Arthrospira (Spirulina) platensis: An effective biosorbent for nutrients

Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Arthrospira (Spirulina) platensis: An effective biosorbent for nutrients Izabela Michalaka,⁎, Małgorzata Mironiuka, Katarzyna Godlewskab, Justyna Tryndac, Krzysztof Maryczc,d a

Department of Advanced Material Technologies, Faculty of Chemistry, Wrocław University of Science and Technology, Smoluchowskiego 25, 50-372, Wrocław, Poland Department of Horticulture, The Faculty of Life Sciences and Technology, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 24A, 50-363, Wrocław, Poland c Department of Experimental Biology, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Norwida 27B, 50-375, Wrocław, Poland d International Institute of Translational Medicine, Jesionowa 11, 55-114, Malin, Poland b

ARTICLE INFO

ABSTRACT

Keywords: Spirulina platensis Biosorption UV–vis ICP-OES SEM-EDX FTIR

Arthrospira (Spirulina) platensis was tested for biosorption properties. Preliminary experiments concerning biosorption kinetics were performed on Cr(III) ions. Equilibrium of biosorption was tested for Cr(III), Mn(II) and Mg (II) ions, since these elements are crucial for animals with metabolic disorders. In our study, Spirulina was proposed as a feed additive for animals suffering from diseases characterized by insulin dysregulation, abnormal adipose distribution and a high risk for laminitis. Maximum biosorption capacity of A. platensis, determined from Langmuir equation, was 45.2 for Cr(III), 44.3 for Mn(II) and 42.0 mg/g for Mg(II) ions. Biosorption of Mg(II) ions by microalga has never been studied so far. Finally, the raw and enriched microalgal biomass was examined by ICP-OES to determine its multielamental analysis before and after biosorption, FTIR to indicate functional groups that participated in biosorption and SEM-EDX to illustrate the binding of metal ions on the surface of algal biomass. ICP-OES showed that the content of elements significantly increased in the enriched A. platensis. FTIR spectroscopy evidenced that biosorption of metal ions was mainly due to carboxylate groups present on the microalgal cell wall. SEM analysis clearly showed that biosorption occurred. Arthrospira platensis turned out to be a good biosorbent of metal ions.

1. Introduction Arthrospira (Spirulina) is a well-known blue-green microalga which is applied in many areas – from human and animal nutrition to the biofuel production. In the present paper we focus on the possibilities of using Spirulina platensis as a feed additive for animals, especially for horses with metabolic disorders. Recent studies indicate that the chemical composition of microalgae can be beneficial in the treatment of many diseases [1]. Spirulina platensis is known as a rich source of biologically active compounds, crucial for feed purposes, especially βcarotene, chlorophyll, astaxanthin, lutein, phycobiliprotein, amino acids, polyunsaturated fatty acids and vitamins [2–6]. According to the Web of Science, there are no papers on the horse feed supplementation with Spirulina. However, in the literature there are some examples on the utilization of this microalga as a livestock supplement and animal feed. Farag et al. (2016) [4], Madeira et al. (2017) [5], Belay et al. (1996) [7], Holman and Malau-Aduli (2013) [8] in their review papers discussed current findings on the application of

⁎

Spirulina as a feed additive and its impact on the animal growth and survival, productivity and health (ruminants, poultry, swine and rabbits). Spirulina can improve the general health especially of animals with diseases like diabetes, arthritis, anaemia, hypertension and cardiovascular disorders [4]. This can result from the biological activity of Spirulina, mainly antioxidative, anti-inflammatory, hypoglycemic, hypolipidimic, hypocholesterolemic, antimicrobial and antiviral [1,4]. Some authors performed feeding experiments on animals with the use of Spirulina as a feed supplement. Abadjieva et al. (2018) examined the effect of Arthrospira platensis on follicular development and related endocrine parameters (estradiol, progesterone, ghrelin levels) in prepubertal gilts [9]. Spirulina maxima as a highly bioavailable source of copper was used as a feed additive for piglets [10,11]. In these works, the enrichment of animal products with microelements was examined, as well as the production performance, metabolical and physiological parameters, for example biochemical indicators in blood serum, carcass slaughter value, daily balance and nitrogen retention, digestibility. Furbeyre et al. (2017) tested the effect of Spirulina on the growth

Corresponding author. E-mail address: [email protected] (I. Michalak).

https://doi.org/10.1016/j.procbio.2019.10.004 Received 1 July 2019; Received in revised form 22 August 2019; Accepted 3 October 2019 1359-5113/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Izabela Michalak, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.10.004

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

performance, nutrient digestibility and gut health in weaned piglets and especially on the appearance of digestive disorders. Spirulina did not cause adverse effects in animals [12]. Therefore, we propose to use Spirulina platensis as a feed additive for horses, suffering from equine metabolic syndrome (EMS). In the course of this disease, an important role plays following elements – chromium (III), manganese(II) and magnesium(II) [13,14]. For this reason, Spirulina platensis was enrich with these elements using biosorption. To the best of our knowledge there are no reports concerning biosorption of Mg(II) ions by S. platensis. Previously, in the works of Saeid et al. (2013a, b) [10,11] and Saeid et al. (2016) [15] it was shown that Spirulina enriched with microelements can serve as a highly bioavailable their source for piglets and laying hens. Moreover, Spirulina is known as a good biosorbent of microelement ions – for example Cr(III) [16–18], Cu(II) [16,19,20], Zn(II) [21,22]. The aim of the present paper was to conduct detailed experiments on biosorption of Cr(III), Mn(II) and Mg(II) ions by Spirulina platensis (kinetics and equilibrium studies) in order to produce a valuable feed additive for horses. We also checked the biosorption properties of the soaked in water Spirulina platensis, because according to the literature soaking enables reduction of the content of non-structural carbohydrates, e.g., simple sugars, starch and fructan, which are not beneficial for horses with diagnosed metabolic syndrome [23,24]. The multielemental characteristics of the raw and enriched biomass was performed using ICP-OES and the morphology of biosorbent using FTIR and SEM-EDX techniques.

between the initial concentration (C0, mg/L) and the concentration of metal ions in the solution (Ct, mg/L) at a given time t (min) divided per the content of the biomass in the solution (CS, g/L). Kinetics of biosorption of Cr(III) ions by S. platensis was described by pseudo-second order model – Eq. (1), which is the most often used in the literature [17,18,20,25–27]:

dqt dt

= k2 (qeq2

qt ) 2

(1)

where qeq2 and qt are amounts of adsorbed metal ions on the biosorbent at equilibrium and at time t, respectively (mg/g) and k2 is the rate constant of pseudo-second order model of biosorption (g/mg·min). From the linearization (2) of the Eq. (1), it was possible to determine these parameters – qeq2 and k2.

t 1 t = + 2 qt k2 qeq 2 qeq2

(2)

2.3.2. Equilibrium of biosorption of metal ions by Spirulina platensis The equilibrium experiments for the raw Spirulina platensis (without soaking) were performed for the following ions: Cr(III), Mn(II) and Mg (II) – as sulphate for the best process conditions (pH 5, CS 1 g/L) determined in kinetic experiments. The concentrations of metal ions in solutions ranged from 10 to 300 mg/L, the biomass content was 0.02 g in 20 mL of the given solution. The contact time was 60 min. Spectrophotometric technique was used to determine the concentration of Cr(III) ions in solutions before and after biosorption, whereas concentrations of Mn(II) and Mg(II) were measured using ICP-OES technique. The presented data are the arithmetic average from three measurements. Langmuir Eq. (3) was used to model the equilibrium between metal ions adsorbed by Spirulina platensis and metal ions remaining in the solution.

2. Materials and methods 2.1. Chemicals Reagents: MgCl2·6H2O, MgSO4·7H2O, Cr(NO3)3·9H2O, MnSO4·H2O, NaOH, HCl, EDTA (thylenediaminetetraacetic acid) were of analytical grade and were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Chemicals necessary for the scanning electron microscopy, as well as micro X-ray analysis which includes 2.5% of glutaraldehyde and ethanol were obtained from Sigma Aldrich (Poland).

qeq =

qmax bCeq 1 + bCeq

(3)

where qmax – maximum biosorption capacity (mg/g), b – constant related to the affinity of binding sites for the metal ions (L/mg), Ceq – concentration of metal ions at equilibrium (mg/L). In order to confirm that biosorption occurred, different analytical techniques: ICP-OES, SEM-EDX and FTIR were used to compare the raw and enriched microalgal biomass. For this reason, 1 g of the raw and soaked S. platensis was suspended in 500 mL of metal ions solution (separately for Mn(II), Cr(III) and Mg(II) ions). The initial concentration of each metal ion was 200 mg/L. Additionally, in the case of magnesium we checked if there is an effect of the type of inorganic salt – sulphate: MgSO4·7H2O and chloride: MgCl2·6H2O), used for the preparation of stock solution, on the biosoption of metal ions by microalga (similarly as in our previous study [14]). The experiments were performed in a shaker – 200 rpm for 3 h.

2.2. Biomass of Spirulina platensis Commercially available microalga – Spirulina platensis (Sp) was purchased from Mühle Ebert Dielheim GmbH (Germany). For soaking, 80 g of Spirulina platensis were suspended in 4 L of tap water for 72 h. After this period, it was filtered and air-dried. The air-dried soaked Spirulina (SSp) was used in biosorption experiments. 2.3. Biosorption process 2.3.1. Kinetics of biosorption of metal ions by Spirulina platensis The preliminary experiments on biosorption properties were performed for the raw Spirulina platensis (without soaking). The biosorption tests were conducted in Erlenmeyer flasks (250 mL) containing 200 mL of Cr(III) ions solution in a shaker (IKA KS 260 basic) at 150 rpm at room temperature. The solutions of Cr(III) ions were prepared in deionized water by dissolving appropriate amounts of Cr (NO3)3·9H2O. pH of solutions was adjusted with 0.1 mol/L NaOH/HCl and was measured with pH meter Mettler-Toledo (Seven Multi; Greifensee, Switzerland) equipped with an electrode InLab413 with compensation of temperature. The kinetic experiments were performed at pH 5 for two different biomass contents in the solution (CS) – 1 and 2 g/L and for different initial concentrations of Cr(III) ions in the solution (C0) – 200, 300 and 400 mg/L. Samples of Cr(III) ions solutions were collected after a given time (till 90 min), filtered and analysed for Cr(III) concentration (a spectrophotometric method) [25]. Biosorption capacity (q, mg/g) was evaluated as the difference

2.4. Analytical techniques 2.4.1. Chemical composition of Spirulina platensis Spirulina platensis was examined in terms of its nutritional value. Protein was determined according to PB-142 I edition (14.05.2012), fibre – PN-EN ISO 6865:2002, ash – PN-R-64795:1976, fat – GAFTA 3:0 edition 2014, starch – HEC81-3. 2.4.2. Spectrophotometric method for Cr(III) determination The concentration of Cr(III) ions in the solution (4 mL) before and after biosorption was determined spectrophotometrically at λ 540 nm using Varian Cary 50 Conc. Instrument (Victoria, Australia). Cr(III) ions formed violet complexes with EDTA (0.095 g) at temperature of 95 °C [25,28]. 2

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

2.4.3. ICP-OES technique The raw, soaked, as well as enriched with metal ions Spirulina platensis (0.5 g of dry mass; d.m.) was mineralized with 5.0 mL of 69% HNO3 (Suprapur, Merck KGaA, Darmstadt, Germany) in a microwave oven – StartD (Milestone MLS-1200 MEGA, Bergamo, Italy). After mineralization, the samples were analyzed using ICP-OES technique (Varian VISTA-MPX ICP-OES, Victoria, Australia). The arithmetic average from three measurements is presented. Uncertainty of measurements is also reported. For the preparation of calibration curve (1.0, 10, 100 mg/L), the multielement reference standards (100 mg/L Agilent, Perlan Technologies Polska Sp. z o. o.) was used. Analyses were performed in the Chemical Laboratory of Multielemental Analysis at Wrocław University of Science and Technology, accredited by International Laboratory Accreditation Cooperation Mutual Recognition Arrangement and Polish Centre for Accreditation (No AB 696) [25,29].

Table 1 The content of fatty acids in Spirulina platensis [6].

2.4.4. Scanning electron microscopy (SEM) combined with energy dispersive X-ray (EDX) Spirulina platensis samples before and after enrichment were fixed in 2.5% glutaraldehyde and stored at room temperature for 48 h. Then samples were dehydrated by ethanol (series from 30 till 100% concentration) and dried at room temperature. Dried biomass was then put on the metal holder and sputtered with gold using gold-sputtered (ScanCoat six equipment–Oxford). After spattering, all samples were visualized under scanning electron microscope (EVO LS15, ZEISS, GERMANY) operating at 20 kV and work distance 11 mm [29].

Fatty acids

Mean ± SD (mg/100 g d.m., N = 3)

C8:0 (caprylic acid) C10:0 (capric acid) C12:0 (lauric acid) C14:0 (myristic acid) 14:1 (n-5; myristoleic acid) 15:0 (pentadecylic acid) C16:0 (palmitic acid) C16:1 (n-7; palmitoleic acid) C18:0 (stearic acid) C18:1 (n-12; petroselinic acid) C18:2 (n-6; linoleic acid) C18:3 (n-3; alpha-linolenic acid; ALA) C18:3 (n-6; gamma-linolenic acid; GLA) C18:4 (n-3; stearidonic acid) C20:0 (arachidic acid) C20:2 (n-6; eicosadienoic acid) C22:0 (behenic acid)

5.07 ± 0.93 3.27 ± 0.57 12.9 ± 1.4 18.1 ± 1.0 n.d. n.d. 2423 ± 241 283 ± 17 42.0 ± 6.1 173 ± 14 1053 ± 61 n.d. 744 ± 24 232 ± 28 12.6 ± 3.0 7.70 ± 0.87 n.d.

n.d. – not detected.

(III) ions (CS 1 g/L, C0 50–300 mg/L). It was shown that it increased with pH and was the highest for pH 5–34.6 mg/g (2 times higher than in pH 4 and 7 times higher than in pH 3) [30]. pH is a crucial parameter in biosorption studies since it influences on the solubility of metal, as well as the dissociation degree of functional groups that are present on the surface of algal cell wall and participate in the biosorption process [26]. The effect of C0 on Cr(III) ions biosorption by S. platensis is presented in Fig. 1. Equilibrium state was reached after ˜50 min, so it was quite fast process. In the work of Chojnacka et al. (2005), the equilibrium of Cr (III) ions sorption by Spirulina sp. was reached after 25 min [16] and in the work of Lodi et al. (2008) after 60 min (for S. platensis) [17]. Rezaei (2016) examined the sorption of Cr(VI) ions by Spirulina sp. and the equilibrium was achieved after 60 min [18]. Fast biosorption by Spirulina was observed by Aneja et al. (2010) for Pb(II) and Zn(II) ions sorption – after 15 min [21] and Zinicovscaia et al. (2018) for Zn(II) ions sorption – after 30 min [22]. As it was shown in the works of AlHomaidan et al. (2014) [19] and Gunasundari and Kumar (2017) [20], the equilibrium of Cu(II) ions biosorption by S. platensis was noted after 90 and 120 min, respectively (much longer than for Cr(III) ions). Determination of the optimal contact time in biosorption kinetic is mandatory. It is beneficial to reach the maximum biosorption of metal ions by biosorbent at the earliest possible time of the biomass suspension in solution in order to achieve efficient biosorption [19]. Table 2 presents the parameters of pseudo-second order model for biosorption of Cr(III) ions by Spirulina platensis. For CS 1 g/L, the

2.4.5. FTIR analysis of the raw and enriched biomass of algae Before analysis, samples were dried for 24 h at 80 °C. For FTIR analysis, KBr discs were prepared, containing 1.5 mg of algal sample and 200 mg of KBr. The spectra were recorded on Bruker spectrophotometer (Bruker FT-IR IFS 66/s; Billerica, Massachusetts, USA) in the mid IR range (4000–400 cm−1). 3. Results and discussion 3.1. Nutritional value of Spirulina platensis Spirulina platensis contains active compounds of high biological value, such as proteins, amino acids, polyunsaturated fatty acids, pigments, vitamins, minerals, antioxidants etc. [2–6]. Spirulina used in this study contains protein 65.37 ± 0.05%, fibre 0.4%, ash 10.73 ± 0.01%, fat 0.2% and starch 2.7% in dry biomass. Taking into account the potential application of S. platensis as a low glycaemic feed additive for horses with EMS, low level of starch is required. According to Marycz et al. (2018), feed for horses with EMS should contain about 3% of starch [13]. In the case of Spirulina this condition is met. More detailed characteristics of this microalga was presented in our previous research [6]. Spirulina platensis can serve as a source of vitamin C (18.37 ± 0.97 mg/100 g d.m.), vitamin E (α-Tc 2.43 ± 0.21 and γ-Tc 1.07 ± 0.15 mg/100 g d.m.), phycocyanin (266 ± 23 mg/100 g d.m.), polyphenols (177 ± 5 mg GAEs/100 g d.m.). The content of fatty acids including polyunsaturated (n-3 and n-6) fatty acids in Spirulina platensis is presented in Table 1. 3.2. Kinetics of biosorption of metal ions by Spirulina platensis Preliminary tests on biosorption properties of Spirulina platensis were performed on Cr(III) ions, due to a simple colorimetric method of Cr(III) ions determination in the solution (in the presence of chelating agent – EDTA). The experiments were conducted for two different biomass contents in the solution – 1 and 2 g/L and different initial Cr (III) ions concentrations (200–400 mg/L). Experiments were performed only at pH 5, because at higher pH (> 5.5), chromium precipitates as hydroxide. Additionally, in our previous study we tested the effect of pH (3, 4 and 5) on the biosorption capacity of Spirulina sp. towards Cr

Fig. 1. The effect of C0 (200, 300 and 400 mg/L) on biosorption of Cr(III) ions by Spirulina platensis (pH 5, CS 1 g/L). 3

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

Table 2 Parameters of the pseudo-second order model for biosorption of Cr(III) ions by Spirulina platensis for different C0 and CS. Parameter

qeq2 (mg/g) k2 (g/mg·min) R2

Spirulina platensis C0 200 mg/L

C0 300 mg/L CS 1 g/L, pH 5

C0 400 mg/L

31.1 0.00670 0.969

45.7 0.00197 0.924

50.5 0.00551 0.992

CS 2 g/L, pH 5

Parameter qeq2 (mg/g) k2 (g/mg·min) R2

30.7 0.00744 0.995

26.6 0.0153 0.992

37.0 0.0193 0.998

Fig. 2. Isotherms carried out for biosorption of Cr(III), Mn(II) and Mg(II) ions by Spirulina platensis at pH 5, CS 1 g/L, C0 10–300 mg/L.

biosorption capacity at equilibrium increased with the increase of C0 and for 400 mg/L it was by 62% higher than for C0 200 mg/L and by 10% higher than for C0 300 mg/L. The small difference between C0 300 and 400 mg/L can result from the saturation of all binding sites on the microalgal surface. Also in the work of Rezaei (2016) it was shown that with the increase of the initial Cr(VI) concentration in the solution, the metal ion uptake capacity by Spirulina sp. also increased [18]. The same was observed for sorption of Cu(II) ions by Spirulina platensis for the concentration range 50–300 mg/L in the work of Gunasundari and Kumar (2017) [20] and for range 10–150 mg/L in the work of Al-Homaidan et al. (2014), who noted that above C0 150 mg/L the saturation of S. platensis functional groups with Cu(II) ions occurred [19]. In the case of CS 2 g/L, the effect of C0 on biosorption capacity was not so evident. Biosorption capacity for CS 2 g/L was comparable for all tested Cr(III) ions concentrations. For C0 400 mg/L, qeq was by 20% higher than for C0 200 mg/L. The results in Table 2 also show that for CS 1 g/L biosorption capacity of S. platensis was higher than for CS 2 g/L (for C0 200 mg/L by 1.3%, for C0 300 mg/L by 72% and for C0 400 mg/L by 36%). This coincides with the literature data – Rezaei (2016) observed also that with the increase of the amount of biosorbent (Spirulina sp.) in the solution, metal ion uptake (Cr(VI)) decreased [18]. The same was for biosorption of Cu(II) ions by S. platensis [19]. Moreover, it was also noted that values of k2 for CS 2 g/L were higher than for CS 1 g/L. With the increase of the amount of biomass in the solution for higher CS, the surface area for sorption of metal ions also increases, therefore the rate of Cr(III) ions sorption can increase [25]. Based on the results from biosorption kinetics, to test biosorption equilibrium of Cr(III), Mn(II) and Mg(II) ions by Spirulina platensis, CS 1 g/L was chosen.

determined qmax for Cr(III) ions for S. platensis from Langmuir equation which was 30.7 mg/g (pH 5, CS 1 g/L, C0 50–200 mg/L) [17]. For the biosorption of Cr(III), Mn(II) and Mg(II) ions by Spirulina platensis we also calculated the dimensionless constant separation factor (RL) which is expressed as: RL=1/1+b·C0

(4)

For RL > 1 – biosorption is unfavourable, for RL = 1 – biosorption is linear, for 0 < RL < 1 – biosorption is favourable and for RL = 0 – is irreversible [18]. In our study, RL for Cr(III) ions was 0.352, Mn(II) ions – 0.187 and for Mg(II) ions – 0.297 what indicates favourable biosorption of all metal ions. 3.4. Analysis of Spirulina by ICP-OES Table 3 presents the multielemental composition of the raw (a) and soaked (b) biomass of Spirulina before and after biosorption of metal ions determined by ICP-OES. The comparison of the results indicates that raw biomass of Spirulina platensis had better biosorption properties than soaked S. platensis – the content of chromium was 2 times higher in Sp-Cr than in SSp-Cr, Mg sorbed as sulphate by 70% higher, Mg sorbed as chloride by 80% higher and Mn by 29% higher. This observation remains unclear, because according to the literature data, pre-soaking of biosorbents should increase the sorption of metal ions, due to the activation of available metal ion binding sites [33]. The same authors found also that in the case of soaking of dealginated seaweeds (waste from the alginate production industry) in water, impaired the subsequent biosorption of Cd(II) and Zn(II) ions. In our study, soaking of Spirulina platensis in water decreased the content of some elements in the biomass, especially K, Na and P – almost 2 times. Similar results were obtained also for the freshwater green macroalga (Cladophora glomerata) – soaking in water reduced the content of K almost three times in the raw biomass and P almost two times [14]. Usually, light metal ions are exchanged with sorbed metal ions in the biosorption due to ion exchange mechanism [34]. As it was shown by Dmytryk et al. (2014), during biosorption process by Spirulina sp. naturally bound light metal ions: K(I), Na(I) and Ca(II) were replaced by sorbed microelements such as Cu(II), Co(II), Mn(II) and Zn(II) [35]. In this work it was also found that inorganic salt (sulphate/chloride) used for the preparation of magnesium stock solution had no significant effect on the amount of Mg(II) ions bound by S. platensis. This is in agreement with our previous results obtained for the freshwater macroalga – Cladophora glomerata [14]. For both tested forms of Spirulina platensis, biosorption allowed for a significant increase in the Cr, Mg and Mn content in the enriched

3.3. Equilibrium of biosorption of metal ions by Spirulina platensis For the description of biosorption equilibrium, many models are used, for example Langmuir, Freundlich, Dubinin–Radushkevich, Temkin, Redlich–Peterson and among them Langmuir is the most often used and biosorption isotherms follow this model. It assumes a monolayer of adsorbate on the surface of biosorbent [18,20,21,25,26,31]. The parameters of Langmuir model for biosorption of Cr(III), Mn(II) and Mg(II) ions by Spirulina platensis (pH 5, CS 1 g/L, C0 10 to 300 mg/ L) are as follows: for Cr(III) ions – qmax 45.2 mg/g, b 0.00729 L/mg, R2 0.985, for Mn(II) ions – qmax 44.3 mg/g, b 0.0152 L/mg, R2 0.971 and for Mg(II) ions – qmax 42.0 mg/g, b 0.00775 L/mg, R2 0.961. Langmuir isotherms are presented in Fig. 2. This is the first study, in which Mg(II) ions were sorbed by the biomass of microalga and qmax was 42.0 mg/g. In the work of Zielińska and Chojnacka (2009), the maximum biosorption capacity determined from Langmuir equation for Spirulina sp. was 26.5 mg/g for Cr(III) ions and 18.4 mg/g for Mn(II) ions (pH 5, CS 1 g/L, C0 50–300 mg/L), which was much lower than in this study [32]. Lodi et al. (2008) also 4

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

Table 3 Multielemental composition of (a) raw Spirulina platensis (Sp) and (b) soaked Spirulina platensis (SSp) before and after biosorption determined by ICP-OES (mg/kg d.m.). (a) Element/ wavelength

Sp

Sp-Cr (as nitrate)

Sp-Mg (as sulphate)

Sp-Mg (as chloride)

Sp-Mn (as sulphate)

B 249.772 Ca 315.887 Cr 267.716 Cu 324.754 Fe 259.940 K 766.491 Mg 285.213 Mn 257.610 Na 588.995 P 213.618 S 181.972 Zn 213.857

14.5 ± 2.2 936 ± 140 2.18 ± 0.33 3.56 ± 0.53 521 ± 78 14 384 ± 2 877 2 301 ± 460 29.6 ± 4.4 14 638 ± 2 928 10 497 ± 2 099 6 645 ± 1 329 11.8 ± 1.9

5.28 ± 0.79 1 242 ± 248 23 993 ± 4 799 6.51 ± 0.98 1 193 ± 239 465 ± 70 723 ± 108 10.3 ± 1.5 990 ± 148 6 167 ± 1 233 7 201 ± 1 440 35.9 ± 5.4

5.27 ± 0.79 2 466 ± 493 17.5 ± 2.6 9.78 ± 1.47 1 504 ± 226 828 ± 124 7 248 ± 1 450 44.7 ± 6.7 1 002 ± 200 2 810 ± 562 8 226 ± 1 645 23.9 ± 3.6

3.47 ± 0.52 2 639 ± 528 1.87 ± 0.28 11.1 ± 1.7 1 484 ± 297 759 ± 114 7 577 ± 1 515 48.9 ± 7.3 731 ± 110 3 049 ± 610 6 872 ± 1 374 43.8 ± 6.6

3.54 ± 0.53 2 061 ± 412 3.93 ± 0.59 3.87 ± 0.58 1 206 ± 241 861 ± 129 1 952 ± 390 11 947 ± 2 389 737 ± 110 7 044 ± 1 409 7 178 ± 1 436 24.2 ± 3.6

Element/ wavelength

SSp

SSp-Cr

SSp-Mg (as sulphate)

SSp-Mg (as chloride)

SSp-Mn

B 249.772 Ca 315.887 Cr 267.716 Cu 324.754 Fe 259.940 K 766.491 Mg 285.213 Mn 257.610 Na 588.995 P 213.618 S 181.972 Zn 213.857

14.1 ± 2.1 1 636 ± 327 3.35 ± 0.50 7.24 ± 1.09 958 ± 144 6 154 ± 1 231 2 054 ± 411 33.4 ± 5.0 6 460 ± 1 292 5 133 ± 1 027 5 936 ± 1 187 23.8 ± 3.57

5.63 ± 0.84 1 336 ± 267 11 935 ± 2 387 8.32 ± 1.25 1 341 ± 268 297 ± 45 673 ± 101 26.5 ± 4.0 614 ± 92 2 324 ± 465 5 955 ± 1 191 33.4 ± 5.0

2.49 ± 0.37 1 764 ± 353 8.81 ± 1.32 9.54 ± 1.43 1 373 ± 275 353 ± 53 4 264 ± 853 39.4 ± 5.9 291 ± 44 1 816 ± 363 6 640 ± 1 328 35.3 ± 5.3

4.39 ± 0.66 1 776 ± 355 8.26 ± 1.24 8.51 ± 1.28 1 374 ± 275 379 ± 67 4 212 ± 842 31.0 ± 4.6 350 ± 52 1 776 ± 355 6 107 ± 1 221 41.6 ± 6.2

3.34 ± 0.50 1 909 ± 382 8.85 ± 1.33 9.65 ± 1.45 1 441 ± 288 376 ± 56 725 ± 109 9 264 ± 1 863 287 ± 43 2 160 ± 432 6 749 ± 1 350 37.3 ± 5.6

(b)

Bold – the biomass enriched with a given element.

biomass. In the raw Spirulina (Sp), the content of Cr increased by 11 000 times, Mg (sorbed as sulphate) about 3 times, Mg (sorbed as chloride) about 3.3 times and Mn about 400 times after biosorption. For the soaked Spirulina (SSp), these values were as follows: about 3 500 times, 2.1 times, 2 times and 277 times, respectively. Spirulina platensis after biosorption can be used as a valuable carrier, especially of Cr(III) ions. In the case of Spirulina platensis, the biomass pre-treatment (soaking) before biosorption is not recommended. It did not increase the biosorbent biosorption capacity. Taking into account the content of examined elements in the enriched biomass it can be stated that very small amounts are necessary to cover the requirement of horses for these elements. For example, according to Kentucky Equine Research (KER) recommendations for working horses are as follows – Cr 0.40.5 mg/kg of dry matter in the total ration, whereas for Mn it is 40–60 mg/kg. This means that for horse feeding we need only 0.20 mg of Spirulina enriched with Cr(III) ions (for 0.5 mg/kg) and 5 mg of Spirulina enriched with Mn(II) ions.

Table 4 The weight percentage (wt %, mean ± SD) of elements in the biomass of raw and soaked Spirulina before and after biosorption determined by SEM-EDX. Element

Spirulina (Sp) before biosorption

Spirulina (Sp) after biosorption

Soaked Spirulina (SSp) before biosorption

Soaked Spirulina (SSp) after biosorption

Mg(II)

0.58 ± 0.098 0.00335

0.57 ± 0.28 0.0212

Cr(III)

0.093 ± 0.011 0.005063 0.177 ± 0.047 0.02303

1.29 ± 0.088* 0.0299 0.826 ± 0.089** 0.0038 1.47 ± 0.036 0.0002 1.32 ± 0.384 0.02711

1.47 ± 0.25* 0.0097 1.1 ± 0.33** 0.0479 1.45 ± 0.04 0.0002 0.92 ± 0.11 0.0005

Mn(II)

0.08 ± 0.02 0.0345 0.28 ± 0.28 0.2318

in italics – statistically significant differences for p < 0.05 (Test t-student). * Mg as sulphate for biosorption. ** Mg as chloride for biosorption.

3.5. Analysis of Spirulina by scanning electron microscopy (SEM) combined with energy dispersive X-ray (EDX)

The results presented in Table 4 confirm binding of metal ions by raw and soaked Spirulina platensis. In the case of raw biomass, the content of Cr in the biomass after biosorption increased 16 times, Mn about 7.5 times and Mg as sulphate about 2.2 times and Mg as chloride about 1.4 times. For the soaked S. platensis, these values were as follows: about 18 times, about 3.3 times, about 2.6 times and about 1.9 times, respectively. The presented trend is in accordance with the results obtained in ICP-OES analysis. SEM-EDX images of the raw/soaked and enriched Spirulina platensis are presented in Figs. 3 and 4. It is worth underlining, that in contrast to Mg(II) and Mn(II) ions, Cr(III) ions are uniformly distributed on the Spirulina surface in both soaked and not soaked biomass. This might indicate on well ability of Spirulina to bind particularly Cr(III) ions. In the case of other elements, the local aggregations of both Mg(II) and Mn

Scanning electron microscopy enabled the visualization of the surface morphology of biosorbents before and after biosorption [14,34,35]. The application of energy dispersive X-ray spectroscopy allowed additionally to identify the elemental composition of biosorbents [34]. Therefore, the combination of SEM with EDX is beneficial in biosorption studies. Table 4 presents the weight percentage of elements in the raw and soaked Spirulina before and after biosorption determined by SEM-EDX. It is worth mentioning that in the case of SEM-EDX only the surface mineral composition of the biosorbent is examined – the area of the sample is as big as the size of the electron beam. In the case of ICP-OES, the whole sample is analysed (after mineralization with nitric acid) [29]. 5

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

Fig. 3. SEM-EDX images of the raw and enriched Spirulina platensis. (A) raw Spirulina platensis (Sp) before biosorption (I, II, III) and enriched with Mg(II) ions as sulphate (IV, V, VI), (B) raw S. platensis (Sp) before biosorption (I, II, III) and enriched with Mg(II) ions as chloride (IV, V, VI), (C) raw S. platensis (Sp) before biosorption (I, II, III) and enriched with Mn(II) ions as sulphate (IV, V, VI) and (D) raw S. platensis (Sp) before biosorption (I, II, III) and enriched with Cr(III) ions as nitrate (IV, V, VI).

(II) ions were observed in the soaked and not soaked biomass. The application of SEM-EDX technique allowed to visualize the surface morphology of microalgal biomass but also the distribution of sorbed metal ions on the surface, what confirmed that biosorption process

occurred. Obtained results clearly correspond with the outcome from ICP as well as FTIR analysis. Michalak at al. (2011) showed that the application of SEM-EDX technique allowed the identification of binding sites

Fig. 4. SEM-EDX images of the soaked and enriched Spirulina platensis. (A) soaked Spirulina platensis (SSp) before process (I, II, III) and enriched with Mg(II) ions as sulphate (IV, V, VI), (B) soaked S. platensis (SSp) before biosorption (I, II, III) and enriched with Mg(II) ions as chloride (IV, V, VI), (C) soaked S. platensis (SSp) before biosorption (I, II, III) and enriched with Mn(II) ions as sulphate (IV, V, VI) and (D) soaked S. platensis (SSp) before biosorption (I, II, III) and enriched with Cr(III) ions as nitrate (IV, V, VI). 6

Fig. 5. FTIR spectra of (a) raw Spirulina (Sp) and (b) soaked Spirulina (SSp) before biosorption and enriched with Cr(III), Mg(II) and Mn(II) ions.

7

stretching vibrations of OeH (polysaccharides, water molecules, proteins) and stretching of N-H (proteins) 2961 asymmetric stretching vibrations of CeH of groups CH3 2927 asymmetric stretching vibrations of CeH of groups CH2 2873 symmetric stretching vibrations of CeH of groups CH3 2855 asymmetric stretching vibrations of C-H of groups CH2O 1731 stretching vibrations of C]O of groups COOH 1657 I amide band (proteins), bending vibrations of OeH of adsorbed water molecules – stretching vibrations of C]C – asymmetric stretching vibrations of CeO of carboxylate ions 1541 II amide band (proteins) 1454 in-plane bending vibrations of OeH, bending vibrations of CeH of groups CH3 and CH2 1398 symmetric stretching vibrations of carboxylate ions 1385 stretching asymmetric vibrations of the ion NO3− (ν2) 1369 in-plane bending vibrations of CeH 1343, 1309 vibrations of CeN 1241 stretching vibrations of CeN of groups COeNH 1152, 1080, 1049, 932, 699 stretching vibrations of CeO and CeC, rocking vibrations of CH2, bending vibrations of a ring ˜ 550 a broad band coming from the water's librational (swaying) band of water molecules

3076, 3214, 3308, 3420 2961 2961 2873 2855 1731 1657 – – 1541 1454 1398 1385 1369 1343, 1309 1241 1152, 1080, 1049, 932,699 ˜ 550

3076, 3214, 3308, 3420 2961 2961 2873 2855 1731 1657 – – 1539 1454 1398 1385 1369 1343, 1309 1236 1152, 1080, 1049, 932,699 ˜ 550

Sp–Mg as sulphate

1398 1385 1369 1343, 1309 1241 1152, 1080, 1049, 932,699 ˜ 550

3076, 3214, 3308, 3420 2961 2961 2873 2855 1731 1657 – – 1541 1454

Sp–Mg as chloride

1398 1385 1369 1343, 1309 1241 1152, 1080, 1049, 932,699 ˜ 550

3076, 3214, 3308, 3420 2961 2961 2873 2855 1731 1657 – – 1541 1454

3.6. FTIR analysis of the raw and enriched biomass of Spirulina platensis

3076, 3214, 3308, 3420

Sp–Cr

Sp–Mn

FTIR analysis was carried out to identify the functional groups of Spirulina platensis on its surface that participated in bisorption of Cr(III), Mn(II) and Mg(II) ions. This analysis was performed for the raw, as well as metal-loaded S. platensis. The main advantage of this method is the possibility to identify the characteristic peaks which are associated with the complex matrix of algae that contains protein, carbohydrates and lipid fractions, as well as functional groups involved in the metal ions biosorption [20,26,34]. FTIR spectra of the raw (Sp) and soaked Spirulina (SSp), as well as enriched with Mn(II), Mg(II) and Cr(III) ions were obtained in the region of 4000–400 cm−1 and are presented in Fig. 5. Table 5 shows FTIR description for the raw/soaked and enriched Spirulina platensis. The spectrum of the raw Spirulina platensis (Sp) shows signals which are characteristic of peptide components and Wavenumber (cm−1) for the enriched biomasses

˜ 600

1400 1385 1370 1343, 1310 1240 1155, 1080, 1050, 930, 700

2961 2920 2873 2850 1730 1660 1630 1565 1540 1455

3050, 3215, 3390, 3420

Soaked Spirulina platensis – raw and enriched

on the surface of Enteromorpha sp. towards Mn(II) ions [29]. The similar effect was observed in the present study, when Mn(II) ions were used as a sorbat and Spirulina as biosorbent. Spirulina platensis enriched with Mg (II) ions exhibited biosorption capacity similar to Cladophora glomerata tested in our previous study [14], what was confirmed by EDX. Dmytryk et al. (2014) showed that SEM-EDX analysis of Spirulina sp. after biosorption revealed the increase in the content of all microelements – Co(II), Cu(II), Mn(II) and Zn(II), which were used in biosorption [35].

Wavenumber (cm−1) for the The origin of the band raw S. platensis (Sp)

Table 5 FTIR description for the raw/soaked and enriched Spirulina platensis.

I. Michalak, et al.

Process Biochemistry xxx (xxxx) xxx–xxx

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al.

polysaccharides. In S. platensis enriched with Cr(III) ions (Sp-Cr), signals characteristic of peptide components and polysaccharides are visible. In comparison to the raw S. platensis, a decrease in the intensity of the carboxylate band (1398 cm−1) is observed. At about 3 cm−1 in the direction of longer wavelengths, the C–N and II amide bands also moved. The change of the shape of a short-wave band at 1541 cm−1 was also visible, which is related to the decrease of the intensity of the signal, hidden under the second amide band, originated from the vibrations of asymmetric carboxylate groups. In the case of S. platensis enriched with Mg(II) ions: Sp-Mg (as sulphate) and Sp-Mg (as chloride) and Mn(II) ions (Sp-Mn), signals characteristic of peptide components and polysaccharides are visible. In comparison to the raw S. platensis, a decrease in the intensity of the carboxylate band (1398 cm−1) is observed. In the case of Sp-Mn, additionally, there is an increase in the signal intensity of approximately 1050 cm−1. This indicates the imposition on the present in this range deformation bands C–H, CeC and C–O, the signal from stretching vibration SO of SO42- ions. In the case of soaked Spirulina platensis (SSp) – raw and enriched, spectra present a number of signals characteristic of protein-rich plant material. All samples, regardless of metal ion enrichment, are generally the same. The only difference is observed between the raw soaked S. platensis and the remaining enriched samples. In the raw soaked S. platensis, there are two intense additional bands at 1565 and 1400 cm−1. They come from free, ionized carboxylate groups. Due to the influence of solutions with metal ions, these bands are disappearing, probably because of washing out low-molecular carboxylate compounds from the material, being enriched. At the same time, the shape of the wide band changes in the 2400-3800 cm−1 range – the centre of the band is shifted towards the higher wavenumbers, which is characteristic for undissociated carboxylic groups. These findings are in agreement with results presented by other authors who examined biosorption of metal ions by Spirulina sp. Ferreira et al. (2011) also underlined the role of carboxyl functional groups in biosorption of Ni(II), Zn(II) and Pb(II) ions by Spirulina platensis dry biomass [26]. In the work of Rezaei (2016), the spectrum of Spirulina sp. before and after biosorption of Cr(VI) ions revealed the participation of carboxylic, carbonyl, hydroxyl and amino groups in sorption [18]. Gunasundari and Kumar (2017) indicated that Spirulina platensis hydroxyl, amine groups amide groups were available for the sorption of Cu(II) ions from aqueous solution [20]. Dmytryk et al. (2014) showed that during biosorption of copper, cobalt, zinc and manganese ions from aqueous solutions by Spirulina biomass, carboxyl, phosphoryl, sulfonate and either hydroxyl or amine groups participated in ion sorption [35]. The broadening and shifting of peaks on spectra proved that there was an interaction of functional groups present on the surface of Spirulina cell wall with metal ions in the aqueous solution.

Contributions The conception and design of the study – IM, KM. Acquisition of data – IM, KG, MM, JT, KM. Analysis and interpretation of data – IM, KG, MM, JT, KM. Drafting the article – IM, KM. Revising it critically for important intellectual content – IM, KM. Final approval of the version to be submitted – IM, KG, MM, JT, KM. Declaration of Competing Interest None. Acknowledgments This research was financed in the framework of grant entitled: “The effect of bioactive algae enriched by biosorption in the certain minerals such as Cr(III), Mg(II) and Mn(II) on the status of glucose in the course of metabolic syndrome horses. Evaluation in vitro and in vivo”; (No 2015/18/E/NZ9/00607); National Science Centre in Poland. References [1] M.F. Frontasyeva, S.S. Pavlov, N.G. Aksenova, L.M. Mosulishvili, A.I. Belokobylskii, E.I. Kirkesali, E.N. Ginturi, N.E. Kuchava, Chromium interaction with blue-green microalga Spirulina platensis, J. Analyt. Chem. 64 (7) (2009) 746–749. [2] K.K. Lum, J. Kim, X.G. Lei, Dual potential of microalgae as a sustainable biofuel feedstock and animal feed, J. Animal Sci. Biotechnol. 4 (53) (2013), https://doi. org/10.1186/2049-1891-4-53. [3] Z. Yaakob, E. Ali, A. Zainal, M. Mohamad, M.S. Takriff, An overview: biomolecules from microalgae for animal feed and aquaculture, J. Biol. Res. Thessaloniki 21 (6) (2014), https://doi.org/10.1186/2241-5793-21-6. [4] M.R. Farag, M. Alagawany, M.E. Abd El-Hack, K. Dhama, Nutritional and healthical aspects of Spirulina (Arthrospira) for poultry, animals and human, Int. J. Pharmacol. 12 (1) (2016) 36–51, https://doi.org/10.3923/ijp.2016.36.51. [5] M.S. Madeira, C. Cardoso, P.A. Lopes, D. Coelho, C. Afonso, N.M. Bandarra, J.A.M. Prates, Microalgae as feed ingredients for livestock production and meat quality: a review, Livestock Sci. 205 (2017) 111–121, https://doi.org/10.1016/j. livsci.2017.09.020. [6] D. Nawrocka, K. Kornicka, A. Śmieszek, K. Marycz, Spirulina platensis improves mitochondrial function impaired by elevated oxidative stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and enhances insulin sensitivity in Equine Metabolic Syndrome (EMS) horses, Mar. Drugs 15 (237) (2017), https://doi.org/10.3390/md15080237. [7] A. Belay, T. Kato, Y. Ota, Spirulina (Arthrospira): potential application as an animal feed supplement, J. Appl. Phycol. 8 (1996) 303–311, https://doi.org/10.1007/ BF02178573. [8] B.W.B. Holman, A.E.O. Malau-Aduli, Spirulina as a livestock supplement and animal feed, J. Animal Physiol. Animal Nutr. 97 (2013) 615–623, https://doi.org/10.1111/ j.1439-0396.2012.01328.x. [9] D. Abadjieva, R. Nedeva, Y. Marchev, G. Jordanova, M. Chervenkov, J. Dineva, A. Shimkus, A. Shimkiene, K. Teerds, E. Kistanova, Arthrospira (Spirulina) platensis supplementation affects folliculogenesis, progesterone and ghrelin levels in fattening pre-pubertal gilts, J. Appl. Phycol. 30 (2018) 445–452, https://doi.org/10. 1007/s10811-017-1263-7. [10] A. Saeid, K. Chojnacka, M. Korczyński, D. Korniewicz, Z. Dobrzański, Biomass of Spirulina maxima enriched by biosorption process as a new feed supplement for swine, J. Appl. Phycol. 25 (2013) 667–675, https://doi.org/10.1007/s10811-0129901-6. [11] A. Saeid, K. Chojnacka, M. Korczyński, D. Korniewicz, Z. Dobrzański, Effect on supplementation of Spirulina maxima enriched with Cu on production performance, metabolical and physiological parameters in fattening pigs, J. Appl. Phycol. 25 (2013) 1607–1617, https://doi.org/10.1007/s10811-013-9984-8. [12] H. Furbeyre, J. van Milgen, T. Mener, M. Gloaguen, E. Labussière, Effects of dietary supplementation with freshwater microalgae on growth performance, nutrient digestibility and gut health in weaned piglets, Animal 11 (2) (2017) 183–192, https://doi.org/10.1017/S1751731116001543. [13] K. Marycz, I. Michalak, K. Kornicka, Advanced nutritional and stem cells approaches to prevent equine metabolic syndrome, Res. Veter. Sci. 118 (2018) 115–125, https://doi.org/10.1016/j.rvsc.2018.01.015. [14] I. Michalak, M. Mironiuk, K. Marycz, A comprehensive analysis of biosorption of metal ions by macroalgae using ICP-OES, SEM-EDX and FTIR techniques, PLoS One 13 (10) (2018) e0205590, https://doi.org/10.1371/journal.pone.0205590. [15] A. Saeid, K. Chojnacka, S. Opaliński, M. Korczyński, Biomass of Spirulina maxima enriched by biosorption process as a new feed supplement for laying hens, Algal Res. 19 (2016) 342–347, https://doi.org/10.1016/j.algal.2016.02.008. [16] K. Chojnacka, A. Chojnacki, H. Górecka, Biosorption of Cr3+, Cd2+ and Cu2+ ions by blue-green algae Spirulina sp.: kinetics, equilibrium and the mechanism of the process, Chemosphere 59 (1) (2005) 75–84, https://doi.org/10.1016/j.

4. Conclusions In this paper, we present a comprehensive analysis of biosorption of metal ions by Spirulina biomass. The experiments on biosorption involved kinetic and equilibrium of this process. Preliminary tests aiming at determination of the optimal experimental conditions for biosorption process (C0, CS, pH) were performed with Cr(III) ions. In order to characterize the effectiveness of biosorption, two types of Spirulina – raw and soaked in water were enriched with Cr(III), Mn(II) and Mg(II) ions in conditions established in the previous step. Spirulina platensis had the highest affinity for Cr(III), then Mn(II) and finally Mg(II) ions. The enriched biomass was analyzed using different techniques, such as ICP-OES, SEM-EDX and FTIR. Their application allowed to confirm that biosorption of elements occurred and Spirulina platensis can serve as a valuable carrier of examined metal ions (for example for the production of bioactive additives for the improvement of insulin resistance in horses). 8

Process Biochemistry xxx (xxxx) xxx–xxx

I. Michalak, et al. chemosphere.2004.10.005. [17] A. Lodi, D. Soletto, C. Solisio, A. Converti, Chromium(III) removal by Spirulina platensis biomass, Chem. Eng. J. 136 (2008) 151–155, https://doi.org/10.1016/j. cej.2007.03.032. [18] H. Rezaei, Biosorption of chromium by using Spirulina sp, Arab. J. Chem. 9 (2016) 846–853, https://doi.org/10.1016/j.arabjc.2013.11.008. [19] A.A. Al-Homaidan, H.J. Al-Houri, A.A. Al-Hazzani, G. Elgaaly, N.M.S. Moubayed, Biosorption of copper ions from aqueous solutions by Spirulina platensis biomass, Arab. J. Chem. 7 (2014) 57–62, https://doi.org/10.1016/j.arabjc.2013.05.022. [20] E. Gunasundari, P.S. Kumar, Adsorption isotherm, kinetics and thermodynamic analysis of Cu(II) ions onto the dried algal biomass (Spirulina platensis), J. Ind. Eng. Chem. 56 (2017) 129–144, https://doi.org/10.1016/j.jiec.2017.07.005. [21] R.K. Aneja, G. Chaudhary, S.S. Ahluwalia, D. Goyal, Biosorption of Pb2+ and Zn2+ by non-living biomass of Spirulina sp, Indian J. Microbiol. 50 (4) (2010) 438–442, https://doi.org/10.1007/s12088-011-0091-8. [22] I. Zinicovscaia, N. Yushin, M. Shvetsova, M. Frontasyeva, Zinc removal from model solution and wastewater by Arthrospira (Spirulina) platensis biomass, Int. J. Phytorem. 20 (9) (2018) 901–908, https://doi.org/10.1080/15226514.2018. 1448358. [23] A.C. Longland, C. Barfoot, P.A. Harris, Effects of soaking on the water-soluble carbohydrate and crude protein content of hay, Vet. Record. 168 (2011) 618–622, https://doi.org/10.1136/vr.d157. [24] C.Mc.G. Argo, A.H.A. Dugdale, C.M. McGowan, Considerations for the use of restricted, soaked grass hay diets to promote weight loss in the management of equine metabolic syndrome and obesity, Veter. J. 206 (2) (2015) 170–177, https://doi. org/10.1016/j.tvjl.2015.07.027. [25] I. Michalak, K. Chojnacka, The new application of biosorption properties of Enteromorpha prolifera, Appl. Biochem. Biotechnol. 160 (2010) 1540–1556, https:// doi.org/10.1007/s12010-009-8635-7. [26] L.S. Ferreira, M.S. Rodrigues, J.C.M. de Carvalho, A. Lodi, E. Finocchio, P. Perego, A. Converti, Adsorption of Ni2+, Zn2+ and Pb2+ onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris. I. Single metal systems, Chem. Eng. J. 173

(2011) 326–333, https://doi.org/10.1016/j.cej.2011.07.039. [27] Z. Aksu, Equilibrium and kinetic modelling of Cd(II) biosorption by C. vulgaris in batch system: effect of temperature, Sep. Purif. Technol. 21 (2001) 285–294, https://doi.org/10.1016/S1383-5866(00)00212-4. [28] Y. Ni, S. Chen, S. Kokot, Spectrophotometric determination of metal ions in electroplating solutions in the presence of EDTA with the aid of multivariate calibration and artificial neural networks, Anal. Chim. Acta 463 (2) (2002) 305–316, https:// doi.org/10.1016/S0003-2670(02)00437-3. [29] I. Michalak, K. Chojnacka, K. Marycz, Using ICP-OES and SEM-EDX in biosorption studies, Microchim. Acta 172 (2011) 65–74, https://doi.org/10.1007/s00604-0100468-0. [30] I. Michalak, A. Zielińska, K. Chojnacka, J. Matula, Biosorption of Cr(III) by microalgae and macroalgae: equilibrium of the process, Am. J. Agricult. Biol. Sci. 2 (4) (2007) 284–290, https://doi.org/10.3844/ajabssp.2007.284.290. [31] N. Tsibakhashvili, T. Kalabegishvili, L. Mosulishvili, E. Kirkesali, S. Kerkenjia, I. Murusidze, H.-Y. Holman, M.V. Frontasyeva, S.F. Gundorina, Biotechnology of Cr (VI) transformation into Cr(III) complexes, J. Radioanalyt. Nuclear Chem. 278 (3) (2008) 1–5, https://doi.org/10.1007/s10967-008-1006-y. [32] A. Zielińska, K. Chojnacka, The comparison of biosorption of nutritionally significant minerals in single‐ and multi-mineral systems by the edible microalga Spirulina sp, J. Sci. Food Agric. 89 (2009) 2292–2301, https://doi.org/10.1002/jsfa. 3723. [33] C.J. Williams, R.G.J. Edyvean, Optimization of metal adsorption by seaweeds and seaweed derivatives, Process Saf. Environ. Prot. 75 (1) (1997) 19–26, https://doi. org/10.1205/095758297528733. [34] I. Michalak, K. Chojnacka, A. Witek-Krowiak, State of the art for the biosorption process – a review, Appl. Biochem. Biotechnol. 170 (2013) 1389–1416, https://doi. org/10.1007/s12010-013-0269-0. [35] A. Dmytryk, A. Saeid, K. Chojnacka, Biosorption of microelements by Spirulina: towards technology of mineral feed supplements, Transfus. Apher. Sci. 356328 (2014), https://doi.org/10.1155/2014/356328.

9