Application of modified green algae Nannochloropsis sp. as 1 adsorbent in the sequential adsorption of methylene blue 2 and Cu(II) cations in solution 3

16 Biomass of algae is a very potent adsorbent for absorbing liquid waste containing 17 heavy metals and organic dyes. This study purposes to confirm the ability of adsorbents 18 from green algae Nannochloropsis sp. modified with silica (ASN) and followed by 19 coating magnetite particles (ASN-MPs) to absorb simultaneously the mixture of 20 methylene blue (ME) and Cu(II) cations in aqueous solution. Simultaneous sorption 21 of ME and Cu(II) cations to ASN and ASN-MPs was carried out by the Batch method 22 with the interaction pH condition 7, contact time (15-120 minutes), and initial 23 concentrations of ME and Cu(II) cations (0.1 - 1.0 mmol·L -1 ). The simultaneous 24 sorption parameters of ME and Cu(II) cations by ASN-MPs trend to comply the 25 pseudo-second-order kinetics model by rate constant values for pseudo-second-order 26 ( k 2 ), respectively 8.98 x 10 -3 and 9.78 x 10 -3 (g·mmol -1 ·min -1 ). The ME adsorption pattern and Cu(II) cations in the competition on ASN-MPs adsorbents, each tends to follow the Freundlich and Langmuir adsorption isotherms. on data, Cu(II) cations have a greater adsorption rate and capacity ( q exp ) compared to ME at the same contact time and initial concentration. that has a high effectiveness in the collective sorption of ME and Cu(II) cations. Therefore, these adsorbents can be used for the adsorption of cation mixtures of heavy metals and organic dyes that are cationic in solution.


Introduction 38
Organic dyes and heavy metals are sources of pollutants that are often found 39 in the environment, especially in waters. Heavy metals and dyes are produced from by-40 products or various industrial wastes such as textiles and petrochemicals. Heavy 41 metals such as Cu exposed to the environment can come from electroplating plants, 42 mining, industrial, and municipal wastes [1] while organic dyes such as methylene blue 43 (ME) are applied considerably as agents of dyes in varied industry such as the 44 pharmaceutical, leather, paper and textile industries [2]. Because of this, the presence 45 of these toxic chemicals needs to be reduced from the environment so that they do not 46 have a negative impact on human health and the surrounding environment. 47 8 magnetite. The mixture was stirred for 30 minutes using a magnetic stirrer. When 118 stirring, the pH of the solution was made to be pH of 2 with the addition of 1 M HCl 119 by dropwise. In another bottle, Nannochloropsis sp. biomass (0.4 g) and ethanol (5 mL) 120 were mixed by a magnetic stirrer for 30 minutes. Then, the two solutions were mixed 121 while stirring until the mixture turns into a gel. The formed gel was filtered with filter 122 paper and allowed for 24 hours. The gel was afterward rinsed using deionized water 123 and ethanol with a ratio of 60/40 to pH≈7. Furthermore, the gel was placed in the oven 124 to be dried at 40 o C for 2-3 hours and crushed using a grinder until smooth with a size 125 of 100 mesh. 126

Characterization of ASN and ASN-MPs adsorbent 127
The ASN adsorbents and ASN-MPs were investigated by Fourier-transform 128 infrared spectroscopy (FTIR) to recognize specific functional groups contained 129 (Shimadzu Prestige-21 IR, Japan). The adsorbent crystallinity level was analyzed by 130 XRD (Shimadzu 6000, Japan). The distribution of particle size from material was also 131 investigated by the particle size analyzer (Fritsch Analysette 22). Surface 132 morphological analysis and element constituents were performed using Scanning 133 Electron Microscopy with Energy Dispersive X-Ray (SEM-EDX) (Zeiss MA10, 9 Germany). 135

Adsorption experiments 136
The experimental procedure to study the adsorption competition between ME 137 and Cu(II) cations in the mixture was carried out under the following adsorption 138 conditions: a dose of 0.1 g ASN-MPs adsorbent was used in a batch system controlled 139 by a shaker (Stuart reciprocating shaker SSL2), the sorption process was held at 27 ºC, 140 interaction pH was 7, and an adsorbate volume was 50 mL. Experiments for 141 investigating adsorption kinetics were performed with various time from 15 to 120 142 minutes while experiments to study the adsorption isotherm by varying the initial 143 concentrations of ME and Cu(II) cations from 0.1 to 1.0 mmol·L -1 , respectively. UV-144 Vis spectrophotometer (Agilent Cary 100, U.S.A) was performed to analyze the 145 concentrations of ME adsorbed on the adsorbent at a maximum wavelength of 664 nm. 146 The concentrations of Cu(II) cations adsorbed by the adsorbent were tested with atomic 147 absorption spectrophotometer (AAS) (Perkins Elmer 3110, U.S.A). 148 The Equations (Eqs.1, 2, and 3) were used to determine an amount of adsorbed 149 ME or Cu(II) cations per unit mass of adsorbent and the percentage of adsorbed ME or 150 Concentrations (mg·L -1 ) of ME or Cu(II) cation solution at initial state, equilibrium, 155 and certain time of t were expressed as Co, Ce, and Ct, respectively. The mass of 156 adsorbent (g), the volume of the solution (L), the amount of ME or Cu(II) cations 157 adsorbed per unit mass (mmol·g -1 ), and the percentage of the ME or Cu(II) cation 158 adsorption are expressed by m, V, q, and R, respectively. 159

Sequential desorption 160
To find out the type of interaction between ASN-MPs adsorbents with ME or 161 Cu(II) cations , a sequential desorption experiment was conducted as follows: 0.1 g of 162 ASN-MPs adsorbents were added to each ME or Cu(II) cation at conditions (pH = 7, T 163 = 27 °C, t = 90 minutes, volume = 50 mL, and adsorbate concentration 0.1 mmol·L -1 ). 164 Adsorbates adsorbed on the AS-MPs adsorbent were sequentially released using 165 several eluents such as aquades, HCl (0.1 M), and Na2EDTA (0.1 M).

Reusability of adsorbent 169
The ability to reuse adsorbents was studied by performing the adsorption 170 process singly at the optimum condition. The adsorbate adsorbed was eluted by an 171 eluent of 0.1 M HCl (50 mL). Then, the distilled water was used to rinse the adsorbent 172 to reach neutral pH. The adsorption-desoption process was repeated several times, up 173 to % adsorption from ME or Cu(II) cations < 80%. 174

Characterization of ASN and ASN-MPs adsorbent 176
To find out the success of the modification process of Nannochloropsis sp. Algae 177 using silica matrix and magnetite particle coating, identification of the adsorbent 178 functional groups was carried out using FTIR. In ASN adsorbents (  The presence of magnetite particles in the modified Nannochloropsis sp. algae 204 can be observed by comparing the XRD pattern between ASN and ASN-MPs (Fig. 3). 205 The presence of magnetite particles (Fe3O4) in ASN-MPs can be seen from the results 206 of the analysis with XRD (Fig. 3b). The XRD diffraction pattern in ASN-MPs has the 207 most intense appearance at 2θ = 35.60 which corresponds to the diffraction pattern of 208 This shows the decrease in particle size after coating with Fe3O4 particles. In Fig. 4,  216 it can be observed that there is a decrease in volume (%) for large particle size diameters 217 from 12.92 in ASN to 11.70% in AS-MPs and an increase in volume (%) in small while the interaction pH in the collective adsorption process from ME and Cu(II) cation 231 solution was conditioned at pH 7.0. This is with consideration, when the pH of the 232 solution is below pHPZC, the adsorbent will be positively charged while the ME solution 233 and Cu(II) cations under these conditions will also be positively charged, so there will 234 be a repulsion between the positive charge of the adsorbent and the adsorbate [42]. At 235 pH 7, the interaction between adsorbent and adsorbate can occur optimally due to at the 236 15 pH, the adsorbent surface charge tends to be neutral and will be negative while the ME 237 and Cu(II) cations solution are positively charged. At pH > 7 adsorption will tend to 238 decrease, because adsorbates such as a solution of ME and Cu (II) cations will undergo 239 hydrolysis which results in a negatively charged species. The same thing happened to 240 the adsorbent surface charge which tends to be negative because of pH > pHpzc [31, 241 43]. Thus in this condition, there will be a repulsion between the negative charge of the 242 adsorbent and adsorbate. 243

Simultaneous adsorption kinetics of ME and Cu(II) cations 244
The impact of interaction time on simultaneous sorption between solution of 245 ME and Cu(II) cations can be seen in Fig. 6 showing that the amount of ME and Cu(II) 246 cations adsorbed on ASN-MPs tends to be greater than ASN. In addition, Fig. 6 shows 247 that Cu(II) cations are more adsorbed than ME dyes in both ASN or ASN-MPs. This 248 shows that Cu(II) cations dominate more adsorbed in ASN and ASN-MPs. 249 To better understand the adsorption competition performance of ME dyes and 250 Cu(II) cations adsorbed simultaneously, the adsorption kinetics model was used.  where qt and qe (mmol g -1 ) are total adsorbate (ME or Cu(II) cation) adsorption capacity 257 at certain time of t (min) and equilibrium, serially. While k1 (min -1 ) and k2 (g·mmol -258 1 ·min -1 ) express the first and second order rate constants, respectively. 259 Table 1 describes the values from R 2 (linear correlation coefficient) of the 260 pseudo-first and -second-order kinetics models compared, then both ME and Cu(II) 261 cations on ASN and ASN-MPs are more likely to take the pseudo-second-order kinetics 262 pattern. If k2 values from ME and Cu(II) cations compared will indicate that Cu(II) 263 cations are more adsorbed on both ASN and ASN-MPs. The presence of Fe3O4 particles 264 increases the adsorption rate of ME or Cu(II) cations in ASN-MPs. 265

Adsorption mechanism 266
The significant section in the investigation of adsorption kinetics is the 267 adsorption mechanism, because it will give an overview of reaction happened between 268 adsorbate and adsorbent. In the process of the adsorption, the amount of adsorbed 269 adsorbate is always expected to be more adsorbed and easily released again 270 17 (desorption). The mechanism of adsorption between ME and Cu(II) cations is really 271 controlled by the surface characteristics of the adsorbent used [46,47].
The 272 mechanism of adsorption to ME and Cu(II) cations on ASN-MPs was analyzed using 273 the proposal of Weber and Morris (Eq. 5) about the intra-particle diffusion pattern 274 (IPD) [ 48,49]. The IPD pattern can be utilized to study the diffusion process of targets 275 absorbed by adsorbents that can be used in simulating kinetics data [50]. 276 Where the rate constant of intra-particle diffusion is stated by kid (mmol·g −1 ·min −0.5 ), 278 and a constant describing resistance for mass transfer in the border layer is represented 279 by C (mmol·g −1 ). Through the slope and intercept of lines resulted from plots of qt 280 versus t 0.5 will be produced kid and C (Fig. 7) and displayed in Table 2. 281 part, this shows that the rate and external mass transfer is not controlled only by the 287 intraparticle diffusion but also it occurs simultaneously [48]. Based on observations 288 in Fig. 7, there are two steps that represent the migration of ME and Cu(II) cations 289 through the solution into the adsorbent external surface and further directed diffusion 290 through the adsorbate target into the adsorbent active site respectively through the pore 291 cavity and the adsorbent active group, according to diffusion theory at adsorption 292 process. The mechanism of adsorption may be illustrated in two dissimilar means 293 namely an electrostatic adsorption and a diffusion. This is because of the porosity and 294 existence of negative charge in the functional groups of adsorbents [42]. This is 295 supported by determining the mechanism of adsorption of ME and Cu(II) cations 296 through sequential desorption using several eluents such as distilled water, HCl solution, 297 and Na2EDTA to release ME and Cu(II) cations which have been adsorbed on ASN-298 MPs through entrapment interactions, electrostatic interaction, and complex formation 299 (Fig. 8). 300 In Fig. 8 can be seen the results of sequential desorption of ME dye and Cu(II) 301 cations contained in ASN-MPs by using an aquades eluent, HCl (0.1 M), and continued 302 with Na2EDTA (0.1 M). Fig. 8 indicates that the percentage of ME dye and Cu(II) 303 cations eluted using 0.1 M HCl is greater than elution using water and 0.1 M Na2EDTA 304 19 solution. This indicates that both ME and Cu(II) solutions adsorbed on ASN-MPs 305 tend to be dominated by electrostatic interactions. ME is an organic cation and Cu(II) 306 cation is positively charged so that it can interact with ASN-MPs which has a negatively 307 charged surface. ASN-MPs tend to be negatively charged because they have functional 308 groups consisting of hydroxyl, carbonyl, and amines from Nannochloropsis sp.. 309 Whereas siloxan and silanol groups are from silica matrix. 310

Adsorption isotherm 311
The simultaneous competition for adsorption of ME and Cu(II) cations with 312 varying initial concentrations of ASN and ASN-MPs can be seen in Fig. 9. The results 313 illustrate that the amount of adsorbate adsorbed goes up with rising initial concentration 314 of adsorbate used. In fact, at the use of high initial concentrations, the amount of 315 adsorbate adsorbed reaches a maximum. In other words, increasing the concentration 316 does not increase the amount of adsorbate adsorbed. This is due to a decrease in the 317 quantity of available active sites accompanied by increasing the concentration of the 318 ME solution and the adsorbed Cu(II) cations. The sorption capacity raises caused by an 319 enhancement in the initial concentration of ME dyes and Cu(II) cations as a booster to 320 20 increase the adsorption capacity because in this condition there are more adsorbates 321 which occupy the active sites on the adsorbent surface [51]. 322 Fig. 9 states that the q value for Cu(II) cations is greater than that of ME in both 323 ASN and ASN-MPs at the same initial concentration. In addition, it can also be seen 324 that the q value of ASN-MPs adsorbents is relatively higher for ME and Cu(II) cations 325 than ASN adsorbent. Furthermore, to investigate the sorption parameters of ME dyes 326 and Cu(II) cations which were adsorbed simultaneously on ASN and ASN-MPs, then 327 Langmuir (Eq. 6) and Freundlich (Eq. 7) adsorption isotherm models were employed 328 [52,53] to describe quantitatively the ME and Cu(II) cations adsorption isotherms 329 (Table 3). using Langmuir and Freundlich adsorption isotherm models can be seen in Fig. 10 and 338 Table 3. 339 Table 3 and Fig. 10 show that the R 2 indicates that the adsorption isotherm 340 pattern of ME is more likely to attend the Freundlich adsorption isotherm. Adsorption  (Table 3). This shows that the active groups in adsorbents derived from algal 352 biomass (hydroxyl, carbonyl, and amines groups) and silica matrix (siloxan and silanol 353 groups) as well as the magnetic properties of adsorbents can raise the quantity of 354