Effects of anions on the biosorption of microelement cations by macroalga Enteromorpha prolifera in single- and multi-metal systems

The results of research on the effects of anions on the biosorption of microelement cations by the edible marine macroalga Enteromorpha prolifera in single- and multi-metal systems are discussed in this paper. It was shown that the maximum biosorption capacity (qmax) in a single-metal system of Co(II) ions decreased in the following sequence: Cl− (46.0 mg g−1) > SO42− (42.8 mg g−1) > NO3− (41.9 mg g−1). In multi-metal systems, in which the ratios of Cl−, NO3−, and SO42− were 0:0:4, 1:1:2, 3:0:1, and 4:0:0, there were clear differences among the biosorption capacities. In all the examined systems (other than the 0:0:4 system), inhibition of the binding of microelement cations by the macroalga was observed. In all the systems, the highest value of qmax was obtained for Cu(II) cations; the value ranged from 31.9 mg g−1 in 0:0:4 (SO42− only) to 18.2 mg g−1 in 4:0:0 (Cl− only).

The biosorption of metal ions by algae has been widely reported in the literature. However, the majority of the published work describes single-metal biosorption systems. Very little information is available on multi-metal biosorption systems such as binary [1][2][3][4][5], ternary [1,[4][5][6], and quaternary systems [7]. There is a necessity to perform experiments on such systems because they better reflect real effluents from industrial operations. Another issue, which is often neglected in the literature, is the investigation of the effects of anions on biosorption processes. This aspect should also be taken into consideration because the presence of anions in aqueous solutions could affect metal cation biosorption [8].
In the literature, two aspects of the effects of anions on biosorption processes are considered: the effects of anions on the maximum biosorption capacity in single-metal systems [9], and the effects of anion concentration on the biosorption of several metal ions in multi-metal systems [8,10,11]. It is important to emphasize that the influence of the anion on the biosorption capacity could differ depending on the biomass used and the biosorbed metal ions. The anions NO 3 and SO 4 2did not significantly influence the removal efficiency of the fungus Aspergillus niger for Cr(VI), Co(II), Ni(II), and Zn(II) ions, whereas the presence of Clanions significantly decreased the efficiency of metal ion biosorption in multi-metal systems [10]. In the case of another fungus, namely Rhizopus arrhizus, the degree of inhibition of the biosorption of La(III), Cd(II), Pb(II), and Ag(I) cations generally followed the order EDTA > SO 4 2-> Cl -> PO 4 3-> glutamate > CO 3 2- [12]. During biosorption of Co(II) cations by the brown macroalga Ascophylum nodosum, the presence of SO 4 2and PO 4 3anions did not result in any change in biosorption, in contrast to NO 3 anions, which were the strongest inhibitor [13]. The opposite situation was observed in the case of biosorption of Zn(II) cations by the cyanobacterium Oscillatoria anguistissim, for which the inhibitory order of the anions was as follows: SO 4 2-> Cl -> NO 3 -(i.e., SO 4 2-anions were the strongest inhibitor) [11].
As mentioned above, the influence of the anion on biosorption capacity could also differ depending on the metal ions biosorbed; for example, Han et al. [8] observed the following inhibitory orders for the biosorption of Cr(VI) and Cr(III) ions, respectively: NO 3 − > Cl − > SO 4 2− and SO 4 2− > Cl − ≈ NO 3 − . In the present paper, these two aspects were taken into consideration to better understand the mechanism of the biosorption of metal ions by Enteromorpha prolifera. This edible marine macroalga, enriched with microelement cations (Zn(II), Cu(II), Co(II), and Mn(II)) via biosorption is used as a biological, mineral feed additive for livestock. Previous experiments on the kinetics and equilibrium of the biosorption revealed that this process is complex and depends not only on the process parameters such as pH, temperature, biomass concentration, and concentration of metal ion [14,15], but also on the physicochemical properties of the metal ions, properties of the biosorbent, and probably on the presence of competing metal ions and the types of anions. For the application of the biosorption process on an industrial scale, detailed experiments are required. The main goal of this paper was to determine whether the type of anion and its concentration in aqueous solution influences the amount of metal cations bound by the Enteromorpha prolifera biomass. The biosorption of microelement cations was carried out in single-and multi-metal systems. In the single-metal system, the effects of anions (Cl − , NO 3 − , and SO 4 2-) on biosorption of Co(II) cations were checked. As an example of a multi-metal system, a quaternary-metal system was chosen because, from the economic point of view, it is beneficial to enrich macroalgal biomass simultaneously with all the cations essential for animals (Cu(II), Zn(II), Co(II), and Mn(II)). These experiments allowed us to determine not only the effects of anions and their concentrations on biosorption performance, but also to investigate the competition between microelement cations for cation binding sites on the surface of macroalgal cells.

Sorbent preparation
The alga Enteromorpha prolifera was collected from the Baltic Sea (Niechorze, Poland). It was identified at the Department of Botany and Plant Ecology of Wrocław University of Environmental and Life Sciences. The collected algal biomass was washed with tap water several times to remove foreign matter, and then three times with deionized water. The biomass was then dried at 60°C until a constant mass was reached (to ensure there would be no bioaccumulation process). The biomass of dry alga was ground and used in biosorption experiments.

Batch biosorption experiments
The biosorption experiments were performed in Erlenmeyer flasks containing 20 mL of microelement cation solution in a thermostated water bath shaken at 100 r/min using the single-and multi-metal systems shown in Table 1.
In the first system (No.1) the concentration of Co(II) cations (chosen as an example) ranged from 25 to 400 mg L -1 . In the multi-metal systems, the concentration of each cation, i.e., Cu(II), Zn(II), Co(II), and Mn(II) in the solution was the same. In systems No.   [14]. The biomass concentration was 1.0 g L -1 .

Analytical methods
The concentrations of metal cations in the solutions before and after the biosorption process were determined by ICP-OES (Inductively Coupled Plasma-optical Emission Spectrometry; Varian VISTA-MPX, Australia) in the Laboratory of Multielemental Analyses at Wrocław University of Technology, which is accredited by the International Laboratory Accreditation Cooperation-Mutual Recognition Arrangement (ILAC-MRA) and the Polish Center for Accreditation (PCA) (No.AB 696). For the preparation of standard solutions (1.0, 10, 50, and 100 mg L -1 ) a multielemental standard (100 mg L -1 Astasol®, Prague, Czech Republic) was used. The samples were analyzed three times and the standard deviations of the measurements did not exceed 5% [16].

Statistical analysis
Statistical analysis of the experimental data was performed with the STATISTICA (v.8) software (StatSoft, Cracow, Poland).

Biosorption in single-metal systems
The Langmuir eq. (1) was used to model the equilibrium between metal ions adsorbed on the biomass and metal ions in the solution at a given temperature [17]: where q eq is the mass of metal ions adsorbed per gram of biomass at equilibrium (mg g -1 ), C eq is the residual equilibrium metal ions concentration in the solution (mg L -1 ), q max is the maximum possible mass of metal ions adsorbed per gram of adsorbent (mg g -1 ), and b is a constant related to the affinities of binding sites for the metal ions (L mg -1 ). Figure  1 shows the influence of the salt anion on the biosorption of Co(II) cations in a single-metal system by Enteromorpha prolifera. From Figure 1, it can be seen that different anions of Co(II) salts did not significantly influence the biosorption capacity of the macroalga. The highest maximum biosorption capacity, qmax (calculated using the Langmuir equation), was obtained for CoCl 2 ·6H 2 O, 46.0 mg g -1 , then for Co-SO 4 ·7H 2 O, 42.8 mg g -1 , and finally for Co(NO 3 ) 2 ·6H 2 O, 41.9 mg g -1 . The average affinity (b) of the macroalgal biomass towards Co(II) cations was (0.062 ± 0.003) L mg -1 .

Figure 1
Influence of anion on the biosorption of Co(II) cations in single-metal system (No.1).
To check whether the values of q max for Co(II) cations differed significantly among the examined groups (Cl -, NO 3 -, and SO 4 2-), the variation coefficient (VC) was calculated and it was equal 4.95%. VC was less than 10%, so the difference was not statistically significant [18]. This was also confirmed by the application of Tukey's test. ; it was assumed that for statistically significant differences P < 0.05.
However, the influence of the anion on the biosorption capacity could differ depending on the type of biomass used and the metal ions biosorbed because the mechanisms differ. Pulsawat et al. [9] showed that q max of microbial extracellular polymeric substances (EPS) towards Mn(II) cations decreased in the sequence SO 4 2-(62.4 mg g -1 ) > NO 3 -(52.5 mg g -1 ) > Cl -(20.5 mg g -1 ) .

Biosorption in multi-metal systems
The experiments on the biosorption of the microelement cations Cu(II), Zn(II), Co(II), and Mn(II) in multi-metal systems were performed according to the scheme presented in Table 1. The systems differ not only in the types of anions, but also in the total anion concentration in the solution.
The first system contained four SO 4 2-, the second contained two SO 4 2-, one Cl -, and one NO 3 -, the third contained three Cland one SO 4 2-, and the fourth contained four Cl -. The biosorption isotherms of the examined microelement cations from salts with different anions are presented in Figure 2.
In all the examined systems (except No.2), inhibition of biosorption, especially of Co(II), Zn(II), and Mn(II) cations, was observed. To describe the experimental data, a modified Langmuir model with inhibition (2) was proposed: where Ki is the inhibition constant (mg L-1). This is a simple, empirical model, with one additional constant, K i , which enables us to model biosorption in multi-metal systems  (1) to compare these two models. The parameters of the Langmuir model with inhibition and those of the Langmuir model are presented in Table 2.
Higher values of R 2 were obtained using the Langmuir model with inhibition; however, the q max values for the microelement cations determined using the Langmuir equation in system No.2 and for Cu(II) cations in the other systems better reflect the experimental data. Figure 3  To find the correlations between the parameters in the Langmuir equation and inhibition, a correlation matrix was performed Table 3.
Additionally, statistically significant differences in the q max values of microelement cations were confirmed by Tukey's test. The obtained results are presented in Table 4. For all examined microelement cations, statistically significant differences were observed between systems Nos.2 and 5.
In all examined systems, the order of the maximum biosorption capacity of Enteromorpha prolifera towards the examined microelement cations was as follows: Cu(II) > Zn(II) > Co(II) > Mn(II). To choose the best system, the q max values of each cation in the studied systems were compared; the orders are as follows: Cu(II), No.2 > No. 3 4. These results showed that the same order was obtained for the cations whose biosorptions were inhibited, i.e., Zn(II), Co(II), and Mn(II). The best results were obtained in a system with different anions, namely No.3 (Cl -: NO 3 -:      Taking Cu(II) cations as an example, the effects of the anions and also of the physicochemical properties of the adsorbed metal cations on the biosorption properties of Enteromorpha prolifera were explained. Comparing the values of q max of macroalga towards Cu(II) cations, calculated from the Langmuir equation, it can be seen that the highest maximum biosorption capacity was obtained in system No.2: 31.9 mg g -1 (SO 4 2only) and the lowest was obtained in system No.5: 19.9 mg g -1 (Clonly). This could be explained by the hypothesis that with increasing concentrations of chlorides in the solution, q max of Enteromorpha prolifera towards Cu(II) cations decreased. In the literature, it has been reported that among all the investigated anions, Clions form the most stable complexes with the examined metal ions [19]. In Figure 4, the effects of increasing concentrations of Cland SO 4 2anions on the biosorption capacity of Cu(II) cations are presented. It was found that with increasing amounts of chloride anions in the solution, qmax of the Cu(II) cations decreased linearly (R = 0.845), and with increasing amounts of sulfate anions, q max of the Cu(II) cations increased linearly (R = 0.955).
As mentioned above, biosorption of microelement cations by algae could be influenced not only by the type of anion and its concentration in the solution, but also by the physicochemical properties of the adsorbed metal ions. The high affinity of Enteromorpha prolifera for Cu(II) cations could result from the ionic characteristics of Cu(II). Nieboer and McBryde et al. [20] proposed a covalent index, which was calculated using the equation: where X m represents the electronegativity of the ion, IR is the ionic radius, and 0.85 is a constant assumed to reflect the ionic radii of O and N donor atoms. In general, the greater the covalent index of the metal ion, the greater is its potential to form covalent bonds with biological ligands such as carboxyl, hydroxyl, amino, and sulfhydryl groups on the biomass surface, and the higher is its biosorption capacity. The covalent indexes of the four metal cations studied are in the order Cu(II) > Co(II) > Zn(II) > Mn(II) (5.70 > 5.65 > 4.36 > 3.65) [21], and the order of the q max values of Enteromorpha prolifera in system No.2 is Cu(II) > Co(II) > Zn(II) > Mn(II) (0.502 mmol g -1 > 0.166 mmol g -1 > 0.157 mmol g -1 > 0.142 mmol g -1 , determined using the Langmuir equation).
Moreover, among all the microelements studied, Cu(II) cations were characterized by the lowest value of |logK OH | (the absolute value of the logarithm of the first hydrolysis constant; |logK OH |: Mn(II), 10.6; Co(II), 9.7; Zn(II), 9.0; Cu(II), 8.0 [21]), which reflects the affinity of metal ions towards ligands [22]. With increasing |logK OH |, q max decreased (e.g., in system No.2, q max of the Cu(II) cations was three times higher than that of the Mn(II) cations, determined using the modified Langmuir equation). Nevertheless, it was found that there was a linear relationship in each system studied between q max (from the modified Langmuir equation) and |logK and PO 4 3anions in a Co(II) solution at pH 4.5 did not result in any change in biosorption, but CO 3 2suppressed the Co(II) uptake capacity of the biosorbent by 4%-14%. NO 3 anions proved to be the strongest inhibitor of biosorptive Co(II) uptake, leading to approximately 35% suppression. In the case of the macroalga examined in our study, i.e., Enteromorpha prolifera, inhibition of the absorption of Co(II) cations was observed in system No.3, where the Co(II) is derived from Co(NO 3 ) 2 6H 2 O, in contrast to system No.2, in which Co(II) was present as CoSO 4 7H 2 O. In system No.2, there was no inhibition of Co(II) ions, because all the cations were presented in the solution as a SO 4 2-. As mentioned above, the effects of anions on biosorption capacity also differ for various biosorbents. For example, Ahuja et al. [11] observed the following inhibitory order of anions on Zn(II) cation biosorption by the cyanobacterium Oscillatoria anguistissim: SO 4 2-> Cl -> NO 3 -. SO 4 2-and Clions in the concentration range 0-10 mmol L -1 decreased Zn(II) biosorption by factors of seven and two, respectively, whereas NO 3 ions did not affect the biosorption of Zn(II) to any significant extent (decreased by 10.5%). Also, Tobin et al. [12] observed inhibition of La(III), Cd(II), Pb(II), and Ag(I) cations uptake by the fungus Rhizopus arrhizus in the presence of anions in the solution. No anion was found to enhance metal uptake levels, and the degree of inhibition generally followed the order EDTA > SO 4 2-> Cl -> PO 4 3-> glutamate > CO 3 2-.

Conclusion
The performed experiments on biosorption of microelement cations in single-and multi-metal systems showed that in the single-metal system the type of anion has no significant influence on biosorption capacity, but in the multi-metal systems, differences were seen. The maximum biosorption capacity of Enteromorpha prolifera towards Co(II) cations decreased in the sequence Cl -(46.0 mg g -1 ) > SO 4 2-(42.8 mg g -1 ) > NO 3 -(41.9 mg g -1 ). The differences in q max values were not statistically significant because the value of VC was smaller than 10% (4.95%). The opposite situation was observed in multi-metal systems. It was confirmed that the differences in the values of q max for each microelement cation were statistically significant. For Cu(II), Zn(II), Co(II), and Mn(II) cations, statistically significant differences were observed between systems No.2 (Cl -: : SO 4 2-= 4 : 0 : 0), inhibition was observed. Therefore, a new modified Langmuir equation with inhibition was proposed. It was observed that the higher value of inhibition constant, the smaller inhibition of binding metal cations by macroalga is. In all examined systems, maximum biosorption capacity of Enteromorpha prolifera towards examined microelement cations was as follows: Cu(II)>Zn(II)> Co(II)>Mn(II).
Using Cu(II) cations as an example, it was shown that Clanions decreased the efficiency of metal ion biosorption in multi-metal systems to the largest extent. The highest value of q max was obtained in system No.2, 31.9 mg g -1 (SO 4 2only) and the smallest in system No.5, 19.9 mg g -1 (Clonly). The total quantities bound by the Enteromorpha prolifera biomass were as follows: system No.3 (1 : 1 : 2), 1.25 mmol g -1 of Cu(II), Zn(II), Co(II), and Mn(II) cations, in No.2 (0 : 0 : 4), 1.12 mmol g -1 , in No.5 (4 : 0 : 0), 1.03 mmol g -1 , and in No.4 (3 : 0 : 1), only 0.761 mmol g -1 . These results indicated that future experiments should be performed using different anions (No.3) or SO 4 2-(No.2). However, system No.2 is more beneficial if the economic aspects are taken into account because microelements in the form of sulfates are much cheaper than those in the form of chlorides or nitrates. (R05 014 01 and N N209 146136).