease than did the addition of NaCl. This decrease was lower, however, when H 2 O 2 was added during the exponential phase of growth (expect in the case of Sporidiobolus and R. aurantica; see Table 1). On the other hand, growth of all tested strains with expection of the R. glutinis in medium with 5-10% NaCl decreased only slightly. Production of carotenoids B-carotene was the major carotenoid in most of the tested strains followed by α- carotene. Producion of lycopene was substantially lower because of its function as an acyclic precursor of carotenes (see Tables 2-4). The highest production of β-carotene (similar to α-carotene) in optimal conditions was observed in strains of S. salmonicolor A and C. capitatum A; where the yield of β-carotene in these strains was more than 1 mg/ g of dry weight (Table 1). Although stress was induced by relatively high concentration of exogenous stress factors, some of strains exhibited a significant increase in carotenoid production (see Tables 2-4), such as R. glutinis B (on average of 1.15 mg/g d.w. of β-carotene, 2.5-fold increase of β-carotene after the addition of 10% NaCl on the beginning of growth), R. mucilaginosa (a 6-fold increase of β-carotene under salt stress) and S. salmonicolor A (on average of 1.88 mg of β-carotene/ g d.w. in the presence of salt). With the aim to comparise influence of oxidative and salt stress on the production of biomass, carotenoids and ergosterol in individual yeast strains, the experiemental data were processed by correlation and principal component analysis (PCA). According to results obtained by statistic analysis it could be concluded, that in most strains the effect of peroxide on the production of carotenoids and ergosterol (either induction or decrease) is very similar, if peroxide is added at the beginning of cultivation or during the exponential phase. 76 I. MAROVA et al.

5 TABLE 1 Growth of yeast strains under stress Condition Growth (D A 660 ; 80 hours cultivation) C. capitatum A C. capitatum B R. glutinis A R. glutinis B R. rubra R. aurantiaca R. minuta S. salmon. A S. salmon. B Control 0.88 ± ± ± ± ± ± ± ± ± 0.31 Salt 1 a 0.80 ± ± ± ± ± ± ± ± ± 0.32 Salt 2 b 0.43 ± ± ± ± ± ± ± ± ± 0.26 Peroxide 1 c 0.07 ± ± ± ± ± ± ± ± ± 0.13 Peroxide 2 d 0.58 ± ± ± ± ± ± ± ± ± 0.15 The data presented are mean ± standard deviation for significance level 0.05 of three independent assays. a Salt 1: addition of 10% NaCl at the beginning of growth; b salt 2: addition of NaCl to the exponentially growing cells (in the strains S. salmonicolor and C. capitatum 5% NaCl was used); c Peroxide 1: addition of 10 mm H2 O 2 at the beginning of growth; d peroxide 2: addition of H 2 O 2 to the exponentially growing cells. Ann. Microbiol., 54 (1), (2004) 77

6 TABLE 2 Production of beta-carotene Condition Beta-carotene (mg/g of dry weight) C. capitatum A C. capitatum B R. glutinis A R. glutinis B R. rubra R. aurantiaca R. minuta S. salmon. A S. salmon. B Control 1.15 ± ± ± ± ± ± ± ± ± 0.20 Salt 1 a 0.04 ± ± ± ± ± ± ± ± 0, ±0.01 Salt 2 b 0.33 ± ± ± ± ± ± ± ± ± 0.18 Peroxide 1 c 0.95 ± ± ± ± ± ± ± ± ± 0.03 Peroxide 2 d 0.68 ± ± ± ± ± ± ± ± ± 0.11 a, b, c, d : See Table I. MAROVA et al.

7 TABLE 3 Production of alpha-carotene Condition Alpha-carotene (mg/g of dry weight) C. capitatum A C. capitatum B R. glutinis A R. glutinis B R. rubra R. aurantiaca R. minuta S. salmon. A S. salmon. B Control 0.36 ± ± ± ± ± ± ± ± ± 0.04 Salt 1 a 0.02 ± ± ± ± ± ± ± ± ± 0.01 Salt 2 b 0.20 ± ± ± ± ± ± ± ± ± 0.10 Peroxide 1 c 0.27 ± ± ± ± ± ± ± ± ± 0.02 Peroxide 2 d 0.13 ± ± ± ± ± ± ± ± ± 0.05 a, b, c, d : See Table 1. Ann. Microbiol., 54 (1), (2004) 79

8 TABLE 4 Production of lycopene Condition Lycopene (mg/g of dry weight) C. capitatum A C. capitatum B R. glutinis A R. glutinis B R. rubra R. aurantiaca R. minuta S. salmon. AS. salmon. B Control 4.2 ± ± ± ± ± ± ± ± ± 0.2 Salt 1 a 0.2 ± ± ± ± ± ± ± ± ± 0.2 Salt 2 b 0.1 ± ± ± ± ± 0, ± ± ± ± 0.6 Peroxide 1 c 0.2 ± ± ± ± ± ± ± ± ± 0.2 Peroxide 2 d 1.4 ± ± ± ± ± ± ± ± ± 1.1 a, b, c, d : See Table I. MAROVA et al.

9 The effect of salt addition to exponentially growing cells was similar to peroxide. However, the salt stress induced by NaCl added at the beginning of cultivation led to substantially different response. In most of the strains studied a significant decrease of pigment as well as ergosterol production was observed in presence of NaCl in medium from the beginning of the growth. The statistical analysis of individual yeast species also revealed, that there are some representatives of the genus Rhodotorula (especially R.glutinis A, R. mucilaginosa and R. aurantiaca) able to produce increased amounts of carotenoids as well as ergosterol in presence of NaCl too. Salt stress is the major PCA component (more than 99 %), that influences pigment production in strains R. glutinis A and R. mucilaginosa. The other Rhodotorula strains exhibited similar increase of carotenoid and ergosterol production also after addition of peroxide during exponential phase. In both Cystofilobasidium strains peroxide was the major component, while in Sporidiobolus the comparable influence of salt and oxidative stress was found. In all strains strong positive correlation between α- carotene and β-carotene production was observed under all conditions tested. Moreover, in all yeast strains the amount of both carotenes correlated with lycopene production under salt and/or oxidative stress induced at the beginning of growth. In all strains grown under salt stress ergosterol production correlated with production of β- carotene, α-carotene and lycopene. Addition of exogenous stress factors led to different responses both between individual strains of the same species as well as between species of the same genus. Responses differed according to type of stress factor and the growth phase during which they were added. In both Cystofilobasidia addition of peroxide led to significant decrease of biomass and pigment production. Addition of NaCl led to a slight increase of β- carotene production in strain B only. According to these findings the yeasts of genus Cystofilobasidium could not be recomended for biotechnological purpose. In all strains of the genus Rhodotorula addition of peroxide led to similar decrease of biomass production, but production of all carotenoids as well as ergosterol significantly increased. In the strain R. glutinis A higher production of carotenoids was induced also by salt stress. This strain exhibited quite high tolerance to exogenous stress combined by increased production of pigments and ergosterol. Thus, it could be acceptable as the candidate strain for potential biotechnological application. On the other side, the strain R. glutinis B exhibited under most of the stress conditions a lower production of β-carotene. Such different response of two strains of the same genus is probably connected with adaptation mechanisms conserved in individual strains dependent on their original environment. The strain A was isolated from the river Donau, where it probably needed more efficient protective mechanisms against UV irradiation than the strain B isolated from a soil. Yeast R. mucilaginosa exhibited similar response to exogenous salt stress as R. glutinis. Biomass poduction in presence of NaCl was not changed, moreover, production of β-carotene and ergosterol increased about twicely. As the limited factor for potential biotechnological use of this strain rather low total yield of pigments and ergosterol could be considered. The strain R. aurantiaca exhibited quite high tolerance to exogenous stress Ann. Microbiol., 54 (1), (2004) 81

10 in biomass production, but amount of carotenoids increased in presence of stress factors only slightly. Stress response of the yeast R. minuta differed from all the other Rhodotorula strains. In presence of stress factors production of all carotenoids as well as ergosterol significantly decreased. Both Sporidiobolus strains exhibited under oxidative stress certain biomass production decrease, the yield is comparable to the strain R. glutinis A. In the strain S. salmonicolor A quite high amount of β-carotene and ergosterol was obtained in optimal conditions. Addition of NaCl to the exponentially growing cells led to the highest yield of β-carotene (1.88 mg per g of d.w.) as well as ergosterol (1.80 mg per g of d.w.) and biomass production kept unchanged. Thus, this strain could be very promissing for biotechnological production of β- carotene as well as of enriched biomass. Similarly to R. glutinis, the strain S. salmonicolor B exhibited substantially different behaviour under stress conditions. Addition of stress factors led in any case to a decrease of β-carotene production and the yield of the other pigments and egosterol was lower too. This difference is probably caused by the same environmental factor as in R. glutinis the strain S. salmonicolor A was isolated from the river Little Donau, while the strain B was gained from soil. Thus, by choice of yeast strain for potential biotechnological production modulated by exogenous stress factors the individual physiological characteristics of each strain should be also noticed. Table 5 summarizes amounts of ergosterol produced by individual yeast strains. In most strains addition of peroxide at the beginning of growth and/or to the exponentially growing cells as well as addition of salt during the exponential phase led to about 2 fold increase of ergosterol production when compared with the control cultivation. The highest yield of ergosterol was obtained in strain S. salmonicolor A stressed by peroxide (1.44 mg per g of d.w.) and salt (1.80 mg per g of d.w., see Table 5). In this strain also the highest increase of ergosterol production in conditions of oxidative and osmotic stress (4.6 times and 5.8 times, respectively) was followed. DISCUSSION It has been estimated that more than 100 million tons of a great variety of natural carotenoids per year are produced. Still it is very difficult to discover not only how the basic carotenoid skeleton is constructed, but also how the great diversity of structural variations is elaborated in plants, algae, fungi and bacteria (Sandmann et al., 1999). Carotenoids are used as colorants, either by direct addition to manufactured foods or by inclusion in the feed of animals, which are then used as food. To date, the commercial demand of carotenoids is mainly met by chemical synthesis and to a minor extent by extraction from natural sources. However, the supply is restricted to a few carotenoids, mainly astaxanthin and β-carotene. Because natural sources contain the whole group of physiologically significant carotenoids, attention is now being focused on the natural production of carotenoids by large-scale cultivation of appropriate plants and algae, or by microbial biotechnology (Sandmann et al., 1999). Factors that influence the efficiency of natural carotenoid biosynthesis, or 82 I. MAROVA et al.

11 TABLE 5 Production of ergosterol Condition Ergosterol (mg/g of dry weight) C. capitatum A C. capitatum B R. glutinis A R. glutinis B R. rubra R. aurantiaca R. minuta S. salmon. A S. salmon. B Control 0.26 ± ± ± ± ± ± ± ± ± 0.09 Salt 1 a 0.12 ± ± ± ± ± ± ± ± ± 0.17 Salt 2 b 0.62 ± ± ± ± ± ± ± ± ± 0.10 Peroxide 1 c 0.57 ± ± ± ± ± ± ± ± ± 0.32 Peroxide 2 d 0.13 ± ± ± ± ± ± ± ± ± 0.07 a, b, c, d : See Table 1. Ann. Microbiol., 54 (1), (2004) 83

12 that determine which carotenoids are accumulated, are therefore important for these commercial applications. The chemical and physical properties of carotenoids in vivo may be modified by interactions with other molecules in the microenvironment and may be significantly different from those of free carotenoids in organic solution. These interactions with other molecules, especially proteins, may be critical to the function or actions of the carotenoid molecule in vivo. Carotenoids are involved in stress responses of microorganisms. Thus, their production could be substantially influenced by several exogenous stress factors. Ability of red yeasts to adapt by means of overproduction of industrially significant metabolites could be of increasing interest. Study of the molecular basis of these effects could make it possible to realize regulated overproduction of carotenoids above all in such industrial applications, when it is not acceptable to use genetically modified strains. In this study all strains R. glutinis and S. salmonicolor grown under salt stress showed the highest production of complex biomass. As the of more complex carotenoids become known pharmaceutical potential, fermentation processes using modified microorganisms will possibly be indispensable in their production. Moreover, biotechnological use of yeasts takes advantage of utilization the whole biomass, efficiently enriched not only for carotenoids, but also for other significant components (e.g. ergosterol). According to our results as well as to those regarding production of carotenoids, it could be possible that some of tested strains could be used for industrial production of enriched biomass and/or β-carotene. Production of these significant metabolites can be effectively controled by exogenous stress factors. The application of stress factors can result in a diversity of carotenoid structures that cannot be synthetized by conventional chemical methods. Presently, only a small number of carotenoids can be obtained as pure compounds in significant amounts in order to study their biological functions. An evaluation of their antioxidant properties, including protection against singlet oxygen or radicals, as well as their potential as anticancer agents will follow. For their large-scale production, further research is necessary, which will also require a more-detailed understanding of growth parameters in fermenters, optimization of the extraction and purification processes, parallelly to experiments focused on construction of vectors for production of many carotenoids in transgenic organisms (Armstrong and Hearst. 1996). Acknowledgements This work was supported by project Nr. 96/ (Kontakt: Czech-Slovak Bilateral Cooperation) of Czech Ministry of Education. REFERENCES Armstrong G.A., Hearst J.E. (1996). Genetics and molecular biology of carotenoid pigment biosynthesis. FASEB J., 10: Britton G., Liaaen-Jensen S., Pfander H. (1995). Carotenoids today and challenges for the future. In: Jensen S., Pfander H., Carotenoids. Vol. 1A: Isolation and Analysis. Birkhäuser Verlag Basel, pp Goodwin T.W., Britton G.(1988). Distribution and analysis of carotenoids. In: Goodwin T.W., Plant Pigments. Academic Press, London, pp I. MAROVA et al.

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