Application of Chitosan/Alginate Nanocomposite Incorporated with Phycosynthesized Iron Nanoparticles for Efficient Remediation of Chromium

Biopolymers and nanomaterials are ideal candidates for environmental remediation and heavy metal removal. As hexavalent chromium (Cr6+) is a hazardous toxic pollutant of water, this study innovatively aimed to synthesize nanopolymer composites and load them with phycosynthesized Fe nanoparticles for the full Cr6+ removal from aqueous solutions. The extraction of chitosan (Cht) from prawn shells and alginate (Alg) from brown seaweed (Sargassum linifolium) was achieved with standard characteristics. The tow biopolymers were combined and cross-linked (via microemulsion protocol) to generate nanoparticles from their composites (Cht/Alg NPs), which had a mean diameter of 311.2 nm and were negatively charged (−23.2 mV). The phycosynthesis of iron nanoparticles (Fe-NPs) was additionally attained using S. linifolium extract (SE), and the Fe-NPs had semispherical shapes with a 21.4 nm mean diameter. The conjugation of Cht/Alg NPs with SE-phycosynthesized Fe-NPs resulted in homogenous distribution and stabilization of metal NPs within the polymer nanocomposites. Both nanocomposites exhibited high efficiency as adsorbents for Cr6+ at diverse conditions (e.g., pH, adsorbent dose, contact time and initial ion concentration) using batch adsorption evaluation; the most effectual conditions for adsorption were a pH value of 5.0, adsorbent dose of 4 g/L, contact time of 210 min and initial Cr6+ concentration of 75 ppm. These factors could result in full removal of Cr6+ from batch experiments. The composited nanopolymers (Cht/Alg NPs) incorporated with SE-phycosynthesized Fe-NPs are strongly recommended for complete removal of Cr6+ from aqueous environments.


Introduction
Biopolymers are biologically synthesized macromolecules of living organisms, e.g., plants, microbes, algae and animals [1]. Many valuable biopolymers are attained from marine sources, e.g., chitosan, alginate, fucoidan, carrageenan, etc., to become extensively involved in numerous products and applications, serving environmental, biomedical, nutritional, pharmaceutical, and industrial sectors [2]. The environmental remediation using biopolymers includes the removal and reduction of pollutants like heavy metals

Biological Samples Collection
The biological samples used, e.g., prawn and macroalgae (Sargassum linifolium), were amassed from the Saudi western coast at the Red Sea, within E 35 • 65 -E 37 • 12 and N 26 • 03 -N 31 • 15 . All samples were identified morphologically by specialized marine biologists at the University of Tabuk in Saudi Arabia.

Alginate (Alg)
The extraction of sodium alginate (Alg) was performed from Sargassum linifolium, using slightly modified protocols [21,43]. Briefly, collected seaweed biomass (100 g) was recurrently cleansed with DW, air-dried and soaked in 1 L of 2% formaldehyde solution for 14 h to eradicate pigments, then washed with DW and immersed in 0.2 M H 2 SO 4 solution (at 1:10 w/v) before keeping for 2 h. Next, samples were further rinsed by DW and extracted with 5% Na 2 CO 3 solution for 6 h under agitation. Acid and alkali treatments were each Polymers 2021, 13, 2481 4 of 13 followed by centrifugation and DW washing. After obtaining Na-Alg gel, it was frozen at −18 • C, and then subjected to lyophilization.

Preparation of Biopolymers Nanoparticles
For preparing chitosan-alginate nanoparticles (Cht/Alg NPs), the microemulsion protocol was implemented [24]. Briefly, both Cht and Alg were dissolved in 2% acetic acid solution and hot DW (at 1% w/v), respectively, for preparing the aqueous phases. For the oil phase, paraffin oil was employed. These 3 solutions were homogenized (equal volumes, 10 mL each) for 30 min (Seward Stomacher, 400, Norfolk, UK) for forming stable emulsions, then cross-linkers were added (10 mL of Na-tripolyphosphate (1 M) and 10 mL of CaCl 2 solutions (1 M)) with constant stirring for 180 min at 25 ± 2 • C. The pellet of NPs formed was attained by centrifugation (SIGMA, 2-16 KL, Osterode, Germany) at 12,000 × g for 30 min, washed once with DW and twice with acetone, then recentrifuged and the acquired NPs pellet was lyophilized and analyzed.

Iron Nanoparticle Phycosynthesis
The S. linifolium algal extract (SE) was prepared using ethanol (70%) as a solvent. First, 50 g of algae powder was immersed in 500 mL of solvent and stirred for 8 h at 320× g at 25 ± 2 • C. The SE was attained by paper filtration to omit algal residues, then the solvent was discarded via vacuum evaporation and the dried SE was re-constituted in 10 folds of DW. For Fe-NPs biosynthesis, 0.1 M solution of FeCl 3 ·6H 2 O was dropped slowly (0.5 mL/min) into SE at a 1:2 ratio (v/v) of FeCl 3 solution:SE, stirred at 425× g, and the stirring was extended for extra 90 min after dropping. The change of the solution color to black indicated the NPs phycosynthesis. The reaction solution was quickly centrifuged at 14,000× g for 33 min and the NPs pellets were resuspended in DW, frozen at −20 • C and lyophilized [44].

Fabrication of Nano-Adsorbents
Aqueous solutions (10 mg/mL) from Cht/Alg NPs and SE-synthesized Fe-NPs were prepared using vigorous homogenization of nanomaterials in DW until achieving clear solutions, which were additionally filtered to eliminate any undisclosed particles. The Cht/Alg NPs solution were stirred 450× g at 25 ± 2 • C and the SE-synthesized Fe-NPs solution (equal volume) was dropped slowly into biopolymers solution under stirring for 120 min. Subsequently, the formed Cht/Alg/Fe-NPs composited particles were separated with centrifugation, washed with DW, frozen and lyophilized.

Structural Analysis of Nanometals
The TEM (transmission electron microscopy, Leo 0430, Leica, Cambridge, UK) imaging was employed for assessing the structural features size, morphology, shape, and distribution of SE-phycosynthesized Fe-NPs.

SEM (Scanning Electron Microscopy) Imaging
The surface structural morphology of the composited Cht/Alg/Fe-NPs was witnessed via SEM (JEOL JSM-IT100, Tokyo, Japan) to validate the composite compatibility and physiognomy. The detection of particle size (Ps) distribution of synthesized nanomaterials/nanocomposites (e.g., Fe-NPs, Cht/Alg NPs and Cht/Alg/Fe-NPs) and their surface charges (Zeta potential) were conducted via DLS (Dynamic Light Scattering, Malvern Zetasizer, Malvern Instruments, Malvern, UK).

Batch Adsorption of Hexavalent Chromium
Batch adsorption experiments were conducted using varying conditions (e.g., pH, adsorbent dose, contact time (CT) and initial ion concentration) to evaluate the potentiality of Cht/Alg NPs and Cht/Alg/Fe-NPs to adsorb hexavalent chromium (Cr 6+ ) from aqueous media [22]. The Cr 6+ aqueous solutions (different concentrations) were prepared via dissolving K 2 Cr 2 O 7 in buffered DW with acetate buffer. The experimented levels from each factor were: for pH. 3-8; for the adsorbent dose, 1-7 g/L; for the contact time, 30-270 min; and for the initial ion concentration, 10-150 ppm. While experimenting a factor, the rest conditions were kept at constant rates. After equilibrium attainment for each trial, the adsorbent was disjointed via vacuum filtration. The initial and final concentrations of Cr 6+ in filtrates were quantified using FAAS (flame atomic absorption spectrometer, AA-680, Shimadzu, Kyoto, Japan). The residual Cr 6+ concentrations were further determined accurately using 1,5-diphenylcarbazide that forms a reddish-violet complex with Cr 6+ . The absorbance of the complex was measured via UV-vis spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) at 540 nm, compared to standard calibration curves.

Phycosynthesis and Characterization of Fe-NPs Using Sargassum Linifolium Extract
The FTIR spectral analysis of SE and SE/Fe-NPs revealed the active characteristic bonds that are involved in SE biochemical activity and their potential role in Fe-NPs synthesis ( Figure 1). The SE spectrum (Figure 1 SE) indicated a strong O-H absorption band at 3421 cm −1 , related to extensive presence of this group in SE phenols and in amine groups, namely hydroxyl amide I, amide II and primary amines, on the Sargassum sp. walls [37].
The moderate peak at 2924 cm −1 reflected the stretched N-H bonding and/or the CH 2 /CH 3 and vibrated stretching in SE aldehydes and aliphatic groups. The intense signal at 1635 cm -1 is assumingly corresponding to stretched C=O and N=O in SE esters and pectin. The absorption bands at frequencies of 1635 and 1384 cm -1 "indicating the combined vibrated stretching of C-O and C-C ring of phenyl", 1042 cm -1 "indicating stretching S=O", and the recorded vibrations signals at 1113 and 829 cm −1 "indicating the existence of SE aromatic polyphenols", validated the main SE biochemical active bonding [45,46].
For the SE/Fe-NPs spectrum ( Figure 1 SE-Fe-NPs), the O-H indicative peak shifted to 3353 cm −1 after Fe-NPs phycosynthesis. Additionally, the shifting and intense variations in SE beaks after Fe-NPs synthesis (from 1635 to 1624 cm −1 , from 829 to 818 cm −1 and from 1384 to 1356 cm −1 ), and the disappearance of the indicative peak at 1113 cm −1 , revealed the involvement of these biochemical bonds in Fe-NPs phycosynthesis and capping [32]. The "Van der Waals" interaction forces are suggested as the main causes of interference between the O and N atoms in algal extract and the phycosynthesized Fe-NPs [31].
The ultrastructure of phycosynthesized Fe-NPs was elucidated via TEM imaging ( Figure 2). The image displayed the majority of NPs in spherical/semispherical shapes, with apparent agglomeration within synthesized particles. The estimated Ps of the phycosynthesized Fe-NPs was in the 13.6−72.4 nm range. The NPs agglomeration is basically attributed to the characteristics of SE, e.g., the thickening features of seaweed extract and the existence of intense O-H groups in it [30]. Moreover, the Fe-NPs agglomeration tendency was somewhat expected because of their minute sizes and potential magnetic characteristics [30,32,47]. For the SE/Fe-NPs spectrum ( Figure 1 SE-Fe-NPs), the O-H indicative peak shifted to 3353 cm −1 after Fe-NPs phycosynthesis. Additionally, the shifting and intense variations in SE beaks after Fe-NPs synthesis (from 1635 to 1624 cm −1 , from 829 to 818 cm −1 and from 1384 to 1356 cm −1 ), and the disappearance of the indicative peak at 1113 cm −1 , revealed the involvement of these biochemical bonds in Fe-NPs phycosynthesis and capping [32]. The "Van der Waals" interaction forces are suggested as the main causes of interference between the O and N atoms in algal extract and the phycosynthesized Fe-NPs [31].
The ultrastructure of phycosynthesized Fe-NPs was elucidated via TEM imaging ( Figure 2). The image displayed the majority of NPs in spherical/semispherical shapes, with apparent agglomeration within synthesized particles. The estimated Ps of the phycosynthesized Fe-NPs was in the 13.6−72.4 nm range. The NPs agglomeration is basically attributed to the characteristics of SE, e.g., the thickening features of seaweed extract and the existence of intense O-H groups in it [30]. Moreover, the Fe-NPs agglomeration tendency was somewhat expected because of their minute sizes and potential magnetic characteristics [30,32,47].

FTIR Analysis
The FTIR spectra of Cht, Alg and their composites (Cht/Alg) are offered in Figure  The Cht spectrum (Figure 3-Cht) appointed the existence of the key Cht characterist groups for amide III, amide I and N-H, and at absorption wavelength of 1425, 1671, an 3418 cm −1 , respectively. The band at 1031 cm −1 corresponds to C-O and C-C stretchin [48].
The distinguishing peaks of Alg (Figure 3-Alg) represented a stretching O-H bon

SEM Imaging
The ultrastructure imaging of constructed Cht/Alg nanocomposite with SE-phycosynthesized Fe-NPs is elucidated in Figure 4. The nanocomposite had ununiformed shapes with estimated Ps of 186.1−542.7 nm range. The incorporated Fe-NPs to Cht/Alg nanocomposite were notably detectable up to the surfaces of nanoparticles, which indicates their potentially strong capability for interacting with the surrounding environment and its components. Additionally, the Fe-NPs appeared in non-agglomerated forms, which confirmed the potency of Cht/Alg nanocomposite for capping and stabilizing the SE-phycosynthesized Fe-NPs [40,52,53].   [49]. The FTIR spectra of extracted Cht and Alg are strongly comparable to the standardized commercial sample of these biopolymers [48,50].
In Cht/Alg (Figure 3-Cht/Alg), the composite spectrum had the most distinctive groups from each of the individual polymers, which validated the polymers crosslinking and biochemical interactions. Additionally, the shifting of C-O-C stretching signal (in Alg spectrum) from 1016 to 1028 cm −1 indicated the cross-linkage of Alg with Ca 2+ and the ionic bonding between Alg carboxyl groups and calcium ions [41,50]. As observed after Cht/Alg cross-link interactions, the stretched -CH vibration peaks of Cht and Alg overlapped; more hydrogen bonds were formed between Cht amino groups "transformed to NH 3+ and produced polyelectrolyte compound under strong electrostatic interaction" and Alg carboxyl anions [51].

SEM Imaging
The ultrastructure imaging of constructed Cht/Alg nanocomposite with SEphycosynthesized Fe-NPs is elucidated in Figure 4. The nanocomposite had ununiformed shapes with estimated Ps of 186.1−542.7 nm range. The incorporated Fe-NPs to Cht/Alg nanocomposite were notably detectable up to the surfaces of nanoparticles, which indicates their potentially strong capability for interacting with the surrounding environment and its components. Additionally, the Fe-NPs appeared in non-agglomerated forms, which

SEM Imaging
The ultrastructure imaging of constructed Cht/Alg nanocomposite with SE-phycosynthesized Fe-NPs is elucidated in Figure 4. The nanocomposite had ununiformed shapes with estimated Ps of 186.1−542.7 nm range. The incorporated Fe-NPs to Cht/Alg nanocomposite were notably detectable up to the surfaces of nanoparticles, which indicates their potentially strong capability for interacting with the surrounding environment and its components. Additionally, the Fe-NPs appeared in non-agglomerated forms, which confirmed the potency of Cht/Alg nanocomposite for capping and stabilizing the SE-phycosynthesized Fe-NPs [40,52,53].

Nanomaterials' Size and Charge
The analysis of Ps and charge of fabricated NPs indicated the minute Ps range of Fe-NPs (12.5-83.1 nm), with a mean Ps diameter of 21.4 nm ( Table 1). The Ps ranged from 162.6-514.6 for Cht/Alg NPs and from 173.9-568.5 for Cht/Alg/Fe nanocomposite, with mean diameters of 311.2 and 342.6 nm, respectively. The integration of Fe-NPs into the polymers' nanocomposite led to a slight increasing in Ps and remarkable increment in nanocomposite negative charging from −23.2 to −34.6 mV (Table 1).

Batch Adsorption of Cr(VI)
The batch adsorption of Cr(VI) was successfully and effectually achieved, using both Cht/Alg NPs and composited Cht/Alg/Fe-NPs, with 100% removal percentages in some experiments ( Figure 5). While examining a factor, the rest conditions were kept at constant rates (e.g., pH = 5, adsorbant dose = 5 g/L, contact time = 200 min and initial ion concentration = 100 ppm). Generally, the composited Cht/Alg/Fe-NPs had higher efficiency of Cr(VI) adsorption than Cht/Alg NPs with each parameter. Due to higher contents from −OH (hydroxyl groups) and-NH 2 (primary amine), which act as powerful sorption sites, Cht-based compositions were advocated as excellent materials for the adsorbing and removal of many heavy metals, including Cr(VI), from solution [54]. The transformation of biopolymers, e.g., Cht and Alg, into nano-forms greatly increased their potentialities and removal capacity of heavy metals due to their enhanced physiochemical properties, providing larger contact area, more functioning groups, and extra sorption sites [53]. These parameters increased with the loading of nanopolymers with metals (e.g., iron nanoparticles), which may explain the higher capacity of Cht/Alg/Fe-NPs composite for absorbing/removing Cr(VI) [44,55].
characteristics. The extra addition of the adsorbents beyond 4 g/L did not trigger significant adsorption increment, which is assumed to be attributed to adsorbent particles overcrowding [24,57]. At the lower concentrations, adsorbent nanocomposites had sufficient surface area, adsorption sites, and amount of Fe-NPs for Cr 6+ uptake [58], whereas at higher adsorbent doses, the adsorption sites of nanopolymers composites are overlapping and their particles are overcrowding, which are suggested to decrease ions adsorption [25,53]. The contact time (CT) is an influential factor in batch adsorption practice; the CT influence on Cr 6+ adsorption, by Cht/Alg NPs and Cht/Alg/Fe-NPs composite, is presented in Figure 5T. The Cr 6+ adsorption experiment was prolonged from 30−270 min, with 30 min intervals. Results indicated that Cr 6+ adsorption significantly increased with CT The consequence of pH variation on Cr 6+ adsorption by Cht/Alg NPs and Cht/Alg/Fe-NPs is obtainable from Figure 5P, at a pH range of 3-8. The highest Cr 6+ removal (%) were 95.66% and 98.90% using Cht/Alg NPs and Cht/Alg/Fe-NPs, respectively, at pH 5. The metal solution pH can affect the surface charging of adsorbents speciation; the active biochemical sites of adsorbents are either deprotonated or protonated with pH alterations, and consequently the ions' adsorption is affected [56]. At lower examined pH values, less Cr(VI) ion uptake was detected, assumingly because of the competitive H + and Cr 6+ adsorption onto the polymers' nanocomposites surface. The protonation of polymer molecules, at lower pH values, can reduce the amount of available sites for metal ion binding. Furthermore, amino groups' protonation generates electrostatic repulsions on metallic cations, henceforth decreasing accessible sites for ion adsorption [57]. The Cr(VI) adsorption increased with pH increment, up to pH = 5, because more active adsorption sites on polymer nanocomposites were deprotonated and had more net attraction forces that enabled higher Cr adsorption from solution. The optimum pH for Cr 6+ adsorption was 5, then further pH increments led to lower ion adsorption, mostly because the precipitation of Cr(OH) 3 complexes, which hinders the adsorption development. The adsorption reduction in higher pH could be a result of development of further solubilized hydroxyl complexes [25]. Although Fe-NPs surface modifications could prevent their oxidation, especially when barriers between target pollutants and Fe-NPs are created which may influence Fe-NPs ability and reactivity for pollutant' removal, the employment of modified coating materials with high reactivity for HM removal can increase the removal potentialities of their composites [53]. The oxidation/reduction potential of Cr 6+ ions was illustrated to increase with pH decline; Cr 6+ ions became more susceptible to reduction in acidic solutions [57]. As Cht and Alg were evidenced as excellent HM adsorption material, they exhibited good affinities for Cr 6+ removal, either as plain composite, or after as active coating for adsorbing NPs [25,54]. Thus, the composited mixture from Cht/Alg/Fe-NPs, and the examined pH values for Cr 6+ adsorption trials, could control the oxidation potentiality of Fe-NPs The adsorbent dose consequence on Cr 6+ adsorption was investigated at 1−7 g/L ratios of Cht/Alg NPs and Cht/Alg/Fe-NPs composite in metal solution ( Figure 5D). This evidenced that Cr 6+ adsorption was at first speedily increased with adsorbent dose increment up to 4 g/L dosage, then remained mostly constant. The highest Cr 6+ removal (%) were 94.26% and 97.78% using Cht/Alg NPs and Cht/Alg/Fe-NPs, respectively, at 4 g/L dose. The initial prompt Cr uptake indicated high affinities between the adsorbate and the adsorbent nanocomposites, which are accredited to the adsorbents' biochemical characteristics. The extra addition of the adsorbents beyond 4 g/L did not trigger significant adsorption increment, which is assumed to be attributed to adsorbent particles overcrowding [24,57]. At the lower concentrations, adsorbent nanocomposites had sufficient surface area, adsorption sites, and amount of Fe-NPs for Cr 6+ uptake [58], whereas at higher adsorbent doses, the adsorption sites of nanopolymers composites are overlapping and their particles are overcrowding, which are suggested to decrease ions adsorption [25,53].
The contact time (CT) is an influential factor in batch adsorption practice; the CT influence on Cr 6+ adsorption, by Cht/Alg NPs and Cht/Alg/Fe-NPs composite, is presented in Figure 5T. The Cr 6+ adsorption experiment was prolonged from 30−270 min, with 30 min intervals. Results indicated that Cr 6+ adsorption significantly increased with CT prolongation up to 210 min, then the increment of removal percentage became insignificant. The highest Cr 6+ removal (%) were 95.42% and 98.93%, using Cht/Alg NPs and Cht/Alg/Fe-NPs, respectively, after a CT of 210 min. Initially, the higher Cr adsorption rate was possibly attributed to large available surface area from polymer composite to adsorb the ions. Subsequently, due to exhaustions of nanopolymers adsorption sites, no improvements could be achieved in Cr 6+ adsorption with CT prolongation beyond 210 min [59].
The effect of initial Cr 6+ concentration on the adsorption potentialities of Cht/Alg NPs and Cht/Alg/Fe-NPs composite is presented in Figure 5I. Both Cht/Alg NPs and Cht/Alg/Fe-NPs could absorb/remove 100% from Cr 6+ at 10 ppm ion concentration. At higher concentrations (25, 50, 75 and 100 ppm), the removal percentages were 98.9%, 97.23%, 96.55% and 90.24% for Cht/Alg NPs, and 100%, 99.42%, 98.96% and 93.61% for Cht/Alg/Fe-NPs composite, respectively. This could be simply explained by the ability of the nanopolymers' composites to possess sufficient binding/adsorption sites to uptake most orall Cr 6+ ions in beginning concentrations. Then, these sites possess partial saturation with metal ions at higher concentrations, which can somewhat lessen their removal capabilities [60,61].

Conclusions
The extraction of Cht from prawn shells and Alg from brown seaweed (Sargassum linifolium) was achieved with standard characteristics, the tow biopolymers was combined and cross-linked to generate Cht/Alg NPs, which had a mean diameter of 311.2 nm. The phycosynthesis of Fe-NPs was successfully attained using S. linifolium extract (SE) and the Fe-NPs had semispherical shapes with a 21.4 nm mean diameter. The conjugation of Cht/Alg NPs with SE-phycosynthesized Fe-NPs resulted in homogenous distribution and stabilization of metal NPs within the polymer nanocomposites. Both nanocomposites exhibited high efficiency as adsorbents for Cr 6+ at diverse conditions; the most effectual conditions for adsorption were a pH value of 5.0, adsorbent dose of 4 g/L, contact time of 210 min and initial Cr 6+ concentration of 75 ppm. These factors could result in the full removal of Cr 6+ from batch experiments. The composited nanopolymers (Cht/Alg NPs), which were innovatively incorporated with SE-phycosynthesized Fe-NPs, are strongly recommended for complete removal of Cr 6+ from aqueous environments. Subsequently, these effectual nanocomposited sorbents are candidates for further HM sorption trials toward extra pollutants and process optimizations.