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Abstract

Batch experiments were conducted to investigate the capacity and mechanisms for adsorbing Cd²⁺ from aqueous solutions by the composite material. The composite material was manufactured with Plesiomonas shigelloides strain H5 and modified polyacrylonitrile-based carbon fiber. Experimental results showed that the surface areas of modified polyacrylonitrile-based carbon fiber increased by 58.54% and pore width increased by 40.19% compared with unmodified polyacrylonitrile-based carbon fiber. Boehm’s titration results show the surface acid sites of composite material were increased by 712% compared with unmodified polyacrylonitrile-based carbon fiber. The field emission scanning electron microscope results show P. shigelloides H5 can be grown on the surface of modified polyacrylonitrile-based carbon fiber closely. The equilibrium removal rate and sorption quantity of composite material were 71.56% and 7.126 mg g⁻¹, respectively. With the pH value of aqueous solution increased, the removal rate of Cd²⁺ ions was also increased, but the change of temperature and ionic strength had no significant effect on the removal rate. Furthermore, the results showed the whole sorption process was a good fit to Lagergren pseudo-second-order model and Freundlich isotherms model. Therefore, the results infer that there was a heterogeneous distribution of active sites, and then the sorption process was chemical adsorption and multilayer adsorption. In a word, microbial composite carbon fiber material can adsorb Cd²⁺ ions from aqueous solution effectively, which might be helpful in wastewater treatment in the future.

Keywords

polyacrylonitrile-based carbon fiber bioremediation wastewater

Introduction

The development of human life and industrial activities, many heavy metals ions are discharged into the tributary rivers. Industrial wastewater is the main cause of heavy metals pollution of shallow lakes in China, and cadmium is the main pollution element. Growing attention is being given to health hazards caused by the presence of heavy metals in aqueous environments (Deng et al., 2016). Previous studies showed metal-contaminated river can lead to the accumulation of metal ions in fish muscle and vegetables, and then affect human health directly (Kwok et al., 2014; Monferran et al., 2016; Sharma et al., 2016). Cadmium ions selectively accumulate in pancreas, bones, liver, and lungs, which affect cell proliferation, differentiation, apoptosis, and cause a series of diseases, including bone-thinning disease, osteoporosis, and respiratory tract problems (Flora et al., 2008). Many methods, including precipitation, membrane filtration, and flocculation are conventional techniques to remove Cd²⁺ ions from wastewater, but more conventional technologies are ineffective and unfavorable as they cause sludge disposal problems, are expensive, and do not lead to complete removal (Bai et al., 2008; Halttunen et al., 2007). Utilization of microbial adsorbents for the treatment of wastewater contaminated with heavy metals has become an alternative method to conventional treatments. In the past two decades, researchers found some microorganisms have the capacity to adsorb metal ions under the metal-contaminated environment (Abbas et al., 2014; Neethu et al., 2015; Roane and Pepper, 1999). Consequently, researchers utilized microorganisms as a new bioremediation technology to remove metal ions (Abbas et al., 2014). Tetraselmis suecica and Clostridium could absorb cadmium ions to generate iron sulfide composites (Pérez-Rama et al., 2010; Yang et al., 2015). Pseudomonas aeruginosa, Micrococcus sp., Plesiomonas sp., and Ochrobactrum sp. could adsorb metals ions in surface through exopolysaccharide (Kılıç and Dönmez, 2008; Vullo et al., 2008). Besides, researchers found active efflux of toxic ions and enzymatic detoxification could be participating in adsorptive process (Malin Mejare, 2001; Nies, 2003; Piddock, 2006; Silver and Le, 2005). The cadmium adsorption bacterium Plesiomonas shigelloides H5 was found in previous research (Xue et al., 2016). The removal rate of Cd²⁺ by P. shigelloides H5 was three times than E. coli DH5α, so it is an excellent adsorbent. But, dry biomass has limitation in wastewater treatment process, which was not easy to retrieve after treatment. Hence, we want to manufacture a composite material to immobilize P. shigelloides on the surface. That is in order to apply in the actual wastewater treatment as soon as possible. The advantages of combined biosorbents with immobilized carrier are obvious. The surface of polyacrylonitrile-based carbon fiber (PAN-CF) contains numerous micropores, –OH, –COOH, –C=O and other functional groups, which can adsorb small molecule’s pollutants like organic contaminant and ions in the environment effectively (Lu, 2005). Carbon fiber has good electrical conductivity and chemical stability, so it has multiple potential advantages in developing biosorbents carrier (Yang et al., 2004). At present, methods for modifying carbon fiber were usually chemical oxidate, heat, and microwave treatment (Park and Jang, 2002; Sun et al., 2013; Valente Nabais et al., 2004; Zhang et al., 2010). The surface free energy and functional groups amounts of carbon fiber were increased after modification and improve the biological compatibility obviously (Dongkai, 2013). The microbial surface is electronegativity (Wilson et al., 2001), so it can combine with the modified carbon fiber closely. Hence, modified carbon fiber is an excellent biological carrier. In the wastewater treatment process, the biological carrier was used to be active sludge immobilization carrier, which can degrade organic contaminant. But when the wastewater contains a large amount of heavy metals ions, it will have a negative effect on the activate sludge microorganisms. Hence, the practical significance of this study is that the composite materials can adsorb heavy metals ions in wastewater effectively and reduce the influence of heavy metals ions in the whole treatment process. The aim of this study was to manufacture a composite material (PAN-CF and P. shigelloides H5) and testify the effectiveness of this combination of biosorbents. Comparing the characteristics of unmodified PAN-CF and modified PAN-CF with Boehm's titration method (Zhu et al., 2014), Brunauer-Emmett-Teller method (BET), field emission scanning electron microscope (FE-SEM), Fourier Transform infrared spectroscopy (FTIR), video-based optical contact angle measuring system (OCA), and automated digital microscope (ADM). The effects of various pHs, temperature, time, adsorbent dosage, and ionic strength on the removal rate in Cd²⁺ solution by composite materials were studied. Subsequently, the biosorption process was systematically studied using a combination of kinetics and isothermal modeling.

Materials and methods

Manufacture composite material

The modified method was first to pretreat PAN-CF in acetone solution for 2 h and then heat material at 200℃ for 1 h. After pretreatment, we used 60% nitric acid, 5% potassium permanganate, and 15% hydrogen peroxide to modify material for 1 h, respectively. The modified PAN-CF was washed with deionized water until the washed water pH at 7. The modified PAN-CF was placed in the culture medium which contains P. shigelloides H5 with 0.5% inoculum concentration to form biofilm for 7 d (35℃, pH = 7). The composite material was washed with deionized water and dried at room temperature after forming biofilm. The dry weight of biofilm strain was calculated using equation (1)

Biofilmstrainsdryweight = \frac{Wf - Wi}{Wi} (g \cdot g^{- 1})

(1) where Wi and Wf are the initial and final PAN-CF dry weight (g), respectively.

The characteristics of experimental materials

Characteristics of unmodified PAN-CF and modified PAN-CF were analyzed with BET, FE-SEM, FTIR, OCA, and ADM. The OCA system was to measure the contact angle of PAN-CF and then calculate the surface free energy by Owens methods (Owens and Wendt, 1969). The surface free energy of PAN-CF was calculated using equations (2) to (4)

γ_{L_{1}} (1 + cos θ_{1}) = 2 \sqrt{γ_{s}^{D} γ_{L_{1}}^{D}} + 2 \sqrt{γ_{s}^{P} γ_{L_{1}}^{P}}

(2)

γ_{L_{2}} (1 + cos θ_{2}) = 2 \sqrt{γ_{s}^{D} γ_{L_{2}}^{D}} + 2 \sqrt{γ_{s}^{P} γ_{L_{2}}^{P}}

(3)

γ_{S} = γ_{s}^{D} + γ_{s}^{P}

(4) where

γ_{L_{1}}^{D}

and

γ_{L_{1}}^{P}

is the surface free energy of water,

γ_{L_{2}}^{D}

and

γ_{L_{2}}^{P}

is the surface free energy of diiodomethane, and

γ_{S}

is the surface free energy of PAN-CF.

Composite material was observed under FE-SEM after freeze-dried overnight. The energy-dispersive X-ray spectrometer (EDX, Oxford, UK) was used to analyze chemical element characteristic after adsorption experiments. In Boehm’s titration experiment, 1.000 g carbon fiber material was added into NaOH, Na₂CO₃, and NaHCO₃ solution (100 ml 0.05 molċl⁻¹) to calculate the number of functional groups on the material surface, respectively. Zeta potential measurements part was obtained by 0.1 g composite material in 0.01 molċl⁻¹ NaNO₃ solution at pH values ranging from 2.0 to 7.0 (adjustment with 1.0 M HNO₃ or NaOH). Zeta potential values were determined for each sample using an Electrokinetic Analyzer (JS94H; ZhongChen, China).

Analysis of affecting factors

The effects of various adsorbent dosages (0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 g), pHs (1 2, 3, 4, 5, 6, and 7), temperature (10, 15, 20, 25, 30, 35, and 40℃), ionic strength (0.01, 0.1, and 1.0 mol l⁻¹ NaCl), and time on the removal rate in Cd²⁺ solution by composite materials were studied. The composite material and the control group (modified PAN-CF) were added into Cd²⁺ solution (50 mg l⁻¹, 100ml) to test the removal rate. The pH of the precipitate appearance of Cd²⁺ ions was 7.41, so the range of detected pHs was set from 1 to 7. Cd²⁺ ions concentrations were analyzed by ICP Optima 8000 inductively coupled plasma (PerkinElmer, USA) at 228.8 nm and then Cd²⁺ removal rate was calculated by using equation (5)

Removalrate = \frac{Ci - Cf}{Ci} \times 100 %

(5) where Ci and Cf are the initial and final Cd²⁺ concentrations (mgċl⁻¹), respectively.

Adsorption kinetic and isotherm model simulation

The composite materials were added into Cd²⁺ ions solution (100 mg l⁻¹, 50 ml) at 20℃ for 24 h. We design 24 h as the final time of adsorption kinetic experiment, because of the adsorption equilibrium time point was at 20 h in the above adsorption experiment. Adsorption kinetic models were analyzed by using Lagergren pseudo-first-order (equation (6)) and pseudo-second-order models (equation (7))

\frac{1}{Q_{t}} = \frac{K_{1}}{Q_{e} t} + \frac{1}{Q_{e}}

(6)

\frac{t}{Q_{t}} = \frac{1}{K_{2} Q_{e}^{2}} + \frac{t}{Q_{e}}

(7) where

Q_{e} t

is the concentration of Cd²⁺ at different time point, and Q_e (mgċg⁻¹) is the quantity of Cd²⁺ adsorbed at equilibrium. K₁ is the kinetic rate constant (min⁻¹), and K₂ is the pseudo-second kinetic rate constant (gċmgċmin⁻¹).

Isotherm sorption model simulations were analyzed by using Langmuir model and Freundlich model. Equations (8) and (9) are mathematical models corresponding to both sorption isotherms, respectively

\frac{C_{e}}{Q_{e}} = \frac{1}{Q_{m} b} + \frac{C_{e}}{Q_{m}}

(8)

lg Q_{e} = \frac{1}{n} lg C_{e} + lg k

(9) where C_e is the concentration of Cd²⁺ in solution at sorption equilibrium (mgċl⁻¹), Q_e is the quantity of Cd²⁺ adsorbed at equilibrium (mgċg⁻¹), Q_m is the maximum sorption capacity at equilibrium (mgċg⁻¹), b is Langmuir constant implying the thermo-state during sorption process (lċmg⁻¹), n is Freundlich constant implying sorption ability, and k is Freundlich constant implying sorption intensity (l mg⁻¹).

Statistical analysis

All experimental data are expressed as mean ± SD and used T-test to analyze significant differences by IBM SPSS Statistics 22.0 (IBM Software Inc, New York, USA). The value of p < 0.05 was considered signiﬁcant.

Results

The characteristics of experimental materials

FE-SEM results reveal the surface of the modified PAN-CF has been oxidized obvious and presented delamination (Figure 1(a) and (b)). The bacteria could be immobilized on the surface of modified carbon fibers, even after deionized water washing (Figure 1(c)). FTIR result shows modified PAN-CF increased one secondary hydroxyl group at about 1082.14 cm⁻¹ and the peak intensity at 1385.21 cm⁻¹ increased obviously (Figure 2). Figure 3 shows the color of PAN-CF became brilliant black into gray black after chemical modification. In addition, the total surface free energy γ_s of modified PAN-CF increased by 31.59% after modification (Table 1). The specific surface data of carbon fiber materials were shown in Table 2 and Figure 4. The specific surface area, pore size, and pore volume of modified PAN-CF were increased after chemical modification. Boehm’s titration results show the surface acid sites of composite material were increased by 712% compared with unmodified PAN-CF (Table 3). Zeta potential analysis showed that when the solution pH value was greater than 3, the absolute electric potential of the composite material increased significantly (Figure 5). The average biofilm dry weight of modified PAN-CF and unmodified PAN-CF was 0.2637 and 0.1487 gċg⁻¹, respectively (Table 2).

Figure 1.

The surface of different materials was observed by FE-SEM. (a) Unmodified PAN-CF, (b) modified PAN-CF, and (c) microbial composite carbon fiber materials. FE-SEM: field emission scanning electron microscope; PAN-CF: polyacrylonitrile-based carbon fiber.

Figure 2.

PAN-CF was analyzed by FTIR system. FTIR: Fourier transform infrared; PAN-CF: polyacrylonitrile-based carbon fiber.

Figure 3.

Surface morphology of PAN-CF was analyzed by ADM system. (a) Unmodified PAN-CF and (b) modified PAN-CF. ADM: automated digital microscope; PAN-CF: polyacrylonitrile-based carbon fiber.

Table 1.

Surface energy analysis of modified PAN-CF and unmodified PAN-CF.

	Contact angle (°)		Surface free energy (mN m⁻¹)
Sample	Water	Diiodomethane	γ_s	γ_d	γ_p
Unmodified PAN-CF	79.62 ± 2.0	18.31 ± 3.0	48.26 ± 0.7	45.72 ± 0.2	2.54 ± 0.6
Modified PAN-CF	30.19 ± 2.0	60.57 ± 2.0	63.51 ± 0.9	15.44 ± 0.3	48.07 ± 0.8

PAN-CF: polyacrylonitrile-based carbon fiber.

Table 2.

Surface areas porosity and biofilm dry weight of PAN-CF samples.

Samples	Surface areas (m² g⁻¹)	Pore width (nm)	Pore volume (cm³ g⁻¹)	Biofilm dry weight (g g⁻¹)
Unmodified PAN-CF	1042 ± 62	1.836 ± 0.109	0.5783 ± 0.0217	0.1487 ± 0.0084
Modified PAN-CF	1652 ± 102	2.574 ± 0.133	1.625 ± 0.0142	0.2637 ± 0.0158

PAN-CF: polyacrylonitrile-based carbon fiber.

Figure 4.

BET experimental data of PAN-CF samples. (a) Nitrogen adsorption–desorption isotherms and (b) pore size distribution. BET: Brunauer-Emmett-Teller; PAN-CF: polyacrylonitrile-based carbon fiber.

Table 3.

Surface acidity sites of PAN-CF by Boehm’ method.

Samples	–COOH (mmol)	–COOR (mmol)	–OH (mmol)	Total acid functional group (mmol)
Unmodified PAN-CF	0.254 ± 0.012	0.152 ± 0.021	0.255 ± 0.018	0.661 ± 0.017
Modified PAN-CF	0.472 ± 0.015	0.484 ± 0.009	0.763 ± 0.026	1.719 ± 0.017
Composite material	1.823 ± 0.056	1.667 ± 0.064	1.878 ± 0.039	5.368 ± 0.053

PAN-CF: polyacrylonitrile-based carbon fiber.

Figure 5.

Zeta potentials of microbial composite carbon fiber material.

Table 4.

Simulation of sorption kinetic equations and corresponding parameters.

Models	K₁ or K₂	Q_e (mg/g)	R ²
Lagergren pseudo-first-order	0.00040	1.5146	0.2436
Pseudo-second-order	0.00059	7.4691	0.9991

Table 5.

Simulation of sorption isotherms equations and corresponding parameters.

	Langmuir			Freundlich
Temperature	Q_m (mg/g)	b	R ²	k	$\frac{1}{n}$	R ²
20℃	11.4667	0.1224	0.9613	1.7877	0.5632	0.9911

Analysis of influencing factors

As can be seen in Figure 6(a), the adsorbent dosage affects removal capacity. We can see that the removal efficiency increased as the adsorbent dosage increased. However, after 0.5 g, the increasing was more stable. Thus, the dosage of 0.5 g was chosen for subsequent experiments. As can be seen in Figure 6(b), the maximum Cd²⁺ ions removal rate was 71.38% at pH 7. However, there was no significant correlation between the removal rate and temperature changes (Figure 6(c)). Ionic strength test results show the adsorption capacity varies no significant with the increase of ionic strength (Figure 6(d)). The consequence of cadmium removal dynamic experiment is shown in Figure 7. The equilibrium removal rate of Cd²⁺ by composite material and the control group was 71.56 and 24.18%, respectively. The sorption of Cd²⁺ ions by composite material was a rapid process and contained three stages. During the initial stage, the adsorption rate increased rapidly; after 60 min, the Cd²⁺ ions removal rate slowed down and then stabilized as it reached equilibrium after 1200 min. The equilibrium adsorption amount of composite material was 7.126 mgċg⁻¹. EDX results showed that Cd²⁺ ions were adsorbed on the surface of composite material (Figure 8).

Figure 6.

pH, temperature, dosages, and ionic strength effect of removal rate patterns. (a) Removal rate under different adsorbent dosages, (b) removal rate under different pH values, (c) removal rate under different temperature, and (d) the effect of ionic strength on Cd²⁺ adsorption on composite material.

Figure 7.

The equilibrium adsorption curve of both material (p < 0.01).

Figure 8.

EDX image of composite material after adsorbing Cd²⁺ ions. EDX: energy-dispersive X-ray spectrometer.

Adsorption kinetics and isotherms model

The linear fitting results show the adsorption process of composite material was in accordance with pseudo-second-order model (R²= 0.9991) (Table 4 and Figure 9). Moreover, the theoretical equilibrium adsorption amounts (Q_e = 7.4691 mgċg⁻¹) inferred from the pseudo-second-order model were closer to the detected experimental equilibrium adsorption amounts (Q = 7.126 mgċg⁻¹). The two most commonly employed models are listed in Table 5. The results indicated that the whole adsorption process is more inclined to the Freundlich model (R²= 0.9911).

Figure 9.

Linear fitting curve plots of isotherms and kinetics model for Cd²⁺ ions removal by composite material. (a) Lagergren pseudo-first-order linear fitting curve plot, (b) pseudo-second-order linear fitting curve plot, (c) Langmuir isotherm linear fitting curve plot, and (d) Freundlich isotherm linear fitting curve plot.

Discussions

The modified PAN-CF was added one secondary hydroxyl group in 1082 cm⁻¹ and the peak intensity increased in 1385 cm⁻¹ which indicates that the modified process can increase the numbers of alkyl, alkene, and hydroxyl groups. Because the chemical oxidant destroyed the PAN-CF surface, so some delamination appeared on the fiber surface. At the same time, a large number of micropores arose in the delamination, which increased the specific surface area, pore width, and pore volume. In addition, the rise of surface free energy and specific surface area can enhance PAN-CF hydrophilic property to promote bacteria fixed on the PAN-CF surface efficiently. Hence, bacteria hanging amount of modified PAN-CF was increased by 77.34% compared with unmodified PAN-CF. These results indicated that the chemical modification can promote the carbon fiber material more biocompatible and increase the bacteria hanging amount. In the affecting factors experiments, we found the removal capacity of composite materials increased as the pH value increased in the solution. The pH of the biosorption solution affects both cell surface metal binding sites and metal chemistry in water. Because when the pH was 1 the H₃O⁺ ions in the solution were at a high concentration and Cd²⁺ ions had to compete with them for biosorption sites. Ionic strength test results indicated the adsorption of cadmium ions on composite material is less affected by ionic strength. The whole adsorption process formed inner sphere surface complexes through surface complexation. The result of Zeta potential data shows that with the increase of pH value, the adsorbent surface absolute potential increased, which means there is more negative charge on the adsorbent surface to promote Cd²⁺ ions adsorbed in solution. Zeta potential results are consistent with the results of pH influencing factor. But, the experimental results of the temperature change were not related to the removal efficiency, which means no need to adjust the temperature in the actual pollution control in the future. The results of dynamic adsorption experiments show that the adsorption process consists of three stages which are same as previous studies (Zhang et al., 2015). The first is fast adsorption stage, a large number of Cd²⁺ ions were bound to the vacant active sites rapidly and the removal rate increased sharply in the first 60 min. The second stage, the removal rate slowed down gradually with vacant active sites decreased. The last stage is the equilibrium stage, which the vacant active sites were all combined with Cd²⁺ ions. This phenomenon may be due to the fact that, initially, all active sites on the surface of the adsorbents were vacant and the solution concentration was high. After that period, many surface active sites were occupied, so no increase in metal uptake was observed. The Cd²⁺ ions removal rate in the equilibrium phase was 71.56%, which is about three times than the control group. We can infer that the modified carbon fiber as carrier for Cd²⁺ ions adsorption is limited. The bacteria are still playing an important role in the whole adsorption process. The Boehm’s titration experiments also validate that conclusion; the surface acidity sites numbers of composite material were much more than modified PAN-CF. EDX results indicated that Cd²⁺ ions were adsorbed on the surface of the composite material as Kim et al. (2015) reported. As can be seen in Figure 9, Lagergren’s first-order kinetic equation and pseudo-second-order kinetic equation were used to simulate the whole process of Cd²⁺ biosorption. For the biosorption of Cd²⁺, the pseudo-second-order kinetic equations (R²= 0.9991) were betterthan Lagergren’s first-order kinetic equations (R²= 0.2436). Therefore, basedon the pseudo-second-order kinetic equation, indicating that the sorption rate was controlled by chemical sorption (Ding et al., 2012). Besides, the equilibrium adsorption amount (Q = 7.126 mgċg⁻¹) of dynamic equilibrium experiment is similar to the theoretical adsorption amount (Q_e = 7.4691 mgċg⁻¹) in pseudo-second-order kinetic model. The comparison between the Langmuir and Freundlich isotherms is provided in Table 5. The value of R² of Freundlich model was determined to be 0.9911, while the Langmuir model yielded 0.9613. This result indicated that the Freundlich model fits better with the experimental data than does the Langmuir model. Freundlich model is an empirical equation; it can explain the whole adsorption capacity in systems and predicting the adsorption behavior. The Freundlich constant $\frac{1}{n}$ was 0.5632, which indicated that the biosorption of Cd²⁺ ions by composite material was favorable under experimental conditions. And thus, the composite material surface active sites are irregular and the adsorption process tends to multilayer adsorption. Although when we manufactured the composite materials has been fully washed with deionized water, but there may be some bacteria shedding in the practical application. So wastewater treatment process should add one disinfection or filtration equipment to avoid bacteria pollution in the future.

Conclusions

In summary, we have manufactured composite material with P. shigelloides H5 and modified PAN-CF, which can adsorb Cd²⁺ ions in solution. Surface free energy and surface areas porosity of PAN-CF were increased after chemical modification. Modified PAN-CF can be used as a microbial carrier to immobilize biosorbents on the surface. Composite materials also can achieve adsorption saturation in a short time and equilibrium removal rate was 71.56%, which was almost three times than the control group (unmodified PAN-CF). And the equilibrium sorption quantity of composite material was 7.126 mgċg⁻¹. Adsorption kinetic models of composite material were in accordance with Lagergren pseudo-second-order models. Isotherm sorption models of composite material were in accordance with the Freundlich model. Therefore, the sorption rate was controlled by chemical sorption and the adsorbent surface active site is irregular and the adsorption process tends to multilayer adsorption. In addition, we can easily take out the composite materials from the solution, which will be applied as an efficient heavy metal adsorbent in bioremediation application.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by the National Science and Technology Major Project of China (No. 2013ZX07201007) and State Key Laboratory of Urban Water Resource and Environment Funding (No. 2017DX15).

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Adsorption of aquatic Cd 2+ using a combination of bacteria and modified carbon fiber

Abstract

Keywords

Introduction

Materials and methods

Manufacture composite material

The characteristics of experimental materials

Analysis of affecting factors

Adsorption kinetic and isotherm model simulation

Statistical analysis

Results

The characteristics of experimental materials

Analysis of influencing factors

Adsorption kinetics and isotherms model

Discussions

Conclusions

Footnotes

Declaration of Conflicting Interests

Funding

References