Light-induced reduction of silver ions to silver nanoparticles in aquatic environments by microbial extracellular polymeric substances (EPS)

Water Research 106 (2016) 242e248

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Light-induced reduction of silver ions to silver nanoparticles in aquatic environments by microbial extracellular polymeric substances (EPS) Xin Zhang, Chuan-Wang Yang, Han-Qing Yu, Guo-Ping Sheng* CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2016 Received in revised form 30 September 2016 Accepted 1 October 2016 Available online 5 October 2016

Microbial extracellular polymeric substances (EPS) widely exist in natural environments and affect the migration and transformation of pollutants in aquatic environments. Previous works report that EPS have some reducing functional groups and can reduce heavy metals. However, because of the weak reducing capability of EPS, the reduction of heavy metals by EPS without cells is extremely slow, and its effect on heavy metals species is insignificant. In this work, the accelerated reduction of silver ions (Agþ) by EPS from Shewanella oneidensis MR-1 under illumination was investigated. UVevisible spectroscopy, transmission electron microscopy (TEM) coupled with an energy dispersive spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS) were used to confirm the formation of silver nanoparticles (AgNPs) via the reduction of Agþ by EPS under light illumination. The Agþ reduction by EPS follows pseudo-firstorder kinetics under both visible and UV light, and the light irradiation can significantly accelerate AgNPs formation. On the one hand, visible light can excite AgNPs for their surface plasma resonance (SPR) and accelerate the electrons from the EPS to adjacent Agþ. On the other hand, EPS molecules may be excited by UV light to produce strong reducing species, which enhance Agþ reduction. Moreover, pH, dissolved oxygen were found to affect the formation of AgNPs by EPS. This work proves the reducing capability of EPS on the reduction of Agþ, and this process can be accelerated under light illumination, which may affect the speciation and transformation of heavy metals in natural waters. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Extracellular polymeric substances (EPS) Kinetics Light illumination Reduction Silver nanoparticles (AgNPs)

1. Introduction Silver nanoparticles (AgNPs) are the most widely used nanomaterials in our daily lives because of their outstanding bactericidal activity and catalytic activity (Adegboyega et al., 2013). The widely used AgNPs are inevitably released into the environment and threaten the ecosystem (Levard et al., 2012). Thus, there are increasing concerns about the formation and transfer of AgNPs in the environment. Apart from the anthropogenic sources, the AgNPs can also form from both biotic and abiotic processes in natural conditions. For instance, some dissimilatory metal reducing bacteria, like Geobacter and Shewanella, can export intracellular electrons to extracellular silver ions (Agþ) to generate AgNPs (Ng et al., 2013; Law et al., 2008). Furthermore, the natural formation of AgNPs by abiotic processes were also observed. Several works have proved that AgNPs can be generated from the reduction of Agþ by dissolved organic matters (Yin et al., 2012; Hou et al., 2013; Akaighe

* Corresponding author. E-mail address: [email protected] (G.-P. Sheng). http://dx.doi.org/10.1016/j.watres.2016.10.004 0043-1354/© 2016 Elsevier Ltd. All rights reserved.

et al., 2011; Adegboyega et al., 2014). Extracellular polymeric substances (EPS), a complex high molecular compound secreted from many organisms in aquatic environments, can be widely found in both fresh water and marine water (Flemming and Wingender, 2010). Particularly for marine environments, microbial EPS can account for up to 40% of the total organic carbon content (Bhaskar and Bhosle, 2005). Many functional groups such as carboxyl, hydroxyl, phosphoryl, sulfhydryl, and phenolic groups are available in microbial EPS to bind heavy metals by electrostatic interaction or complexing bonds (Sheng et al., 2013). Thus, microbial EPS can affect the fate of these heavy metals in the environment. For example, EPS can accumulate cooper and affect its entry into the environmental food chain (Mittelman and Geesey, 1985). In recent years, several groups have reported that EPS can reduce heavy metals (e.g., Cr6þ) (Harish et al., 2012), radionuclides (e.g., U6þ) (Cao et al., 2011a), and organic pollutants (Kang and Zhu, 2013). The hemiacetal reducing ends in polysaccharides and phenol groups may be responsible for the reducing capacity of EPS (Kang and Zhu, 2013). In addition, some redox proteins in EPS may act as electron donors to reduce some

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pollutants such as U(VI) (Cao et al., 2011a,b). Consequently, more attention should be paid to the roles of EPS on the fate of heavy metals in the environment considering the reducing characteristics of EPS, particularly in natural waters. Natural sunlight is reported to affect the redox of many metal ions (Fe(III), Hg(II), Cr(VI), Ag(I)) in the presence of humic substances because some redox-active species are generated under light irradiation (Yin et al., 2012; Fukushima and Tatsumi, 1999; Matthiessen, 1998; Gaberell et al., 2003). Many reducing functional groups in humic substances also exist in EPS such as hydroxyls, phenolic-OH, thiols and aldehydes etc (Akaighe et al., 2011; Sheng et al., 2010). Therefore, it is hypothesized that light may stimulate the EPS molecules and affect the Agþ reduction to AgNPs in natural environments, which has not been investigated. This investigation will be useful to better understand the effects of EPS on Ag transformation in natural waters. The objective of this study is to investigate the formation of AgNPs in the presence of microbial EPS under light illumination (290e800 nm). The formation kinetics of AgNPs under visible and UV light irradiation were investigated. To better understand the reduction of Agþ by EPS in aquatic environments, the solution chemistry conditions such as pH and dissolved oxygen (DO) were also investigated. With the experimental results, the enhancement mechanisms of AgNPs formation through Agþ reduction in the presence of EPS under illumination were proposed. The results from this work are useful to understand the role of EPS in the formation of silver nanoparticles in aquatic environments and broaden our knowledge on the transformation of pollutants in the environments. 2. Materials and methods 2.1. Microbial strain and EPS extraction Microbial EPS was harvested from Shewanella oneidensis MR-1, a bacterial strain widely existing in natural environments. The strain was cultivated in 0.5 mL of LB medium at 30  C for 10 h and inoculated in 250 mL of fresh LB (1‰, v/v) for additional 12 h. The bacterial cells were collected, washed three times with HEPES buffer, and transferred into 1 L of M1 medium (Nealson and Scott, 2006) containing 30 mM sodium lactate and 30 mM fumarate as the electron donor and acceptor for bacteria growth, respectively (full medium compositions see Table S1). The initial bacterial content was controlled at an OD600 of 0.1. After 48 h, the bacterial cells were separated from the medium by centrifugation (5000  g, 5 min) and washed three times with DI water to remove the residual medium. The bacteria pellets were re-suspended for EPS extraction using a Naþ-form cation exchange resin (CER) according to our previous study (Sheng and Yu, 2006) with a slight modification. The CER was added into the bacteria suspended solution with a dosage of 60 g/(g-dry cell weight). Then, the suspensions were stirred at 900 rpm and 4  C for 5e6 h. The solution was subsequently settled for 3 min to remove the CER. Then, the EPS and bacterial cells were separated by centrifugation at 13 000  g for 10 min. After that, the crude EPS solution was filtered through a 0.22-mm membrane and stored at 4  C and oxygen free condition for subsequent experiments. The portion of EPS reduced by direct electrochemical reduction on glassy carbon working electrodes at 0.6 V (Aeschbacher et al., 2011) was denoted as reduced EPS. No special statement, the EPS extracted under ambient condition was used as experimental EPS. 2.2. Characterization of EPS The contents of proteins and carbohydrates in the extracted EPS

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were measured using the Lowry method with bovine serum albumin (BSA) as a standard and the anthrone method with glucose as a standard, which were 29.97 and 38.53 mg/g-dry cell weight, respectively. The content of humic substances (1.02 mg/g-dry cell weight) in the extracted EPS was measured using the modified Lowry method with humic acids (Sigma aldrich) as the standard. The content of DNA (0.31 mg/g-dry cell weight) in the extracted EPS was measured using diphenylamine colorimetric method with calf thymus DNA as the standard. The low content of DNA in EPS implied that the cell lysis was negligible (Liu and Fang, 2002). The total carbon content (TOC) of the extracted EPS was determined using a TOC analyzer (Multi N/C 2100, Analytik Jena, Germany). In addition, the Fourier transform infrared (FTIR) spectrum and 3dimensional excitationeemission matrix (EEM) were used to characterize the extracted EPS. The FTIR and EEM were recorded from a VERTEX 70 FTIR (Bruker Co., Germany) and an LS55 fluorescence spectrophotometer (PerkinElmer Co., USA), respectively. The EPS characterization results are provided in the Supporting Information (Fig. S1 and Table S2). 2.3. Formation of AgNPs To investigate the formation of AgNPs in the presence of EPS under illumination and the effects of the solution chemistry, the experiments were performed in 50-mL transparent vials. Before the experiments, the EPS and AgNO3 solutions were purged with nitrogen gas for 30 min to remove the dissolved oxygen and were subsequently added into the vials in an anaerobic glove box. The vials were wrapped in two layers of aluminum foil to maintain dark conditions before the experiments. Eight general fluorescent lamps (PHILIPS, T8) were used as the light source. The illumination intensity was 500 mW/cm2 (400e800 nm). The temperature was maintained at 26  C in all experiments; a 1.2-mL solution was sampled at specific time to monitor the formation of AgNPs. To explore the effect of light with different wavebands (UV and visible) on the Agþ reduction by EPS, a 250/350 W windrefrigerated Xe lamp with adjustable brightness was used to simulate sunlight, and 50-mL quartz tubes were used. Two filters were used to obtain visible (400e800 nm) and UV light (290e400) nm, (UVA and UVB, no UVC output). The incident intensity of visible and UV light was measured using a solar power photometer (TENMARS, TM-207) and a UV light photometer (LUTRON, UV340A), respectively. The incident intensity of visible light was 500 mW/cm2 on the surface of the quartz tubes by using a UV-block filter (>400 nm), and the corresponding UV light intensity under 400 nm was 23 mW/cm2. By adjusting the distance between the lamp and the samples, the enhanced UV light intensity in our experiments with the vis-block filter (<400 nm) was 70 mW/cm2. The tubes covered by two layers of aluminum foil were the dark control in the experiment. To explore the Agþ reduction mechanism by EPS under visiblelight illumination, the effects of two monochromatic lights at 415 nm and 600 nm were investigated using band-pass filters. The surface plasmon resonance (SPR) peak of AgNPs is near the 415 nm light and far from the 600 nm light. The reaction system was initially irradiated under visible light for 6 h. Then, the reaction solution was divided into two parts under anaerobic and dark conditions, which were then irradiated with 415 nm and 600 nm monochromatic light, respectively. 2.4. Characterization of AgNPs The UVevis spectra of the formed AgNPs were recorded at 200e800 nm using a UV-2450 spectrometer (Shimadzu Co., Japan). The morphology of the AgNPs was characterized using

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transmission electron microscopy (TEM), which was coupled with an energy dispersive spectrometer (EDS) (H-7650, Hitachi Co., Japan). X-ray photoelectron spectroscopy (XPS) (PHI 5600, PerkinElmer Inc.) was used to determine the valence state of the produced AgNPs.

3. Results and discussion 3.1. Formation and characterization of AgNPs The reduction of Agþ to AgNPs by EPS under illumination was investigated, and the generation of AgNPs were confirmed using UVeVis, TEM, EDS and XPS. Because of the SPR of AgNPs, the generated silver colloid absorbed visible light and the suspension displayed a yellow color. The UVevis spectrum in Fig. 1a is the typical absorption spectrum of spherical AgNPs (Jin et al., 2001); the maximum absorption peak was approximately at 410 nm, whereas no absorption peak was observed in the control conditions without EPS or Agþ even after 72 h of illumination (Fig. S2). The morphology of the produced AgNPs was nearly spherical, as indicated in the TEM image (Fig. 1b). The size of the AgNPs was not uniform, and most of them were in the range of 8e10 nm. The EDS and XPS further confirmed the generation of AgNPs from Agþ in the presence of EPS (Fig. 1c and d). The Ag3d5/2 and Ag3d3/2 peaks were at 368.3 eV and 374.3 eV, respectively, which are the characteristic peaks of Ag0 (Dong et al., 2009). Besides, the potential of the microbial cells before and after EPS extraction for Agþ reduction was also investigated. Fig. S3 shows that the Agþ could be reduced to AgNPs by Shewanella oneidensis MR-1 cells no matter whether the EPS existed or not. However, the reduction of Agþ by cells with EPS was obviously faster than that without EPS, which also demonstrated that the Agþ could be reduced to AgNPs mediated by microbial EPS. With increasing irradiation time, the absorbance of AgNPs increased (Fig. 2). At the initial stage, the absorbance quickly

Fig. 2. Plot of the maximum UVeVis absorption of the produced Ag colloids versus time. Solution conditions: 0.1 mM Agþ, 30 mg C/L EPS, pH ¼ 7.6, 26  C. Inset: Plot of ln([Agþ]t/[Agþ]0) ¼ kt versus time. General fluorescent lamps were used as the light source.

increased, subsequently slowed and finally reached a balance. The reduction of Agþ by EPS to AgNPs under illumination can be described using a pseudo-first-order kinetic equation:


þ Ag Ln
þ  t ¼ kt Ag 0

(1)

where [Agþ]t and [Agþ]0 are the concentrations of Agþ at time t and initially, respectively; k is the pseudo-first-order rate constant. The assumed average agglomeration number of AgNPs is n (Huang et al., 1993). [Agn]t is the concentration of AgNPs at time t; then

Fig. 1. Characterization of AgNPs produced via the reduction of Agþ (0.2 mM) by EPS (20 mg C/L) under fluorescent-lamp illumination for 12 h (a) UVevis spectrum, (b) TEM image, (c) corresponding EDS spectrum and (d) XPS spectrum. The temperature and pH were 26  C and 7.6, respectively.

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i i h h n½Agn t þ Ag þ ¼ Agþ t

0

245

(2)

Comparing Equations (1) and (2) gives:

  n½Agn  Ln 1 
þ  t ¼ kt Ag 0

(3)

The absorbance of Ag colloid satisfies Lambert-Beer's law (Huang et al., 1993):

Abs ¼ a½Agn t

(4)

Thus, the change in absorbance of the produced AgNPs with time is described in Equation (5):

i h Abs ¼ m Agþ ½1  expð  ktÞ 0

(5)

where Abs is the maximum absorbance value of Ag colloid; a and m are constants, and m ¼ a/n. Fitting the data with Equation (5) gives the predicted line (Fig. 2) and rate constant k (0.056 h1). The linear relationship of ln([Agþ]t/[Agþ]0) vs. reaction time, which is regressed by Equation (1) (inset of Fig. 2), also indicates that the reduction of Agþ in the presence of EPS follows the pseudo-first-order kinetics. The rate constant of AgNPs formation under illumination was much higher than that in the dark. 3.2. Effect of visible and UV light on AgNPs formation To investigate the effect of sunlight of different wavelengths on the AgNPs formation, the reduction of Agþ by EPS under simulated sunlight irradiation (290e800 nm) was explored. Exposure to full simulated sunlight, the AgNPs colloids with yellow color quickly formed, and the pseudo-first-order rate constant was 0.174 h1. However, the formation rate of AgNPs decreased to 0.143 h1 under visible light irradiation when a 400-nm cut-off filter was used (Fig. 3a, Table S3). Almost no AgNPs were formed under dark conditions in the test. The visible or UV light its own had very little influence on the reduction of Agþ (Fig. S4). This result indicates that both visible light and UV light can induce the reduction of Agþ by EPS. To confirm the role of UV light in the reduction of Agþ by EPS, UV light was specially adopted to irradiate the reaction system. Significant formation of AgNPs was observed in the EPS solution after several hours of exposure to UV light (Fig. 3a), which further confirms that UV light can catalyze the reduction of Agþ by EPS. Compared to the formation rate of AgNPs under visible-light illumination, the reaction rate was much faster under UV light (p < 0.05), although the UV light intensity was much lower than the visible light intensity (Fig. 3a). The pseudo-first-order rate constant was higher under UV light (k ¼ 0.188 h1) than under visible light conditions (k ¼ 0.143 h1) (Table S3), which suggests the higher catalytic efficiency of UV light. No apparent increase in absorbance was observed in the control under dark conditions within 60 h (Fig. 3b), and obvious SPR peak of AgNPs appeared until 14 days (Fig. S2). This result implies that the reduction of Agþ by EPS in the dark requires a relative high activation energy (Yin et al., 2012). Light illumination can stimulate this process. 3.3. Effects of pH and dissolved oxygen (DO) on Agþ reduction To better understand the transformation of Agþ in natural waters, the effects of pH and DO on the reduction of Agþ by EPS were

Fig. 3. Plot of the maximum SPR absorbance of Ag colloid versus time in the EPS solution under illumination. Experimental conditions: (a) 0.2 mM Agþ, 20 mg C/L EPS; (b) 0.4 mM Agþ, 30 mg C/L EPS. All experiments were performed at 26  C, and the solution pH was 7.6. Error bar indicates the standard deviation of mean, n ¼ 2. Detailed captions of light intensity of different wavebands are shown in Table S3. A Xe lamp was used as the light source.

investigated. Fig. 4a shows that the generation rate of AgNPs accelerated with the increase in pH in the weak alkaline range (p < 0.05). Because there are many functional groups in EPS, such as carboxyl, phosphoryl, phenolic and hydroxyl groups, EPS have a high binding capacity for heavy metals (Sheng et al., 2010). With the increase in pH from 7.0 to 9.0, more groups in EPS deprotonated and facilitated the combination between the negatively charged EPS and the positively charged Agþ to form the metal-organic complex. Furthermore, with an increased pH, the proteins in EPS would transform from helical to random coil with extended chain structure because of the increased electrostatic repulsion of the intra and inter-colloids, which might result in more available binding sites in EPS for Agþ (Li and Yu, 2014). Therefore, more Agþ ions were adsorbed and aggregated by EPS at higher pH. The electrons from EPS are more easily transferred to adsorbed Agþ than to free Agþ in the solution. Thus, a higher solution pH leads to a higher reduction rate. In addition, a higher pH may decrease the redox potential of some components or functional groups in EPS, such as humic acids (Aeschbacher et al., 2011), tyrosine and tryptophan residues in the proteins and phenolic groups (Harriman, 1987; Song and Hu, 2010), which reduces the reaction energy barrier and promotes the reduction of Agþ. Fig. S5 shows that Agþ reduction by EPS extracted under ambient condition was slower than Agþ reduction by EPS

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Fig. 4. Effects of pH and DO on the reduction of Agþ by EPS (20 mg C/L) at 26  C under fluorescent-lamp illumination (500 mW/cm2). UVeVis spectrum of the produced AgNPs in the EPS solution (a) at different pH values (7.0, 8.0, and 9.0) and (b) with and without dissolved oxygen and.

after electrochemical reduction. Some reducing groups in EPS may also be oxidized by dissolved oxygen to decrease the Agþ reduction rate by EPS. Previous works also reported that a high pH could enhance AgNPs formation through glucose reduction. (Wang et al., 2005; Khan et al., 2011). As shown in Fig. 4b, the formation rate of AgNPs in the solution that was purged with atmospheric oxygen was obviously slower than that under anaerobic conditions (p < 0.05) (Fig. 4b). The produced AgNPs can be oxidized by dissolved oxygen to release Agþ again (Liu and Hurt, 2010; Chaki et al., 2004; Xiu et al., 2012; Siriwardana et al., 2015). The decreased concentration of AgNPs further weakened the Agþ reduction on their surface (Xue et al., 2008) and decreased the reduction rate. In addition, hydroxyl radicals were detected under aerobic conditions under light illumination (400e800 nm) (Fig. S6), whereas no OH was detected in the dark control. For the SPR of AgNPs under visible light, the excited AgNPs could reduce dissolved O2 to OH (Kamat, 2002), and H2O2 might also produce from the reduction of dissolved O2 by the generated AgNPs (Song and Hu, 2010). These strong oxidizing ROS competed for electrons from EPS and oxidized the produced AgNPs to Agþ, which decreased the Agþ reduction rate (Fraser and Sims, 1984).

3.4. Enhancement mechanisms of AgNPs formation under visible and UV light A higher formation rate of AgNPs was observed under visiblelight irradiation than in the dark control. The reduction rate

drastically decreased when the light was shut off, and the reduction rate was almost equal to that in the dark (Fig. 3b). However, the AgNPs formation via Agþ reduction began to increase again after the light irradiation was switched on. This result demonstrates that visible light indeed can enhance the reduction of Agþ by EPS possibly because of the light-induced autocatalysis of produced AgNPs (Xue et al., 2007, 2008), which is similar to the photodeposition of noble metals like Ag, Au and Pt. The visible light could induce the reduction of these ions to elementary metals (Liang et al., 2015). For the SPR of AgNPs excited by visible light, the adsorbed Agþ on the surface of AgNPs could be more easily reduced by the surrounding EPS, which resulted in the faster formation rate of AgNPs. To verify this mechanism, two monochromatic irradiation wavelengths (415 nm and 600 nm) were used. Fig. S7 shows that the absorbance of AgNPs rapidly increased under 415 nm monochromatic light irradiation, which is near the SPR peak of AgNPs. However, the SPR peak slowly increased under 600 nm monochromatic light irradiation. This result shows that the light of different wavelengths in the visible region has different catalytic efficiencies to enhance the AgNPs formation, implying that the SPR of AgNPs excited by visible light contributes to the reduction of Agþ by EPS. As shown in Fig. 3a, UV could also enhance the Agþ reduction compared to the dark conditions. The reason may be that under UV light illumination, some active species with strong reducing ability formed, e.g., hydrated electrons and reducing radicals. Many previous works have reported that the hydrated electrons can be photoproduced from the ionization of aromatic compounds in natural organic matter under UV irradiation (Zepp et al., 1987; Wang et al., 2007; Kumamoto et al., 1994; Thomas-Smith and Blough, 2001). The components of EPS were similar with that of natural organic matter, and the aromatic compounds were also found in our extracted EPS (Fig. S1, Table S2). Therefore, the hydrated electrons might be produced in EPS solution under UV irradiation to enhance the Agþ reduction. For example, the tryptophan residues in EPS could be excited by UV light to eject hydrated electrons (Sherin et al., 2006). Furthermore, the alcoholic and carbonyl compounds in EPS may also be excited to produce strong reducing radicals (Hada et al., 1976; Kometani et al., 2002; Yonezawa et al., 1991; Henglein, 1998) to enhance Agþ reduction. Fig. 5 shows possible enhancement mechanisms of Agþ reduction by EPS under light irradiation. Initially, the reducing groups in EPS, such as aldehyde and phenolic groups (Kang and Zhu, 2013), can directly reduce Agþ to form Ag0n seeds. This process is independent of light. Then, these Ag0n seeds grow to form larger AgNPs. If visible light is available, the AgNPs can be further excited to produce “hot electrons” on their surface, which subsequently transfer to adjacent Agþ, whereas the “hot holes” are filled with electrons from the reducing groups in EPS (Xue et al., 2008) and accelerate the reduction of Agþ by EPS. In addition, UV light can excite the EPS solution to produce hydrated electrons or reducing radicals to accelerate the reduction of Agþ. Microbial EPS have complex components including proteins and carbohydrates (Sheng et al., 2010). For the challenge to separate the various components, alginate and BSA were selected as models to represent the polysaccharides and proteins in EPS, respectively, to clarify which EPS components contribute to the reduction of Agþ. The SPR peak and TEM image of AgNPs in Fig. 6a show that AgNPs were generated in alginate solution under both visible and UV light irradiation. Furthermore, the reduction of Agþ in the presences of alginate under UV light was faster than that under visible light. Fig. 6b also demonstrates the formation of AgNPs in BSA solution under both visible and UV light irradiation. Under dark control, the reduction of Agþ was slower compared to that under light

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Fig. 5. Proposed mechanism of the effect of visible and UV light on the reduction of Agþ by EPS.

can also contribute to the reduction of Agþ under illumination. In brief, both visible and UV light could enhance the reduction of Agþ in alginate and BSA solution compared to dark control, implying that both polysaccharides and proteins in EPS might contribute to the reduction of Agþ under natural light irradiation. Various polysaccharides were reported to be able to reduce Agþ (Park et al., 2011). The hemiacetal reducing ends of polysaccharides account for the reduction of Agþ (Vigneshwaran et al., 2006), and abundant hydroxyl groups in polysaccharides may also be involved in the Agþ reduction (Donati et al., 2009). In addition, the reducing capacity of proteins for Agþ may result from the reducing amino acid residues such as tyrosine and tryptophan (Selvakannan et al., 2004; Si and Mandal, 2007).

4. Conclusions

Fig. 6. UVeVis spectra and TEM images (inset) of the produced AgNPs in (a) alginate solution (50 mg/L) and (b) BSA solution (50 mg/L). The initial concentration of Agþ was 0.2 mM. All experiments were performed at 26  C, and the solution pH was 7.6. Detailed captions of light intensity of different wavebands are shown in Table S3. A Xe lamp was used as the light source.

irradiation, and small SPR peak of AgNPs was formed in alginate solution after 28 days. However, no AgNPs were formed in BSA solution even for 28 days (Fig. S8), suggesting that polysaccharides in EPS dominated the reduction of Agþ under dark conditions and this result was consistent with previous reports (Kang and Zhu, 2013; Kang et al., 2014). It should be noticed that proteins in EPS

In this work, the AgNPs formation through Agþ reduction by EPS under light illumination has been confirmed. It was found that this process followed the pseudo first order kinetics under both visible and UV light. AgNPs formation could be enhanced with light irradiation (290e800 nm) significantly, and the enhancement mechanism was proposed. It was also proved that solution chemical conditions, like pH and dissolved oxygen, would influence the AgNPs formation through Agþ reduction by microbial EPS. These results are useful to better understanding the role of EPS on the Agþ transformation in natural waters. The species of Agþ changes after the reduction by microbial EPS in natural waters and subsequently shifts the ecotoxicity of Ag, implying that the ecotoxicity of some other similar heavy metals might also change after reducing by EPS. Thus, microbial EPS reducing capacity may play an important role in the transformation and ecotoxicity of heavy metals in natural waters or wastewater treatment process. More works on the transformation of heavy metals through adsorption, complexation and reduction by microbial EPS should be investigated.

Acknowledgments The authors wish to thank the Natural Science Foundation of China (21377123 and 51322802), the Fundamental Research Funds for the Central Universities, and Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC020) for the partial support of this study.

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