Nature and Value of Freely Dissolved EPS Ecosystem Services

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Nature and Value of Freely Dissolved EPS Ecosystem Services: Insight into Molecular Coupling Mechanisms for Regulating Metal Toxicity Weijun Shou, Fuxing Kang, and Jiahao Lu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04834 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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Environmental Science & Technology

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Nature and Value of Freely Dissolved EPS Ecosystem Services: Insight into Molecular

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Coupling Mechanisms for Regulating Metal Toxicity

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Weijun Shou, Fuxing Kang*, and Jiahao Lu

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College of Resources and Environmental Sciences, Nanjing Agricultural University, Jiangsu 210095,

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China

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Manuscript prepared for Environmental Science & Technology

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November 22, 2017

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ACS Paragon Plus Environment


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ABSTRACT: Extracellular polymeric substances (EPS) dispersed in natural waters play a

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significant role in relieving impacts to microbial survival associated with heavy metal release; yet,

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little is known about the association of freely dissolved EPS ecosystem services with metal

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transformation in natural waters. Here, we demonstrate that dispersive EPS mitigate the metal

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toxicity to microbial cells through an associative coordination reaction. Microtitrimetry coupled

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with fluorescence spectroscopy ascribes the combination of freely dissolved EPS from Escherichia

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coli (E. coli) with Cu2+/Cd2+ to a coordination reaction associated with chemical static quenching.

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Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and

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computational chemistry confirm that carboxyl residues in protein-like substances of the EPS are

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responsible for the coordination. Frontier molecular orbitals (MOs) of a deprotonated carboxyl

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integrate with the occupied d orbitals of Cu2+ and/or d, s orbitals of Cd2+ to form metal-EPS

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complexes. Microcosmic systems show that because the metal-EPS complexes decrease cellular

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absorbability of metals, E. coli survivals increase by 4.3 times for Cu2+ and 1.6 times for Cd2+,

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respectively. Based on bonding energies for six metals-EPS coordination, an associative toxic effect

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further confirms that increased bonding energies facilitate retardation of metals in the EPS matrix,

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protecting against E. coli apoptosis.

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Table of Contents

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INTRODUCTION

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Extracellular polymeric substances (EPS) not only form three-dimensional architectures for

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cell adhesion to surfaces but also constitute a freely dissolved, healthy broth associated with

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microbial ecosystem services1. Owing to their ubiquitous dispersion2 in natural waters, freely

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dissolved EPS are likely to encounter various chemicals, such as toxic heavy metals, and defuse

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their toxicity through coordination reactions3, reduction4, 5, and antagonism6. Previous studies have

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shown that when microbial cells encounter toxic Cr6+, the normalized accumulation of excreted

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dissolved microbial products (SMP) in natural water increases from 2% to 20%7. This

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“defusing-accretion” response relationship shows that initiative EPS ecosystem services are

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associated with heavy metal coordination. Nevertheless, little information is available to explain

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microbial EPS ecosystem services in natural waters, and a proper investigation is necessary.

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Ecosystem services regarding microbial survival are associated with freely dissolved

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EPS-mediated metal transformation in surface waters. Due to charge anisotropy, electronegative

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functionalities in EPS, such as carboxyl, phenolic, and phosphate groups, can coordinate with

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electropositive metals8, mobilizing free metals in the form of metal-EPS complexes. Additionally,

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freely dissolved EPS can also rely on active functionalities (e.g., hemiacetal and sulfhydryl) to

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reduce high-oxidation-state Ag+, Au3+, Cr6+, and U6+ species4,

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activities against oxidative stress to microbial cells. Therefore, complexation and reduction are two

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key ways to defuse metal toxicity to microorganisms. Nevertheless, most heavy metals are

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subordinate to transition metals with low oxidation states and do not easily capture electrons from

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active EPS through the reductive reaction. Coordination with EPS appears to be particularly

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important to mitigate the metal toxicity. Thus, freely dissolved EPS ecosystem services coupled

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with metal coordination is worthy of in-depth study for the improvement of natural water 4


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, inactivating their chemical

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environments. Metal coordination mediated by sludge-engineered EPS has been frequently studied12,

13

.

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Studies based on spectral technologies (e.g., Fourier transform infrared spectroscopy, FTIR and

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X-ray photoelectron spectroscopy, XPS) elucidate the numerous functionalities in freely dissolved

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EPS, including phosphate, phenolic, carboxyl, and amino groups, responsible for coordination14, 15.

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Titration technology is also used to identify the electrostatic binding sites associated with amino,

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carboxyl, and phenolic groups in sludge-engineered EPS, implying that these functionalities are

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crucial to the biosorption of metals16. Therefore, these technologies provide a qualitative description

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of the importance of the EPS functionalities. As the precise and molecular roles in variously freely

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dissolved EPS components have not been defined and their contributions to ecosystem services are

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poorly understood at a more precise level17, it is difficult to assume that some inappreciable

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functionalities (e.g., low-content phosphoric groups18) make a critical contribution to coordination

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and the associated ecosystem services. These results may prohibit a much deeper understanding of

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nature and value of freely dissolved EPS ecosystem services.

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Currently, a pair of critical priorities are to quantitatively describe the key functionalities

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responsible for metal coordination and to discriminate the bonding mechanisms between

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complicated EPS mixtures and metals. On the basis of multiple technologies (e.g. spectral

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recognition and computational chemistry), it has been found that EPS overlaying bacterial cells can

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protect against cell damage from exogenous stressors4, 6. These successful cases provide important

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and convincing information on the discrimination of bonding sites, which is helpful to

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quantitatively describe the association of freely dissolved EPS ecosystem services with metal

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coordination. Meanwhile, empirical studies have determined that adhesive EPS attached to the cell

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surface forms a barrier to relieve the toxicity of ZnO nanoparticles19 and Au3+9. Metal coordination 5



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with freely dissolved EPS and its potential effect on metal toxicity are not well understood,

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although toxic metals in natural waters is a mature topic.

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Thus, it is expected that the combined approach of fluorescence microtitrimetry, batch

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experiment, and computational chemistry can offer deeper and more comprehensive insight into

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freely dissolved EPS ecosystem services coupled with heavy metal coordination. In the present

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study, copper (Cu2+) and cadmium ions (Cd2+), as a pair of model metals due to their high

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abundance in the environment, are adopted to investigate the EPS-metal coordination. To further

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characterize key functionalities, X-ray photoelectron spectroscopy (XPS) and Fourier transform

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infrared spectroscopy (FTIR) are used to investigate the functional structures of freely dissolved

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EPS. A group of batch experiments are performed to explore the association of E. coli survival with

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metal coordination and the cellular absorbability of metals. The results obtained from the

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computational chemistry are used to predict the key functionalities and analyze the electron-shared

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orbital interactions associated with metal-EPS coordination. To obtain a statistical significance, six

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“metal-EPS” structures (Cd2+-EPS, Cu2+-EPS, Cr3+-EPS, Ca2+-EPS, Zn2+-EPS, and Al3+-EPS) are

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used to determine the mutual relation between E. coli survival and bonding energy.

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MATERIALS AND METHODS

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Materials. Six sulfates (analytical pure > 98.0%) – cadmium sulfate (CdSO4), copper sulfate

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(CuSO4), chromium sulfate (Cr2(SO4)3), calcium sulfate (CaSO4), zinc sulfate (ZnSO4), and

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aluminum sulfate (Al2(SO4)3) – were purchased from Sinopharm Chemical Reagent Co., Ltd.

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(China). Cadmium, copper, chromium, and zinc were subordinate to the transition metals, and

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aluminum and calcium were subordinate to the main group metals, which could also coordinate

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with freely dissolved EPS. Peptone, yeast extract, and biotechnology-grade NaCl, purchased from

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Oxoid Co., Ltd. (England), were used to prepare the Luria-Bertani (LB) medium (10 g/L of peptone, 6


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5 g/L of yeast extract, and 10 g/L of NaCl). When necessary, solid agar medium (Oxoid, England)

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was prepared with 15 g of agar powder for each 1 L of LB medium. Sodium hydroxide (NaOH, ≥

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98%) and sulfuric acid (H2SO4, ≥ 96%) were used to adjust the acidity and/or alkalinity

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(Sigma-Aldrich, St. Louis, MO). Ultrapure water was used in all experiments (Millipore, Bedford,

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MA).

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Preparation of Freely Dissolved EPS. A Gram-negative Escherichia coli (E. coli) DH5a was

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chosen to extract the freely dissolved EPS. The bacterial strain, stored at -60°C, was recovered in 20

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mL of LB medium for 12 h at 37°C and 120 rpm. The recovered bacterial suspension (about 1.0 ×

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109 cells/mL) was inoculated to 1 L of fresh LB medium and grown for another 48 h to reach a

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stable phase (37°C and 120 rpm). The bacterial cells were separated from the suspension by

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centrifugation for 6 min at 4°C and 6000g, followed by washing for 3 times with Milli-Q water to

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fully remove the medium. The bacteria were then suspended in Milli-Q water (approximately 9.0 ×

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109 cells/mL). Freely dissolved EPS were extracted from the bacterial suspension using a modified

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method20. The cell suspension was processed by ultrasounication at 60 W and a frequency of 40

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kHz at 4°C for 6 min and then centrifuged at 15000g at 4°C for 20 min to separate dissolved EPS

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from the bacterial cells. The supernatant was filtered through a 0.22-µm membrane (Millipore,

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USA). The filtrate acted as a freely dissolved EPS solution and was stored at 4°C for the following

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analyses.

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The dry weight (DW) of freely dissolved EPS was measured after drying for 48 h at 105°C

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(36.7 mg/L). Constituents of EPS, including proteins (36.7 mg/g), saccharides (7.2 mg/g), and

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nucleic acid (2.1 mg/g), were determined according to the literature21-23. The low nucleic acid

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concentration in EPS, which was less than that in previous reports14, indicated negligible cell lysis

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during the EPS extraction, because its content was less than that in previous reports14. The carboxyl 7



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group content in freely dissolved EPS was determined by a previous method24 (carboxyl equivalent:

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15.1 µmol/g).

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Fluorescence Quenching Combined with Microtitrimetry. Fluorescent chromophores in

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EPS are generally related to tyrosine, tryptophan, and quinoid structures25. In this study, there was

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only a fluorescence peak, which was associated with tryptophan in the protein-like substances25 in

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EPS (shown in Figure 1). Three-dimensional excitation-emission matrix (3DEEM) fluorescence

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spectroscopy combined with microtitrimetry26 was used to quantitatively explore the metal-EPS

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association26 through using natural tryptophan probe. Specifically, an aqueous metal stock solution

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(10 mmol/L) was titrated into 20 mL of dissolved EPS solution (0.5 mg/L, dry weight basis) using a

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chromatographic injector (25-mL scale, Agilent, USA), followed by magnetic stirring for 20 min at

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160 revolutions per minute (rpm, pH 7.0, and 25°С). Both the fluorescence spectrum and intensity

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were recorded over an excitation wavelength (EX) of 200 ~ 370 nm (5-nm band width) and an

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emission wavelength (EM) of 280 ~ 500 nm (5-nm bandwidth) at a scanning speed of 3000 nm/min

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(F96PRO, Lengguang, China). The titration and detection procedures were performed for eight

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reciprocating cycles. The relationship between the fluorescence intensity and added metal can be

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described by the following Stern-Volmer equation27,

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⁄ = 1 + = 1 +

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where F0 and F are the relative fluorescence intensity of the chromophore in the absence and

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presence of the quencher (metal ion), respectively; [Q] is the concentration of the quencher; KSV is

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the Stern-Volmer quenching constant; Kq is the bimolecular quenching rate constant; and τ0 is the

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average lifetime of the fluorophore in the absence of the quencher. For the static quenching process,

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the following equation can be used to determine the association constant (KA) and number of

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bonding sites (n)28, 8


(1),

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log − ⁄ = log + log

(2).

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Absorption of Metals by E. coli and E. coli Survival. Two types of microcosmic system

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experiments – with and without addition of freely dissolved EPS – were performed to explore the

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effect of metals on E. coli survival. For the microcosmic system with freely dissolved EPS, E. coli

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cells were suspended in 50 mL of freely dissolved EPS solution (73.4 mg/L, dry weight basis) to

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obtain approximately 2.4 × 106 cells/mL, followed by the addition of the desired metal

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concentration (Cu2+: 0–35 µmol/L; Cd2+: 0–350 µmol/L). The mixed solutions were incubated for 6

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h at 25°C and 170 rpm; afterwards, 100 µL of each solution was uniformly spread onto the surface

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of a LB solid culture medium. Each petri dish (90 mm in diameter) was left upright for 30 min to

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ensure that the E. coli solution was fully imbibed by the solid culture medium and was then inverted

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for 36 h at 37°C in the dark. The bacterial survival (%) was calculated as the ratio of colonies in the

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presence and absence of metal. Likewise, a separate set of experiments were performed to explore

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the effect of metals on E. coli survival in the absence of freely dissolved EPS. Each sample (20 mL)

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was treated by centrifugation at 6000 rpm to obtain a suspension containing metal and freely

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dissolved EPS. The concentrations of Cu2+ and Cd2+ in aqueous solution were determined by atomic

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absorption spectrometry (Thermo, USA), following previous methods29, 30. In the microcosmic

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system without addition of freely dissolved EPS, the operational procedures were the same as those

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with addition of freely dissolved EPS.

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FTIR and XPS Analyses. A separate set of experiments was performed to characterize the

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structural information of the metal-EPS complexes (EPS: 73.4 mg/L; metal: 1.0 mg/L). After

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reaction for 6 h at 25°C, the sample was freeze-dried at -65°C for one week (Labconco, UK).

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Fourier transform infrared (FTIR) spectroscopy was performed to characterize changes in the

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functional groups in EPS. The FTIR spectra of the freeze-dried samples mixed with KBr (mass ratio 9



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of 1:100) were acquired on a Nicolet NEXUS870 spectrometer (Nicolet). X-ray photoelectron

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spectroscopy (XPS) was further performed on the metal-EPS complexes with a 30.0-eV pass energy

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for the broad survey scan and a 70.0-eV pass energy for the high-resolution scan using a PHI 5000

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VersaProbe spectrometer (UlVAC-PHI, Japan).

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Computational Chemistry. Computation can be used to elucidate three important aspects of

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the complexes –bonding sites, shared electrons, and bonding energies – to demonstrate metal-EPS

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coordination mechanisms through mutual “theory-experiment” exploration. Specifically, based on

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the fluorescence and FTIR spectral data, we built the most compact oligopeptide (GlyTrp) including

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a tryptophan residue and glycine. The tryptophan residue was linked to the carboxyl in glycine via a

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condensation reaction associated with the formation of a peptide bond. Here, glycine was chosen on

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the basis of its high abundance in EPS and simple structure31, 32. This compact oligopeptide had a

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completely functional protein structure, containing a carboxyl residue, amino residue, and peptide.

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In addition, the carboxyl residue was kept some distance apart from the amino residue. The Gabedit

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software (Version 2.4.5)33 was used to build the molecular models, which included the six listed

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hydrated metallic ions and GlyTrp. Each metallic ion was completely surrounded by six water

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molecules, followed by the computation allowing the free movement of all atoms at the

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B3LYP/def2TZVP level. After the computation, oxygen atoms in the water molecules were

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associated with the central metal atom to form a hydrated metallic ion. The hydrated metallic ions

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were manually docked to GlyTrp using Gabedit software (Version 2.4.5)33. All molecular structures

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were optimized at the B3LYP/def2TZVP level34 using the Gaussian 09 package35. The hydrated

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metallic ions were allowed to freely move around the carboxyl residue, amino residue, and peptide.

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After the computation, the bonding site was determined based on a comparison between the

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theoretically predicted and experimental FTIR spectra. In addition, the molecular energies were 10


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analyzed at the same computational level, and the bonding energies were calculated by the

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following equation,

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Bonding energy Δ =

!"#$%&⋯()*+"

−

!"#$%&

+

()*+"

(3),

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where Gmetal···GlyTrp, Gmetal, and GGlyTrp are the Gibbs free energies (G) of the metal···GlyTrp, metal,

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and GlyTrp residue in EPS, respectively.

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Charge-Transfer Properties. The wave function obtained from computation was analyzed by

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the Multiwfn software36 to further elucidate the electron-shared mechanism regarding metal-EPS

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coordination at the key carboxyl site. The process was described by a generalized charge

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decomposition analysis (GCDA)37, as follows:

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,- = ∑;∈A ∑=∈@ /-

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B- = ∑;∈A ∑=∈@ 2

34 51 34 | |12 6

1789

:;,- :=,- >;,=

34 ,1 34 (DE12 6

1789

/- :;,- :=,- >;,=

(4), (5),

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where m, n, and i denote the fragment orbital (FO) of fragment A, the FO of fragment B and the MO

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of complex, respectively; C is the coefficient matrix of complex MO in the FO basis; S is the

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overlap matrix between FOs; η denotes the orbital occupation number; ηref equals 1.0 or 2.0 for

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GH GH open-shell and closed-shell cases, respectively; and min/( , /E denotes the minimum of the

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GH two values. In the calculation of t, if only /( > /EGH terms are considered, then t corresponds to

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GH the amount of electron donation from A to B, while if only /( < /EGH terms are considered, then t

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can be interpreted as back-donation from B to A. The r term essentially represents the overlap

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population between the occupied FOs of the two fragments.

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RESULTS AND DISCUSSION

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Metal Coordination to Dissolved EPS. Three-dimensional excitation-emission matrix (EEM)

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fluorescence spectroscopy was applied to characterize metal coordination to freely dissolved EPS.

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In the EEM spectra, the locations of the fluorescence peaks of freely dissolved EPS were as 11



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follows25: EX = 220 – 250 nm/EM = 280 – 380 nm: tyrosine and aromatics in protein-like

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components; EX > 250 nm/EM = 280 – 380 nm, tryptophan in protein-like components; EX = 220

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– 250 nm/EM > 380 nm: fulvic acid-like components (phenol/quinone structure); and EX > 250

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nm/EM > 380 nm: humic acid-like components (phenol/quinone structure). In Figure 1a, there is

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only one fluorescence peak (EX/EM = 280/340 nm), which is associated with tryptophan in freely

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dissolved EPS. Fulvic acids, humic acids, and tyrosine were not observed in Figure 1a. This result

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suggests that tryptophan in freely dissolved EPS acts as a natural fluorescence probe, which is

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critical to explore metal-EPS coordination.

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3DEEM fluorescence spectroscopy combined with quenching titration was used to

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quantitatively probe metal-EPS coordination. Figure 1b shows the final 3DEEM fluorescence

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spectrum of freely dissolved EPS after a Cu2+ titration (9.7 × 10-5 mol/L). The fluorescence

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intensities in the 3DEEM spectrum decreased from 372 (pristine EPS in Figure 1a) to 206 a.u. To

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reveal the formation of the Cu2+-EPS complex, we exhibit the continuous change in the

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fluorescence intensity. Figure 1d shows that as Cu2+ increased from 0, 1.7 × 10-5, 2.7 × 10-5, 3.6 ×

240

10-5, to 9.7 × 10-5 mol/L, the fluorescence intensity regarding the tryptophan probe continuously

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declined (corresponding to 372, 333, 308, 228 and 206 a.u.), suggesting that fluorescence

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quenching resulted from formation of the Cu2+-EPS complex. Likewise, Figure 1c also shows that

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the fluorescence intensity declined to 240 au, suggesting Cd2+-EPS coordination. Figure 1d further

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shows that with the addition of Cd2+, the fluorescence intensity also continuously declines,

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confirming the formation of the Cd2+-EPS complex. Overall, these results regarding the formation

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of metal-EPS complexes indicate that tryptophan in freely dissolved EPS can be quenched by the

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addition of Cu2+ and Cd2+ and is appropriate to serve as a probe to describe the formation of

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metal-EPS complexes. 12


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The type of fluorescence quenching, including diffusion-controlled and chemical static

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quenching, can be discriminated by changes in the fluorescence spectrum. Figure 1d shows a series

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of redshifts in the emission wavelength. In pristine EPS, the maximum intensity is located at 337

252

nm. As the Cu2+ concentration increased to 9.7 × 10-5 mol/L, the fluorescence wavelength redshifted

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by 9 nm to 346 nm. Likewise, as the Cd2+ concentration increased to 9.7 × 10-5 mol/L, the emission

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wavelength was redshifted by 8 nm to 345 nm. Generally, diffusion-controlled dynamic quenching

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is associated with the interactive collision of molecules/ions, while chemical static quenching is

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closely related to the formation of coordination compounds38. In this study, the redshifts are caused

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by chemical static quenching, conclusively confirming the formation of the metal-EPS coordination

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compounds.

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From the above microtitration and equation 1, we calculated the Stern-Volmer plots of F0/F

260

versus added Cu2+ (pH 7.0 and 25 °C). Figure 1e shows the good linear correlation between them,

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and the quenching constant (KSV) is 1.0 × 104 L/mol. The KSV for Cd2+-EPS coordination is 2.5 ×

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103 L/mol. The KSV for the Cu2+-EPS complex is almost one order of magnitude higher than that for

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the Cd2+-EPS complex. Apparently, compared to the latter, Cu2+ more efficiently quenches the

264

dissolved EPS due to its stronger coordination capacity. It is well known that the average lifetime of

265

a tryptophan-related chromophore is ~5 × 10-9 s19. Based on equation 1, the bimolecular quenching

266

rate constant (Kq) was calculated to be 2.0 × 1012 for Cu2+-EPS and 5.0 × 1011 L/mol/s for Cd2+-EPS.

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Generally, the maximum Kq for a diffusion-controlled quenching process is approximately 2.0 ×

268

1010 L/mol/s39. Higher Kq values in the present study further confirm the occurrence of a static

269

quenching process involving in metal-EPS coordination40. Furthermore, based on equation 3, Figure

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1f shows a pair of good linear correlations between log[(F0-F)/F] and log[Q]. Number of bonding

271

sites (n) are 0.93 for the Cu2+-EPS complex and 0.99 for the Cd2+-EPS complex, respectively. The 13



272

values of n are close to 1.0, indicating a single bonding site in the metal-EPS complexes. Based on

273

the intercept (logKA), the bonding constant (KA) are 5.0× 103 L/mol for Cu2+-EPS and 2.2 × 103

274

L/mol for Cd2+-EPS, respectively, reconfirming that Cu2+-EPS coordination is stronger.

275

Coordination Site at Freely Dissolved EPS. The coordination site in freely dissolved EPS

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can be discriminated through changes of the functionalities in FTIR. The black plots in Figure 2a-c

277

show the experimental FTIR spectra; the blue bar charts and red plots represent the Lorentz

278

oscillators and computed FTIR spectra, respectively. In the experimental FTIR spectra, the

279

absorption band of pristine EPS (Figure 2a) at 1655 cm-1 is assigned to C=O stretching (amide I)41,

280

and the band near 1547 cm-1 is assigned to N-H bending/C-N stretching (amide II) in peptides4, 8.

281

The band at 1452 cm-1 is ascribed to the deformation vibration of CH241. The bands at 1402 and

282

1232 cm-1 are related to the stretching of C-O and C=O in carboxylates, respectively, while that at

283

1082 cm-1 is related to the deformation vibration of C-O in the carboxyl group42.

284

After reaction with Cu2+ (Figure 2b), two bands at 1082 cm-1 and 1402 cm-1 disappear or

285

weaken compared to those of pristine EPS (Figure 2a). These changes, associated with carboxyl

286

groups, show that carboxyl groups (C-O) contribute more to Cu2+ coordination with dissolved EPS.

287

Likewise, Figure 2c also shows a pair of weaker vibrations at 1082 and 1402 cm-1, reconfirming

288

that carboxyl group in EPS are involved in formation of Cd2+-EPS coordination. This conclusion is

289

supported by previous results13. Notably, Figure 1 shows that there are no humics in freely

290

dissolved EPS. Therefore, carboxyl groups from humics can be rules out as the metal coordination

291

site. Carboxyl terminals in amino acids, rather than in humics of freely dissolved EPS, are

292

responsible for metal-EPS coordination.

293

The GlyTrp model was used to examine metal-EPS coordination at the carboxyl sites of the

294

amino acids in freely dissolved EPS. Figure 2a-c shows the computed results of the metal model 14


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(blue plots) based on the Lorentz oscillator (red plots). It exhibits a high precision, and suggests that

296

protein-like substances in EPS contain the majority of functionalities. Furthermore, the result also

297

shows that after reaction with Cu2+ and/or Cd2+, the vibrations of carboxyl terminals (1402 and

298

1082 cm-1) in structured GlyTrp weaken or disappear (blue plots in Figure 2b and c). Reappearance

299

of these peaks show the reliability of the functional appointment, reconfirming that the carboxyl

300

terminals of amino acids in freely dissolved EPS are mainly responsible for metal coordination.

301

XPS analysis was used to characterize the oxygen valence-electron changes in EPS (Figure 2d).

302

For pristine EPS, the O1s peak at 530.7 eV corresponds to C=O in the terminal carboxyl. There are

303

two prominent peaks at 532.4 and 533.2 eV, assigned to C-O-C in glycoside and C-OH in hydroxyl,

304

respectively. After reaction with metals, the oxygen signals (C=O) in Cu2+-EPS and Cd2+-EPS shift

305

to 531.1 eV, and their C-OH signals also shift to 533.7 eV. These increased bonding energies

306

reconfirm that metal coordination at the carboxyl terminals is closely related to the oxygen

307

functionalities, and underline the importance of the carboxyl groups of the amino acids for

308

EPS-metal coordination.

309

Bacterial Survival Facilitated by Metal-EPS Coordination. Metal coordination with freely

310

dissolved EPS in surface waters changes the metal toxicity. This process was verified through a 6-h

311

water suspension experiment (25°C and 160 rpm). Bacterial survival was calculated as the ratio of

312

E. coli densities with and without metals. Figure 3a shows that freely dissolved EPS has an

313

important effect on E. coli survival under Cu2+ stress. Although E. coli survivals with different EPS

314

manipulations show an overall decline with increased Cu2+, the survivals in freely dissolved EPS

315

are higher over a moderate Cu2+ range of 2.5 – 25 µg/L. Therefore, when Cu2+ is 10.0 µg/L, the E.

316

coli survival without freely dissolved EPS reduces to 7%; in the presence of freely dissolved EPS,

317

the E. coli survival is enhanced to 30%. Clearly, freely dissolved EPS in surface waters can relieve 15



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318

the Cu2+ toxicity. Likewise, Figure 3b shows that the E. coli survivals with the two EPS

319

manipulations decline from 100% to approximately 0%; however, at moderate Cd2+ concentrations

320

(20 – 250 µmol/L), freely dissolved EPS can enhance E. coli survival. Therein, a maximal contrast

321

shows that when the Cd2+ concentration is 100 µmol/L, the survival with freely dissolved EPS is

322

approximately 47%, whereas that without dissolved EPS is lower at 31.0%. The finding further

323

confirms that freely dissolved EPS weaken the metal toxicity through metal-EPS coordination.

324 325 326

Bacterial survival can be influenced by the cellular absorbability of toxic metals. We further analyzed the extra- and intra-cellular distribution of metals using an isothermal model43, as follows, ; K = L :M

(6),

327

; where q and :M represent the equilibrium concentrations of metal absorbed in E. coli cells

328

(µmol/106 cells) and dissolved in EPS-water phase (µmol/L), respectively, and L (µmol1-mLm/106

329

cells) is the affinity coefficient, where m (unitless) is the linearity index (larger departures from m =

330

1 imply more nonlinear absorption).

331

Figure 3c shows the relationship between Cu2+ absorbed by E. coli and Cu2+ in EPS-water

332

phase. In the absence of freely dissolved EPS, the linearity index (m) is 1.0, while in the presence

333

of freely dissolved EPS, the index is 0.93. These two linearity indexes are equal or close to m = 1.0,

334

suggesting that linearity index is not influenced by freely dissolved EPS (Table S1). The

335

distribution coefficients (Kd, calculated as a ratio of q versus Cw for each datum) fall in a narrow

336

range from 0.10 to 0.11 L/106 cells without addition of the EPS and from 0.03 to 0.05 L/106 cells

337

with addition of the EPS, respectively. It is noteworthy that the Kd values in the absence of EPS are

338

generally 2.2 – 3.3 times larger than those in the presence of EPS, which suggests that the

339

coordination of metal to freely dissolved EPS suppresses the cellular absorption of Cu2+. Overall,

340

combined with Figure 1, it directly demonstrates that freely dissolved EPS in surface waters can 16


Page 17 of 35


341

coordinate with Cu2+ to enhance E. coil survival through restraining cellular absorption of the toxic

342

metal.

343

Figure 3d shows that the linearity index is 0.81 without addition of the EPS and 0.60 with

344

addition of the EPS (Table S1), suggesting the nonlinear absorption of E. coli cells to Cd2+. The

345

up-convex curve reflects the attraction between the E. coli surface and Cd2+44. Furthermore,

346

nonlinear absorption corresponds to a wider Kd range, from 0.08 – 0.15 L/106 cells without addition

347

of the EPS and from 0.03 – 0.08 L/106 cells with addition of the EPS. Here, Kd in the absence of

348

EPS is generally 1.9 – 2.6 times larger than that in the presence of freely dissolved EPS. This

349

difference shows that although there is a poor linear correlation between Cd2+ absorbed by E. coli

350

and Cd2+ in EPS-water phase, freely dissolved EPS restrains the cellular absorption of Cd2+,

351

enhancing the E. coil survival through Cd2+-EPS coordination.

352

The relationship between E. coli survival and metal-EPS coordination was further elucidated

353

by the following quantitative analysis. In Figure 4, the x-coordinate shows the ratio of metal-EPS

354

complex to free metal in aqueous solution, while the y-coordinate represents the ratio of the cell

355

density with and without EPS. For the x-coordinate, the metal-EPS complex (ME) and free metal

356

(Mn+) are calculated based on the association constant (KA) as follows,

357

M =O + E=5 ⇌ ME

(7),

358

= :RS ⁄ :RTU × :STW

(8),

359 360 361 362 363

Because :STW is the difference between the initial concentration (:XYTW ) and the metal-EPS complex (:RS ), equation 8 can be altered as follows,

:RS \:ZRTU [ = :XYTW − :RS

(9),

where ]=5 is the initial EPS concentration (carboxyl equivalent, 15.1 µmol/g). Figure 4 shows that as the ratio of Cu2+-EPS complex to free Cu2+ increased to 0.19, the cell 17



364

density ratio rapidly increases to 12.8, indicating that free Cu2+ greatly contributes to the toxicity

365

and the formation of the Cu2+-EPS complex recovers the E. coli survival. As the ratio of Cu2+-EPS

366

to free Cu2+ increases to 0.35, the cell density ratio sharply declined to approximately 1.52. Actually,

367

with an addition of Cu2+, concentrations of both free Cu2+ and EPS-Cu complex will concurrently

368

increase in bacterial solution, although their increments are out of synchronization (x-axis in Figure

369

4). The excessive free Cu2+ will weaken the freely dissolved EPS protectiveness until it completely

370

kill off the E. coli cells. Additionally, an entirely different plot pattern is observed, in which the cell

371

density ratios gradually increase from 1.08 to 2.57 as ratio of Cd2+-EPS to free Cd2+ increases to

372

0.88 (Figure 4). This result underlines the importance of formation of Cd2+-EPS complex in

373

bacterial survival. Overall, combined with Figure 3, these results suggest that free metal ions are

374

responsible for bacterial apoptosis and the formation of metal-EPS coordination compounds

375

restrains the cellular absorption of metals, recovering the E. coli survival.

376

Orbital Interactions Associated with Metal-EPS Coordination. Orbital interaction diagrams

377

are commonly used to explore the electron-shared mechanism associated with metal-EPS

378

coordination. As is shown in Figure 5, the carboxyl cluster in EPS and the metal ions are taken as

379

the two fragments. The left side and right side in Figure 5 show the carboxyl ligand and metal ions,

380

respectively; the middle shows a metal-EPS complex orbital through orbital combination of the two

381

fragments. To avoid visual interference, the water molecules in the computational system are hidden.

382

Figure 5 shows that Cu2+-EPS coordination is linked to a pair of oxygen atoms from deprotonated

383

carboxyl residues in freely dissolved EPS. The occupied MOs (53 and 54) of carboxyl substantially

384

mix with the occupied MO (11, d orbital) of Cu2+ to yield a new higher-energy occupied MO

385

(orbital 59 in the complex); these MOs from two fragments attributed to 57% of the complex.

386

Significantly, there are two different occupied MOs (53 and 54), suggesting that the pair of oxygen 18


Page 18 of 35

Page 19 of 35


387

atoms in the deprotonated carboxyl (COO-) are chemically un-symmetric. In addition, one branch of

388

the occupied MO (53) in the carboxyl group mixes with the occupied MO (12, d orbital) of Cu2+ to

389

form a new higher-energy MO (57) in the complex. An occupied MO (52), generated by the pair of

390

oxygen atoms in the carboxyl group, mixes with an occupied MO (10, d orbital) of Cu2+ to form a

391

new higher-energy MO (58) in the complex, accounting for 27% of the complex. Overall, these

392

results confirm that a Cu2+-carboxyl hybrid orbital is responsible for metal-EPS coordination.

393

Figure 5b exhibits a slightly different hybrid orbital. The positive phases of the lowest

394

unoccupied MO (10, s orbital) of Cd2+ mixes with three occupied MOs (51, 48, and 42) of the two

395

oxygen atoms to generate the occupied MOs (63, 61, and 55) of the Cd2+-carboxyl complex. These

396

two fragments make the largest contribution (68%) to the formation of the complex. The

397

negative-phase orbital (32) of the carboxyl associates with the positive-phase orbital (6, d orbital) of

398

Cd2+ to generate an occupied MO (38) of the Cd2+-carboxyl complex through negative-positive

399

phase attraction. Two occupied MOs, containing positive (29) and negative phases (27) from a pair

400

of oxygen atoms, synchronously mix with an occupied MO (6, d orbital) of Cd2+ to form an

401

occupied MO (32) in the Cd2+-carboxyl complex. Comparing the Cu2+-carboxyl and Cd2+-carboxyl

402

complexes, the latter presents a series of more intricate hybrid orbitals that contain both unoccupied

403

and occupied orbitals. In short, the orbital interaction diagram analyses clearly show that the

404

frontier MOs of the deprotonated carboxyl cluster (C-COO-) in amino acid residues in dissolved

405

EPS are distinctly linked to the unoccupied and occupied orbitals in the metals to produce

406

metal-EPS coordination.

407

Bonding Energy Associated with Bacterial Survival. To quantitatively illuminate the

408

association of bacterial survival with metal-EPS coordination, two parameters – the association

409

constants and bonding energy – are used to determine the correlation with bacterial survival. The 19



410

association constants (KA) of six metals (Ca2+, Zn2+, Al3+, Cd2+, Cu2+, and Cr3+) and freely dissolved

411

EPS were calculated by the fluorescence microtitration method described above (equation 2, results

412

can be found in Table S2 of the Supporting Information). In Figure 6a, the bonding energy of the

413

metal-carboxyl complex, calculated at the density functional theory (DFT) B3LYP/def2TZVP level,

414

is considered to be the change in Gibbs free energy before and after metal-EPS coordination.

415

An apparent linear correlation between logKA and the computed bonding energies (correlation

416

coefficient, R2 = 0.61; slope = -19.3) can be observed in Figure 6a. As the association constant

417

(logKA) increased, the bonding energy between the carboxyl site and metals linearly increased

418

(absolute value, the negative sign represents a spontaneous binding process). Additionally, in Figure

419

6a, the computed bonding energies for the six metal-carboxyl complexes are shown to be greater

420

than 20 kJ/mol, suggesting that all carboxyl sites in EPS are able to undergo spontaneous

421

chemisorption45 associated with the chemical coordination. Most importantly, the good linear

422

correlation verifies that the carboxyl sites of the amino acid residues in dissolved EPS are mainly

423

responsible for metal-EPS coordination.

424

Figure 6b shows the linear correlation (R2 = 0.98) between the computed △G and the ratio of E.

425

coli density with (T) and without (T0) dissolved EPS at the median lethal dose of each metal. The

426

T/T0 ratios follow a descending order (on the Y-axis) as follows: Cr3+ > Al3+ > Cu2+ > Ca2+ > Cd2+ >

427

Zn2+. Compared to other metals, the toxicity of trivalent Cr3+ and Al3+ is more easily suppressed by

428

freely dissolved EPS in natural waters because of the stronger sequestering capacity associated with

429

the increased positive charge. With an increase in the computed △G at the major carboxyl site, the

430

T/T0 ratios exhibit a clear linear reduction. This finding indicates that a continuous increase in

431

bonding energy at the key carboxyl terminal residue of freely dissolve EPS can lead to the further

432

retardation of metals in the EPS matrix, which facilitates E. coli survival. 20


Page 20 of 35

Page 21 of 35


433

Environmental Implications. Freely dissolved microbial EPS widely exist in surface waters

434

and play an important role in transformation of metal speciation46 associated with microbial

435

ecosystem services47. Whereas the combinative mechanisms of freely dissolved EPS with heavy

436

metals have been extensively studied and exploited for engineering applications, little information

437

is available to explore the quantitative association of metal coordination with its toxicity in freely

438

dissolved EPS-enriching surface waters. Here, we demonstrate that freely dissolved EPS produced

439

by bacteria constitute an important active mediator that mitigates the bio-/eco-toxicity of toxic

440

metals. Specifically, a major carboxylic residue in freely dissolved EPS is responsible for Cu2+ and

441

Cd2+ coordinations, which restrains the cellular absorption of free metal ions and increases the E.

442

coli survival. Combined computational chemistry with XPS, frontier MOs of deprotonated carboxyl

443

cluster (C-COO-) in amino acid residues in freely dissolved EPS are distinctly linked to unoccupied

444

and occupied orbitals in metals to form the metal-EPS coordination compounds. Analysis regarding

445

six metals-to-EPS binding energies further suggests a continuously increase in bonding energy lead

446

to a more retardation of metals in freely dissolved EPS, which is beneficial to bacterial survival in

447

natural waters. The findings further our understanding of the nature and value of freely dissolved

448

EPS ecosystem services in natural waters.

449

21



450

ASSOCIATED CONTENT

451

Supporting Information

452

The Supporting Information is available free of charge on the ACS Publications website at DOI:

453

******. Fitted parameters related to the extra- and intracellular distribution of metals in the absence

454

and presence of freely dissolved EPS (Table S1) and fitted parameters related to the coordination of

455

metal with freely dissolved EPS on the basis of fluorescence microtitrimetry (Figure S2).

456

AUTHOR INFORMATION

457

Corresponding Author

458

*Telephone/Fax: +86-25-8439-5860; e-mail: [email protected]/[email protected].

459

Notes

460

The authors declare no competing financial interest.

461

ACKNOWLEDGMENTS

462

This work was supported by the National Science Foundation of China (Grant Nos. 21777071 and

463

41401543) and the National Science Foundation for Postdoctoral Scientists of China (Grant No.

464

2014M561662).

22


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465

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573 574

27



575

FIGURE LEGENDS

576

Figure 1. EEM characterization of dissolved EPS with Cu2+ and Cd2+: (a) EPS only, (b) Cu2+-EPS,

577

and (c) Cd2+-EPS. (d) Changes in the fluorescence emission spectrum of EPS due to Cu2+-EPS and

578

Cd2+-EPS coordination. (e) Stern-Volmer plot. (b) Plot of log [(F0 -F)/F] vs. log[Q]. In b and c, the

579

concentration of added metals is 9.7 × 10-5 mol/L. In d, for better discrimination, five of the added

580

points were selected to draw the spectral plots. The concentrations of Cu2+ and Cd2+ are shown by

581

different colors (mol/L): black line (0), red line (2.7 × 10-5), blue line (3.6 × 10-5), blue line (5.0 ×

582

10-5), purple line (6.9 × 10-5 mol/L), and brown line (9.7 × 10-5 mol/L).

583 584

Figure 2. Comparison of the FTIR spectra and XPS-O spectra of dissolved EPS before and after

585

reaction with Cu2+/Cd2+. (a, b, and c) FTIR spectra of pristine EPS, Cu2+-EPS, and Cd2+-EPS,

586

respectively. The blue bar charts and red plots represent the Lorentz oscillators and FTIR spectra

587

computed at the B3LYP/def2TZVP level, respectively. The black plot is the experimental FTIR. The

588

red arrows show the absorption bands related to the carboxyl groups in EPS. (d) Change in oxygen

589

signals in the XPS spectra of freely dissolved EPS before and after reaction with metals.

590 591

Figure 3. Bacterial survival associated with the extra- and intracellular distribution of metals: (a)

592

EPS-facilitated E. coli survival under Cu2+ stress, (b) EPS-facilitated E. coli survival under Cd2+

593

stress, (c) extracellular and intracellular distribution of Cu2+, and (d) extracellular and intracellular

594

distribution of Cd2+.

595 596

Figure 4. Relative survival of bacterial cells facilitated by dissolved EPS-metal coordination.

597 28


Page 28 of 35

Page 29 of 35


598

Figure 5. Orbital interaction diagrams for (a) Cu2+-carboxyl and (b) Cd2+-carboxyl. The vertical

599

axis shows the MO energies (eV). The solid and dashed lines (black) correspond to the occupied

600

and unoccupied MOs, respectively. All MOs are plotted as isosurface graphs with an isovalue of

601

0.03. The numbers beside the red lines indicate the contribution (purple percentage) of fragmental

602

MOs (metal or EPS) to the metal-EPS complex. Contributions below 20% are not labeled. The blue

603

and green mesh contours on the surface of the molecules (or ions) represent the negative- and

604

positive-phase orbital wave functions, respectively.

605 606

Figure 6. Correlation of the metal-carbonyl bonding energy at a major tryptophan (Trp) residue in

607

dissolved EPS (computed △Gcarbonyl-metal) with the association constant (KA, log-transformed) and

608

growth rate of E. coli cells (T/T0). (a) Computed △Gmetal-carbonyl versus logKA. (b) Growth rate (T/T0)

609

versus computed △Gmetal-carbonyl. The growth rate (T/T0) was determined based on the cell density

610

ratio with and without dissolved EPS at the medial lethal dose for each metal.

611 612 613

29



614 615

Figure 1.

30


Page 30 of 35

Page 31 of 35


616 617

Figure 2.

618 619

31



620 621

Figure 3.

622

32


Page 32 of 35

Page 33 of 35


623

624 625

Figure 4.

626 627

33



628 629

Figure 5.

630

34


Page 34 of 35

Page 35 of 35


631

632 633

Figure 6.

634

35


Nature and Value of Freely Dissolved EPS Ecosystem Services

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