Remediation of Methyl Orange Dye in Aqueous Solutions by Green Microalgae (Bracteacoccus sp.): Optimization, Isotherm, Kinetic, and Thermodynamic Studies

Abstract

This study aims to assess the ability of old, immobilized fresh, and free fresh green microalgae (a Bracteacoccus sp.) to remove methyl orange (MO) dye from aqueous solutions. The effects of four factors, including initial MO concentration (5–25 mg L⁻¹), adsorbent dose (0.02–0.10 g mL⁻¹), temperature (4–36 °C), and contact time (5–95 min), were examined. The Box–Behnken design (BBD) was used to determine the number of required experiments and the optimal conditions expected to provide the highest removal percentage of MO dye from aqueous solutions. The experimental data were applied to four isotherm models (Langmuir, Freundlich, Dubinin–Radushkevich (D–R), and Temkin isotherm models) and three kinetic models (pseudo–first–order, pseudo–second–order, and Elovich kinetic models). The results indicate that the highest removal of MO (97%) could be obtained in optimal conditions consisting of an initial MO concentration of 10.0 mg L⁻¹, an adsorbent dose of 0.10 g mL⁻¹, a temperature of 20 °C, and a contact time of 75 min. Moreover, the experimental data were best fitted by the Langmuir and Temkin isotherm models and followed a pseudo-second-order kinetic model. The interaction between MO and the Bracteacoccus sp. was confirmed by UV and ESI/MS analyses, indicating that MO removal occurred via both sorption and degradation processes.

1. Introduction

Industrial activities are significantly increasing, resulting in the discharge of massive amounts of effluent into the environment. This leads to water pollution, a critical issue that requires significant attention [1]. Many industries, such as textile, cosmetic, leather, paper, plastic, printing, rubber, and pharmaceutical industries, produce effluents containing synthetic dyes [2]. These synthetic dyes are classified into ionic (cationic and anionic) and nonionic (vat and disperse) dyes. In terms of toxicity, ionic dyes are more toxic because they are more reactive and carcinogenic [3]. Methyl orange (MO) dye is an example of ionic dye and is widely used in the textile industry and in research laboratories as an indicator in acid–base titrations [4]. MO dye can cause skin allergies, death (in the case of exposure to a high MO concentration), vomiting, and diarrhea [5]. Therefore, the uptake of synthetic dyes from contaminated water is a necessary step to protect the aquatic environment from pollution. Several techniques, such as membrane filtration, coagulation–flocculation, ozonation, photocatalytic oxidation, electrochemical oxidation, and adsorption, have been used to remove synthetic dyes from aqueous media [6]. Among these techniques, adsorption is considered the most appropriate for its several advantages, such as simplicity, low cost, environmental friendliness, effectiveness, and ease of use [7].

In general, the adsorbents used for adsorption of MO dye from aqueous solutions include biosorbents, activated carbon, biochar, minerals and clays, resins and polymers, nanoparticles, and composites [8]. Among them, biosorbents are preferred for being easily available, environmentally friendly (they do not form intermediate products), highly efficient, regeneratable, and low-cost. Based on biosorbent sources, biosorbents can be classified into (i) natural biosorbents (e.g., straw, peels, chitosan, zeolites, etc.); (ii) microbial biomass (e.g., algae, bacteria, yeasts, and fungi); (iii) agricultural and industrial wastes (e.g., wheat bran, sawdust, etc.) [9]. Using algae in bioremediation has been described as an attractive and promising method because algae grow quickly and are highly effective in removing pollutants from aquatic systems. [10]. In previous studies, algal biosorbents such as Fucus vesiculosus [11], Oedogonium subplagiostomum-AP1 [12], Chlorella species, and algal blooms [13] were used to remove MO from aqueous solutions with reasonable removal efficiency (>50%). However, the optimization of MO removal to obtain a high removal efficiency at room temperature within a short time, the confirmation of MO removal/degradation, and the study the effect of immobilization of a biosorbent on the efficiency of MO removal from aqueous solutions require further investigation. In the literature, there are few studies related to the use of green microalgae for the treatment of wastewater polluted by dyes. Therefore, the current study adds new results regarding MO removal using green microalgae (a Bracteacoccus sp.) as a green method for the treatment of wastewater containing synthetic dyes.

In fact, adsorptive removal mainly depends on factors such as initial adsorbate concentration, adsorbent dose, temperature, and contact time [14]. Therefore, the optimization of these experimental variables is an essential step that can be achieved by the traditional experimental method (i.e., studying the effect of each factor while keeping the other factors constant), but the main drawbacks of this strategy are ignoring the interactions between the variables and needing a larger number of experiments as the number of variables increases. Consequently, an alternative experimental strategy is using response surface methodology (RSM) as a statistical and mathematical tool to design the experiments. RSM takes into account the interactions between the experimental variables and easily determines the optimum conditions that can provide the best response [15]. RSM can use a full or fractional factorial design, a central composite design (CCD), a Box–Behnken design (BBD), and a Doehlert matrix design (DMD) [16]. Amongst them, the BBD is favored for achieving better results [17]; therefore, it was used in the current study.

The main objectives of the current study were (i) to evaluate the ability of the Bracteacoccus sp. (old, fresh, and immobilized) to remove MO dye from aqueous solution; (ii) to optimize the experimental variables, including initial MO concentration, adsorbent dose, temperature, and contact time using the BBD; (iii) to describe the equilibrium and the adsorption kinetics of MO removal by a Bracteacoccus sp.; (iv) to investigate the reusability of the Bracteacoccus sp. for MO removal from aqueous solutions.

2. Materials and Methods

2.1. Materials

All chemicals used in the current study were of analytical grade and were used without further purification. MO dye (

Table 1. Characteristics of MO dye [18].

Parameter	Value
Chemical structure
Chemical formula	C14H14N3SO3Na
Molecular weight (g mol−1)	327.33
Molecular size (Å)	15.8 × 6.5 × 2.6
λmax (nm)	464
Density at 20 °C	1.28
pKa	3.42
Solubility (mg L−1)	5.0 × 103

) was selected as a model anionic dye and purchased from Sigma Aldrich. A stock solution of MO dye (1000 mg L⁻¹) was prepared by dissolving a precise amount (1.000 g) of MO dye in 1000 mL of distilled water and was then used to prepare a set of MO diluted solutions with concentrations in the range of 5–25 mg L⁻¹. A calibration curve using the MO solutions in the concentration range of 5–25 mg L⁻¹ was established.

2.2. Sampling of Green Microalgae

Green microalgae samples were collected from ground water in the west part of Mafraq city in Jordan and directly transferred to the laboratory. Subsequently, green microalgae cultivation was carried out in flasks containing Bold Basal Medium (BBM). Furthermore, the isolation of algal colonies was conducted by establishing a series of subcultures in BBM agar plates. A pure culture was prepared from algal colonies and then examined microscopically. Routine cultivation was conducted at 25 ± 2 °C under a light intensity of 20.25 µEm⁻²s⁻¹ for 25 days. A pure culture was chosen and was identified as consisting of a Bracteacoccus sp.

2.3. Immobilization of the Bracteacoccus sp.

The immobilization of the Bracteacoccus sp. was accomplished based on the procedure described in a previous study [19]. In detail, a solution of sodium alginate (3%) was well mixed with a suspension of the Bracteacoccus sp. in a 2:1 ratio (v/v). The mixture was then transferred dropwise into a calcium chloride (CaCl₂) solution (0.03 M) to form adsorbent beads, remaining for 30 min into the CaCl₂ solution to become more solid. Thereafter, the beads were washed three times with distilled water and stored in the fridge at 4 °C for later use.

2.4. Characterization of the MO Solutions

The analysis of the MO dye solutions before and after adding the Bracteacoccus sp. was accomplished using (i) a UV Vis diode-array spectrophotometer (Specord S 600-Molecular Spectroscopy, Germany) operated in the wavelength range of 210–790 nm, with a quartz cuvette; (ii) a high-resolution TOF-MS/MS system (impact II Bruker, Bruker Daltonik, Bremen, Germany) and an impact II ESI-Q-TOF system equipped with a Bruker Daltonik Elute UPLC system (Bremen, Germany), as shown in Supplementary Materials.

2.5. Adsorption Experiments

The experiments of MO adsorption from an aqueous solution using the free fresh Bracteacoccus sp. were conducted in batch mode. The effect of several experimental factors, such as initial MO concentration, adsorbent dose, temperature, and contact time, on MO removal from aqueous solutions was investigated. In detail, 20.0 mL of MO solution (5–25 mg L⁻¹) was transferred to a 50.0 mL test tube, and then a desired adsorbent dose (0.02–0.10 g mL⁻¹) was added at a specific temperature in the range of 4–36 °C. The mixture was kept in a dark place on the lab bench for a desired time (5–95 min), and then a small portion (~2.0 mL) of the mixture was withdrawn, and its absorbance was measured at 464 nm. All the experiments were conducted in triplicate. The initial pH of the MO solutions was 4, which was described as the optimum pH value for acquiring the highest removal % of MO from aqueous solutions [11].

Subsequently, the removal efficiency percentage (R%) of MO dye and the adsorption capacity ($q_m$), which represents the amount of dye that can be absorbed by 1.0 g of adsorbent (mg g⁻¹), was calculated using Equations (1) and (2), respectively [20].

$$R\%=\frac{C_o-C_t}{C_o}\times 100\%$$

(1)

$$q_m=\frac{{(C}_o-C_t)V}{m}$$

(2)

where $C_o$ (mg L⁻¹) is the initial concentration of the MO dye solution, $C_t$ (mg L⁻¹) is the concentration of the MO dye solution at time t, V (L) is the volume of the MO dye solution, and m (g) is the adsorbent amount used.

Design Expert version 8 was used to determine the required experiments, and a BBD based on RSM with a quadratic model was used. These experiments allowed for studying the effects of four experimental variables, namely, initial MO concentration (A), adsorbent dose (B), temperature (C), and contact time (D), each set at three levels (−1, 0, +1), as summarized in

Table 2. Four experimental factors and their levels and actual values using a Box-Behnken design for MO dye removal using green microalgae (a Bracteacoccus sp.).

Parameter	Factor		Level
		Low (−1)	Medium (0)	High (+1)
Initial MO concentration (mg L−1)	A	5	15	25
Adsorbent dose (g mL−1)	B	0.02	0.06	0.10
Temperature (°C)	C	4	20	36
Contact time (min)	D	5	45	95

. Consequently, the overall required experiments were 29, as shown in

Table 3. BBD matrix for the four examined experimental factors (initial MO concentration, adsorbent dose, temperature, and contact time) including their coded values.

Exp. #	Factor 1 (Initial Concentration, mg L−1)	Factor 2 (Adsorbent Dose, g mL−1)	Factor 3 (Temperature, °C)	Factor 4 (Time, min)
1	0	−1	−1	0
2	−1	0	0	−1
3	0	−1	0	−1
4	+1	0	−1	0
5	0	0	0	0
6	+1	0	0	−1
7	0	+1	0	+1
8	+1	0	+1	0
9	0	0	−1	−1
10	0	0	−1	+1
11	−1	−1	0	0
12	0	+1	0	−1
13	0	0	0	0
14	−1	0	0	+1
15	−1	+1	0	0
16	0	−1	+1	0
17	+1	−1	0	0
18	−1	0	+1	0
19	0	0	0	0
20	+1	+1	0	0
21	0	0	0	0
22	−1	0	−1	0
23	0	0	+1	−1
24	0	+1	+1	0
25	+1	0	0	+1
26	0	0	+1	+1
27	0	+1	−1	0
28	0	−1	0	+1
29	0	0	0	0

, and the number of experiments (N) was determined using the following equation (Equation (3)) [21]:

N = 2K(K − 1) + M_o

(3)

where K and M_o are the number of experimental variables and the center points, respectively.

2.6. Isotherm Study

Four common isotherm models, namely, Langmuir [22] (Equation (4)), Freundlich [23] (Equation (6)), Dubinin–Radushkevich (D-R) [24] (Equation (7)), and Temkin [25] (Equation (10)), were applied to the experimental data.

$$\frac{C_e}{q_e}=\frac{1}{K_L{q}_e^{max}}+\frac{C_e}{q_e^{max}}$$

(4)

$$R_L=\frac{1}{1+K_L{C}_o}$$

(5)

where $q_e$ (mg g⁻¹) is the MO amount adsorbed at equilibrium, $q_e^{max}$ is the maximum adsorption capacity (mg g⁻¹), $K_L$ (L mg⁻¹) is the Langmuir constant related to the adsorbent–adsorbate affinity and associated with sorption energy, and R_L, expressed in Equation (5), is a dimensionless equilibrium constant used to describe the adsorption process as irreversible (R_L = 0), linear (R_L = 1), favorable (0 < R_L < 1), or unfavorable (R_L > 1). The parameters $q_e^{max}$ and K_L are calculated by plotting a linear relationship between C_e/q_e and C_e to obtain a slope of 1/$q_e^{max}$ and an intercept of 1/(K_L$q_e^{max}$).

$$log{q}_e=log{K}_f+\frac{1}{n}log{C}_e$$

(6)

where $K_f$ ((mg g⁻¹)(L mg⁻¹)^1/n) is the Freundlich isotherm constant, and n represents the heterogeneity factor (i.e., the non-linearity degree between the concentration of the dye solution and the absorption process), with the adsorption process being favorable when n > 1 [26]. By plotting a linear relationship between logC_e and $log{q}_e$, the n and K_f values are calculated from the slope and intercept, respectively.

$$ln{q}_e=ln{q}_m+K_D {\epsilon }^2$$

(7)

$$E=\frac{1}{\sqrt{2K_D}}$$

(8)

$$\epsilon =RTln (1+\frac{1}{C_e})$$

(9)

where K_D is the absorption energy constant (mol²/kJ²) used to calculate the average of sorption free energy E (KJ mol⁻¹) provided by Equation (8), with adsorption being physical (E < 8 KJ mol⁻¹), based on ion exchange (E = 8–16 KJ mol⁻¹), or chemisorption (E = 20–40 KJ mol⁻¹) [27]. In Equation (9), ε is the Polanyi potential (KJ mol⁻¹), R is the universal gas constant (8.314 × 10⁻³ KJ mol⁻¹), and T is the temperature (K). By plotting a linear relationship between lnq_e and ε², the slope (K) and intercept (lnq_m) are obtained.

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

(10)

$$B_T=\frac{RT}{b_T}$$

(11)

where b_T is the Temkin constant and refers to the sorption heat (J mol⁻¹), and A_T is the Temkin isotherm constant (L g⁻¹). The A_T, B_T, and b_T values can be calculated by plotting a liner relationship between q_e and lnC_e to obtain the slope (B_T) and the intercept (B_TlnA_T).

2.7. Kinetic Study

The experimental data were analyzed using three common kinetic models, namely, the pseudo–first–order (Equation (12)) [28], pseudo–second–order (Equation (13)) [29], and Elovich (Equation (14)) [30] kinetic models.

$$\mathit{ln}\left(q_e-q_t\right)=\mathit{ln}{q}_e-t{K}_1$$

(12)

$$\frac{t}{q_t}=\frac{1}{K_2{q}_e^2}+\frac{t}{q_e}$$

(13)

$$q_t=\frac{1}{\beta }\mathit{ln}\left(\alpha ·\beta \right)+\frac{1}{\beta }\mathit{ln}t$$

(14)

where $q_t$ (mg g⁻¹) is the MO amount adsorbed at time t (min), K₁ is the adsorption rate constant of the pseudo–first–order model (min⁻¹), K₂ is the adsorption rate constant of the pseudo–second-order model (g mg⁻¹ min⁻¹), β is the desorption constant (g mg⁻¹), and α is the initial adsorption rate (mg g⁻¹ min⁻¹).

2.8. Thermodynamics

Three common thermodynamic parameters, namely, the standard changes in Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) were determined to identify the adsorption type [31]. Therefore, the ΔG° value of MO adsorption by the Bracteacoccus sp. at three different temperatures (4, 20, and 36 °C) could be calculated by Equation (15) while keeping the other three factors constant at their optimum values (i.e., C_o = 10 mg L⁻¹, adsorbent dose = 0.10 g mL⁻¹, and contact time = 75 min). In addition, the ΔH° and ΔS° values were determined using the Van’t Hoff equation (Equation (16)) by plotting lnk_d vs. 1/T, and ΔG° could be calculated at a specific temperature using Equation (17) [28].

$$\mathsf{\Delta }G°=-RTln{K}_d$$

(15)

$$ln{k}_d=\mathsf{\Delta }SoR+\frac{\mathsf{\Delta }H°}{RT}$$

(16)

$$\mathsf{\Delta }G°=\mathsf{\Delta }H°-T\mathsf{\Delta }S°$$

(17)

where R is the universal gas constant (8.314 J mol⁻¹ K⁻¹), T is the absolute temperature in kelvin (K), and $K_d$ is the distribution coefficient, which equals the ratio between the amount of MO dye adsorbed and the MO concentration at equilibrium ($K_d$ = q_e/C_e).

2.9. Regeneration

The reusability of an adsorbent is a significant factor in reducing the cost of the removal process [32]. Therefore, the Bracteacoccus sp. used in the current study was reused up to five times after its separation from an aqueous solution by centrifugation and washing for three times with distilled water.

3. Results

3.1. UV-Vis Analysis

The MO concentration before and after treatment with the Bracteacoccus sp. could be easily quantified using UV-vis spectrophotometry. Herein, the mixture containing the MO dye solution and the Bracteacoccus sp. was left on the lab bench in the dark for a sufficient contact time of 75 min. Then, the mixture was centrifuged, filtered, and analyzed using a UV spectrophotometer in continuous mode in the range of 200–800 nm at 10 nm wavelength intervals. The analysis results are shown in Figure 1, indicating maximum absorbance of MO at 464 nm and a great reduction in the area under the curve at 464 nm for the MO solution after treatment with the Bracteacoccus sp. (i.e., the 464 nm peak vanished). In addition, clear changes were observed for the 230 and 270 nm peaks associated with benzene ring absorption, with the 230 nm peak disappearing, and the 270 nm peak becoming broader. These findings could be due to the destruction of the azo bond in the MO molecules and the formation of intermediate compounds comprising benzene rings [33]. Another crucial observation was that the color of the MO solution that initially was reddish orange gradually changed after the addition of the Bracteacoccus sp., and the solution became entirely colorless after 75 min. Therefore, the decolorization of the MO solution by the Bracteacoccus sp. could occur as a result of both degradation and sorption processes (binding of the dye molecules by functional groups such as hydroxyl, carboxylate, phosphate, amino, and sulfate groups on the algal surface) [19].

In addition, a microscopic view of the Bracteacoccus sp. before and after the addition of MO dye is shown in Figure 2. Before MO absorption, the surface morphology of the Bracteacoccus sp. was clear and homogeneous, but after MO absorption, the surface morphology became rough and heterogenous.

3.2. A Comparison between Old, Immobilized Fresh, and Free Fresh Bracteacoccus sp.

The Bracteacoccus sp. as a biosorbent was used in three statuses, namely, old, immobilized fresh, and free fresh, to investigate its ability to remove the MO dye from aqueous solutions. Based on Figure 3, the free fresh Bracteacoccus sp. achieved the highest removal efficiency of MO (97%) from aqueous solutions, followed by the immobilized fresh (85%) and the old (71%) Bracteacoccus sp., respectively. This behavior may be due to the high ability of the free fresh Bracteacoccus sp. to interact with the MO dye molecules. In contrast, the immobilized fresh Bracteacoccus sp. showed a lower removal efficiency because the Bracteacoccus sp. molecules were shielded on the beads, which hindered the direct interaction between the MO molecules and the Bracteacoccus sp., resulting in a lower efficiency of MO removal. The old Bracteacoccus sp. achieved the lowest removal efficiency, as its removal activity was lower, and that adversely reflected on its ability to remove MO from the aqueous solutions.

3.3. Optimization of Experimental Factors Using a BBD

The results of the experiments are listed in

Table 4. Responses are presented as removal % values for the experiments listed in Table 3.

Exp. #	Response (Removal %)	Exp. #	Response (Removal %)
1	76	16	80
2	52	17	76
3	44	18	96
4	83	19	85
5	85	20	86
6	48	21	86
7	94	22	77
8	86	23	60
9	48	24	99
10	82	25	80
11	75	26	91
12	60	27	89
13	84	28	74
14	84	29	86
15	96

. The analysis of the experimental data related to MO dye removal using the free fresh Bracteacoccus sp. was achieved using Design Expert version 8, which provided a plot for each factor, displayed in Figure 4. In addition, 3D plots, including illustrations of the interactive effects of the experimental variables, are shown in Figure 5. The effects of these experimental factors can be explained in more detail as follows.

3.4. Effect of the Initial MO Concentration

Figure 4a shows the effect of the initial MO concentration on the removal percentage of MO dye from aqueous solutions. Clearly, an increase in the initial concentration of MO in the range of 5–15 mg L⁻¹ did not lead to any significant changes in removal %, and the MO removal efficiency (~85%) remained approximately constant. In contrast, a slight decrease in removal % was observed when the MO concentration was in the range of 15–25 mg L⁻¹, which might be due to the saturation of the active sites onto the adsorbent surface by the adsorbate molecules [34]. In addition, the interactive effect between the initial MO concentration and temperature, adsorbent dose, and contact time is shown in Figure 5a,b,e, respectively. The general trend indicated that a decrease in the MO concentration in the presence of a higher adsorbent dose, a higher temperature, and a longer contact time up to 75 min increased the removal of MO dye. Similar trends were described for MO removal in previous studies [35,36].

3.5. Effect of the Adsorbent Dose

The adsorbent dose has a clear effect on both the process cost and the removal % [7]. Therefore, studying the effect of the adsorbent dose to determine the optimal adsorbent dose is an essential step in the optimization process. In Figure 4b, the removal % of MO increases from 77 to 94% with an increasing adsorbent dose from 0.02 to 0.10 g mL⁻¹. This can be attributed to the increase in available sites on the adsorbent, as the adsorbent dose increases [37]. In contrast, the adsorption capacity decreases from 3.45 to 0.9 mg g⁻¹ with an increasing adsorbent dose from 0.02 to 0.10 g. This behavior is due to the fact that the adsorption sites remain unsaturated during adsorption, and there is a less than proportional increase in adsorption due to the lower adsorptive capacity of the adsorbent, though the amount of adsorbent is increased [38]. These results are in agreement with results reported in previous studies [39,40].

3.6. Effect of the Temperature

The temperature affects the chemical composition of a solution, as an increase or a decrease in temperature may increase or decrease the adsorption % [41]. Based on Figure 4c, an increase in temperature from 4 to 36 °C increased the removal % of MO dye from 81 to 90%. This behavior is also shown in Figure 5a,d,f to explain the interactive effect between temperature and the remaining three factors (initial MO concentration, adsorbent dose, and contact time). In general, increasing the temperature up to 36 °C in the presence of a higher adsorbent dose and with sufficient contact time resulted in a high removal of MO from the aqueous solutions. This behavior could be due to several reasons, accompanied by high temperatures, which are (i) the increasing swelling of the dye molecules into the adsorbent structure [42]; (ii) the increasing solubility of the dye; (iii) the increasing collisions between the adsorbent and the adsorbate molecules; (iv) the increasing pore size in the adsorbent [43]. This finding related to the effect of temperature on MO removal is similar in trend to that reported in previous studies for MO removal using Zn-Al layered double hydroxide [42] and Bacillus stratosphericus SCA1007 [37].

3.7. Effect of the Contact Time

Figure 4d shows the influence of the contact time on MO removal from aqueous media using the Bracteacoccus sp. The removal % was low in the beginning (~40%), then it gradually started to increase, reaching the maximum removal (~90%) after 75 min. Initially, the number of available sites of adsorption was large; therefore, the adsorption rate was large, but with the time passing, these adsorptive sites became saturated, and the adsorption rate decreased; a plateau was observed at equilibrium [44]. In addition, the interactive effect between the contact time, adsorbent dose, initial MO concentration, and temperature is shown in Figure 5c,e,f.

3.8. Analysis of Variance (ANOVA)

In fact, the F-value and p-value are crucial to determining the significance of the used model, and a model with a high F-value and a low p-value (<0.05) is significant [45]. The results of the ANOVA analysis of the surface quadratic model for MO removal using the Bracteacoccus sp. are listed in

Table 5. ANOVA analysis of the surface quadratic model for MO removal using green microalgae.

	Sum of		Mean	F	p-Value
Source	Squares	df	Square	Value	Prob > F
Model	6347.89	14	453.42	286.71	<0.0001	significant
A—Initial concentration	38.16	1	38.16	24.13	0.0002
B—Adsorbent dose	806.88	1	806.88	510.21	<0.0001
C—Temperature	261.33	1	261.33	165.25	<0.0001
D—Contact time	3084.81	1	3084.81	1950.62	<0.0001
AB	29.70	1	29.70	18.78	0.0007
AC	68.06	1	68.06	43.04	<0.0001
AD	0.000	1	0.000	0.000	1.0000
BC	7.56	1	7.56	4.78	0.0462
BD	4.00	1	4.00	2.53	0.1341
CD	3.24	1	3.24	2.05	0.1743
A2	19.51	1	19.51	12.33	0.0035
B2	1.52	1	1.52	0.96	0.3435
C2	19.10	1	19.10	12.08	0.0037
D2	1854.62	1	1854.62	1172.73	<0.0001
Residual	22.14	14	1.58
Lack of Fit	19.87	10	1.99	3.50	0.1195	not significant
Pure Error	2.27	4	0.57
Cor Total	6370.03	28

, which evidently indicates a high F-value (286.71), a low p-value (<0.0001), and a non-significant lack of fit. Adequate precision, which represents the ratio of signal to noise, had a value of 62.028, indicating a desirable ratio (i.e., an adequate signal), being higher than 4 [46]. In addition, the adjusted R² of 0.9930 was in reasonable agreement with the predicted R² of 0.9815, with a difference of less than 0.2. Therefore, the quadratic model used in the current study is valid.

Considering the effects of the different factors, the following equation can be used to determine the predicted value of the removal % of MO given the values of the experimental factors:

MO removal = + 17.83024
                + 1.26629 × Initial concentration
               + 272.75347 × Adsorbent dose
               + 0.34388 × Temperature
               + 1.18298 × Contact time
                        − 6.81250 × Initial concentration × Adsorbent dose
                        − 0.025781 × Initial concentration × Temperature
                          − 6.77242 × 10⁻¹⁸ × Initial concentration × Contact time
                       + 2.14844 × Adsorbent dose × Temperature
                       + 0.55556 × Adsorbent dose × Contact time
                       − 1.25000 × 10⁻³ × Temperature × Contact time
                 − 0.017342 × Initial concentration²
                 − 302.60417 × Adsorbent dose²
                + 6.70247 × 10⁻³ × Temperature²
                − 8.35021 × 10⁻³ × Contact time²

3.9. Desirability Function

The desirability function is used as an optimization method; the desirability range is 0–1 (1 is the favored value of the response, while 0 means that the response is far from the accepted one) [47]. Figure 6 shows the optimal predicted values of the experimental variables to obtain the best response (removal %). The results of the desirability function are shown in Figure 6, where the maximum response is displayed with all experimental factors varying in the indicated ranges (i.e., C_o = 5–25 mg L⁻¹, temperature = 4–36 °C, and contact time = 5–95 min). Consequently, the maximum removal of MO (99%) could be achieved at the predicted values of 10 mg L⁻¹ as the initial MO concentration, 0.10 g L⁻¹ as the adsorbent dose, 20 °C, and 75 min of contact time. These predicted values were experimentally tested in triplicate, and the MO removal was 97%, which indicated that the experimental result was in agreement with the predicted value, with a 2% relative error. Thus, these optimum conditions were used in the subsequent isotherm, kinetic, and thermodynamic experiments.

3.10. Isotherm Study

The aim of isotherm models is to understand the mechanisms of interaction between adsorbent and adsorbate molecules at constant temperature [48]. Herein, the results of the isotherm study using the Langmuir, Freundlich, D–R, and Temkin isotherm models are shown in Figure 7. Additionally, the calculated values of the isotherm models’ parameters are listed in

Table 6. The values of the isotherm terms of the Langmuir, Freundlich, D–R, and Temkin isotherm models for MO dye adsorption using the Bracteacoccus sp.

Isotherm Model	Parameters	Values
Langmuir	$q_e^{max}$ (mg g−1)	5.4171
	KL (L mg−1)	1.2067
	RL	0.07635
	R2	0.9991
	RMSE	0.04197
Freundlich	Kf ((mg g−1)(L mg−1)1/n)	2.3977
	n	2.8514
	R2	0.9717
	RMSE	61.0529
D-R	Qm (mg g−1)	3.8129
	KD (mol2/kJ2)	0.0653
	E (J mol−1)	2.7671
	R2	0.9518
	RMSE	14.4135
Temkin	AT (L g−1)	33.869
	BT (J mol−1)	1.1639
	bT (J mol−1)	3225.58
	R2	0.9946
	RMSE	0.07198

, where the experimental data obeying the Langmuir and Temkin isotherm model indicated R² of > 0.99 and small RMSE values (RMSE < 1). Note that the Langmuir isotherm model indicated that the adsorbate (MO dye) molecules were adsorbed onto the homogeneous surface of the adsorbent (the Bracteacoccus sp.), forming a monolayer of adsorbate molecules [22]. The value of q_m was 5.4171 mg g⁻¹, B_T was 1.1639 J mol⁻¹, and b_T was 3225.58 J mol⁻¹. Moreover, MO adsorption onto the Bracteacoccus sp. algae was favorable (R_L = 0.07635, between 0 and 1). These findings are in agreement with the results described in previous studies [4,29]. Considering the D-R mode results, the value of E was 2.7671 J mol⁻¹, i.e., <8 J mol⁻¹; therefore, MO adsorption by the Bracteacoccus sp. can be considered physisorption. The n value in the Freundlich isotherm model was 2.8514 (n > 1), which confirmed that the sorption of MO by the Bracteacoccus sp. was adequate.

3.11. Kinetic Study

The kinetic study helped to understand the adsorption mechanism, calculate the adsorption rate constant, and evaluate the adsorption capacity. The results obtained after applying the pseudo–first–order, pseudo–second–order, and Elovich kinetic are depicted in Figure 8, and the calculated values of the corresponding kinetic parameters are summarized in

Table 7. The calculated values of parameters in pseudo-first-order, pseudo-second-order, and Elovich kinetic models. (C_o = 10 mg L⁻¹, adsorbent dose = 0.1 g, and temperature = 20 °C).

Kinetic Model	Parameters	Values
Pseudo-first-order	qe (mg g−1)	0.9307
	K1 (min−1)	0.0354
	R2	0.9363
	RMSE	2.0556
Pseudo-second-order	qe (mg g−1)	2.0588
	K2 (g mg−1 min−1)	0.0766
	R2	0.9991
	RMSE	0.04569
Elovich	β (g mg−1)	66.6667
	α (mg g−1 min−1)	5.1476 × 1011
	R2	0.9674
	RMSE	1.0074

. Obviously, the experimental data were best fitted by the pseudo-second-order model (R² = 0.9999 and RMSE = 0.0457). This finding is in agreement with previous studies of MO removal using Oedogonium subplagiostomum AP1 [12] and Chlorella species [13]. The adsorption capacity was 2.0588 mg g⁻¹, and the rate constant K₂ was 0.0766 g mg⁻¹ min⁻¹. Compared to the pseudo-second-order kinetic model, the Elovich kinetic model achieved a lower fit (R² = 0.9363) for MO adsorption onto the Bracteacoccus sp., indicating that the MO adsorption mechanism diverged from chemisorption [49]. Among these kinetic models, the pseudo-first-order kinetic model provided the lowest value.

Algae mainly consist of polysaccharides, including three groups, namely, polycolloids, alginates, and carrageenans, which have a key role in binding pollutants to the algal surface [50]. For example, the Bracteacoccus sp., a green alga, consists of protein and cellulose groups bonded to polysaccharides in the wall of cells [51]. Therefore, the adsorptive removal of a pollutant (e.g., MO) from aqueous solutions using a biosorbent (e.g., the Bracteacoccus sp.) can occur through two approaches as follows: (i) surface sorption, in which the adsorbate molecules migrate from the bulk aqueous solution toward a surrounding layer on the adsorbent surface, then establishing a direct interaction with the functional groups on the adsorbate surface; (ii) interstitial sorption, where the adsorbate molecules enter the pores in the adsorbent [52]. Consequently, the proposed mechanism for dye adsorption onto green microalgae (e.g., the Bracteacoccus sp.) includes the interaction between the functional groups (e.g., amine, hydroxyl, carbonyl, carboxyl, phosphate, and sulfate) of the green microalgae and the dye molecules [53]. Thus, the biosorption of MO may occur via H-bonding, electrostatic, ion exchange, and π-π interactions [54].

3.12. Thermodynamic Study

The thermodynamic study of the current work was carried out at three different temperatures: 4 °C (277 K), 20 °C (293 K), and 36 °C (309 K). The thermodynamic results are depicted in Figure 9, and the values of the thermodynamic parameters are listed in

Table 8. The calculated values of thermodynamic parameters for MO removal using the Bracteacoccus sp. (C_o = 10 mg L⁻¹, adsorbent dose = 0.1 g, and contact time = 75 min).

ΔH° (KJ/mol)	ΔS° (KJ/mol)	ΔG° (KJ/mol)
		277 k	293 k	309 k
+49.469	+0.205	−7.345	−10.627	−13.909

. The positive ΔH° and the negative ΔG° values indicated that MO adsorption by the Bracteacoccus sp. was endothermic and spontaneous, respectively. The absolute value of ΔG° increased with increasing temperature, and the maximum value of ΔG° was −13.909 KJ mol⁻¹, in the range from 0 to −20 KJ mol⁻¹; therefore, MO adsorption by the Bracteacoccus sp. is physisorption [55]. In addition, the positive value of ΔS° indicated an increase in entropy (randomness) at the solid–solution interface during MO adsorption [56]. Similar trends were reported in previous studies of MO adsorption using activated carbon [57] and magnetic resin in chitosan microspheres [55].

3.13. Regeneration

The reusability of the Bracteacoccus sp. is an important property that should be investigated to estimate the cost effectiveness of MO removal from aqueous solutions, especially if the current procedure is applied on a large scale. Therefore, five regeneration and reusability cycles of the immobilized and free fresh Bracteacoccus sp. were performed to remove MO from aqueous solutions. The results are shown in Figure 10 and indicate that the Bracteacoccus sp. can be reused up to five times without any significant deficiency in the removal efficiency of MO from aqueous solutions, considering that the free fresh Bracteacoccus sp. removed approximately 96% of MO from the aqueous solutions, while the removal efficiency of the immobilized alga was lower (~83%).

3.14. Comparison with Other Adsorbents

Based on previous studies, some biosorbents such as algae (e.g., Fucus vesiculosus [11], Chlorella species [13], and Oedogonium subplagiostomum AP1 [12]) and microbial cultures (e.g., of M. yunnaenensis [58] and Bacillus stratosphericus SCA1007 [37] and anaerobic–aerobic microbial culture/coconut fiber systems [59]) are effective in removing/degrading MO dye (

Table 9. Some biosorbents used for MO removal from aqueous solutions.

Biosorbent	Experimental Conditions	qm (mg g−1)	R%	Ref.
Alga (Fucus vesiculosus)	10 mL of a 60 mg L−1 MO solution, pH = 7.0, 3 g L−1 of adsorbent dose, and 60 min of contact time	0.10	50%	[11]
Chlorella species	1.0–2.2 mg L−1 initial MO concentration and 0.7 g of adsorbent in 50 mL of aqueous solution	0.74–0.27	-	[13]
Alga (Oedogonium subplagiostomum AP1)	500 mg L−1 initial MO concentration, pH = 6.5, 400 mg L−1 algal dose, and 5.5 days of contact time	1213	97%	[12]
Microbial culture (bacterial strain, M. yunnaenensis)	100 mL of a 100 mg L−1 MO solution, pH 7.0, 3 mL (culture), and 5.0 days of contact time	-	98%	[58]
Microbial culture (Bacillus stratosphericus SCA1007)	100 mL of a 150 mg L−1 MO solution, optical density 1.0 (λmax = 600 nm, 1.50 × 106 cells mL−1), pH 7.0, T = 35 °C, 3 mL (culture), and 12 h of contact time	-	100%	[37]
Biofilm reactor containing anaerobic–aerobic microbial culture/coconut fiber	3 L reactor with anaerobic–aerobic biofilm (7.2 cm dimeter × 75 cm height) filled with 1 L of coconut fiber, 200 mg L−1 initial MO concentration, contact time of 36 days with aeration for 24 h	1.20	81%	[59]
Green microalga (Bracteacoccus sp.)	20 mL of MO solution (10 mg L−1), 0.10 g L−1 adsorbent dose, 20 °C temperature, and 75 min of contact time	5.42	97%	The current study

). Algae (e.g., Fucus vesiculosus) have a reasonable efficiency (50%) of MO removal in 60 min. In addition, the highest MO removal (>95%) was observed with Oedogonium subplagiostomum AP1, M. yunnaenensis, and Bacillus stratosphericus SCA1007, but the required contact time was relatively high and varied based on the adsorbent used, in the following order: Oedogonium subplagiostomum AP1 (5.5 days) > M. yunnaenensis (5.0 days) > Bacillus stratosphericus SCA1007 (1/2 days).

In contrast, the Bracteacoccus sp. used in the current study needed a shorter contact time (~75 min) to remove 97% of MO dye from an aqueous solution. Therefore, the Bracteacoccus sp. is an effective biosorbent, able to extensively remove MO dye from an aqueous medium within a relatively short time.

4. Conclusions

The Bracteacoccus sp. (green microalga) showed a high efficiency (97%) of MO dye removal from aqueous solutions in a relatively short time (75 min). The Box–Behnken design (BBD)-based response surface methodology (RSM) is a useful mathematical tool that can be successfully used to optimize MO removal from aqueous solutions and reduce the operation cost. Experimental factors such as initial MO concentration, adsorbent dose, temperature, and contact time have clear effects on MO removal using the Bracteacoccus sp. MO is adsorbed and forms a monolayer onto the Bracteacoccus sp. surface in an endothermic and spontaneous process. Compared to old and immobilized fresh Bracteacoccus sp. algae, free fresh Bracteacoccus sp. algae showed a higher efficiency of MO removal from aqueous solutions. The mechanism of MO removal using the Bracteacoccus sp. was found to include both sorption and degradation processes, where MO is converted to smaller compounds. The Bracteacoccus sp. can be regenerated and reused up to five times to remove MO from aqueous solutions with a high removal efficiency. Moreover, the removal process of MO from an aqueous solution using the Bracteacoccus sp. has several advantages, such as simplicity, low cost, ease of use, and use of an environmentally friendly adsorbent. Thus, the current procedure is a promising method in environmental remediation for protecting water sources from pollution by synthetic dyes, using an abundant natural biosorbent (e.g., the Bracteacoccus sp.).