Effect of oxidative stress and exogenous β-carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95

Authors


Jian-Rong Han, School of Life Science and Technology, Shanxi University, Taiyuan 030006, China (e-mail: hjr@sxu.edu.cn).

Abstract

Aims:  To determine the effect of oxidative stress and exogenous β-carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95.

Methods and Results:  In this experiment, high oxidative stress was applied by inclusion of FeCl3 (10 μmol l−1) in the growth medium and by light exposure. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous β-carotene (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 72 h), decrease of sclerotial biomass (up to 43%) and reduction of carotenoid yield (up to 92%). On the contrary, the exogenous β-carotene also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus.

Conclusions:  Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 141 mg and 30·03 μg, which were 1·53 and 3·51 times higher respectively, than that at low oxidative stress growth condition.

Significance and Impact of the Study:  These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants.

Introduction

Sclerotia are compact hyphal structures produced by some fungi that belong to Ascomycetes, Basidiomycetes and Deuteromycetes. They play a vital role in the survival of these organisms for long periods in unfavourable conditions because of their high resistance to chemical and biological degradation. Because of the biological and agricultural significance of the sclerotiogenic fungi, many efforts have been directed towards elucidating the mechanism of sclerotial biogenesis. It was shown that in Sclerotium rolfsii, sclerotial differentiation is accompanied by a high degree of lipid peroxidation, suggesting for the first time a possible relationship between sclerotial differentiation and reactive oxygen species (ROS) such as hydroxyl radicals, alkoxyl and alkoperoxyl radicals and singlet oxygen (Georgiou 1997). Based on these data and considering that ROS can act as signal transducers (Lander 1997), and also that ROS-generated lipid peroxides and their aldehydic decomposition products can affect cell proliferation and differentiation (Esterbauer et al. 1991), Georgiou (1997) had advanced a theory proposing that sclerotial differentiation in fungi is triggered by oxidative stress. Additional evidence was presented to support this theory by showing that certain antioxidants inhibited sclerotial differentiation in S. rolfsii, Sclerotinia sclerotiorum, Sclerotinia minor and Rhizoctonia solani (Georgiou et al. 2001a,b).

The antioxidant mechanisms in organisms are generally manifested as a concerted interplay between antioxidant molecules and enzymes. Carotenoids are antioxidants as they are known to reduce oxidative stress by acting as scavengers of ROS (mainly singlet oxygen) (Stratton and Liebler 1997). The β-carotene and other carotenoids have been associated with fungal photomorphogenesis and development (Mohr and Schopfer 1995). Georgiou et al. (2001a,b) had shown that S. sclerotiorum and S. rolfsii produce β-carotene in response to light-induced oxidative stress and that exogenous β-carotene decreases their differentiation.

To date, almost all the industrial production of carotenoids using filamentous fungi and yeasts has involved the use of liquid-state fermentation (Vandamme 1992). The use of solid-state fermentation (SSF) for carotenoid production is still in the exploratory stage (Han 1998). Many well-known carotenoid-accumulating micro-organisms such as Blakeslea trispora, Aspergillus giganteus and Phaffia rhodozyma are not suitable for SSF because of the difficulty in separating their mycelia or cells from the fermented substrate, making it difficult to collect mycelia or cells for pigment extraction. We isolated a strain of Penicillium sp. PT95 from a soil sample. This strain can form abundant orange, sand-shaped sclerotia (c. 300 μm in diameter) in which carotenoid (mainly β-carotene) is accumulated. Because the sclerotia can be separated easily from their solid medium, strain PT95 is suitable for SSF for carotenoid production (Han 1998). Accordingly, SSF of PT95 is a promising alternative for the efficient production of carotenoid. However, the pigment yield from this strain was lower when compared with other fungi that are well known for their high carotenogenic power (Han and Yuan 2003). In order to meet the needs of commercial production, both sclerotial biomass and pigment content of strain PT95 must be increased still further.

The question arose whether the use of high oxidative stress growth condition and supplementation of exogenous β-carotene into medium would result in a significant increase (or decrease) in both the sclerotial biomass and carotenoid yield of strain PT95.The aim of this study was to determine how high and low oxidative stress growth conditions affect carotenoid biosynthesis and sclerotial differentiation of strain PT95. In addition, the effects of exogenous β-carotene on sclerotial biomass, carotenoid yield and lipid peroxidation of this fungus were examined.

Materials and methods

Chemicals

β-Carotene was obtained from Merck (Darmstadt, Germany). Trichloroacetic acid (TCA), tetrahydrofuran (THF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA) and Coomassie Brilliant Blue G250 (CBB-G250) were supplied from Sigma (St Louis, MO, USA). Other chemicals were of analytical grade and purchased from domestic companies.

Preparation of inocula

The strain PT95 was isolated from soil, collected close to Fenyang, Shanxi Province, and identified as a species of the Penicillium thomii series described by Raper and Thom (1949). See Han et al. (1998) for additional details and a complete bibliography. Strain PT95 was cultured on Czapek's agar plate in a dark incubator at 25°C. The sclerotia as inocula were obtained from 14-day-old Czapek's agar plate cultures of the strain. To purify sclerotia, the plate cultures were centrifuged, rinsed aseptically five times with sterilized water to remove mass spores.

Growth conditions

Strain PT95 was grown in 9-cm dishes on cellophane membrane discs placed on top of 25-ml agar medium (Georgiou et al. 2001a), consisting of NH4NO3, 15 mmol l−1; NaCl, 2 mmol l−1; MgSO4, 0·5 mmol l−1; FeCl3, 10 μmol l−1; MnCl2, 15 μmol l−1; ZnCl2, 70 μmol l−1; thiamine, 1 μmol l−1; glucose, 0·1 mol l−1; yeast extract, 0·1% (w/v) and agar, 1·5% (w/v) in 10 mmol l−1 potassium phosphate buffer (pH 7·0). Basal growth medium and glucose (10 X stock solution) were sterilized separately to avoid formation of oxidizing Maillard reaction products. Cellophane membranes were boiled in EDTA (10 mmol l−1) and washed four times in deionized-distilled water before sterilization. The medium constituents and the membrane were sterilized at 121°C for 20 min.

A grain of sclerotium was inoculated at the centre of Petri dish. The fungus was grown in single dish layers in an incubator at 25°C at high, middle and low oxidative stress. In this experiment, high oxidative stress was applied by inclusion of FeCl3 (10 μmol l−1) in the growth medium and by light exposure (12-h period of light alternating with 12-h period dark), with light intensity 40 μE m−2 s−1 provided by fluorescence lamps (Philips TLD 36W/965, 400–800 nm emission range; Philips Electronics, Zhuhai, China). Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Middle oxidative stress was applied by light minus iron or by dark plus iron.

Effect of exogenous β-carotene

The role of exogenous β-carotene on PT95 strain differentiation at high oxidative stress was studied following a procedure reported by Georgiou et al. (2001b) with minor modifications. The growth medium was supplemented with various concentrations of β-carotene up to 8 μmol l−1 (in 0·2% THF and 1 μmol l−1 BHT final concentration). Appropriate volume of β-carotene/THF solution was filter-sterilized (with a cellulose acetate syringe filter, 25 mm in diameter and 0·2 μm in pore size) and was mixed with 25-ml growth medium. The resulting mixture was poured into 9-cm Petri dish. A cellophane membrane disc was placed on the surface of agar medium. Then, a grain of sclerotium was inoculated on the centre of plate. All the plates were incubated at 25°C at high oxidative stress.

Lipid peroxidation and protein assays

We determined the antioxidant effect of 2 and 6 μmol l−1 exogenous β-carotene on lipid peroxidation of a 4-day-old undifferentiated colony and an 8-day-old differentiated colony. As a control, we measured lipid peroxidation in same-day colonies of the fungus grown in growth medium without supplementation of β-carotene. Fungal samples removed from the cellophane membrane by a pair of tweezers were ground in a porcelain mortar in liquid nitrogen to prevent artificial lipid peroxidation. The resulting powder was mixed with 100 μmol l−1 EDTA solution at a ratio of 1 : 6 (fungal fresh weight : EDTA solution volume) and further homogenized on ice. The homogenate was centrifuged at 25 000 g for 30 min and the EDTA-supernatant was assayed for lipid peroxidation by a modification of the TBA method (Castilho et al. 1995). For the assay, 0·5-ml EDTA-supernatant was mixed with 0·5 ml TBA reagent (0·67% w/v TBA dissolved in 5% w/v TCA). To the resulting mixture was added 5 μl 2% BHA (w/v, dissolved in absolute ethanol). BHA was used as lipid antioxidant to prevent artificial lipid peroxidation during the assay. The resulting mixture was incubated at 100°C for 20 min and was centrifuged at 15 000 g for 3 min. Absorbance of the supernatant was measured at 535 and 600 nm against sample blank (0·5 ml sample mixed with 0·5 ml 5% w/v TCA and treated as above). The absorbance difference A535−600nm was converted to micromoles of malondialdehyde (MDA) equivalents using extinction coefficient for MDA 1·55 × 105 mol−1 cm−1. Lipid peroxidation was expressed in micromoles MDA mg−1 of protein.

Protein concentration was determined by a modification of a CBB-based method (Sedmak and Grossberg 1977). Briefly, 0·1 ml EDTA-supernatant was mixed with 0·9 ml 0·033% (w/v) CCB-G250 (dissolved in 0·5 mol l−1 HCI), and after 5 min of incubation at room temperature its absorbance was measured at 620 nm and converted to milligram protein from a BSA standard curve (0–50 μg).

Carotenoid extraction and determination

The sclerotia in different developmental stages grown on the agar surfaces in Petri plates were separated and washed thoroughly with distilled water and lyophilized. The lyophilites were weighed to determine dry sclerotia weight before carotenoid analysis. The extraction and determination of pigments were performed as a modified procedure described by An et al. (1989). One gram of the dry sclerotia was manually ground with a glass homogenizer and extracted three times with 10-ml aliquots of acetone. The combined acetone extracts were combined in a separatory funnel, and c. 10 ml of chloroform and few milliliters of a solution of saturated NaCl to help break emulsions. The chloroform extract was collected after removal of the acetone layer, which was reextracted. Absorbance of the chloroform extract was measured at 475 nm. The content of carotenoid was calculated by using the 1% extinction coefficient = 2500 by the formula:

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Experimentation and analysis

All experiments were replicated in three plates and the data are presented as the arithmetic mean ± standard error. Duncan's multiple range test (Ray 1985) was used to determine significant differences among mean values at the 1% level of confidence.

Results

Effect of oxidative stress on sclerotial biomass and carotenoid yield

Iron and light are well known contributors of oxidative stress. We determined the biomass of matured sclerotia (14-day-old) and the content of carotenoid in sclerotia of PT95 strain under high, middle and low oxidative stress (Table 1). Both the sclerotial biomass and the amount of carotenoid accumulated in sclerotia of this strain were strongly dependent on oxidative growth conditions. When the fungus was grown at high oxidative stress, its sclerotial biomass (i.e. dry sclerotia weight) increased by 1·53-fold with respect to low oxidative stress. Carotenoid content in sclerotia under high oxidative stress increased by 2·29-fold with respect to sclerotia formed under low oxidative stress. As the total amount of caroteniod in each plate culture was affected by both the sclerotial biomass and the content of carotenoid in sclerotia, we tested the effect of oxidative stress on carotenoid yield. The growth condition of high oxidative stress caused a statistically significant increase (by 3·51-fold) in carotenoid yield as compared with the growth condition of low oxidative stress. Middle oxidative stress growth condition, regardless of whether the oxidative stress originated from light or iron, also caused a significant increase in both sclerotial biomass and carotenoid content as compared with the growth condition of low oxidative stress (P < 0·05). However, single factor effect of light was found higher than that of iron (P < 0·05).

Table 1.  Sclerotial biomass and carotenoid yield of Penicillium sp. PT95 grown under oxidative growth conditions
Oxidative growth conditionsSclerotial biomass (mg per plate)Content of carotenoid (μg g−1 of dry sclerotia)Carotenoid yield (μg per plate)
  1. *Light intensity was 40 μE m−2 s−1 provided by fluorescence lamps (Philips TLD 36W/965, 400–800 nm emission range). Iron means supplementation of 10 μmol l−1 FeCI3 in the growth medium.

  2. Data are mean values from three independent experiments ± standard errors.

  3. Mean values in the same column followed by different letters are significantly different at the P < 0·05 level according to Duncan's multiple range test.

Light plus iron*141 ± 12 d213 ± 17 d30·03 ± 0·20 d
Light minus iron130 ± 11 c189 ± 15 c24·57 ± 0·16 c
Dark plus iron118 ± 10 b126 ± 11 b14·87 ± 0·11 b
Dark minus iron92 ± 8 a93 ± 9 a8·56 ± 0·07 a

One conclusion from these results was that light and iron was more favourable to sclerotial differentiation and carotenogenesis of PT95 than dark. Light was a stronger stress factor than iron. The interaction of light and iron achieved a better degree of oxidative stress than any either factor alone.

Carotenoid production by this fungus is not solely dependent on light as it is also produced in the dark, although in lower levels. Furthermore, β-carotene is known to act as antioxidant by scavenging ROS (mainly singlet oxygen), which can cause lipid peroxidation and protein inactivation via protein peroxidation (Palozza and Krinsky 1992). The ROS can be generated from light-sensitization reactions as well as from light-insensitive reactions (i.e. lipid peroxidation reactions) and other oxidative stress growth conditions (Palozza et al. 1996). Therefore, ROS can form in PT95 strain in dark as well, although to a lesser degree, especially when the fungus uses up its nutrients. The ROS, then may induce PT95 strain antioxidant defenses by producing carotenoid (mainly β-carotene) in concentration levels related to the degree of oxidative stress.

Effect of exogenous β-carotene on lipid peroxidation and sclerotial differentiation

Georgiou (1997) had advanced a hypothesis that fungi survive in nature under adverse (high ROS causing) conditions by differentiating (forming sclerotia) as a result of their inability to reduce oxidative stress. This theory predicts that antioxidants are expected to decrease sclerotial differentiation by lowering oxidative stress. As β-carotene can be incorporated into eucaryotic cells in culture and can lower oxidative stress by its lipid antioxidant action (Palozza et al. 1996), we studied its effect on lipid peroxidation and on sclerotial differentiation of PT95 strain at high oxidative growth condition.

The effect of exogenous β-carotene on sclerotial differentiation of PT95 strain was concentration-dependent, resulting in a gradual decrease in the biomass of produced matured sclerotia (14-day-old) per plate as the concentration of β-carotene in the growth medium up to 8 μmol l−1 (Table 2). At the maximum tested concentration of β-carotene, the sclerotial biomass decreased by 43%, as compared with the sclerotial biomass formed per plate in the absence of β-carotene in the growth medium. Similar results were obtained at low oxidative stress (data not given). Additionally, β-carotene delayed sclerotial differentiation in a concentration-dependent manner (Table 2). At β-carotene concentrations up to 8 μmol l−1 there was a gradual delay of differentiation, reaching 72 h. However, the time of sclerotial maturation was not changed.

Table 2.  Effect of exogenous β-carotene on sclerotial differentiation of Penicillium sp. PT95
Concentrations of exogenous β-carotene (μmol l−1)Time of sclerotial initial (d)Time of sclerotial maturation (d)Sclerotial biomass (mg per plate)
  1. *Concentration-point zero means the absence of exogenous β-carotene in medium and serves as the control.

  2. Data are mean values from three independent experiments ± standard errors.

  3. Mean values in the same column followed by different letters are significantly different at the P < 0·05 level according to Duncan's multiple range test.

2614120 ± 10 d
4614105 ± 9 c
6714 93 ± 8 b
8814 81 ± 6 a
0*514141 ± 12 e

Lipid peroxidation is an important indicator of oxidative stress and is accompanied by the formation of aldehydic lipid hydroperoxide decomposition products such as MDA (Esterbauer et al. 1991). The MDA formation is considered evidence that free radical-mediated reactions have taken place and its concentration is used as a measure of the degree of this stress (Yazdanpanah et al. 1997). Therefore, we tested the effect of exogenous β-carotene on lipid peroxidation in an undifferentiated (4-day-old) and a differentiated (8-day-old) colony grown at high oxidative stress (Fig. 1). The medium without supplementation of β-carotene served as the control. Exogenous β-carotene at 2 and 6 μmol l−1 decreased lipid peroxidation levels in the undifferentiated colony to 83 and 50% (of the control) respectively. Furthermore, exogenous β-carotene decreased lipid peroxidation levels of the differentiated colony also, to a greater degree (83 and 42% respectively). This result showed that the effect of exogenous β-carotene on lipid peroxidation of PT95 strain was also concentration dependent.

Figure 1.

Effect of exogenous β-carotene on lipid peroxidation of 4-day-old (bsl00000) and 8-day-old (bsl00001) colonies of Penicillium sp. PT95. Concentration-point zero means the absence of exogenous β-carotene and serves as the control. Columns represent mean values (n = 3) and bars represent standard errors

Effect of exogenous β-carotene on content of carotenoid accumulated in sclerotia

The content of carotenoid accumulated in matured sclerotia (14-day-old) was strongly dependant on the amount of exogenous β-carotene added to the medium (Fig. 2). The 0 μmol l−1 means the absence of exogenous β-carotene in medium and serves as the control. Figure 2 showed that the content of carotenoid accumulated in sclerotia responded to the treatment with exogenous β-carotene in a dose-dependent manner. When plotting the carotenoid content versus the concentration of exogenous β-carotene in medium, a negative linear relation (r = −0·9905) was found. The higher the concentration was, the lower the carotenoid content. Exogenous β-carotene at 2, 4, 6 and 8 μmol l−1 decreased the content of carotenoid accumulated in sclerotia to 85, 49, 31 and 14% of the control respectively. Consequently, the carotenoid yield in each plate culture was also decreased to 72, 37, 20 and 8% of the control respectively.

Figure 2.

Effect of exogenous β-carotene on content of carotenoid accumulated in sclerotia of Penicillium sp. PT95. Concentration-point zero means the absence of exogenous β-carotene and serves as the control. Columns represent mean values (n = 3) and bars represent standard errors

Discussion

According to Georgiou's theory, antioxidants are expected to decrease sclerotial differentiation by lowering oxidative stress. The data of this study are in agreement with the criteria that establish β-carotene's antioxidant role (Halliwell 1997): its effect on degree of differentiation and on lipid peroxidation was concentration-dependent. It is worth noting that exogenous β-carotene partially inhibits sclerotial differentiation of PT95 strain, possibly by lowering the levels of some of the elements of oxidative stress (possibly singlet oxygen). Georgiou et al. had observed similar differentiation-inhibiting effects by β-carotene on S. sclerotiorum and S. rolfsii differentiation (Georgiou et al. 2001a,b), as well as by various noncarotenoid antioxidants on S. minor and on other sclerotia-producing fungi.

The lipid peroxidation- and differentiation-inhibiting effect of exogenous β-carotene on PT95 strain may suggest the following possible conclusions. As the fungus uses up its carbon source, it may fail to administer effectively its antioxidant defense mechanisms during the initial stages of its development, and it differentiates forming sclerotia for long-term survival. Carotenoid (mainly β-carotene) may be produced by PT95 strain to counter ROS formation, although via its antioxidant action, and possibly to protect from oxidative stress the mycelium as well as the cortex and the medulla of its sclerotium. Carotenoid production during differentiation seems to be the consequence of oxidative stress development during this developmental stage, and its antioxidant protection seems to be only partial, resulting in partial inhibition of differentiation. This is indicated by the less than 100% decrease of sclerotial biomass and lipid peroxidation, even at high levels of exogenously administered β-carotene. The data of this study support Georgiou's theory that sclerotial differentiation in fungi is induced by oxidative stress, and they are also in accordance with the general theory of microbial differentiation, which also postulates that this phenomenon is induced by oxidative stress (Hansberg and Aguirre 1990).

The results from the experiments mentioned above indicate that growth condition of high oxidative stress caused a statistically significant increase (by 3·51-fold) in carotenoid yield as compared with the growth condition of low oxidative stress and, hence, has profound commercial significance. These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants.

Acknowledgements

Support for this research by the Chinese National Natural Science Fund (grant no. 30070021) and the Shanxi Province Science Foundation (no. 20041076) is gratefully acknowledged.

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