Effect of biosorption of Fe2+ by Rhodotorula mucilaginosa YMM19 on the physiology of Lupinus albus, Triticum aestivum, Vicia faba, and Zea mays seedlings

The goal of this study was to look into growth changes and a number of metabolic events in Fe2+ treated Lupinus albus, Triticum aestivum, Vicia faba, and Zea mays plants and assess the role of biosorption of Fe2+ by Rhodotorula mucilaginosa YMM19 to alleviate these changes. The effect of Fe2+ concentrations (untreated and treated with biosorbent) on seed germination was studied. T. aestivum plumule was stimulated with a low dose of Fe2+. However, the application of Fe2+ reduced the elongation of plumule, and radicle of all seeds during germination. High doses of Fe2+ treated with biosorbent significantly increased seedling weights (fresh and dry) of all plants. Also, after 20 days, the height, and weight of seedlings of L. albus, T. aestivum, and Z. mays were increased with biosorbed Fe2+ solution. In addition, biosorption of Fe2+ enhanced total carbohydrate and protein accumulation in both T. aestivum and Z. mays radicles. Moreover, Fe2+ caused slight suppression of α- and β-amylase in L. albus and Z. mays seeds after 2 days, but the opposite effect was observed in T. aestivum. Thus, the biosorption by R. mucilaginosa YMM19 is an efficient system for removing the negative effect of excess Fe2+ from water.


Introduction
Because of their long-term persistence in the environment and poisonous nature, heavy metals constitute a severe environmental issue (Ali et al. 2019). They include iron, silver, lead, cadmium, nickel, and cobalt, among others (Nagajyoti et al. 2010). Some of the heavy metals (e.g., Zn, Fe, Cu, Ni and Co) are micronutrients for plants (Reeves and Baker 2000;Wintz et al. 2002). Iron is a necessary element for all plants and has many vital roles. It is also a significant component of cellular oxidation systems (Marschner 1995). Also, Fe is a micronutrient for all plants. It performs vital task in respiration, photosynthesis, and as enzymes cofactor (Rout and Sahoo 2015). However, excessive absorption of these plant micronutrients leads to toxic effects (Blaylock 2000;Monni et al. 2000). Although Fe 2+ is abundant in soil minerals, signs of iron poisoning appear in flood conditions due to high plant absorption of Fe 2+ (Becker and Asch 2005). Fe 2+ toxicity includes the formation of toxic radicals that destroy cellular structures such as proteins, DNA, and membranes and reduce photosynthesis (Arora et al. 2002;De Dorlodot et al. 2005;Sinha et al. 1997). Therefore, removing excess Fe 2+ from irrigation water is an important issue (Shamshuddin et al. 2013;Shokoohi et al. 2009). Several systems have been aimed to eliminate Fe 2+ from the aqueous solution, including physical, chemical, and biological processes (Shamshuddin et al. 2013;Shokoohi et al. 2009). However, new eco-friendly methods have emerged with cost-effective and straightforward implementation (Quintelas and Tavares 2002;Shamshuddin et al. 2013;Shokoohi et al. 2009). Several microbial isolates are used as bio-sorbents (Aksu et al. 1999;Kanamarlapudi and Muddada 2019;Quintelas and Tavares 2002;Verma et al. 2017;Wang and Chen 2006). Moreover, several studies have used R. mucilaginosa in the bioremediation of heavy metals (Jiang et al. 2013;Jin et al. 2020;Lopez-Fernandez et al. 2019;San and Dönmez 2012).
The present study aimed to study the biosorption of Fe 2+ from solutions by R. mucilaginosa YMM19. In addition, factors governing the biosorption, such as biomass dosage, pH, adsorption period, and initial Fe 2+ concentration were analyzed. Moreover, to evaluate the influence of bio-treated water on the germination, growth, and development of Lupinus albus, Triticum aestivum, Zea mays, and Vicia faba compared to untreated water.

Yeast biosorbent
The fungus (strain YMM19) was isolated and purified from the fruits of Morus nigra L. at Beheira governorate, Egypt. According to their morphological and molecular characteristics (GenBank accession numbers of MN960158) the fungus (YMM19) was recognized as R. mucilaginosa via a significant identity of 99.28%.

Preparation of yeast biosorbent
The PDB medium was used for yeast biosorbent formation and was made according to the manufacturer's instructions (Oxoid, Basingstoke, Hampshire, RG24 8PW, UK). An aliquot of 1% (v/v) of yeast strain (10 5 cell/ml), grown in PDB at 200 rpm for 3 days, 28 ℃ and pH 6.5, were used as an inoculum for biosorbent production. To prepare the biosorbent, R. mucilaginosa strain YMM19 was cultured in a flask (Erlenmeyer) of 250 mL size (contains 50 mL of PDB) at pH 6.5, 200 rpm, and 28 ℃ for three days. First, yeast cell masses were obtained by centrifugation of the PDB (at 6000 rpm for 15 min) and washed well twice with distilled water. Then, the harvested live yeast cells were dried for one day in 60 ℃ in an electric oven and powdered with a mortar and pestle.

Biosorption of Fe 2+ using different doses of yeast biosorbent
The dried yeast biomass (1-7 g/L) was subjected to direct contact with aqueous solutions (20 ml) containing 125 mg/L of Fe 2+ at 28 °C, pH 7 and 200 rpm. Metal solution without biomass addition was used as a control. After 3 h of contact time, the biomass was aseptically filtered using Whatman (No. 1) paper. Then, the residual Fe 2+ ions in the supernatants were analyzed after dilution with a Perkin-Elmer 2380 atomic absorption spectrophotometer. Finally, the biosorption (%) of the Fe 2+ ions was calculated by the equation: where B = Fe 2+ ion biosorption (percentage), C i = Fe 2+ in solution initial concentration (mg/L), and C f = Fe 2+ in solution final concentration (mg/L).

Effect of time on Fe 2+ biosorption
The dried biomass (1 g/L) of the YMM19 strain was examined for its biosorption of Fe 2+ from aqueous solutions (20 ml) containing 125 mg/L of Fe 2+ ions at 28 ℃, pH 7 and 200 rpm for 3 and 6 and 8 h.

Effect of pH on Fe 2+ biosorption
The YMM19 strain was tested for its Fe 2+ (125 mg/L) biosorption using a 1 g/L biosorbent under a pH of 3-8 at 200 rpm, 28 ℃ for 3 h. The tested pHs were regulated by freshly prepared 0.1 N HCl or 0.1 N NaOH solutions.

Effect of Fe 2+ on its biosorption
The YMM19 strain was tested for its biosorption of 125 and 500 mg/L Fe 2+ using 1 g/L biosorbent at pH 7, 28 ℃ and 200 rpm for 3 h.

Germination and seedling experiment
Germination of four seedlings (Lupinus albus, Triticum aestivum, Zea mays, and Vicia faba) was carried out in 0.02 m 2 plastic plates containing 780 g acid-washed sterilised sandy soil at ambient temperature (23 ℃). According to the Fe2 + concentrations used, seeds were divided into 5 groups. (0.0, 125, 125B, 500 and 500B mg/L); "B" meaning biosorption of Fe 2+ by R. mucilaginosa. Each dish included ten seeds that were irrigated with various iron solutions that were either biosorption treated or not. The light/darkness photoperiod was 10:14 h. The germinating seeds were collected at 10-days-old to determine germination (percentage) and growth characteristics (radicle and plumule lengths, fresh and dry weights), and activity of alpha-and beta-amylases were assayed in seeds at 2-day old (MacGregor 1978) (Fig. 1). The seedlings (Lupinus albus, Triticum aestivum, and Zea mays) were harvested at 20 days old to discover pigment content, total carbohydrate content, and total protein content are all growth metrics

Estimation of pigments
Freshly weighed leaves were homogenised in 5 ml 85 percent cold acetone right away, centrifuged for 15 min at 3000×g, and kept in the refrigerator overnight. The colour intensity of the acetone extract was measured at 663, 644, and 452.5 nm once it was completed to the required volume (Metzner et al. 1965). Pigment fractions were expressed as μg/g fresh weight.
Estimation of carbohydrates and total proteins. Aliquots (100 mg) of fine powdered dry plumules and radicles were ground into a fine powder (100 mg), extracted in borate buffer ( 28.63 g boric acid + 29.8 g potassium chloride + 3.5 g sodium hydroxide in a liter of distilled water). The mixture pH was regulated to 8.0 and kept standing for 24 h at 4 ℃ before centrifugation for 15 min at 3000 × g. The residues were cleaned different times and dried at 80 ℃ for polysaccharide estimation. The supernatant and residue washings were collected and used to estimate soluble sugars and proteins.
Carbohydrates were extracted in a 0.1 dry mass (10 cm 3 buffer) −1 borate buffer with a pH of 8. Carbohydrates were estimated quantitatively using Nelson (1944) together with certain amendments was attained by Naguib (1963). Briefly, 10 mg of dry residues of the plant were extracted in a borate solution and mixed with acetate (0.1 ml of mixture containing 6 ml acetic acid (0.2 N) and 4 mL 0.2 N sodium acetate) and 0.2 ml amylase (0.1%; w/v), completed until 3 ml using distilled water, and left for one day at 25 ℃, the starch can then be estimated quantitatively after centrifugation for 15 min at 3000 rpm. According to Lowry et al. (1951) total proteins were measured.

Statistical analysis
To evaluate the degree of significance between treatments, the data were statistically examined. The data was analysed using a one-way analysis of variance (ANOVA; factorial). The least-significant difference method (LSD) at 5% was used to compare means. All the experiments were run in triplicates.

Germinating percentage and growth criteria
The current results shown that biosorption of Fe 2+ at 125 mg/L induced the same trend applied to the seed germination (%) of Lupinus albus, Triticum aestivum, Zea mays, and Vicia faba seedlings of 10-day old ( Table 1). The seedlings' fresh weight data demonstrated that biosorption of Fe 2+ at 500 mg/L induced a significant raise in Vicia faba seedlings compared to control. Also, the results of Table 1 denoted that in seedling's dry weight, the biosorption of Fe 2+ at both concentrations induced a significant raise in Lupinus albus, Vicia faba, and Zea mays seedlings. The data of plumule length more prominently at biosorption of Fe 2+ at 125 mg/L in Triticum aestivum compared to control, but in Zea mays the plumule length is more pronounced at biosorption of Fe 2+ at 125 mg/L compared to the same concentration without biosorption. Under all conditions, the plumule radicle lengths were increased in Vicia faba seedlings regardless, at the treatment of biosorption of Fe 2+ at 125 mg/L compared to control. The Table 1 exposed a considerable decrease in radicle length at all concentrations of Lupines albus, Triticum aestivum, and Zea mays seedlings compared to control. Figure 4a, b shows the shoot height besides root size of Lupinus albus, Triticum aestivum, and Zea mays seedlings of 20-day old. It found that biosorption of Fe 2+ at 125 mg/L induced a considerable growing in plumule height and radicle length compared to the same concentration without biosorption in all three mentioned seedlings above. Concerning Fig. 5a, b the undried and dehydrated weights of the three seedlings, the data revealed that biosorption of Fe 2+ at 125 mg/L induced a significant increase compared to control. In certain circumstances, high amounts of iron may be a risk. However, It's probably toxic and may stimulate the development of reactive oxygen-based radicals, which can cause lipid peroxidation, which can disrupt essential cellular constituents (e.g., membranes). Acidity, bronzing, and change of color of the plant roots indicate treatment with upper levels of Fe 2+ (Laan et al. 1991). The bioavailability of iron is greatly influenced by microorganisms. The catalytic chemical reaction of Fe 2+ by microorganisms takes occur in an acidic environment on their cell walls. The existence of chelating-producing bacteria that create iron chelate complexes through the production of metabolites, in turn, is a crucial component of the Fe 3+ bioreduction process (Wetzel 2001). Various oxide reduction processes occur as a result of microorganism activity, which, along with fungous organic metabolites, play a vital role in metal circulation biogeochemical cycles, increasing their toxicity while also inactivating them (Urík et al. , 2018.

Total carbohydrates content
The data of plumule, and radicle total carbohydrates (Fig. 6a, b), using the control as a reference, demonstrated that at biosorption of Fe 2+ at 125 mg/L slightly increase in shoot total carbohydrates of Lupinus albus, regardless the same treatment at Triticum aestivum, and Zea mays. However, the root total carbohydrates slightly increased in Triticum aestivum, and Zea mays. In the cells of those fungi, Fe 3+ ions resulted in a decrease in monosaccharide content. when compared to control, proving that high levels of Fe 3+ are toxic to R. mucilaginosa. Fe 3+ ions are required for yeast metabolism and perhaps pathogenicity, indicating that at lower Fe 3+ concentrations, the quantity of each saccharide and protein within the cell is kept at a manageable level. Glucose and its amine derivatives have been shown to cause virulence in other yeasts, such as Candida albicans (Konopka 2012;Rodaki et al. 2009). Therefore, it looks that waters with a high quantity of Fe 3+ ought to show explicit sanitary threats.

Total proteins content
It is clearly shown from Fig. 7a that, using the control as a reference, the plumule total proteins were decreased in all plants at 125 mg/L Fe 2+ treated with or without biosorption. Still, from Fig. 7b, the total radicle proteins were increased in Triticum aestivum, and Zea mays seedlings with a slight decrease in the case of Lupinus albus. Because of the properties of iron, oxidation-reduction characteristics and its capability to make complexes with numerous ligands, this part may be a component of enzymes and many-electron carriers; thus, It plays a crucial part in the metabolism of plants. On the opposite hand, the low Fe 2+ solubility in physiological conditions hydrogen ion concentration levels and its high reactivity within the presence of an element that generates hydroxyl harmful radicals denote a serious problem (Hell and Stephan 2003). Fe plays also an indispensable job in protein metabolism. Through iron shortage, the proteins decrease instantaneously and a rise in soluble organic nitrogen compounds. Nitric oxide is a messenger in various cellular physiological routes in plants (Neill et al. 2003). N 2 -fixation is aided by Fe. and deoxyribonucleic acid repair (Møller et al. 2007). It causes several important functional teams to be replaced at high doses, including lipid peroxidation, cellular damage, and the formation of reactive gas species (Lowry et al.), disturbance within the numerous metabolic reaction by sterilization the catalyst activity (Anjum et al. 2015;de Oliveira Jucoski et al. 2013). Iron is cytotoxic once high levels accumulate. It will be catalyzed by the reaction of Fenton to develop radicals, which might injure lipids, proteins, and DNA. Therefore, plants should reply to iron stress regarding each iron deficiency and iron-storage disease. The incidence of iron toxicity is also influenced by plant species and growth media-related parameters such as plant age, sulphide buildup, organic acids, and various reduction products (De Datta et al. 1993;Sahrawat 2005). Figure 8a shows the chloroplast pigments of Lupinus albus of 20-day old, at the treatment of biosorption of Fe 2+ at 125 mg/L neither Chl a nor Chl b exhibit any increasing the slight increase was observed in carotenoids. Figure 8b shows the chloroplast pigments of Triticum aestivum of 20-day old, at the treatment of Fe 2+ at 125 mg/L a noticeable increase in Chl b and total pigments were observed but, the response was reserved with the treatment of biosorption of Fe 2+ at 125 mg/L regardless, the quantity of carotenoids was increase comparing to control. Figure 8c shows the chloroplast pigments of Zea mays of 20-day old, there is a slight decrease in all results at the treatment of Fe 2+ at 125 mg/L compared to control, but at biosorption of Fe 2+ at 125 mg/L the same trend was observed. Photosynthesis cells contain approximately eightieth of Fe 2+ wherever it's essential for the biogenesis of cytochrome molecules, pigment, the electron transport mechanism, and so Fe-S cluster formation (Briat et al. 2007;Hänsch and Mendel 2009). Within the photosynthetic equipment, in compounds directly related to photosystem II (PS-II), two or three iron atoms are detected, whereas photosystem I has twelve atoms (PS-I), 2 within the ferredoxin and 5 within the hemoprotein complicated (Varotto et al. 2002). Iron allocations are directly concerned with the chemical action of plant's productivity (Briat et al. 2007). Iron may be probably harmful at high concentrations. Because of iron's ability to provide and accept electrons, if it is present in the cell, it will convert peroxide molecules into free harmful radicals. These radicals will harm a healthy form of cellular structure, eventually leading to cell death (Crichton et al. 2002). To prevent this type of harm, life forms have evolved a metabolic protective mechanism that involves protein-iron atom interaction. Plants use antioxidant enzymes and metabolites like ascorbic acid and carotenoids to guard against the harmful effects of reactive radicals on cellular and subcellular structures (Alscher et al. 1997). Excess iron quantities, according to Monteiro and Winterbourn (1988), should have stimulated the creation of active O 2 atom species, which might have changed the chlorophyll and led to a reduction in chlorophyll content. With increased iron concentrations, there is a decrease in chlorophyll and antioxidant content as reported by Arunachalam  (Wilkinson and Ohki 1988). Carotenoids also serve as antioxidants vital aiding metabolites saving the subcellular and cellular organizations from the toxic properties of those reactive oxygen species (Sarika et al. 2010) by inhibiting the singlet oxygen (Prasad and Bisht 2011). Hemalatha and Venkatesan (2011) found that as iron levels dropped, pigments like chlorophyll and carotenoid levels dropped as well.

Alpha and beta-amylase activities
It is clear from results indicated in Fig. 9a, b that activity of alpha and beta amylase of 2-day old seeds at 125 mg/L Fe 2+ treated with or without biosorption were higher than control in Triticum aestivum seeds but, in Zea mays, besides Lupinus albus with little adverse effect in both treatments. The existence of a Fenton's catalyst, such as iron or copper ions, which gives birth to highly reactive OH − radicals in the occurrence of H 2 O 2 and O 2− , determines the toxicity of reactive oxygen species (Chen et al. 1999). DNA, proteins, lipids, chlorophyll, and practically every other organic component of living cells are destroyed by harmful oxygen reactive species (Becana et al. 1998). Amylase is an enzyme involved in the metabolism of carbohydrates. Hofner (1970) discovered that as iron concentrations increased, amylase activity reduced, which he attributed to iron forming complexes with carbohydrate molecules like maltose in plants. Excess iron concentrations also harmed the enzymes amylase, invertase, aspartate aminotransferase, and glutamate synthase in tea plants, according to Hemalatha and Venkatesan (2011). It appears that for yeast, 5 mg/L Fe 3+ ions are already venomous and cause defensive responses to worry. Extracellularly secreted proteins most likely bind metal ions in the atmosphere or transformed the metals into harmless forms. As a result, they reduce the quality of their products and avoid penetrating cells. The active creation of various exo-metabolites by fungus led to the distribution of metal ions in the aquatic environment varies dramatically as a result of this, as well as their quality, bioavailability, and toxicity (Kolenčík et al. 2014). The creation of reactive   atomic number 8 species is one of the processes by which cells respond to anxiety produced by heavy metals (Lowry et al.1951) and therefore the connected aerophilic stress (Azevedo et al. 2007;Pradhan et al. 2015). It was discovered that when higher quantities of Fe3 + ions were present in the media, the activity of specified inhibitor enzymes increased in yeast R. mucilaginosa cells. This indicates that Fe3 + ions have a poisonous effect on plant cells. Moreover, Fe 3+ ions within the medium within which strains of yeast ended up in stress responses (an increase in inhibitor enzyme activity and a decrease in the quantity of proteins and simple carbohydrates), and biosorption of Fe 3+ by cells significantly decreases. In R. mucilaginosa cells, intense absorption of Fe 3+ ions with no stress impact was determined at low concentrations of Fe 3+ ions in the culture media (up to 1 mg/L). Consequently, Fe 3+ ions at a level of 1 mg/L enhance R. mucilaginosa metabolism. At levels higher than 5 mg/L in the medium, the yeast produced a cellular mechanism that prevented the intake ions of Fe 3+ (Cudowski and Pietryczuk 2019).