A comprehensive analysis of biosorption of metal ions by macroalgae using ICP-OES, SEM-EDX and FTIR techniques

In the present study, a comprehensive approach to the biosorption process was proposed. Biosorption of Cr(III), Mn(II) and Mg(II) ions by a freshwater macroalga Cladophora glomerata was examined using several advanced techniques including FTIR (Fourier Transform Infrared Spectroscopy), ICP-OES (Inductively Coupled Plasma–Optical Emission Spectrometry) and SEM-EDX (Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy). The enriched biomass can become a valuable, bioactive feed additive for different breeds of animals. Additionally, the collected algal biomass was soaked in water in order to reduce the content of carbohydrate, what is especially important for animals with metabolic disorders. The content of starch was reduced by 22% but additionally some elements–mainly Si, K and P were removed from the biomass. It was shown that the natural macroalga had better biosorption properties than soaked. Cr(III) ions were sorbed by the biomass in the highest extent, then Mn(II) and finally Mg(II) ions. The content of chromium in the enriched algal biomass increased almost ~200 000 times, manganese ~75 times and magnesium ~4.5 times (both for Mg(II) ions used from magnesium sulphate, as well as from magnesium chloride) when compared to the natural Cladophora glomerata. In the case of the soaked biomass the increase of the content of elements in the enriched biomass was as follows ~17 165 times for Cr, ~25 times for Mn and for Mg ~3.5 times for chloride and 3.8 times for sulphate. The type of magnesium salt (chloride or sulphate) had no significant effect on the algal sorption capacity. The proposed mechanism of the biosorption is ion exchange in which mainly potassium participated. The applied FTIR analysis enabled the identification of the functional groups that participated in the biosorption process–mainly carboxyl and hydroxyl. The main changes in the appearance of the spectra were observed for the following wavenumbers– 3300–3400; 2900; 1700; 1400–1500 and 1200–1300 cm-1. The application of SEM-EDX proved that the metal ions were sorbed on the surface of both tested algae.

a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Nowadays, the novel feeding strategies that will improve animals production, as well as their health are developed and implemented. A series of promising innovations and practices in feed production and feeding includes a balanced and phased feeding, the increase of the quality and level of use of forages in diets, reduction of grains use, a targeted mineral feeding etc. [1,2]. These proposals are especially important in terms of horse feeding, where a creation of a well-balanced diet is of particular importance. A forage diet, low in non-structural carbohydrates, with a proper content of minerals and vitamins is highly recommended [3]. The second issue is to search for alternative/additional feed ingredients, mainly due to the global demand for grains and the competition between man and the livestock industry with the existing food and feeds [2]. There are several promising new feed resources, rich in biologically active compounds, that can be used as feed additives for animals. One of them is the biomass of algae, both micro-and macroalgae (also called seaweeds). In the literature, most of the attention is paid to marine macroalgae, however freshwater algae, being the result of eutrophication of water reservoirs, are also a rich source of biologically active compounds, such as unsaturated and saturated fatty acids, carbohydrates, amino acids and proteins, phenolic compounds, minerals, carotenoids, vitamins etc., and can constitute a very useful raw material for nutritional applications [3,4]. The proposed approach is consistent with the concept of the Food and Agriculture Organization of the United Nations (FAO) of the sustainable animal diet, "which integrates the importance of efficient use of natural resources, protection of the environment, socio-cultural benefits and ethical integrity" [2].
In order to obtain a valuable feed additive with micro-and macroalements based on algal biomass, biosorption process can be applied. This is a surface phenomenon in which metal ions from aqueous solutions are passively bound by a non-living biomass [5][6][7][8][9][10][11]. Several operating conditions, such as solution pH, metal ions concentration, biomass size and its content in the solution, contact time, etc., influence the amount of metal ions bound with the biomass [8,9,11,12,13]. The choice of metal ion that will be sorbed by the biomass depends on the animal species and animal's requirement for a given nutrient.
The ability of the biomass to bind metal ions can be examined with the use of several advanced techniques such as FTIR, SEM-EDX and ICP-OES. In the available literature, such a comprehensive approach to the biosorption process is randomly presented, e.g., see in [12,14,15]). Among tested techniques, FTIR is the most often used in biosorption studies. According to the Web of Science database (accessed on 15 th May 2018), "FTIR" and "biosorption" appeared in the topic of the publications 1885 times, whereas in their title only 15 times. For "SEM" and "biosorption" it was 1634 and 7 times, respectively, for "SEM-EDX" and "biosorption"-189 and 4 times, respectively and finally for "ICP" and "biosorption" 170 and 4 times, respectively. These techniques, used together, provide a full insight into biosorption process. FTIR is used to determine changes in the vibration frequency of functional groups of biosorbents before and after biosorption process, thus indicating which groups participated in biosorption [8,9,12,13,15,16,17]. ICP-OES is a precise and sensitive technique which allows to determine mineral composition of the whole natural and enriched biomass, after its digestion in mineral acids [6,12,14], whereas SEM-EDX enables determination of the elemental composition of the biomass surface and the distribution of metal ions [8,9,16,12,15,17].
It is important to emphasize that the enriched with microelement ions algal biomass was tested both in vitro and in vivo. The enriched with Cr(III) ions freshwater macroalga Cladophora glomerata (C. glomerata) reduced apoptosis and inflammation in adipose derived mesenchymal stromal stem cells (ASCs) isolated from EMS horses (with Equine Metabolic Syndrome) through the improvement of mitochondrial dynamics, decreasing of PDK4 (Pyruvate Dehydrogenase Kinase 4) expression and reduction of the endoplastic reticulum stress. It was showed, that C. glomerata enriched with Cr(III) ions served antioxidative protection coming from enhanced SOD (Superoxide Dismutase) activity [18]. In in vivo tests which were conducted on pigs and laying hens it was shown that the algal biomass rich in microelements positively influenced the health of animals, their productivity, the quality of products (the content of micro-and macroelements), as well as the decrease of the excretion of elements what can lead to the decrease of the environmental burden [19][20][21][22][23].
The aim of the present paper was to obtain and characterise a feed additive for animals, especially for equine nutrition based on the biomass of a freshwater macroalga-Cladophora glomerata. The biomass was enriched separately with Cr(III), Mn(II) and Mg(II) ions, because they seem to be crucial in an equine clinical nutrition [3,12,18,24]. In a dietary management of horses, especially with a diagnosed metabolic syndrome, soaking of hay in water is also recommended. It results in a significant reduction of the content of non-structural carbohydrates, e.g., simple sugars, starch and fructan [25,26,27]. Therefore, the algal biomass was also subjected to the soaking before biosorption process. The characteristics of the obtained feed additives was performed using ICP-OES, FTIR and SEM-EDX techniques.

Biomass of freshwater macroalgae
Cladophora glomerata (C)-a freshwater macroalga was collected by hand from the surface of pond in a village Tomaszówek, Poland (51˚27 0 21@N 20˚07 0 43@E) twice-in August (C A ) and October (C O ) 2016. No specific permissions were required for this location/activity. The study was carried out on private land and the owner of the land gave permission to conduct the study on this site. These studies did not involve endangered or protected species. Then, the air-dried biomass was milled using a grinding mill (Retsch GM300, Haan, Germany). For the biosorption experiments, the biomass with the particle size lower than 500 μm was chosen.

Soaking of Cladophora glomerata
For the soaking, 800 g of a dried Cladophora glomerata (C A ; August 2016) was suspended in 40 L of tap water for 72 h. After this period, it was filtered and the air-dried soaked Cladophora biomass (SC A ) was used in biosorption.

Biosorption process
The biosorption process of Mn(II), Cr(III) and Mg(II) ions was performed in Erlenmeyer flasks containing 1 g of the biomass (the natural collected in October 2016 -C O and soaked-SC A ) and 500 mL of the metal ions solution in a shaker (IKA KS 260 basic) at 150 rpm in room temperature. The concentration of each metal ion was 200 mg/L. The solutions were prepared in deionized water by dissolving separately, appropriate amounts of inorganic salts-MnSO 4 �H 2 O, Cr(NO 3 ) 3 �9H 2 O, MgCl 2 �6H 2 O; MgSO 4 �7H 2 O (POCh S.A. Gliwice, Poland). pH 5 of solutions was adjusted with 0.1 mol/L solutions of NaOH/HCl (POCh S.A. Gliwice, Poland) using a pH meter Mettler-Toledo (Seven Multi; Greifensee, Switzerland) equipped with an electrode InLab413 with the compensation of temperature. The contact time was 3 hours determined in our previous experiments [12]. A general scheme of preformed experiments is presented in   SEM-EDX technique. The natural (C O ), soaked (SC A ), as well as enriched with metal ions biomass (EC O and ESC A ) was analyzed using SEM-EDX technique. For a morphological observation, SEM was applied according to the previously described method [6]. The samples of the biomass were fixed in 2.5% of glutaraldehyde (Sigma) and were dehydrated by ethanol (from 30% till 100% concentration). Next, samples were covered with gold using a sputter coater (Scancoat six, Oxford) and observed in a scanning electron microscope (EVO LS15, ZEISS, Oberkochen, Germany) operating at 15 kV. The elemental analysis, as well as ions distribution (mapping) on investigated samples were performed using BRUCKER energy dispersive X-ray system according to the previously published methodology [28,29]. The X-ray spectrum of each Cladophora glomerata loaded with a given microelement was obtained.

Results
This manuscript provides a comprehensive insight into biosorption properties of a freshwater macroalga-Cladophora glomerata, natural and additionally soaked in tap water before this process. The biomass was enriched separately with Cr(III), Mn(II) and Mg(II) ions in a batch system. The initial concentration of metal ions in the aqueous solution (C 0 ) was 200 mg/L, pH 5, the biomass content (C S ) was 2 g/L and contact time 3h. The experimental conditions were established in our previous study [12]. After biosorption, the enriched biomass was examined using several techniques such as ICP-OES, FTIR and SEM-EDX. As a control group, the natural and soaked biomass was used. A nutritional value of the natural and soaked Cladophora glomerata was also determined.

The nutritional value of the natural and soaked Cladophora glomerata
The treatment of metabolic disorders in animals requires a proper diet, which should be low in non-structural carbohydrates. Soaking in water components of forage can partially remove them [26,27]. In the present study we wanted to check how soaking of macroalgae in water affects their nutritional value. The obtained results are presented in Table 1. It is important to note that the soaking of freshwater macroalgae in tap water resulted in the reduction of starch by 22% in the soaked macroalga when compared with the natural one.
The nutritional value of Cladophora glomerata can vary depending on the conditions in which it grows. Khuantrairong [30]. The tested in the present study Cladophora glomerata, grown in the natural freshwater environment, was a richer source of proteins and ash. Moreover, it was found that the soaking of the algal biomass in water reduced the content of starch by 22%. Longland et al. (2011) also showed that the soaking of hay reduced the content of carbohydrate, whereas crude protein was not leached from the hay during soaking [25]. Our research showed that the content of protein decreased by 4% in the soaked alga. This issue is very important taking into account a proper feeding of horses with metabolic disorders, which should not be fed with high-starch concentrates [3].

ICP-OES technique in biosorption process
ICP-OES is a precise and sensitive analytical technique with low detection limits. It enables determination of elements in the whole biomass, not only on the surface as it is in the case of SEM-EDX. However, it must be proceeded by the biomass mineralization with concentrated inorganic acids. This technique is used for the analysis both solutions, as well as the biomass before and after biosorption [6].
In the biosorption process, examined metal ions from the aqueous solution are exchanged with metal ions which are naturally bound with functional groups on the surface of the biomass cell wall. Therefore, the dominating mechanism of biosorption is ion exchange [5,6,9,10,31]. The untreated biomass generally contains light metal ions such as K, Na, Ca and Mg which are acquired from the water [5,9]. In this paper we wanted to check if the freshwater biomass of Cladophora glomerata is able to bind light metal ions, for example magnesium. This element has never been used in the biosorption studies as a sorbate. Moreover, magnesium was chosen since is thought to elicit its positive effect on insulin sensitivity what is very important in the case of laminitic obese horses [32]. Additionally we checked if there is an effect of the magnesium inorganic salt (sulphate and chloride) used for the preparation of a stock solution for the biosoption process on its sorption by algae. In the literature it is noted that metal anions (e.g., sulphate, nitrate, chloride) can influence the biosorption capacity of green algae [33].
In Table 3, the multielemental composition of the natural biomass of Cladophora glomerata (C O ) before and after biosorption (EC O -Cr (as nitrate), EC O -Mg (as sulphate), EC O -Mg (as chloride), EC O -Mn (as sulphate)) determined by ICP-OES is presented. Comparing the content of Cr, Mg and Mn in the natural and in the enriched biomass it can be noticed that the content of chromium increased in the enriched biomass by about 200 000 times when compared with the natural, Mn increased by about 75 times and Mg about 4.4 times when it was used as a chloride and about 4.6 times when Mg was used as a sulphate. In the case of chromium biosorption, main metal ions which participated in the ion exchange and were removed from the biomass to the aqueous solution were: K, Si, Ca, Mg and Na. After biosorption, the content of K decreased~25 times in the enriched biomass, when compared to the natural, Si~24 times, Mg~4 times, Ca~3 times and Na~2.2 times.
Magnesium which was sorbed by macroalga from solutions prepared from magnesium sulphate and chloride showed similar properties. The decrease of the content of elements in the enriched biomass when compared to the natural, concerned the following micro-and macroelements: Si decreased by~26 times, K~15 times, Na~2.2 times, Ca~1.4 times-for Mg used as a sulphate salt and Si decreased by~17 times, K~17 times, Na~2.7 times, Ca~1.3 times-for Mg used as a chloride salt.
The biosorption of manganese resulted in the release mainly of Si, which was~20 times lower in the enriched when compared to the natural biomass, K was~15 times lower, Na was~4 times lower, Mg was~2.1 times lower and Ca was~1.4 times lower. From the results it can be noted that beside light metal ions-alkali metal cations (Na, K) and alkaline earth metals (Mg, Ca), also silicon participated in this process. This element is known to be a nutrient required for the growth of Cladophora glomerata, but also an essential component of the algal cell wall [34]. Table 4 presents a multielemental composition of a natural Cladophora glomerata (collected in August 2016 -C A ) which was later soaked for 48 hours (SC A ) and finally enriched with Cr

(III)-ESC A -Cr, Mg(II)-ESC A -Mg (separately as magnesium sulphate and magnesium chloride) and Mn(II) ions-ESC A -Mn.
The results showed that the biomass soaking in tap water led to the removal of some elements. The main differences were obtained mainly for Si, where its content in the soaked biomass was~52 times lower than in the natural biomass, for K the decrease was~2.9 times, P 1.9 times, for Mg decrease was by 21% and for Mn by 19%.
Comparing the content of Cr, Mg and Mn in the soaked and in the enriched biomass it can be noticed that the content of chromium increased by about 17 165 times in the enriched biomass when compared to the soaked, Mn increased~25 times and Mg increased~3.5 times when it was sorbed as a magnesium chloride and increased~3.8 times when it was sorbed as a magnesium sulphate. During the biosorption of Cr(III) ions, Ca, K and Mg were the main elements which were released from the soaked biomass to the aqueous solution (due to cation exchange). The content of K decreased~30 times in the enriched biomass, when compared to the soaked biomass before biosorption, Mg decreased~5.6 times, Ca~3 times, Mn~2.1 times and Si~1.6 times. Biosorption of Mg(II) ions in the form of sulphate and chloride salts by the soaked C. glomerata showed similarities. The main metal ions which were released from the biomass during biosorption of Mg(II) ions were K, which content in the enriched biomass decreased by~16.6 times (for sulphate) and~18.8 times (for chloride) when compared to the soaked biomass before biosorption, Na decreased by~3.8 times (for sulphate) and~7.5 times (for chloride), Mn by~29% (for sulphate) and~31% (for chloride) and P by~17% (for sulphate) and~14% (for chloride). During biosorption of Mn(II) ions, in the cation exchange participated mainly K-a decrease of the content in the enriched soaked biomass by~17.2 times when compared to the soaked biomass before biosorption), Na-a decrease by~9.0 times and Mg-a decrease by~3.3 times.

Fig 2. A comparison of a multielemental composition of a natural Cladophora glomerata collected in August (C A ) and in October 2016 (C O ).
In this study it was also found that the best biosorption properties showed the biomass of the natural, as well as the soaked Cladophora glomerata towards Cr(III), then Mn(II) and finally Mg(II) ions. The extent to which a metal ion will be bound to functional groups on the surface of biomass cell wall largely depends on the chemistry of the sorbed metal ion (oxidation state, electrochemical potential, ionization potential, electronegativity, atomic radius, ionic radius, atomic weight, atomic number, covalent index etc., [9,35,36]) and its preference to form bonds [36]. The most important parameters of metal ion are a covalent index (the greater the covalent index value of a metal ion, the greater its potential to form covalent bonds with biological ligands) and ionization potential [35,36]. In the case of Cladophora glomerata, the amount of metal ions bound with the biomass increased (Cr > Mn > Mg) with the increase of a covalent index (X m  (Tables 3 and 4). The content of K decreased in the highest extent among other light metal ions in the enriched biomass, when compared to the raw material used in the biosorption process. This is in accordance with the literature data. Pouya and Behnam (2017) based on ICP analysis suggested that the ion exchange can be considered as a main mechanism for copper removal by a red alga-Jania adhaerens, because ions of Na (considerably higher than other ions), K, B, Al and Fe were released from the biomass into the solution upon biosorption process [15]. Also Dmytryk et al. (2014) showed the replacement of naturally bound by Spirulina sp. light metal ions: Na(I), K(I), or Ca(II) with biosorbed microelements such as Co(II), Cu(II), Mn(II) and Zn(II) [14].
Additionally it was presented that for both tested algae-natural (C O ) and soaked (SC A ), the type of magnesium salt (sulphate/chloride) did not influence significantly the sorption of Mg (II) by the biomass. Slightly higher content of magnesium in the enriched biomass was noted for the sulphate. Similar results were obtained for a green marine macroalga-Enteromorpha prolifera, where biosorption capacity of Cu(II) ions used as chloride was 46.0 mg/g, whereas for copper sulphate it was 42.8 mg/g [33].
In the present research it was also shown that soaking of algae in water decreased the content of mainly K, P, Mg, Mn in the biomass. The ash content in a soaked C. glomerata was by 4% lower than in the raw material (Table 1). This is in agreement with the literature data that  [12] https://doi.org/10.1371/journal.pone.0205590.t005

Insight into biosorption process
indicates that the soaking of hay significantly reduced the level of sodium, potassium, magnesium, phosphorus and copper (but presumably not nitrogen, calcium, zinc, manganese and iron which were also tested) [37].

The effect of the season on the multielemental composition of freshwater macroalgae
In this paper we also examined the effect of the season on the multielemental composition of freshwater macroalgae. The results from ICP-OES analysis showed that the content of elements in freshwater macroalgae depended on the season-it was higher in summer (August) than in autumn (October) -Fig 2. Cladophora glomerata collected in August was a richer source of most of the elements than C. glomerata collected in October, for example contained~4.8 times more of Mn,~3 times more of Fe,~2.4 times more of P, by~69% more of Mg, by~56% more of S, by~23% more of Si, by~22% more of K and by~12% more of Na. Only the content of Ca in algal biomass collected in October was much more higher (~3 times) than in alga from August. The mineral composition of algae depends on many factors, for example the type of algae species, the place of their harvest (ocean, sea, pond, river etc.,) and its geography, residence time in a water reservoir and other seasonal, annual, environmental and physiological factors [4,38]. In the work of Michalak et al. (2009) it was shown that a green macroalga Enteromorpha sp. collected from the Gulf of Gdańsk (Poland) in June (summer) contained higher amounts of Mn, Mg, Ca, K Insight into biosorption process and Fe than the alga collected in April (spring-lower temperatures) [38]. For the future research it will be beneficial to collect algae mainly in the summer season because they are a richer source of micro-and macroelements than algae harvested in the autumn, especially taking into account their potential application as feed additves with elements for animals. Additionally, after biosorption of metal ions, the enriched biomass can constitute a concentrated form of micro-and macronutrients. Moreover, the summer is the most favourable season for algae growth what ensures their availability.

Scanning electron microscopy (SEM) combined with energy dispersive Xray (EDX) in biosorption process
The SEM-EDX investigation allows the examination in details the surface concentration and distribution of Cr(III), Mn(II) and Mg(II) ions in the natural and enriched Cladophora glomerata. In our and other previous research it was shown, that SEM-EDX becomes a valuable tool for a precise investigation of the elemental composition, as well as distribution of elements in different biomasses [6,18,39]. In the present study, the SEM-EDX elemental analysis revealed the increased quantity of Mg, Cr, as well as Mn in the course of biosorption process in Cladophora glomerata biomass (Table 5). The biosorption process resulted in the increased amount of Mg, Cr, as well as Mn in both soaking and non-soaking biomass what is shown in Figs 3 and 4, when compared to the native form of Cladophora glomerata. However, the non-soaking form of the investigated biomass was characterized by higher biosorption properties in the case of Cr(III) and Mg(II) ions. This can be probably due to the removal of native elements from algal biomass during soaking process, what finally could have reduced the biosorption properties of the soaked algal biomass.
The EDX mapping clearly showed, that Cr was equally distributed on the surface of the investigated biomass in the both soaking and non-soaking form (Figs 3 and 4D). It was found, that the soaking process negatively affected biosorption properties of Cladophora glomerata in the case of Cr(III) and Mg(II) ions, but surprisingly enhanced absorption of Mn(II) ions. The decreased quantity of Mg was observed in the biomass, that was soaked before biosorption process. The EDX mapping confirmed the equal surface distribution of Mg and Mn on the investigated Cladophora glomerata, regardless it was soaked or not (Figs 3 and 4).
In this study it was shown, that the algal biomass is characterized by good biosorption properties and can actively bind to its surface particular elements, including Cr(III), Mn(II) and Mg(II). In our research, it was demonstrated that among all the tested elements, Cr(III) ions were sorbed in the highest extent and chromium content in a non-soaking form was equal to 4.78±1.17%, when compared to the soaking form (2.07±1.14%). Using EDX method it was also demonstrated that all tested ions were successfully bound to the surface of Cladophora glomerata, what was shown using a mapping technique.

FTIR analysis of the natural and enriched biomass of algae
FTIR technique was used to examine a surface characterization of the biosorbent and to observe the chemical modification after biosorption of metal ions by Cladophora glomerata. Analysis of the natural and enriched biomass enables identification of the functional groups that participate in the biosorption process. These groups originate from macromolecules (polysaccharides, proteins and lipids) which are a part of the algal cell wall. The cell wall offers several functional groups such as carboxyl (e.g., fatty acid and amino acids), hydroxyl (e.g., polysaccharides), amine, phosphate or sulfonate etc., [14,31]. FTIR spectra of the natural (C O ) and soaked (SC A ) Cladophora glomerata, 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 Figs 5 and 6. Table 6 presents a FTIR description of groups according to their wavelengths for the natural and soaked Cladophora glomerata before and after biosorption process.
The FTIR spectra of natural and soaked algae before and after biosorption show a complex nature of the biomass due to the presence a large number of peaks. The broadening and shifting of peaks was observed after interaction of a raw biomass with the examined metal ions. The largest differences in the appearance of the spectrum were observed between the natural/ soaked Cladophora glomerata and enriched with chromium ions. In Fig 5 it can be seen, that the spectra for the natural and enriched algae generally have similar shape, however especially in the case of EC O -Cr the increase in the transmittance of peaks in a chromium loaded biomass when compared to the metal free biomass is observed in the region~1000-2000 cm -1 and decrease in the region~2400-3600 cm -1 . For biomasses: EC O -Mg as sulphate and chloride and EC O -Mn, transmittances of peaks in metal loaded biomasses are generally lower than in the https://doi.org/10.1371/journal.pone.0205590.g006 Table 6. FTIR description for the natural and enriched Cladophora glomerata.

Wavenumber (cm -1 )
for the natural C. glomerata (C O ) The origin of the band Wavenumber (cm -1 ) for the enriched C. glomerata (sulphate/chloride) and Cr(III) biomass and in the region of~2400-3600 cm -1 with the exception of ESC A -Cr (Fig 6). Main differences between the tested natural and enriched biomass were observed for the wavenumber range from approximately 3300-3400 cm -1 (overlapping of amine N-H and hydroxyl O-H stretching vibrations, especially in EC O -Cr and EC O -Mg (sulphate)), at~2900 cm -1 for all enriched biomasses (-CH stretching vibration of C-CH 3 ), at~1700 cm -1 (C = O stretching mode of the amide I band), at~1400-1500 cm -1 (stretching of C = O bond from carboxylic acids), in the range 1200-1300 cm -1 (bending vibrations of C-H and O-H, rocking vibrations of CH 2 (polysaccharides), III amide band (proteins)) ( Table 6). From Figs 5 and 6, it can be seen that transmittance values were shifted mainly for carboxyl and hydroxyl groups. It was also noted that differences in the transmittance of peaks of the unloaded and loaded biomass were higher for the natural Cladophora glomerata, (C O ) rather than for soaked (SC A ), what is in agreement with results obtained by ICP-OES.
In this study it was shown that hydroxyl and carboxyl groups (~1200-1280 cm -1 ;~1400-1500 cm -1 ) were effective for capturing Cr(III), Mg(II) and Mn(II) ions (Figs 5 and 6). This is in line with results obtained in the previous work-biosorption of Cr(III) ions by Vaucheria sp. [31] and Cladophora glomerata [12]. Deng et al. (2007) also indicated from FTIR analysis that amino or hydroxy, C = O, and C-O groups of Cladophora fascicularis could combine strongly with Cu(II) ions [40].

Conclusions
Summarizing, in the present research, we investigated biosorption properties of Cladophora glomerata in a native, as well as in a soaked form and then enriched with Cr(III), Mn(II) and Mg(II) ions. We chose these ions, because they are recognized as insulin sensitizers, which in turn in the form of the enriched biomass can serve as a potential clinical feed additive for horses that suffer from EMS. Cladophora glomerata showed the best biosorption properties towards Cr(III) ions, then Mn(II) and finally Mg(II) ions. In both macroalgae-natural and soaked, potassium was the main light metal ion that participated in the ion exchange in biosorption process. FTIR analysis showed that carboxyl and hydroxyl groups were effective for capturing Cr(III), Mg(II) and Mn(II) ions. SEM-EDX technique confirmed binding of metal ions on the surface of algal biomass. Our research clearly showed, that soaked Cladophora glomerata was characterized by the lower ability to bind Cr(III), Mn(II) and Mg(II) ions. Additionally it was found that soaking of algae in water decreased the content of mainly K, P, Mg, Mn in the biomass. Although, biosorption properties of a soaked Cladophora glomerata are reduced, when compared to the native form, it can serve as a low glycaemic component for the production of advanced feed supplements for horses (soaking of algae resulted in the reduction of starch by 22% when compared with natural macroalga). Moreover, the additional advantage of low glycaemic index components in particular produced from Cladophora glomerata might be the possibility to increase its quantity in a daily ration, which in turn will serve the higher amounts of bioactive factors. To summarize, it might be highlighted, that soaking of Cladophora glomerata negatively affected its biosorption properties, but reduced the glycemic index, which is crucial in the feeding of insulin resistance animals.