3.2. Identification of bacteria and EC50 determination
The spread of diluted samples from the soil on medium containing Pb and Cd resulted in six distinct bacterial colonies. Amplification of the 16S rRNA gene and BLAST search indicated the identity of the bacterial isolates as Nocardia cyriacigeorgica strain 4F, Bacillus sp. strain 1F20, Streptomyces rochei strain 8F, Priestia megaterium strain 5F, Bacillus paralicheniformis strain 1F, and Bacillus licheniformis strain 6F. The phylogenetic tree of these bacteria is depicted in Fig. 1 and revealed that they belong to the phyla Firmicutes and Actinobacteria.
Bacterial isolates were studied for their resistance to Pb and Cd (Tables 1 and 2). At low concentrations of Cd, bacteria 6F and 8F displayed greater resistance, whereas bacterial strains, 1F and 1F20, were more tolerant at concentrations of 10, 20, and 50 mg L− 1. Similarly, strains 1F and 1F20 exhibited greater resistance to Pb, as indicated by the EC50 values for both metals. These values were the highest for both metals and corresponded to isolate 1F and then to isolate 1F20. Overall, the bacteria were found to be more tolerant to Pb than to Cd.
Table 1
The growth inhibition (%) of bacteria at different concentrations of Pb in culture medium, and EC50 values of Pb for each isolate.
Code | 20 | 50 | 100 | 200 | EC50(mg/L) | Isolate name | Accession Number |
1F | 30 ± 1.7 | 35 ± 1.7 | 36.6 ± 2.0 | 41.1 ± 1.6 | 1261.8 | Bacillus paraliceniformis | OR287132 |
4F | 38 ± 3.3 | 46.6 ± 4.6 | 59.6 ± 3.1 | 68 ± 3.4 | 53.34 | Nocardia cyriacigeorgica | OQ198017 |
5F | 44.6 ± 4.6 | 51 ± 2.3 | 58 ± 3.0 | 69 ± 2.0 | 40.92 | Priestia megaterium | OQ912902 |
6F | 54 ± 1.1 | 60 ± 2.6 | 65.3 ± 1.7 | 76 ± 2.3 | 15.47 | Bacillus licheniformis | OQ283982 |
8F | 42.3 ± 1.45 | 60.6 ± 3.4 | 62.6 ± 3.9 | 65.3 ± 2.0 | 35.57 | Streptomyces rochei | OQ198233 |
1F20 | 28 ± 1.1 | 38 ± 1.1 | 54 ± 1.15 | 64.7 ± 2.3 | 79.66 | Bacillus sp. | OR287153 |
Table 2
The growth inhibition (%) of bacteria at different concentrations of Cd in culture medium, and EC50 values of Cd for each isolate.
Code | 2.5 | 5 | 10 | 20 | 50 | EC50(mg/L) |
1F | 31.3 ± 1.8 | 44 ± 1.1 | 72.5 ± 1.7 | 87 ± 4.0 | 94.3 ± 3.7 | 5.15 |
4F | 36.3 ± 2.0 | 64.5 ± 2.9 | 81 ± 3.4 | 90 ± 2.3 | 97 ± 2.1 | 3.53 |
5F | 45 ± 2.2 | 84 ± 2.9 | 91 ± 1.7 | 93 ± 2.3 | 95 ± 1.5 | 2.66 |
6F | 22 ± 1.5 | 67 ± 1.4 | 92 ± 1.6 | 93 ± 1.7 | 95 ± 3.1 | 3.92 |
8F | 28 ± 1.3 | 48 ± 4.0 | 91.3 ± 2.3 | 94 ± 2.9 | 97 ± 1.7 | 4.49 |
1F20 | 34 ± 2.0 | 44 ± 2.3 | 80 ± 1.0 | 86 ± 2.3 | 94 ± 2.3 | 4.71 |
3.2.1. FT-IR analysis of selected bacterial biomass
The findings of the FT-IR analysis for Bacillus paraliceniformis strain 1F and Bacillus sp. strain 1F20 (with the highest EC50 vales for Cd and Pb) grown in Nutrient broth with and without metals are illustrated in Fig. 1. The band at 3407 (Fig. 2A), which changed to 3388 (Fig. 2B), and 3427 (Fig. 2C) in the Pb- and Cd-exposed 1F strain, and the peak at 3413 (Fig. 2D), which changed to 3422 (Fig. 2F) in the Pb- and Cd-exposed 1F20 strain, indicating the stretching mode of the hydroxyl groups.
The changes in the bands in the regions 2880 cm− 1 (Fig. 2A) and 2877 cm− 1 (Fig. 2D) showed the presence of asymmetrical and symmetrical C-H stretching in both strains, which disappeared in the Cd-exposed bacteria (Fig. 2C and F). The band at 2958 cm− 1 is related to CH3 antisymmetric stretching, which is present in the spectra of both strains exposed to Pb (Fig. 2B and E), and indicates the presence of lipids and protein side chains, with minor contributions from carbohydrates and nucleic acids.
The peaks at 2930 cm− 1 and 2932 cm− 1 in Fig. 2A and D changed to 2927 cm− 1 (Fig. 2B) and 2926 cm− 1 (Fig. 2F), which shows CH2 antisymmetric stretching of mainly lipids, with minor contributions from proteins, carbohydrates, and nucleic acids. The peaks at 2877 cm− 1 (Fig. 2B) and 2872 cm− 1 (Fig. 2E) in Pb-exposed bacteria indicate CH3 symmetric stretching, which is mainly due to protein side chains, with minor contributions from lipids, carbohydrates, and nucleic acids [32].
The results suggested that the peak at 2930 cm− 1 (Fig. 2C) was associated with C-H stretching in the aliphatic cell wall, as highlighted by Patel et al. [33]. Furthermore, the peaks at 1656 cm− 1 (Fig. 2A) and 1655 cm− 1 (Fig. 2D) in both bacteria shifted to 1645 cm− 1 (Fig. 2C) and 1641 cm− 1 (Fig. 2F), respectively, in Cd-exposed bacteria. This shift indicates NH2 bending, and C = O and C = N stretching related to the functional group of the amide I band, in accordance with the findings of Patel et al. [34]. Additionally, the band at 1544 cm− 1 (Fig. 2D) shifted to 1515 cm− 1 (Fig. 2F) in Cd-exposed 1F20, suggesting the presence of an amide II functional group, as previously reported by Gurbanov et al. [32]. The peak at 1232 cm− 1 (Fig. 2A) in strain 1F shifted to 1241 cm− 1 (Fig. 2C) in Cd-exposed 1F, which suggests PO2- antisymmetric stretching. This is primarily due to nucleic acids with minor contributions from phospholipids, as mentioned by Gurbanov et al. [32]. Small peaks at 1163 cm− 1 and 1110 cm− 1 (Fig. 2F) appeared in the Cd-exposed 1F20 strain. The absence of the peak at 1153 cm− 1 in the FTIR spectrum of Cd-exposed strain 1F (Fig. 2D) might indicate that the presence of Cd ions disrupts CO–O–C stretching [32]. The appearance of the peak at 975 cm− 1 in the Pb-exposed strain 1F (Fig. 2B) suggests P-O-P stretching of phospholipids and ribose phosphate chain pyrophosphate [33]. The disappearance of the peak at 701 cm− 1, which is related to C-O in the plane bending of carbonate [34], in both Pb and Cd-exposed strain 1F and Cd-exposed strain 1F20 suggests that this region was affected by these metals. The appearance of bands at 872 cm− 1 (Fig. 2C) and 876 cm− 1 (Fig. 2F) in Cd-exposed 1F and 1F20 indicates the presence of Cd in the medium. These changes in the peaks and the appearance or disappearance of certain peaks suggest that functional groups in the bacterial cell wall are involved in absorbing metals. The negative charges on the cell wall of bacteria, including hydroxyl, alcohol, phosphoryl, amine, carboxyl, ester, sulfhydryl, sulfonate, thioether, and thiol groups [35], may be involved in the absorption of metal cations. The amide, carbonyl, hydroxyl, ethyl, and phosphate groups on bacterial biomass have been suggested as the main Cd binding sites [36]. Some of these groups are present on the cell surfaces of 1F and 1F20 and interact with metal ions. Metals accumulate in microorganisms due to metabolic processes such as exchanging metals in the cell wall, complex formation in the cell wall, or intracellular precipitation [37]. The changes in the peaks are due to the interaction of metals with functional groups on the bacterial cell surface, which exhibit biosorption activity. However, they do not modify the structural (i.e., hydrogen bond) components of bacteria, as reported for Pseudomonas aeruginosa cells in the presence of heavy metals [34].
3.3. The equilibriums study of Cd and Pb adsorption by sorbents
The initial concentrations of both metals affected their adsorption. As depicted in Fig. 3A, the adsorption capacity for Cd increased with increasing initial metal concentration. The q values for Cd sorption at an initial concentration of 200 mg L− 1 by CbB, Cb, ClB, and Cl were 52.72, 30.18, 31.97, and 24.42 mg g− 1, respectively. The maximum adsorption was obtained using CbB, followed by ClB. The increased biosorption capacity was attributed to the increased number of collisions between the ions and active sites on the sorbent. However, the Cd-removal efficiency decreased with increasing metal concentration (Fig. 3B). This can be attributed to the increased competition for active sites on the adsorbent with an increasing ion concentration [38]. The adsorption capacity and removal efficiency of Pb by the adsorbents improved with increasing metal concentrations in the solution. This result suggests that the adsorbents have a higher potential for Pb adsorption than for Cd, even at the maximum concentration of Pb. The q values for CbB, Cb, ClB, and Cl were 58.68, 55.32, 58.12, and 54.0 mg g− 1, respectively, at a Pb concentration of 200 mg L− 1 in solution (Fig. 3C). The maximum adsorption was achieved with CbB, followed by ClB. The parameters of adsorption isotherms are listed in Table 3. The Freundlich model was used to fit the data for both metals, resulting in the highest determination coefficient and lower standard error. The n value ranging from 1 to 10 is indicative of proper adsorption, reflecting the strong affinity between the adsorbent and metal ions [39]. Additionally, the Langmuir isotherm was found to be an effective model for describing the adsorption of both the metals. This model assumes that adsorption occurs on homogenous active sites as a monolayer surface, with each active site adsorbing only a single molecule until all the sites are filled [40]. In contrast, the Freundlich isotherm model assumes that heterogeneous surface adsorption occurs as both monolayer and multilayer [40]. Previous studies have reported the monolayer adsorption of heavy metals by chitosan [20, 41]. In a study conducted by Dong and Xiao [42], the Pb-adsorption capacity of dry calcium cross-linked water-soluble chitosan-based beads was measured to be 39.28 mg g− 1. Another investigation by Lv et al. [43] discovered that the adsorption capacity of heavy metals from aqueous solutions could be increased by modifying chitosan (CS) with xanthate in the CS/PVA system, resulting in an adsorption capacity of approximately 35 mg g− 1 that remained high even after three cycles.
Table 3
The constants, correlation coefficients and standard errors (SE) of Langmuir, Freundlich, and Temkin isotherms for adsorption of Pb and Cd on sorbents. qmax: maximum adsorption capacity by the adsorbent (mg g− 1), KL (L mg− 1): Langmuir isotherm constant, KF (mg1 − 1/n L1/n g− 1) and n: Freundlich isotherm constants, B and KT (L mg− 1): heat of sorption and Temkin isotherm constant, respectively. Cb (chitosan-biochar beads), CbB (bacterial immobilized chitosan-biochar beads), Cl (chitosan- lignosulfonate beads), and ClB (bacterial immobilized chitosan- lignosulfonate beads).
Freundlich isotherm model | Langmuir isotherm model | Temkin isotherm model | | |
KF | 1/n | R2 | SE | qmax | KL | R2 | SE | B | KT | R2 | SE | Sorbent | Metal |
0.57 | 0.86 | 1.00 | 0.56 | 142.5 | 0.0026 | 1.00 | 0.68 | 5.53 | 0.53 | 0.75 | 5.56 | Cb | Cd |
3.00 | 0.90 | 1.00 | 0.41 | 309.8 | 0.01 | 1.00 | 0.45 | 9.70 | 1.97 | 0.73 | 10.37 | CbB |
1.96 | 0.53 | 1.00 | 0.57 | 35.28 | 0.017 | 0.97 | 1.69 | 3.80 | 1.44 | 0.86 | 3.26 | Cl |
2.54 | 0.56 | 1.00 | 0.82 | 49.31 | 0.019 | 0.99 | 1.46 | 4.36 | 2.78 | 0.79 | 5.49 | ClB |
1.82 | 1.24 | 1.00 | 0.42 | 3194.9 | 0.0011 | 0.98 | 2.92 | 16.29 | 0.84 | 0.80 | 9.30 | Cb | Pb |
9.31 | 1.26 | 0.97 | 3.78 | 6140.2 | 0.0021 | 0.92 | 5.44 | 27.62 | 1.50 | 0.92 | 6.45 | CbB |
0.74 | 1.43 | 1.00 | 0.81 | 3653.2 | 0.0007 | 0.94 | 4.42 | 14.96 | 0.65 | 0.74 | 10.38 | Cl |
3.48 | 1.54 | 0.99 | 2.13 | 7483.8 | 0.0011 | 0.89 | 6.46 | 23.21 | 1.11 | 0.81 | 9.65 | ClB |
In a study, polymerized sodium lignosulfonate and chitosan (LS/CS) have been used as adsorbents for Pb2+ removal from water. The researchers found adsorption of 345 mg Pb2+ g− 1 adsorbent at 0.01 g loading of the adsorbent from a 100 mg L− 1 Pb solution in 120 min. [17]. The Langmuir isotherm adsorption model could explain the adsorption of Pb2+ to the LS/CS adsorbent as single-molecular-layer chemical adsorption. The adsorption of metals by CS can occur by physical process such as by van der Waals attraction between the adsorbate and the surface of the adsorbent, chemical forces such as by covalent bonds between adsorbate and adsorbent or through ion exchange mechanisms [44]. Electrostatic interaction, ionic exchange, metal chelation, and ion-pair formation have been identified as CS and its nanocomposite mechanisms to remove heavy metal ions [45, 46]. A batch adsorption experiment was conducted to remove Cd from water using Gasifier Biochar (GBC) and Chitosan-Coated Gasifier Biochar (CGBC) derived from pine. The maximum capacity of Cd adsorption by GBC was calculated with the Langmuir model by 68.6 mg g− 1 and this was 85.8 mg g− 1 on CGBC. Adsorption of Cd on CGBC and GBC followed pseudo-second order kinetics and equilibrium occurred in 2 h for Cd adsorption [47]. Chitosan enhances Cd removal through the strong coordination of metal ions and amides [47]. In another study B. drentensis MG21831T biomass was immobilized on polysulfone. The porous structure of the biosorbent was determined by using SEM analysis. Pb ions were adsorbed by the formation of plaque-type solid crystals on the surface and inside the pore walls. The maximum biosorption capacity of 0.3332 mg g− 1 was calculated by the Langmuir model (initial metal ion concentration 0.01–100 mg L− 1, 20°C, biosorbent dosage 40 g L− 1, contact time 440 min).
The adsorption capacity (q) and removal efficiency (RE) of the adsorbents for Cd and Pb from aqueous solutions at a concentration of 100 mg L− 1 and at different contact times are depicted in Fig. 4. The maximum adsorption of Cd achieved was 24.81, 31.17, 29.19, and 35.57 mg g− 1 for the adsorbents Cb, CbB, Cl, and ClB, respectively, at a contact time of 240 minutes (Fig. 4A). The removal efficiencies (RE) of Cd by Cb, CbB, Cl, and ClB were 99.2%, 99.7%, 99.3%, and 99.6%, respectively (Fig. 4B). The maximum adsorption of Pb attained was 27.14, 32.93, 30.77, and 37.88 mg g− 1 for Cb, CbB, Cl, and ClB, respectively, at a contact time of 240 minutes (Fig. 4C). The removal efficiency (RE) of Pb by Cb, CbB, Cl, and ClB were 98.90%, 99.40%, 99.20%, and 99.30%, respectively (Fig. 4D). The rate of adsorption increased as the contact time was prolonged from 1 to 240 min by slow steep, which indicates the potential of the sorbents to adsorb Cd and Pb at the initial contact times. The Pb adsorption capacity was higher than that of Cd. The adsorption kinetics demonstrate the adsorption rate, which helps to design the adsorption process at a proper contact time. The pseudo-first-order kinetic model shows that the binding of heavy metal ions to adsorbents occurs via physical adsorption, whereas the pseudo-second-order kinetic model shows that chemical adsorption is the binding mechanism. In addition, the intra-particle diffusion model illustrates the mass diffusion within the pores, which may limit the adsorption rate [40, 48]. Table 4 lists the kinetic parameters of Pb and Cd adsorption. All kinetic models had a high determination coefficient (R2) value related to the adsorption of both metals. The results suggest that the intra-particle diffusion model yielded smaller standard errors for both metals than the other models, indicating a more accurate estimation of adsorption kinetics. The larger intercept (Ci) may indicate that surface diffusion played a more significant role in the adsorption rate-controlling step for both metals. As shown in Table 4, the rate constant of intra-particle diffusion (K) was higher for Pb adsorption by chitosan-lignosulfonate (Cl) and chitosan-lignosulfonate-bacteria (ClB), indicating the larger role of surface diffusion in the adsorption of Pb. The first-order rate constants for Pb and Cd adsorption were greater than those of the pseudo-second-order model, suggesting that Pb2+ and Cd2+ ions require less time to reach adsorption equilibrium.
Table 4
Kinetic parameters for Pb and Cd adsorption by sorbents. qe(cal.) (mg g− 1), K(min− 1), K (g mg− 1 min− 1) and K (mg g− 1 min− 0.5): rate constant of models of Pseudo-first order, Pseudo-second order, Intra-particle diffusion, respectively, Ci (mg g− 1): intercept value, a (mg g− 1 min− 1), β (g mg− 1), R2: correlation coefficient and SE: standard error. Cb (chitosan-biochar beads), CbB (bacterial immobilized chitosan-biochar beads), Cl (chitosan- lignosulfonate beads), and ClB (bacterial immobilized chitosan- lignosulfonate beads).
Elovich | Intra-particle diffusion | Pseudo-second order | Pseudo-first order | | |
β | a | R2 | SE | Ci | K | R2 | SE | qe | K | R2 | SE | qe | K | R2 | SE | Sorbent | Metal |
0.63 | # | 1.00 | 0.62 | 23.39 | 0.11 | 1.00 | 0.20 | 24.36 | 0.72 | 1.00 | 0.36 | 24.25 | 4.01 | 1.00 | 0.43 | Cb | Cd |
0.75 | # | 1.00 | 0.77 | 29.41 | 0.13 | 1.00 | 0.17 | 30.52 | 0.71 | 1.00 | 0.50 | 30.41 | 4.35 | 1.00 | 0.58 | CbB | |
0.70 | # | 1.00 | 0.74 | 27.69 | 0.12 | 1.00 | 0.24 | 28.71 | 0.77 | 1.00 | 0.48 | 28.59 | 4.42 | 1.00 | 0.56 | Cl | |
0.78 | # | 0.99 | 1.13 | 34.63 | 0.07 | 1.00 | 0.12 | 35.24 | 1.27 | 1.00 | 0.26 | 35.17 | 5.23 | 1.00 | 0.30 | ClB | |
0.61 | # | 1.00 | 0.76 | 26.28 | 0.07 | 1.00 | 0.23 | 26.94 | 0.89 | 1.00 | 0.17 | 26.86 | 4.32 | 1.00 | 0.24 | Cb | Pb |
0.75 | # | 0.99 | 0.98 | 31.84 | 0.08 | 1.00 | 0.18 | 32.59 | 0.90 | 1.00 | 0.25 | 32.51 | 4.60 | 1.00 | 0.31 | CbB | |
1.06 | # | 1.00 | 0.53 | 27.06 | 0.28 | 1.00 | 0.67 | 29.65 | 0.24 | 0.99 | 0.96 | 29.33 | 2.98 | 0.99 | 1.19 | Cl | |
1.13 | # | 1.00 | 0.81 | 34.11 | 0.30 | 1.00 | 0.75 | 36.70 | 0.29 | 0.99 | 1.30 | 36.39 | 3.56 | 0.99 | 1.49 | ClB | |
In the adsorption process, ions are transferred to the surface of sorbents and diffuse into their pores [49]. Biosorbents, such as bacteria, cyanobacteria, yeast, and fungi, can be utilized in this process. For instance, free-living cells of Bacillus infantis and Pseudomonas fluorescens accumulated 90 µg mL− 1 and 81 µg mL− 1 of Cd, respectively, after a 24-hour contact period. Meanwhile, 2% sodium alginate immobilized cells accumulated 93 µg mL− 1 and 85 µg mL− 1 Cd, respectively. The Pb removal capacities of Bacillus infantis and Pseudomonas fluorescens were 82.3%, 91.5%, and 94.1% within 24, 48, and 72 h, respectively, at 200 µg mL− 1. In addition, Pb concentrations were reduced by 38.5%, 40.7%, 61.3%, 73.6%, 69.6%, and 94.1% after 72 h from media containing 1200, 1000, 800, 600, 400, and 200 µg mL− 1, respectively [50].
The processes involved in biosorption include physical adsorption, ion exchange, complex formation, reduction, and precipitation, as identified by Fomina et al. [51]. Microbial cell walls are composed of functional groups, such as carboxyl, hydroxide, amino, imidazole, sulfate, and sulfhydryl groups, which play a critical role in the removal of metal ions from aqueous solutions [52]. The thick peptidoglycan layer on the cell walls of Gram-positive bacteria leads to a higher sorption capacity owing to the presence of teichoic and teichuronic acids [53]. However, challenges in regenerating microbial biomass, separating biomass from liquid, and sorbent clogging pose difficulties in using microorganisms for biosorption processes [54, 55]. These issues can be resolved by immobilizing microorganisms using appropriate carriers, allowing for straightforward removal of the immobilized biomass from solutions and their reuse.
3.3. 1. FTIR analysis of adsorbents
A shift in the FT-IR spectrum peaks was observed, indicating the presence of Cd and Pb in the adsorbents (Fig. 5). The peaks at 3448 cm− 1 at ClB and CbB shifted to 3440 cm− 1, 3434 cm− 1 (ClB-Cd and ClB-Pb, respectively) and 3440 cm− 1 and 3443 cm− 1 (CbB-Cd and CbB-Pb, respectively) which are attributed to stretching of OH group of chitosan overlapping NH group stretching [56]. The peaks at 2939 cm− 1 and 2932 cm− 1 correspond to aliphatic C–H stretching vibrations that changed to 2932 cm− 1 and 2925 cm− 1 (ClB-Cd and ClB-Pb, respectively) and 2885 cm− 1 and 2977 cm− 1 (CbB-Cd and CbB-Pb, respectively) [57]. The peaks at 1651 cm− 1 and 1650 cm− 1 shifted to 1635 cm− 1 and 1630 cm− 1 (ClB-Cd and ClB-Pb, respectively) and 1642 cm− 1 and 1640 cm− 1 (CbB-Cd and CbB-Pb, respectively) related to the carboxylated functional group NH2 due to the amide carbonyl stretching vibration of the unhydrolyzed amide functional groups in chitosan (Nguyen and Nguyen, 2019; Burk et al., 2020) and supported the amide groups of chitosan and metals. New peaks at 1761 cm− 1 and 1763 cm− 1 appeared in ClB after the binding of Cd and Pb, respectively, representing the carboxyl group (–COO). The peaks at 1543 cm− 1 and 1541 cm− 1 changed to 1540 cm− 1 and 1531 cm− 1 (ClB-Cd and ClB-Pb, respectively) and 1539 cm− 1 (CbB-Cd) corresponding to the quaternary amine groups [58]. The peak at 1384 cm− 1 changed to 1382 cm− 1 and its intensity increased for ClB-Pb and ClB-Cd indicating the interaction of metals with C–H groups [58]. The asymmetric stretching of C-O bands at 1088 cm− 1 and 1091 cm− 1 in ClB and CbB, respectively shifted to 1076 cm− 1 (ClB-Cd and ClB-Pb) and 1078 cm− 1 and 1074 cm− 1 in CbB-Cd and CbB-Pb, respectively [56]. The peaks at 1154 cm− 1 (ClB-Cd and ClB-Pb) and 1157 cm− 1 (CbB-Cd and CbB-Pb) appear to be related to the asymmetric stretching of C-O-C and C-N. The peak 1029 cm− 1 in ClB disappeared after binding Cd and Pb which is related to the stretching of the C-O bond of C6 from chitosan (primary OH) [56]. The peaks at 512 cm− 1 and 519 cm− 1 in ClB and CbB, respectively, shifted to 558 cm− 1, 560 cm− 1 (ClB-Cd and ClB-Pb) and 542 cm− 1 in CbB-Cd because of the metal binding of M-O [58]. The peaks at 869 cm− 1 and 877 cm− 1 in ClB and CbB, respectively, changed to 892 cm− 1and 893 cm− 1 (ClB-Cd and ClB-Pb) and 889 cm− 1and 887 cm− 1(CbB-Cd and CbB-Pb) due to metal binding to the adsorbents. According to these results, carboxyl, amino, and hydroxyl groups are involved in binding of metals.
The hydroxyl and amino groups in CS act as sorption sites for metal ions, and the CS polymer is flexible which leads to the formation of complexes with metal ions [59]. Complexation between chitosan and metal ions in the adsorption process can occur via bonding metal ions with amino groups in CS chains through inter- or intramolecular reactions or in a hanging manner [46]. However, under acidic conditions, the main functional groups (-OH and –NH2) in CS are protonated and metal adsorption by CS is reduced. Electrostatic interactions and anion exchange are adsorption mechanisms that occur at acidic pH [60].
In the experiment on Pb2+ sorption from water by LS/CS, the absorption peaks of –NH2, –OH, C–O–C and C = C changed significantly after Pb adsorption. Probably a Pb- 𝝅 structure between Pb2+ and aromatic functional groups on LS/CS [61], and the Pb–O and Pb–N structures with hydroxyl [62] and amino groups [63] were formed in Pb ions adsorption process. Electrostatic interactions between Pb2+ and the LS/CS surface are also involved in ion adsorption [17]. The mechanism of Pb ion adsorption by LS/CS showed a negative charge on the LS/CS surface according to zeta potential analysis. The SEM–EDS analysis of the loaded Pb2+-LS/CS indicated a surface layer on the LS/CS, which was assumed to be Pb2+ [17].
3.3.2. SEM- EDX analysis
The SEM images of the adsorbents with and without-metals loading are presented in Fig. 6, which demonstrate obvious differences in surface morphology. The adsorbents without loading metals were observed to be transparent and without any particles, while the rough and shaggy forms with some particles were observed on the surface of the adsorbents in the presence of Cd and Pb. The carrier surface without cells was smoother than that with cells, which showed curly and wavy surfaces. The elemental composition of applied immobilized chitosan accompanying biochar or lignosulfonate in the result of EDX analysis confirmed Cd and Pb presence in each adsorbent (Fig. 7).