Mycelial Growth , Biomass Production and Iron Uptake by Mushrooms of Pleurotus species Cultivated on Urochloa decumbens ( Stapf ) R . D .

Mushrooms absorb minerals and are able to accumulate them as functional organic compounds during growth. Iron is one of the elements required for various metabolic processes but low intake has resulted to some nutritional deficiencies. This study investigated the effect of different iron concentrations in the culture media on mycelial growth, biomass production, hyphae diameter and distance between septa of six species of Pleurotus, namely Pleurotus ostreatus; Pleurotus cornucopiae; Pleurotus djamor; Pleurotus pulmonarius, and Pleurotus djamor v. roseus. These macrofungi were further grown on substrate based Urochloa decumbens with addition of Fe (0, 500 and 1000 mg Kg). The mycelial growth rate and biomass produced by Pleurotus species decreased (P<0.05) as iron concentration increased from (0 100) mg L. This shows an inhibitory interaction between the fungi and iron at higher concentration. The hyphae diameter and distance between septa of the examined fungi were respectively ranged from 45 μm to 80 μm and 40 μm to 119 μm at iron concentration of (0 -100) mg L. The estimated iron uptake in cultivated Pleurotus species ranged from 37.8 μg g to 96.6 μg g when 500 mg Kg and 1000 mg Kg of Fe was added to the substrate. The cultivation of edible macrofungi enriched with iron could improve the socio economic status and play a nutritional role in decreasing anemia.


Introduction
Iron is an essential element that is involved in synthesis of many important cell components such as adenine tri-phosphate (ATP), transport of oxygen, deoxyribonucleic acid (DNA) and electron transport (Abbaspour et al., 2014).In human, iron is a component of hemoglobin and myoglobin but when it is low in supply, it leads to nutritional deficiency called iron deficiency anemia (IDA).Iron deficiency anemia has been one of the major public health problems in under developing and developing countries, particularly among pregnant women, nursing mothers and the newborn babies (Kassebaum et al., 2014).
Iron is one of the most abundant metals on the earth but it is not readily available (Philpott, 2006).Although, most of the microbes have developed various regulated system for iron uptake, utilization and storage (Haas, 2004) but some food crops show low absorption capability for iron.The diminishing quantity of iron in food has tremendously contributed to the increase in malnutrition (Zhang et al., 2015), which therefore require a reliable and alternative source of iron supply in foods to enhance the promotion of good health.
Edible mushrooms are source of nutrient that enhances health benefits of individual.The ability of Pleurotus species to breakdown complex organic compounds and transform metals into biologically active compounds has significantly influenced their nutritional status, and also serves as complementary medicine to prevent degenerative diseases (Da Fonseca et al., 2015).Mushroom biotechnology is a renewed interest towards producing edible mushrooms fortified with minerals, contributing to the bioavailability of essential nutrients (De Assunção et al., 2012;Silva et al, 2012;Nunes et al., 2014) and thus, prevents the symptoms of micronutrient deficiencies.
Mushrooms of Pleurotus spp.can be easily cultivated on many cellulolytic substrates (Da Luz et al., 2012).Hence, Pleurotus spp grown on agro-waste residues with addition of minerals is not only an alternative means of recycling the wastes residues containing metals but of producing mineral-enriched mushrooms in an ecofriendly environment.The production of Pleurotus spp.mushrooms have been an integral part of food security due to the presence of essential food components such as proteins, carbohydrates, minerals; calcium, phosphorus, nitrogen, iron and vitamins (Ganeshpurkar et al., 2010).In essence, Pleurotus spp could be a source of macro and micronutrient that can be substituted for food deficient in some essential minerals (Falandysz & Borovička, 2013).The bioaccumulation of essential minerals in Pleurotus species had built a comprehensive understanding toward strengthening the bioavailability of nutrients in edible mushrooms.This study therefore investigates the effect of iron on the mycelial growth, biomass production and uptake of iron by six species of Pleurotus cultivated on iron-fortified substrate Urochloa decumbens.

Effect of Iron on Mycelial Growth Rate and Biomass of Fungi
The fungi were exposed to iron at different concentrations.Briefly, one agar disc (8.0 mm) of each actively grown fungi was cut and transferred to the center of culture medium (PDA, Merck, Darmstadt, Germany) containing 0, 50.0 or 100.0 mg L −1 of iron in form of FeSO 4 .7 H 2 O, pH 5.5 ± 0.1.The plates were incubated at 25 °C ± 1°C for seven days.The fungal growth rate was determined after seven days by measuring the colony's diameter in two directions that were perpendicular to each other (Kim et al., 2005).The biomass (dry mass) was determined by emptying the entire contents of Petri dish (mycelium + culture media) into a bottle with distilled water and heated in a water bath (5 -10 min) to dissolve culture medium.Then, the solution was filtered and the mycelium was dried in an oven at 60 °C until a constant weight was reached (Reeslev & Kjøller, 1995).

Hyphae Diameter and Distance between Septa
The hyphae diameter and septa distance was determined.Briefly, a slide was inserted in a fungal plate at angle 45 o in order for the mycelia to spread on it.The slide was removed, stained with calcofluor (F3543 Tinopal UNPA-GX, Sigma-Aldrich, USA) and observed under epifluorescence microscopy (Olympus BX 50).Pictures were taken with a digital camera FUJIX HC-300Z, processed with the software Image Pro Plus to calculate hyphae diameter and distance between septa.

Mushroom Production and Iron Uptake
We investigated the effect of iron supplementation in substrate based Urochloa decumbens on iron level found in cultivated mushrooms.Urochloa decumbens; a grass-like plant was obtained from a farmland in Unai, Minas Gerais State, Brazil, which was dried at room temperature and milled into particle of different sizes, which passed through a sieve of 6.0 mm.The U. decumbens used in present assay have the following composition (mg Kg -1 ): N (10.2),P (0.5), K (15.2),Ca (3.4), Mg (4.3), S (0.11), Zn (9.0), Fe (121.0),Mn (19.0), Cu (3.0) and B (6.5).The milled U. decumbens was soaked in lime water solution of 2% Ca(OH) 2 for 24 h and centrifuged at 1800 rpm for 5 min to remove excess water (Kasuya et al., 2014).Then, two experimental units of each fungus were cultivated on U. decumbens (150 g) supplemented with 0, 500 and 1000 mg Kg -1 of iron.The experimental units were incubated at 25 ± 3 ºC for 30 d.After this time, the bags were transferred to a room and incubated for fructification with light, at temperature of 25 ± 3 ºC in 90% air humidity for 10 to 20 d.The mushrooms produced were duly harvested and prepared for further analysis.

Determination of Iron Content in Cultivated Mushrooms
Dried mushroom samples (200 mg) were ground and subjected to digestion with a mixture of nitric acid (HNO 3 ) and perchloric acid (HClO 4 ) (3:1, v:v) and 500 μL hydrogen peroxide (H 2 O 2 ) at 200 °C (Tedesco et al., 1995).Complete oxidation of the organic matter occurred when no further darkening of the solution occurred upon continued heating and when a clear yellow or white solution was obtained.The mixture was cooled, quantitatively transferred to a volumetric flask and diluted with ultra-pure water (Milli-Q system, Millipore, USA) to a final volume of 15 mL.The resulting solutions were used for direct spectrophotometric analysis.Three blank digests were processed in the same way.The concentration of iron (Fe) was determined using an In order to calculate the efficiency of Iron absorption by the fungi, we make a relation between the iron in the substrate and iron present in the mushrooms, using the following formula: K 0 = total iron uptake by the mushrooms at 0 mg Kg -1 / 121 (total iron content in the substrate) K 500 = total iron uptake by the mushrooms at 500 mg Kg -1 / 621 (total iron content in the substrate) K 1000 = total iron uptake level in mushrooms at 1000 mg Kg -1 / 1121(total iron content in the substrate) ND = not determined

Discussion
Edible fungi were cultivated on iron-fortified agro waste substrate to enrich the mineral content of the Pleurotus species.Microorganisms require some trace elements for their growth rate, metabolic processes and sustenance in other to overcome severe physiological consequences (Seth & Taga, 2014).The ability of an organism to survive in an environment with over accumulated or under secretion of metals gives competitive advantage in biotransformation.This has been the fundamental importance of Pleurotus in transforming organic and inorganic compound present in agro waste residues to various valuable metabolic products (Philippoussis & Diamantopoulou, 2011, Da Luz et al., 2014).
This study shows that the mycelial growth, hyphae diameter and distance between septa of tested Pleurotus spp was obviously reduced at higher concentration of iron 100 mg L -1 (Table 1).Optimum mycelial growth and biomass production of P. ostreatus had reported by Almeida et al. (2015) at 150 mg L -1 but reduced at 175 mg L -1 of iron concentration using a special medium at pH of 4.5 or 6.5.The varying response of fungi at different iron concentrations could be the choice of medium and the growth factors.The reduction observed in mycelial growth indicates that iron displayed inhibitory action against the Pleurotus species.Fleurat-Lessard et al. (2011) had reported the inhibitory action of FeSO 4 on the mycelial growth of grapevine fungal pathogens such as Botrytis cinerea, Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Diplodia seriata, and Neofusicoccum parvum.In essence, iron compounds could be suggested as source of preservatives and antimicrobial agents against some pathogenic fungi.
Despite the toxic action of iron at higher concentration, the ability of Pleurotus spp to survive and produce biomass indicated that these macrofungi possess extracellular enzymes, which can be recommended for the bioconversion of waste containing mineral residues.Therefore, the biotechnological applications and medicinal potentials of Pleurotus species could be attributed to their higher biomass production and presence of accumulated bioactive molecules, which are highly demanded and efficiently used for pharmaceutical and food products (Papaspyridi et al., 2011).Kim et al. (2002) had so far reported the mycelia growth and exo-biopolymer of some edible mushrooms such as Pleurotus spp, Flammulina velutipes, Lentinus edodes, Grifola frondosa, Auricularia polytricha, Cordyceps militaris.These are the medicinal mushrooms that are responsible for human health benefits by preventing diseases.
The iron contents obtained in cultivated Pleurotus species (Table 2) is slightly different from the findings of Vieira et al. (2013).These authors reported decrease in iron content due to low concentration of iron (0.8 mg Kg -1 ) fortified with coffee husk during the cultivation of Pleurotus ostreatus.The low content of iron was also recovered from studied Pleurotus species when compared to the concentrations (500 and 1000) mg Kg -1 applied into U. decumbens.This could be possibly due to the insolubility of iron into the substrate and other growth factors such as pH, oxygen and temperature required for the cultivation of Pleurotus species, which may slow down the absorption rate of iron from substrate into Pleurotus species.The bioavailability of iron content is lower in aerobic environments, primarily because ferric iron reacts with oxygen to form insoluble ferric hydroxides, which are highly insoluble, and thus it is not readily available for uptake by organisms (Linda, 2011).
The fungal cell wall is dynamic in structure and composition but its permeability under different growth factors and condition may alter the uptake of minerals (Philpott, 2006).
The available state of iron compound in substrate may contribute to the amount of iron assimilated by Pleurotus species, this need to be considered in any further studies on iron bioavailability in edible mushrooms, crops, fruits and vegetables.The previous statement conform to Hass (2004) who reveals that iron is not readily available in ionized states but in valence states, which therefore require some enzymatic processes during electron transfer in respiration, redox reactions by numerous oxygenase, hydrogenase and DNA-synthesis.Therefore, the unsuitable position of this metal to microorganisms could be associated to low availability of iron.
The presence of other metals in the substrate could also affect the uptake level of iron in Pleurotus spp.Metal uptake is known to pass through specific transporters for the acquisition of essential metal and these transporters may cross react with other metals ( Harms et al., 2011), which might difficult for the easy assimilation of iron for Pleurotus species.Hence, fungi face some challenges in accumulating iron in the presence of other trace metals whose aqueous and redox chemistry make iron not readily bioavailable (Kosman, 2003).
Iron content recovered in cultivated Pleurotus species were inconsistent base on the concentration of iron added to the substrate.Hoa and Wang (2015); and Alam et al. (2008) have reported that the mineral and nutritional values of cultivated mushrooms are being affected by differences among strains, the composition of growth substrate, the method of cultivation, stage of harvesting, pH of the substrate, concentration of supplemented mineral in the enriched substrate and the specific portion of the fruiting bodies used for analysis.
The findings of Saha et al. (2012) had stated that the biosynthesis of siderophores in microorganisms is induced by intracellular iron deficiency in the environment, this statement therefore justify that at low concentration of iron; some microorganisms can still survive.So the availability of much iron concentration in the substrate might not be directly proportional to the amount of iron assimilated and further increase could be toxic to the organisms.Halliwell and Gutteridge (1992) supported the previous statement by revealing that excess amount of iron is deleterious to organisms, which allow free iron to catalyze the production of cell-damaging reactive oxygen species through Fenton reaction.Hence, some microorganisms will not be able to assimilate much quantity of iron even at optimum conditions.
Conclusively, iron metabolism is sometime difficult to absorb by microorganisms at higher dose therefore, several strategies still need to be adopted to make iron easily bioavailable for organisms and in food products.This will definitely combat the problem of iron deficiency in human.

Table 2 .
Iron uptake level (µg g -1 ) in Pleurotus species cultivated on substrate based Urochloa decumbens at different concentrations