All chemicals: Bisphenol A (99% purity) reagent, ferric chloride hexahydrate (FeCl36H2O), ferrous sulfate heptahydrate (FeSO47H2O), and sodium hydroxide (NaOH) (AR) utilized in this study were of analytical grade and purchased from Sigma-Aldrich (West Chester, PA, USA) and applied without additional purification. Distilled water (DW) was utilized for the preparation of all the aqueous solutions during the entire experiment. The BPA solution utilized in the experiment was carefully prepared prior to each experiment to avoid possible microbial degradation. The chemical and physical properties of BPA are presented in Table 1. The PKS biochar was obtained from AMR Environmental Sdn Bhd, located at Johor, Bahru, Malaysia. All chemicals: Bisphenol A (99% purity) reagent, ferric chloride hexahydrate (FeCl36H2O), ferrous sulfate heptahydrate (FeSO47H2O), and sodium hydroxide (NaOH) (AR) utilized in this study were of analytical grade and purchased from Sigma-Aldrich (West Chester, PA, USA) and applied without additional purification. Distilled water (DW) was utilized for the preparation of all the aqueous solutions during the entire experiment. The BPA solution utilized in the experiment was carefully prepared prior to each experiment to avoid possible microbial degradation. The chemical and physical properties of BPA are presented in Table 1. The PKS biochar was obtained from AMR Environmental Sdn Bhd, located at Johor, Bahru, Malaysia.

**Table 1.** Properties of Bisphenol A (BPA). **Table 1.** Properties of Bisphenol A (BPA).

#### *2.2. Synthesis of Magnetic Biochar 2.2. Synthesis of Magnetic Biochar*

The impregnation of biochar with magnetite using co-precipitation of ferric and ferrous salts on carbonaceous materials method was according to the procedure employed in previous studies [16]. Initially, the PKS-BC sample was grounded and modified using high energy ball milling (HEBM) for 3 hrs to achieve a nano-sized biochar. The obtained ball-milled powdered BC was passed through 50–63 µm sieves and washed with distilled water and ethanol three times. Afterwards, the PKS-BC was oven-dried overnight at 110 °C. The obtained dried BC was collected in a bottle container, sealed, and labelled as NBC and kept in a desiccator for further use. In the next phase, 8.5 g of the dried PKS-BC was soaked in a 100 mL of FeCl3 of 0.25 mol·L−1 and 0.125 mol·L−1 of FeSO4 solution and uniformly mixed via the magnetic stirrer. The mixtures were subjected to heat at 60 °C with constant stirring to ensure that Fe3+ and Fe2+ could saturate into BC and later allowed to cool to 40 °C. Subsequently, 100 mL of 1 mol·L−1 of sodium hydroxide (NaOH) solution was dropwisely added to the solution and stirred until the pH attained the range of (10– The impregnation of biochar with magnetite using co-precipitation of ferric and ferrous salts on carbonaceous materials method was according to the procedure employed in previous studies [16]. Initially, the PKS-BC sample was grounded and modified using high energy ball milling (HEBM) for 3 hrs to achieve a nano-sized biochar. The obtained ball-milled powdered BC was passed through 50–63 µm sieves and washed with distilled water and ethanol three times. Afterwards, the PKS-BC was oven-dried overnight at 110 ◦C. The obtained dried BC was collected in a bottle container, sealed, and labelled as NBC and kept in a desiccator for further use. In the next phase, 8.5 g of the dried PKS-BC was soaked in a 100 mL of FeCl3 of 0.25 mol·L −1 and 0.125 mol·L −1 of FeSO4 solution and uniformly mixed via the magnetic stirrer. The mixtures were subjected to heat at 60 ◦C with constant stirring to ensure that Fe3+ and Fe2+ could saturate into BC and later allowed to cool to 40 ◦C. Subsequently, 100 mL of 1 mol·L −1 of sodium hydroxide (NaOH) solution was dropwisely added to the solution and stirred until the pH attained the range of (10–11), and the colour of the solution transform from brownish green to black. Fe3O4 precipitation was formed on the surface of carbon materials under this alkaline condition. Upon the completion of the reaction, the NaOH-impregnated biochar solution was adequately stirred for 60 min to achieve better dispersion and homogenous mixture. The resultant mixture was separated by an external magnet and rinsed with distilled water until the solution pH closed to 7 and then oven-dried at 70 ◦C for 12 h. The final modified BC sample was grounded and collected into a bottle container, sealed, and labelled as MBC and kept in a desiccator thereafter.

#### *2.3. Characterization of Neat and Magnetic Biochar*

The magnetic properties and magnetization curve of MBC was determined using a vibrating sample magnetometer (Lakeshore 7404, Westerville, OH, USA) with an applied field between −10,000 and 10,000 Oe at room temperature, from American Quantum.

Surface functional groups of the BC samples (before and after modification) were confirmed using Fourier transform infrared (FTIR) spectroscopy (Perkin Elmer, 1650 Spectrometer) within the scanned range of 400 to 4000 cm−1 using the attenuated total reflection method.

The textural properties such as surface area, pore volume, and pore size distribution of the samples were determined by the standard N2 physisorption procedures using Micrometrics analyzer (Tristar II Plus model) and determined in line with the Brunauere Emmentte Teller (BET) technique with degassed temperature of 350 ◦C.

X-ray diffraction (XRD) was used to analyse the phase and chemical composition of the prepared BC adsorbents at Cu Kα radiation (2θ spectrum = 20–80◦ ; phase = 0.05◦ 2θ; time per step = 0.2 s) via X-ray powder diffractometer (Rigaku MiniFlex 600). The compositions and surface morphologies of the samples were detected by field emission scanning electron microscopy (FESEM, Zeiss ULTRA 55) and energy dispersive spectroscopy (EDS, Bruker/Quanta 200), Westerville, OH, USA).

Also, the pH of BC was assessed viz: BC was blended with distilled water at (1:10) mass ratio, agitated magnetically for 30 min, and subsequently kept for 60 min. Afterwards, the pH of BC was determined by a pH5S Spear pH tester (T531009086, Shanghai, China).

### *2.4. Analysis of Surface Chemistry of Biochar (pHpzc)*

The surface chemistry of neat and magnetically-modified biochar was performed experimentally using the pH at the point of zero charge according to the procedure of [56] with slight modifications. Ten samples of varying pH (2.0–11.0) were prepared using a 0.01 M aqueous solution of sodium acetate (C2H3NaO2) as the base electrolyte. The pH of the solution was adjusted using either 0.1 M of NaOH or 0.1 M HCl aqueous solution. Then, 0.1 g of magnetic and neat samples of BC were carefully added to each 20 mL of the prepared solutions in a conical flask placed in a swing agitator at a speed of 120 rpm at 298 K room temperature and stirred for 48 h. Afterwards, the resultant supernatant was then decanted, and its pH was determined. The value of pHpzc was computed from a plot of pH of the initial solution against pH of the supernatant [57,58]. Thus, the zeta potentials of neat biochar (NBC) and magnetic biochar (MBC) were examined via zeta potential analyzer (Zetasizer Nano Plus 3, Zeta/nano particle analyzer, Malvin, Austin, TX, USA).

### *2.5. Adsorption Experiment*

Definite amount of NBC and MBC adsorbents (50.0 mg) and 125.0 mL of BPA solution were weighed and added to a set of 250 mL; Erlenmeyer conical flasks sealed externally using aluminium foil to avoid probable photodegradation. The mixture was then placed on an orbital shaker (HY-8, Shanghai, China) and agitated mechanically at 160 rpm at room temperature for 24 h to achieve equilibrium condition. Upon the completion of the agitation process and at pre-specified times, the solution was rapidly removed and filtered using 0.4-µm membrane and the absorbance of the supernatants were analyzed spectrophotometrically at a wavelength of 276 nm using a double beam UV-visible spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) with a pair of 10 mm matched quartz cells, and the concentrations were converted into the established BPA calibration (standard) curve [59,60].

In the single-variable experiment, the effects of adsorbents (BC) doses (0.2–0.8 g; BPA = 20 ppm), pH range were adjusted by 0.01 mol/L HCl and 0.01 mol/L NaOH solution (3.0–12.0; BPA = 20 ppm), contact time (20–240 min; BPA = 20 ppm), ionic strength (Na+ concentration: 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mol/L, BPA = 20 ppm); initial BPA concentrations (8–150 ppm), and reaction temperature (298.15, 318.15, and 338.15 K), on the adsorption of

BPA by NBC and MBC were studied. The amount of BPA adsorbed on BC adsorbent was determined using the following Equation (1):

$$q\_t = \frac{(\mathbb{C}\_S - \mathbb{C}\_t)}{M} \times V \tag{1}$$

where, *Cs* and *Ct* are the starting and residual concentrations of BPA in mg/L, *M* is the mass of the adsorbent (g); and *V* represent the solution volume (L).

For the adsorption thermodynamic tests, 0.5 g of the adsorbent and 100 mL of BPA solution were added to 250 mL conical flask to study the adsorption thermodynamic of MBC for BPA. The mixtures were stirred in a temperature-regulated incubator shaker (Excella E24 Incubator shaker series, USA) at a speed of 150 rpm, pH (6) for 60 min adsorption time, and the adsorption temperature were varied between (298.15 K–338.15 K).

#### *2.6. Statistical Analysis*

For the adsorption experiment, each experimental samples and group were carried out in triplicate, and the average value of the data was considered. The removal efficiency of BPA (*Re%*) and quantity of BPA adsorbed (*qe*) onto BC adsorbent were determined.

The adsorption isotherms, kinetics (20–240 min), and thermodynamics (298.15, 318.15, and 338.15 K) were studied. Each test was carried out in triplicate, and the average results were considered.

#### *2.7. Reusability and Regeneration of MBC*

The reusability studies on the prospect of desorbing of BPA compounds from MBC are indispensable based on environmental and economical perspective, in addition to industrial applicability [61]. With the purpose of achieving regeneration of MBC, an efficient and green eluent was considered. The reusability of MBC at the starting BPA concentration of 20 ppm was assessed by rinsing BPA-ladened MBC with 0.1-M NaOH and distilled water and subjected to ultrasonication, and then followed by oven-drying at 60 ◦C overnight after magnetic field separation [49]. The MBC was repetitively used five times, and the values of *qe* was noted accordingly.

#### *2.8. Adsorption Isotherm*

Adsorption isotherm was employed to compute the quantity of adsorbed BPA on BC based on BPA concentration at a steady temperature. The application is based on standardizing the amount of BPA adsorbed by the mass of BC adsorbent and this confirms comparison with Isotherm models.

#### *2.9. Langmuir Adsorption Isotherm*

The Langmuir model relies on the assumption that uptake of BPA takes place on a homogenous surface via monolayer adsorption with no interaction between adsorbed materials. It also presumes that all sorption sites are 'correspondingly active', and the surface is robustly homogeneous [62]. Principally, the larger values of *R* 2 signify the significance of the adsorption model for contaminants removal in water. The value of *RL* between 0 and 1 implies that the isotherm is advantageous. The isotherm is unsuitable if *RL* > 1, linear if *RL* = 1, irreversible if *RL* = 0, and favorable if *RL* lies between 0 to 1. Arithmetically, the Langmuir isotherm model is denoted in Equation (2):

$$\frac{1}{q\_{\varepsilon}} = \frac{1}{\mathcal{K}\_{L} q\_{\max}} \cdot \frac{1}{\mathcal{C}\_{\varepsilon}} + \frac{1}{q\_{\max}} \tag{2}$$

where *Ce* represent BPA concentration at equilibrium (mg/L); *qmax.*(mg/g) is the maximum single-layer adsorption capacity of the BC adsorbent, *qe* (mg/g) is the quantity of BPA adsorbed; *KL* (mg/g) represent the Langmuir constant closely related to adsorption capacity for overall monolayer coverage; the intercept (1/*KL*), slope (1/*qmax.*), and *qmax.* could be evaluated from a plot of 1/*qe* versus 1/*Ce* for BPA adsorption onto NBC and MBC.

#### *2.10. Freundlich Adsorption Isotherm*

Freundlich isotherm model was employed to study the uneven distribution on the surface of the absorbent, which is heterogeneousness in the adsorption process [63]. The model indicates that the adsorption energy proportionally declines on the endpoint of the adsorption centres of an adsorbent [64]. The degree of correlation between adsorption and solution concentration relies on the adsorption intensity, n. The adsorption conditions can be chemical (n < 1), favourable physical process (n > 1) and favourable linear (n = 1). The *KF* and n are Freundlich constants and were found from the graph as the intersection and slope, respectively. Scientifically, the Freundlich isotherm model can be described in Equation (3). The graph of ln *qe* against ln *Ce* provide a linear plot with an intercept log*KF* and slope 1/n, from which *R* 2 , *KF* and *n*, can be computed, respectively [65].

$$
\log q\_{\varepsilon} = \log K\_f + \frac{1}{n} \log \mathbb{C}\_{\varepsilon} \tag{3}
$$

#### **3. Results**

### *3.1. Results and Discussion*

Characterization and Analysis of Synthesized Biochar Adsorbents

The surface chemistry and morphological characterization of BC sorbents are excellent indicators of adsorption propensity. Hence, VSM, FTIR, FESEM, EDX, and BET surface analyses, together with the point of zero charge of the adsorbents (NBC and MBC) were also evaluated.

#### *3.2. Morphological Analysis of Synthesized Biochar*

FESEM analysis was conducted to examine the surface morphology of the BC samples. The presence of abundant pores were observed on the surface of the BC, which was substantiated by the FESEM micrographs (Figure 1a–c). Images of NBC, MBC (before and after adsorption) are described in Figure 1b,c. The FESEM analysis of the MBC (prior to adsorption and after adsorption) was performed to observe the morphological structure along with the particle size and size distribution of MBC, as presented in (Figure 1b–e), respectively. From Figure 1a–c, the images showed the particles obtainable are spherical in shape with a single uniform aggregate and small non-uniform agglomerates. In Figure 1a, larger particles with a porous and rougher surface were noticed for NBC. Also, the particles possess more cleavages with few connected to bulks. Furthermore, it can be observed from the FESEM micrographs (Figure 1a,b) of NBC and MBC, that the surface of NBC is relatively coarse and rough since the spherical shaped particles with particle size more than 28.32 nm were agglomerated and attached to each other and the pore form is not fully developed.  The dispersal of Fe3O4 nanoparticles on the surface of BC is relatively uniform. The surface of MBC developed shinier and smoother surface after adsorbing BPA (Figure 1c). The average particle size of MBC prior to adsorption as depicted in Figure 1d was computed to be 28.32 nm. Conversely, after adsorption of BPA, the average particle size rose to 46.65 nm as showed in Figure 1e, confirming that the adsorption process was taking place.

#### *3.3. Elemental Analysis of Synthesized PKS Biochar*

For this procedure, 1.0 g of each of the samples (NBC and MBC) was utilized for the EDX analysis. This test studied different component elements existing in each of the BC samples. The EDX test performed reveal the elemental compositions and distribution both in the NBC and MBC. Figure 2 presents the elemental analysis of the NBC and MBC. As revealed in Figure 2, the existence of peaks matches with the carbon (C), oxygen (O), iron (Fe), and silicon (Si) elements. The intensity of C peak is greater than O, Si, Al, and Fe, demonstrating the elevated content of C in NBC (Figure 2a). Also, three peaks displayed at

0.2, 0.5, and 6.40 keV to substantiate the binding energies of Fe3O4 nanoparticles [66]. The elemental composition of novel MBC was presented in Table 2 with the mass ratio of C, O, and Fe was 32.52, 27.23, and 38.14%, respectively. was computed to be 28.32 nm. Conversely, after adsorption of BPA, the average particle size rose to 46.65 nm as showed in Figure 1e, confirming that the adsorption process was taking place.

(Figure 1c). The average particle size of MBC prior to adsorption as depicted in Figure 1d

**Figure 1.** FESEM (field emission scanning electron microscopy) images of NBC (**a**), MBC before adsorption (**b**), magnetic biochar after adsorption (**c**), average particle size distribution before (**d**), and after adsorption (**e**). **Figure 1.** FESEM (field emission scanning electron microscopy) images of NBC (**a**), MBC before adsorption (**b**), magnetic biochar after adsorption (**c**), average particle size distribution before (**d**), and after adsorption (**e**).

revealed in Figure 2, the existence of peaks matches with the carbon (C), oxygen (O), iron (Fe), and silicon (Si) elements. The intensity of C peak is greater than O, Si, Al, and Fe, demonstrating the elevated content of C in NBC (Figure 2a). Also, three peaks displayed at 0.2, 0.5, and 6.40 keV to substantiate the binding energies of Fe3O4 nanoparticles [66]. The elemental composition of novel MBC was presented in Table 2 with the mass ratio of C, O, and Fe was 32.52, 27.23, and 38.14%, respectively. **Table 2.** Elemental constituents of magnetic biochar. **Elements C O Si Fe Total** Mass ratio (%) 32.52 27.23 2.11 38.14 100.00 Atom ratio (%) 52.40 32.93 1.45 13.22 100.00 The elements of NBC include C, O, Si, Al, and Fe, while the element observed in MBC were C, O, Si, and Fe. Noticeably, from Figure 2b,c, the Al element entirely disappeared after the magnetic modification, and also C, and Si weight components were significantly reduced. This indicates that chemical reactions occur during the modification. Conversely, Fe and O weight composition were substantially increased in modified MBC as compared to NBC. Particularly, the oxidation process considerably influences the obtainability of C active sites, in addition to the structural reformation of the adsorbent [67]. This indicates that the oxidation activity of the carbonaceous material justified the reduction in the weight composition of the trace elements, and also the formation of more acidic oxygencontaining functional groups on the surface [68]. These processes play a significant role in the development of more active sites for effectual adsorption process. The mass fraction of Fe in MBC was 38.14%. The high composition of Fe3O4 further proved the success of the magnetic modification of BC, which agreed with the FTIR and XRD analysis.

The elements of NBC include C, O, Si, Al, and Fe, while the element observed in MBC were C, O, Si, and Fe. Noticeably, from Figure 2b,c, the Al element entirely disappeared after the magnetic modification, and also C, and Si weight components were significantly reduced. This indicates that chemical reactions occur during the modification. Conversely, Fe and O weight composition were substantially increased in modified MBC as compared to NBC. Particularly, the oxidation process considerably influences the obtainability of C active sites, in addition to the structural reformation of the adsorbent [67]. This indicates that the oxidation activity of the carbonaceous material justified the reduction in the weight composition of the trace elements, and also the formation of more acidic oxygencontaining functional groups on the surface [68]. These processes play a significant role in the development of more active sites for effectual adsorption process. The mass fraction of Fe in MBC was 38.14%. The high composition of Fe3O4 further proved the success of the

magnetic modification of BC, which agreed with the FTIR and XRD analysis.

**Figure 2.** EDX spectra of the as-synthesized NBC (**a**), and MBC prior to adsorption (**b**), and after adsorption (**c**). **Figure 2.** EDX spectra of the as-synthesized NBC (**a**), and MBC prior to adsorption (**b**), and after adsorption (**c**).

#### *3.4. BET Surface Area Analysis of Biochar*

The nitrogen adsorption isotherm of NBC and MBC is presented in Figure 3. Essentially, the physical factors of BC samples that can influence the adsorptive removal of organic contaminants comprises of effective surface area, total pore diameter and pore volume. Surface area properties of the NBC and MBC were assessed via BET analysis. The BET results illustrate the adsorbate–adsorbent relationships of that adsorbed molecules which are gathered around on the surface of MBC [69]. The results of effective BET surface area, pore diameter and pores volume for both NBC and MBC are presented in Table 3. Noticeably from Table 3, the NBC exhibited a superior surface area of 536.54 m2/g, while MBC had a surface area of 362.07 m2/g. The decline in the surface area of MBC was possible since the MBC was comprised of a moderate surface area of Fe3O4 and an elevated surface area carbonaceous, and superfluous iron (Fe) nanoparticles loading could capture and clog some of the active sorption sites and pores of the NBC [70,71]. This reduction in

surface area of MBC composites as compared to NBC has also been reported in previous studies [72,73]. Similarly, the pore size and pore volume of MBC are higher than that of NBC, which is better beneficial for the sorption of BPA. relative pressures, the adsorption isotherm rose relatively than at relatively low pressures. This implies that the major adsorption takes place at moderately increased pressures and suggests that the material is highly porous with a narrow size distribution.

The nitrogen adsorption isotherm of NBC and MBC is presented in Figure 3. Essentially, the physical factors of BC samples that can influence the adsorptive removal of organic contaminants comprises of effective surface area, total pore diameter and pore volume. Surface area properties of the NBC and MBC were assessed via BET analysis. The BET results illustrate the adsorbate–adsorbent relationships of that adsorbed molecules which are gathered around on the surface of MBC [69]. The results of effective BET surface area, pore diameter and pores volume for both NBC and MBC are presented in Table 3.

/g. The decline in the surface area of MBC was possi-

/g, while

Noticeably from Table 3, the NBC exhibited a superior surface area of 536.54 m2

ble since the MBC was comprised of a moderate surface area of Fe3O4 and an elevated surface area carbonaceous, and superfluous iron (Fe) nanoparticles loading could capture and clog some of the active sorption sites and pores of the NBC [70,71]. This reduction in surface area of MBC composites as compared to NBC has also been reported in previous studies [72,73]. Similarly, the pore size and pore volume of MBC are higher than that of

Similarly, the total pore volume of NBC and MBC found were, respectively, 0.416260

presents a substantial capacity of pollutant adsorption, while after modification with Fe3O4, the existence of Fe3O4 between layers of MBC upsurges the heterogeneity of the adsorbent, thereby resulting in a superior porosity [74]. The impregnation of magnetite on BC has no significant influence on the pore-volume, surface area, and mean aperture. Iron-amendment can either decrease or increase the surface area of an adsorbent and this relies on the initial surface area value of the adsorbent and the proportions of the Fe3O4 particles [75]. The infrequent blocking of surface micropores from magnetite groups may also be responsible for the insignificant reduction in surface area. Thus, the adsorption capacity of modified biochar material is improved. This finding was further corroborated by the FESEM images and EDS spectra. Figure 3 displays the N2 adsorption–desorption isotherms of N2 at 77 K of NBC and MBC. The findings revealed that at moderately high

/g, while MBC modified by Fe3O4 exhibits a better porosity, and

*Polymers* **2021**, *13*, 3781 10 of 31

NBC, which is better beneficial for the sorption of BPA.

*3.4. BET Surface Area Analysis of Biochar*

MBC had a surface area of 362.07 m2

/g, and 0.442203 cm3

cm3

**Figure 3. Figure 3.**  Nitrogen adsorption–desorption isotherm. Nitrogen adsorption–desorption isotherm.

**Table 3.** Physico-chemical properties of neat biochar and magnetic biochar.

Similarly, the total pore volume of NBC and MBC found were, respectively, 0.416260 cm3/g, and 0.442203 cm3/g, while MBC modified by Fe3O4 exhibits a better porosity, and presents a substantial capacity of pollutant adsorption, while after modification with Fe3O4, the existence of Fe3O4 between layers of MBC upsurges the heterogeneity of the adsorbent, thereby resulting in a superior porosity [74]. The impregnation of magnetite on BC has no significant influence on the pore-volume, surface area, and mean aperture. Iron-amendment can either decrease or increase the surface area of an adsorbent and this relies on the initial surface area value of the adsorbent and the proportions of the Fe3O4 particles [75]. The infrequent blocking of surface micropores from magnetite groups may also be responsible for the insignificant reduction in surface area. Thus, the adsorption capacity of modified biochar material is improved. This finding was further corroborated by the FESEM images and EDS spectra. Figure 3 displays the N2 adsorption–desorption isotherms of N2 at 77 K of NBC and MBC. The findings revealed that at moderately high relative pressures, the adsorption isotherm rose relatively than at relatively low pressures. This implies that the major adsorption takes place at moderately increased pressures and suggests that the material is highly porous with a narrow size distribution.

#### *3.5. Magnetic Properties of As-Synthesized Magnetic Biochar*

The vibrating sample magnetometer (VSM) procedure was used to determine the hysteresis loop at room temperature. Most importantly it is employed to quantify the magnetic properties of a materials with respect to magnetic field, time, and temperature. Besides, VSM analysis provides information about whether the magnetization is perpendicular or parallel to the plane described by the substrate. The hysteresis loop of the synthesized MBC was obtained by plotting the magnetization (emu/g) against the magnetic field (Oe). Figure 4 reveals the magnetic hysteresis loops used to analyze the magnetic properties of MBC. As illustrated in the loop observed in Figure 4, the magnetization sharply increased with the decrease of the mass ration of BC. The curve signified distinctive super-paramagnetic

properties. The synthesized MBC exhibited magnetization M values of 6.4882 emu/g which revealed that the magnetization of MBC can be altered by the mass ration of BC: magnetite (Fe3O4) nanoparticles. The super-paramagnetic properties was triggered by the alteration of BC with magnetite (Fe3O4) nano-particles, which could ensure the MBC can be readily recovered from the suspended solution through external magnetic field, which makes the replicated use of the MBC in the actual wastewater purification system viable [72]. Thus, novel MBC was easily detached using an external magnetic field, as illustrated by the inset. The result of magnetization obtained in this study is consistent with previous studies reported from [76–78]. values of 6.4882 emu/g which revealed that the magnetization of MBC can be altered by the mass ration of BC: magnetite (Fe3O4) nanoparticles. The super-paramagnetic properties was triggered by the alteration of BC with magnetite (Fe3O4) nano-particles, which could ensure the MBC can be readily recovered from the suspended solution through external magnetic field, which makes the replicated use of the MBC in the actual wastewater purification system viable [72]. Thus, novel MBC was easily detached using an external magnetic field, as illustrated by the inset. The result of magnetization obtained in this study is consistent with previous studies reported from [76–78].

**/g) BET Specific Surface Area (m2**

The vibrating sample magnetometer (VSM) procedure was used to determine the hysteresis loop at room temperature. Most importantly it is employed to quantify the magnetic properties of a materials with respect to magnetic field, time, and temperature. Besides, VSM analysis provides information about whether the magnetization is perpendicular or parallel to the plane described by the substrate. The hysteresis loop of the synthesized MBC was obtained by plotting the magnetization (emu/g) against the magnetic field (Oe). Figure 4 reveals the magnetic hysteresis loops used to analyze the magnetic properties of MBC. As illustrated in the loop observed in Figure 4, the magnetization sharply increased with the decrease of the mass ration of BC. The curve signified distinctive super-paramagnetic properties. The synthesized MBC exhibited magnetization M

**/g) pHpzc**

*Polymers* **2021**, *13*, 3781 11 of 31

**Table 3.** Physico-chemical properties of neat biochar and magnetic biochar.

*3.5. Magnetic Properties of As-Synthesized Magnetic Biochar*

NBC 15.516 0.416260 536.5398 4.829 MBC 24.427 0.442203 362.0673 5.612

**Materials Pore Diameter (Å) Pore Volume (cm3**

**Figure 4.** Magnetization curve for MBC. **Figure 4.** Magnetization curve for MBC.

#### *3.6. XRD Analysis of Synthesized Biochar 3.6. XRD Analysis of Synthesized Biochar*

X-ray diffraction analysis of a material describe the size and the nature of the planes of the synthesized material. The crystalline structures of NBC and MBC were analyzed X-ray diffraction analysis of a material describe the size and the nature of the planes of the synthesized material. The crystalline structures of NBC and MBC were analyzed using XRD as indicated in Figure 5. XRD is an efficient technique to verify the presence of Fe3O4 in the synthesized MBC [79]. Figure 5 reveals the XRD patterns of both NBC and MBC. The diffraction spectra of the synthesized BC samples showed the existence of magnetite (Fe3O4). The diffraction peaks at 2θ of 30.044◦ , 36.66◦ , 36.57◦ , 42.45◦ , 57.26◦ , 61.54◦ , and 62.76◦ are indexed to the (200), (016), (220), (232), and (040) hkL planes, respectively, which correspond satisfactorily with the database of Fe3O4 standard card Inorganic Crystal Structure Database (ICSD No. 98-007-7864) with a space group of P12/c1 and lattice parameter (a = b = c) of 28.644 Å and confirms the signature peaks of a hexagonal unit cell Fe3O4, respectively. No impurity peak is observed in the XRD pattern, which indicates that the Fe3O4 particles are highly crystalline hexagonal spinel structure. As observed from the spectra, all the diffraction peaks are designated to the magnetic hexagonal structure. There are no other peaks associated with another material detected from the XRD result, which confirmed that the nano-magnetite is pure magnetite (Fe3O4). The XRD spectra of Fe3O4 in this study is analogous to other studies from [80,81], and both Fe3O4 and BC patterns were overlapped in a XRD spectra of MBC, demonstrating successful synthesis of MBC composite.

using XRD as indicated in Figure 5. XRD is an efficient technique to verify the presence of Fe3O4 in the synthesized MBC [79]. Figure 5 reveals the XRD patterns of both NBC and MBC. The diffraction spectra of the synthesized BC samples showed the existence of magnetite (Fe3O4). The diffraction peaks at 2θ of 30.044°, 36.66°, 36.57°, 42.45°, 57.26°, 61.54°, and 62.76° are indexed to the (200), (016), (220), (232), and (040) hkL planes, respectively, which correspond satisfactorily with the database of Fe3O4 standard card Inorganic Crystal Structure Database (ICSD No. 98-007-7864) with a space group of P12/c1 and lattice parameter (a = b = c) of 28.644 Å and confirms the signature peaks of a hexagonal unit cell Fe3O4, respectively. No impurity peak is observed in the XRD pattern, which indicates that the Fe3O4 particles are highly crystalline hexagonal spinel structure. As observed from the spectra, all the diffraction peaks are designated to the magnetic hexagonal structure. There are no other peaks associated with another material detected from the XRD result, which confirmed that the nano-magnetite is pure magnetite (Fe3O4). The XRD spectra of Fe3O4 in this study is analogous to other studies from [80,81], and both Fe3O4 and BC patterns were overlapped in a XRD spectra of MBC, demonstrating successful synthesis of MBC compo-

**Figure 5.** X-ray diffraction (XRD) patterns of NBC (a) and MBC (b). **Figure 5.** X-ray diffraction (XRD) patterns of NBC (**a**) and MBC (**b**).

#### *3.7. Analysis of Functional Group 3.7. Analysis of Functional Group*

site.

The surface functional groups are the central chemical variable of BC material that influence the BPA adsorption. FTIR spectroscopy was employed to substantiate the modification process and acquire the information on the existence of different functional groups on the surface of the material [82]. The spectrum results and various bands in the spectra signifying vibration of functional groups were illustrated in Figure 6 for NBC and MBC, respectively. During the Fe3+/Fe2+/NaOH process, the surface of the char aids the nucleation of iron oxide precipitation. As indicated in Figure 6, the existence of functional groups of –COOH and –OH group are responsible for the binding of the iron oxide and iron hydroxide particles in the solution, and then bonded to the char [83]. This could be symbolized as char-O-FexOy (Fe3O4/Fe2O3). In this context, the bold 'O' was initially a hydroxyl group on the surface and signify a chemical bond between iron oxide particle and the char phases. This bonding may conceivably be combined with some mechanical interlocking between H-bonding (such as between Fe–OH at the metal oxide surface with C– OH of the char surfaces) and phases in addition to some columbic interfaces. These interactions could strongly bind magnetic iron oxide to the BC materials [84]. Also, as indicated in Figure 6 of the FTIR spectra, there are limited peaks that surfaced for both NBC and MBC. The peaks at 2834, and 2878 cm−1 were distinctive peaks of The surface functional groups are the central chemical variable of BC material that influence the BPA adsorption. FTIR spectroscopy was employed to substantiate the modification process and acquire the information on the existence of different functional groups on the surface of the material [82]. The spectrum results and various bands in the spectra signifying vibration of functional groups were illustrated in Figure 6 for NBC and MBC, respectively. During the Fe3+/Fe2+/NaOH process, the surface of the char aids the nucleation of iron oxide precipitation. As indicated in Figure 6, the existence of functional groups of –COOH and –OH group are responsible for the binding of the iron oxide and iron hydroxide particles in the solution, and then bonded to the char [83]. This could be symbolized as char-O-FexOy (Fe3O4/Fe2O3). In this context, the bold 'O' was initially a hydroxyl group on the surface and signify a chemical bond between iron oxide particle and the char phases. This bonding may conceivably be combined with some mechanical interlocking between H-bonding (such as between Fe–OH at the metal oxide surface with C–OH of the char surfaces) and phases in addition to some columbic interfaces. These interactions could strongly bind magnetic iron oxide to the BC materials [84]. *Polymers* **2021**, *13*, 3781 13 of 31 the C-H bond [82]. Similarly, the peak at 1081 cm−1 was the C-O stretching vibration in the composition of carbohydrate, polysaccharide, or aromatic ether. The peaks close to 1578 and 1579 cm−1 were the stretching vibration peaks of C=C, C-H, and of C=O on the aromatic ring [85]. The results go along with the BC characteristics that not only contain porous structure but also abundant active adsorption sites for BPA removal [86]. Besides, a noticeable peak at 652 cm−1 was observed for MBC, which correspond to the typical stretching peak of Fe-O [87]. This indicated that the Fe3O4 nanoparticles have been efficaciously loaded on the surface of BC as revealed by the FESEM results. The spectra results revealed that magnetic alteration improved the varieties and number of functional groups of the BC, which may influence the adsorption of BPA.

pHpzc is the pH at which the net surface charge on the surface of an adsorbent is zero.

or attracting organic contaminants, in line with their cationic or anionic functional groups [88]. The point of zero charges was investigated to describe the surface chemistry of MBC and NBC. The values of point of zero charge (pHpzc) of neat and MBC in relation to the solution pH were computed and displayed in Figure 7. According to Figure 7, the pHpzc at the point where the change in pH (initial pH-final pH) equal to zero for the MBC and NBC were 5.61 and 4.81, respectively. It is observed that the pHpzc of MBC (5.61) is higher than that of NBC (4.81). This increase may be due to the introduction of iron oxides (Fe3O4) on the surface of NBC [89]. Hence, higher pHpzc is more beneficial for the adsorption of BPA anions, which are often existed in acidic solution [90]. The zeta potential of all samples steadily reduced with rising pH indicating more net negative surface charge at elevated pH. Similarly, the zeta potential of both NBC and MBC was both positive and negative within the whole pH range studied (2–11) as can be seen from Figure 7. Evidently, both MBC and NBC are positively charged when the pH is lower than 6.0. Once the pH is increase, the surface charge of the two biochar materials swiftly transformed from positive to negative. As pH consistently increase, the surface charges of both NBC and MBC risen and, noticeably, the zeta potential of NBC and MBC were −28.56 mV and −22.47 mV, which

**Figure 6.** Fourier transform infrared spectra of NBC (a) and MBC (b). **Figure 6.** Fourier transform infrared spectra of NBC (**a**) and MBC (**b**).

*3.8. Determination of Electrokinetic Charge (pHpzc) of Biochar*

Also, as indicated in Figure 6 of the FTIR spectra, there are limited peaks that surfaced for both NBC and MBC. The peaks at 2834, and 2878 cm−1 were distinctive peaks of the C-H bond [82]. Similarly, the peak at 1081 cm−1 was the C-O stretching vibration in the composition of carbohydrate, polysaccharide, or aromatic ether. The peaks close to 1578 and 1579 cm−1 were the stretching vibration peaks of C=C, C-H, and of C=O on the aromatic ring [85]. The results go along with the BC characteristics that not only contain porous structure but also abundant active adsorption sites for BPA removal [86]. Besides, a noticeable peak at 652 cm−1 was observed for MBC, which correspond to the typical stretching peak of Fe-O [87]. This indicated that the Fe3O4 nanoparticles have been efficaciously loaded on the surface of BC as revealed by the FESEM results. The spectra results revealed that magnetic alteration improved the varieties and number of functional groups of the BC, which may influence the adsorption of BPA.

### *3.8. Determination of Electrokinetic Charge (pHpzc) of Biochar*

pHpzc is the pH at which the net surface charge on the surface of an adsorbent is zero. It is a critical variable in examining the efficacy of the adsorption systems. When the pH is greater than the pHpzc, the adsorbent surface acquires negative charge, hence repelling or attracting organic contaminants, in line with their cationic or anionic functional groups [88]. The point of zero charges was investigated to describe the surface chemistry of MBC and NBC. The values of point of zero charge (pHpzc) of neat and MBC in relation to the solution pH were computed and displayed in Figure 7. According to Figure 7, the pHpzc at the point where the change in pH (initial pH-final pH) equal to zero for the MBC and NBC were 5.61 and 4.81, respectively. It is observed that the pHpzc of MBC (5.61) is higher than that of NBC (4.81). This increase may be due to the introduction of iron oxides (Fe3O4) on the surface of NBC [89]. Hence, higher pHpzc is more beneficial for the adsorption of BPA anions, which are often existed in acidic solution [90]. The zeta potential of all samples steadily reduced with rising pH indicating more net negative surface charge at elevated pH. Similarly, the zeta potential of both NBC and MBC was both positive and negative within the whole pH range studied (2–11) as can be seen from Figure 7. Evidently, both MBC and NBC are positively charged when the pH is lower than 6.0. Once the pH is increase, the surface charge of the two biochar materials swiftly transformed from positive to negative. As pH consistently increase, the surface charges of both NBC and MBC risen and, noticeably, the zeta potential of NBC and MBC were −28.56 mV and −22.47 mV, which shows that both biochar samples were negatively charged (see Figure 7). Though, the surface of NBC is slightly more negatively charged, and the electrostatic attraction between the negatively charged NBC and HBPA improves the adsorption. This finding is in good agreement with previous studies [16,91]. *Polymers* **2021**, *13*, 3781 14 of 31 shows that both biochar samples were negatively charged (see Figure 7). Though, the surface of NBC is slightly more negatively charged, and the electrostatic attraction between the negatively charged NBC and HBPA improves the adsorption. This finding is in good agreement with previous studies [16,91].

**Figure 7.** Zeta potential of NBC and MBC at different pH. **Figure 7.** Zeta potential of NBC and MBC at different pH.

*3.9. Influence of Working Conditions on the Adsorption of BPA*

*3.10. Effect of pH on BPA Adsorption*

The operating variables for instance contact time, solution pH, adsorbent dosage, ionic strength, BPA concentration, and other variables can influence the surface properties

The pH is one of the main parameters that control the removal of compounds present in aqueous environment using solid adsorbents. The solution pH is among the most essential variables that determine the elimination of various compounds existing in aqueous environment utilizing solid adsorbent materials and the optimization of adsorption process. The effect of pH on adsorption was reliant on the target contaminants and nature of absorbent [42]. It influences not only the speciation of the adsorbate, but also the level of ionization and adsorbent surface charge [90,93,94]. Accordingly, most of the investigations involved in contaminants adsorption onto BC strongly considered the effect of solution pH. To investigate the influence of different solution pH upon adsorption of BPA on the surface of NBC and MBC, the tests were performed in the pH range of 3.0–12.0, BPA concentration 20 ppm, adsorbent dose 0.5 g, and temperature 20 °C, and the results were illustrated in Figure 8a. As indicated in the figure, the sorption of BPA by NBC and MBC is clearly pH reliant. The highest adsorption capacity of NBC to BPA take place at pH (3.0), which is attributable to the development of electron receiver-giver interaction (ERG) between BPA and NBC, together with a robust hydrogen bond [95,96]. When pH rose from 6.0 to 7.0, the adsorption capacity of NBC to BPA slightly increased further, since BPA starts to moderately dissociate, and BPA in the solution is no more in molecular form, nevertheless few HBPA- still occurs. The electrostatic attraction between NBC and HBPAenhances the adsorption process due to the positively charged surface of NBC. Further

was carried out on the as-synthesized BC adsorbents to study the influence of these operating variables on the adsorption capacity and removal efficiency for BPA uptake.

#### *3.9. Influence of Working Conditions on the Adsorption of BPA*

The operating variables for instance contact time, solution pH, adsorbent dosage, ionic strength, BPA concentration, and other variables can influence the surface properties of the adsorbent surface and its BPA binding ability [88,92]. Hence, a batch adsorption test was carried out on the as-synthesized BC adsorbents to study the influence of these operating variables on the adsorption capacity and removal efficiency for BPA uptake.

#### *3.10. Effect of pH on BPA Adsorption*

The pH is one of the main parameters that control the removal of compounds present in aqueous environment using solid adsorbents. The solution pH is among the most essential variables that determine the elimination of various compounds existing in aqueous environment utilizing solid adsorbent materials and the optimization of adsorption process. The effect of pH on adsorption was reliant on the target contaminants and nature of absorbent [42]. It influences not only the speciation of the adsorbate, but also the level of ionization and adsorbent surface charge [90,93,94]. Accordingly, most of the investigations involved in contaminants adsorption onto BC strongly considered the effect of solution pH. To investigate the influence of different solution pH upon adsorption of BPA on the surface of NBC and MBC, the tests were performed in the pH range of 3.0–12.0, BPA concentration 20 ppm, adsorbent dose 0.5 g, and temperature 20 ◦C, and the results were illustrated in Figure 8a. As indicated in the figure, the sorption of BPA by NBC and MBC is clearly pH reliant. The highest adsorption capacity of NBC to BPA take place at pH (3.0), which is attributable to the development of electron receiver-giver interaction (ERG) between BPA and NBC, together with a robust hydrogen bond [95,96]. When pH rose from 6.0 to 7.0, the adsorption capacity of NBC to BPA slightly increased further, since BPA starts to moderately dissociate, and BPA in the solution is no more in molecular form, nevertheless few HBPA− still occurs. The electrostatic attraction between NBC and HBPA− enhances the adsorption process due to the positively charged surface of NBC. Further surge in pH led to a subsequent reduction in the adsorption capacity of NBC to BPA, owing to weakness in the interaction of the hydrogen bonding and π-π electron receiver–giver(ERG) between BPA and NBC when the solution pH is higher than the BPA acid dissociation constant [97]. Conversely, the adsorption capacity of BPA by MBC rose initially and after that it declined with the upsurge of pH greater than 6.0, which implied that the adsorption of BPA on BC was largely dependent on the solution pH of the system. Thus, the considerable rise in BPA adsorption on MBC between pH 3.0–6.0 was possibly attributed to the electrostatic interaction between the positively charged HBPA+ species and negatively charged surface of MBC in the solution. The surface of MBC may develop positively charged at low pH because of the protonation reaction (H+ (aq) + −ROH(surf) ↔ −ROH2+ (surf)) on the surface of MBC [98]. When the solution pH is increased, a negatively charged surface of MBC with plentiful active biding sites emerge owing to deprotonation reaction (OH− (aq) + −ROH(surf) ↔ H2O + –RO− (surf)) on the surface of MBC. Hence, the enhanced BPA adsorption by MBC at pH 3.0–6.0 was not only controlled by the electrostatic interaction mechanism [99], but possibly caused by the surface reduction/complexation of BPA species onto MBC, and the decline of BPA adsorption at pH >6.0 following the repulsion interaction between negatively charged MBC and dissociated bisphenolate anions (BPA2− and HBPA−) species. The maximum adsorption capacity take place at pH of 6.0.

**Figure 8.** Effect of solution pH (**a**), and ionic strength (**b**) on the adsorption of BPA by NBC and MBC. (BPA concentration: 20 ppm; contact time: 60 min; biochar dose: 0.5 g; and temperature: 293.15 K).

#### *3.11. Effect of Ionic Strength on BPA Adsorption*

Generally, the water body comprises such a complex system that salts and organic contaminants frequently coexist in wastewater which may influence the removal of the contaminants. Hence, the profile of ionic strength influence on BPA adsorption by the assynthesized BC was also investigated using 0–1.0 mol/L NaCl, BPA concentration 20 ppm, and the experimental findings are presented in Figure 8b. Initially, it could be noticed in Figure 8b, the increase in NaCl concentration led to a decline in the adsorption capacity of NBC to BPA and later a rise. Noticeably, the surge in high ionic strength (Na+ concentration) considerably increased the adsorption capacity of BPA to MBC (Figure 8b). The BPA adsorption capacity of BC increased when the Na+ concentration rose from 0 to 1.0 mol/L. Analogously, Zhou et al. [100], found that the upsurge in ionic strength with various ionic species (CaCl2 or NaCl) yielded increase in the adsorption capacity of BPA when peat was applied as an adsorbent. Similarly, erstwhile study has also reported that the increased ionic strength could improve the adsorption of organic contaminants into carbonaceous adsorbents, such as BC, owing to the screening effect of the surface charge generated by

the addition of salt [101]. The BPA adsorption capacity by BC adsorbents rose steadily with the increase in the NaCl concentration. The plausible explanations for this surge could be due to the penetration of ions into the diffusion dual layer around NBC and MBC surfaces and lessen the repulsion between the adsorbents, thereby stimulating the squeezing-out effect (i.e., nanoparticle aggregation), which caused a decline in the adsorption capacity of BPA [102], enhanced activity coefficient of hydrophobic organic contaminants, which leads to a salting-out effect (reduction in solubility), and, therefore, was favorable to the adsorption of BPA [103]. Thus, the decreased adsorption capacity when the NaCl concentration is at 0.2 mol./L might be caused by the competition between BPA and the low concentration of salt solution for the accessible active sites of adsorbents, corroborating that the squeezing-out effect was stronger than the salting-out effect. Conversely, as the NaCl concentration increases, the adsorption capacity enhanced evidently, suggesting that the salting-out effect improved unceasingly [104].

### *3.12. Effects of Biochar Dose on the Removal of BPA*

Adsorbent dosage (AD) is an important parameter in an adsorption process. It controls the adsorbate–adsorbent equilibrium of the adsorption procedure. The removal efficiency based on the adsorbent dosage was studied at other test conditions, BPA concentration 20 ppm, varying dosages (0.2–0.8 g), pH 6.0, contact time (60 min), and adsorption temperature 20 ◦C. The influence of BC dose on the adsorption of BPA by NBC and MBC is illustrated in Figure 9. The removal rate of BPA enhanced with the rise in BC dose. When BC dosage was 0.2 g, the removal efficiencies of BPA by NBC and MBC were 48.45% and 60.0%, respectively. Similarly, when the BC dose rose to 0.5 g, the removal efficiency substantially rose to 85.97% and 94.2%, respectively. The increased removal rate is because of the augmented BC, which also expands the operational specific surface area of adsorption, together with increase in the active pore (binding) site of adsorption [105]. This outstanding adsorption performance was attributable to the presence of plentiful active sites and various interactions between BC adsorbent and BPA, specifically ion exchange and complexation electrostatic interaction. Afterward, further increase in BC dosage has no influence on the removal rate. This is because surplus adsorbent overlaps the effective active sites on BC and competes for limited solutes [42]. *Polymers* **2021**, *13*, 3781 17 of 31 complexation electrostatic interaction. Afterward, further increase in BC dosage has no influence on the removal rate. This is because surplus adsorbent overlaps the effective active sites on BC and competes for limited solutes [42]**.**

**Figure 9.** Effects of BC (a: NBC, B: MBC) dose on the removal of BPA. BPA removal efficiency by BC under varying doses: 0.2–0.8 g; BPA concentration: 20 ppm; temperature: 293.15 K; pH: 6.0; and contact time: 60 min. **Figure 9.** Effects of BC (a: NBC, b: MBC) dose on the removal of BPA. BPA removal efficiency by BC under varying doses: 0.2–0.8 g; BPA concentration: 20 ppm; temperature: 293.15 K; pH: 6.0; and contact time: 60 min.

The influence of initial BPA concentration and contact time on the adsorption of BPA

The test conditions evaluating the influence of contact time on the adsorption of BPA at the test conditions, BPA concentration 20 ppm, adsorbent dosage 0.5, and pH 6.0. Similarly, the result of the effects of contact time on the adsorption capacity and the removal efficiency are illustrated in Figure 11. Noticeably, more than 93% of BPA became adsorbed in about 60 min. For contact time beyond 60 min, the per cent removal of BPA remains stable, because the active sorption sites have been saturated on the adsorbent surface.

initial concentrations of BPA in the stock solution varied between 8.0 to 150.0 ppm, at different time intervals up to 240 min. The result of effect of initial concentration on BPA removal is presented in Figure 10. The initial concentration offers the stimulating force required to overcome the mass transfer wall between the adsorbate and the adsorbent media [106]. Thus, a higher initial concentration may enhance the efficacy of the adsorption process. Evidently, BPA removal is lower at a small concentration due to smaller amount of adsorbates in the solution to dominate active sites on the adsorbent and the amount of BPA adsorbed rises with the upsurge in BPA concentration. Thus, increase in initial BPA concentration, led to gradual rise in the removal efficiency of BPA, since the increased BPA concentration can improve the adsorption drive between the solute and the adsorbent [107,108]. The rise in the concentration of BPA conforms with an increase in BPA removal as shown in Figure 9. When equilibrium is attained, the adsorbent turns out to be saturated. This result is in line with previous study reported by Wang and Zhang

[16].

*3.13. Effects of Initial Concentration and Contact Time on the Removal of BPA*

#### *3.13. Effects of Initial Concentration and Contact Time on the Removal of BPA*

The influence of initial BPA concentration and contact time on the adsorption of BPA from aqueous solution was investigated. The experiments were performed in the presence of a constant dose of adsorbent (0.5 g) of BC at room temperature, at pH (6.0) with various initial concentrations of BPA in the stock solution varied between 8.0 to 150.0 ppm, at different time intervals up to 240 min. The result of effect of initial concentration on BPA removal is presented in Figure 10. The initial concentration offers the stimulating force required to overcome the mass transfer wall between the adsorbate and the adsorbent media [106]. Thus, a higher initial concentration may enhance the efficacy of the adsorption process. Evidently, BPA removal is lower at a small concentration due to smaller amount of adsorbates in the solution to dominate active sites on the adsorbent and the amount of BPA adsorbed rises with the upsurge in BPA concentration. Thus, increase in initial BPA concentration, led to gradual rise in the removal efficiency of BPA, since the increased BPA concentration can improve the adsorption drive between the solute and the adsorbent [107,108]. The rise in the concentration of BPA conforms with an increase in BPA removal as shown in Figure 9. When equilibrium is attained, the adsorbent turns out to be saturated. This result is in line with previous study reported by Wang and Zhang [16]. *Polymers* **2021**, *13*, 3781 18 of 31

**Figure 10.** Effects of initial BPA concentrations on the removal efficiency under varying initial concentrations (8–150 ppm) at constant pH (6.0), dosage (0.5 g), and varying contact times (20–240 min). **Figure 10.** Effects of initial BPA concentrations on the removal efficiency under varying initial concentrations (8–150 ppm) at constant pH (6.0), dosage (0.5 g), and varying contact times (20–240 min).

The test conditions evaluating the influence of contact time on the adsorption of BPA at the test conditions, BPA concentration 20 ppm, adsorbent dosage 0.5, and pH 6.0. Similarly, the result of the effects of contact time on the adsorption capacity and the removal efficiency are illustrated in Figure 11. Noticeably, more than 93% of BPA became adsorbed in about 60 min. For contact time beyond 60 min, the per cent removal of BPA remains stable, because the active sorption sites have been saturated on the adsorbent surface.

**Figure 11.** Effects of contact time (20–240 min) on the per cent removal of BPA, at 0.5 g dosage, BPA

Sorption isotherm models are indispensable for recognizing the mechanisms of the adsorption process based on fundamental characteristics and numerical derivations. Also, the isothermal models are used to study the interrelatedness between the adsorbate and the adsorbent. The Freundlich and Langmuir isotherm models are normally employed

concentration 20 ppm, temperature (293.15 K), and pH (6.0).

*3.14. BPA Sorption Isotherm Study*

**Figure 11.** Effects of contact time (20–240 min) on the per cent removal of BPA, at 0.5 g dosage, BPA concentration 20 ppm, temperature (293.15 K), and pH (6.0).

#### *3.14. BPA Sorption Isotherm Study*

Sorption isotherm models are indispensable for recognizing the mechanisms of the adsorption process based on fundamental characteristics and numerical derivations. Also, the isothermal models are used to study the interrelatedness between the adsorbate and the adsorbent. The Freundlich and Langmuir isotherm models are normally employed and were fitted to the adsorption experimental data. The Freundlich isotherm is appropriate to both multilayer and monolayer adsorption and assumes that the adsorbates are adsorbed onto the heterogeneous surface of an adsorbent [109]. The Langmuir isotherm assumes monolayer adsorption on a uniform surface with a limited amount of adsorption sites [110].

The Langmuir isotherm model presumes that every molecule has a stable adsorption enthalpy and activation energy and signifies homogeneous adsorption. Correspondingly, the Freundlich isotherm model is experimental and regards the surface to be heterogeneous [111]. Two regular isotherm models were applied to fit the BPA adsorption isotherms on the as-synthesised BC (Figures 12 and 13). The models were employed to better investigate the BPA adsorption mechanism and performance. The fitting data are illustrated in Figures 12 and 13, and the applicable fitting parameters of these models were summarized in Table 3. The BPA adsorption experimental data were fitted to the Freundlich and Langmuir isotherm models with Origin 8.0.

The fitting of the experimental data into the isotherm models illustrates the adsorption process by the correlation coefficient R2 and constants. To accomplish this, log *qe* is plotted against log Ce based on the linear form of the Freundlich model (Figure 12) [112]. For the Langmuir model linear expression, 1/*qe* versus 1/*Ce* is plotted (Figure 13) [113,114]. The computed qmax values of the Langmuir model from the graph of 1/*qe* against 1/*Ce* was lower than the *qe* values from the experiments (see Table 4), indicating that the experimental adsorption data fit this model. However, the R2 value of Langmuir is lower than that of the Freundlich model.

The variables revealed that the Freundlich model (R2 = 0.88964–0.9195) offers a superior fit to the data than the Langmuir model (R2 = 0.7535–0.85608), as showed by the linear regression values. Additionally, the heterogeneity coefficient (1/n) of the two BC falls above 1 (1/n >1), which implied satisfactory physical adsorption process [115]. This trend

sites [110].

muir isotherm models with Origin 8.0.

was also corroborated by several studies [116,117] using BC synthesized from eucalyptus forest residues, sugarcane bagasse, castor meal, water hyacinth, green pericarp of coconut, and MBC to remove various contaminants. Hence, BC generated from various biomasses at different temperatures were observed to have various adsorption mechanisms and adsorption abilities [118]. In this work, BPA adsorption onto NBC and MBC was excellently fit using the Freundlich model in the determination of the efficacy of NBC and MBC for the removal of BPA, which demonstrates that the adsorption take place as a heterogeneous surface multilayer. A synopsis of the isotherm variables is presented in Table 4. The fitting of the experimental data into the isotherm models illustrates the adsorption process by the correlation coefficient R2 and constants. To accomplish this, log *qe* is plotted against log Ce based on the linear form of the Freundlich model (Figure 12) [112]. For the Langmuir model linear expression, 1/*qe* versus 1/*Ce* is plotted (Figure 13) [113,114]. The computed qmax values of the Langmuir model from the graph of 1/*qe* against 1/*Ce* was lower than the *qe* values from the experiments (see Table 4), indicating that the experimental adsorption data fit this model. However, the R2 value of Langmuir is lower than that of the Freundlich model.

and were fitted to the adsorption experimental data. The Freundlich isotherm is appropriate to both multilayer and monolayer adsorption and assumes that the adsorbates are adsorbed onto the heterogeneous surface of an adsorbent [109]. The Langmuir isotherm assumes monolayer adsorption on a uniform surface with a limited amount of adsorption

The Langmuir isotherm model presumes that every molecule has a stable adsorption enthalpy and activation energy and signifies homogeneous adsorption. Correspondingly, the Freundlich isotherm model is experimental and regards the surface to be heterogeneous [111]. Two regular isotherm models were applied to fit the BPA adsorption isotherms on the as-synthesised BC (Figures 12 and 13). The models were employed to better investigate the BPA adsorption mechanism and performance. The fitting data are illustrated in Figures 12 and 13, and the applicable fitting parameters of these models were summarized in Table 3. The BPA adsorption experimental data were fitted to the Freundlich and Lang-

*Polymers* **2021**, *13*, 3781 19 of 31

**Figure 12.** Freundlich isotherm for BPA adsorption onto NBC (a) and MBC (b) at pH 6.0, dose 0.5 g, contact time 240 min, and initial BPA concentration 50 ppm. **Figure 12.** Freundlich isotherm for BPA adsorption onto NBC (a) and MBC (b) at pH 6.0, dose 0.5 g, contact time 240 min, and initial BPA concentration 50 ppm. the removal of BPA, which demonstrates that the adsorption take place as a heterogeneous surface multilayer. A synopsis of the isotherm variables is presented in Table 4.

) R2⁎ qmax(mg/g) KL(L·mg−1

**Figure 13.** Langmuir isotherm on BPA adsorption onto NBC and MBC at 0.5 g dose, pH 6.0, contact time 240 min, and initial BPA concentration 50 ppm. **Figure 13.** Langmuir isotherm on BPA adsorption onto NBC (a) and MBC (b) at 0.5 g dose, pH 6.0, contact time 240 min, and initial BPA concentration 50 ppm.

Kinetic study offers essential information on the mechanism of adsorption and influencing mechanism of adsorption process as either chemical reaction or mass transfer to attain optimal working conditions for industrial scale [119]. With a view to examine the kinetic mechanism triggering BPA adsorption, experimental data were fitted to the pseudo-first order (PFO) [120] and pseudo-second order (PSO) [121] linearized models

( −) = ( − 1

1 2 2 × 1 + 1

where *qt* and *qe* are the amounts of BPA adsorbed at time t and equilibrium, respectively (mg/g), t is the contact time (min), and *K1* and *K2* are the rate constants of the PFO and PSO kinetic models, respectively (1/min). In batch process, sorption kinetics is described by different models based on adsorption equilibrium including the PSO (pseudo-first order) and PSO (pseudo-second order) kinetic models. In this context, it is used to model the

Figure 14a,b illustrates the graphs of (ln(*qe* − *qt*) against time) and (time/*qt* against time) for the pseudo-first order and pseudo-second order kinetic models, respectively. The computed variables and the experimental data of the two models are presented in Table 5. Noticeably the coefficient of correlation value (R2 = 0.9937) for the PSO model is of better linearity and higher in comparison with the PFO (R2 =0.8515) for BPA adsorption,

1 = ) RL R2⁎⁎

) (4)

(5)

NBC 1.02974 9.718964 0.88964 5.438033607 3.95207 0.00509 0.7535 MBC 1.05679 11.62947 0.9195 4.72701489 4.01652 0.005 0.85608 Note: 1/n, KF, and R2⁎ were computed from a plot of logqe versus logCe (Equation (5), while KL, qmax, and R2⁎⁎ were

**Table 4.** Adsorption Isotherms variables.

**Adsorbents Freundlich Model Langmuir Model**

*3.15. BPA Adsorption Kinetic Studies*

using Equations (4) and (5), respectively:

kinetics of the adsorption of BPA onto BC.

calculated by plotting 1/qe against 1/Ce (Equation (4), respectively, using the data from Figures 10 and 11.

1/n KF(L·mg−1

Note: 1/n, *KF*, and R2 \* were computed from a plot of log*qe* versus logCe (Equation (5)), while *KL*, qmax, and R2 \*\* were calculated by plotting 1/*qe* against 1/Ce (Equation (4)), respectively, using the data from Figures 10 and 11.

#### *3.15. BPA Adsorption Kinetic Studies*

Kinetic study offers essential information on the mechanism of adsorption and influencing mechanism of adsorption process as either chemical reaction or mass transfer to attain optimal working conditions for industrial scale [119]. With a view to examine the kinetic mechanism triggering BPA adsorption, experimental data were fitted to the pseudo-first order (PFO) [120] and pseudo-second order (PSO) [121] linearized models using Equations (4) and (5), respectively:

$$\ln(q\_{\varepsilon} - q\_{t}) = \ln(\ln q\_{\varepsilon} - \mathcal{K}\_{1} \, t) \tag{4}$$

$$\frac{1}{q\_t} = \frac{1}{K\_2 q\_\varepsilon 2} \times \frac{1}{t} + \frac{1}{q\_\varepsilon} \tag{5}$$

where *qt* and *qe* are the amounts of BPA adsorbed at time t and equilibrium, respectively (mg/g), t is the contact time (min), and *K*1 and *K*2 are the rate constants of the PFO and PSO kinetic models, respectively (1/min). In batch process, sorption kinetics is described by different models based on adsorption equilibrium including the PSO (pseudo-first order) and PSO (pseudo-second order) kinetic models. In this context, it is used to model the kinetics of the adsorption of BPA onto BC.

Figure 14a,b illustrates the graphs of (*ln*(*qe* − *qt*) against time) and (time/*qt* against time) for the pseudo-first order and pseudo-second order kinetic models, respectively. The computed variables and the experimental data of the two models are presented in Table 5. Noticeably the coefficient of correlation value (R2 = 0.9937) for the PSO model is of better linearity and higher in comparison with the PFO (R2 =0.8515) for BPA adsorption, which was also noticed for a similar compound (sulfamethoxazole) adsorption by functionalized BC [122]. Similarly, the computed *qe* (mg/g) for the PSO model is close to the experimental *qe* value (see Table 5). Hence, PSO model satisfactorily fits better the experimental results than the PFO kinetic model. The PSO kinetic model suggests that the chemisorption can be regarded as a rate-controlling phase during the adsorption procedure [3,123]. Similarly, the chemisorption takes place via electron-exchange or sharing between BPA and BC [124]. The elevated rate constants of the PSO model for BPA can be attributed to rapid interaction with the active adsorption sites of MBC. This finding agrees with previous studies on the BPA adsorption on the following adsorbents: algal BC [48], magnetic CuZnFe2O4–BC composite [125], modified organo-montmorillonites [126].

**Table 5.** Kinetic study result for BPA adsorption.

NB: PSO is pseudo-second order; PFO is pseudo-first order.

**Figure 14.** (**a**) Pseudo-first order model for adsorption of BPA by MBC, and (**b**) pseudo-second order model for adsorption of BPA by MBC. **Figure 14.** (**a**) Pseudo-first order model for adsorption of BPA by MBC, and (**b**) pseudo-second order model for adsorption of BPA by MBC.

#### **Table 5.** Kinetic study result for BPA adsorption. *3.16. Adsorption Thermodynamics*

*3.16. Adsorption Thermodynamics*

PSO

PFO

1 2 2

1 =

**Kinetic Models Parameters Value Linear Regression** × 1 + 1 where K2 is rate constant (g·mg−1·min−1 ) K2(g·mg−1·min−1 ) 0.005289 = 1.556 +0.0583 Comp. qe (mg/g) 47.15266 Exp. qe (mg/g) 37.75 R2 0.99376 ln(qe − qt) = ln(qe) − K1t K1 (min−1 ) 3.50 × 10−6 = 0.00383 + 8.4 Comp. qe (mg/g) 12.003837 Exp. qe (mg/g) 37.75 R2 0.8515 NB: PSO is pseudo-second order; PFO is pseudo-first order. Thermodynamic studies are employed to decipher any reaction in a better approach and reveals whether the adsorption process is an intended process or a spontaneous process [127]. It also indicates the influence of temperature on the adsorption process. Generally, exothermal and endothermal sorption processes are the two common processes. If the sorption declines with increasing temperature, it implies the exothermal sorption process whereas if the sorption increases with rising temperature, it signifies that the sorption is an endothermal process. In this study, the temperature influence on BPA adsorption onto MBC was studied by performing adsorption tests at varying temperatures, viz., 298.15, 318.15, and 338.15 K, at optimum pH (6.0) and adsorbent dosage 0.5 g, respectively. The thermodynamic variables such as enthalpy change (∆*H*◦ ), standard Gibb's free energy change (∆*G* ◦ ), and change in entropy (∆*S* ◦ ), were computed and summarized in Table 6 using the following equations:

which was also noticed for a similar compound (sulfamethoxazole) adsorption by functionalized BC [122]. Similarly, the computed *qe* (mg/g) for the PSO model is close to the experimental *qe* value (see Table 5). Hence, PSO model satisfactorily fits better the experimental results than the PFO kinetic model. The PSO kinetic model suggests that the chemisorption can be regarded as a rate-controlling phase during the adsorption procedure [3,123]. Similarly, the chemisorption takes place via electron-exchange or sharing between BPA and BC [124]. The elevated rate constants of the PSO model for BPA can be attributed to rapid interaction with the active adsorption sites of MBC. This finding agrees with previous studies on the BPA adsorption on the following adsorbents: algal BC [48], magnetic

CuZnFe2O4–BC composite [125], modified organo-montmorillonites [126].

$$
\Delta G\_O = -RT\ln K\_L \tag{6}
$$

$$K\_L = \frac{q\_\varepsilon}{\mathcal{C}\_\varepsilon} \tag{7}$$

$$
\Delta \text{S}^{\circ} = \Delta H^{\circ} - \Delta \text{G}^{\circ} / T \tag{8}
$$

the sorption declines with increasing temperature, it implies the exothermal sorption process whereas if the sorption increases with rising temperature, it signifies that the sorption is an endothermal process. In this study, the temperature influence on BPA adsorption onto MBC was studied by performing adsorption tests at varying temperatures, viz., where *R* is the gas constant (8.314 J/mol−1K−1 ), *qe* is the adsorption capacity (mg/g), *Ce* is the equilibrium concentration (mg/L) and T is the actual temperature (*K*). The y-intercept and the slope of the linear fit following a plot of ln *KL* versus 1/T were employed to calculate the values of ∆*H*◦ and ∆*S* ◦ via the Van't Hoff plot (Figure 15).

**Table 6.** Computed thermodynamic variables for BPA adsorption on MBC at varying temperature.

BPA

**Figure 15.** Thermodynamic plot of lnKL versus 1/T for adsorption of BPA on MBC. **Figure 15.** Thermodynamic plot of lnKL versus 1/T for adsorption of BPA on MBC.

should be the primary leading force for the adsorption of BPA on MBC [131].

*3.17. Investigation of Biochar Regeneration and Reusability* As the sorption temperature rises, the values of ∆*G* ◦ turn out to be more negative, which implies that BPA is better effectively adsorbed to MBC at elevated temperatures. The negative ∆*G* ◦ indicate that all adsorption processes were spontaneous [128]. The positive ∆*H*◦ (51.23 kJ·mol−1 ) indicated that the interaction between BPA and MBC was an endothermic process which possibly attributed to the competitive dissolution of BPA in aqueous solution [129]. The positive ∆*S* ◦ and negative ∆G0 values suggested the spontaneity of the adsorption process. The higher positive ∆*S* ◦ (176.13 J·mol−1 ·K−1 ) as indicated in Table 5, further demonstrate increasing randomness at the adsorbate-adsorbent interface as a result of free water molecules [130]. Thus, the determined positive ∆*S* ◦ and negative ∆*G* ◦ values at experimented temperatures substantiated the BPA adsorption on MBC and indicated the spontaneity of the sorption process, besides the entropy effect should be the primary leading force for the adsorption of BPA on MBC [131].

298.15, 318.15, and 338.15 K, at optimum pH (6.0) and adsorbent dosage 0.5 g,respectively. The thermodynamic variables such as enthalpy change (*ΔH*°), standard Gibb's free energy change (*ΔG*°), and change in entropy (*ΔS*°), were computed and summarized in Table 6

∆ = − ln

° − ∆

**) ΔH°(kJmol−1**

 =

° = ∆

298.15 0.526093278 −1.304090065 51.22768066<PHONE_NUMBER>

As the sorption temperature rises, the values of *ΔG°* turn out to be more negative, which implies that BPA is better effectively adsorbed to MBC at elevated temperatures. The negative *ΔG°* indicate that all adsorption processes were spontaneous [128]. The positive *ΔH°* (51.23 kJ·mol−1) indicated that the interaction between BPA and MBC was an endothermic process which possibly attributed to the competitive dissolution of BPA in aqueous solution [129]. The positive *ΔS°* and negative ΔG0 values suggested the spontaneity of the adsorption process. The higher positive *ΔS°* (176.13 J·mol−1·K−1) as indicated in Table 5, further demonstrate increasing randomness at the adsorbate-adsorbent interface as a result of free water molecules [130]. Thus, the determined positive *ΔS°* and negative *ΔG°* values at experimented temperatures substantiated the BPA adsorption on MBC and indicated the spontaneity of the sorption process, besides the entropy effect

where *R* is the gas constant (8.314 J/mol−1K−1), *qe* is the adsorption capacity (mg/g), *Ce* is the equilibrium concentration (mg/L) and T is the actual temperature (*K*). The y-intercept and the slope of the linear fit following a plot of ln *KL* versus 1/T were employed to calculate

∆

the values of *ΔH°* and *ΔS°* via the Van't Hoff plot (Figure 15).

**Table 6.** Computed thermodynamic variables for BPA adsorption on MBC at varying temperature.

318.15 1.803593997 −4.770684859 338.15 2.971634746 −8.354391819 (6)

**)**

⁄ (7)

°⁄ (8)

**) ΔS°(J**·**mol·K−1**

using the following equations:

**Adsorbate Temperature (K) lnKL ΔG°(kJmol−1**

#### *3.17. Investigation of Biochar Regeneration and Reusability*

Recyclability of the adsorbents is strongly essential to preserve process costs down and for the industrial-scale application. Five phases of adsorption–desorption tests were undertaken to examine the reusability of the as-synthesized MBC in accordance with previously adopted experimental procedure [125]. The MBC was repetitively applied five times, and the *qe* values were recorded, as illustrated in Figure 16. The result indicated that *qe* was slightly reduced during the adsorption–desorption experiment. Upon the completion of five cycles of reusability test, the removal efficiency of BPA only reduced by 12.85% as compared with the first cycle. The slight reduction in adsorption capacity during multiple adsorption–desorption tests, was due to the partial desorption of BPA in MBC surface, competitive available sorption sites, loss of solid in solution, and the elution of iron oxide nanoparticles and energetic substances on MBC surface [49,132]. This reveals that the MBC could be an effectual, economical, and environmental benign adsorbent with superior re-usability, which can be practically applied in BPA removal procedures. The recyclability test reveals that MBC can be applied repeatedly in wastewater purification as an efficient adsorbent.

Table 7 presents the comparison of specific surface area characteristics, adsorption capacity of BPA, and the magnetic intensity of the synthesized MBC material in the current study in comparison with various studies.

as an efficient adsorbent.

**Figure 16.** Regeneration of magnetic biochar for BPA removal. Experimental conditions: m = 0.5 g; [BPA] = 20 ppm; and temperature (293.15 K). **Figure 16.** Regeneration of magnetic biochar for BPA removal. Experimental conditions: m = 0.5 g; [BPA] = 20 ppm; and temperature (293.15 K).

Recyclability of the adsorbents is strongly essential to preserve process costs down and for the industrial-scale application. Five phases of adsorption–desorption tests were undertaken to examine the reusability of the as-synthesized MBC in accordance with previously adopted experimental procedure [125]. The MBC was repetitively applied five times, and the *qe*values were recorded, as illustrated in Figure 16. The result indicated that *qe* was slightly reduced during the adsorption–desorption experiment. Upon the completion of five cycles of reusability test, the removal efficiency of BPA only reduced by 12.85% as compared with the first cycle. The slight reduction in adsorption capacity during multiple adsorption–desorption tests, was due to the partial desorption of BPA in MBC surface, competitive available sorption sites, loss of solid in solution, and the elution of iron oxide nanoparticles and energetic substances on MBC surface [49,132]. This reveals that the MBC could be an effectual, economical, and environmental benign adsorbent with superior re-usability, which can be practically applied in BPA removal procedures. The recyclability test reveals that MBC can be applied repeatedly in wastewater purification

dried pineapple 84.89 12.83 34.93 101.16 [47] NA = Not Available; ND = Not determined.

corn straw 313.88 14.5 ND 46.90 [111]

Though few studies reported higher adsorption capacity than the current study despite their low surface area as indicated in Table 7, this is because previous studies have described the performance of MBC to be significantly influenced by the nature of auxiliary and raw materials, starting contaminant concentration, pyrolysis temperature, competitive anions, nature of modifier, reaction temperature, sorption time, and various synthesis methods [86,136,137]. As highlighted in Table 7, the maximum adsorption capacity recorded in the current study is comparable and in line with previous studies using different BC adsorbents. Hence, it can be inferred that the MBC synthesized from palm kernel shell is a promising, efficient, and essential precursor (adsorbent) for the removal of BPA from aqueous environment. The sorption capacity recorded from the current study using magnetic PKS-BC could be stems from highly developed pore structure, superfluous surface area, smaller pore size, considerable surface functional group, high sorptive capacity, and super paramagnetism. It is noteworthy that studies on the adsorption of BPA onto MBC synthesized in-house from palm kernel shell biomass is still very limited. Besides, the

adsorbent demonstrated superior regeneration efficacy, and the resultant MBC could be reclaimed numerous times.

#### *3.18. Controlling Mechanism for BPA Removal*

The schematic diagram of mechanisms of adsorption of BPA (adsorbate) onto assynthesized NBC and MBC is depicted in Figure 17. The average pore size of the assynthesized BC falls below 50 nm, indicating mesoporous. Also, BPA molecules can penetrate the pores of the as-synthesized BC via pore filling. As stated earlier, the adsorption capacity of NBC and MBC to BPA is largely influenced by pH. In acidic environments, the phenolic hydroxyl groups in the chemical structure of BPA protonate, and thereby producing electrostatic repulsion with the positively charged surfaces of both NBC and MBC. Also, since BC adsorbent comprises carbon, silicon, iron, and oxygen groups, these elements C, Si, Fe, and O groups are vastly electronegative because of the sufficiency of available lone pair of electrons, which exhibits binding capabilities toward the BPA as indicated in Figure 2. The carboxyl, hydroxyl, carbonyl, and amine functional groups (as identified in Figure 5) facilitate the affinity of BPA molecules and their adsorption on the surface of BC. Such sorption mechanism is an electron giver–receiver type according to the unbalanced electrons' supply between BPA compound and the BC functional groups. The hydroxyl functional group (−OH) on the BC's surface also develops potent hydrogen bonds with the C-H and −OH on the molecular composition of BPA. Also, the C=O and −COOH acidic functional groups of the as-synthesized BC can serves as electron receivers, producing π-π electron giver–receiver interactions (EGR) with BPA. In alkaline environments, the surface of MBC becomes negatively charged, which retains the electrostatic repulsion with the dissociated BPA2− and HBPA−, whereas NBC acts oppositely. Similarly, the π-π electron giver-receiver interaction (EGR), as well as hydrogen bonds between BPA and as-synthesized BC, would be severely weakened, thereby making it hard for BC to adsorb BPA. Since, BPA exhibits robust hydrophobicity and can be intermixed with the hydrophobic site on NBC and MBC surfaces. Hence, a hydrophobic interaction is correspondingly a central influential force for the adsorption of BPA onto NBC and MBC. *Polymers* **2021**, *13*, 3781 25 of 31

**Figure 17.** Schematic coordination of BPA removal mechanism using biochar. **Figure 17.** Schematic coordination of BPA removal mechanism using biochar.

solution with benefit of being sustainable, and abundantly available.

thors have read and agreed to the published version of the manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

Nigeria, is also hereby thankfully acknowledged.

#### **4. Conclusions 4. Conclusions**

5540204.

corresponding author.

In this work, a novel magnetic palm kernel shell biochar was efficiently synthesized via magnetic modification and applied to remove BPA from aqueous solution. The results In this work, a novel magnetic palm kernel shell biochar was efficiently synthesized via magnetic modification and applied to remove BPA from aqueous solution. The re-

of VSM, XRD, FTIR, BET, FESEM/EDX, and Zeta potential revealed that the Fe3O4 nanoparticles was effectually engrafted on the surface of biochar. The experimental findings

whereas the adsorption kinetic data was controlled by pseudo-second order model. The as-synthesized BC also demonstrated a good magnetic strength for facile recovery, superior recyclability, high selectivity, and eco-friendly. It was noticed that BPA removal is greatly influenced by adsorbent dose, pH, and contact time. The BPA adsorption does increases with reduction of pH, with a maximum adsorption at pH 6 for MBC. Thermodynamic study revealed that BPA adsorption on MBC is endothermic process and spontaneous due to +ΔH and −ΔG, respectively. The principal mechanisms for BPA adsorption on the prepared BC comprised of electrostatic interactions (π-π electron acceptor– donor interactions), hydrophobic interaction, and H-bonding. Therefore, it can be deduced from this study that novel MBC is efficient and practical for the removal of BPA from aqueous

**Author Contributions:** K.K.K. designed the project, performed the study, and wrote the manuscript; K.F.Y. supervised the study, and helped acquire funding; R.S.A. contributed to nanoparticles synthesis and characterization; thanks M.Z.M.N., for the software support; A.Z.A. and H.C.M. were principal investigators and helped acquire funding; and A.Z.A. contributed to chemicals. All au-

**Funding:** This work was funded by Universiti Putra Malaysia under FRGS with grant number

**Data Availability Statement:** The data presented in this study are available on request from the

**Acknowledgments:** The Universiti Putra Malaysia is hereby appreciated. The sponsorship received from the Tertiary Education Trust Fund (TETFund) through the Kwara State University, Malete,

sults of VSM, XRD, FTIR, BET, FESEM/EDX, and Zeta potential revealed that the Fe3O4 nanoparticles was effectually engrafted on the surface of biochar. The experimental findings demonstrated the adsorption isotherm could be better fitted by Freundlich model, whereas the adsorption kinetic data was controlled by pseudo-second order model. The as-synthesized BC also demonstrated a good magnetic strength for facile recovery, superior recyclability, high selectivity, and eco-friendly. It was noticed that BPA removal is greatly influenced by adsorbent dose, pH, and contact time. The BPA adsorption does increases with reduction of pH, with a maximum adsorption at pH 6 for MBC. Thermodynamic study revealed that BPA adsorption on MBC is endothermic process and spontaneous due to +∆H and −∆G, respectively. The principal mechanisms for BPA adsorption on the prepared BC comprised of electrostatic interactions (π-π electron acceptor– donor interactions), hydrophobic interaction, and H-bonding. Therefore, it can be deduced from this study that novel MBC is efficient and practical for the removal of BPA from aqueous solution with benefit of being sustainable, and abundantly available.

**Author Contributions:** K.K.K. designed the project, performed the study, and wrote the manuscript; K.F.Y. supervised the study, and helped acquire funding; R.S.A. contributed to nanoparticles synthesis and characterization; thanks M.Z.M.N., for the software support; A.Z.A. and H.C.M. were principal investigators and helped acquire funding; and A.Z.A. contributed to chemicals. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by Universiti Putra Malaysia under FRGS with grant number 5540204.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The Universiti Putra Malaysia is hereby appreciated. The sponsorship received from the Tertiary Education Trust Fund (TETFund) through the Kwara State University, Malete, Nigeria, is also hereby thankfully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Cytocompatibility of Polymethyl Methacrylate Honeycomb-like Pattern on Perfluorinated Polymer**

**Klaudia Hurtuková 1 , Veronika Juˇricová 1 , Klára Fajstavrová 1 , Dominik Fajstavr 1 , Nikola Slepiˇcková Kasálková 1 , Silvie Rimpelová 2 , Václav Švorˇcík 1 and Petr Slepiˇcka 1,\***

**Abstract:** In this study, we present a simple approach for developing a biocompatible polymer scaffold with a honeycomb-like micropattern. We aimed to combine a plasma treatment of fluorinated ethylene propylene (FEP) substrate with an improved phase separation technique. The plasma exposure served for modification of the polymer surface properties, such as roughness, surface chemistry, and wettability. The treated FEP substrate was applied for the growth of a honeycomb-like pattern from a solution of polymethyl methacrylate (PMMA). The properties of the pattern were strongly dependent on the conditions of plasma exposure of the FEP substrate. The physico-chemical properties of the prepared pattern, such as changes in wettability, aging, morphology, and surface chemistry, were determined. Further, we have examined the cellular response of human osteoblasts (U-2 OS) on the modified substrates. The micropattern prepared with a selected combination of surface activation and amount of PMMA for honeycomb construction showed a positive effect on U-2 OS cell adhesion and proliferation. Samples with higher PMMA content (3 and 4 g) formed more periodic hexagonal structures on the surface compared to its lower amount (1 and 2 g), which led to a significant increase in the pattern cytocompatibility compared to pristine or plasma-treated FEP.

**Keywords:** polymer; plasma modification; PMMA; material morphology; honeycomb-like pattern; cell growth; cytocompatibility; FEP; cell adhesion

### **1. Introduction**

Honeycombs are one of the most studied naturally occurring shapes/structures, which is evidenced by many new reported studies [1]. The impressive hexagonal shape of a honeycomb structure has fascinated many scientists for several decades, especially the research groups of Francois [2,3], Pirk, Zhang [1,4–7], or Bui et al. [8–10]. There were a lot of theories that competed for periodic structures. Two of them have got considerable attention in this field. The first theory states that a bee's six legs ideally fit onto a hexagon. On the contrary, the second theory supports a mathematical explanation that their geometry enables the coverage of the largest possible area using a minimum of building material [11,12]. However, the latest new theory confirms that hexagonal structures are formed due to highly elastic wax, elevated temperature, and surface tension, transforming circular shapes into hexagonal [12–14]. Based on this study, honeycomb-like patterns (HCP) have been identified as unique surfaces due to thousands of interconnected and oriented pores with high mechanical strength [15,16]. Based on the pore size and porosity, three-dimensional (3D) scaffolds resembling the in vivo shape and conditions can be created. This has great potential in biomedical applications, especially in tissue engineering. Porous scaffolds are essential for tissue nutrition, cell proliferation, and the formation of new viable tissues [15,17,18].

**Citation:** Hurtuková, K.; Juˇricová, V.; Fajstavrová, K.; Fajstavr, D.; Slepiˇcková Kasálková, N.; Rimpelová, S.; Švorˇcík, V.; Slepiˇcka, P. Cytocompatibility of Polymethyl Methacrylate Honeycomb-like Pattern on Perfluorinated Polymer. *Polymers* **2021**, *13*, 3663. https:// doi.org/10.3390/polym13213663

Academic Editors: Antonio M. Borrero-López, Concepción Valen-cia-Barragán, Esperanza Cortés Triviño, Adrián Tenorio-Alfonso and Clara Delgado-Sánchez

Received: 30 September 2021 Accepted: 22 October 2021 Published: 24 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Moreover, the porous scaffolds have also protective and storage functions, the latter of which serves for depositing of adhesion molecules and growth factors. Another possible way of utilization is the formation of specific systems for drug delivery [19]. Otherwise, the HCP structures have found a great use in macro-, micro-, and nano-applications in electronics [20], electro-optics [21,22], bioanalytics [23], or in nanofabrication [24].

In several studies, the processes of HCP preparation by various methods including lithography or soft lithography [25–27], template techniques [27], emulsions, and plasma etching [28] have been described. However, one of the fundamental techniques invented by Francois et al. has become the so-called breath figure (BF) method. This method has proven to be cost-effective, uncomplicated, flexible, fast, and does not require specific skills to create a micro-/nano-pattern of honeycombs [2,3]. The principle consists of applying a polymer solution in a non-polar solvent to a substrate located in an environment with a relative humidity higher than 50%. A humid atmosphere can be ensured in two ways: static or dynamic. The static method requires atmosphere saturation with water vapor in a closed vessel, while in the dynamic method, the environment is created by the flow of moist air onto the substrate surface [29–31]. At the beginning of the process, the solvent is endothermically evaporated from the prepared solution. As the surface temperature decreases, water vapor begins to condense in the form of heterogeneous droplets on the surface of the substrate. After equilibration between the substrate and the environment, the condensed droplets begin to evaporate, thus, spontaneously leaving the hexagonal HCP structures on the substrate surface. The change of key units such as soluble substance, solvent, and humid air creates a wide range of highly ordered porous films with different pore sizes [28,32]. While the BF method requires a humid atmosphere for HCP formation, the newer method of improved phase separation (IPS) invented by Bui et al. [8] requires typical air conditions. The IPS technique uses two steps to prepare thin films on the surface of economically undemanding polymeric materials [8,33,34]. The method is based on a tertiary system: solvent (chloroform)–polymer–"no" solvent (methanol), in the correct volume ratio. To achieve the preparation of HCP structures is necessary to immerse the substrate in the prepared solution and then, spontaneously evaporate it in the air [16,35–37]. The advantage of this method, except for the humid environment and other active substances, is the exact ratio of solvents [8].

The development of new methods for creating scaffolds that are structurally and functionally similar to the extracellular matrix is one of the main challenges of tissue engineering. One possibility is to use polymeric materials, mainly due to their simplicity, flexibility, and low cost compared to other materials, and to prove their usefulness as extracellular matrix (ECM) replacements [38]. These properties and many others such as low density, environmental friendliness, and good physical and mechanical properties have been found in polymethyl methacrylate (PMMA) and its derivatives [35], which have been approved as bioinert and biocompatible polymers used in biomedical applications [36,37,39]. Nanocomposites based on PMMA exhibit excellent results in a body environment and have a protective role against corrosion and, thus, increase the durability of biomedical implants [40]. PMMA is commonly used in dentistry in dental prostheses [38,41] as well as bone cement [42–44]. Moreover, PMMA has been also used in the field of drug delivery or for the production of contact and implanted lenses, blood pumps and reservoirs, intravenous sets, and blood dialysis membranes [40,45].

In this study, we have focused on the preparation of HCP structures with the addition of PMMA in different amounts/ratios. Surface morphology, chemical composition, wettability, and cytocompatibility of the formed material were investigated for the particular HCP-coated foils. Samples with higher PMMA content formed more periodic hexagonal structures on the FEP surface, as well as cytocompatibility testing of cell culture was more efficient.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

In this study, we used a PMMA polymer foil with a thickness of 50 µm and a density of 1.19 g·cm−3 , which was purchased from Goodfellow Ltd. (Cambridge, Huntingdon, UK). As solvents, we used methanol (MeOH, HPLC purity, Mr = 32.04 g·mol−1 , the density of 0.791 g·cm−3 ; purchased from Penta, Prague, The Czech Republic), and chloroform (CHCl3, stabilized with ~1% ethanol A.G., Mr = 119.38 g·mol−1 , the density of 1.48 g·cm−3 ; purchased from Penta, Prague, The Czech Republic). As a substrate, we used fluorinated ethylene propylene polymer (FEP, the thickness of 50 µm, the density of 2.15 g·cm−3 ; purchased from Goodfellow Ltd., Cambridge, Huntingdon, UK).

#### *2.2. Pattern Preparation and Modification*

The polymer film was treated by Ar+ plasma using the SCD 050 sputtering device from BAL-TEC (Baltec AG, Balzers, Liechtenstein). The purity of gas in the chamber was 99.997%, and the pressure was 8 Pa. The samples were placed onto a circular holder (an anode) with a diameter of 10 cm and a distance of 5 cm from the cathode. They were modified at 8 W for 240 s.

In the next step, HCP-like structures were formed using the IPS method via immersion of plasma-modified polymeric substrates in the prepared 100 mL solution for 10 s. The solution was a mixture of two solvents, namely chloroform and methanol, at a volume ratio of 85:15. Then, the amount of 1, 2, 3, and 4 g of PMMA foils was added to the mixture while stirring for 2–3 h at room temperature (22 ◦C), which gave a homogeneous solution. Then, the HCP-like structures were removed and left to air dry in Petri dishes. After complete evaporation of the solvents, the samples were prepared for further modification and subjected to examination by various analytical methods (see Section 2.3).

#### *2.3. Analytical Methods*

The wettability of all samples was studied via goniometric measurements of the contact angles, with a drop of distilled water applied to the surface of each sample (6 positions). The contact angles of the samples were determined using a See System goniometer (Advex Instruments, Brno, Czech Republic) at room temperature. A drop of water (8 µL) was applied onto each sample with a Transferpette® automatic pipette (Brand, Wertheim, Germany).

Atomic force microscopy (AFM) was used to study the surface morphology of the FEP substrate with HCP microstructures. The movement of the tip as it passed over the sample was recorded, and a point-by-point image of the surface was compiled. Sample surface morphology was examined by atomic force microscope (AFM) Dimension ICON (Bruker Corp., Billerica, MA, USA); the ScanAsyst mode in the air was used for determination. A silicon tip on a nitride lever SCANASYST-AIR with the spring constant of 0.4 N·m−1 was used. NanoScope Analysis software was applied for data processing. The mean roughness values (Ra) represent the average of the deviations from the center plane of the sample.

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were used for detailed analysis of the morphology and chemical characterization of the FEP substrate with HCP microstructures. We used a scanning electron microscope LYRA3 GMU (Tescan, Brno, The Czech Republic) with the accelerating voltage of 10 kV for the electrons that bombarded the samples and an F-MaxN analyzer and SDD (Silicon Drift Detector) (Oxford Instruments, Abingdon, UK) with an applied accelerating voltage of 10 kV for EDS. Platinum (target, purity 99.999%, Safina, Vestec, The Czech Republic) was sputtered onto the samples before analysis using a Quorum Q300T sputtering device (Quorum Technologies, Laughton, East Sussex, England) at a current of 30 mA for 400 s.

#### *2.4. Cell Culture*

VAMPIRO U-2 OS cells supplied by Innoprot (Derio, Spain) were used to evaluate the material cytocompatibility. The VAMPIRO U-2 OS are cells derived from human osteosarcoma developed through stable transfection of U-2 OS cell line with tFP602 under puromycin resistance gene. The VAMPIRO U-2 OS cells exhibit red fluorescence since they express the red fluorescent protein gene sequence as free cytoplasmatic protein. The cells were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing a stable L-glutamine dipeptide. The medium was supplemented with 10% fetal bovine serum (FBS; certified, Thermo Fisher Scientific, Waltham, MA, USA). The cells were cultivated in a sterile cell culture incubator at 37 ◦C, atmosphere with 5% CO2 and 95% humidity. The cells were regularly passaged when they were at the exponential phase of growth. First, the cells were washed with 5 mL of sterile phosphate-buffered saline (PBS, pH 7.4). After its removal, the cells were detached from the underlying cell culture dish (VWR International, Radnor, PA, USA) with 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid).

#### *2.5. Cell Seeding and Staining*

To evaluate the material cytocompatibility, the VAMPIRO U-2 OS cells were used from passages 4–8 (counted from defrosting after the cell delivery). The prepared material samples were subjected to sterilization by immersion into 70% (volume percent) ethanol in water for 30 min. Then, the samples were inserted into cell culture plates with 12 wells (the diameter of 2.14 cm; VWR, Radnor, PA, USA), washed twice with sterile PBS, and weighted down by hollow PMMA cylinders (Zenit, Prague, The Czech Republic). Then, 1 mL of DMEM containing 15,000 cells per one squared centimeter was added to each well onto the evaluated samples in four biological replicates. The endpoints of cultivation were 24 and 96 h, after which the medium was removed, the cells were washed with prewarmed PBS, and fixed with a 4% methanol-free formaldehyde solution in PBS as described in [46]. The fixation occurred for 20 min. at 22 ◦C in the dark, then, the fixative was removed, and the cells were washed twice with cold PBS. This was followed by staining of the cell structures. Cell nuclei were stained with 0.5 µg·mL−1 of 40 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) and cell cytoskeleton was stained with 2 µg·mL−1 of phalloidin conjugated to Atto 488 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min. in the dark at 22 ◦C. Then, the staining solution was removed, and the cells were washed with cold PBS. The standard deviation was calculated from four biological replicates each including twenty regions of interest, on which the number of grown cells was counted.

#### *2.6. Fluorescence Microscopy*

The fixed and stained VAMPIRO U-2 OS cell samples were subjected to wide-field fluorescence microscopy using an inverse fluorescence microscope Olympus IX-81 (Olympus, Tokyo, Japan) operated with xCellence software. The fluorescently labeled cells were examined at 100× (10× objective, NA of 0.30), 200× (20× objective, NA of 0.45), and 400× (40× objective, NA of 0.60) magnification. Cell cytoplasm, cytoskeleton, and nuclei were imaged by a triple quad filter DAPI/FITC/TRITC (Olympus, Tokyo, Japan). The images were captured by an EM-CCD camera (Hamamatsu, Honshu, Japan). Then, the fluorescence images were background-corrected, deconvolved (50%), and all three fluorescence channels (DAPI, FITC, TRITC) were merged.

#### **3. Results**

### *3.1. Surface Morphology Analysis Using SEM and AFM Method*

In this study, we have aimed to prepare HCP structures with incorporated PMMA (different content of 1, 2, 3, and 4 g) on plasma-modified FEP matrices. Plasma treatment affects polymer structure resulting in free radical formation. This phenomenon can increase the movement of molecular particles (the ability of a polymer to diffuse) and stimulate the etching rate of the surface layer. The duration of Ar+ particle deposition (plasma) significantly affects the polymer topography. Short exposure of a polymer to the plasma treatment creates nanometric structures while changing the polymer surface into a porous form for a longer time [47]. Based on previous research [16,25], the plasma treatment

parameters for creating the suitable surface of HCP structures were selected: duration 240 s at the power of 8 W. The changes on the prepared HCP structures' surface morphology were analyzed by two different methods, AFM and SEM. Figure 1 represents the scans of the prepared samples acquired by the SEM analysis. The analyzed area is divided into two regions for samples with an amount of PMMA higher than 1 g. The first area shows the major heterogeneous site (herein called "major"), which confirms the occurrence of inhomogeneous hexagonal pores. While the second area (herein called "minor") detected 2 mm from the sample edges of the formed regular hexagonal microstructures. The phenomenon of the split surface is probably caused by different evaporation rates of the solvent mixture on the sample surface, and even though that the difference in patterns was not significant, it was still present on all examined samples, therefore, we wanted to point this out also in the description of Figure 1. treatment creates nanometric structures while changing the polymer surface into a porous form for a longer time [47]. Based on previous research [16,25], the plasma treatment parameters for creating the suitable surface of HCP structures were selected: duration 240 s at the power of 8 W. The changes on the prepared HCP structures' surface morphology were analyzed by two different methods, AFM and SEM. Figure 1 represents the scans of the prepared samples acquired by the SEM analysis. The analyzed area is divided into two regions for samples with an amount of PMMA higher than 1 g. The first area shows the major heterogeneous site (herein called "major"), which confirms the occurrence of inhomogeneous hexagonal pores. While the second area (herein called "minor") detected 2 mm from the sample edges of the formed regular hexagonal microstructures. The phenomenon of the split surface is probably caused by different evaporation rates of the solvent mixture on the sample surface, and even though that the difference in patterns was not significant, it was still present on all examined samples, therefore, we wanted to point this out also in the description of Figure 1.

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affects polymer structure resulting in free radical formation. This phenomenon can increase the movement of molecular particles (the ability of a polymer to diffuse) and stimulate the etching rate of the surface layer. The duration of Ar+ particle deposition (plasma) significantly affects the polymer topography. Short exposure of a polymer to the plasma

**Figure 1.** Scanning electron microscopy images of honeycomb-like pattern major and minor areas formed on plasmamodified fluorinated ethylene propylene containing different amounts (1–4 g) of incorporated polymethyl methacrylate. The scanned areas were 100 × 100 µm2. **Figure 1.** Scanning electron microscopy images of honeycomb-like pattern major and minor areas formed on plasma-modified fluorinated ethylene propylene containing different amounts (1–4 g) of incorporated polymethyl methacrylate. The scanned areas were 100 × 100 µm2 .

From the SEM images, it can be concluded that a sample containing 1 g of PMMA was the least favorable substrate for forming hexagonal structures. The remaining concentrations of PMMA confirmed the formation of HCP structures. As the amount of PMMA increased, the pore size also increased as well as the pattern homogeneity, and other smaller cavities were layered below each other to different levels. A slight difference in pore sizes was observed when comparing the major and minor areas of HCP on the sample surface. The minor area of the HCP was detected up to 2 mm from the outer diameter. Even the difference in pattern properties in the outer and inner areas was not significant for samples

prepared with 3 and 4 g of PMMA, we wanted to introduce this fact in this article for all studied patterns. If we focus specifically on images containing 4 g of PMMA, we conclude that the major area provided pores of up to 4 µm in diameter and the minor area contained also larger pores of up to 10 µm in diameter. In the minor areas, when compared to the major areas, there was a more precise distribution of cavities of almost the same size. These trends were maintained also for samples with a polymer layer containing 2–4 g of PMMA. However, no considerable variation in pore formation was observed for samples formed from a solution containing 3 and 4 g of PMMA. significant for samples prepared with 3 and 4 g of PMMA, we wanted to introduce this fact in this article for all studied patterns. If we focus specifically on images containing 4 g of PMMA, we conclude that the major area provided pores of up to 4 µm in diameter and the minor area contained also larger pores of up to 10 µm in diameter. In the minor areas, when compared to the major areas, there was a more precise distribution of cavities of almost the same size. These trends were maintained also for samples with a polymer layer containing 2–4 g of PMMA. However, no considerable variation in pore formation was observed for samples formed from a solution containing 3 and 4 g of PMMA.

From the SEM images, it can be concluded that a sample containing 1 g of PMMA was the least favorable substrate for forming hexagonal structures. The remaining concentrations of PMMA confirmed the formation of HCP structures. As the amount of PMMA increased, the pore size also increased as well as the pattern homogeneity, and other smaller cavities were layered below each other to different levels. A slight difference in pore sizes was observed when comparing the major and minor areas of HCP on the sample surface. The minor area of the HCP was detected up to 2 mm from the outer diameter. Even the difference in pattern properties in the outer and inner areas was not

*Polymers* **2021**, *13*, x FOR PEER REVIEW 6 of 15

Minor areas of the samples with a polymer layer of 1–4 g PMMA were also studied by the AFM analysis as shown in Figure 2. From the results obtained with AFM and SEM, it can be concluded that the pore sizes on the substrates with the same concentration of PMMA remained similar for both studied methods. Therefore, both dimensions and shape of the pattern could be considered as verified. Furthermore, it should be added that the analyzed surface roughness and the height of the pores formed did not change dramatically with the increasing amount of PMMA. The only significant difference among the samples was on the substrate containing the highest content of PMMA (4 g). On the AFM images, we can observe the formation of pores with a slightly smaller diameter in the major area, compared to the edges of the sample (minor area), however, the differences were not significant for samples prepared with 3 and 4 g of PMMA. Even though that the AFM analysis of the sample's surface was repeated several times. We would like to point out that the SEM scans were acquired on larger sample areas than AFM scans, therefore, the measurement can be considered as more "accurate". Minor areas of the samples with a polymer layer of 1–4 g PMMA were also studied by the AFM analysis as shown in Figure 2. From the results obtained with AFM and SEM, it can be concluded that the pore sizes on the substrates with the same concentration of PMMA remained similar for both studied methods. Therefore, both dimensions and shape of the pattern could be considered as verified. Furthermore, it should be added that the analyzed surface roughness and the height of the pores formed did not change dramatically with the increasing amount of PMMA. The only significant difference among the samples was on the substrate containing the highest content of PMMA (4 g). On the AFM images, we can observe the formation of pores with a slightly smaller diameter in the major area, compared to the edges of the sample (minor area), however, the differences were not significant for samples prepared with 3 and 4 g of PMMA. Even though that the AFM analysis of the sample's surface was repeated several times. We would like to point out that the SEM scans were acquired on larger sample areas than AFM scans, therefore, the measurement can be considered as more "accurate".

**Figure 2.** The surface morphology of honeycomb-like pattern minor areas on plasma-modified fluorinated ethylene propylene with different amounts (1–4 g) of incorporated polymethyl methacrylate. The scanned areas were 30 *×* 30 µm2. Ra represents the average roughness in nm. **Figure 2.** The surface morphology of honeycomb-like pattern minor areas on plasma-modified fluorinated ethylene propylene with different amounts (1–4 g) of incorporated polymethyl methacrylate. The scanned areas were 30 × 30 µm2 . Ra represents the average roughness in nm.

#### *3.2. Surface Chemical Analysis*

Qualitative and quantitative data on the elemental composition of the prepared substrates were determined by the EDS method and are summarized in Table 1 and Figure 3. The samples were analyzed before any modification (pristine FEP), after plasma deposition at 8 W for 240 s (plasma FEP), and after preparation of HCP structures from PMMA solution of various content 1–4 g (HCP + 1 g PMMA). The chemical distribution of major and minor areas of the prepared samples was studied by the EDS technique and described in Section 3.1.

**Table 1.** The atomic concentrations of fluorine, oxygen, and carbon in at. % were determined using the energy-dispersive X-ray spectroscopy for unmodified pristine fluorinated ethylene propylene (FEP), plasma-treated FEP, and with a honeycomb-like pattern (HCP) major and minor structure with incorporated polymethyl methacrylate (PMMA) of various content (1–4 g).

From Table 1, it is evident that no oxygen element was detected on the pristine FEP compared to other modified samples. The explanation is the characteristic of the FEP polymer chain containing only C and F elements [48]. One of the reasons for the increased oxygen concentration is plasma treatment, which causes that the ionized Ar ions to hit the polymer surface. This process induces new functional groups that bind oxygen from the surrounding atmosphere after the sample is removed from the chamber or during the process if a slight amount of oxygen is present in the atmosphere in the chamber [49,50]. Another reason is the presence of PMMA honeycombs, which contain a large amount of oxygen in their structure [51]. The subsequent phase separation increases the carbon content and, conversely, decreases the fluorine concentration, confirming a polymeric PMMA layer formation. The presence of PMMA may limit the content of the F element, as shown in Table 1. The highest tested amount of PMMA (4 g) formed surface with minimum fluorine content, which was only 6.4%. It is interesting to note that if we observed a minor area of the sample (minor), the F element almost disappeared, i.e., the substrate was fully covered with a PMMA polymer layer. For a better interpretation of the surface's chemical composition, the scans from EDS of the sample with 2 g PMMA are attached in Figure 3.

#### *3.3. Surface Wettability*

Wettability, i.e., the contact angle values, is a crucial property for material characterization. It plays an essential role in applications of technological (cleaning, gluing, dyeing) [52,53] or medical processes (cell or bacterial adhesion) [54]. The value of a contact angle has a significant impact on the material surface and characterizes the hydrophilicity/hydrophobicity of a polymer [55].

The graph shown in Figure 4 provides data on the surface wettability of all evaluated samples during an aging period of 34 days. The aging study was performed on the unmodified FEP substrate (pristine FEP), plasma-modified FEP substrate at 8 W for 240 s (plasma FEP), and the substrate after plasma modification applied with a layer of PMMA at various amounts (1–4 g). The value of the contact angle of the unmodified FEP sample was 98.9◦ . As it is obvious from Figure 4, the samples with HCP had contact angles in the region of slight hydrophobicity ranging from 90–110◦ with fluctuation during the aging period. However, the slightly hydrophobic character remained unchanged and stable during the aging period. The most apparent change in contact angle values was detected for the plasma-modified FEP substrate. On the first day, the plasma FEP had a contact angle of 50.2◦ , and on the third day, it increased rapidly to 96.8◦ . Contact angle did not change significantly throughout the measurement and was around the values of the pristine FEP. This could be caused by the redirection of polar groups from the material's surface to its volume due to the preservation of samples in the ambient atmosphere. On the other hand, an atmosphere with relatively low humidity and temperature can slow down the material aging process. All FEP samples covered with PMMA maintained a hydrophobic surface

with contact angles within 10◦ of the value of unmodified FEP (98.9◦ ). The influencing of contact angle by various patterns including honeycomb structure was reported in [56], where also superhydrophibic effect was observed. *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 15

**Figure 3.** A spectrum from energy-dispersive X-ray spectroscopy (EDS) analysis and elemental maps with layered EDS images for the honeycomb-like pattern major and minor structures on plasma-treated fluorinated ethylene propylene. **Figure 3.** A spectrum from energy-dispersive X-ray spectroscopy (EDS) analysis and elemental maps with layered EDS images for the honeycomb-like pattern major and minor structures on plasma-treated fluorinated ethylene propylene.

Wettability, i.e., the contact angle values, is a crucial property for material character-

has a significant impact on the material surface and characterizes the hydrophilicity/hy-

*3.3. Surface Wettability*

drophobicity of a polymer [55].

where also superhydrophibic effect was observed.

The graph shown in Figure 4 provides data on the surface wettability of all evaluated

samples during an aging period of 34 days. The aging study was performed on the unmodified FEP substrate (pristine FEP), plasma-modified FEP substrate at 8 W for 240 s (plasma FEP), and the substrate after plasma modification applied with a layer of PMMA at various amounts (1–4 g). The value of the contact angle of the unmodified FEP sample was 98.9°. As it is obvious from Figure 4, the samples with HCP had contact angles in the region of slight hydrophobicity ranging from 90–110° with fluctuation during the aging period. However, the slightly hydrophobic character remained unchanged and stable during the aging period. The most apparent change in contact angle values was detected for the plasma-modified FEP substrate. On the first day, the plasma FEP had a contact angle of 50.2°, and on the third day, it increased rapidly to 96.8°. Contact angle did not change significantly throughout the measurement and was around the values of the pristine FEP. This could be caused by the redirection of polar groups from the material's surface to its volume due to the preservation of samples in the ambient atmosphere. On the other hand, an atmosphere with relatively low humidity and temperature can slow down the material aging process. All FEP samples covered with PMMA maintained a hydrophobic surface with contact angles within 10° of the value of unmodified FEP (98.9°). The influencing of contact angle by various patterns including honeycomb structure was reported in [56],

**Figure 4.** Water contact angles measurements over 34 days for pristine fluorinated ethylene propylene (FEP), plasma-treated FEP (plasma FEP), and honeycomb-like pattern (HCP) structure with incorporated polymethyl methacrylate (PMMA) of various content (1–4 g). **Figure 4.** Water contact angles measurements over 34 days for pristine fluorinated ethylene propylene (FEP), plasma-treated FEP (plasma FEP), and honeycomb-like pattern (HCP) structure with incorporated polymethyl methacrylate (PMMA) of various content (1–4 g).

#### *3.4. Cytocompatibility*

*3.4. Cytocompatibility*  Human cells derived from osteosarcoma stably transfected with a red fluorescent protein tFP602 (VAMPIRO U-2 OS cells) were chosen as a model cell line for cytocompatibility measurement of the prepared materials. Samples of pristine FEP, plasma-modified FEP, and FEP with a PMMA layer were subjected to this study. Tissue culture polystyrene (TCPS) was chosen as a control since this material is routinely used for tissue culture. From the plot in Figure 5, it is evident that after 24 h post-seeding, there was no statistically significant difference in the number of cells adhered on modified or unmodified samples compared to the control sample of TCPS. It can be caused by the cell adaptation Human cells derived from osteosarcoma stably transfected with a red fluorescent protein tFP602 (VAMPIRO U-2 OS cells) were chosen as a model cell line for cytocompatibility measurement of the prepared materials. Samples of pristine FEP, plasma-modified FEP, and FEP with a PMMA layer were subjected to this study. Tissue culture polystyrene (TCPS) was chosen as a control since this material is routinely used for tissue culture. From the plot in Figure 5, it is evident that after 24 h post-seeding, there was no statistically significant difference in the number of cells adhered on modified or unmodified samples compared to the control sample of TCPS. It can be caused by the cell adaptation process to the samples after their seeding [57]. After additional cultivation, 96 h post-seeding, cell proliferation was obvious on each evaluated sample. The lowest number of cells was detected on the unmodified FEP sample, which is consistent with our previous results, e.g., [58–60]. The cell number was increased on plasma-treated FEP 96 h after seeding when compared to pristine FEP. This was caused by the plasma exposure, which creates new oxygen-containing groups (carbonyl and carboxyl mainly) in combination with significant wettability change, enabling the design of excellent scaffolds with increased biocompatibility [60]. However, the best cytocompatibility was observed on FEP samples with HCP structures and PMMA layers, since a markedly pronounced increase in the cell number was detected. The number of cells of these samples was equal to that of the control TCPS sample. The improved VAMPIRO U-2 OS cell proliferation on the modified samples might be caused by the porous character of the HCP-modified sample surface or the presence of PMMA. The presence of 3D HCP structure mimics the extracellular matrix necessary as scaffolds for proper cell growth and proliferation [61], the effect also observed for laser induced periodic surface structures [24,62]. On the other hand, the PMMA material exhibits biocompatible properties, such as surface chemistry in combination with morphology prop-

erties [36,37,39,40] that could increase cell proliferation on the substrate surface compared to pristine and plasma-modified FEP. surface of the unmodified FEP. The shape of the cells after day 4 can no longer be recognized in all sample types.

Another evaluation of the cellular response, morphology, was studied on samples with cultured U-2 OS cells for 1 and 4 days, shown in Figure 6. Images of the cell samples were captured by fluorescence microscopy. After the first day, round cells were detected with irregular spreading on pristine FEP samples and with a layer of 3 g and 4 g of PMMA, which did not correspond to the shape of osteoblasts. In comparison, the control sample showed a cell morphology similar to the shape of osteoblasts. Samples with plasma modification and 1 g and 2 g PMMA had a cell shape similar to the control, but their distribution was not regular. After day 4, all prepared samples were completely covered with cells, except for the pristine FEP sample. This phenomenon could be due to the smooth

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surface compared to pristine and plasma-modified FEP.

process to the samples after their seeding [57]. After additional cultivation, 96 h post-seeding, cell proliferation was obvious on each evaluated sample. The lowest number of cells was detected on the unmodified FEP sample, which is consistent with our previous results, e.g., [58–60]. The cell number was increased on plasma-treated FEP 96 h after seeding when compared to pristine FEP. This was caused by the plasma exposure, which creates new oxygen-containing groups (carbonyl and carboxyl mainly) in combination with significant wettability change, enabling the design of excellent scaffolds with increased biocompatibility [60]. However, the best cytocompatibility was observed on FEP samples with HCP structures and PMMA layers, since a markedly pronounced increase in the cell number was detected. The number of cells of these samples was equal to that of the control TCPS sample. The improved VAMPIRO U-2 OS cell proliferation on the modified samples might be caused by the porous character of the HCP-modified sample surface or the presence of PMMA. The presence of 3D HCP structure mimics the extracellular matrix necessary as scaffolds for proper cell growth and proliferation [61], the effect also observed for laser induced periodic surface structures [24,62]. On the other hand, the PMMA material exhibits biocompatible properties, such as surface chemistry in combination with morphology properties [36,37,39,40] that could increase cell proliferation on the substrate