# Al2O3 Nanoparticles Promote the Removal of Carbamazepine in Water by Chlorella vulgaris Immobilized in Sodium Alginate Gel Beads

Authors: Wu Yi-cheng; Yang Ai-li; Gao Wei; Fu Hai-yan; Wang Ze-jie
Journal: Journal of Chemistry
 (2020)

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## Abstract

The roles of Al2O3 nanoparticles on the removal of carbamazepine (CBZ) by Chlorella vulgaris immobilized in sodium alginate gel beads were for the first time investigated. The optimum conditions to prepare immobilized C. vulgaris beads with addition of Al2O3 nanoparticles were determined as follows: C. vulgaris density was 3.0 × 106 cells for 1 mL sodium alginate solution, Al2O3 nanoparticle concentration was 0.5 g/L, and concentrations of sodium alginate and CaCl2 were 1.6% and 1%, respectively. The results showed that the proposed algae beads achieved the highest CBZ removal rate of 89.6% after 4 days of treatment, relative to 68.84%, 48.56%, and 17.76% in sodium alginate-immobilized C. vulgaris, free microalgae, and Al2O3 nanoparticle alginate beads, respectively. The results also showed that the CBZ removal rate increased with more proposed algae beads, while decreased with increased bead diameter. The algae beads exhibited excellent CBZ removal ability even after three recycles. This work provided an economical and effective approach to remove CBZ from water.


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## Body

## 1. Introduction

The presence of pharmaceutical and personal care products (PPCPs) in surface water has been well documented in recent years [1–4]. Carbamazepine (CBZ) as an important species of PPCPs is widely used for the treatment of epilepsy and neuralgia. As early as in 1997, the consumption of CBZ had reached 6.33 tons per year in Austria [4]. After being consumed, CBZ is predominantly metabolized in the liver, and 2-3% of the given dose would be excreted via urine in its original form [5]. The excreted CBZ was suspected to enter rivers, streams, and surface waters through the effluent of waste water treatment plants due to its low removal efficiency [6–8]. The accumulation of CBZ in surface wasters was found to pose a great threat for aquatic environment and human health [9–11].Owing to high photosynthesis efficiency and removal efficiency towards CBZ, microalgae are considered to be one of the most promising ways to polish CBZ-contaminated water [12–14]. It was revealed that immobilized microalgae demonstrated higher resistance to hazardous materials and faster reaction kinetics relative to free microalgae because of the higher cell density [15,16]. However, immobilization with traditional carriers such as sodium alginate has the disadvantage of poor mass transfer process [17–19]. Because of the large specific surface area and perfect biocompatibility, addition of nanoparticles to a sodium alginate-immobilized microalgae system demonstrated promising advantage to promote the contact between immobilized microorganisms and pollutants [20–22].Al2O3 nanoparticles possess high specific surface area and surface energy, which provided sufficient space for the immobilized microorganism and promoted the contact of the microorganism and the pollutant and thus enhanced the removal efficiency of pollutants [23]. In the current study, Al2O3 nanoparticles were adopted as additives into sodium alginate-immobilized C. vulgaris (termed as ANFICV) for the purpose of promoting the removal of CBZ.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

CBZ (purity ≥ 98%) and Al2O3 nanoparticles (purity ≥ 99.99%) were purchased from Aladdin Industrial Corporation (California, USA), and methanol (HPLC grade) was provided by Fisher Chemical (USA). All the other chemicals used in this study were of analytical grade.


### 2.2. Microalgae Cultures

TheC. vulgaris was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The microalga was cultivated with the autoclaved BG11 medium in an Erlenmeyer flask under illumination at 28°C and with a light/dark period of 18 h/6 h [24]. Fluorescent lamps (36 W; Lifemax Super 80, Philips, Shanghai, China) were employed as the light source.


### 2.3. Immobilization ofC. vulgaris

ANFICV was prepared according to previous studies [25, 26]. C. vulgaris in the logarithmic growth phase was harvested by centrifuging at 4500 rpm for 5 min. The cell residues were washed with distilled water and then resuspended in the BG11 medium to form a microalgae solution with high cell density. Both Al2O3 nanoparticles and the resuspended C. vulgaris were added to the sodium alginate solution and then mixed evenly in 100 ml Erlenmeyer flasks, resulting in a volume of 30 mL. After that, the BG11 medium was added to the resulted mixture. The mixture was then added to the CaCl2 solution drop by drop with a syringe to form immobilized beads. Beads without C. vulgaris cells or Al2O3 nanoparticles were prepared to carry out control experiments. The prepared beads were washed with sterile distilled water and stored at 4°C for further experiments.To gain optimal immobilization condition, the microalgae density and concentration of Al2O3 nanoparticles, CaCl2, and sodium alginate were adopted as factors to design a four-factor and three-level orthogonal experiment L9(34) (Table 1).Table 1
Factors and levels of the orthogonal experiment design.
LevelsFactor AFactor BFactor CFactor DAl2O3 nanoparticles (g/L)Sodium alginate concentration (%)Microalgae density (cell/ml)CaCl2 concentration (%)10.511 × 106121.01.62 × 106231.52.43 × 1063


### 2.4. Determination of Cell Density in ANFICV

The cell density of suspendedC. vulgaris was determined at the wavelength of 680 nm by an F-4600 spectrophotometer (Hitachi, Japan). Optical density was converted to cell density using a previously prepared calibration curve. Cell density in ANFICV was measured as follows: ANFICV beads were dissolved in 50 g/L sodium citrate solution and then centrifuged at 4500 rpm for 10 min, and the cell residues were resuspended in BG11 for the subsequent optical density determination [27].


### 2.5. Determination of CBZ

The concentration of CBZ was determined by high pressure liquid chromatography (HPLC) equipped with a UV-VIS detector (LC-20, Shimadzu, Japan) [28, 29]. Prior to analysis, the samples were filtrated through a 0.22 μm membrane filter (Bandao, China) and then injected into a C18 column (250 mm × 4.6 mm, 5 μm, Shimadzu, Japan), maintaining a column temperature of 30°C and a pressure of 25 MPa. The mobile phase was the mixture of HPLC grade methanol and water (65 : 35 v/v) and was introduced into the C18 column at a flow rate of 1 mL/min. The CBZ in the column effluent was detected at 285 nm.


### 2.6. Statistical Analysis

The experiments were carried out in triplicates, and data were processed by OriginPro8.0 and the statistical software package of SPSS (Ver.19.0). One-way analysis of variance was performed to determine the significant difference between means (n = 3) at a significant level of P<0.05.


## 2.1. Chemicals and Reagents

CBZ (purity ≥ 98%) and Al2O3 nanoparticles (purity ≥ 99.99%) were purchased from Aladdin Industrial Corporation (California, USA), and methanol (HPLC grade) was provided by Fisher Chemical (USA). All the other chemicals used in this study were of analytical grade.


## 2.2. Microalgae Cultures

TheC. vulgaris was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The microalga was cultivated with the autoclaved BG11 medium in an Erlenmeyer flask under illumination at 28°C and with a light/dark period of 18 h/6 h [24]. Fluorescent lamps (36 W; Lifemax Super 80, Philips, Shanghai, China) were employed as the light source.


## 2.3. Immobilization ofC. vulgaris

ANFICV was prepared according to previous studies [25, 26]. C. vulgaris in the logarithmic growth phase was harvested by centrifuging at 4500 rpm for 5 min. The cell residues were washed with distilled water and then resuspended in the BG11 medium to form a microalgae solution with high cell density. Both Al2O3 nanoparticles and the resuspended C. vulgaris were added to the sodium alginate solution and then mixed evenly in 100 ml Erlenmeyer flasks, resulting in a volume of 30 mL. After that, the BG11 medium was added to the resulted mixture. The mixture was then added to the CaCl2 solution drop by drop with a syringe to form immobilized beads. Beads without C. vulgaris cells or Al2O3 nanoparticles were prepared to carry out control experiments. The prepared beads were washed with sterile distilled water and stored at 4°C for further experiments.To gain optimal immobilization condition, the microalgae density and concentration of Al2O3 nanoparticles, CaCl2, and sodium alginate were adopted as factors to design a four-factor and three-level orthogonal experiment L9(34) (Table 1).Table 1
Factors and levels of the orthogonal experiment design.
LevelsFactor AFactor BFactor CFactor DAl2O3 nanoparticles (g/L)Sodium alginate concentration (%)Microalgae density (cell/ml)CaCl2 concentration (%)10.511 × 106121.01.62 × 106231.52.43 × 1063


## 2.4. Determination of Cell Density in ANFICV

The cell density of suspendedC. vulgaris was determined at the wavelength of 680 nm by an F-4600 spectrophotometer (Hitachi, Japan). Optical density was converted to cell density using a previously prepared calibration curve. Cell density in ANFICV was measured as follows: ANFICV beads were dissolved in 50 g/L sodium citrate solution and then centrifuged at 4500 rpm for 10 min, and the cell residues were resuspended in BG11 for the subsequent optical density determination [27].


## 2.5. Determination of CBZ

The concentration of CBZ was determined by high pressure liquid chromatography (HPLC) equipped with a UV-VIS detector (LC-20, Shimadzu, Japan) [28, 29]. Prior to analysis, the samples were filtrated through a 0.22 μm membrane filter (Bandao, China) and then injected into a C18 column (250 mm × 4.6 mm, 5 μm, Shimadzu, Japan), maintaining a column temperature of 30°C and a pressure of 25 MPa. The mobile phase was the mixture of HPLC grade methanol and water (65 : 35 v/v) and was introduced into the C18 column at a flow rate of 1 mL/min. The CBZ in the column effluent was detected at 285 nm.


## 2.6. Statistical Analysis

The experiments were carried out in triplicates, and data were processed by OriginPro8.0 and the statistical software package of SPSS (Ver.19.0). One-way analysis of variance was performed to determine the significant difference between means (n = 3) at a significant level of P<0.05.


## 3. Results and Discussion

### 3.1. Optimization of ANFICV Preparation

An orthogonal experiment was carried out in order to determine the optimal factors to prepare ANFICV (Table2). It was demonstrated that A3B2C3D1 achieved the best results towards CBZ removal, for which Al2O3 nanoparticle concentration was determined as 0.5 g/L, sodium alginate concentration as 1.6%, the density of microalga cell as 3.0 × 106 cell/mL, and the CaCl2 concentration as 1%. Under such conditions, the removal efficiency of CBZ reached 94.68 ± 0.81% after 5 days; meanwhile, the mechanical strength of the ANFICV bead was the best. According to the range value (R), the concentration of CaCl2 was the most important factor for the removal of CBZ, followed by microalgae density, the concentration of Al2O3 nanoparticles, and sodium alginate concentration.Table 2
Orthogonal experimental results.
Group numberFactor levelRemoval efficiency (%)StrengthABCD10.51.01182.43 ± 1.49+20.51.62282.66 ± 2.99++30.52.43378.53 ± 0.80+++41.01.03290.28 ± 0.51+51.01.61380.97 ± 0.28++61.02.42189.78 ± 5.18+++71.51.02376.36 ± 0.78+81.51.631394.68 ± 0.81+++91.52.41278.58 ± 0.93++K178.7989.2989.9290.92K279.4581.6377.3580.39K386.3483.2869.4165.31R5.8003.8067.1689.215The number of “+” indicates the level of mechanical strength of the prepared microalgae beads. More “+” means higher level.K1, K2, and K3 represent the value of CBZ removal under different factors at leve1, 2, and 3, respectively. R represents the range of CBZ removal under different factors.


### 3.2. Al2O3 Nanoparticles Promoted the Growth of Immobilized Microalgae

The specific surface area was measured by an automatic specific surface area and porosity analyzer (ASAP2020, Micromeritics Instrument Corporation, USA). The results showed that the specific surface area of the beads prepared by the sodium alginate alone was 2.64 m2/g, which was increased to 3.57 m2/g with the addition of Al2O3 nanoparticles. The effect of addition of Al2O3 nanoparticles into sodium alginate-immobilized beads on the growth of microalgae was evaluated through determining the cell density of the microalgae. It was demonstrated that the difference in cell density was enlarged over inoculation time (P<0.05). After 5 days inoculation, the cell density with Al2O3 nanoparticles was determined as 4.80 × 106 cells/mL, larger than 4.21 × 106 cells/mL with no addition of Al2O3 nanoparticles (Figure 1). The results confirmed that Al2O3 nanoparticles could effectively promote the metabolism of C. vulgaris.Figure 1
The growth curves ofC. vulgaris immobilized in beads with and without Al2O3 nanoparticles.


### 3.3. ANFICV Enhanced the Removal Efficiency of CBZ

The removal of CBZ from water by ANFICV and three control treatments (sodium alginate-immobilizedC. vulgaris, free algae, and Al2O3 nanoparticles alginate bead) was compared. It was illustrated that the concentration of CBZ decreased rapidly on the first day for ANFICV and sodium alginate-immobilized microalgae beads, followed by free microalgae and Al2O3 nanoparticle alginate beads (Figure 2). It might be related to the strong adsorption of CBZ by the alginate bead and C. vulgaris. The concentration of CBZ in water decreased slightly in Al2O3 nanoparticle alginate beads over time probably due to the saturated adsorption of CZB.Figure 2
The concentration of CBZ with time under different treatment proposals.With prolonged treatment, the concentration of CBZ decreased continuously and the treatment efficiency for ANFICV, sodium alginate-immobilizedC. vulgaris, and free C. vulgaris became larger and larger (P<0.05). After 4 days of treatment, the CBZ concentration decreased from 100 to 10.40 ± 3.01 mg/L treated by ANFICV, followed by 31.16 ± 10.69 mg/L and 51.44 ± 2.91 mg/L for sodium alginate-immobilized C. vulgaris and free algae, respectively, while it was decreased to 82.34 ± 5.08 mg/L for Al2O3 nanoparticle alginate beads. The largest removal efficiency of CBZ by ANFICV might be because of the promoted metabolism of C. vulgaris caused by the addition of Al2O3 nanoparticles, as well as the adsorption of CBZ by Al2O3 nanoparticles.


### 3.4. Effect of Initial Concentration on CBZ Removal

The effect of initial concentration of CBZ on the removal efficiency by ANFICV was evaluated. It was observed that the removal efficiency of CBZ decreased with increased CBZ concentration (Figure3). The removal efficiency of CBZ for 4 days of treatment by ANFICV reached 86.73 ± 3.06% for an initial concentration of 40 mg/L, which was reduced to 72.83 ± 3.46%, 65.29 ± 3.57%, and 65.25 ± 2.75% for 80, 120, and 160 mg/L CBZ, respectively. The result showed the removal efficiency of CBZ decreases with the increase in initial concentration (P<0.05). The reason might be that the increase in CBZ concentration inhibited the growth and the physiological characteristics of C. vulgaris, especially when the concentration of CBZ was increased to 160 mg/L.Figure 3
Removal efficiency of CBZ with different initial concentrations by ANFICV.


### 3.5. Effect of ANFICV Bead Diameter on CBZ Removal

Bead diameter could affect the mass transfer performance of the bead, which in turn affects pollutant absorption by the microorganism immobilized within the bead [30]. In order to investigate the effect of bead size on CBZ removal by ANFICV, three bead diameters of 6 mm (big bead), 4 mm (middle bead), and 2 mm (small bead) were prepared with the same Al2O3 nanoparticles concentration, C. vulgaris cell density, and sodium alginate. It was shown that the bead diameter significantly affected the removal of CBZ, and its effects were enlarged over time (P<0.05). After 4 days of inoculation, the concentration of CBZ reduced from 100 to 60.02 ± 7.93 mg/L with big beads, which was 16.89 mg/L higher than that for middle beads and was 5.91 times of that for small beads (Figure 4). The larger removal efficiency of CBZ by the smaller bead might be because the smaller bead could result in a larger surface/volume ratio, which was beneficial for microalgae to obtain light and pollutant.Figure 4
The effect of bead size of ANFICV on CBZ removal.


### 3.6. Effect of ANFICV Bead Dosage on CBZ Removal

Moreover, the present research evaluated the effects of the ANFICV bead dosage on the removal of CBZ. The results showed that the concentration of CBZ was reduced from 100 mg/L to 50.14 ± 3.54 mg/L when 100 ANFICV beads were adopted (Figure5). It was further decreased to 38.77 ± 4.40 mg/L, 21.71 ± 5.43 mg/L, and 7.44 ± 2.42 mg/L when the dosage of ANFICV bead was increased to 300, 500, and 700, respectively. The promoted removal performance of CBZ with increased ANFICV dosage might be mainly because more ANFICV beads could increase the number of microalgae cells to remove CBZ, and it could also increase the quantity of ANFICV to absorb CBZ.Figure 5
Effect of the dosage of the ANFICV bead on the removal of CBZ.


### 3.7. Effect of Recycling Times on the Performance of CBZ Removal

The recycling of ANFICV bead can significantly reduce the cost and promote its practical application. The effect of recycling times of the ANFICV bead on the removal efficiency of CBZ was evaluated. As shown in Figure6, the removal efficiency of CBZ was 87.67% for the first cycle, while decreased to 52.86% for the third cycle. The removal efficiency of CBZ decreased with the increased recycling times, which was mainly due to the expansion and pyrolysis of partial ANFICV beads during operation.Figure 6
Effect of recycling times on the performance of CBZ removal.


## 3.1. Optimization of ANFICV Preparation

An orthogonal experiment was carried out in order to determine the optimal factors to prepare ANFICV (Table2). It was demonstrated that A3B2C3D1 achieved the best results towards CBZ removal, for which Al2O3 nanoparticle concentration was determined as 0.5 g/L, sodium alginate concentration as 1.6%, the density of microalga cell as 3.0 × 106 cell/mL, and the CaCl2 concentration as 1%. Under such conditions, the removal efficiency of CBZ reached 94.68 ± 0.81% after 5 days; meanwhile, the mechanical strength of the ANFICV bead was the best. According to the range value (R), the concentration of CaCl2 was the most important factor for the removal of CBZ, followed by microalgae density, the concentration of Al2O3 nanoparticles, and sodium alginate concentration.Table 2
Orthogonal experimental results.
Group numberFactor levelRemoval efficiency (%)StrengthABCD10.51.01182.43 ± 1.49+20.51.62282.66 ± 2.99++30.52.43378.53 ± 0.80+++41.01.03290.28 ± 0.51+51.01.61380.97 ± 0.28++61.02.42189.78 ± 5.18+++71.51.02376.36 ± 0.78+81.51.631394.68 ± 0.81+++91.52.41278.58 ± 0.93++K178.7989.2989.9290.92K279.4581.6377.3580.39K386.3483.2869.4165.31R5.8003.8067.1689.215The number of “+” indicates the level of mechanical strength of the prepared microalgae beads. More “+” means higher level.K1, K2, and K3 represent the value of CBZ removal under different factors at leve1, 2, and 3, respectively. R represents the range of CBZ removal under different factors.


## 3.2. Al2O3 Nanoparticles Promoted the Growth of Immobilized Microalgae

The specific surface area was measured by an automatic specific surface area and porosity analyzer (ASAP2020, Micromeritics Instrument Corporation, USA). The results showed that the specific surface area of the beads prepared by the sodium alginate alone was 2.64 m2/g, which was increased to 3.57 m2/g with the addition of Al2O3 nanoparticles. The effect of addition of Al2O3 nanoparticles into sodium alginate-immobilized beads on the growth of microalgae was evaluated through determining the cell density of the microalgae. It was demonstrated that the difference in cell density was enlarged over inoculation time (P<0.05). After 5 days inoculation, the cell density with Al2O3 nanoparticles was determined as 4.80 × 106 cells/mL, larger than 4.21 × 106 cells/mL with no addition of Al2O3 nanoparticles (Figure 1). The results confirmed that Al2O3 nanoparticles could effectively promote the metabolism of C. vulgaris.Figure 1
The growth curves ofC. vulgaris immobilized in beads with and without Al2O3 nanoparticles.


## 3.3. ANFICV Enhanced the Removal Efficiency of CBZ

The removal of CBZ from water by ANFICV and three control treatments (sodium alginate-immobilizedC. vulgaris, free algae, and Al2O3 nanoparticles alginate bead) was compared. It was illustrated that the concentration of CBZ decreased rapidly on the first day for ANFICV and sodium alginate-immobilized microalgae beads, followed by free microalgae and Al2O3 nanoparticle alginate beads (Figure 2). It might be related to the strong adsorption of CBZ by the alginate bead and C. vulgaris. The concentration of CBZ in water decreased slightly in Al2O3 nanoparticle alginate beads over time probably due to the saturated adsorption of CZB.Figure 2
The concentration of CBZ with time under different treatment proposals.With prolonged treatment, the concentration of CBZ decreased continuously and the treatment efficiency for ANFICV, sodium alginate-immobilizedC. vulgaris, and free C. vulgaris became larger and larger (P<0.05). After 4 days of treatment, the CBZ concentration decreased from 100 to 10.40 ± 3.01 mg/L treated by ANFICV, followed by 31.16 ± 10.69 mg/L and 51.44 ± 2.91 mg/L for sodium alginate-immobilized C. vulgaris and free algae, respectively, while it was decreased to 82.34 ± 5.08 mg/L for Al2O3 nanoparticle alginate beads. The largest removal efficiency of CBZ by ANFICV might be because of the promoted metabolism of C. vulgaris caused by the addition of Al2O3 nanoparticles, as well as the adsorption of CBZ by Al2O3 nanoparticles.


## 3.4. Effect of Initial Concentration on CBZ Removal

The effect of initial concentration of CBZ on the removal efficiency by ANFICV was evaluated. It was observed that the removal efficiency of CBZ decreased with increased CBZ concentration (Figure3). The removal efficiency of CBZ for 4 days of treatment by ANFICV reached 86.73 ± 3.06% for an initial concentration of 40 mg/L, which was reduced to 72.83 ± 3.46%, 65.29 ± 3.57%, and 65.25 ± 2.75% for 80, 120, and 160 mg/L CBZ, respectively. The result showed the removal efficiency of CBZ decreases with the increase in initial concentration (P<0.05). The reason might be that the increase in CBZ concentration inhibited the growth and the physiological characteristics of C. vulgaris, especially when the concentration of CBZ was increased to 160 mg/L.Figure 3
Removal efficiency of CBZ with different initial concentrations by ANFICV.


## 3.5. Effect of ANFICV Bead Diameter on CBZ Removal

Bead diameter could affect the mass transfer performance of the bead, which in turn affects pollutant absorption by the microorganism immobilized within the bead [30]. In order to investigate the effect of bead size on CBZ removal by ANFICV, three bead diameters of 6 mm (big bead), 4 mm (middle bead), and 2 mm (small bead) were prepared with the same Al2O3 nanoparticles concentration, C. vulgaris cell density, and sodium alginate. It was shown that the bead diameter significantly affected the removal of CBZ, and its effects were enlarged over time (P<0.05). After 4 days of inoculation, the concentration of CBZ reduced from 100 to 60.02 ± 7.93 mg/L with big beads, which was 16.89 mg/L higher than that for middle beads and was 5.91 times of that for small beads (Figure 4). The larger removal efficiency of CBZ by the smaller bead might be because the smaller bead could result in a larger surface/volume ratio, which was beneficial for microalgae to obtain light and pollutant.Figure 4
The effect of bead size of ANFICV on CBZ removal.


## 3.6. Effect of ANFICV Bead Dosage on CBZ Removal

Moreover, the present research evaluated the effects of the ANFICV bead dosage on the removal of CBZ. The results showed that the concentration of CBZ was reduced from 100 mg/L to 50.14 ± 3.54 mg/L when 100 ANFICV beads were adopted (Figure5). It was further decreased to 38.77 ± 4.40 mg/L, 21.71 ± 5.43 mg/L, and 7.44 ± 2.42 mg/L when the dosage of ANFICV bead was increased to 300, 500, and 700, respectively. The promoted removal performance of CBZ with increased ANFICV dosage might be mainly because more ANFICV beads could increase the number of microalgae cells to remove CBZ, and it could also increase the quantity of ANFICV to absorb CBZ.Figure 5
Effect of the dosage of the ANFICV bead on the removal of CBZ.


## 3.7. Effect of Recycling Times on the Performance of CBZ Removal

The recycling of ANFICV bead can significantly reduce the cost and promote its practical application. The effect of recycling times of the ANFICV bead on the removal efficiency of CBZ was evaluated. As shown in Figure6, the removal efficiency of CBZ was 87.67% for the first cycle, while decreased to 52.86% for the third cycle. The removal efficiency of CBZ decreased with the increased recycling times, which was mainly due to the expansion and pyrolysis of partial ANFICV beads during operation.Figure 6
Effect of recycling times on the performance of CBZ removal.


## 4. Conclusions

The present research confirmed that ANFICV with Al2O3 nanoparticles as additives significantly promoted the growth of immobilized C. vulgaris and improved the performance of CBZ removal by immobilized C. vulgaris. The CaCl2 concentration was revealed with the most significant impact on ANFICV preparation for CBZ removal. The results demonstrated that both dosage and diameter of the ANFICV bead could affect the removal efficiency of CBZ. Relative smaller ANFICV beads benefited CBZ removal, and CBZ removal efficiency was increased with the increased dosage of ANFICV beads. This study provided a low-cost and efficient method for the treatment of CBZ-contaminated water.



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