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Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction
Accepted Manuscript Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction Jiaying Cui, Nengwu Zhu, Naixin Kang, Chitam Ha, Chaohong Shi, Pingxiao Wu PII: DOI: Reference:
S1385-8947(17)31272-X http://dx.doi.org/10.1016/j.cej.2017.07.124 CEJ 17390
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
2 June 2017 19 July 2017 20 July 2017
Please cite this article as: J. Cui, N. Zhu, N. Kang, C. Ha, C. Shi, P. Wu, Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.07.124
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Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction Jiaying Cuia, Nengwu Zhua,b,c,*, Naixin Kanga, Chitam Haa,d, Chaohong Shia, Pingxiao Wua,b,e a
School of Environment and Energy, South China University of Technology, Guangzhou
510006, PR China b
The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of
Education, Guangzhou 510006, PR China c
Guangdong Environmental Protection Key Laboratory of Solid Waste Treatment and
Recycling, Guangzhou 510006, PR China d
Resources and Environment Department, Vinh Long City, Vinh Long Town, Viet Nam
e
Guangdong Engineering and Technology Research Center for Environmental Nanomaterials,
Guangzhou 510006, PR China
* Corresponding author at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. Tel./fax: +86 20 3938 0522. E-mail address: [email protected] (N. Zhu).
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Abstract In this study, the biorecovery mechanism of palladium ions (Pd(II)) as palladium nanoparticles by Enterococcus faecalis (E. faecalis), a typical Gram-positive bacterium, was explored from acting force, participated substance and reaction process. The XRD and TEM analysis showed that the deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption, and the process was invested by effect factors and theory models of isotherm, thermodynamics, and kinetics, which was a spontaneous endothermic process fitted well by Langmuir isotherm and pseudo-second order kinetics. In addition, the surface characteristic changes of E. faecalis during biorecovery of Pd(II) were compared by FTIR and XPS, and it found that carboxyl groups, hydroxyl groups and amine groups were the main participated functional groups in biorecovery. Furthermore, the contrary pH change between biosorption and bioreduction combined with above results proved that electrostatic interaction, complex formation of Pd(II) on the cell and intracellular uptake were the acting forces for biosorption, and the bioreduction of adsorbed Pd(II) was via the hydrolysis and oxidation of sodium formate to deliver electron. Therefore, the biorecovery mechanism of Pd(II) can be divided into the biosorption from solution to cells, and then the bioreduction and crystallization in situ by the electron donor and the link frame of E. faecalis. Keywords: Biorecovery mechanism; Biosorption; Bioreduction; Palladium nanoparticles; Enterococcus faecalis
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1. Introduction The application of precious metals is increasing in various industries resulted in the research urgency for recycle [1]. Palladium (Pd) as a significant precious metal has been applied in many industries such as catalyst, chemical, electronic, and glass [2]. Moreover, the biorecovery methods of Pd using different microorganisms have been widely explored [3]. It is considered as an eco-friendly alternative to the conventional physical and chemical methods [4]. The biorecovery of Pd(II) from solution usually divided two main types, namely the biosorption and accumulation of Pd(II) [5] and the bioreduction and recycle as Pd-NPs [6]. Therefore, for the biorecovery system of Pd(II), especially as Pd-NPs loaded on cells, both the biosorption and the bioreduction could be related to biorecovery mechanism, but the existing researches seldom clarified the whole process. The biosorption of Pd(II) from solution aimed to transfer Pd(II) from solution to biomass, and adsorption amount and selective biorecovery are the core of research [7, 8], but the bioreduction and applicability of recovering Pd(II) is also limited. In addition, adsorption isotherm models (Langmuir and Freundlich models), thermodynamics model and kinetics models (pseudo-first order, pseudo-second order and Weber-Morris intraparticle diffusion models) usually explained the adsorption process and mechanism for traditional adsorbent [9-12]. For different biomass, the calculated adsorption parameters are different due to the various surface structure and functional groups, and some new biosorbent like live bacterium biomass is seldom studied for biosorption mechanism by these typical models [13]. Furthermore, the researches on biorecovery of Pd(II) as Pd-NPs mainly focused on the control 3
of environment conditions and the characterization of nanoparticles [14-16], and the deep insight into the biosorption process is incomplete [17, 18]. And the precise mechanisms of the Pd(II) bioreduction was not well understood due to the difference of biomass [4]. Enterococcus faecalis (E. faecalis), a model microorganism of gram-positive bacteria, appears common in various environment. Its genome has been decoded, and its cell membrane structure also has been analyzed which is more simply than gram-negative bacteria [19]. Therefore, the metal biorecovery property of E. faecalis could give abundant information for the study of gram-positive bacteria. In our previous study [6], the biorecovery of Pd by E. faecalis and the catalysis of Pd-NPs for chromate reduction have obtained satisfactory results, but the biorecovery process has not been explained in detail. In this study, the biorecovery mechanism was explored by the characterization of Pd-loaded cell, the transfer of Pd(II) in biosorption, surface characteristic of E. faecalis during biorecovery of Pd(II), and the transform of Pd(II) in bioreduction.
2. Materials and methods 2.1. Bacteria preparation E. faecalis (CCTCC M2012445) was cultured in Luria Broth culture and centrifuged to harvest cells as reported previously [6]. The cell samples were resuspended in the buffer solution of 20 mM 3-[N-morpholino] propanesulfonic acid (MOPS) (pH 7.0) to an optical density at 600 nm of 1.6 (corresponding to cell dry weight (CDW) of 1.2 g L-1), and then stored at 4 ℃ to keep the cellular morphology and activity for experiments within 48 h. 2.2. Biorecovery of Pd(II) as Pd-NPs 4
The solution of Pd(II) was prepared in distilled water (pH 7.0) from Na2PdCl4 salts (Aladdin Industrial Corporation, China), using sulfuric acid and sodium hydroxide for pH control. The cell suspension in MOPS buffer solution was centrifuged (10621 g, 5 min) as E. faecalis biomass. Biomass dosage of 1.2 g/L contacted to 10 mL Na2PdCl4 solution (pH 3.0) with final Pd(II) concentration of 210 mg/L, which was cultured at 40 ℃ in a 50 mL serum bottle. After 30 min, the Pd(II)-loaded cell samples were analysis as the result of biosorption. Then 2 M sodium formate (final concentration of 25 mM) as the electron donor was added to the Pd(II)-loaded cell solution in serum bottle for 48 h to form Pd-NPs, and the Pd-NPs-loaded cell samples were analysis as the result of bioreduction. The design of experiment parameters for the formation process of Pd-NPs has been reported at our recent research [6]. The pH value of solution was measured using a pH meter (INESALucy PHS-3C, China). Comparing with Pd(II)-loaded cells in biosorption, the crystallization of Pd-NPs after bioreduction of Pd(II) were confirmed by using XRD method (Bruker D8 ADVANCE X-ray diffractometer, USA). The deposit locations of Pd(II) in biosorption and Pd-NPs in bioreduction were compared by TEM equipment (HITACHI H-7650, Japan). The samples for XRD and TEM analysis were prepared and analyzing as reported previously [6]. 2.3. Biosorption experiments of Pd(II) The transfer of Pd(II) from solution to cell was described by biosorption. The factors affecting the Pd(II) biosorption efficiency without the addition of electron donor were studied including:
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(1) Effect of the biomass dosage was studied from 0.6 to 3.6 g/L with 210 mg/L Pd(II) (pH 3.0) under incubation temperature 30 oC for 24 h. (2) Effect of the initial Pd(II) concentration (pH 3.0) was studied from 100 to 300 mg/L with the biomass dosage of 1.2 g/L under incubation temperature 30 oC for 24 h. (3) Effect of the incubation temperature was studied from 20 to 60 oC within 60 min with the biomass dosage of 1.2 g/L and 210 mg/L Pd(II) (pH 3.0). (4) Effect of the initial pH value of Pd(II) solution was studied from 1.5 to 3.5 with biomass dosage of 1.2 g/L and 210 mg/L Pd(II) under incubation temperature 30 oC for 24 h. The concentration of Pd(II) was measured using AAS (Hitachi Z-2000 Zeeman, Japan). Experiments were performed in triplicate, and the mean relative errors of Pd (II) concentration were estimated to be less than 5%. The Pd(II) biosorption capacity of E. faecalis was calculated through Eq. (S.1) and the biosorption efficiency of Pd(II) in solution were calculated through Eq. (S.2) (Supplementary materials S1). Adsorption equilibrium is usually described by Langmuir model (Eq. (S.3)) and Freundlich model (Eq. (S.4)) to draw adsorption isotherms (Supplementary materials S2) [20]. The thermodynamic parameters for biosorption reaction can be determinate by fitting experiment data [21-23] (Supplementary materials S3). The transfer mechanism of Pd(II) can be explored by the pseudo-first order kinetics model (Eq. (S.8)), the pseudo-second order model (Eq. (S.9)), and Weber-Morris intraparticle diffusion model (Eq. (S.10)) to fit sorption rate of E. faecalis (Supplementary materials S4). 2.4. Characterization of functional groups for Pd(II)biorecovery 6
Infrared spectra of the E. faecalis cells before and after the Pd(II) biorecovery were recorded by FTIR (Bruker VERTEX 70, Germany) for identifying the change of the potential functional groups on the cell. The samples were firstly thoroughly dried in a vacuum freezing desiccator (4KBTXL-75, US), mixed with dry KBr powder (a sample:KBr ratio of about 1:20), then pressed into discs (0.2 mm thick) for FTIR examination. The FTIR spectra were recorded at a resolution of 4 cm−1 in the transmission mode 4000−400 cm−1. The binding energy of the elements on the cell surface during the Pd(II) biorecovery were obtained using XPS (PHI X-Tool, Japan). For XPS analysis, a PHI X-Tool instrument equipped with Alkα X-ray source operating at 15kV (51W) was used to analyze the chemical composition of the cell surface with an analysis area of 202 µm and the take-off angle of 45o. Binding energies were obtained in the wide energy survey scans over the range 1000−0 eV with pass energy of 140 eV. High-resolution spectra were recorded for Pd 3d, Cl 2p, C 1s, O 1s and N 1 at step size of 0.25 eV.
3. Results and discussion 3.1. Characterization of Pd-loaded cells XRD analysis (Fig. 1) indicated that the Pd-NPs loaded on E. faecalis cells had four characteristic diffraction peaks at 2θ = 40.1, 46.7, 68.1 and 82.1o, corresponding to the (111), (200), (220) and (311) facets of crystalline Pd particles. In contrast, the Pd(II) species loaded on cells in biosorption without adding sodium formate were amorphous state. TEM analysis (Fig. 2) was carried out for exploring the locations of Pd on E. faecalis cells. These TEM images showed that with the addition of sodium formate, the locations of Pd-NPs by 7
biorecovery were the same as the locations of Pd(II) by biosorption that were at the membrane and inside of the cell via intracellular uptake. Therefore, the biosorption promoted the transfer of Pd(II) from solution to cells, which determined the deposit locations of Pd-NPs, and with the addition of electron donor, the bioreduction promoted the transformation from Pd(II) species to Pd-NPs by biochemical reactions. Moreover, the formation process of Pd-NPs could be divided into two steps, namely the biosorption of Pd(II) and the bioreduction, nucleation and crystallization of the adsorbed Pd(II) in situ. 3.2. Transfer of Pd(II) in Biosorption 3.2.1. Effect factors on the biosorption of Pd(II) The critical factors for the biorecovery of Pd(II) included biomass dosage, initial Pd(II) concentration, incubation temperature, initial pH of Pd(II) solution as reported previously [6], which were further investigated in the biosorption process of Pd(II) by E. faecalis. The effect of biomass dosage on the Pd(II) biosorption by E. faecalis was presented in Fig. 3A, which indicated that the rising biomass dosage of E. faecalis cells, with larger surface area, increased the number of binding sites and improved the biosorption efficiency of Pd(II). However, when the biosorption efficiency had reached a high level, the increase rate of efficiency was slow, which might be owing to the mass ratio of adsorbent and adsorbate that determined the touch probability between each other. Therefore, it was uneconomic for the recovery of Pd(II) to add more biosorbent at over 95% of biosorption efficiency. Nevertheless, the biosorption efficiency decreased at higher initial Pd(II) concentrations, with higher residual Pd(II) concentrations, indicating the saturation of all binding sites and the establishment of biosorption equilibrium 8
(Fig. 3B). The effect of incubation temperature (20−60 °C) on the Pd(II) biosorption by E. faecalis within 60 min was shown in Fig. 3C, which indicated that 40 ℃ was the optimal incubation temperature for the biosorption of Pd(II). In addition, a high incubation temperature varying from 50 to 60 oC for more than 30−40 min would cause the decrease of metal biosorption capacity of the E. faecalis cells owing to the damage of cells [6], which might also be explained by the comparison between adsorption force and molecule thermal motion. Furthermore, the optimum initial pH for the Pd(II) biosorption by E. faecalis was found in the range of 3.0−3.5, and the final pH would decline (Fig. 3D). Moreover, the Gram-positive strains such as E. faecalis with the thick cell wall containing 90% peptidoglycan uaually presents a positive electrical property [24]. Therefore, the Pd(II) in solution were present in the form of PdCl42− or PdCl3− (negative charge), which could explain that there was no competition between the proton (H+) and Pd(II) for the same binding site on the surface of cells. It also proved that electrostatic interaction was a significant force to promote the transfer of Pd(II) in biosorption. 3.2.2. Biosorption isotherm To analyze the experimental data obtained from biosorption process, the isotherm models such as Langmuir and Freundlich were used (Supplementary materials S2). The result from these two models was shown in Fig. 4, and Langmuir model could describe the biosorption process better than Freundlich model. From the calculated parameters, the biosorbent prepared from E. faecalis biomass had excellent biosorption property with 189.361 mg/g of the maximum biosorption capacity of Pd(II). According to the theory assumption of Langmuir 9
model, the surface properties of E. faecalis was uniform, each biosorption site could adsorb a molecule of Pd(II) with the same adsorption heat, and there was no interacting force between Pd(II) species. 3.2.3. Biosorption thermodynamics Table 1 Thermodynamics parameters for Pd(II) biosorption by E. faecalis. ∆G (kJ/mol) 20 ℃ −2.822
25 ℃ −6.550
30 ℃ −10.279
35 ℃ −14.008
∆H (kJ/mol)
∆S (J/(mol∙K))
215.790
745.733
40 ℃ −17.736
The dependence of Pd(II) biosorption efficiency by E. faecalis cells on solution temperatures was investigated with thermodynamic model (Supplementary materials S3) as shown in Fig. 5. The temperature range was selected for the investigation of biosorption thermodynamics from 20−40 oC (293.15−313.15 K) eliminating the effect from the thermal damage for cell structures. The obtained thermodynamic data for biosorption of Pd(II) by using E. faecalis cells were calculated in Table 1. The data reflected the promotion to biosorption efficiency from the increase of temperature. All the change in Gibbs free energy (∆G) in table 1 were negative value, and with the increase of temperature, the data would decrease, which reflected the improvement of the driving force for biosorption reaction at high temperature. Under constant pressure condition, the biosorption reaction system exchanged heat with environment that could be calculated by the change in enthalpy (∆H). In our experiments. the value of ∆H was 215.790 kJ/mol higher than 62.7 kJ/mol, an empirical heat value demarcation 10
of chemical adsorption, which meant chemical reactions occurred in the biosorption process of Pd(II) onto E. faecalis cells. The positive value of the change in entropy (∆S, 745.733 J/(mol K)) indicated the increase of arbitrariness at the solid−liquid interface during the biosorption process. According to these parameters, the biosorption was spontaneous endothermic process in the temperature range of 20−40 oC. 3.2.4. Biosorption kinetics Table 2 Kinetics parameters for Pd(II) biosorption by E. faecalis. Pseudo-first order
Pseudo-second order
Intraparticle diffusion
K1 (1/min)
Qe (mg/g)
K2 (g/(mg∙min))
Qe (mg/g)
Kb (mg/g)
K3 (mg/(g∙min1/2))
0.0468
169.341
0.000601
174.520
151.085
0.552
The interaction process between Pd(II) and the biomass of E. faecalis could be evaluated through biosorption kinetics models (Supplementary materials S4) via the potential rate-controlling steps including material transport and physical-chemical reaction as shown in Fig. 6, and the calculated parameters were listed in Table 2. According to R2 value, the pseudo-second order model was perfect for fitting the biosorption process of Pd(II), which meant that the main rate-controlling step was the chemical reactions, and that the share and the transfer of electron for chelate formation of Pd(II) were the critical factors for the biosorption rate. Other kinetics models were also widely applied to describe various adsorbents, but it could not explain this biosorption process, especially Weber-Morris intraparticle diffusion model, which meant that the liquid film between the solution and the surface of live cell cannot be 11
ignored for metal ions to transfer into cells. Therefore, chemical adsorption for Pd(II) was the dominant comparing with physical adsorption, which was also supported by the value of ∆H from thermodynamics model. 3.3. Surface characteristic of E. faecalis during biorecovery of Pd(II) The functional groups in E. faecalis cells, during Pd(II) biosorption and bioreduction, were explored to reveal the substance provided by cell to act on Pd(II) and synthesize Pd-NPs through FTIR analysis shown in Fig. 7. Comparing FTIR spectra, the adsorption peak at 3384 cm−1 was intensified into 3428 cm−1 after biosorption and 3408 cm−1 after bioreduction, indicating that the chemical reaction between Pd(II) and H atoms of hydroxyl groups (−OH) from saccharides and amine groups (−NH) from proteins formed the complex, then to realease Pd [22]. The unchanged adsorption peaks at 2961 and 2930 cm−1 were determined as the –CH groups, and the peak at 1454 cm−1 was characteristic of –CH2 groups, all of which were common occurrence in peptidoglycan, teichoic acid, and phospholipids of cells [25, 26]. The peaks at 1654 cm−1 decreased to 1650 cm−1 assigned for amine groups (–CON−), and at 1548 cm−1 decreased to 1541 cm−1 assigned for amine groups (–CN−, –NH) from the peptidoglycan layer [25] in biosorption, and then both went back the original value in bioreduction, which proved that the ion-exchange occurred, namely that N and O atoms from these groups formed the complex with Pd(II) for biosorption to release H atoms, and these groups reappeared with the decrease of Pd(II) in bioreduction. In addition, the peaks at 1398 and 1312 cm−1, assigned for carboxylic group (–COOH) from amino-acid on cells, reappeared in bioreduction after vanishing in biosorption, which meant that Pd(II) formed complex with –C=O from the cell in 12
biosorption, and then was released in bioreduction. After biosorption and bioreduction of Pd(II) by E. faecalis, the peak at 1240 cm−1 corresponding to the phosphodiester band [27] was shifted into a lower energy at 1234 cm−1, indicating that the character of phosphate group (−P=O) was weakened [26]. And the peak at 1081 cm−1 was shifted into a lower frequency at 1070 cm−1, and the peak at 916 cm−1 disappeared, which indicated that the complex formation between Pd(II) with O atoms of the hydroxyl group (–OH). Therefore, carboxyl groups, hydroxyl groups and amine groups were the main participated functional groups in biorecovery by FTIR analysis. The XPS method was used to analyze the further change of substance on E. faecalis during Pd(II) biorecovery as shown in Fig. 8. Comparing the XPS spectra in Fig. 8A found that during Pd(II) biorecovery, the changes of C 1s, N 1s and O 1s core level were negligible in three XPS spectra, but the changes of Pd 3d and Cl 2p core level were significant. E. faecalis cells without Pd 3d peak were observed in Fig 8B that after the biosorption of Pd(II), the binding energies of Pd 3d peaks on Pd(II)-loaded cells were 336.7 and 341.9 eV, but after the addition of the electron donor to Pd(II)-loaded cells, these changed into 334.4 and 339.7 eV, which indicated the reduction of Pd(II) to Pd (0) at the lower binding energy. The peak of Cl 2p found in biosorption was disappeared in bioreduction confirmed that chemical bond between Pd(II) and chlorine ions (Cl−) has been broken (Fig. 8C). In addition, the core level spectra of C 1s, O 1s and N 1s were shown in Fig. 8 (D-F), which were similarly found as mentioned above before and after the Pd(II) bioreduction by E. faecalis cell. The peaks of C 1s spectrum at 283.82, 285.11 and 287.81 eV were assigned to chemical bond of carbon, nitrogen and hydroxyl such as (C−C, C−H), (C−O, C−N) and (C=O, O=C−O), respectively, which were commonly found on 13
peptidoglycan, amide, teichoic acid and phospholipids groups of cells. Three distinct peaks of O 1s at 503.01, 531.77 and 532.49 eV could be assigned to N－C=O, O=C (or O−C=O) and O−C−H (or C−O−C, C−O−C=O), respectively, which were from amide, carboxylate, hydroxide or acetal functional groups. The bonding energies of N 1s core level were centered at 398.74, 399.28 and 399.98 eV corresponding to chemical bonds of carbon, nitrogen and hydroxyl as C−N and N−H from amide functional groups. Therefore, the XPS results proved that the surface of E. faecalis was rich in various functional groups, but only part of these could participate the biorecovery of Pd(II). Moreover, the functional groups had a negligible change that was mainly leaded by carboxyl groups, hydroxyl groups and amine groups as the same as the FTIR results, but most of these chemical reactions between Pd(II) and functional groups had a little change of binding energies, which indicated that the ion exchange interaction was the dominant relative to other combination reaction with the generation of new covalent bonds. 3.4. Transform of Pd(II) in bioreduction According to the results from biosorption, the factors that effected the biosorption efficiency would also effect the biosynthesis of Pd-NPs as our previous research [6]. Therefore, the most significant factor, solution pH value, was further investigated as shown in Fig. 9. The pH value, related to the concentration of H+ and OH−, directly affected the success or failure for the reduction of Pd(II). The biosorption would release H+ into the solution (Fig. 9). Combined with isotherm, thermodynamics and kinetics models and the characterization by FTIR and XPS, it further proved the ion-exchange between palladium(II) with carboxyl groups, hydroxyl groups and amine groups on cells in biosorption. However, after 14
bioreduction, part of these functional groups reappeared as FTIR analysis. Without the addition of sodium formate (HCOONa), Pd(II) only can be adsorbed by E. faecalis, but cannot be reduced. Therefore, the main function for E. faecalis was to provide a wide and uniform place for the deposit and crystallization of Pd-NPs. The reduction with the increase of pH by the addition of sodium formate proved the hydrolysis of sodium formate, and without E. faecalis, the reduction also occurred with large Pd particles as our previous research. However, with the initial pH at 1.0, the pH value cannot increase to 8.0, and Pd(II) also cannot be reduced indicating the significance of the hydrolysis of sodium formate. Therefore, the bioreduction of Pd(II) was via the hydrolysis of sodium formate and then the oxidation of hydrolysate to deliver electron, and the link frame of E. faecalis cell was the dominant reasons for the nanoscale of Pd-NPs.
4. Conclusion In this study, we clarified the biorecovery mechanism for Pd(II) by E. faecalis as nanoparticles from acting force, participated substance and reaction process. The deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption. According to the change of pH value, typical biosorption models and the analyses of XRD, TEM, FTIR and XPS, the acting forces for biosorption were electrostatic interaction, complex formation of Pd(II) on the cell and intracellular uptake. The main participated functional groups in the biorecovery for Pd(II) were carboxyl groups, hydroxyl groups and amine groups. The bioreduction of adsorbed Pd(II) was via the hydrolysis and oxidation of sodium formate to deliver electron. Therefore, the biorecovery mechanism of Pd(II) can be divided into the 15
biosorption from solution to cells, and then the bioreduction and crystallization in situ by the electron donor and the link frame of E. faecalis.
Acknowledgements The authors would like to thank South China University of Technology for providing equipment to perform this research. The authors would like to thank National Natural Science Foundation of China (51178191), the Fundamental Research Funds for the Central Universities (2017PY012), Guangdong Provincial Science and Technology Project (2017A020216013), and Guangzhou Science and Technology Project (201604020055) for financial support.
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[19] S. Mohan, A. Gurtu, K.K. Dixit, A. Mehrotra, Enterococcus faecalis- an endodontic enigma, J. Dent. Sci. & Oral Rehabilitation Oct-Dec (2013) 09-11. [20] J.S. He, J.P. Chen, A comprehensive review on biosorption of heavy metals by algal biomass: Materials, performances, chemistry, and modeling simulation tools, Bioresour. Technol. 160 (2014) 67-78. [21] E. Mildan, M. Gülfen, Equilibrium, kinetics, and thermodynamics of Pd(II) adsorption onto poly(m-aminobenzoic acid) chelating polymer, J. Appl. Polym. Sci. (2015) 42533(42531-42510). [22] A. Sari, D. Mendil, M. Tuzen, M. Soylak, Biosorption of palladium(II) from aqueous solution by moss (Racomitrium lanuginosum) biomass: equilibrium, kinetic and thermodynamic studies, J. Hazard. Mater. 162 (2009) 874-879. [23] D. Park, Y.-S. Yun, J.M. Park, The past, present, and future trends of biosorption, Biotechnol. Bioproc. E. 15 (2010) 86-102. [24] M. Tariq, C. Bruijs, J. Kok, B.P. Krom, Link between culture zeta potential homogeneity and Ebp in Enterococcus faecalis, Appl. Environ. Microbiol. 78 (2012) 2282-2288. [25] R.M. Silverstein, F.X. Webster, D.J. Kiemle, Spectroscopic Identification of Organic Compound, John Wiley and Sons, United States of America, 2005. [26] W. Jiang, A. Saxena, B. Song, B.B. Ward, T.J. Beveridge, S.C.B. Myneni, Elucidation of Functional Groups on Gram-Positive and Gram-Negative bacterial surfaces using infrared, Langmuir 20 (2004) 11433-11442.
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[27] D. Naumann, Infrared Spectroscopy in Microbiology, in: R.A. Meyers (Ed.) Encyclopedia of Analytical Chemistry, John Wiley & Sons Ltd, Chichester, 2000, pp. 102–131.
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Fig. 1. XRD pattern of Pd-NPs-loaded cells and Pd(II)-loaded cells.
Fig. 2. TEM images of E. faecalis (A), the biosorption of Pd(II) at the membrane (B) and inside of the cell (C), and the biorecovery of Pd-NPs (D), at the membrane (E) and inside of the cell (F).
Fig. 3. The biosorption efficiency of Pd(II) by effects of biomass dosage (A), initial Pd(II) concentration (B), incubation temperature (C), and initial pH (D).
Fig. 4. The biosorption isotherm of Pd(II) by E. faecalis (contact time: 24 h; incubation temperature: 30 ℃; biomass dosage: 1.2 g/L; initial pH: 3.0).
Fig. 5. The biosorption thermodynamics of Pd(II) by E. faecalis (contact time: 24 h; biomass dosage: 1.2 g/L; initial pH: 3.0; initial Pd(II) concentration: 210 mg/L). Fig. 6. The biosorption kinetics of Pd(II) by E. faecalis (incubation temperature: 30 ℃; biomass dosage: 1.2 g/L; initial pH: 3.0; initial Pd(II) concentration: 210 mg/L).
Fig. 7. FTIR spectrum of the E. faecalis cells, the Pd(II)-loaded cells and Pd-NPs-loaded cells.
Fig. 8. XPS spectra of the E. faecalis cells before and after bioreduction (A), Pd 3d core level (B), Cl 2p core level (C), C 1s core level (D), O 1s core level (E) and N 1s core level (F). Fig. 9. The pH change of Pd(II) solution in biosorption, bio-reduction, and chem-reduction.
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Highlights 1 The deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption. 2 The biosorption was a spontaneous endothermic process fitted well by Langmuir isotherm and pseudo-second order kinetics. 3 The main functional groups for Pd(II) biorecovery were carboxyl groups, hydroxyl groups and amine groups. 4 The bioreduction was via the hydrolysis and oxidation of sodium formate to deliver electron. 5 The biorecovery of Pd(II) was via biosorption to cells and then bioreduction in situ.
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S1385-8947(17)31272-X http://dx.doi.org/10.1016/j.cej.2017.07.124 CEJ 17390
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
2 June 2017 19 July 2017 20 July 2017
Please cite this article as: J. Cui, N. Zhu, N. Kang, C. Ha, C. Shi, P. Wu, Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.07.124
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Biorecovery mechanism of palladium as nanoparticles by Enterococcus faecalis: From biosorption to bioreduction Jiaying Cuia, Nengwu Zhua,b,c,*, Naixin Kanga, Chitam Haa,d, Chaohong Shia, Pingxiao Wua,b,e a
School of Environment and Energy, South China University of Technology, Guangzhou
510006, PR China b
The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of
Education, Guangzhou 510006, PR China c
Guangdong Environmental Protection Key Laboratory of Solid Waste Treatment and
Recycling, Guangzhou 510006, PR China d
Resources and Environment Department, Vinh Long City, Vinh Long Town, Viet Nam
e
Guangdong Engineering and Technology Research Center for Environmental Nanomaterials,
Guangzhou 510006, PR China
* Corresponding author at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. Tel./fax: +86 20 3938 0522. E-mail address: [email protected] (N. Zhu).
1
Abstract In this study, the biorecovery mechanism of palladium ions (Pd(II)) as palladium nanoparticles by Enterococcus faecalis (E. faecalis), a typical Gram-positive bacterium, was explored from acting force, participated substance and reaction process. The XRD and TEM analysis showed that the deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption, and the process was invested by effect factors and theory models of isotherm, thermodynamics, and kinetics, which was a spontaneous endothermic process fitted well by Langmuir isotherm and pseudo-second order kinetics. In addition, the surface characteristic changes of E. faecalis during biorecovery of Pd(II) were compared by FTIR and XPS, and it found that carboxyl groups, hydroxyl groups and amine groups were the main participated functional groups in biorecovery. Furthermore, the contrary pH change between biosorption and bioreduction combined with above results proved that electrostatic interaction, complex formation of Pd(II) on the cell and intracellular uptake were the acting forces for biosorption, and the bioreduction of adsorbed Pd(II) was via the hydrolysis and oxidation of sodium formate to deliver electron. Therefore, the biorecovery mechanism of Pd(II) can be divided into the biosorption from solution to cells, and then the bioreduction and crystallization in situ by the electron donor and the link frame of E. faecalis. Keywords: Biorecovery mechanism; Biosorption; Bioreduction; Palladium nanoparticles; Enterococcus faecalis
2
1. Introduction The application of precious metals is increasing in various industries resulted in the research urgency for recycle [1]. Palladium (Pd) as a significant precious metal has been applied in many industries such as catalyst, chemical, electronic, and glass [2]. Moreover, the biorecovery methods of Pd using different microorganisms have been widely explored [3]. It is considered as an eco-friendly alternative to the conventional physical and chemical methods [4]. The biorecovery of Pd(II) from solution usually divided two main types, namely the biosorption and accumulation of Pd(II) [5] and the bioreduction and recycle as Pd-NPs [6]. Therefore, for the biorecovery system of Pd(II), especially as Pd-NPs loaded on cells, both the biosorption and the bioreduction could be related to biorecovery mechanism, but the existing researches seldom clarified the whole process. The biosorption of Pd(II) from solution aimed to transfer Pd(II) from solution to biomass, and adsorption amount and selective biorecovery are the core of research [7, 8], but the bioreduction and applicability of recovering Pd(II) is also limited. In addition, adsorption isotherm models (Langmuir and Freundlich models), thermodynamics model and kinetics models (pseudo-first order, pseudo-second order and Weber-Morris intraparticle diffusion models) usually explained the adsorption process and mechanism for traditional adsorbent [9-12]. For different biomass, the calculated adsorption parameters are different due to the various surface structure and functional groups, and some new biosorbent like live bacterium biomass is seldom studied for biosorption mechanism by these typical models [13]. Furthermore, the researches on biorecovery of Pd(II) as Pd-NPs mainly focused on the control 3
of environment conditions and the characterization of nanoparticles [14-16], and the deep insight into the biosorption process is incomplete [17, 18]. And the precise mechanisms of the Pd(II) bioreduction was not well understood due to the difference of biomass [4]. Enterococcus faecalis (E. faecalis), a model microorganism of gram-positive bacteria, appears common in various environment. Its genome has been decoded, and its cell membrane structure also has been analyzed which is more simply than gram-negative bacteria [19]. Therefore, the metal biorecovery property of E. faecalis could give abundant information for the study of gram-positive bacteria. In our previous study [6], the biorecovery of Pd by E. faecalis and the catalysis of Pd-NPs for chromate reduction have obtained satisfactory results, but the biorecovery process has not been explained in detail. In this study, the biorecovery mechanism was explored by the characterization of Pd-loaded cell, the transfer of Pd(II) in biosorption, surface characteristic of E. faecalis during biorecovery of Pd(II), and the transform of Pd(II) in bioreduction.
2. Materials and methods 2.1. Bacteria preparation E. faecalis (CCTCC M2012445) was cultured in Luria Broth culture and centrifuged to harvest cells as reported previously [6]. The cell samples were resuspended in the buffer solution of 20 mM 3-[N-morpholino] propanesulfonic acid (MOPS) (pH 7.0) to an optical density at 600 nm of 1.6 (corresponding to cell dry weight (CDW) of 1.2 g L-1), and then stored at 4 ℃ to keep the cellular morphology and activity for experiments within 48 h. 2.2. Biorecovery of Pd(II) as Pd-NPs 4
The solution of Pd(II) was prepared in distilled water (pH 7.0) from Na2PdCl4 salts (Aladdin Industrial Corporation, China), using sulfuric acid and sodium hydroxide for pH control. The cell suspension in MOPS buffer solution was centrifuged (10621 g, 5 min) as E. faecalis biomass. Biomass dosage of 1.2 g/L contacted to 10 mL Na2PdCl4 solution (pH 3.0) with final Pd(II) concentration of 210 mg/L, which was cultured at 40 ℃ in a 50 mL serum bottle. After 30 min, the Pd(II)-loaded cell samples were analysis as the result of biosorption. Then 2 M sodium formate (final concentration of 25 mM) as the electron donor was added to the Pd(II)-loaded cell solution in serum bottle for 48 h to form Pd-NPs, and the Pd-NPs-loaded cell samples were analysis as the result of bioreduction. The design of experiment parameters for the formation process of Pd-NPs has been reported at our recent research [6]. The pH value of solution was measured using a pH meter (INESALucy PHS-3C, China). Comparing with Pd(II)-loaded cells in biosorption, the crystallization of Pd-NPs after bioreduction of Pd(II) were confirmed by using XRD method (Bruker D8 ADVANCE X-ray diffractometer, USA). The deposit locations of Pd(II) in biosorption and Pd-NPs in bioreduction were compared by TEM equipment (HITACHI H-7650, Japan). The samples for XRD and TEM analysis were prepared and analyzing as reported previously [6]. 2.3. Biosorption experiments of Pd(II) The transfer of Pd(II) from solution to cell was described by biosorption. The factors affecting the Pd(II) biosorption efficiency without the addition of electron donor were studied including:
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(1) Effect of the biomass dosage was studied from 0.6 to 3.6 g/L with 210 mg/L Pd(II) (pH 3.0) under incubation temperature 30 oC for 24 h. (2) Effect of the initial Pd(II) concentration (pH 3.0) was studied from 100 to 300 mg/L with the biomass dosage of 1.2 g/L under incubation temperature 30 oC for 24 h. (3) Effect of the incubation temperature was studied from 20 to 60 oC within 60 min with the biomass dosage of 1.2 g/L and 210 mg/L Pd(II) (pH 3.0). (4) Effect of the initial pH value of Pd(II) solution was studied from 1.5 to 3.5 with biomass dosage of 1.2 g/L and 210 mg/L Pd(II) under incubation temperature 30 oC for 24 h. The concentration of Pd(II) was measured using AAS (Hitachi Z-2000 Zeeman, Japan). Experiments were performed in triplicate, and the mean relative errors of Pd (II) concentration were estimated to be less than 5%. The Pd(II) biosorption capacity of E. faecalis was calculated through Eq. (S.1) and the biosorption efficiency of Pd(II) in solution were calculated through Eq. (S.2) (Supplementary materials S1). Adsorption equilibrium is usually described by Langmuir model (Eq. (S.3)) and Freundlich model (Eq. (S.4)) to draw adsorption isotherms (Supplementary materials S2) [20]. The thermodynamic parameters for biosorption reaction can be determinate by fitting experiment data [21-23] (Supplementary materials S3). The transfer mechanism of Pd(II) can be explored by the pseudo-first order kinetics model (Eq. (S.8)), the pseudo-second order model (Eq. (S.9)), and Weber-Morris intraparticle diffusion model (Eq. (S.10)) to fit sorption rate of E. faecalis (Supplementary materials S4). 2.4. Characterization of functional groups for Pd(II)biorecovery 6
Infrared spectra of the E. faecalis cells before and after the Pd(II) biorecovery were recorded by FTIR (Bruker VERTEX 70, Germany) for identifying the change of the potential functional groups on the cell. The samples were firstly thoroughly dried in a vacuum freezing desiccator (4KBTXL-75, US), mixed with dry KBr powder (a sample:KBr ratio of about 1:20), then pressed into discs (0.2 mm thick) for FTIR examination. The FTIR spectra were recorded at a resolution of 4 cm−1 in the transmission mode 4000−400 cm−1. The binding energy of the elements on the cell surface during the Pd(II) biorecovery were obtained using XPS (PHI X-Tool, Japan). For XPS analysis, a PHI X-Tool instrument equipped with Alkα X-ray source operating at 15kV (51W) was used to analyze the chemical composition of the cell surface with an analysis area of 202 µm and the take-off angle of 45o. Binding energies were obtained in the wide energy survey scans over the range 1000−0 eV with pass energy of 140 eV. High-resolution spectra were recorded for Pd 3d, Cl 2p, C 1s, O 1s and N 1 at step size of 0.25 eV.
3. Results and discussion 3.1. Characterization of Pd-loaded cells XRD analysis (Fig. 1) indicated that the Pd-NPs loaded on E. faecalis cells had four characteristic diffraction peaks at 2θ = 40.1, 46.7, 68.1 and 82.1o, corresponding to the (111), (200), (220) and (311) facets of crystalline Pd particles. In contrast, the Pd(II) species loaded on cells in biosorption without adding sodium formate were amorphous state. TEM analysis (Fig. 2) was carried out for exploring the locations of Pd on E. faecalis cells. These TEM images showed that with the addition of sodium formate, the locations of Pd-NPs by 7
biorecovery were the same as the locations of Pd(II) by biosorption that were at the membrane and inside of the cell via intracellular uptake. Therefore, the biosorption promoted the transfer of Pd(II) from solution to cells, which determined the deposit locations of Pd-NPs, and with the addition of electron donor, the bioreduction promoted the transformation from Pd(II) species to Pd-NPs by biochemical reactions. Moreover, the formation process of Pd-NPs could be divided into two steps, namely the biosorption of Pd(II) and the bioreduction, nucleation and crystallization of the adsorbed Pd(II) in situ. 3.2. Transfer of Pd(II) in Biosorption 3.2.1. Effect factors on the biosorption of Pd(II) The critical factors for the biorecovery of Pd(II) included biomass dosage, initial Pd(II) concentration, incubation temperature, initial pH of Pd(II) solution as reported previously [6], which were further investigated in the biosorption process of Pd(II) by E. faecalis. The effect of biomass dosage on the Pd(II) biosorption by E. faecalis was presented in Fig. 3A, which indicated that the rising biomass dosage of E. faecalis cells, with larger surface area, increased the number of binding sites and improved the biosorption efficiency of Pd(II). However, when the biosorption efficiency had reached a high level, the increase rate of efficiency was slow, which might be owing to the mass ratio of adsorbent and adsorbate that determined the touch probability between each other. Therefore, it was uneconomic for the recovery of Pd(II) to add more biosorbent at over 95% of biosorption efficiency. Nevertheless, the biosorption efficiency decreased at higher initial Pd(II) concentrations, with higher residual Pd(II) concentrations, indicating the saturation of all binding sites and the establishment of biosorption equilibrium 8
(Fig. 3B). The effect of incubation temperature (20−60 °C) on the Pd(II) biosorption by E. faecalis within 60 min was shown in Fig. 3C, which indicated that 40 ℃ was the optimal incubation temperature for the biosorption of Pd(II). In addition, a high incubation temperature varying from 50 to 60 oC for more than 30−40 min would cause the decrease of metal biosorption capacity of the E. faecalis cells owing to the damage of cells [6], which might also be explained by the comparison between adsorption force and molecule thermal motion. Furthermore, the optimum initial pH for the Pd(II) biosorption by E. faecalis was found in the range of 3.0−3.5, and the final pH would decline (Fig. 3D). Moreover, the Gram-positive strains such as E. faecalis with the thick cell wall containing 90% peptidoglycan uaually presents a positive electrical property [24]. Therefore, the Pd(II) in solution were present in the form of PdCl42− or PdCl3− (negative charge), which could explain that there was no competition between the proton (H+) and Pd(II) for the same binding site on the surface of cells. It also proved that electrostatic interaction was a significant force to promote the transfer of Pd(II) in biosorption. 3.2.2. Biosorption isotherm To analyze the experimental data obtained from biosorption process, the isotherm models such as Langmuir and Freundlich were used (Supplementary materials S2). The result from these two models was shown in Fig. 4, and Langmuir model could describe the biosorption process better than Freundlich model. From the calculated parameters, the biosorbent prepared from E. faecalis biomass had excellent biosorption property with 189.361 mg/g of the maximum biosorption capacity of Pd(II). According to the theory assumption of Langmuir 9
model, the surface properties of E. faecalis was uniform, each biosorption site could adsorb a molecule of Pd(II) with the same adsorption heat, and there was no interacting force between Pd(II) species. 3.2.3. Biosorption thermodynamics Table 1 Thermodynamics parameters for Pd(II) biosorption by E. faecalis. ∆G (kJ/mol) 20 ℃ −2.822
25 ℃ −6.550
30 ℃ −10.279
35 ℃ −14.008
∆H (kJ/mol)
∆S (J/(mol∙K))
215.790
745.733
40 ℃ −17.736
The dependence of Pd(II) biosorption efficiency by E. faecalis cells on solution temperatures was investigated with thermodynamic model (Supplementary materials S3) as shown in Fig. 5. The temperature range was selected for the investigation of biosorption thermodynamics from 20−40 oC (293.15−313.15 K) eliminating the effect from the thermal damage for cell structures. The obtained thermodynamic data for biosorption of Pd(II) by using E. faecalis cells were calculated in Table 1. The data reflected the promotion to biosorption efficiency from the increase of temperature. All the change in Gibbs free energy (∆G) in table 1 were negative value, and with the increase of temperature, the data would decrease, which reflected the improvement of the driving force for biosorption reaction at high temperature. Under constant pressure condition, the biosorption reaction system exchanged heat with environment that could be calculated by the change in enthalpy (∆H). In our experiments. the value of ∆H was 215.790 kJ/mol higher than 62.7 kJ/mol, an empirical heat value demarcation 10
of chemical adsorption, which meant chemical reactions occurred in the biosorption process of Pd(II) onto E. faecalis cells. The positive value of the change in entropy (∆S, 745.733 J/(mol K)) indicated the increase of arbitrariness at the solid−liquid interface during the biosorption process. According to these parameters, the biosorption was spontaneous endothermic process in the temperature range of 20−40 oC. 3.2.4. Biosorption kinetics Table 2 Kinetics parameters for Pd(II) biosorption by E. faecalis. Pseudo-first order
Pseudo-second order
Intraparticle diffusion
K1 (1/min)
Qe (mg/g)
K2 (g/(mg∙min))
Qe (mg/g)
Kb (mg/g)
K3 (mg/(g∙min1/2))
0.0468
169.341
0.000601
174.520
151.085
0.552
The interaction process between Pd(II) and the biomass of E. faecalis could be evaluated through biosorption kinetics models (Supplementary materials S4) via the potential rate-controlling steps including material transport and physical-chemical reaction as shown in Fig. 6, and the calculated parameters were listed in Table 2. According to R2 value, the pseudo-second order model was perfect for fitting the biosorption process of Pd(II), which meant that the main rate-controlling step was the chemical reactions, and that the share and the transfer of electron for chelate formation of Pd(II) were the critical factors for the biosorption rate. Other kinetics models were also widely applied to describe various adsorbents, but it could not explain this biosorption process, especially Weber-Morris intraparticle diffusion model, which meant that the liquid film between the solution and the surface of live cell cannot be 11
ignored for metal ions to transfer into cells. Therefore, chemical adsorption for Pd(II) was the dominant comparing with physical adsorption, which was also supported by the value of ∆H from thermodynamics model. 3.3. Surface characteristic of E. faecalis during biorecovery of Pd(II) The functional groups in E. faecalis cells, during Pd(II) biosorption and bioreduction, were explored to reveal the substance provided by cell to act on Pd(II) and synthesize Pd-NPs through FTIR analysis shown in Fig. 7. Comparing FTIR spectra, the adsorption peak at 3384 cm−1 was intensified into 3428 cm−1 after biosorption and 3408 cm−1 after bioreduction, indicating that the chemical reaction between Pd(II) and H atoms of hydroxyl groups (−OH) from saccharides and amine groups (−NH) from proteins formed the complex, then to realease Pd [22]. The unchanged adsorption peaks at 2961 and 2930 cm−1 were determined as the –CH groups, and the peak at 1454 cm−1 was characteristic of –CH2 groups, all of which were common occurrence in peptidoglycan, teichoic acid, and phospholipids of cells [25, 26]. The peaks at 1654 cm−1 decreased to 1650 cm−1 assigned for amine groups (–CON−), and at 1548 cm−1 decreased to 1541 cm−1 assigned for amine groups (–CN−, –NH) from the peptidoglycan layer [25] in biosorption, and then both went back the original value in bioreduction, which proved that the ion-exchange occurred, namely that N and O atoms from these groups formed the complex with Pd(II) for biosorption to release H atoms, and these groups reappeared with the decrease of Pd(II) in bioreduction. In addition, the peaks at 1398 and 1312 cm−1, assigned for carboxylic group (–COOH) from amino-acid on cells, reappeared in bioreduction after vanishing in biosorption, which meant that Pd(II) formed complex with –C=O from the cell in 12
biosorption, and then was released in bioreduction. After biosorption and bioreduction of Pd(II) by E. faecalis, the peak at 1240 cm−1 corresponding to the phosphodiester band [27] was shifted into a lower energy at 1234 cm−1, indicating that the character of phosphate group (−P=O) was weakened [26]. And the peak at 1081 cm−1 was shifted into a lower frequency at 1070 cm−1, and the peak at 916 cm−1 disappeared, which indicated that the complex formation between Pd(II) with O atoms of the hydroxyl group (–OH). Therefore, carboxyl groups, hydroxyl groups and amine groups were the main participated functional groups in biorecovery by FTIR analysis. The XPS method was used to analyze the further change of substance on E. faecalis during Pd(II) biorecovery as shown in Fig. 8. Comparing the XPS spectra in Fig. 8A found that during Pd(II) biorecovery, the changes of C 1s, N 1s and O 1s core level were negligible in three XPS spectra, but the changes of Pd 3d and Cl 2p core level were significant. E. faecalis cells without Pd 3d peak were observed in Fig 8B that after the biosorption of Pd(II), the binding energies of Pd 3d peaks on Pd(II)-loaded cells were 336.7 and 341.9 eV, but after the addition of the electron donor to Pd(II)-loaded cells, these changed into 334.4 and 339.7 eV, which indicated the reduction of Pd(II) to Pd (0) at the lower binding energy. The peak of Cl 2p found in biosorption was disappeared in bioreduction confirmed that chemical bond between Pd(II) and chlorine ions (Cl−) has been broken (Fig. 8C). In addition, the core level spectra of C 1s, O 1s and N 1s were shown in Fig. 8 (D-F), which were similarly found as mentioned above before and after the Pd(II) bioreduction by E. faecalis cell. The peaks of C 1s spectrum at 283.82, 285.11 and 287.81 eV were assigned to chemical bond of carbon, nitrogen and hydroxyl such as (C−C, C−H), (C−O, C−N) and (C=O, O=C−O), respectively, which were commonly found on 13
peptidoglycan, amide, teichoic acid and phospholipids groups of cells. Three distinct peaks of O 1s at 503.01, 531.77 and 532.49 eV could be assigned to N－C=O, O=C (or O−C=O) and O−C−H (or C−O−C, C−O−C=O), respectively, which were from amide, carboxylate, hydroxide or acetal functional groups. The bonding energies of N 1s core level were centered at 398.74, 399.28 and 399.98 eV corresponding to chemical bonds of carbon, nitrogen and hydroxyl as C−N and N−H from amide functional groups. Therefore, the XPS results proved that the surface of E. faecalis was rich in various functional groups, but only part of these could participate the biorecovery of Pd(II). Moreover, the functional groups had a negligible change that was mainly leaded by carboxyl groups, hydroxyl groups and amine groups as the same as the FTIR results, but most of these chemical reactions between Pd(II) and functional groups had a little change of binding energies, which indicated that the ion exchange interaction was the dominant relative to other combination reaction with the generation of new covalent bonds. 3.4. Transform of Pd(II) in bioreduction According to the results from biosorption, the factors that effected the biosorption efficiency would also effect the biosynthesis of Pd-NPs as our previous research [6]. Therefore, the most significant factor, solution pH value, was further investigated as shown in Fig. 9. The pH value, related to the concentration of H+ and OH−, directly affected the success or failure for the reduction of Pd(II). The biosorption would release H+ into the solution (Fig. 9). Combined with isotherm, thermodynamics and kinetics models and the characterization by FTIR and XPS, it further proved the ion-exchange between palladium(II) with carboxyl groups, hydroxyl groups and amine groups on cells in biosorption. However, after 14
bioreduction, part of these functional groups reappeared as FTIR analysis. Without the addition of sodium formate (HCOONa), Pd(II) only can be adsorbed by E. faecalis, but cannot be reduced. Therefore, the main function for E. faecalis was to provide a wide and uniform place for the deposit and crystallization of Pd-NPs. The reduction with the increase of pH by the addition of sodium formate proved the hydrolysis of sodium formate, and without E. faecalis, the reduction also occurred with large Pd particles as our previous research. However, with the initial pH at 1.0, the pH value cannot increase to 8.0, and Pd(II) also cannot be reduced indicating the significance of the hydrolysis of sodium formate. Therefore, the bioreduction of Pd(II) was via the hydrolysis of sodium formate and then the oxidation of hydrolysate to deliver electron, and the link frame of E. faecalis cell was the dominant reasons for the nanoscale of Pd-NPs.
4. Conclusion In this study, we clarified the biorecovery mechanism for Pd(II) by E. faecalis as nanoparticles from acting force, participated substance and reaction process. The deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption. According to the change of pH value, typical biosorption models and the analyses of XRD, TEM, FTIR and XPS, the acting forces for biosorption were electrostatic interaction, complex formation of Pd(II) on the cell and intracellular uptake. The main participated functional groups in the biorecovery for Pd(II) were carboxyl groups, hydroxyl groups and amine groups. The bioreduction of adsorbed Pd(II) was via the hydrolysis and oxidation of sodium formate to deliver electron. Therefore, the biorecovery mechanism of Pd(II) can be divided into the 15
biosorption from solution to cells, and then the bioreduction and crystallization in situ by the electron donor and the link frame of E. faecalis.
Acknowledgements The authors would like to thank South China University of Technology for providing equipment to perform this research. The authors would like to thank National Natural Science Foundation of China (51178191), the Fundamental Research Funds for the Central Universities (2017PY012), Guangdong Provincial Science and Technology Project (2017A020216013), and Guangzhou Science and Technology Project (201604020055) for financial support.
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Fig. 1. XRD pattern of Pd-NPs-loaded cells and Pd(II)-loaded cells.
Fig. 2. TEM images of E. faecalis (A), the biosorption of Pd(II) at the membrane (B) and inside of the cell (C), and the biorecovery of Pd-NPs (D), at the membrane (E) and inside of the cell (F).
Fig. 3. The biosorption efficiency of Pd(II) by effects of biomass dosage (A), initial Pd(II) concentration (B), incubation temperature (C), and initial pH (D).
Fig. 4. The biosorption isotherm of Pd(II) by E. faecalis (contact time: 24 h; incubation temperature: 30 ℃; biomass dosage: 1.2 g/L; initial pH: 3.0).
Fig. 5. The biosorption thermodynamics of Pd(II) by E. faecalis (contact time: 24 h; biomass dosage: 1.2 g/L; initial pH: 3.0; initial Pd(II) concentration: 210 mg/L). Fig. 6. The biosorption kinetics of Pd(II) by E. faecalis (incubation temperature: 30 ℃; biomass dosage: 1.2 g/L; initial pH: 3.0; initial Pd(II) concentration: 210 mg/L).
Fig. 7. FTIR spectrum of the E. faecalis cells, the Pd(II)-loaded cells and Pd-NPs-loaded cells.
Fig. 8. XPS spectra of the E. faecalis cells before and after bioreduction (A), Pd 3d core level (B), Cl 2p core level (C), C 1s core level (D), O 1s core level (E) and N 1s core level (F). Fig. 9. The pH change of Pd(II) solution in biosorption, bio-reduction, and chem-reduction.
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Highlights 1 The deposit locations of Pd-NPs depended on the transfer of Pd(II) in biosorption. 2 The biosorption was a spontaneous endothermic process fitted well by Langmuir isotherm and pseudo-second order kinetics. 3 The main functional groups for Pd(II) biorecovery were carboxyl groups, hydroxyl groups and amine groups. 4 The bioreduction was via the hydrolysis and oxidation of sodium formate to deliver electron. 5 The biorecovery of Pd(II) was via biosorption to cells and then bioreduction in situ.
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