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Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation
Accepted Manuscript Protein corona between nanoparticles and bacterial proteins in activated sludge: characterization and effect on nanoparticle aggregation Peng Zhang, Xiao-Yan Xu, You-Peng Chen, Meng-Qian Xiao, Bo Feng, KaiXun Tian, Yue-Hui Chen, You-Zhi Dai PII: DOI: Reference:
S0960-8524(17)31959-4 https://doi.org/10.1016/j.biortech.2017.11.008 BITE 19158
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
13 September 2017 31 October 2017 4 November 2017
Please cite this article as: Zhang, P., Xu, X-Y., Chen, Y-P., Xiao, M-Q., Feng, B., Tian, K-X., Chen, Y-H., Dai, YZ., Protein corona between nanoparticles and bacterial proteins in activated sludge: characterization and effect on nanoparticle aggregation, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.11.008
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Protein corona between nanoparticles and bacterial proteins in activated sludge: characterization and effect on nanoparticle aggregation
Peng Zhang1,2, Xiao-Yan Xu1, You-Peng Chen2, Meng-Qian Xiao1, Bo Feng1, Kai-Xun Tian1, Yue-Hui Chen1, You-Zhi Dai1,*
1
College of Environment and Resources, Xiangtan University, Xiangtan, 411105,
China 2
Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environments of
MOE, Chongqing University, Chongqing, 400045, China
*Corresponding Author: Prof. You-Zhi Dai; Tel: +86-731-58292231, Fax: +86-731-58292231 Email: [email protected]
1
Abstract: In this work, the protein coronas of activated sludge proteins on TiO2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation.
Keywords: Protein corona, Aggregation, Nanoparticles, Shotgun proteomics, Proteins with high affinity to nanoparticles
2
1. Introduction The increasing discharge of metal and metal oxide nanoparticles (NPs) into water environments leads to water pollution and potentially threats to aquatic ecological communities (Skjolding et al., 2016). NPs undergo transference and transition in natural water environment, and enter into biological wastewater treatment system (Lin et al., 2016). The diffusion and migration of NPs within the microbial aggregates, can penetrate cell membrane and result in cytotoxicity, which reduce the removals of COD, nitrogen, and phosphorus (Wang et al., 2016; Zheng et al., 2011). Soluble proteins, humic substances, and polysaccharides of microbial extracellular polymeric substances (EPS) presented in wastewater treatment system can absorb onto the surfaces of most NPs, and alter the stability of NPs (Lin et al., 2016). Proteins are the most primary component in extracellular matrix of activated sludge flocs and had high binding ability to NPs. (Mahendran et al., 2012; Park et al., 2008; Sheng et al., 2016). The content of extracellular protein are substantially increased after microbes exposing on TNPs and ZNPs (He et al., 2017; Li et al., 2015). Furthermore, proteomics researches demonstrated that proteins in soluble EPS and bound EPS were mainly (more than 60%) originated from the release of cell lysis (Wu et al., 2017; Zhang et al., 2015). A better understanding of the roles of microbial proteins in activated sludge in the aggregation and migration of NPs is therefore critical. The adsorbed proteins form protein corona on the surface of NPs, which change the surface properties of NPs, e.g., zeta potential and hydrophobicity (Jain et al., 2015). 3
The composition of protein corona can control the interaction of NPs with outer membrane receptors for specific cell uptake (Gao et al., 2017). The adsorption of proteins onto NPs is a dynamic process, which relies on the affinity (Fleischer and Payne, 2014). The proteins with higher affinity to NPs can displace those with lower affinity to NPs, and irreversibly bind on the surface of NPs (Saptarshi et al., 2013). Therefore, these proteins absorbed on NPs are the key components that affect the aggregation, transference, and intracellular uptake of NPs. Previous investigations on the effect of proteins on NPs aggregation focused on the model proteins, such as cytochrome and albumin (Levak et al., 2017; Sheng et al., 2016a; Sheng et al., 2016b). While hundreds of proteins with various isoelectric points, molecular weights, and structures presented in activated sludge have different effects to NPs (Herbst et al., 2016). It is indispensable for studies the function of mixed proteins from activated sludge in the aggregation and fate of NPs. In activated sludge, EPS are the major component, which exhibit a large three-dimensional network structure at the microscopic scale (Sheng et al., 2010). The diffusion of NPs into the microbial aggregates is dependent upon the surface properties of both the NPs and the aggregates. NPs interact with the proteins in microbial aggregates when they migrate in the three-dimensional network structure. The proteins with various functions presented in activated sludge have different affinities to NPs, among which the proteins with higher affinity to NPs can immobilize the NPs and limit the NPs dispersal (Herbst et al., 2016; Moreau et al., 2007). Therefore, identifying these proteins with higher affinity to NPs, and analyzing 4
of their functions and located regions could be helpful for understanding the migration of NPs in activated sludge flocs. Here, the total proteins were extracted from the microbes in activated sludge. The protein coronas of the proteins on TiO2 NPs (TNPs) and ZnO NPs (ZNPs) were observed using TEM, and the binding mechanism of the proteins to TNPs and ZNPs was investigated. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and the effect of these proteins on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of the proteins on the aggregations of TNPs and ZNPs were evaluated.
2. Materials and Methods 2.1. NPs TNPs (99.8%, 25 nm, anatase type) was purchased from Aladdin, Shanghai Jingchun Biochemistry Technology Co., China. ZNPs (99.8%, 30 ± 10 nm) was purchased from Micxy Chemical Co., Chengdu, China. The TNPs and ZNPs powders were dispersed in deionized water, and the suspension was treated by ultrasound for approximately 30 min before further test.
2.2. Microbial culture and protein extraction Microbes were derived from the supernatant of a municipal wastewater treatment plant. One mL supernatant was incubated in 250 mL of LB-Lennox medium in 500 mL flasks (approximately 40 h, 30 °C, 120 rpm) (Guo et al., 2015). The bacterial 5
suspension was centrifuged at 5000 g for 20 min at 4 °C to remove growth medium. The cell pellet was washed three times with deionized water followed by a second centrifugation. The bacterial proteins were extracted using total protein extraction kit (Jiancheng Bioengineering Institute, Nanjing, China). Ten microliters phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of 100 mM of PMSF were added in per liter of cold lysis buffer, and mixed at 4 °C. The mixture was added to cell pellet and mildly shocked for 15 min at 4 °C. After centrifugation at 12000 rpm for 15 min at 4 °C, the supernatant was collected and quantified using total protein assay kit (BCA method, Jiancheng Bioengineering Institute, Nanjing, China). The required protein solution was separately stored at –70 °C before subsequent research.
2.3. FT-IR TNPs and ZNPs mixed with bacterial proteins solution for 3 h followed by centrifugation at 4000 g for 10 min and wash three times with deionized water to remove unbound protein on NPs surfaces. Both the sediments and protein were freeze-dried at –50 °C, and the powder was mixed with IR grade KBr powders to prepare the pellets. The FT-IR of all the samples were obtained by an FT-IR spectrophotometer (Nicolet 380, USA) within the scanning range 4000–400 cm−1. The pure TNPs and ZNPs powders were directly prepared the pellets to test.
2.4. Protein adsorption to NPs 6
Adsorption isotherms of the proteins on TNPs and ZNPs were measured in solution at pH approximately 7.0. The initial concentrations of NPs were prepared to 400 mg/L. The protein solution was prepared with the concentrations of 6.7, 13.3, 26.5, 53, 106, and 212 mg/L. One mL of NPs suspension was mixed completely with equal volume of each protein solution. And thus both the concentrations of the NPs and proteins were reduced by half. The samples were incubated in a shaker operating at 150 rpm and 25 ºC for 3 h to reach adsorption equilibrium, followed by centrifugation for 10 minutes at 4000 g. Protein concentrations in supernatants were determined using total protein assay kit.
2.5. Identification of proteins in the corona 2.5.1. Protein corona isolation The proteins were incubated with 3 mg of ZNPs or TNPs for 3 h at 4 °C (Cai et al., 2013). After centrifugation at 4000 g for 10 min at 4 °C to remove unbound proteins, the pellets were thoroughly washed with deionized water. The bound proteins were eluted by two centrifugations at 15000 g for 1 h at 4 °C (Ali et al., 2016). The supernatant was collected for proteomics analysis.
2.5.2. Digestion Protein digestion was conducted according to a corrected method described by Wisniewski et al. (2009). In general, approximately 30 μg of the protein sample was dissolved in 30 μL of SDT buffer (4% sodium dodecyl sulfate, 100 mM dithiothreitol, 7
150 mM Tris-HCl and pH 8.0) and kept boiling water bath for 5min. Then 200 μL of UA buffer (8 M urea, 150 mM Tris-HCl and pH 8.0) was added and mixed. The mixture was transferred to an ultrafiltration device (30 kD) for centrifugation, and the supernatant was removed. Then 100 μL of 50 mM iodoacetamide in UA buffer was added to sample pellet and shocked for 1 min. The samples were incubated for 30 min in darkness at room temperature. One hundred μL of UA buffer was added and centrifuged, and the procedure was repeated twice. Then 100 μL of 25 mM of NH4HCO3 was added and centrifuged, repetition twice. The protein suspension with 2 μg of trypsin in 40 μL of 25 mM NH4HCO3 incubated overnight at 37 °C followed by centrifugation. At last, 40 μL of 25 mM of NH4HCO3 was added, and the mixture was through centrifugation and acidification. The resulting peptides were collected for subsequently analysis.
2.5.3. Mass spectrometry (MS) Samples were loaded on a Thermo scientific EASY column (2 cm × 100 mm, 5 μm-C18, USA), and then separated on a Thermo scientific EASY column (75 μm × 100 mm, 3 μm-C18, USA). The mobile phases were prepared as follows: A (0.1% formic acid in HPLC-grade water) and B (0.1% formic acid in acetonitrile). Twenty μg of tryptic peptide mixtures was separated at a flow rate of 2 μL/min with a linear gradient (4–50% solution B, 0–50 min; 50–100% solution B, 50–54 min; 100% solution B, 54–60 min) in the columns. The eluted peptides were detected using a MS (Q exactive, Thermo Scientific, USA) equipped with a micro-spray interface. The 8
MS/MS spectra were set so that one full scan mass spectrum (m/z 300-1800) was followed by ten MS/MS events.
2.5.4. Data analysis MS/MS spectra were automatically searched against the SwissProt database using the Mascot 2.2 (Thermo Electron, San Jose, USA), and the protein identification results were obtained. Classifications of identified proteins were carried out using gene ontology annotation (GOA; http://www.uniprot.org/uniprot/) according to the protein accession numbers.
2.6. Aggregation and settlement assay The UV-vis spectra of NPs were scanned by Cary 60 (Agilent, USA), and the maximum absorption bands of TNPs and ZNPs were 349 nm and 361 nm, respectively. Then the effect of proteins on the aggregation and settlement of NPs was evaluated. One milliliter of 70, 106, 212 mg/L of proteins were mixed completely with equal volume of TNPs (200mg/L) and ZNPs (400 mg/L) and transferred to quartz colorimetric cuvette. Thus both the concentrations of the NPs and proteins were reduced by half. As the time elapsed, the NPs aggregated and settled to the bottom of the colorimetric cuvette. Therefore, the absorbance change was used to indirectly characterize the NPs aggregation. The optical density (OD) was determined after 5, 10, 20, 40, 80, 120, 160, 220, 240, and 320 min. The aggregation percentage (At) of NPs was calculated using Eq. (1). 9
–
(1)
The kinetic of particles aggregation and settlement can be described by pseudo first-order equation (Eq. (2)) (Brunelli et al., 2013; Quik et al., 2012; Zhang et al., 2014). (2) Where At is the aggregation percentage at a certain time (%), Ae is the aggregation percentage at equilibrium, and k1 is the pseudo first-order rate constant (min−1).
2.7. Atomic absorption spectra and TEM The dissolution of ZNPs was researched, and Zn ion was determined by atomic absorption spectrometer (AA-6300C, Shimazu, Japan). TNPs and ZNPs dispersed in deionized water and in protein solution were collected and dropped onto carbon-coated copper grids for TEM analysis (JEM-2100, JEOL, Japan).
3. Results and Discussion 3.1. Formation of protein corona 3.1.1. TEM characterization The images and morphologies of the proteins, and the NPs before and after protein adsorption were characterized by TEM. The protein images exhibited the spherical shape with different diameters. The results demonstrated that the proteins shape was reconstruction after adsorption on NPs. The absorbed proteins presented a variety of shapes on TNPs. Both the thin film-shape with the thickness of 2 nm and the 10
aggregated-shape proteins were found on TNP surfaces, whereas the ZNP surfaces were only observed the aggregated-shape proteins.
3.1.2. FT-IR characterization FT-IR spectra was used to reveal the binding of the proteins on NPs. The IR bands were assigned according to the existing literatures (Comte et al., 2006; Liu et al., 2010; Maquelin et al., 2002). The broad band around 3431 cm–1 was assigned to the stretching vibrations of O–H and N–H, whereas the bands at 2931 cm–1 and 2870 cm–1 were attributed to the C–H stretching vibrations. The C=O stretching vibration of amide I exhibited at 1631 cm–1, whereas N–H deformation vibrations of amide II were observed at 1563 cm–1 and 1512 cm–1. The band near 1384 cm–1 was assigned to C=O symmetric stretching vibration of carboxyl group. The C–N stretching vibrations of amide III with broad band was found at 1247 cm–1, and the band also could be related to P=O asymmetric stretching vibration of >PO2– phosphodiesters. The broad bands around 1105 cm–1 and 1050 cm–1 were attributed to ring vibrations of P=O, C–O–C, C–OH, and C–O–P stretching. The peak at 943 cm–1 was observed due to O–P–O asymmetric stretching vibration, whereas the weak peak presents at 831 cm–1 owing to C–C and C–OH stretching vibrations. The results revealed that the protein sample contained nucleoproteins and glycoproteins. The FT-IR spectra showed the change in TNPs before and after absorbing proteins. Before proteins adsorption, the bands of TiO2 nanoparticles were mainly found at 3431 cm–1 (O–H), 1634 cm–1 (O–H), and 570 cm–1 (Ti–O). The new peaks were 11
appeared at 1559/1512 cm–1 (N–H), 1247 cm–1 (C–N/P=O), 943 cm–1 (O–P–O), and the peak intensities at 3441 (O–H/N–H), 1634 cm–1 (C=O) and 1102 cm–1 (C–OH/P=O/C–O–C/C–O–P) were increased after proteins adsorption. This demonstrated that proteins were bound onto TNPs. The infrared absorption bands of ZNPs were observed at 3441 cm–1 (O–H), 1631 cm–1 (O–H), and 555/425 cm–1 (Zn–O). After absorbing proteins, the new peaks were presented at 1510 cm–1 (N–H), 948 cm–1 (O–P–O), 831 cm–1 (C–C/C–OH), and the peak intensities at 3441 (O–H/N–H), 1631 cm–1 (C=O) and 1105/1041 cm–1 (C–OH/P=O/C–O–C/C–O–P) were enlarged. The difference of absorption bands between ZNPs and TNPs indicated that the absorbed proteins were various.
3.1.3. Adsorption isotherm Langmuir isotherm model was examined to describe the equilibrium data (Matharu et al., 2009). (3) Where Qe and Qm are the equilibrium and theoretical maximum adsorbed proteins on NPs surfaces, respectively (mg/mg), Ce is the equilibrium concentration of proteins (mg/L), KL is the Langmuir constant (L/mg), also called the adsorption constant (L/mg), reflecting the affinity of proteins to NPs surfaces. The sizes, shape, and specific surface area between these two kinds of NPs were changed with the aggregation and dissolution of NPs, which could affect the adsorption of protein onto NPs. The results showed that the theoretical maximum adsorbed proteins on TNPs 12
and ZNPs were similar, whereas the binding affinity of proteins to TNPs was higher than that of proteins to ZNPs (Fig. 1 and Table 1). The results indicated that the protein layer on TNPs surface was denser than that on ZNPs surface. The previous research demonstrated that the adsorption of TNPs to activated sludge is very strong (Walden and Zhang, 2016). The extracellular matrix of activated sludge was main proteins, which also indicated the high affinity of proteins to TNPs. The proteins adsorption onto NPs can be promoted through ion-bridging (Ji et al., 2010). The dissolution of ZNPs in the protein solution was also investigated, and the Zn ion contents after ZNPs interacting with different concentrations of proteins are determined. The Zn ion contents after ZNPs interacting with the concentrations of 0, 35, 53, and 106 mg/L of protein were 0.164, 1.111, 1.339, and 1.001 mg/L, respectively. The results showed that the proteins accelerated the dissolution of ZNPs, which was attributed to the binding of Zn ion to proteins (Xu et al., 2016). Therefore, the adsorption of the proteins onto ZNPs could be enhanced by the dissolved Zn ion. The absorbed proteins on ZNPs surface could be stack or aggregation by ion bridging, whereas more proteins directly absorbed on TNPs surface and exhibits greater coverage compared with ZNPs. These results were validated by TEM images.
(Fig. 1)
3.2. Proteins with high affinity to NPs The proteins with high affinity to TNPs and ZNPs were identified using shotgun 13
proteomic. Thirty and nine absorbed proteins with high affinity to TNPs and ZNPs were identified, respectively, which demonstrated that the protein coronas between activated sludge proteins and NPs were dependent upon the types of NPs (Gunawan et al., 2014). The identified lipoproteins and nucleoproteins were consistent with the corresponding IR peaks. The proteomic results showed the isoelectric points of the proteins with high affinity to TNPs and ZNPs were ranged from 4.0 to 12.0, and over 70% of them were lower than 7.0, suggesting that most proteins absorbed on TNPs and ZNPs were negative charge in the neutral solution. Four shared proteins were found in the two samples, indicating these proteins could have high affinity to multiple NPs. The cellular component and biological process of the identified proteins were classified based on the gene ontology annotation (Ashburner et al., 2000). The location of the proteins with high affinity to NPs could affect the distribution of NPs in activated sludge floc. The cellular component indicates the location of proteins. As shown in Fig. 2a, a total of 8 catalogues of cellular component including cell out membrane, cytoplasm, bacterial-type flagellum, extracellular region, pore complex, nucleus, and nucleosome, were classified in the proteins with high affinity to ZNPs. Three out membrane proteins were identified, implying that the ZNPs could bind on cell out membrane when they penetrated to cell wall. IR spectra indicated that both TNPs and ZNPs changed the secondary structures of the absorbed proteins, which could damage the integrity of cell membrane. Fig. 2b shows that a total of 14 catalogues of cellular component, including cytoplasm, cell out membrane, nucleus, 14
and so on, were classified in the proteins with high affinity to TNPs. More locations suggested abundant binding sites to TNPs, which could be conducive to bind TNPs in cell internal. The top ten of the most abundant proteins absorbed on TNPs were obtained according to coverage, numbers of peptides and peptide spectra matches (PSMs), and are highlighted in table. The data showed that 6, 2, and 1 proteins were located in cytoplasm, cell out membrane, and extracellular region, respectively. The contact between NPs and organelle could affect the biological process. The proteins with high affinity to ZNPs participated in 10 biological processes, whereas those with high affinity to TNPs involved in much more biological processes, among which protein refolding and translational elongation were the majority. Therefore, these relative processes could be interfered when the NPs entered into intracellular region and bound to their high affinity proteins. The cellular component of the proteins predicted the intracellular distribution of the NPs. However, most of the NPs were aggregated with EPS in the extracellular region of activated sludge (Park et al., 2013). Especially, the proteins interacted with TNPs much stronger compared with polysaccharides and phospholipids (Huang et al., 2017). The abundant proteins in extracellular region played an important role in the absorption of NPs (Walden and Zhang, 2016). The results showed that tens of proteins in activated sludge can strongly bind TNPs, whereas only several proteins can strongly adsorb ZNPs. This indicated that activated sludge proteins provided much more binding sites to TNPs, demonstrating activated sludge could have a higher affinity to TNPs compared with that to ZNPs. It was consistent with the results of 15
adsorption isotherm. In addition, it is noted that 2 proteins with high affinity to TNPs and ZNPs located in the extracellular region were identified. Flagellin 2 was presented in the bacterial-type flagellum, and involved the biological process of bacterial-type flagellum-dependent cell motility. This suggested that the ZNPs and TNPs can easily adsorb onto flagellum, which could affect the motility of the flagellated bacteria. The other protein, enolase, involved in glycolytic process, which as associated with phosphopyruvate hydratase activity and magnesium ion binding activity. TNPs in the extracellular region can absorb enolase and disturb the glycolytic process.
(Fig. 2)
3.3. Effect of protein corona on NPs aggregation Fig. 3 shows the effect of different concentrations (0, 35, 53, and 106 mg/L) of proteins on the aggregation of NPs. The aggregation process was described by pseudo first-order equation. The kinetic parameters (aggregation percentages and rate constants) of the aggregations of ZNPs and TNPs at different concentrations of proteins are listed in Table 3. The aggregation rate constant of ZNPs was 0.003 min–1 in the absence of proteins, and the aggregation percentage was 17% at the equilibrium. The aggregation rate constant was increased when ZNPs was presented in the proteins solution, whereas the aggregation percentage was almost invariable. In the presence of proteins, the aggregation process of ZNPs could be associated with Zn ion. Proteins 16
promoted the dissolution of ZNPs, and thus the aggregation of ZNPs absorbed by proteins can be prompted by the dissolved Zn ion acting as ion-bridging (Philippe and Schaumann, 2014). Therefore, the aggregation potentials of ZNPs were obviously enhanced at 35 and 53 mg/L of proteins. As the proteins increased to 106 mg/L, the binding of Zn ion to the protein interior would weaken the ion-bridging and reduced the aggregation rate.
(Fig. 3)
Compared with ZNPs, TNPs exhibited the distinct aggregation process (Fig. 3b). The aggregation percentage was closed to 90% at 320 min in the absence of proteins. The high aggregation potential of TNPs was attributed to their point of zero charge ranging in neutral pH values (Schaumann et al., 2015). Contrary to ZNPs, the aggregation extents of TNPs were substantially reduced by proteins because the proteins tightly bound on TNPs surface and changed the surface property. As the protein concentrations increased, both the aggregation rates and aggregation percentages were elevated, demonstrating that increasing proteins gradually promoted TNPs aggregation and consequently formed larger hetero-agglomeration (Huang et al., 2017). The results revealed that the TNPs aggregation in water was dependent upon the concentrations of protein. On one hand, the proteomic results revealed that the isoelectric points of the major absorbed proteins were lower than 7.0, which implied that the TNP surfaces modified by proteins were negative charge in neutral aqueous 17
solution (Jain et al., 2015). The increasing electrostatic forces enlarged the TNPs stability. On the other hand, high surface coverage of the proteins on TNPs or overabundance proteins in solution could dominate the TNPs aggregation by bridging or repulsive interaction (Sheng et al., 2016b). In this work, with the increasing protein concentrations, macromolecule bridging flocculation induced by the proteins could promote the TNPs aggregation (Philippe and Schaumann, 2014). Therefore, high density of proteins in extracellular region tightly aggregated with TNPs (Park et al., 2013). These findings demonstrated the various mechanisms of the aggregations of TNPs and ZNPs under bacterial proteins in activated sludge, which were related to the protein species, the binding types of proteins on NPs, and ions in solution, etc.
3.4. Environmental Implications Current work showed the formation and composition of the protein coronas between NPs and bacterial proteins in activated sludge, and their effects on the aggregation and migration of NPs. The adsorption isotherms demonstrated that the proteins had a higher affinity to TNPs than to ZNPs. The proteins conduced to dissolution of ZNPs, and the dissolved Zn ion promoted to the ZNPs aggregation by ion-bridging. The aggregation extents of TNPs were substantially reduced by the proteins. However, the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Proteins are the major component in the extracellular region of activated sludge system, which composition and function were complicated. Thirty and nine proteins 18
with high affinities to TNPs and ZNPs were identified, respectively. More abundant proteins of high affinity indicated that proteins provided much more binding sites to TNPs, and also implied that activated sludge have a higher binding ability to TNPs compared with ZNPs. On the other hand, the intracellular distributions of the TNPs and ZNPs were predicted. Therefore, this study deepened our understanding of the roles of proteins in the aggregation and migration of NPs in activated sludge system.
4. Conclusions Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively, and the intracellular distributions of the NPs were predicted. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than ZNPs. The proteins conduced to the dissolution of ZNPs, which promoted to the ZNPs aggregation by dissolved Zn ion-bridging. The aggregations of TNPs were substantially reduced by the proteins. However, the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging.
Acknowledgments
The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (51708475, 21777135 and 51578527) and Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization. 19
Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online.
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26
Fig. 1 Adsorption isotherms of the proteins on TNPs and ZNPs at 25 ºC.
27
Fig. 2 The cellular components of the proteins with high affinity to ZNPs (a) and TNPs (b). PHC: phosphopyruvate hydratase complex; PMSDC: plasma membrane succinate dehydrogenase complex; PT-ATP-SC: proton-transporting ATP synthase complex, catalytic core F(1).
28
Fig. 3 The effects of 35, 53, and 106 mg/L of proteins on the aggregations of ZNPs (a) and TNPs (b).
29
Table 1 Langmuir isotherm model of the adsorption of the proteins on TNPs and ZNPs. Qm (mg/mg)
K (L/mg)
R2
TNPs
0.246
0.028
0.93
ZNPs
0.258
0.013
0.98
30
Table 2 The aggregation percentages and rate constants of ZNPs and TNPs at different concentrations of proteins. R2
Proteins
k1 (1/min)
Ae (%)
(mg/L)
TiO2
ZnO
TiO2
ZnO
TiO2
ZnO
0
0.003
0.003
149
17
0.98
0.95
35
0.001
0.012
21
13
0.98
0.94
53
0.004
0.011
30
14
0.98
0.91
106
0.008
0.006
41
18
0.98
0.99
31
Graphical abstract
32
Highlights
30 and 9 proteins absorbed on TNPs and ZNPs were identified, respectively. Activated sludge had a higher affinity to TNPs than to ZNPs. The proteins promoted to ZNPs aggregation through dissolved Zn ion-bridging. The increasing protein enhanced the TNPs aggregation by macromolecule bridging.
33
S0960-8524(17)31959-4 https://doi.org/10.1016/j.biortech.2017.11.008 BITE 19158
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
13 September 2017 31 October 2017 4 November 2017
Please cite this article as: Zhang, P., Xu, X-Y., Chen, Y-P., Xiao, M-Q., Feng, B., Tian, K-X., Chen, Y-H., Dai, YZ., Protein corona between nanoparticles and bacterial proteins in activated sludge: characterization and effect on nanoparticle aggregation, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.11.008
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Protein corona between nanoparticles and bacterial proteins in activated sludge: characterization and effect on nanoparticle aggregation
Peng Zhang1,2, Xiao-Yan Xu1, You-Peng Chen2, Meng-Qian Xiao1, Bo Feng1, Kai-Xun Tian1, Yue-Hui Chen1, You-Zhi Dai1,*
1
College of Environment and Resources, Xiangtan University, Xiangtan, 411105,
China 2
Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environments of
MOE, Chongqing University, Chongqing, 400045, China
*Corresponding Author: Prof. You-Zhi Dai; Tel: +86-731-58292231, Fax: +86-731-58292231 Email: [email protected]
1
Abstract: In this work, the protein coronas of activated sludge proteins on TiO2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation.
Keywords: Protein corona, Aggregation, Nanoparticles, Shotgun proteomics, Proteins with high affinity to nanoparticles
2
1. Introduction The increasing discharge of metal and metal oxide nanoparticles (NPs) into water environments leads to water pollution and potentially threats to aquatic ecological communities (Skjolding et al., 2016). NPs undergo transference and transition in natural water environment, and enter into biological wastewater treatment system (Lin et al., 2016). The diffusion and migration of NPs within the microbial aggregates, can penetrate cell membrane and result in cytotoxicity, which reduce the removals of COD, nitrogen, and phosphorus (Wang et al., 2016; Zheng et al., 2011). Soluble proteins, humic substances, and polysaccharides of microbial extracellular polymeric substances (EPS) presented in wastewater treatment system can absorb onto the surfaces of most NPs, and alter the stability of NPs (Lin et al., 2016). Proteins are the most primary component in extracellular matrix of activated sludge flocs and had high binding ability to NPs. (Mahendran et al., 2012; Park et al., 2008; Sheng et al., 2016). The content of extracellular protein are substantially increased after microbes exposing on TNPs and ZNPs (He et al., 2017; Li et al., 2015). Furthermore, proteomics researches demonstrated that proteins in soluble EPS and bound EPS were mainly (more than 60%) originated from the release of cell lysis (Wu et al., 2017; Zhang et al., 2015). A better understanding of the roles of microbial proteins in activated sludge in the aggregation and migration of NPs is therefore critical. The adsorbed proteins form protein corona on the surface of NPs, which change the surface properties of NPs, e.g., zeta potential and hydrophobicity (Jain et al., 2015). 3
The composition of protein corona can control the interaction of NPs with outer membrane receptors for specific cell uptake (Gao et al., 2017). The adsorption of proteins onto NPs is a dynamic process, which relies on the affinity (Fleischer and Payne, 2014). The proteins with higher affinity to NPs can displace those with lower affinity to NPs, and irreversibly bind on the surface of NPs (Saptarshi et al., 2013). Therefore, these proteins absorbed on NPs are the key components that affect the aggregation, transference, and intracellular uptake of NPs. Previous investigations on the effect of proteins on NPs aggregation focused on the model proteins, such as cytochrome and albumin (Levak et al., 2017; Sheng et al., 2016a; Sheng et al., 2016b). While hundreds of proteins with various isoelectric points, molecular weights, and structures presented in activated sludge have different effects to NPs (Herbst et al., 2016). It is indispensable for studies the function of mixed proteins from activated sludge in the aggregation and fate of NPs. In activated sludge, EPS are the major component, which exhibit a large three-dimensional network structure at the microscopic scale (Sheng et al., 2010). The diffusion of NPs into the microbial aggregates is dependent upon the surface properties of both the NPs and the aggregates. NPs interact with the proteins in microbial aggregates when they migrate in the three-dimensional network structure. The proteins with various functions presented in activated sludge have different affinities to NPs, among which the proteins with higher affinity to NPs can immobilize the NPs and limit the NPs dispersal (Herbst et al., 2016; Moreau et al., 2007). Therefore, identifying these proteins with higher affinity to NPs, and analyzing 4
of their functions and located regions could be helpful for understanding the migration of NPs in activated sludge flocs. Here, the total proteins were extracted from the microbes in activated sludge. The protein coronas of the proteins on TiO2 NPs (TNPs) and ZnO NPs (ZNPs) were observed using TEM, and the binding mechanism of the proteins to TNPs and ZNPs was investigated. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and the effect of these proteins on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of the proteins on the aggregations of TNPs and ZNPs were evaluated.
2. Materials and Methods 2.1. NPs TNPs (99.8%, 25 nm, anatase type) was purchased from Aladdin, Shanghai Jingchun Biochemistry Technology Co., China. ZNPs (99.8%, 30 ± 10 nm) was purchased from Micxy Chemical Co., Chengdu, China. The TNPs and ZNPs powders were dispersed in deionized water, and the suspension was treated by ultrasound for approximately 30 min before further test.
2.2. Microbial culture and protein extraction Microbes were derived from the supernatant of a municipal wastewater treatment plant. One mL supernatant was incubated in 250 mL of LB-Lennox medium in 500 mL flasks (approximately 40 h, 30 °C, 120 rpm) (Guo et al., 2015). The bacterial 5
suspension was centrifuged at 5000 g for 20 min at 4 °C to remove growth medium. The cell pellet was washed three times with deionized water followed by a second centrifugation. The bacterial proteins were extracted using total protein extraction kit (Jiancheng Bioengineering Institute, Nanjing, China). Ten microliters phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of 100 mM of PMSF were added in per liter of cold lysis buffer, and mixed at 4 °C. The mixture was added to cell pellet and mildly shocked for 15 min at 4 °C. After centrifugation at 12000 rpm for 15 min at 4 °C, the supernatant was collected and quantified using total protein assay kit (BCA method, Jiancheng Bioengineering Institute, Nanjing, China). The required protein solution was separately stored at –70 °C before subsequent research.
2.3. FT-IR TNPs and ZNPs mixed with bacterial proteins solution for 3 h followed by centrifugation at 4000 g for 10 min and wash three times with deionized water to remove unbound protein on NPs surfaces. Both the sediments and protein were freeze-dried at –50 °C, and the powder was mixed with IR grade KBr powders to prepare the pellets. The FT-IR of all the samples were obtained by an FT-IR spectrophotometer (Nicolet 380, USA) within the scanning range 4000–400 cm−1. The pure TNPs and ZNPs powders were directly prepared the pellets to test.
2.4. Protein adsorption to NPs 6
Adsorption isotherms of the proteins on TNPs and ZNPs were measured in solution at pH approximately 7.0. The initial concentrations of NPs were prepared to 400 mg/L. The protein solution was prepared with the concentrations of 6.7, 13.3, 26.5, 53, 106, and 212 mg/L. One mL of NPs suspension was mixed completely with equal volume of each protein solution. And thus both the concentrations of the NPs and proteins were reduced by half. The samples were incubated in a shaker operating at 150 rpm and 25 ºC for 3 h to reach adsorption equilibrium, followed by centrifugation for 10 minutes at 4000 g. Protein concentrations in supernatants were determined using total protein assay kit.
2.5. Identification of proteins in the corona 2.5.1. Protein corona isolation The proteins were incubated with 3 mg of ZNPs or TNPs for 3 h at 4 °C (Cai et al., 2013). After centrifugation at 4000 g for 10 min at 4 °C to remove unbound proteins, the pellets were thoroughly washed with deionized water. The bound proteins were eluted by two centrifugations at 15000 g for 1 h at 4 °C (Ali et al., 2016). The supernatant was collected for proteomics analysis.
2.5.2. Digestion Protein digestion was conducted according to a corrected method described by Wisniewski et al. (2009). In general, approximately 30 μg of the protein sample was dissolved in 30 μL of SDT buffer (4% sodium dodecyl sulfate, 100 mM dithiothreitol, 7
150 mM Tris-HCl and pH 8.0) and kept boiling water bath for 5min. Then 200 μL of UA buffer (8 M urea, 150 mM Tris-HCl and pH 8.0) was added and mixed. The mixture was transferred to an ultrafiltration device (30 kD) for centrifugation, and the supernatant was removed. Then 100 μL of 50 mM iodoacetamide in UA buffer was added to sample pellet and shocked for 1 min. The samples were incubated for 30 min in darkness at room temperature. One hundred μL of UA buffer was added and centrifuged, and the procedure was repeated twice. Then 100 μL of 25 mM of NH4HCO3 was added and centrifuged, repetition twice. The protein suspension with 2 μg of trypsin in 40 μL of 25 mM NH4HCO3 incubated overnight at 37 °C followed by centrifugation. At last, 40 μL of 25 mM of NH4HCO3 was added, and the mixture was through centrifugation and acidification. The resulting peptides were collected for subsequently analysis.
2.5.3. Mass spectrometry (MS) Samples were loaded on a Thermo scientific EASY column (2 cm × 100 mm, 5 μm-C18, USA), and then separated on a Thermo scientific EASY column (75 μm × 100 mm, 3 μm-C18, USA). The mobile phases were prepared as follows: A (0.1% formic acid in HPLC-grade water) and B (0.1% formic acid in acetonitrile). Twenty μg of tryptic peptide mixtures was separated at a flow rate of 2 μL/min with a linear gradient (4–50% solution B, 0–50 min; 50–100% solution B, 50–54 min; 100% solution B, 54–60 min) in the columns. The eluted peptides were detected using a MS (Q exactive, Thermo Scientific, USA) equipped with a micro-spray interface. The 8
MS/MS spectra were set so that one full scan mass spectrum (m/z 300-1800) was followed by ten MS/MS events.
2.5.4. Data analysis MS/MS spectra were automatically searched against the SwissProt database using the Mascot 2.2 (Thermo Electron, San Jose, USA), and the protein identification results were obtained. Classifications of identified proteins were carried out using gene ontology annotation (GOA; http://www.uniprot.org/uniprot/) according to the protein accession numbers.
2.6. Aggregation and settlement assay The UV-vis spectra of NPs were scanned by Cary 60 (Agilent, USA), and the maximum absorption bands of TNPs and ZNPs were 349 nm and 361 nm, respectively. Then the effect of proteins on the aggregation and settlement of NPs was evaluated. One milliliter of 70, 106, 212 mg/L of proteins were mixed completely with equal volume of TNPs (200mg/L) and ZNPs (400 mg/L) and transferred to quartz colorimetric cuvette. Thus both the concentrations of the NPs and proteins were reduced by half. As the time elapsed, the NPs aggregated and settled to the bottom of the colorimetric cuvette. Therefore, the absorbance change was used to indirectly characterize the NPs aggregation. The optical density (OD) was determined after 5, 10, 20, 40, 80, 120, 160, 220, 240, and 320 min. The aggregation percentage (At) of NPs was calculated using Eq. (1). 9
–
(1)
The kinetic of particles aggregation and settlement can be described by pseudo first-order equation (Eq. (2)) (Brunelli et al., 2013; Quik et al., 2012; Zhang et al., 2014). (2) Where At is the aggregation percentage at a certain time (%), Ae is the aggregation percentage at equilibrium, and k1 is the pseudo first-order rate constant (min−1).
2.7. Atomic absorption spectra and TEM The dissolution of ZNPs was researched, and Zn ion was determined by atomic absorption spectrometer (AA-6300C, Shimazu, Japan). TNPs and ZNPs dispersed in deionized water and in protein solution were collected and dropped onto carbon-coated copper grids for TEM analysis (JEM-2100, JEOL, Japan).
3. Results and Discussion 3.1. Formation of protein corona 3.1.1. TEM characterization The images and morphologies of the proteins, and the NPs before and after protein adsorption were characterized by TEM. The protein images exhibited the spherical shape with different diameters. The results demonstrated that the proteins shape was reconstruction after adsorption on NPs. The absorbed proteins presented a variety of shapes on TNPs. Both the thin film-shape with the thickness of 2 nm and the 10
aggregated-shape proteins were found on TNP surfaces, whereas the ZNP surfaces were only observed the aggregated-shape proteins.
3.1.2. FT-IR characterization FT-IR spectra was used to reveal the binding of the proteins on NPs. The IR bands were assigned according to the existing literatures (Comte et al., 2006; Liu et al., 2010; Maquelin et al., 2002). The broad band around 3431 cm–1 was assigned to the stretching vibrations of O–H and N–H, whereas the bands at 2931 cm–1 and 2870 cm–1 were attributed to the C–H stretching vibrations. The C=O stretching vibration of amide I exhibited at 1631 cm–1, whereas N–H deformation vibrations of amide II were observed at 1563 cm–1 and 1512 cm–1. The band near 1384 cm–1 was assigned to C=O symmetric stretching vibration of carboxyl group. The C–N stretching vibrations of amide III with broad band was found at 1247 cm–1, and the band also could be related to P=O asymmetric stretching vibration of >PO2– phosphodiesters. The broad bands around 1105 cm–1 and 1050 cm–1 were attributed to ring vibrations of P=O, C–O–C, C–OH, and C–O–P stretching. The peak at 943 cm–1 was observed due to O–P–O asymmetric stretching vibration, whereas the weak peak presents at 831 cm–1 owing to C–C and C–OH stretching vibrations. The results revealed that the protein sample contained nucleoproteins and glycoproteins. The FT-IR spectra showed the change in TNPs before and after absorbing proteins. Before proteins adsorption, the bands of TiO2 nanoparticles were mainly found at 3431 cm–1 (O–H), 1634 cm–1 (O–H), and 570 cm–1 (Ti–O). The new peaks were 11
appeared at 1559/1512 cm–1 (N–H), 1247 cm–1 (C–N/P=O), 943 cm–1 (O–P–O), and the peak intensities at 3441 (O–H/N–H), 1634 cm–1 (C=O) and 1102 cm–1 (C–OH/P=O/C–O–C/C–O–P) were increased after proteins adsorption. This demonstrated that proteins were bound onto TNPs. The infrared absorption bands of ZNPs were observed at 3441 cm–1 (O–H), 1631 cm–1 (O–H), and 555/425 cm–1 (Zn–O). After absorbing proteins, the new peaks were presented at 1510 cm–1 (N–H), 948 cm–1 (O–P–O), 831 cm–1 (C–C/C–OH), and the peak intensities at 3441 (O–H/N–H), 1631 cm–1 (C=O) and 1105/1041 cm–1 (C–OH/P=O/C–O–C/C–O–P) were enlarged. The difference of absorption bands between ZNPs and TNPs indicated that the absorbed proteins were various.
3.1.3. Adsorption isotherm Langmuir isotherm model was examined to describe the equilibrium data (Matharu et al., 2009). (3) Where Qe and Qm are the equilibrium and theoretical maximum adsorbed proteins on NPs surfaces, respectively (mg/mg), Ce is the equilibrium concentration of proteins (mg/L), KL is the Langmuir constant (L/mg), also called the adsorption constant (L/mg), reflecting the affinity of proteins to NPs surfaces. The sizes, shape, and specific surface area between these two kinds of NPs were changed with the aggregation and dissolution of NPs, which could affect the adsorption of protein onto NPs. The results showed that the theoretical maximum adsorbed proteins on TNPs 12
and ZNPs were similar, whereas the binding affinity of proteins to TNPs was higher than that of proteins to ZNPs (Fig. 1 and Table 1). The results indicated that the protein layer on TNPs surface was denser than that on ZNPs surface. The previous research demonstrated that the adsorption of TNPs to activated sludge is very strong (Walden and Zhang, 2016). The extracellular matrix of activated sludge was main proteins, which also indicated the high affinity of proteins to TNPs. The proteins adsorption onto NPs can be promoted through ion-bridging (Ji et al., 2010). The dissolution of ZNPs in the protein solution was also investigated, and the Zn ion contents after ZNPs interacting with different concentrations of proteins are determined. The Zn ion contents after ZNPs interacting with the concentrations of 0, 35, 53, and 106 mg/L of protein were 0.164, 1.111, 1.339, and 1.001 mg/L, respectively. The results showed that the proteins accelerated the dissolution of ZNPs, which was attributed to the binding of Zn ion to proteins (Xu et al., 2016). Therefore, the adsorption of the proteins onto ZNPs could be enhanced by the dissolved Zn ion. The absorbed proteins on ZNPs surface could be stack or aggregation by ion bridging, whereas more proteins directly absorbed on TNPs surface and exhibits greater coverage compared with ZNPs. These results were validated by TEM images.
(Fig. 1)
3.2. Proteins with high affinity to NPs The proteins with high affinity to TNPs and ZNPs were identified using shotgun 13
proteomic. Thirty and nine absorbed proteins with high affinity to TNPs and ZNPs were identified, respectively, which demonstrated that the protein coronas between activated sludge proteins and NPs were dependent upon the types of NPs (Gunawan et al., 2014). The identified lipoproteins and nucleoproteins were consistent with the corresponding IR peaks. The proteomic results showed the isoelectric points of the proteins with high affinity to TNPs and ZNPs were ranged from 4.0 to 12.0, and over 70% of them were lower than 7.0, suggesting that most proteins absorbed on TNPs and ZNPs were negative charge in the neutral solution. Four shared proteins were found in the two samples, indicating these proteins could have high affinity to multiple NPs. The cellular component and biological process of the identified proteins were classified based on the gene ontology annotation (Ashburner et al., 2000). The location of the proteins with high affinity to NPs could affect the distribution of NPs in activated sludge floc. The cellular component indicates the location of proteins. As shown in Fig. 2a, a total of 8 catalogues of cellular component including cell out membrane, cytoplasm, bacterial-type flagellum, extracellular region, pore complex, nucleus, and nucleosome, were classified in the proteins with high affinity to ZNPs. Three out membrane proteins were identified, implying that the ZNPs could bind on cell out membrane when they penetrated to cell wall. IR spectra indicated that both TNPs and ZNPs changed the secondary structures of the absorbed proteins, which could damage the integrity of cell membrane. Fig. 2b shows that a total of 14 catalogues of cellular component, including cytoplasm, cell out membrane, nucleus, 14
and so on, were classified in the proteins with high affinity to TNPs. More locations suggested abundant binding sites to TNPs, which could be conducive to bind TNPs in cell internal. The top ten of the most abundant proteins absorbed on TNPs were obtained according to coverage, numbers of peptides and peptide spectra matches (PSMs), and are highlighted in table. The data showed that 6, 2, and 1 proteins were located in cytoplasm, cell out membrane, and extracellular region, respectively. The contact between NPs and organelle could affect the biological process. The proteins with high affinity to ZNPs participated in 10 biological processes, whereas those with high affinity to TNPs involved in much more biological processes, among which protein refolding and translational elongation were the majority. Therefore, these relative processes could be interfered when the NPs entered into intracellular region and bound to their high affinity proteins. The cellular component of the proteins predicted the intracellular distribution of the NPs. However, most of the NPs were aggregated with EPS in the extracellular region of activated sludge (Park et al., 2013). Especially, the proteins interacted with TNPs much stronger compared with polysaccharides and phospholipids (Huang et al., 2017). The abundant proteins in extracellular region played an important role in the absorption of NPs (Walden and Zhang, 2016). The results showed that tens of proteins in activated sludge can strongly bind TNPs, whereas only several proteins can strongly adsorb ZNPs. This indicated that activated sludge proteins provided much more binding sites to TNPs, demonstrating activated sludge could have a higher affinity to TNPs compared with that to ZNPs. It was consistent with the results of 15
adsorption isotherm. In addition, it is noted that 2 proteins with high affinity to TNPs and ZNPs located in the extracellular region were identified. Flagellin 2 was presented in the bacterial-type flagellum, and involved the biological process of bacterial-type flagellum-dependent cell motility. This suggested that the ZNPs and TNPs can easily adsorb onto flagellum, which could affect the motility of the flagellated bacteria. The other protein, enolase, involved in glycolytic process, which as associated with phosphopyruvate hydratase activity and magnesium ion binding activity. TNPs in the extracellular region can absorb enolase and disturb the glycolytic process.
(Fig. 2)
3.3. Effect of protein corona on NPs aggregation Fig. 3 shows the effect of different concentrations (0, 35, 53, and 106 mg/L) of proteins on the aggregation of NPs. The aggregation process was described by pseudo first-order equation. The kinetic parameters (aggregation percentages and rate constants) of the aggregations of ZNPs and TNPs at different concentrations of proteins are listed in Table 3. The aggregation rate constant of ZNPs was 0.003 min–1 in the absence of proteins, and the aggregation percentage was 17% at the equilibrium. The aggregation rate constant was increased when ZNPs was presented in the proteins solution, whereas the aggregation percentage was almost invariable. In the presence of proteins, the aggregation process of ZNPs could be associated with Zn ion. Proteins 16
promoted the dissolution of ZNPs, and thus the aggregation of ZNPs absorbed by proteins can be prompted by the dissolved Zn ion acting as ion-bridging (Philippe and Schaumann, 2014). Therefore, the aggregation potentials of ZNPs were obviously enhanced at 35 and 53 mg/L of proteins. As the proteins increased to 106 mg/L, the binding of Zn ion to the protein interior would weaken the ion-bridging and reduced the aggregation rate.
(Fig. 3)
Compared with ZNPs, TNPs exhibited the distinct aggregation process (Fig. 3b). The aggregation percentage was closed to 90% at 320 min in the absence of proteins. The high aggregation potential of TNPs was attributed to their point of zero charge ranging in neutral pH values (Schaumann et al., 2015). Contrary to ZNPs, the aggregation extents of TNPs were substantially reduced by proteins because the proteins tightly bound on TNPs surface and changed the surface property. As the protein concentrations increased, both the aggregation rates and aggregation percentages were elevated, demonstrating that increasing proteins gradually promoted TNPs aggregation and consequently formed larger hetero-agglomeration (Huang et al., 2017). The results revealed that the TNPs aggregation in water was dependent upon the concentrations of protein. On one hand, the proteomic results revealed that the isoelectric points of the major absorbed proteins were lower than 7.0, which implied that the TNP surfaces modified by proteins were negative charge in neutral aqueous 17
solution (Jain et al., 2015). The increasing electrostatic forces enlarged the TNPs stability. On the other hand, high surface coverage of the proteins on TNPs or overabundance proteins in solution could dominate the TNPs aggregation by bridging or repulsive interaction (Sheng et al., 2016b). In this work, with the increasing protein concentrations, macromolecule bridging flocculation induced by the proteins could promote the TNPs aggregation (Philippe and Schaumann, 2014). Therefore, high density of proteins in extracellular region tightly aggregated with TNPs (Park et al., 2013). These findings demonstrated the various mechanisms of the aggregations of TNPs and ZNPs under bacterial proteins in activated sludge, which were related to the protein species, the binding types of proteins on NPs, and ions in solution, etc.
3.4. Environmental Implications Current work showed the formation and composition of the protein coronas between NPs and bacterial proteins in activated sludge, and their effects on the aggregation and migration of NPs. The adsorption isotherms demonstrated that the proteins had a higher affinity to TNPs than to ZNPs. The proteins conduced to dissolution of ZNPs, and the dissolved Zn ion promoted to the ZNPs aggregation by ion-bridging. The aggregation extents of TNPs were substantially reduced by the proteins. However, the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Proteins are the major component in the extracellular region of activated sludge system, which composition and function were complicated. Thirty and nine proteins 18
with high affinities to TNPs and ZNPs were identified, respectively. More abundant proteins of high affinity indicated that proteins provided much more binding sites to TNPs, and also implied that activated sludge have a higher binding ability to TNPs compared with ZNPs. On the other hand, the intracellular distributions of the TNPs and ZNPs were predicted. Therefore, this study deepened our understanding of the roles of proteins in the aggregation and migration of NPs in activated sludge system.
4. Conclusions Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively, and the intracellular distributions of the NPs were predicted. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than ZNPs. The proteins conduced to the dissolution of ZNPs, which promoted to the ZNPs aggregation by dissolved Zn ion-bridging. The aggregations of TNPs were substantially reduced by the proteins. However, the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging.
Acknowledgments
The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (51708475, 21777135 and 51578527) and Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization. 19
Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online.
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26
Fig. 1 Adsorption isotherms of the proteins on TNPs and ZNPs at 25 ºC.
27
Fig. 2 The cellular components of the proteins with high affinity to ZNPs (a) and TNPs (b). PHC: phosphopyruvate hydratase complex; PMSDC: plasma membrane succinate dehydrogenase complex; PT-ATP-SC: proton-transporting ATP synthase complex, catalytic core F(1).
28
Fig. 3 The effects of 35, 53, and 106 mg/L of proteins on the aggregations of ZNPs (a) and TNPs (b).
29
Table 1 Langmuir isotherm model of the adsorption of the proteins on TNPs and ZNPs. Qm (mg/mg)
K (L/mg)
R2
TNPs
0.246
0.028
0.93
ZNPs
0.258
0.013
0.98
30
Table 2 The aggregation percentages and rate constants of ZNPs and TNPs at different concentrations of proteins. R2
Proteins
k1 (1/min)
Ae (%)
(mg/L)
TiO2
ZnO
TiO2
ZnO
TiO2
ZnO
0
0.003
0.003
149
17
0.98
0.95
35
0.001
0.012
21
13
0.98
0.94
53
0.004
0.011
30
14
0.98
0.91
106
0.008
0.006
41
18
0.98
0.99
31
Graphical abstract
32
Highlights
30 and 9 proteins absorbed on TNPs and ZNPs were identified, respectively. Activated sludge had a higher affinity to TNPs than to ZNPs. The proteins promoted to ZNPs aggregation through dissolved Zn ion-bridging. The increasing protein enhanced the TNPs aggregation by macromolecule bridging.
33