Chitosan-silanol groups functional magnetic nanoparticles for heavy metals and bacteria removal

Studies have shown that there are multiple co-existence pollutants in environmental water over recent years. In this study, we report the design and synthesis of magnetic adsorbents incorporating chitosan-silanol groups (Fe 3 O 4 @Si-OH@CS) with improved physicochemical properties for the removal of various heavy metals and bacteria from polluted water. Fe 3 O 4 @Si-OH@CS was synthesised using the co-precipitation method. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), Brunauer-Emmett-Teller (BET), Vibrating sample magnetometer (VSM), and Zeta techniques were used to characterise. The effects of factors such as pH, adsorption time, and adsorbent dosage were optimised. The results indicated that Fe 3 O 4 @Si-OH@CS had high adsorption eciency and adsorption capacity for Cr (VI), As, Hg and Se. Moreover, Cr (VI) and As have a preferential adsorption effect when multiple metal ions coexist. The adsorption performance of Fe 3 O 4 @Si-OH@CS to bacteria was veried using E. coli (gram-negative) and S. aureus (gram-positive). The developed adsorbent also showed good adsorption eciency for both gram-negative and gram-positive bacteria. Overall, the synthesised Fe 3 O 4 @Si-OH@CS adsorbent showed high removal eciency and adsorption capacity with a stable structure and easy separation. It has promising applications for the removal of heavy metals and bacteria from water.


Introduction
Water pollution caused by illegal discharge of wastewater, accidental leakage of raw materials, and poor water management or monitoring has severely affected water quality safety (Gothandam et al., 2020).
The pollutants can include organic pollutants, heavy metals, pharmaceuticals, and drug-resistant bacteria (Chen et al., 2020). Among these contaminants, heavy metals are toxic, accumulative, non-degradable, and carcinogenic; therefore, their potential risk to people and the ecosystem is immense (Ma et al., 2011). Antibiotics and other chemicals induce the development of resistance-harbouring resistance genes.
Because of horizontal gene transfer, numerous drug-resistant bacteria have emerged, posing a risk to human health (Na et al., 2021).
Presently, there are many methods for removing heavy metals or bacteria from water. The commonly used methods include membrane ltration, wet heat sterilisation, chemical precipitation, and electrochemical treatment (Ince et al., 2019; Bairagi et al., 2020). The adsorption method has been widely used for the removal of pollutants from water because of its advantages such as low cost, high adsorption e ciency, simple operation, and short adsorption cycle (Liu et al., 2019;Panda et al., 2020).
The quality of the adsorption method depends on the adsorbent; therefore, it is important to select a suitable adsorbent to eliminate pollutants from water (Hussain et al., 2021). In recent years, a variety of materials for removing heavy metals and bacteria have been reported, including carbon-based materials such as activated carbon (Yuan et al., 2021), carbon nanotubes (Zhu et al., 2020), and graphene (Perumal et al., 2020), as well as natural polymers such as cellulose (Pei et al., 2020) and chitosan . Chitosan, derived from chitin, is one of the most abundant biopolymers in nature (Gabriel et al., 2020).
Because chitosan contains amino, acetylamino, and hydroxyl groups, it is relatively active and can be modi ed, activated, and coupled (Tanhaei et al., 2015;Bala et al., 2017). Moreover, its biodegradability, cell a nity, biological effects, and many other unique properties, have contributed to chitosan's extensive applications in the pharmaceutical and light industries and for environmental protection (Nayak et  In this study, chitosan-silanol functional magnetic nanoparticles (Fe 3 O 4 @Si-OH@CS) were synthesised by a co-precipitation method with improved physicochemical properties for the removal of various heavy metals and bacteria (Fig. 1). The magnetic composite exhibits high adsorption capacity for both heavy metals such as, Hg, Se, and Cr(VI), and bacteria including Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive). were dispersed in a chitosan solution (60 mL, 1%). Then, 3 mL of glutaraldehyde solution (5%) was added, and the mixture was stirred at 60°C for 30 min. The products were separated by an external magnetic eld, washed with acetic acid (3%), and dried at 80°C. Fe 3 O 4 @Si-OH@CS was obtained and stored at 25 ℃.

Characterisation
The adsorbent was dispersed in 3% acetic acid and ultrasonicated. The particle size and particle size distribution of the obtained uniform suspension were determined using a laser scattering particle size analyser (LS13-320, Beckman Coulter, USA). The conductive adhesive was coated on the surface of the sample in a vacuum environment, and the adsorbent was observed by scanning electron microscopy (SEM) with SU-1510 (Japan) at a voltage of 5 kV. The X-ray diffraction (XRD) patterns were measured at 100 mA in the 2θ range of 5-80° with a Smartlab (Neo-Confucianism, Japan) using Cu Kα radiation. A Nicolet Nexus 6700 FTIR (USA) spectrometer was used to determine the Fourier transform infrared (FTIR) spectra of the Fe 3  was used to determine the pore size, speci c surface area, and pore volume of the adsorbent surface by nitrogen adsorption-desorption analysis using the Brunauer-Emmett-Teller (BET) method. The surface potentials (zeta) of Fe 3 O 4 @ Si-OH and Fe 3 O 4 @Si-OH@CS were measured using a nanoparticle potentiometer (Zetasizer Nano ZS90, Malvern, UK). A Versalab vibrating sample magnetometer (VSM, Quantum Design, USA) was used for the magnetisation measurements of the adsorbents. To study the adsorption e ciency of the adsorbent on heavy metal ions, 20 µg/L As, 1 µg/L Hg, 50 µg/L Se, 40 µg/L Pb, and 7 µg/L Cd were used for adsorption. To study the adsorption capacity of the adsorbent on heavy metal ions, 50 mg Fe 3 O 4 @Si-OH@CS was added, and the concentrations of As, Hg, Se, Pb, and Cd were changed from 0 to 800 mg/L, respectively. Furthermore, 200 µg/L Cr(VI), 20 µg/L As, 10 µg/L Hg, and 50 µg/L Se were used for the cation adsorption competition experiments. The concentration of Cr(VI) was determined using a UV/visible spectrophotometer (UV-2550, Tianjin, China). The concentrations of As, Hg, and Se were determined using an atomic uorescence photometer (AFS-930, Beijing Jitian Instrument Co., Ltd., China). The adsorption capacity of the adsorbents toward heavy metal ions q e (mg/g) and the removal percentage η (%) were determined based on the following equations:

Batch adsorption experiments
(1) (2) where q e (mg/g) is the adsorption capacity of heavy metals, C 0 (µg/L) is the initial concentration of heavy metal ions, C e (µg/L) is the equilibrium concentration of heavy metal ions, V (mL) is the volume of the adsorbate solution, m (mg) is the amount of adsorbent, and η (%) is the percentage of heavy metal ions adsorbed from the solution.

Fe 3 O 4 @Si-OH@CS application for bacteria
Escherichia coli was cultured in accordance with the standard test method (GB/T 5750. . Nutritional AGAR medium was used instead of beef extract peptone medium to reduce the pH adjustment steps. First, 2.5 mg Fe 3 O 4 @Si-OH@CS was added to a certain volume of bacterial liquid for vibration adsorption. After magnetic separation, 200 µL of the supernatant was collected and coated on the plate. This step was carried out in a biosafety cabinet. The coated plate was then placed in an electric thermostatic incubator and cultured at 37°C for 24-48 h. The number of colonies that grew on the plates was counted. Finally, 200 µL of bacterial solution from the bacterial solution diluted 1000 times was coated onto the plate to calculate the blank colony. This step was carried out in a biosafety cabinet. The adsorption rate (AR) was calculated using the following equation: where AR indicates the removal e ciency (%), N a is the number of bacteria after adsorption, and N 0 is the number of bacteria in the control group.

Characterisation of adsorbents
The synthesis of Fe 3 O 4 @Si-OH@CS involves three steps (Fig. S1). SEM and SEM mapping were used to characterise the size, shape, and surface element distribution of Fe 3 O 4 , Fe 3 O 4 @Si-OH, and Fe 3 O 4 @Si-OH@CS. As shown in Fig. 2 (a), Fe 3 O 4 has an irregular blocky structure. In Fig. 2 (b), Fe 3 O 4 @Si-OH is a regular spherical structure with a smooth surface and a particle size of approximately 125 nm, possibly because Si-OH is coated on the surface of Fe 3 O 4 . As shown in Fig. 2 (c), the particle size of Fe 3 O 4 @Si-OH@CS increased slightly compared with that of Fe 3 O 4 @Si-OH, approximately 170 nm, likely because the coating of chitosan increased the particle size of the material. Fe 3 O 4 @Si-OH@CS exhibited an agglomeration phenomenon, which may be the result of the uneven dispersion of chitosan. The local elemental information of Fe 3 O 4 @Si-OH@CS is shown in Fig. 2 (d) BET and Barrett-Joyner-Halenda (BJH) methods were used to analyse the speci c surface area, pore volume, and pore size of the adsorbents with N 2 desorption and adsorption isotherms. As shown in Fig. 2(g), a typical characteristic of the type-III isotherm accompanying by H3 type hysteresis loop, indicating that Fe 3 O 4 @Si-OH@CS is a mesoporous material (Kruk and Jaroniec, 2001). The pore parameters of the three materials are listed in Table S1. The speci c surface area of Fe 3 O 4 is 141.86 m 2 /g, which is favourable for Si-OH coating. The speci c surface area of Fe 3 O 4 @Si-OH is 21.82 m 2 /g and may be due to the addition of the Fe 3 O 4 surface Si-OH. The speci c surface area of Fe 3 O 4 @Si-OH@CS is 27.34 m 2 /g, which may be because the -NH 2 coating increases the speci c surface area and provides more adsorption sites for adsorbents.
The zeta potential was used to determine the charge type on the surface of the adsorbent. As shown in Fig. S2, the surface of Fe 3 O 4 @Si-OH is negatively charged (-41.3 mV), which may be caused by the addition of Si-OH on the surface of Fe 3 O 4 @Si-OH. However, the surface of Fe 3 O 4 @Si-OH@CS is positively charged (+28.89 mV), indicating that -NH 2 successfully modi ed Fe 3 O 4 @Si-OH, which is more conducive to electrostatic adsorption.
The magnetic properties of the three materials were studied by examining their magnetic hysteresis loops. As seen in Fig. S3, the remanent magnetism and coercivity of these three particles are close to zero, indicating that they have superparamagnetic properties (

Effect of pH
The pH is a signi cant factor that can affect adsorption e ciency. Cr(VI) was selected as the model heavy metal for further condition optimisation. As shown in Fig. 3(a), the removal e ciency increased when the pH changed from 1 to 2.5. This is mainly because Cr (

Effect of adsorption time
The adsorption time in uenced the adsorption equilibrium between the adsorbents and the targets. Fig. 3(b) shows the relationship between the adsorption time and the adsorption e ciency. The adsorption e ciency increased rapidly when the adsorption time was below 15 min. This may be due to the fact that the surface of adsorbents can initially provide a large number of adsorption sites for targets. When the adsorption time was changed from 15 to 90 min, the adsorption e ciency increased slowly. This may be due to a large number of occupied active sites on the surface of the adsorbent as well as complexation playing a dominant role. This process is slow, and and the maximum adsorption capacity is 82.5 mg/g after adsorption for 180 min (Zhou et al., 2019). Combined with the factors of time cost and adsorption e ciency, an adsorption time of 15 min was selected.

Effect of absorbent dosage
The weight of the adsorbent is suitable for adsorption and elution. Fig. 3(c) shows the relationship between the absorbent dosage and the adsorption e ciency. The adsorption e ciency increased from 62.5-90.5% when the adsorbent dosage was increased from 25 to 100 mg. This is because the adsorption sites increase with increasing adsorbent dosage. The adsorption e ciency increased from 90.5-94.5% when the adsorbent dosage was increased from 100 to 150 mg. The reason may be that the mass ratio of adsorbent to Cr( ) decreases with the increased dosage of adsorbent, which leads to the under-utilisation of Fe 3 O 4 @Si-OH@CS surface adsorption sites (Zeng et al., 2020). Based on our results, we inferred that a good adsorption e ciency can be achieved with 100 mg adsorbents.

Adsorption of other heavy metals
Various heavy metal ions present in water pose severe risks to environmental and public health. Therefore, the performance of materials for removing multiple heavy metal ions is very important for practical applications. The adsorption e ciencies of Fe 3 O 4 @Si-OH@CS for several typical heavy metals, including As, Hg, and Se, were further studied. As shown in Fig. 3(d), the proposed material also exhibited excellent adsorption performance under the optimum adsorption conditions for Cr( ). The adsorption e ciencies for As, Hg, and Se were 73.5%, 91.6%, and 100.0%, respectively. Under the optimum adsorption conditions for Cr( ), the adsorption capacities of Fe 3 O 4 @Si-OH@CS for Cr( ), As, Hg, and Se were also studied. As shown in Fig. 3(e), the adsorption capacity is not directly proportional to the adsorption e ciency. As shown in Table S2, the adsorption capacity of Fe 3 O 4 @Si-OH@CS for heavy metals was also higher than that of other materials reported in the literature. Therefore, Fe 3 O 4 @Si-OH@CS can potentially be applied for the simultaneous removal and enrichment of heavy metal ions.

Effect of competing metal ions
When multiple metal ions coexist in the environment, the adsorption capacity of the adsorbent may be reduced, because they compete with each other for the adsorption site. Therefore, a series of metal cations were selected as the research objects to explore the Fe 3 O 4 @Si-OH@CS priority adsorption order of heavy metals. The adsorption e ciency of Fe 3 O 4 @Si-OH@CS for various metal cations was also studied.
As shown in Fig. 3 conditions. The oxygen anion possesses preferential electrostatic adsorption with protonated amino groups on the surface of the Fe 3 O 4 @Si-OH@CS surface. Therefore, the adsorption e ciencies of Cr( ) and As were higher than those of Hg and Se when they coexist.

Effect of pH
Antibiotic resistance, especially that of gram-negative bacteria, is one of the greatest public health threats worldwide. Moreover, as the most signi cant microbial habitat, aquatic environments are known to be favourable for antibiotic gene transfer. It has been reported that they play a crucial role in the spread of drug resistance in the environment (Cherak, Z et al., 2021). As a typical representative of gram-negative bacteria, the adsorption ability of Fe 3 O 4 @Si-OH@CS for E. coli was evaluated. First, the pH of the system was optimised. As shown in Fig. 4(a)

Effect of adsorption time
The adsorption time plays an important role in practical applications. As shown in Fig. 4(b), when the amount of magnetic material was 1.0 mg and the adsorption time was 5 min, the AR reached 74.95%. When the adsorption time was changed from 10 to 180 min, the adsorption e ciencies changed from 79.94-97.41%. Combined with the need for practical application, 5 min was selected for further experiments.

Effect of adsorbent dosage
The adsorption sites of Fe 3 O 4 @Si-OH@CS increased with increasing adsorbent dosage. Various amounts including 0.001, 0.0025, 0.005, 0.0075, and 0.01 g of Fe 3 O 4 @Si-OH@CS were studied (Fig. 4(c)). The initial concentration of E. coli was 656500 CFU/ml, and the adsorption time was 5 min. The adsorption e ciency of 0.001 g of Fe 3 O 4 @Si-OH@CS reached 88.4%. When the dosage of adsorbent was changed from 0.0025 to 0.01 g the adsorption e ciency was further increased from 96.82% to approximately 100%.

Effect of adsorption time
As reported in the literature, all active sites on the material would be occupied when a large number of bacteria are present in the samples, and excessive bacterial uid would not be adsorbed (Rihayat, et al., 2020). Therefore, multiple adsorption experiments were conducted using 0.0025 g of adsorbent. As shown in Fig. 4(d), the adsorption e ciency was 94.6% at an adsorption time of 5 min when the concentration of E. coli was 451500 CFU/ml. The adsorption e ciency was 100% after four adsorption cycles. Therefore, the proposed materials can be used to e ciently remove bacteria through repeated adsorption.

Adsorption of gram-positive bacterium
The performance of the proposed materials was further veri ed for gram-positive bacteria. Staphylococcus aureus is a typical gram-positive bacterium. It can cause various illnesses, from minor skin infections to life-threatening diseases (Tong S et al., 2010.). Moreover, S. aureus is widespread in the environment, including air and sewage. Therefore, S. aureus was used as a model bacterium to demonstrate this adsorption method. As shown in Fig. 5(b), under the optimal conditions for E. coli, the proposed material also showed excellent adsorption and removal performance for S. aureus. With a 5 min adsorption time and 25 mg adsorbent, S. aureus could be removed completely after two adsorption cycles, because the isoelectric point of most gram-positive bacteria was estimated to be 2-3, which was lower than that of gram-positive bacteria. Therefore, the proposed adsorption method exhibited good adsorption performance.

Conclusions
In summary, we developed a magnetic adsorbent (Fe 3 O 4 @Si-OH@CS) incorporating chitosan-silanol groups. The proposed strategies showed good performance for both heavy metals (Cr(VI), As, Hg, and Se) and bacteria (gram-negative and gram-positive). The adsorbent is low cost, has a high adsorption capacity, is readily synthesised, and can be widely applied. It is expected to be an effective adsorbent for the removal of heavy metals and bacteria from environmental water.

Declarations
Funding: This study was supported by the National Key R&D Program of China (grant no. 2017YFC1601101). The funders played no role in study design, collection, analysis or interpretation of data, the writing of the report, or the decision to submit the article for publication.
Competing Interests: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Supplementary Files
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