Appl Biochem Biotechnol DOI 10.1007/s12010-015-1510-9
Improvement of Zinc Bioaccumulation and Biomass Yield in the Mycelia and Fruiting Bodies of Pleurotus florida Cultured on Liquid Media Nasser Poursaeid & Abas Azadbakht & Gholam Reza Balali
Received: 25 August 2014 / Accepted: 21 January 2015 # Springer Science+Business Media New York 2015
Abstract The effect of different concentrations of zinc on the bioaccumulation of zinc and biomass yield in both mycelium and fruiting body of Pleurotus florida cultivated in liquid medium was studied. The results showed that the optimum yield of mycelia (11.33±0.44 g/L) and fruiting bodies (7.70±0.19 g/L) dry biomass was obtained in a liquid medium containing 100 mg/L of zinc. At a zinc concentration of 200 mg/L, the highest concentration of zinc in the mycelia and fruiting bodies reached 1.869±0.115 and 0.151±0.008 mg/g dry weight, respectively. The addition of zinc to the culture media significantly reduced zinc bioaccumulation factor in mycelia (from 24.64±0.52 to 3.35±0.24) and fruiting bodies (from 36.71±0.30 to 0.49±0.02) dry weight. Our findings indicated that the ability of zinc bioaccumulation in the mycelia is much higher than in the fruiting bodies. The fundamental information obtained in this study will be useful for the improvement of zinc bioaccumulation and biomass yield in mycelia and fruiting bodies of P. florida cultivated in liquid media to obtain maximum zincenriched biomass. Keywords Pleurotus florida . Mycelium . Fruiting body . Zinc bioaccumulation . Biomass yield . Liquid culture medium
N. Poursaeid (*) Technology Incubator Center of Medicinal Plants, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan 81595-158, Iran e-mail: [email protected] A. Azadbakht Deputy of Food and Drug, Isfahan University of Medical Sciences, Isfahan 81745-313, Iran e-mail: [email protected] G. R. Balali Department of Biology, University of Isfahan, Isfahan 81746-73441, Iran e-mail: [email protected]
Appl Biochem Biotechnol
Introduction The importance of micronutrients in living organism nutrition is undisputable, and among them, zinc is an essential trace element for the growth of plants and mushrooms and for physiological processes in humans and animals. Most biochemical roles of zinc reflect its involvement in a large number of enzymes or as a stabilizer of the molecular structure of subcellular constituents and membranes [1]. Zinc is required for proteins, carbohydrates, mucopolysaccharides, lipids, and nucleic acids metabolism [1]. It is necessary for cell division, growth, and repair; for example, zinc is required for the activity of both DNA and RNA polymerase. Zinc also serves as a ligand, binding to and stabilizing various compounds [1]. It plays an important biological role in insulin resistance, antioxidant properties, hepatic injury, and sperm physiology [2]. Zinc has critical effects in homeostasis, immune function, oxidative stress, apoptosis, and aging, and significant disorders of great public health interest are associated with zinc deficiency [3–5]. Edible mushrooms are beneficial for health because of their functional food character, i.e., they have a high nutritional value and contain substances that help in prevention, treatment, and recovery from illness [6–8]. Moreover, they may present an excellent dietary source of some microelements because of their ability to absorb them from medium [9]. In recent years, several studies have tried to improve on the production of functional ingredients, such as polysaccharides [10] and certain essential minerals [11–13] in yeast strains and mushrooms by supplementing certain materials to cultures. The purpose of some of these studies is to obtaining organisms enriched with essential minerals, like selenium [14, 15], copper, and zinc [16–18] for production of natural nutritional supplements. An essential step for successful commercial cultivation of mushrooms is to determine the nutritional factors that are necessary for better mycelium growth, fruiting body formation, and the enzymes involved in the biological processes. Fruiting body induction is influenced by different factors, including the genetic makeup of the strain, environmental parameters, and the nutrition of the growth medium [19]. Mushrooms require one or more minerals for vegetative growth and fruiting body development [20]. The number of studies has showed that trace elements such as zinc in the submerged culture media had significant effects on the cell growth of Pleurotus tuber-regium [21], Ganoderma lucidum [10], Grifola frondosa [22], Lentinula edodes [23], and Cordyceps sinensis [24]. Trace elements at tolerable concentrations are either part of the active site or act as an activity modulator of ligninolytic enzymes of white rot mushrooms [25]. Baldrian et al. [26] demonstrated that the activity of laccase, endo-1,4-βglucanase, and 1,4-β-glucosidase enzymes of Pleurotus ostreatus was increased in the presence of zinc and copper. It was also reported that zinc, copper, selenium, iron, and chromium in culture medium exerted potent effects on the composition of the mushroom cell wall and semipermeable membrane and on the content of polyphenolics and polysaccharides that are involved in antioxidant, antitumor, immunomodulating, and insulin-resistant activities [27–32]. Studies have shown that the ability of adsorption and bioaccumulation of minerals, especially heavy metals in mushrooms, is considerably higher than those in agricultural crop plants, vegetables, and fruits. They tend to bioaccumulate more molecules because of their mycelia [33]. Copper and zinc contents in 28 species of edible mushrooms were studied, and it was concluded that their concentrations are species-dependent: some are bioaccumulators (i.e., Agaricus spp.), whereas others are bioexcluders [34]. Zinc bioaccumulation in Agaricus blazei (=Agaricus brasiliensis) [18] and G. frondosa [16] mycelia was recently studied in the presence of different zinc contents in liquid culture media. In the presence of 400 mg/L zinc, the mycelia accumulated 163 and 103 times the basal content of zinc, respectively. It was
Appl Biochem Biotechnol
demonstrated that G. lucidum mycelia, cultivated in a liquid medium with nonmycotoxic concentrations of 25 and 50 mg/L zinc, could bioaccumulate these metal [17]. Other studies have documented the maximum zinc bioaccumulation of 5.27 [35], 1.72 [36], and 4.97 mg/g in dry yeast (Saccharomyces cerevisiae) strains [37]. In recent years, the number of essential minerals bioaccumulation in mycelia (cultivated in submerged media) and/or fruiting bodies (cultivated in solid media) of mushrooms has been investigated [9, 13, 38, 39], while the bioaccumulation of essential minerals such as zinc in both mycelium and fruiting body cultivated in liquid media has not been reported yet up to now. This is the first report about the evaluation of different zinc concentrations on the absorption and bioaccumulation of zinc, mycelium growth, and fruiting body production in an edible-medicinal mushroom cultivated in liquid medium. The mushroom used in this study, Pleurotus florida, is well known as an edible and medicinal mushroom (some have important medicinal properties: reduction of blood sugar and cholesterol levels, antitumor, anticancer, antioxidant activity, and immunomodulating activity) [8, 20, 40, 41]. The aim of this research is the enhancement of zinc bioaccumulation and biomass yield in both mycelia and fruiting bodies of P. florida cultivated in liquid media to obtain maximum zinc-enriched biomass.
Materials and Methods Mushroom Strain P. florida edible-medicinal mushroom was obtained from the Mushroom Culture Collection of Mycological Institute, Fujian Agriculture and Forestry University (FAFU), China. Seed Culture Preparation The seed culture was grown in 500 mL Erlenmeyer flasks containing 200 mL of basal medium (glucose 50.0 g/L, yeast extract 10.0 g/L, casein hydrolysate 10.0 g/L, KH2PO4 1.0 g/L (Merck, Germany), distilled water 1 L, and pH 6.0) at 25 °C, on a rotary shaker, at 110 rev/ min for 7 days. The obtained seed culture was used for inoculation [13, 15]. Preparation of Basal Liquid Media Containing Zinc The basal liquid medium used by Turlo et al. [13, 15], which has been reported to promote good mycelia growth, was modified in this study. The composition of the basal liquid medium for mycelium and fruiting body cultivation was beet molasses 50 g/L (beet-sugar factory, Isfahan, Iran), soybean flour 5 g/L (IPP company, Isfahan, Iran), KH2PO4 1 g/L, MgSO4 ·7H2O 1 g/L (Merck, Germany), and distilled water 1 L with initial pH 6.0. The liquid media was enriched with zinc by the addition of different concentrations of zinc sulfate (ZnSO4) (Merck, Germany). The concentrations of zinc were in the range 0 to 300 mg/L (0, 50, 100, 150, 200, and 300 mg/ L). All liquid culture media were sterilized by autoclaving at 121 °C for 20 min. After cooling, cultivation had been carried out in polypropylene containers (12 cm diameter in×7 cm high) containing 100 mL of liquid medium and inoculated with 5 % (v/v) of the seed culture. The Mycelium Growth and Fruiting Body Formation Conditions The polypropylene containers were incubated at 25 °C under stationary conditions until total colonization with the P. florida mycelium. After incubation at 25 °C in the dark for 15 days, all
Appl Biochem Biotechnol
polypropylene containers containing liquid culture medium were further incubated at 20±2 °C under illumination with LED lamps at about 500 lx/day, and relative humidity was maintained at 90–95 % during the initiation of primordial and at 80–90 % during fruiting body formation (Fig. 1). In addition, the plastic netting as a physical support for fruiting body formation was used [42, 43]. Determination of Mycelium and Fruiting Body Biomass (Fresh and Dry Weight) The fresh and dry mycelium and fruiting body biomass were determined at 10 and 25–30 days after inoculation to analyze the mycelia growth and fruiting bodies production, respectively. The mycelium was separated from the liquid medium by filtration under vacuum (using a Whatman no. 4 filter paper), and washing the precipitated mycelia three times with distilled water. The fresh mycelium weight was determined and then dried at 60 °C for 6 h until a constant dry weight (DW) [13]. For measurement of fruiting body dry weight, the fruiting bodies were harvested from the liquid medium with a scalpel. The fresh weight was determined, then samples were dried at 60 °C for 12 h until a constant dry weight. Analysis of Zinc Concentration Atomic absorption spectroscopy (AAS) was used to determine the concentration of zinc in mycelia and fruiting bodies. For the digestion of samples, wet digestion procedure was performed. An amount of 1 g of sample (mycelium or fruiting body dried in 60 °C) was placed in a quartz crucible and mineralized for 4 h at 500 °C. When cooled, the sample was
Fig. 1 Different stages of mycelium growth and fruiting body formation of Pleurotus florida cultivated in liquid medium. Mycelium growth at 10 days after inoculation (a); mycelium growth at 15 days after inoculation (b); primordial formation at 20 days after inoculation (c); fruiting body formation at 25 days after inoculation (d)
Appl Biochem Biotechnol
dissolved in 5 mL of 25 % HNO3 (Merck, Germany) solution. Liquid samples before mineralization were evaporated to dryness. Two-milliliter samples were evaporated, mineralized at 500 °C for 4 h, and dissolved in 5 mL of 25 % HNO3 solution. Afterwards, samples were diluted to 25 mL with deionized water and then filtered using filter paper Whatman no. 1 and a 0.45-μm Milex-HA membrane filter (Nihon Millipore Japan). If necessary, the solutions were diluted several times in a few steps with deionized water. A blank digest was conducted the same way. Zinc standard solutions of concentrations 20 and 50 mg/L were prepared under the same conditions. Zinc atomic absorption standard solution in 1 % HNO3, 0.998 mg/L (Buck Scientific), was used for the preparation of zinc standards. The content of zinc was performed using flame atomic absorption spectrophotometer (Perkin Elmer Model AAnalyst 3110, CT, USA). Ten milliliters of zinc standard or sample solution was wet-injected in the graphite furnace. Slow solution uptake and slow solution injection conditions were selected. Two standard additions (three replications of each) and peak height measurements were used for quantification [13]. Determination of Zinc Bioaccumulation Factor The zinc bioaccumulation (or bioconcentration) factor from medium to the cultivated mycelium and fruiting body was calculated from the ratios of the zinc bioaccumulation in mycelia and/or fruiting bodies dry weight and concentration in the medium [13]. Statistical Analysis All analyses were carried out in triplicates with replication. The mean and standard deviation of the data obtained were calculated. The data were evaluated for significant differences in their means with analysis of variance (ANOVA) (p
Improvement of Zinc Bioaccumulation and Biomass Yield in the Mycelia and Fruiting Bodies of Pleurotus florida Cultured on Liquid Media Nasser Poursaeid & Abas Azadbakht & Gholam Reza Balali
Received: 25 August 2014 / Accepted: 21 January 2015 # Springer Science+Business Media New York 2015
Abstract The effect of different concentrations of zinc on the bioaccumulation of zinc and biomass yield in both mycelium and fruiting body of Pleurotus florida cultivated in liquid medium was studied. The results showed that the optimum yield of mycelia (11.33±0.44 g/L) and fruiting bodies (7.70±0.19 g/L) dry biomass was obtained in a liquid medium containing 100 mg/L of zinc. At a zinc concentration of 200 mg/L, the highest concentration of zinc in the mycelia and fruiting bodies reached 1.869±0.115 and 0.151±0.008 mg/g dry weight, respectively. The addition of zinc to the culture media significantly reduced zinc bioaccumulation factor in mycelia (from 24.64±0.52 to 3.35±0.24) and fruiting bodies (from 36.71±0.30 to 0.49±0.02) dry weight. Our findings indicated that the ability of zinc bioaccumulation in the mycelia is much higher than in the fruiting bodies. The fundamental information obtained in this study will be useful for the improvement of zinc bioaccumulation and biomass yield in mycelia and fruiting bodies of P. florida cultivated in liquid media to obtain maximum zincenriched biomass. Keywords Pleurotus florida . Mycelium . Fruiting body . Zinc bioaccumulation . Biomass yield . Liquid culture medium
N. Poursaeid (*) Technology Incubator Center of Medicinal Plants, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan 81595-158, Iran e-mail: [email protected] A. Azadbakht Deputy of Food and Drug, Isfahan University of Medical Sciences, Isfahan 81745-313, Iran e-mail: [email protected] G. R. Balali Department of Biology, University of Isfahan, Isfahan 81746-73441, Iran e-mail: [email protected]
Appl Biochem Biotechnol
Introduction The importance of micronutrients in living organism nutrition is undisputable, and among them, zinc is an essential trace element for the growth of plants and mushrooms and for physiological processes in humans and animals. Most biochemical roles of zinc reflect its involvement in a large number of enzymes or as a stabilizer of the molecular structure of subcellular constituents and membranes [1]. Zinc is required for proteins, carbohydrates, mucopolysaccharides, lipids, and nucleic acids metabolism [1]. It is necessary for cell division, growth, and repair; for example, zinc is required for the activity of both DNA and RNA polymerase. Zinc also serves as a ligand, binding to and stabilizing various compounds [1]. It plays an important biological role in insulin resistance, antioxidant properties, hepatic injury, and sperm physiology [2]. Zinc has critical effects in homeostasis, immune function, oxidative stress, apoptosis, and aging, and significant disorders of great public health interest are associated with zinc deficiency [3–5]. Edible mushrooms are beneficial for health because of their functional food character, i.e., they have a high nutritional value and contain substances that help in prevention, treatment, and recovery from illness [6–8]. Moreover, they may present an excellent dietary source of some microelements because of their ability to absorb them from medium [9]. In recent years, several studies have tried to improve on the production of functional ingredients, such as polysaccharides [10] and certain essential minerals [11–13] in yeast strains and mushrooms by supplementing certain materials to cultures. The purpose of some of these studies is to obtaining organisms enriched with essential minerals, like selenium [14, 15], copper, and zinc [16–18] for production of natural nutritional supplements. An essential step for successful commercial cultivation of mushrooms is to determine the nutritional factors that are necessary for better mycelium growth, fruiting body formation, and the enzymes involved in the biological processes. Fruiting body induction is influenced by different factors, including the genetic makeup of the strain, environmental parameters, and the nutrition of the growth medium [19]. Mushrooms require one or more minerals for vegetative growth and fruiting body development [20]. The number of studies has showed that trace elements such as zinc in the submerged culture media had significant effects on the cell growth of Pleurotus tuber-regium [21], Ganoderma lucidum [10], Grifola frondosa [22], Lentinula edodes [23], and Cordyceps sinensis [24]. Trace elements at tolerable concentrations are either part of the active site or act as an activity modulator of ligninolytic enzymes of white rot mushrooms [25]. Baldrian et al. [26] demonstrated that the activity of laccase, endo-1,4-βglucanase, and 1,4-β-glucosidase enzymes of Pleurotus ostreatus was increased in the presence of zinc and copper. It was also reported that zinc, copper, selenium, iron, and chromium in culture medium exerted potent effects on the composition of the mushroom cell wall and semipermeable membrane and on the content of polyphenolics and polysaccharides that are involved in antioxidant, antitumor, immunomodulating, and insulin-resistant activities [27–32]. Studies have shown that the ability of adsorption and bioaccumulation of minerals, especially heavy metals in mushrooms, is considerably higher than those in agricultural crop plants, vegetables, and fruits. They tend to bioaccumulate more molecules because of their mycelia [33]. Copper and zinc contents in 28 species of edible mushrooms were studied, and it was concluded that their concentrations are species-dependent: some are bioaccumulators (i.e., Agaricus spp.), whereas others are bioexcluders [34]. Zinc bioaccumulation in Agaricus blazei (=Agaricus brasiliensis) [18] and G. frondosa [16] mycelia was recently studied in the presence of different zinc contents in liquid culture media. In the presence of 400 mg/L zinc, the mycelia accumulated 163 and 103 times the basal content of zinc, respectively. It was
Appl Biochem Biotechnol
demonstrated that G. lucidum mycelia, cultivated in a liquid medium with nonmycotoxic concentrations of 25 and 50 mg/L zinc, could bioaccumulate these metal [17]. Other studies have documented the maximum zinc bioaccumulation of 5.27 [35], 1.72 [36], and 4.97 mg/g in dry yeast (Saccharomyces cerevisiae) strains [37]. In recent years, the number of essential minerals bioaccumulation in mycelia (cultivated in submerged media) and/or fruiting bodies (cultivated in solid media) of mushrooms has been investigated [9, 13, 38, 39], while the bioaccumulation of essential minerals such as zinc in both mycelium and fruiting body cultivated in liquid media has not been reported yet up to now. This is the first report about the evaluation of different zinc concentrations on the absorption and bioaccumulation of zinc, mycelium growth, and fruiting body production in an edible-medicinal mushroom cultivated in liquid medium. The mushroom used in this study, Pleurotus florida, is well known as an edible and medicinal mushroom (some have important medicinal properties: reduction of blood sugar and cholesterol levels, antitumor, anticancer, antioxidant activity, and immunomodulating activity) [8, 20, 40, 41]. The aim of this research is the enhancement of zinc bioaccumulation and biomass yield in both mycelia and fruiting bodies of P. florida cultivated in liquid media to obtain maximum zinc-enriched biomass.
Materials and Methods Mushroom Strain P. florida edible-medicinal mushroom was obtained from the Mushroom Culture Collection of Mycological Institute, Fujian Agriculture and Forestry University (FAFU), China. Seed Culture Preparation The seed culture was grown in 500 mL Erlenmeyer flasks containing 200 mL of basal medium (glucose 50.0 g/L, yeast extract 10.0 g/L, casein hydrolysate 10.0 g/L, KH2PO4 1.0 g/L (Merck, Germany), distilled water 1 L, and pH 6.0) at 25 °C, on a rotary shaker, at 110 rev/ min for 7 days. The obtained seed culture was used for inoculation [13, 15]. Preparation of Basal Liquid Media Containing Zinc The basal liquid medium used by Turlo et al. [13, 15], which has been reported to promote good mycelia growth, was modified in this study. The composition of the basal liquid medium for mycelium and fruiting body cultivation was beet molasses 50 g/L (beet-sugar factory, Isfahan, Iran), soybean flour 5 g/L (IPP company, Isfahan, Iran), KH2PO4 1 g/L, MgSO4 ·7H2O 1 g/L (Merck, Germany), and distilled water 1 L with initial pH 6.0. The liquid media was enriched with zinc by the addition of different concentrations of zinc sulfate (ZnSO4) (Merck, Germany). The concentrations of zinc were in the range 0 to 300 mg/L (0, 50, 100, 150, 200, and 300 mg/ L). All liquid culture media were sterilized by autoclaving at 121 °C for 20 min. After cooling, cultivation had been carried out in polypropylene containers (12 cm diameter in×7 cm high) containing 100 mL of liquid medium and inoculated with 5 % (v/v) of the seed culture. The Mycelium Growth and Fruiting Body Formation Conditions The polypropylene containers were incubated at 25 °C under stationary conditions until total colonization with the P. florida mycelium. After incubation at 25 °C in the dark for 15 days, all
Appl Biochem Biotechnol
polypropylene containers containing liquid culture medium were further incubated at 20±2 °C under illumination with LED lamps at about 500 lx/day, and relative humidity was maintained at 90–95 % during the initiation of primordial and at 80–90 % during fruiting body formation (Fig. 1). In addition, the plastic netting as a physical support for fruiting body formation was used [42, 43]. Determination of Mycelium and Fruiting Body Biomass (Fresh and Dry Weight) The fresh and dry mycelium and fruiting body biomass were determined at 10 and 25–30 days after inoculation to analyze the mycelia growth and fruiting bodies production, respectively. The mycelium was separated from the liquid medium by filtration under vacuum (using a Whatman no. 4 filter paper), and washing the precipitated mycelia three times with distilled water. The fresh mycelium weight was determined and then dried at 60 °C for 6 h until a constant dry weight (DW) [13]. For measurement of fruiting body dry weight, the fruiting bodies were harvested from the liquid medium with a scalpel. The fresh weight was determined, then samples were dried at 60 °C for 12 h until a constant dry weight. Analysis of Zinc Concentration Atomic absorption spectroscopy (AAS) was used to determine the concentration of zinc in mycelia and fruiting bodies. For the digestion of samples, wet digestion procedure was performed. An amount of 1 g of sample (mycelium or fruiting body dried in 60 °C) was placed in a quartz crucible and mineralized for 4 h at 500 °C. When cooled, the sample was
Fig. 1 Different stages of mycelium growth and fruiting body formation of Pleurotus florida cultivated in liquid medium. Mycelium growth at 10 days after inoculation (a); mycelium growth at 15 days after inoculation (b); primordial formation at 20 days after inoculation (c); fruiting body formation at 25 days after inoculation (d)
Appl Biochem Biotechnol
dissolved in 5 mL of 25 % HNO3 (Merck, Germany) solution. Liquid samples before mineralization were evaporated to dryness. Two-milliliter samples were evaporated, mineralized at 500 °C for 4 h, and dissolved in 5 mL of 25 % HNO3 solution. Afterwards, samples were diluted to 25 mL with deionized water and then filtered using filter paper Whatman no. 1 and a 0.45-μm Milex-HA membrane filter (Nihon Millipore Japan). If necessary, the solutions were diluted several times in a few steps with deionized water. A blank digest was conducted the same way. Zinc standard solutions of concentrations 20 and 50 mg/L were prepared under the same conditions. Zinc atomic absorption standard solution in 1 % HNO3, 0.998 mg/L (Buck Scientific), was used for the preparation of zinc standards. The content of zinc was performed using flame atomic absorption spectrophotometer (Perkin Elmer Model AAnalyst 3110, CT, USA). Ten milliliters of zinc standard or sample solution was wet-injected in the graphite furnace. Slow solution uptake and slow solution injection conditions were selected. Two standard additions (three replications of each) and peak height measurements were used for quantification [13]. Determination of Zinc Bioaccumulation Factor The zinc bioaccumulation (or bioconcentration) factor from medium to the cultivated mycelium and fruiting body was calculated from the ratios of the zinc bioaccumulation in mycelia and/or fruiting bodies dry weight and concentration in the medium [13]. Statistical Analysis All analyses were carried out in triplicates with replication. The mean and standard deviation of the data obtained were calculated. The data were evaluated for significant differences in their means with analysis of variance (ANOVA) (p
