Complexation Of Amino Acid With Cadmium And Its Application To Remove Cadmium From Contaminated Soil

Low molecular organic acids, such as amino acid, play an important role in cadmium (Cd) mobility. However, its complexation ability with Cd was not well studied. The complexation structure of amino and cadmium was investigated by theory calculation based on B3ly/SDD and detecting by FTIR spectrum. The conformers were found to be [CO c , CO c ] for fatty amino-cadmium and PheCd 2+ , [CO c , CO c , CO s ] for GluCd 2+ and ThrCd 2+ , respectively. The complex energy of these conformers was calculated in water phase by SMD model and the order of chelation energy was; PheCd 2+ > AlaCd 2+ > LeuCd 2+ > GluCd 2+ > GlyCd 2+ > ThrCd 2+ . All the dissolving energy of complexes was below zero, indicating these complexes was easily dissolved in water. In aqueous solution experiment, the Cd 2+ concentration decreased with increasing amino acid concentration. The order of logβ (Complex stability constant) was: PheCd 2+ > AlaCd 2+ > LeuCd 2+ > GluCd 2+ > GlyCd 2+ > ThrCd 2+ , consisting with the order of calculated chelation energy. The Cd removal eciency by Thr, Glu, Gly, Ala, Leu and Phe were 38.88%, 37.47%, 35.5%, 34.72%, 34.04% and 31.99%, respectively. From soil batch experiment, the total Cd in soil was decreased in present of amino acid with the concentration of Cd in water increased from 231.97 µg/L to 652.94-793.51 µg/L. The results of BCR sequential extraction showed that the Cd in acid soluble and reducible fraction sharply decreased. From all the results, the amino acid has potential to be used as a chelation to remedy the Cd contaminated soil.


Introduction
The "Itai-Itai disease" happened in Japan during the 1950s was attributed to the prolonged intake of cadmium-contaminated rice and aroused worldwide concern. (Huang et al. 2009) Gummuluru et al (2004) claimed that Metal bioavailability is considerably depended on chemical speciation of metal and correlates with the activity of free Cd ion in soil solution. (Gummuluru S. R. Krishnamurti et al. 2004) Voets et al (2004) reported that the type and concentration of organic ligands play a critical role on the activity of Cd. (Voets et al. 2004) Thus, washing method with organic ligand was used to remove Cd from contaminated soil. However, the synthesized organic ligand might introducing ecological damage to environment, causing harm for plant and prevent growth of plants, so the natural organic ligand was aroused widely concentration. (Borggaard et al. 2019, Hosseini et al. 2020, Yu et al. 2019) As reported by previous study, plant root exudates have a great impact on the bio-availability and mobility of heavy metals in the soil. (Sun et al. 2020, Tao et al. 2019, Ubeynarayana et al. 2021, Vranova et al. 2013) Among these exudates, amino acid with amounts of function group like carboxyl, hydroxyl and amino group, have important effects on the mobility and bio-availability of heavy metals by forming stable complexing with heavy metal.(CHANGEr- Hua 2009, Ghasemi et al. 2013, Rogiers et al. 2016 The study of Ghasemi et al.(2013) indicated that amino acid could produce stable complex with heavy metal and affect the activity of Cd. Thus, it could be concluded that the natural amino acid might have great potential to be used to remedy the Cd contaminated soil and worth being studied.
According to Jones et al (2005), (Jones et al. 2005) the common amino acids in soil were: Glutamic (Glu), Alanine (Ala), Glycine (Gly), Threonine (Thr), Leucine (Leu) and Phenylalanine (Phe). Among them, the carboxyl group is an electron-donating group capable of complexing metal ions, and the amino group is an amphoteric group capable of donating and obtaining electrons (Bell et al. 2016). Therefore, the conformers of amino-Cd complexes are diverse and complex. According to Dudev and Lim (2009), different complexes structure caused by different side chain, might affect metal-binding a nity. (Dudev and Lim 2009) Theoretical calculation was widely used method to study the complexes' structure and a nity. (Bingyu et al. 2018, Close et al. 2018 Comparing the calculated theoretical spectrum with the experimental infrared spectrum could con rm the existence of the calculated structure and avoid computational error. (Dunbar et al. 2009) The metal-binding a nity of complexes could be studied by chelation energy. (Pearson 2005) However, the effects of amino acid complexes on the Cd removal and fraction transfer in soil are not clear.
Thus, the objectives of this study were: (1) to identify the possible conformers of those amino-cadmium; (2) to conform the complex ability of the amino-cadmium complexes; (3) to investigate the effect of ligand concentration and cation on the chelation of amino-cadmium; (4) to evaluate the remove effective and the fraction transfer of Cd in soil by amino acids.

Calculation method
Most of amino acids would exist as zwitterions in which the N-terminus is protonated and the C-terminus is deprotonated (Jockusch et al. 1999). Manual of chemical analysis shows most pK a of carboxyl ranging from 2.0 to 3.0 and the pK a of -NH 3 + ranging from 9.0 to 10.0, which support this point indicating the cadmium may bind with amino and form salt-bridge structure. According to the study of Armentrout, four conformers of calculated PheCs 2+ were considered as starting points for geometry and vibrational frequency calculations. (Armentrout et al. 2013, Phillips 2002 The conformers of metal-Gly complex calculated in previous work were used as starting points for geometry and vibrational frequency calculations for fatty amino acid. (Bowman et al. 2010) And the conformers ThrCd 2+ were considered according to the study of Bowman and P. B. Armentrout (Armentrout et al. 2013, Bowman et al. 2010. For GluCd 2+ , the conformers of GluBa 2+ and GluLi + complexes studied by Jeremy T. O'Brien were considered . All theoretical calculations were done using Gaussian09. According to previous study, the SDD was suitable for calculate Cd complexes. (Dudev andLim 2009, Frisch andFrisch 1999) To ensure the accuracy of SDD method, a few known compound was calculated and compared the bond length with the actual bond length in Table 1. There is only a little error from the actual M-L bond distances with calculated M-L bond distances and this result indicated the SDD method could be used in this study (Table 1). Thus, Relative energies were determined for the geometries by using single point energies calculated at B3LYP levels using SDD basis set in this study. In order to produce enough complexes and avoid the interference of amino acids, the concentration of cadmium is much higher than that of amino acids. 10 ml of 0.01 mol/L amino acid solution was reacted with 10 ml of 0.1 mol/L Cd solution for 48 hours and the pH was adjusted to 7.0 (with 0.01 mol/L NaOH and 0.01 mol/L HCl). The reacted solution was dried in a water bath at 343K. The precipitation was centrifuged at 10000 r/min and separated. All the IR spectrum of separated precipitation was determined by FTIR.

Experiment in water phase 2.2.2.1 Effect of amino concentrations on chelation
The experiment in water phase was carried out in 100 ml amino acid (concentration from 0 to 0.1 mmol/L) with 0.01 mmol/L CdCl 2 . The pH value was adjusted to 7.0 with 0.1 mol/L HCl and NaOH solution. The cation concentration was adjusted to 0.1 mol/L by 1 mol/L KCl solution. The equation of chelation process was shown as follow: Where the LpCd was the concentration of complexes; C Cd 2+ was concentration of Cd ions; log β was complex stability constant;p was the numbers of amino acid complexed with Cd L was the concentration of amino acid. When the concentration of amino acid was much more than concentration of Cd, the concentration of L p Cd is approximately equal to the concentration of total Cd in solution and the concentration of amino acid is approximately equal to the concentration of total amino acid. The concentration of Cd 2+ was measured by Cd ion meter (Orion 9648BNWP) at 296 K.

Effect of cation concentration on chelation
100 ml of 0.01 mmol/L CdCl 2 was mixed with a series of 0.1 mmol/L amino acid at 7.0 pH value (adjusted by 0.01 mol/L NaOH and HCl). The cation concentration was prepared by KCl solution with concentration varying from 0.05 mol/L to 0.5 mol/L. The Cd 2+ concentration was detected by Cd ion meter at 296 K.  Table 2. A serial of amino acid solution from 0.1 to 1 mmol/L (adjusted with 0.1 mol/L NaOH and 0.1 mol/L HCl to soil pH 6.5) was prepared. The amino acid solution was mixed with soil at 10:1 of liquid to soil ratio. The mixture solution was shaken for 48 hours and centrifuge separation at 8000 r/min for 10 minutes. After centrifuging, Cd concentration in the separated solution was tested by ICP-OES (Agilent 5100). The BCR (Fajković et al. 2017) sequential extraction method was used to analyze the Cd transfer among different fractions in soil treated by amino acids. The heavy metals were was mainly distributed in four fractions: HOA C -extractable, reducible, oxidizable, and residual fraction, which were extracted with four extractants: (1) 0.11 mol/L acetic acid (AcOH) was added into soil to extract exchangeable species and the weak acid soluble fraction (HOAC-extractable fraction); (2) 0.5 mol/L hydroxylamine hydrochloride was adjusted to pH 2 with HNO 3 to extract the reducible metal species bound to Fe-Mn oxyhydroxides (reducible fraction); (3) 8.8 mol/L hydrogen peroxide and 1 mol/L ammonium acetate (AcONH4) was employed to extract oxidizable metal species bound to organics and sulphides (oxidizable fraction); and (4) aqua regia was used to obtain the residual fraction. The Cd concentrations in extractants were analyzed with an ICP-OES (Agilent 5100). The Gly, Leu and Ala was classi ed as fatty amino acid for the similar side chain. The possible conformers of Gly-Cd 2+ after calculated is shown in Fig. 1 Table 2. The length of Cd-O of [CO c , CO c ] were shorter than other structures with 2.18 and 2.13 Å and the < OCdO was 59°. Due to the similar structure, the results of Ala and Leu was similar with Gly. As depicted in Fig.S1 and Fig.S2, the conformer of AlaCd 2+ and LeuCd 2+ also was [CO c , CO c ]. From the detail information in Table 3, the length of O-Cd bond was 2.23 and 2.28 Å. Though, the O-Cd was longer than N-Cd, the previous study found the strength of O-Cd was much stronger than N-Cd, indicating [CO, CO] conformer was more stable than others. (Remelli et al. 2016) Thus, the GlyCd 2+ , AlaCd 2+ and LeuCd 2+ were mainly exist as [CO, CO] conformer.

Conformers of Glu with Cd 2+
Glu has a carboxyl group in side chain with 4.25 dissociation PK constants, thus the dehydrogenation of side chain and the tridentate conformer should be considered. (Sarkowski 1982  For PheCd 2+ , the benzene (P) contain electron, thus the bond of Cd and benzene was also considered.
From the IR spectra of PheCd 2+ (Fig. 4) and 2.54 Å, which was attributed to the attractivity of benzene ring (Table 3).

Theory calculation of chelation energy
From the result of FTIR spectrum, the structure of Glu and Thr was chelation tridentate, while the others were bidentate. Based on the conformers, the frees energy of dissolving process (Eq. 4) and chelation process (Eq. 5) was calculated in water phase.
As depicted in Table 4, all the dissolving energy of complexes was negative, indicating all these complexes were easily dissolved in water. All the chelation energy was also below zero, indicating the chelation process was spontaneous reaction in water phase. The order of Cd and amino acid chelation energy was: Thr < Glu < Gly < Ala < Leu < Phe. The energy of Cd and Thr chelation process was the lowest, indicating ThrCd 2+ was the most stable complex. In addition, chelation energy of Glu and Glu was − 24.33 kcal/mol and − 23.87 kcal/mol in water phase, which was much higher than other amino acid. This result might be attributed to the different conformer of Thr and Glu. Compared to other amino acids, the additional -OH an -COOH in side chain of Thr and Glu might donor more electron to Cd 2+ . Thus, the Thr and Glu is better electron-donor for heavy metal ion. Besides, the structure of Gly, Ala, Leu and Phe was bidenate conformer and the energy increased with the increasing molecule weight. In water phase, the water molecule would prevent the mobility of ligand agents. Thus, the larger molecule introduced more entropy penalty.

Chelation in water phase
In fact, the forming of Cd complexes might be affected by various factors in water. As shown in Fig. 5, the Cd 2+ increased with the increasing cation concentration, implying the decrease of ligand-Cd complexes concentration. In fact, the concentration of cation might affect the chelation by compete the function group. According to previous study, natural groundwater contained ions (such as Na + ,andK + ), which compete with Cd 2+ for ligand. (Xie et al. 2020) The concentration of chelation agent also affects the concentration of Cd 2+ . As shown in Fig. 5  (3), and the t curve was shown in Fig. 6. The constants "p" was greater than 1, indicating the that cadmium exists in the form of multidentate complex in high concentration amino acid solution( Table 5). The constant logβ of these complexes were 7.06, 6.99, 6.69, 6.62, 6.67 and 6.30 for Thr, Glu, Gly, Leu, Ala and Phe, respectively. The order of logβ of these complexes was Thr > Glu > > Gly > Ala > Leu > > Phe, consisting with the order of chelation energy. The result implied that the Thr and Glu has greater ability to chelate Cd 2+ to form the stable conformer of ThrCd 2+ and GluCd 2+ . Unlike in water phase, the condition in the soil was complicated. As shown in Fig. 7 the leachable Cd concentration increased with the increasing amino acid concentration from 0 to 1000 µmol/L. In distilled water (control treatment), the leachable Cd was only 231.97 µg/L. Maxium leachable Cd of these amino acid were 793.51 µg/L, 764.75 µg/L, 724.62 µg/L, 708.72 µg/L, 694.66 µg/L and 652.94 µg/L for 1000 µmol/L Thr, Glu, Gly, Ala, Leu and Phe, respectively. The Cd removal e ciency by Thr, Glu, Gly, Ala, Leu and Phe were 38.88%, 37.47%, 35.5%, 34.72%, 34.04% and 31.99%, respectively. Thus, it could be concluded that Cd was complex with the function groups of amino acid to formed soluble complexes, inhibiting the adsorption of Cd on soil surface. Besides, the Thr was found have much more ability to remove Cd from soil, which was consistent with the previous study. (Dolev et al. 2020) 3.6 Distribution of Cd in soil fraction To elucidate the fraction distribution of heavy metals after treated by amino acid, the BCR extraction was implemented. These acids soluble and reducible Cd with higher bio-availability are easily taken up by plants, while the oxidable and residual Cd are hardly taken up. In un-treated soil, distribution of Cd was mainly in acid soluble fraction. (Fernández-Ondoo et al. 2016) As shown in Fig. 8, the Cd in acid soluble fraction, reducible fraction, oxidable fraction and residual fraction were 33.81%, 21.36%, 13.73% and 31.1%,respectively. After treated by amino acid, the acid soluble and reducible Cd sharply decreased. In present of Thr, the Cd in acid soluble fraction decreased to 16.48% and the reducible fraction decreased to 14.72%. Similarly, in present of the Glu, Cd in acid soluble fraction decreased to 16.58% and the reducible fraction decreased to 15.24%. These results indicate the Cd in acid soluble fraction and reducible fraction have strong mobility and chelate with amino acid to be washed out from soil. According to previous studies, the heavy metals bound with acid soluble fraction has highly mobility and was easily extractable from soil in present of chelation agents. The Cd in residual fraction was mainly exist in crystal of mineral, which was hardly removed by chelating with organic agents. (Li et al. 2018, Xing et al. 2021 The decreased trend of Cd in acid soluble and residual fraction after treated by amino acid implied that Cd can be released into solution to be taken up by plant or washed out by.

Conclusion
In this study, the complexation effectiveness of amino acid and Cd 2+ was invetigated by theory calculate, FTIR spectrum, chelation experiment in water and soil batch experiment. indicating the GluCd 2+ and ThrCd 2+ were more stable than the others. And the dissolving energy of these complexes was below zero, indicating these complexes were easily dissolved in water. The order of logβ of chelation was: PheCd 2+ < AlaCd 2+ < LeuCd 2+ < GlyCd 2+ < GluCd 2+ < ThrCd 2+ , consisting with the order of chelation energy. The pH and cation concentration has negative relationship with the chelation process. The increased pH value and cation concentration prevent the chelation reaction for competing. In contaminated soil, the remove e ciency of Cd increased with the increasing amino acid concentration. At 1000 µmol/L amino acid solution, the remove e ciency of Cd was from 31.99-38.84%. the Thr has the best ability to remove Cd from soil for the stronger a nity of ThrCd 2+ with the lowest chelation energy and the highest logβ. With amino acid supplying, the Cd in acid soluble and reducible fraction in soil sharply decreased, indicating that Cd was transferred into more stable oxidable and residual fractions. Thus, amino acid was found having ability to be used to remedy the Cd contaminated soil. The (a) leachable Cd concentration and (b) remove e ciency of Cd in contaminated soil Figure 8