ADSORPTION OF METHYLENE BLUE FROM AQUEOUS SOLUTION USING FREE AND IMMOBILIZED ALGAE CELLS

This study was aimed to investigate the potential adsorptive behavior of mixed algae for decolorization of methylene blue dye from aqueous solution in the form of free and immobilized. Effects of initial pH value (3-9), biosorbent dosage (0.02-0.5)g/L, and initial concentration (10-50) mg/L for different contact time (0-180) min at 200 rpm shaking speed were investigated. In addition, experimental data were analyzed using adsorption (Langmuir and Freundlich) isotherms as well as kinetic models. The results demonstrated that the maximum decolorization percentage was 93% and 91% by free and immobilized algae, respectively for pH 6, 10 initial concentration, 0.3g/100mL algae dosage, and 90 min contact time. The results revealed that the Langmuir isotherm model was adequate for describing the dye removal process (R 2 = 0.99) for both forms. Results of pseudo-second order kinetic model revealed best fitting with the sorption data indicating that chemisorption process is the dominant mechanism controlling the dye removal. Furthermore, the characterization study performed using FT-IR and scanning electron micrograph techniques, revealed that the used algal biomass has good biosorption capabilities associated to active groups and surface structure and confirming that the immobilization was successful.


INTRODUCTION
Dyes or pigments used to color the end products in the textile, paper, plastics, leather, food, and other sectors are present in their effluents, and light penetration and photosynthesis are reduced when these contaminants are present in water.Additionally, some dyes are toxic, mutagenic, or carcinogenic.Different methods have been used for the removal of dyes from wastewater including sorption (8), coagulation and flocculation (25), advanced oxidations (31), biological treatment and nanofltration (33).Among them adsorption is a simple, versatile and feasible process (34;16).The commonly used adsorbent is the activated carbon.However, the price of activated carbon is relatively expansive.In recent years, many studies have carried out to use low-cost adsorbent, through using industrial and agricultural solid wastes (4;10).The use of biological materials, natural substance or biomass as a sorbent in the pollutant removal called biosorption .Biomass has been utilized to adsorb pollution from a variety of sources, including bacteria, yeast, algae, fungi, and plants.Algal biomass (both living and nonviable) is one of the most promising biosorbent under investigation (18).It has been found in many kinds of research that algal biomass is rich in functional groups, such as carboxyl, hydroxyl, phosphate, amine groups, and so on (1).Immobilization is an attractive technique to fix and retain biomass on suitable natural or synthetic support materials.The key advantages of immobilization include biomass retention within the working environment, easy separation of products from cells and relatively, high local cell density (2).Wastewater treatment by immobilized algae has been shown to offer numerous advantages over algal free cells.For example, it takes up less space and overcomes issues during the cell harvesting procedure.Immobilized algae are also more stable and resistant to severe conditions with high salinity, toxicity, and pH value, and recovering the concentrated algal cells for subsequent processes in a less harmful manner can improve cost-effectiveness by reusing the regenerated process (20 Stock solution of MB dye was prepared by dissolving 1.0 g of MB dye in 1L of distilled water to obtain1000 mg/L.Further, from the stock solution, dye solutions were prepared, by dilution.Moreover, the HCl and NaOH solutions of 0.1M enabled the adjustment and control of the pH of the prepared solutions throughout the experimental work.However, for beads preparation sodium alginate of the chemical structure (NaC 6 H 7 O 6 ) and main source algal polysaccharides derivatives (marine-seaweed algae) was manufactured in China and supplied from amazon and CaCl 2 were used.

Algae collection and preparation
A mixture of three classes of algae (abbreviated as MA), was chosen as a model of biosorbent in its nonliving form, and was gathered from the canal near College of Engineering at University of Baghdad where the algae floated at the canal water.The major proportions of the classes of MA samples under analysis are Chlorophyceae (37.77٪),Cyanophyceae (32.88٪), and Bacillariophyceae (29.34%).Thereafter MA was cleaned many times with distilled water to get rid of impurities, dirt, and other undesired elements such as (non-vertebrate animals, small worms, crustaceans, bird feathers), then sun drying for three days, crashed and sieved at mesh no.63 µm, Fig 1a.Agitation of flasks in the form of shaking performed for a definite time using orbital shaker (Edmund Buhler SM25, German).Volume of 10 mL of the contaminated solution was withdrawn from each flask, (filtered for free MA), then the concentration of MB was determined by using UV-visible spectrophotometer (Cary-100 conc., Varian, USA) at a wavelength corresponding to the maximum absorbance of the dye solution (λmax= 665nm).Further, the results obtained from the effects of the biosorbent content and contact time were subsequently applied, for the isotherm and kinetic studies, respectively.During the biosorbent phase, the percentage removal of MB dye, in addition to, the biosorption capacity (q t , mg/g) at any time of experiment (min), can be calculated using Eqs.( 1) and (2), respectively.
) Where: C i and C t refer to initial and equilibrium dye concentration (mg/L), respectively;   ; represent the contaminant uptake, (mg/g), m is the biosorbent mass (g) and V is the contaminant solution volume (L).The experiments were done with duplicate for more accurate result, thereby determining the average results.

RESULTS AND DISCUSSION
Parameters for sorption process Ph: The pH of industrial effluent is a determining element, and its variation can have a direct impact on dye uptake and adsorption.The mechanism of the process alters when the pH of the reaction media changes on a regular or irregular basis.The textile industry wastewater discharge criteria are expected to be in the pH ranges of 6 to 9 (23).The solution pH is a significant issue impacting the subtraction of contaminant from aqueous solutions, affect the functional groups on the adsorbent surface, and determine the solubility of contaminant in the aqueous medium (13).Different pH value (3,5,6,9) were studied while keeping other parameters constant (MB dye of 30mg/L , 0.1g of MA dosage free or immobilized for every 100 mL of dye solution and for contact time not exceeded 120 min).And there results were plotted in Fig. 2. It was observed that, when the pH increased from 3 to 6, an increase in the dye removal efficiency from 75 to 89% by free MA and from 70 to 88% by immobilized MA.Further increase in the pH value to 9 decreases the removal percentage to 77 and 72 by free and immobilized MA, respectively.The mechanism of the process alters when the pH of the reaction media changes on a regular or irregular basis.The textile industry wastewater discharge criteria are expected to be in the pH ranges of 6 to 9.

Fig.2.The removal efficiency of MB dye as a function of pH Biomass dosage
The biomass dosage is a significant parameter used to determine the capacity of biosorbent for specific initial concentration, it is one of the most essential factors that effected the biosorption process (5).The effect of different dosage including (0.02, 0.1, 0.3, 0.5, 0.7) g/100 mL were studied while keeping other parameters constant (pH 6, 30 mg/ L initial MB concentration and for 120 min contact time) and their results were plotted in Fig. 3.It can be observed from this figure that removal efficiency of MB improved with increasing biosorbent dosage.By increasing the amount of algal biomass, the pollutants may be fully adsorb or reach an equilibrium state when reaching a plateau at a fixed concentration of each pollutant (3).The maximum removal efficiency was 93 and 91 for free and immobilized MA, respectively.

Fig. 3. The removal efficiency of MB dye as a function of adsorbent dose Initial concentration and sorption time
Fig. 4 shows the profiles of MB dye removal at various initial concentrations of 10, 30, and 50 mg/l, as well as contact time (0-180min).The analysis indicates that as the initial concentration of the MB dye was increased, the percentage elimination reduced.The decline in dye removal percentage can be attributed to the absence of binding groups on the surface of algal biomass at greater concentrations or to the cell offering an infinite number of surface binding sites, resulting in saturation at higher species concentrations (6).This figure also shows that as contact time increase, the percentage of MA removed rose until it reached 90 minutes, at which point equilibrium was reached for both free and immobilized MA.Adsorption by the outer surface of the sorbent-free and immobilized MA could explain the fast rate of sorption during the first 90 minutes of contact time.The adsorption isotherms are required for the design of adsorption systems (15).At a given temperature, equilibrium isotherm is the equilibrium relationship between the adsorbate concentration in the adsorbent particles and the adsorbate concentration in the fluid phase.The widely used isotherm models Langmuir (Eq.3), and Freundlich (Eq.4), were used in this study for modeled the obtained data from the experiments of MB dye biosorption by free and immobilized MA.The following publications could be reviewed for more information on the physical background of the application of these two formalisms in pollutant sorption research (30)... (4) Where:   (mg/g) is the dye sorbed, q m (mg/g) represents the maximum biosorption capacity of the used biosorbent for target pollutant (uptake); K L (L/mg) is a Langmuir model constant; n(g/L) and K F (mg/g) are the two Freundlich equation constants denoting the biosorption process intensity and biosorbent capacity toward the pollutant's molecules, respectively.The equilibrium parameter, R L , is used to predict if an adsorption system is ''favorable" or ''unfavorable".It is obtained by the following relationship: The value of R L indicates whether the form of the isotherm to be either is unfavorable (R L > 1), linear (R L = 1), favorable (0 < R L < 1) or irreversible (R L = 0).The non-linear isotherm model was used to examine the experimental data.Origin software was used to estimate the nonlinear isotherm model parameters.These models are plotted in Fig. 5 and their parameters with correlation coefficients are presented in Table 2. Based on the value of the coefficient of determination (R 2 ), the obtained data were found to fit well with the Langmuir model for MB dye, indicating that the process was controlled by chemisorption.Furthermore, the equilibrium isotherm for MB dye was of the favorable type (0 <R L < 1) ( 7).

Kinetic parameters
Biosorption kinetic modeling describes the response of a biosorption system caused by variations in experimental conditions, by the properties of biosorbents.The parametric sensitivity of the model to process parameters is also assessed by biosorption kinetic modeling ( 14).In this study, the sorption kinetic was modelled using the pseudo firstorder model (Eq.6) and pseudo second-order kinetic model (Eq.7), and their results were plotted in Fig. 6 and their parameters were tabulated in Table 1, the results present that the two models are capable of explaining the links between the experimental and predicted values; however, the pseudo-second order model for sorption provided a better fit based on R 2 values.This suggests that the sorption process was chemisorption (17).  =   ( −  −   ) ....( 6) Where :q e (mg/g) represents equilibrium amount of MB dye onto CA; q t (mg/g) sorbed amount from the metal at time t; and k 1 and k 2 represent pseudo first order equilibrium rate sorption constant (1/min) and rate constant of the pseudo-second order sorption.(g/mg min), respectively.Algae characteristics (sem)/ (eds): SEM images combined with EDS are of importance to detect the changes in the surface of the MA as powder and beads form before and after loaded with MB dye.The SEM/EDS analysis was carried out in Tehran University using SEM/EDS device (ARYA Electron Optic, FE-SEM).The representative images are shown in Fig. 7.It can be present from this figure that there are a number of cavities and pores found on the surface of algae cell, whereas immobilized cells after treatment of these pores were filled with unknown molecules of colored compounds and calcium present in the aqueous solution which indicated ,that the surface morphology of algae was heterogeneous.The EDS analysis for free and immobilized algae shows C, O, Mg, Al, Ca, and Si elements are present, which alkali and alkaline earth metals that are usually available in algal biomass (12).Increasing the peaks of Ca, C and O after immobilization can be explained by the chemical composition of the algal beads.Alginate, i.e. alginic acid, is a heteropolysaccharide consisting of 1→ 4 linked b-D-mannuronic acid and its C-5 epimer a-L-guluronic acid (26).While increasing Ca 2+ ions was from the CaCl 2 used during the preparation procedure of the biosorbent as the hardening agent of the alginate beads.After adsorption of MB dye, it can be noticed from EDS analysis that the percentage of the C increased after adsorption of MB, while the percentage of Al and Mg decreases slightly.Adsorption capacity of solid adsorbents not only depends on surface area but also on chemical surface functional groups.The purpose behind the FT-IR analysis is to identify the different functional groups found in algae biomass that is responsible for the adsorption process (19).The FT-IR analysis was carried out at Tehran University using a Jasco 4100 device in the spectral range 400-5000 cm -1 and their results were plotted in Fig. (8), it can be noticed from this figure that the peaks for unloaded MA free and immobilized form are as follows, respectively: the bands around (3412.08, 3751.55)cm -1 and (3425.58,3755.4)cm -1 are the indication of the existence of the -OH groups.While the bands around 2927.94 cm -1 and 2926.01cm-1 confirm the existent of C-H aldehyde stretch.However, the band around 1649.14 and 1616.35confirms that the presence of -C=O stretching, which could be attributed to amide I and II.The band around (1425.4,1087.85)cm -1 and (1429.25,1091.71cm -1 signifies the presence of Amines (18).However, the band around (873.75, 707.88, 468.7) cm-1 and (875.68,709.80,474.49) cm -1 indicates the presence of Aromatic and alkyl halides(C-Cl) and Alkyl halides.This suggested that the -OH, C-H, and -C=O groups were major contributors in the mechanism for the biosorption MB onto algae and alginate support matrix thus verifying that immobilization had successfully been performed (9).The free algae have more transmittance intensity than the immobilized algae, this indicated the higher removal efficiency by free MA.After MB biosorption, some peaks for free and immobilized MA disappeared, shifting or decreasing in their intensity, the peaks that disappeared after MB biosorption showing active involvement of these peaks in the biosorption (21).The specific surface area (m 2 /g), specific pore volume (cm 3 /g), average pore diameter was measured by Brunauer Emmett Teller (BET) analysis at the University of Tehran, by using Barrett Joyner and Halden (BJH) method (32).
The pore volume (V), the pore diameter (D), and the pore size distribution were determined by the graph drawn between quantity adsorbed (cm 3 /g STP), and their results were tabulated in Table 2.

1 .
Mixed algae immobilized Immobilized algal cells were prepared by entrapping a selected amount of dried MA biomass in 2% alginate solution.The mixture was filled into a syringe and injected into 5% CaC1 2 solution to form the immobilized granular cells with 4±0.1 mm diameter.Caalginate gel immobilized MA beads were stored in calcium chloride solution at 4 • C for 4 h to complete gelation before used, Fig 1b.For blank alginate beads, similar procedures were followed, but without MA.a-free MA b-immobilized MA Fig. Free and immobilized MA Experimental work Batch sorption experiments were carried out in 250 mL conical-flasks, each flask was filled with 100 mL of MB dye solution of the desired concentration and sorbent amount.Experiments carried out with; initial pH (3-9), biosorbent dosage of (0.02-0.7) g/100 mL, contact time (0-180) min, initial concentration of MB (10-50) mg/L at 200 rpm agitation speed and room temperature.The sorption mechanisms were specified through the identification of the active functional groups responsible for MB binding on the immobilized MA by application of Fourier transform infrared (FT-IR) analysis, and Scanning Electron Microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS).

Fig. 4 .
The removal efficiency of MB dye as a function of initial concentration Equilibrium and kinetic studies Equilibrium isotherms: =     1  ⁄ (a)free MA (b ) immobilized MA Fig. 5. Equilibrium of MB dye adsorption onto MA