Application of Chitosan-Clay Biocomposite Beads for Removal of Heavy Metal and Dye from Industrial Effluent

: In recent years, there has been increasing interest in developing green biocomposite for industrial wastewater treatment. In this study, prawn-shell-derived chitosan (CHT) and kaolinite rich modified clay (MC) were used to fabricate biocomposite beads with different compositions. Prepared composite beads were characterized by FTIR, and XRD, and SEM. The possible application of the beads was evaluated primarily by measuring the adsorption efficiency in standard models of lead (II) and methylene blue (MB) dye solution, and the results show a promising removal efficiency. In addition, the composites were used to remove Cr (VI), Pb (II), and MB from real industrial effluents. From tannery effluent, 50.90% of chromium and 39.50% of lead ions were removed by composites rich in chitosan and 31.50% of MB was removed from textile effluent by a composite rich in clay. Moreover, the composite beads were found to be activated in both acidic and basic media depending on their composition, which gives a scope to their universal application in dye and heavy metal removal from wastewater from various industries. adsorbent to remove Cr (VI) and Pb (II) ions and methylene blue dye from both standard solution and real effluent. FTIR and XRD studies confirmed the successful formation of the composites. SEM micrographs clearly explained the surface morphology of the prepared composites. The composite which was rich in clay (SB-5) showed a better performance for cationic dye (MB) uptake, whereas heavy metals (Cr (VI) and Pb (II)) were better adsorbed on the SB-1 composite, which is rich in chitosan. In addition, adsorption studies were performed for real tannery and textile effluent showing a lower removal percentage compared to the standard solution. From tannery effluent, SB-1 can remove 50.90% of Cr (VI) and 39.50% of Pb (II) ions. A methylene blue dye removal efficiency of 31.50% was experienced in the case of textile effluent. The results suggest that a wide range of efficient composite formulations could be developed based on the adsorbates present in the effluent. However, a detailed study should be performed on the adsorption isotherm and adsorption efficiency of the composites for different heavy metals and dyes present in tannery effluent and textile effluent.


Introduction
In modern times wastewater released from industries is a major concern for environmentalists. Industrial effluents contain various toxic metals, harmful gases, and many organic and inorganic compounds. The discharge of these untreated toxic effluents has deteriorated natural flora and fauna and causes a risk to human health. A human who is exposed to such an environment in the long term can be attacked by fatal diseases like cancer, delayed nervous responses, mutagenic changes, and neurological disorders, etc. [1]. Thus, there is a current demand to maintain the standard permissible limit of such elements in industrial effluents before discharge to the environment. Hence, a fair number of physical, chemical, and biological methods have been employed to embark upon the problem of the removal of these materials from wastewater [2][3][4][5][6][7][8]. Compared to many expensive techniques, adsorption is preferred due to its flexibility, compatibility, low cost, and regeneration ability [9][10][11]. Considering the recent trends in attaining environmental sustainability, scientists are concentrating on utilizing naturally available materials to produce low-cost green adsorbents for the efficient removal of hazardous materials from wastewater [12,13].
Chitosan, obtained from chitin, is a relatively inexpensive material as chitin is the second most abundant polymer in nature next to cellulose [14,15]. It has shown many potential applications in the fields of wastewater treatment [14,16,17], tissue engineering [18], agriculture [19,20], biomedicine [21], drug delivery [22], and so on. Chitosan can be extracted from the exoskeletons of crustacean fungi and some insects. Bangladesh exports large quantities of shrimp/prawn every year. The respective processing plants produce large quantities of shells that remain unused and become treated as waste, showing no potential scope for profitability or reuse. In addition, these shells generate another issue for environmental pollution. Hence, waste prawn shells could be a good source of chitosan extraction. A large and growing body of literature has investigated the use of chitosan for heavy metal and dye removal from industrial effluents [17,23]. One major drawback of this approach is that chitosan is dissolved under acidic conditions, while several metals are preferentially adsorbed in acidic media. Researchers have attempted to solve this problem by incorporating chitosan into other materials that could improve its performance under a wide range of environmental conditions [24].
Recent research has revealed clay minerals as a natural inorganic material with definite structural adsorption, rheological, and thermal properties [25]. These materials originally have a hydrophilic character due to the presence of their surface hydroxyl (-OH) groups which can link water molecules very easily [26]. Clay mineral has a long enchanting history of metal binding [25], dye removal [27], and fruit packaging [28], while being used independently or combined with other natural or synthetic polymers. However, it is sometimes necessary to purify and modify clay for better compatibility with other polymers [29].
In our previous studies, we developed a biocomposite, both in film form and bead form, based on chitosan and modified Bijoypur clay, and evaluated adsorption isotherms and efficiency for chromium (VI) removal from a standard water model [24,30]. The results suggested better adsorption performance for the bead form compared to the film form of the composites. Hence, in the present study, we attempt to fabricate chitosan (CHT)-modified clay (MC) biocomposite beads and evaluate their applicability in removing Pb (II) and methylene blue dye from a standard model solution. In addition, as a preliminary study, the efficiency performance of these composites for removing Cr (VI), Pb (II), and methylene blue (MB) from two real industrial effluents (tannery and textile effluents) is investigated.

Materials
Waste prawn shell was collected from the local hatchery of Satkhira district, Khulna division, Bangladesh. Bijoypur clay was collected from Netrokona, Bangladesh (supplied by Bangladesh Insulator and Sanitaryware Factory (BISF), Dhaka, Bangladesh). Sodium hydroxide, ethanol, and acetic acid were supplied by E. Merck, Germany; hydrochloric acid, methylene blue dye, Pb (II) salt, nitric acid, and sulfuric acid, etc., were collected from Merck KGaA 64271, Dastadt, Germany. Sigma-Aldrich, Stockholm, Sweden, supplied the clay modifying agent dodecylamine. Chromium (VI) salt was collected from Loba Chemie Pvt. Ltd., Mumbai, India. Tannery and textile effluents were collected from industries located in Dhaka city.

Extraction of Chitosan from Waste Prawn Shell
CHT was extracted from the waste prawn shell by following a modified version of the method of Rashid et al. [31]. Dried prawn shell was washed with hot water several times and dried in an oven. The sample was deproteinated with 4% (w/w) NaOH solution followed by demineralization with 3 N HCl to obtain chitin. The chitin was then deacetylated by 50% (w/w) NaOH at 80-100 °C to produce chitosan.

Purification and Modification of Bijoypur Clay
Raw Bijoypur clay was screened through a 150-mesh screen and then purified with concentrated hydrochloric acid to remove unbound silica and other impurities. The purified clay was modified by dodecylamine to facilitate the interaction of chitosan with clay and then used in the biocomposite fabrication as MC [32].

Fabrication of Biocomposite Beads
Following the method described by Wang et al. and Biswas et al., biocomposite beads of the MC and CHT were prepared [30,33]. At first, 1% CHT solution (in 1% acetic acid solvent) and 1% MC dispersion (in 1% acetic acid solvent) were mixed at various amounts (by weight) at a temperature of 60 °C to form a concentrated homogeneous mixture ( Table 1). The mixture was then transferred, in small droplets, into a mixture (1:4) of 15% (w/w) sodium hydroxide and 95% (w/w) ethanol. Small beads were formed and decanted from the liquid. The separated beads were dried at 60 °C for 24 h. A detailed fabrication process of the composite is illustrated in Figure 1.

Characterization of Biocomposite Beads
The composite beads (SB-1, SB-3, and SB-5) were examined using an FTIR spectrophotometer (8400S spectrophotometer, Shimadzu Corporation, Kyoto, Japan) to investigate the interaction between chitosan and modified clay, which helps to develop the composite structure. An XRD (BRUKER AXS Diffractometer D8, Karlsruhe, Germany) analyzer was used to obtain preliminary information about the presence of chitosan and modified clay in the composite beads from their crystalline natures. Surface morphology of the raw clay, acid-treated clay, modified clay, and composite beads was compared by SEM (JSM-6490). Detailed descriptions of all of these experimental methods have been reported in our previous article [24].

Adsorption Studies for Standard Solution and Industrial Effluents
A stock solution of 100 ppm was separately prepared for each of Pb (II) and MB. After diluting the stock solution to 25 ppm, 20 mL was taken in Erlenmeyer flasks with 0.05 g of biocomposite adsorbent and agitated at 27 °C on a reciprocating shaker at a fixed rate of 180 rpm for 2 h. The pH was adjusted to 6 for Pb (II) and 9.1 for MB by adding 0.1 N NaOH solution, keeping all other parameters constant. MB concentration after and before the removal was determined by a UV-visible spectrophotometer (Shimadzu 1700 UV, Kyoto, Japan) by proper calibration (λmax = 663 nm), whereas an atomic absorption spectrophotometer (AAS) (Varian AA 240 FS) was used for determination of the Pb (II) concentration by fixing the wavelength at 283.3 nm. In every aspect, the following equation (Equation (1)) was employed to determine the adsorption capacity, i.e., where qe is the amount of the species (Pb (II) or MB) adsorbed (mg/g) by the adsorbent, Co is the initial concentration of the solution (mgL −1 ), Ce is the final (in some cases equilibrium) concentration of the species solution (mgL −1 ) after 2 h of contact, V is the volume of the solution used (in liters), and W is the weight of the adsorbent used (in grams).
In the case of the tannery and textile effluents, the samples were digested by concentrated nitric acid and diluted to adjust to the concentration range of the instrument [34,35]. Adsorption studies for Cr (VI), Pb (II), and MB present in the samples were performed using the same procedure followed for standard solution.

FTIR Analysis
The composite fabrication starts with chitosan extraction and clay modification. The conversion of chitin to chitosan is a sophisticated task that requires extra attention at the deacetylation step. Moreover, modification of clay requires sophisticated handling of materials under certain conditions. Successful extraction of chitosan, modification of clay, and fabrication of the composite film were confirmed by detailed FTIR analysis, as discussed in our previous work [24]. However, in this work, the fabrication of the three composites (SB-1, SB-3, and SB-5) in bead form was analyzed by FTIR. While studying the FTIR spectra of the composites, the characteristic peaks of chitosan (at 1601 cm −1 (N-H bending) and 3402 cm −1 (O-H stretching overlapping the N-H stretching of primary amine) and modified clay (at approximately 2900 cm −1 for C-H stretching of dodecylamine in MC) were observed, which confirmed the existence of these two components in the composites ( Figure 2). The changes in intensities observed at these three peak positions support the corresponding ratio of chitosan and MC in the three composites. Moreover, the red shifts of the peak for N-H bending (from 1638 cm −1 in pure chitosan to 1601 cm −1 in the composite) and the peak for overlapping of O-H stretching and the N-H stretching of the primary amine (from 3448 cm −1 in pure chitosan to 3402 cm −1 in the composite) indicate the possible interaction between functional groups of -NH2 and -OH with the -OH of MC. All other important groups from CHT and MC were observed in the FTIR spectra of the composites, resulting in a comparable interaction of the elements. However, it is also noticeable that the FTIR spectra for the bead form composite is similar to that of film form composites (presented in our previous study [24]).

XRD Analysis
Comparative XRD analyses of the chitosan, raw clay, and composites were performed to obtain preliminary information about the presence of chitosan and modified clay in the composite beads ( Figure 3). Chitosan has a semi-crystalline nature and clay is very crystalline. However, the observed composite XRD data showed in-between crystallinity, indicating that each component was well-dispersed within the composite system. The results observed from the XRD pattern of the composites reveal that after addition of chitosan into the MC, a peak at 40°, a characteristic peak of kaolinite, disappeared in all the composites. Moreover, a broadening and shifting of another peak at 20°, responsible for clay, was also observed. We assume this was because of the presence of chitosan, which interrupted the overall crystallinity of the clay.

SEM Analysis
The morphologies of the raw clay, acid-treated clay, modified clay and composites (SB-1 and SB-5) were evaluated by SEM images at ×1000 magnification (Figures 4 and 5). In Figure 4, the crystalline nature of the clay can be observed in the image of the raw clay, whereas a clear image of the acid-treated clay (purified clay) reveals the removal of unbound silica and other impurities incorporated into the raw clay with clay minerals [24]. In the last image of Figure 4, in modified clay, smooth but discrete spherical particles can be observed that confirm the incorporation of the organic modifying agent into the inorganic clay materials.
In Figure 5, two SEM micrographs of composite samples SB-1 and SB-5 are shown at ×1000 magnification. As SB-1 contains a higher percentage of chitosan, a comparatively smooth surface can be seen. In contrast, SB-5 possesses a relatively rough surface due to a lower percentage of CHT and a higher percentage of MC.

Comparative Adsorption Studies on a Standard Solution
The details of the adsorption technique used, including the isotherm of chromium (VI) from standard solution, have been discussed in our previous work [30]. Better adsorption of Cr (VI) ions on the SB-1 composite was observed because of the presence of a higher amount of chitosan in the SB-1 samples compared to SB-3 and SB-5 ( Table 2). The electrostatic interaction between the Cr2O7 2− ions (a form of Cr (VI) in the acidic solution) and NH3 + (a form of an amine group of chitosan under acidic conditions) helped ensure a higher uptake of chromium by SB-1 from the solution. In SB-5, meanwhile, the percentage of clay was higher than that of chitosan, and fewer free amine sites were available for adsorbing chromium. Being a cationic dye, MB was adsorbed in the highest amount on composite SB-5 compared to CHT, MC, and the other two composites (Table 2) ( Figure 6). The lowest adsorption capacity was observed for SB-1. The better adsorption of MB by SB-5 is attributed to the cationic nature of MB and the anionic nature of the clay-rich composite in alkaline solution. The -NH2 of chitosan in basic solution became neutral and in acidic media, it became positively charged (NH3 + ). Hence, cationic dye adsorption on chitosan is believed to be lowered due to the absence of any interaction between the -NH2 and dye in solution. However, the presence of an -OH group in chitosan might result in some adsorption of MB, which was lower than the -OH group in clay. In SB-5, the amount of clay was higher than that of chitosan, and hence more anionic -OH clay sites were available for dye adsorption, in addition to the rough surface structure of the bead. In SB-1 the amount of chitosan was higher and, resulting in poor adsorption of methylene blue. A similar kind of phenomenon has been documented by many other researchers over the last decade [36][37][38]. A schematic diagram of the mechanism of MB adsorption by composite beads is depicted in Figure 7.  Based on the adsorption analysis, in the case of the Pb (II) solution, with an increase in the available amine groups in the composite (from SB-1 to SB-3), the adsorption capacity was observed to increase. The lowest adsorption capacity among the composites was found for SB-5 (Table 2) (Figure 8). This composite was prepared by the addition of a higher percentage of chitosan compared to clay. Hence, higher amine groups from the chitosan were available, which in turn allowed for the formation of a co-ordination bond with the Pb 2+ ion. In the case of SB-5, the percentage of clay was higher than that of the chitosan. Hence, fewer free amine sites were available for adsorbing Pb (II) ions. A detailed adsorption mechanism of Pb 2+ ions on beads (both on the surface and in the interior part of the composite) has been illustrated in Figure 9. However, to understand the removal pattern of MB and Pb (II) from standard solution, a complete protocol of their isotherm studies is needed, which is currently ongoing in our research lab.

Removal of Cr (VI) and Pb (II) from Tannery Effluent
The amount of chromium and lead in the original tannery sample was found to be 55 ppm (the United States Environmental Protection Agency (EPA) approved ermissible limit for drinking water is 0.00001 ppm) and 40.5 ppm (the EPA permissible limit for drinking water is 0.015 ppm), respectively, and was measured using a UV-visible spectrophotometer and AAS, respectively. The tannery sample was diluted to 0.110 ppm for chromium and 0.081 ppm for lead to match the instrument specifications. The adsorbent selected for effluent treatment was based on the results obtained from the standard solution adsorption. As the composite sample SB-1 showed the best adsorption performance for Cr (VI) and Pb (II), it was selected for the treatment of tannery effluent. After 2 h of treatment, the concentration was reduced to 0.054 ppm for Cr (VI) and 0.049 ppm for Pb (II). Hence, 50.90% of the Cr (VI) and 39.50% of the Pb (II) were removed from the tannery effluent (Table 3). The composite sample SB-5 was selected for the removal of methylene blue dye from real textile effluent. The initial concentration of MB in the effluent was found to be 0.19 ppm; the concentration decreased to 0.13 ppm after treatment with composite beads. The percent removal was around 31% at slightly acidic pH (6) following a 2 h shaking period with the adsorbent.

Discussion
After analyzing all the above results, it can be inferred that the fabricated composites have potential applicability for industrial wastewater treatment. The removal percentage for Pb (II) and MB for the standard solution presented here is lower than that for Cr (VI) because these adsorption performances have not yet been optimized ( Table 4). The results showed a lower value for removal percentages of Cr (VI), Pb, and MB from real effluent ( Table 3) compared to that from the standard solution (Table 4). The reason for this can be said to be the interference of other dissolved species present in real effluents like salts, surfactants, other dye molecules, and heavy metals, etc. Moreover, as the adsorption process was highly pH-dependent, the deviation from standard pH for effluents was also responsible for this reduced performance. Some pretreatments used to exclude these interfering species, as well as adjust the pH for maximum adsorption efficiency, may increase the performance.
According to recent reports focusing on potential adsorbents (Table 5), more than 93% of MB can be removed from wastewater by a chitosan-based composite, which is a much higher percentage than that relating to our composite under investigation [39]. In the case of removing Pb(II) from groundwater, maximum efficiency of 94% has been reported by Badmus et al. [40]. However, all these data were obtained under optimized conditions and present maximum efficiency. Our preliminary non-optimized studies show promising results compared to many other standard absorbents while removing the selected metal or dye ions. Optimizing the adsorption conditions (especially pH, adsorbent dose, and the temperature used for obtaining maximum efficiency for chitosan-MC biocomposite beads) may make this composite competitive compared to all other adsorbents.
We are in the process of studying a detailed adsorption isotherm and the performance evaluation for Pb (II) and MB (as we did for Cr (VI)) and hope that the adsorption efficiency for these two species will be promising compared to other available adsorbents. We will present detailed outcomes in our next report.  * Non-optimized condition for specific species removal from standard aqueous solution. ** Non-optimized condition for specific species removal from effluent.

Conclusions
From the present study, it can be concluded that the biocomposite beads of chitosan with modified Bijoypur clay act as a promising adsorbent to remove Cr (VI) and Pb (II) ions and methylene blue dye from both standard solution and real effluent. FTIR and XRD studies confirmed the successful formation of the composites. SEM micrographs clearly explained the surface morphology of the prepared composites. The composite which was rich in clay (SB-5) showed a better performance for cationic dye (MB) uptake, whereas heavy metals (Cr (VI) and Pb (II)) were better adsorbed on the SB-1 composite, which is rich in chitosan. In addition, adsorption studies were performed for real tannery and textile effluent showing a lower removal percentage compared to the standard solution. From tannery effluent, SB-1 can remove 50.90% of Cr (VI) and 39.50% of Pb (II) ions. A methylene blue dye removal efficiency of 31.50% was experienced in the case of textile effluent. The results suggest that a wide range of efficient composite formulations could be developed based on the adsorbates present in the effluent. However, a detailed study should be performed on the adsorption isotherm and adsorption efficiency of the composites for different heavy metals and dyes present in tannery effluent and textile effluent.