Algae Modified Alginate Beads for Improved Cd(II) Removal from Aqueous Solutions

Abstract

The aim of this research was to synthesize alginate beads. The beads were modified with a mixture of three different species of algae. Both synthesized beads were evaluated for the efficiency of Cd(II) removal from aqueous solutions as one of the currently most sustainable metal removal methods. The focus was on the characterization of synthesized beads and their stability. The characterization was performed using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The specific surface area was determined. Cd(II) ion standard solutions were brought into contact with unmodified and modified beads. The experimental results showed that the most influential factors on biosorption are pH value and temperature. The maximum biosorption of Cd(II) ions was achieved at 181.0 mg/g. Kinetic and thermodynamic studies were carried out. The data obtained followed pseudo-second-order kinetics.

1. Introduction

The heavy metals, including Cd(II), are persistent environmental pollutants with significant health risks. Various physico-chemical methods are used for their removal from water. Traditional physico-chemical methods such as ion exchange, membrane separation, adsorption, coagulation, and extraction are commonly used but have notable drawbacks, including high cost, energy consumption, and environmental harm due to the input of various chemicals [1]. Consequently, there is a pressing need for novel, more sustainable treatment methods. Biosorption, which utilizes bacteria or algae immobilized on various natural materials, has emerged as a promising alternative. The advantages are low cost, high efficiency, and sustainability [2]. The algae are particularly effective in biosorption due to their functional groups, such as hydoxy, carboxy, and sulphate, which are able to bond metal ions [3]. Microalgae-derived biochar pyrolyzed at over 500 °C showed good potential as an adsorbent with high removal efficiencies for Cu(II), Cd(II), and Zn(II) ions in aqueous solution [4]. The removal capacity of microalgae is significantly influenced by several factors, including the concentration of heavy metals, temperature, biomass concentration, and pH [5]. Biosorption capacity could be increased by the immobilization of algae [6]. Some examples of natural immobilization media that have been used for algal cell immobilization include chitosan, alginates, cellulose material, etc. Na-alginate is among the most commonly used media. Microbial biomass cannot be used directly in a standard metal sorption process. Cell immobilization may enhance biosorption capacity due to increased surface area [1]. It was reported that the elevated content of organic material did not result in enhanced bioremoval efficiency due to exceptionally low adsorption capacity under those conditions [7]. Cd(II) removal efficiency was negatively correlated with microalgal specific growth rate. Therefore, dead biomass in the form of powder can also be used, though it may have lower efficiency compared to live biomass [8]. Research on Cd(II) removal using algae-based Chitosan bio-composites in model solution has shown promising results [9]. Under optimized conditions (pH 5.5; contact time, 120 min), an adsorption capacity of 22.34 mg/g of Cd(II) was achieved. A much higher capacity of 215.8 mg/g of Cd(II) on calcium alginate has been reported [10]. Other studies have developed composite gel balls of ball-milled biochar and calcium alginate for efficient Cd(II) removal from water. The World Health Organization (WHO) sets limit for Cd(II) in drinking water at 0.005 mg/L, underscoring the critical need for effective removal techniques to ensure water safety [11].

The main objective of the work presented was to study Cd(II) removal from aqueous solutions. The novelty here was to synthesize the Na-alginate beads immobilized with a powdered mixture of three different algae species, Spirulina, Chlorella and Lithothamnium, commercially available as capsules. The synergistic effect of such an algae mixture on Cd(II) removal was investigated, while in our previous study, the toxicity effect of metals on cells of living algae was noticed [1]. It is expected that the algae modification of alginate would alter its surface properties and hence increase Cd(II) removal efficiency. The beads were characterized by specific surface area measurement, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) analyses. The efficiency of Cd(II) removal from aqueous solutions was determined and kinetic and thermodynamic studies were performed.

2. Materials and Methods

2.1. Beads Synthesis

Alginate beads, non-immobilized and immobilized with powdered algae mixture were prepared. An algae mixture of three different types was applied: Chlorella, Lithotamnium and Spirulina. They were combined together into a powdered material mixture (NutriLab 3 Alge, Slovenia). Sodium alginate was used for algae immobilization. Firstly, 2% Na-alginate solution was prepared, and 2 g of algae was added. Na-alginate was added into 2% CaCl₂ solution and spherical beads were formed. The immobilized were green-colored and those without algae were colorless as seen in Figure 1. Both beads were left in jars for 24 h and then washed with 0.85% NaCl and distilled water.

The Na- alginate shown in Figure 2 is a polysaccharide of brown algal extract. It is a widely used and inexpensive material as well as non-toxic [6]. The polymer is composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) in different properties. M and G sequences can be seen in the composition, with an occasional MG sequence [12]. Among its advantages, its solubility in cold water is important, as well as the fact that it forms thermally stable gels [12]. The gel is formed with polysaccharides, usually Na-alginate [6]. Sodium alginate in the form of dry powder can be stored without degradation in a cool, dry place and away from sunlight for several months.

2.2. Characterization

The Brunauer, Emmett and Teller method (BET) based on gas adsorption measurements was used to generate the surface area results. The data were evaluated to determine the surface area and pore size distributions of the material. A Micromeritics ASAP 2020MP apparatus was applied for gas adsorption at −196 °C after degassing the sample at 343 K for 11 h under vacuum.

FTIR was used for characterization. Samples were dried at 60 °C and cooled to room temperature and FTIR was performed. The samples were analyzed with the spectrometer ATR FTIR Perkin Elmer SpectrumGX (Perkin Elmer FTIR, Omega, Ljubljana, Slovenia). The ATR accessory (supplied by Specac Ltd., Orpington, Kent, UK) contained a diamond crystal. A total of 16 scans were taken of each sample with a resolution of 4 cm⁻¹. All spectra were recorded at ambient temperature over a wavenumber range between 4000 cm⁻¹ and 400 cm⁻¹.

The specimens were observed using a Quanto 2003D FEI scanning electron microscope, an energy dispersive X-ray microanalysis was performed on an Sirion 400 FEI instrument (Oxford Instruments, UK) at 15 kV, and SEM was performed.

2.3. Adsorption Experiments

Working solutions of Cd(II) ions were prepared by diluting standard stock solution (γ= 1 g/L Cd(II)), using Cd(NO₃)₂.4 H₂O (Sigma-Aldrich, Hohenems, Germany). In a 250 mL flask, a series of concentrations from 1 mg/L up to 100 mg/L were prepared by dilution with MilliQ water. The determined mass of algae was added into the flask and shaken for 24 h at 250 rpm. For the sorption tests, 15.0 ± 1.5 g of wet algal beads was weighed. In the next step, the samples were filtered through a GF-3 filters (glass fiber). Then, the samples were used in the analysis. The concentration of Cd(II) ions was determined using a Perkin-Elmer Aanalyst 400 atomic absorption spectrometer following standard procedure [13]. A calibration curve was prepared using standard Cd(II) solutions. pH was adjusted using 0.1 M HCl or 0.1 M NaOH when required using a Multi 3410 WTW pH meter. The amount of Cd(II) adsorbed q_e (mg g⁻¹) was determined by using Equation (1) as follows:

$$q_{\mathrm{t}}={\displaystyle \frac{(c_0-c_e)·V}{m}}$$

(1)

where c_o and c_e represent the initial and equilibrium concentration of Cd(II) ion (mg/L), V is the volume of the Cd(II) ion in solution (L) and the m is the mass of dry adsorbent (g). The Cd(II) removal percentage can be calculated as follows:

$$q_{\mathrm{t}}={\displaystyle \frac{(c_0-c_e)}{c_0}}·100$$

(2)

The influence of pH, initial Cd(II) ions concentration, contact time and temperature was studied to optimize the adsorption process. The influence of the initial pH was examined by pH values adjusted to 4 and 5 based on the literature results [1] at a Cd(II) concentration of 25 mg/L, temperatures of 298, 308 and 318 K, and four different contact times (1, 2, 3, and 4 h). The influence of initial Cd(II) concentration on adsorption was examined using Cd(II) solutions of different concentrations: 25, 50, 75, 100, 150, 200, and 250 mg/L.

Several biosorption–desorption cycles were performed under the same conditions. During the biosorption experiments, the pH was adjusted to 5. The initial metal concentration was 25 mg/L and the contact time was 3 h. The desorption of each metal was performed by using appropriate amounts of 0.02 M EDTA solution in contact with the biosorbent at room temperature and stirred at 250 rpm for 2 h. The algal beads were then washed with MilliQ water. In the next step, the regeneration was performed with 2% CaCl₂ solution (60–120 min). This step was important to strengthen the beads. Before being used in the next biosorption cycle, the algal beads were washed firstly with MilliQ water, secondly with 0.85% NaCl, and the third time with MilliQ water. All experiments were conducted in triplicate and the results were reported as an average.

2.4. Kinetic Studies

Kinetic studies involved the optimum determined pH and initial concentration in different contact time units. The parameter q_e of the pseudo-second-order kinetic model was determined from the slope and the parameter k₂ from the intercept of the linear graph t/qt versus t, whilst for the pseudo-first-order model, the linear graph of log(q_e − q_t) against t was applied [14].

q_t = q_e(1 − e^−k1t)

(3)

where

• q_t sorption capacity at time t (mg/g);
• q_e sorption capacity at equilibrium (mg/g);
• k₁ pseudo-first-order reaction rate constant.

  $$q_{\mathrm{t}}={\displaystyle \frac{k^2{q_e}^{2}t}{{1+k}^2{q}_{e}t}}$$

  (4)
• k₂ pseudo-second-order reaction rate constant [14].

Adsorption isotherms were constructed using Langmuir, Freundlich and Temkin (Equation (7)) models. The Langmuir isotherm is based on the assumption that maximum adsorption occurs when a saturated monolayer of solute molecules is present on the adsorbate surface (Equation (5)) [3]:

$${\displaystyle \frac{1}{q_e}}={\displaystyle \frac{1}{Q_0}}+{\displaystyle \frac{1}{b{Q}_0{c}_e}}$$

(5)

where

• c_e the equilibrium concentration of adsorbate (mg/L);
• Q₀ the Langmuir constant regarding maximum specific uptake (mg/g);
• b the Langmuir constant related to the affinity of binding sites to the metal ion (L/mg) [14].

• The Freundlich isotherm describes non-ideal and reversible adsorption (Equation (6)):

$$ln{q}_e=ln{K}_F+{\displaystyle \frac{1}{n}}ln{c}_e$$

(6)

where

• q_e the amount of Cd(II) adsorbed at equilibrium;
• K_F Freundlich constant ((mg/g)/(mg/L)ⁿ);
• 1/n the adsorption intensity of surface heterogeneity that ranges between 0 and 1, becoming more heterogeneous as its value gets closer to zero [14].

• The Temkin isotherm contains a factor that explicitly takes into account adsorbent–adsorbate interactions (Equation (7)):

$$q_e={\displaystyle \frac{RT}{b_T}}ln{A}_T+{\displaystyle \frac{RT}{b_T}}ln{c}_e$$

(7)

where

• q_e the amount of Cd(II) adsorbed at equilibrium;
• b_T the Temkin constant related to the heat of sorption (kJ/mol);
• T the absolute temperature (K);
• R the universal gas constant (8.314 J/mol K);
• A_T the Temkin isotherm constant (L/g) [15].

2.5. Thermodynamics of Sorption

Based on the thermodynamics, the relation between Gibbs free energy (ΔG^o) and the Langmuir constant (b) is given by Equation (8) [3]:

ΔG^o = −RT Ln (b)

(8)

Enthalpy (ΔH^o) and entropy changes (ΔS^o) in the reaction at constant temperature are related to the ΔG^o according to Equations (9) and (10) as follows:

ΔG^o = ΔH^o − TΔS^o

(9)

$$\mathrm{Ln}(b)={\displaystyle \frac{{\mathsf{\Delta }S}^{\mathrm{o}}}{R}}-{\displaystyle \frac{{\mathsf{\Delta }H}^{\mathrm{o}}}{RT}}$$

(10)

where b can be considered as the Langmuir constant [16]. The parameters in Reaction (10) determine whether the reaction is spontaneous or energy is required, as well as the random particles introduced into a system during the biosorption process.

The ΔH^o and ΔS^o values of sorption were measured from the slope and the intercept of the plot between Ln (b) and 1/T. Here, the b value was taken as Langmuir constant b, obtained for sorption by non-immobilized alginate beads and powdered algae mixture-immobilized alginate beads (

Table 1. Freundlich, Langmuir and Temkin constants for immobilized beads.

Model	Konstants	Value
Freundlich	KF[(mg/g)(mg/L)n]	4.498
	n	1.595
	R2	0.975
Langmuir	qm (mg/g)	181.0
	b (L/mg)	0.0108
	R2	0.998
Temkin	bT	13.9
	A (mol/g)	99.8
	R2	0.866

).

3. Results

3.1. Characterization Results Study

All surface area analyses indicate the enhancement of the surface area due to algae immobilization. The specific surface area of the algae-immobilized alginate beads was measured at 578 m²/g, while alginate beads had 4.4 m²/g. Immobilization thus enabled a much larger surface area. The pore sizes of the beads with or without algae amounted to 15 nm. The results were very close to those reported in the literature [1]. It was reported that algal cell polysaccharide compounds may be dissolved in the outer layer of the algal cell wall and therefore produce additional binding sites [16].

Figure 3 represents the immobilized bead with an algae micrograph. The surface seemed to be uniform and very homogeneous. Algae beads contain different chemical elements that form the cell such as peptidoglycan, teichoic acid, polysaccharides, and proteins with numerous functional groups [1].

Figure 4a represents the FTIR spectra of alginate beads and biosorption on alginate beads. From the FTIR spectra, the peaks at 1594 cm⁻¹, 1416 cm⁻¹ and 1013 cm⁻¹ typical of alginate are evident. At 1594 cm⁻¹ there are COO^- group stretching vibrations, and at 1416 cm⁻¹, OH- group stretching vibrations [17]. The peaks at 1073 cm⁻¹ and 1013 cm⁻¹ correspond to the C-O-C glycosidic linkage of polysaccharide in alginate [18]. The difference in both spectra is in peak at 669 cm⁻¹, which disappeared after adsorption, probably due the adsorption of Cd(II) on a free functional group. The peaks were slightly shifted after adsorption, e.g., from 564 cm⁻¹ to 573 cm⁻¹, indicating the adsorption of Cd(II).

Figure 4b represents the spectra of immobilized beads and biosorption on immobilized beads. The same peaks as seen in the FTIR spectra of alginate beads remained in these spectra and several similarities could be seen. Further, on both spectra with immobilized beads, there are much more stronger peaks at 2356 cm⁻¹, 1416 cm⁻¹, 1075 cm⁻¹, 1018 cm⁻¹ and 616 cm⁻¹, which are due to the different groups present in the algae, such as CH₃, primary alcohols, halogen groups, etc. Additional peaks were observed at 3600–3000 cm⁻¹ and at 2914 cm⁻¹. A broad and strong band was observed at 3600–3000 cm⁻¹, corresponding to the stretching of -OH and -NH vibrations. The functional groups, such as -OH or -NH, are capable of binding heavy metal ions via ion exchange [17]. The peak at 2914 cm⁻¹ corresponds to CH₂ asymmetric stretching vibration in algal polysaccharides [19]. Comparing the peaks before and after sorption, the spectra are similar, except at 551 cm⁻¹, where an additional peak appeared, which could be due the adsorption of Cd(II) on immobilized beads.

3.2. Study of Influential Factors

pH influence was studied in the pH range from 2 to 8. The results in Figure 5a showed the optimum at pH = 5 in accordance with the literature [20]. If the pH was adjusted to acidic values, the removal was high due to the protonation of functional groups. The initial step is mostly affected by Cd(II) ion concentration, the time of stirring and rate Cd(II) ions at pH = 5. Our results are comparable with previous studies on green, red and brown algae, where the maximum Cd(II) uptake was obtained within the range 0.5–1.17 mmol g⁻¹ at pH values between 4.0 and 5.5 [21].

The form of Cd(II) species that is mainly present in water solutions is Cd(II), which is the most represented species at an acid pH ranging from 3.5 to 5 [22]. When the pH value gradually increases, more reactive groups will be negatively charged, thereby providing more adsorption sites [23]. At basic pH values above 7, Cd(II) ions have a tendency to precipitate as Cd(OH)_2. At increasing pH values, more amino and carboxyl algae groups are negatively charged and metal ions are positively charged, and hence, biosorption onto the cell surface increase. When the pH of the solution exceeds the metal ion solubility product constant, a large amount of Cd(II) ions in the solution will generate oxide precipitation Cd(OH)₂, making the adsorption process impossible. The pK_a value of carboxylic groups in the alginate ranged from 3.4 to 3.9, meaning that the alginate became positively charged if the pH was below these values [10]. Thus, all subsequent sorption experiments in this study were conducted at pH 5.0.

The results of contact time influence are shown in Figure 5b. Equilibrium was not reached until 240 min; however, the difference to the value at 180 min was negligible. The biosorption capacity was almost constant after 240 min, reaching equilibrium conditions. The reason for this is its relative inert surface and the fact that does not favor chemical bonds. The adsorption capacity increased rapidly after the first 30 min on both non-immobilized and immobilized beads. In equilibrium, the removal efficiency reached 80% of the initial Cd(II) concentration. The removal efficiency of Cd(II) without microalgae was significantly lower than that with microalgae treatment. The equilibrium was attained in 180 min at an adsorption efficiency of 34%. Its degree of increase in the next hour was negligible.

The results are in accordance with other studies, where in the first phase, the transport of the metal ions from the solution to the sorbent takes place [22]. Vacant sites are mainly on the surface of the algae layer and become more saturated with time.

The influence of the temperature was studied at 298 K, 308 K and 318 K. The sorption capacity of alginate beads and adapted bacterial consortia-immobilized in alginate beads was maximum at 318 K (Figure 6). The effect of temperature on Cd(II) solubility was tested by experiments, which were performed without the sorbent and were noted as control experiments. The non-significant difference in Cd(II) concentration at all three measured temperatures indicates the negligible impact of temperature on Cd(II) solubility (p > 0.05). The optimal temperature at 303 K was previously determined for Cd(II) adsorption onto alginate beads where a lipid extraction algae residue was tested [14].

Previous experiments have shown the Cd(II) concentration was at least toxic to the algae; therefore, we decided to study concentrations ranging from 25 mg/L to 400 mg/L [1]. The results are seen in Figure 7. The sorption capacity of alginate beads did not increase much with increasing concentrations, only for up to 5%, meaning a capacity of 44 mg/g, while that of the bacteria-immobilized alginate beads increased to 181 mg/g, with an increase in sorbent concentration at 250 mg/L. With the increase in Cd(II) concentration from 250 to 400 mg/L, a decreasing trend in sorption capacity was observed.

3.3. Adsorption Isotherms

Langmuir, Freundlich and Temkin isotherm models were applied to investigate the adsorption process. Figure 7 shows the equilibrium biosorption by plotting q_e vs. C_e. The shape of the curve suggested that the active vacant sites for adsorption were gradually filled and eventually became saturated after increasing the metal concentrations of Cd(II), resulting in the plateau nature of the curve. Such an adsorption isotherm type suggest monolayer adsorption [23]. From the data shown in Figure 7, the constants of the Langmuir, Freundlich and Temkin isotherms were calculated according to Equations (5)–(7). The calculated constants are seen in Table 1.

A comparison of the results in Table 1 demonstrated that the Langmuir isotherm better fits the adsorption, with R² = 0.998 compared to the Freundlich isotherm (R² = 0.975) and Temkin isotherm (R² = 0.866), respectively. The maximum adsorption capacity q_m according to the Langmuir model was very high q_m = 181 mg/gas, as seen in Table 1. The suitability of the Langmuir isotherm model prescribes monolayer coverage of the surface of the immobilized alginate by Cd(II) [10]. This assumption is valid because the surface area of immobilized alginate beads for metal adsorption is high. On the other hand, the surface area of alginate was much lower, and thus, a more than four times lower maximum adsorption capacity was determined, as seen in Figure 7.

3.4. Adsorption Kinetic Studies

The interparticle model and pseudo-first-order and pseudo-second-order kinetics were applied to evaluate the kinetics data. The rate constant values, k₁ and k₂, were obtained from the plots presented in Figure 8 and calculated according to Equations (3) and (4). The correlation coefficient R² = 0.9976 suggests that a pseudo-second order for bacteria-immobilized alginate beads was a better fit than a pseudo-first order with R² = 0.8618. The pseudo-second order of adsorption occurred due to the migration of ions from the bulk to the interface, with chemisorptions involving valence forces through the sharing or exchange of electrons between alginate beads/powdered algae mixture-immobilized alginate beads and the Cd(II) species [24]. This indicates that, in the presence of a large excess of Cd(II), the rate of sorption highly depends on the capacity of the sorbent, which in turn depends on the available sites for binding or ion exchange [10]. Therefore, the rate limiting step for sorption is the sorbent capacity. Some studies showed that, firstly, during the rapid phase, physical adsorption on the surface occurs, followed by slower chemisorption in the second phase, known as the interparticle diffusion model [25]. In our experiments, the data showed better fit to the pseudo-second-order model then pseudo-first-order and interparticle model. The results are in accordance with Wang’s study [10]. The pseudo-second-order adsorption kinetics is used to accommodate some adsorption processes that require a longer time to fill the adsorption sites [26]. The results showed that 180 min is required to reach equilibrium, which is indeed much longer compared with, e.g., activated carbon. Compared to the unmodified alginate, the pseudo-second-order model was found to be the most suitable.

3.5. Thermodynamic Study

The changes in ΔG^o, ΔS^o, and ΔH^o are gathered in

Table 2. Thermodynamic parameters at different temperatures.

T (K)	ΔGo (kJ/mol)	ΔSo (J/molK)	ΔHo (kJ/mol)
298	−10.9	6.1	−12.7
308	−10.4
318	−10.1

, calculated from Equations (6) and (7).

ΔG^o was negative at all three studied temperatures, and the process was spontaneous [3,27]. The most negative value was achieved at the temperature 298 K. A negative ΔH^o value shows that the adsorption reactions were exothermic in nature, confirming more efficient biosorption at lower temperatures [1]. If the value of ΔS^o is positive, this indicates that the particles are randomly introduced at the solid–liquid interface due to the release of water molecules, ion exchange and Cd(II) ions binding in agreement with the literature [28]. A positive ΔS^o reflects that a non-significant change occurs in the internal structure and the affinity of the adsorbent material toward Cd(II) adsorption [29]. The adsorption mechanism is primarily physisorption due to the low value of enthalpy. The biosorption of Cd(II) by algae-immobilized beads is predominantly monolayer bio-adsorption, and positively charged Cd(II) can be adsorbed onto algae via an electrostatic interaction with negative functional groups [30].

3.6. Desorption Study

The reusability of biosorbents is gaining interest. The regeneration of alginate beads with algae was performed using 0.02 M EDTA. The study was conducted by five cycles of alternating desorption/sorption tests. The desorption capacities of the cycles were 92, 90, 88%, and 87% in the sequence of four cycles, and then a decrease in desorption for 10% was observed. The change in the beads was also visible after sixth cycle (Figure 9). These results indicate that the immobilized algae could be used repeatedly in Cd(II) sorption studies without any measurable loss in the total sorption capacity. It could be concluded that their reuse up to four times is recommendable. The results are comparable with those reported [31].

4. Conclusions

The synthesis of alginate beads and algae-modified beads was performed for the removal of Cd(II) from aqueous solutions. The characterization of synthesized beads was performed by SEM, FTIR and BET analyses. The SEM image showed the uniform surface of the algae-immobilized alginate beads. FTIR indicated the successful immobilization of algae on alginate beads. The surface area of immobilized beads was more than 1000-fold higher compared to alginate beads. The adsorption capacities of the adsorbents were predicted by the Freundlich, Langmuir and Temkin isotherm models. The maximum adsorption capacities as predicted by a pseudo-second-order reaction were 181 mg/g and 44 mg/g for immobilized alginate beads and alginate beads, respectively. The thermodynamic study showed that the adsorption of Cd(II) onto algae-immobilized beads was spontaneous.