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Adsorption Behavior of Extracellular Polymeric Substances on Graphene Materials Explored by Fluorescence Spectroscopy and TwoDimensional Fourier Transform Infrared Correlation Spectroscopy Bo-Mi Lee, and Jin Hur Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01286 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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Environmental Science & Technology

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Adsorption Behavior of Extracellular Polymeric Substances on Graphene

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Materials Explored by Fluorescence Spectroscopy and Two-Dimensional Fourier

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Transform Infrared Correlation Spectroscopy

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Bo-Mi Lee and Jin Hur*

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Department of Environment and Energy, Sejong University, Seoul, 143-747, South Korea

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Revised and Re-submitted to Environmental Science & Technology, June, 2016

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* Corresponding author: Tel. +82-2-3408-3826. E-mail: [email protected]

Fax +82-2-3408-4320.

ACS Paragon Plus Environment


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Abstract Adsorption isotherms of extracellular polymeric substances (EPS) on graphene oxide (GO)

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and reduced GO (rGO) were studied using fluorescence excitation-emission matrix - parallel

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factor analysis (EEM-PARAFAC) and two dimensional correlation spectroscopy (2D-COS)

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combined with Fourier transform infrared spectroscopy (FTIR). Chemical reduction of GO

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resulted in a greater extent of carbon adsorption with a higher degree of isotherm nonlinearity,

33

suggesting that heterogeneous adsorption sites were additionally created by GO reduction.

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Two protein-like and two humic-like components were identified from EPS by EEM-

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PARAFAC. Adsorption of protein-like components was greater than that of humic-like

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components, and the preferential adsorption was more pronounced for GO versus rGO.

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Adsorption of protein-like components was more governed by site-limiting mechanisms than

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humic-like components as shown by the higher isotherm nonlinearity. 2D-COS provided

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further information on the adsorption of secondary protein structures. Adsorption of the EPS

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structures related to amide I and aromatic C-C bands was greater for rGO versus GO. Protein

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structures of EPS were more favorable for adsorption in the order of α-helix → amide II → β-

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sheet structures with increasing site limitation. Our results revealed successful applicability of

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EEM-PARAFAC and 2D-COS in examining the adsorption behavior of heterogeneous

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biological materials on graphene materials.

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1. Introduction Graphene, a mono-layered and sp2 structured carbon material, has been used for a variety

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of applications including electrodes, semiconductors, sensors, and hydrogen storage, owing to

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its robust structures and excellent conductivity for heat and electricity.1-5 A recent report has 1


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projected that graphene will lead a billion-dollar industry over the next ten years.6 While a

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number of studies focused on producing cost-effective and efficient graphene materials to

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extend the applicability to many fields, public interest has rapidly grown over the

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environmental concerns with respect to their fate and transport in natural and engineered

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systems, which requires in-depth studies on related topics.4

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Graphene oxide (GO), a common form of graphene materials, can be produced in massive

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amounts through the so-called Hummer’s method, which renders GO to retain partially

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defected sp2 structures and various functional groups. Often, GO is intentionally modified

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into a reduced form to strengthen the unique physical/chemical functions, causing GO to have

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more defected sp2 structures and less abundance of oxygen-containing functional groups.

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Such modifications can even occur in natural systems via sulfur-containing reductants and/or

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bacterial activities,5 leading to the existence of various forms of graphene materials in aquatic

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environments.

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It is known that adsorption of organic chemicals to GO materials are driven by several

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adsorption mechanisms such as π bonding interaction, hydrophobic interaction, electrostatic

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interaction, and hydrogen bonding, which may cause competitive adsorption among different

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types of organic compounds.7 For example, π- π interaction is an important mechanism for

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aromatic compounds and macromolecules, while the adsorption of oxygen-containing

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compounds onto GO may be governed by hydrogen bonding.7 The adsorption behavior of

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organic matter is affected by its structure as well as co-existing species. It was reported that

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aromatic fractions in organic matter showed a higher affinity to adsorb on GO surfaces than

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non-aromatic fraction.8 Such a preferential adsorption can occur for humic acids with

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relatively heterogeneous characteristics with respect to molecular size. Lee et al.9 have shown

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that large sized molecules of a humic acid exhibited a greater adsorption affinity than smaller 2



75 76

sized molecules. Recently, the interplay between graphene materials and microbial products has received a

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great deal of attention concerning biological responses (e.g., toxicity) of microbes after

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contact with graphene materials and/or the production of bio-functionalized graphene.10-12

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Many studies reported that graphene materials could absorb many types of biological

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products.10,13-15 Ahmed and Rodrigues (2013) using a scanning electron microscope (SEM)

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confirmed the interaction of graphene materials with activated sludge in wastewater.6 Proteins,

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peptides, and lipids are known to adsorb graphene surfaces via hydrophobic interactions, π-π

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interaction, electrostatic interactions, and/or hydrogen bonding.10,16 Polyanionic structures of

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nucleic acids may create electrostatic repulsive forces between microbial products and

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graphene surfaces.16 It was previously reported that positively charged side chains and

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aromatic structures of amino acids may promote their adsorption onto graphene materials.10

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The related adsorption behavior and the involved mechanisms may be affected by the types

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of graphene materials (i.e., surface characteristics). For example, Zhang et al (2013) reported

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that reduced forms of GO (rGO) were more effective in adsorbing model enzymes (e.g.,

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horseradish peroxidase and oxalate oxidase) than untreated GO due to a greater contribution

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of hydrophobic interactions.13

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Extracellular polymeric substances (EPS), which are placed outside of microbial cells and

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in the interior of microbial aggregates, relate to many biological properties of wastewater

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such as settling of sludge, cell protection, and membrane fouling.17-20 EPS consists of a

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variety of organic matter including polysaccharides, proteins, uronic acids, humic substances,

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lipids, and DNA.21 The heterogeneous composition of EPS can make its adsorption behavior

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much complicated. For example, Omoike and Chorover (2004, 2006) found that certain EPS

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constituents might be preferentially adsorbed onto mineral surfaces.22,23 They observed that 3


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the infrared spectra of EPS were red-shifted with the decrease of amide Ι/amide ΙΙ peak area ratio after adsorption. Considering the environmental importance of EPS in biological treatment systems and the

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growing demand of graphene materials for industry, it is surprising that there was no prior

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study on the interactions between graphene materials and EPS. Although it is easily

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postulated that the heterogeneous EPS structures make their adsorption behavior onto

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graphene materials very complicated, the complication is possibly resolved by employing

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advanced EPS characterization methods. Fluorescence excitation emission matrix (EEM)-

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parallel factor analysis (PARAFAC) can separate dissimilar fluorescence components from

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bulk dissolved organic matter (DOM), helping to trace different behaviors/responses of the

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individual components upon a given process.24-26 Our previous study has demonstrated the

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successful application of EEM-PARAFAC in evidencing adsorptive fractionation

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phenomenon of humic acids on the GO surface.9 Two-dimensional correlation spectroscopy

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(2D-COS) is another valuable mathematical tool for identifying the subtle responses of a

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heterogeneous mixture (e.g., EPS) to changing conditions, in which the variations of the

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correlations among different spectral variables are visualized in a two-dimensional space.27-29

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2D-COS could also provide the extent and the order of the sequence in the variable changes

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upon external factors such as solution pH and surface coverage by adsorption.30 Although

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such spectroscopic characterization has some limitations, there is a high hope that applying

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EEM-PARAFAC and 2D-COS could provide valuable information to untangle the complex

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adsorption behavior of EPS on graphene materials and to explain the associated mechanisms,

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which have not yet been explored.

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The objectives of this study were (1) to track the compositional changes of EPS upon their adsorption onto differently reduced graphene materials, and (2) to compare the 4



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individual adsorption isotherm behavior of different EPS constitutes via the two advanced

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mathematical tools - fluorescence EEM-PARAFAC and 2D-COS based on Fourier transform

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infrared spectroscopy (FTIR).

126 127

2. Materials and Methods

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2.1. Sludge collection and EPS extraction

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Aerobic sludge was sampled from an aerobic tank in a municipal wastewater treatment

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plant, located in the city of Seoul (Jungrang-gu), Korea. The facility has a treatment capacity

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of 1,590,000 ton/day, and it is operated by two advanced biological treatment processes,

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namely, A2/O (Anaerobic-anoxic/aerobic) and MLE (Modified Ludzack Ettinger). The

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collected sludge was stored at 4˚C in the dark. Total suspended solids (TSS) and volatile

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suspended solids (VSS) were measured immediately in a laboratory after sampling.

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EPS of the collected sludge were extracted based on a modified formaldehyde/NaOH

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method suggested in Domíguez et al.31, which is known to be beneficial for the protection of

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cell lysis. A volume (700 mL) of sludge was centrifuged at 5000 rpm for 15 min at 4˚C

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before the sludge pellet was re-suspended in 0.05% NaCl solution (350 mL). An aliquot (2.1

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mL) of formaldehyde (37%) was then injected to the NaCl solution containing sludge, and

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the mixture was stirred for 1 hour at 900 rpm at 4˚C. In the next step, 120 mL of 1N NaOH

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was added and stirred for 3 hours under the same conditions. The extracted EPS was

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separated by centrifugation (5000 rpm, 10 minutes) followed by filtration using a 0.2 µm

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pre-washed cellulose acetate membrane filter (Advantec). The EPS was further purified by

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using regenerated cellulose tubular dialysis membrane (3500 Da, Membrane Filtration

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Products, Inc.) for 24 hours at 4 ˚C in Milli-Q water to remove inorganic species,

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formaldehyde, and small organic molecules. A portion of the final EPS solutions was freeze5


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dried for FTIR analysis.

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2.2. Preparation of GO and rGO GO was prepared based on a modification of Hummer’s method, generally following the

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procedure reported in Marcano et al (2010).32 For pre-oxidation, 12 g of graphite flakes

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(Sigma Aldrich) were mixed with 50 mL of concentrated sulfuric acid containing potassium

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peroxydisulfate (10 g) and phosphorous pentoxide (10 g) at 80˚C. After 5 hours, the slurry

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was added to 2 L of Milli-Q water and kept overnight in ambient temperature. The pre-

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oxidized graphite flakes were then washed in Milli-Q water, filtered through 5 µm pore sized

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filter paper to remove residuals, and dried overnight in ambient temperature. For the next

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step, 10 g of the pre-oxidized graphite flakes were stirred in 230 mL of concentrated sulfuric

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acid containing sodium nitrate (1 g) at 0˚C. Potassium permanganate (30 g) was very slowly

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added to the mixture with the temperature maintained at under 10˚C. The mixture was

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reacted at 35˚C for 2 hours before 2 L of Milli-Q water was added under 50˚C. It was then

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stored overnight after adding 40 mL of hydrogen peroxide (30%). Precipitated GO was

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finally washed with 6 L of hydrochloric acid (4 N) to remove the remaining manganese. The

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prepared GO solution was sonicated for 30 minutes in a bath sonicator to separate un-

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exfoliated graphene sheets. Single-sheet GO was finally obtained by centrifugation at 5000

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rpm for 10 minutes after washing with Milli-Q water.

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Hydrazine monohydrate (1 µL per 15 mg of GO) was added to a GO solution to induce

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the chemical reduction of GO. The solution was then stirred at 80˚C for 2 hours (rGO-2h)

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and 8 hours (rGO-8h) to obtain partially reduced GO. The obtained rGO was washed

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repetitively (five times) with Milli-Q water followed by centrifugation at 8000 rpm for 15

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minutes. The final graphene materials were characterized by using attenuated total reflection 6



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(ATR)-FTIR (Perkin-Elmer spectrum 100), X-ray diffraction (XRD, D/MAX-2500/PC,

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Rigaku), Raman spectroscopy (Renisshaw 633 nm), X-ray photoelectron spectroscopy (XPS,

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K-alpha, Thermo VG). The specific surface area (TriStar II 3020) and the zeta potentials

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(NICOMP 380 ZLS) were also measured. All the characteristics are described in the

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supporting information (Table S1, Figures S1 and S2).

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2.3. Adsorption isotherm experiments The obtained EPS solution was diluted in a range of dissolved organic carbon (DOC)

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concentrations from 10 mg C/L to 50 mg C/L for adsorption isotherm experiments at pH 5.0

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± 0.02. The net surface charges of the GO materials were negative at pH 5.0 (Figure S1d).

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The ionic strength was fixed at 0.1 M NaCl. Three types of graphene stock solutions (GO,

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rGO-2h, and rGO-8h) were added to the EPS solutions to achieve the final adsorbent’s

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concentrations of 300 mg/L, 300 mg/L, and 200 mg/L, respectively. The equilibrium time

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was set at 24 hours based on a preliminary kinetic adsorption test (Figure S5). No change in

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EPS (e.g., biodegradation) was confirmed for a control test without the graphene materials

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during the equilibration (Figure S6). The residual EPS after adsorption was separated from

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the adsorbents (i.e., graphene materials) by centrifugation at 8000 rpm followed by filtration

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through a 0.2 µm cellulose acetate membrane (Advantec).

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Adsorption parameters were estimated based on the following Freundlich model, a commonly used isotherm model: 1

Qe =k F Cen 191

where Qe (mg C/g) and Ce (mg C/L) are the adsorbed EPS amounts per graphene and the

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EPS concentrations in solution at equilibrium, respectively. The model parameters of kF and

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1/n refer to the model capacity factor and the isotherm nonlinearity, respectively. 7


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2.4. EPS characterization DOC concentrations of EPS samples were determined using a TOC analyzer (Shimadzu

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V-series, TOC-V CPH). Fluorescence EEMs of EPS were measured by a luminescence

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spectrometry (LS-55, Perkin-Elmer) with the excitation and emission wavelengths set at 220

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nm to 500 nm with 5 nm increments and at 280 nm to 550 nm with a 0.5 nm increment,

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respectively. ATR-FTIR (Perkin-Elmer spectrum 100) was used to determine EPS structures

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and the distributions of the functional groups. The original EPS was fractionated into

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hydrophobic acids (HPO-A), hydrophobic neutrals (HPO-N), transphilic acids (TPI-A),

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transphilic neutrals (TPI-N), and hydrophilic acids (HPI) based on the resins of Supelite

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DAX-8 (Supelco) and Amberite XAD-4 (Sigma).33 The compositions of the resin-fractioned

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EPS were quantified by the measurements of DOC and fluorescence EEM (Table S3 and

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Figure S4). The ATR-FTIR spectra of the freeze dried GO and rGO-8h were measured

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before and after adsorption at a wavenumber range of 2000 to 450 cm-1.

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2.5. EEM-PARAFAC modeling and 2D FTIR correlation spectroscopy Following Stedmon and Bro (2008) tutorial,26 EEM-PARAFAC modeling was conducted

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for the EEM data set (n = 43) using MATLAB7.1 and the free-downloaded DOMFluor

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toolbox (www.models.life.uk.dk). The number of the PARAFAC components was validated

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by split-half analysis and a core consistency test. The maximum fluorescence intensities (Fmax)

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of the identified fluorescent components were used to represent their concentrations.

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Synchronous and asynchronous maps of 2D FT-IR correlation spectroscopy were generated

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using 2Dshige software version 1.3 (https://sites.google.com/site/Shigemorita/home/2dshige).

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In a synchronous map, auto peaks located at the diagonal line represent the variation of the 8



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changes in the spectral intensities corresponding to the locations upon an external

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perturbation. Cross peaks, which are located off the diagonal line, refer to the simultaneous

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changes of the spectral variables observed at two different locations (i.e., x- and y-axes).

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Positive cross peaks indicate the two variables changing in the same direction, while negative

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cross peaks imply the opposite trend in the changes. Meanwhile, cross peaks in an

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asynchronous map represent the sequence of the events (or variable changes) by given

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perturbations. If the signs of cross peaks are the same for synchronous and asynchronous

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maps, the spectral change of one variable (e.g., x-axis) precedes that of the other (e.g., y-axis).

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In contrast, if the signs are different, the orders of the changes are reversed. The general

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interpretation of the event’s sequence is based on Noda’s rules.28

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3. Results and Discussion

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3.1. EPS characterization and adsorption of EPS

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The TSS and VSS concentrtions of the sludge were 5991 and 3912 mg/L, respectively

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(Table S2). The EPS yield per VSS was 53.2 mg/g (Table S2), which was lower than the

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ranges previously reported (79-129 mg/g VSS).31,34 The ATR-FTIR showed the presence of

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proteins (amide I and amide II bands), polysaccharides, and nucleic acids as the major

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structures of EPS, which was based on the assignments following Omoike and Chorover 22

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(Figure 1a). Meanwhile, the most abundant resin fraction of EPS was HPI, followed by HPO-

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N → HPO-A → TPI-A → TPI-N (Figure 1b).

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Irrespective of graphene type, all adsorption behaviors of EPS were well described by the

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Freundlich model (R2 > 0.94), and the corresponding parameters are presented in Table 1.

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Overall, an increasing and a decreasing trend were observed for the KF (i.e., adsorption

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capacities) and the 1/n values (i.e., isotherm nonlinearity), respectively, on the order of GO, 9


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rGO-2h, and rGO-8h. The chemical reduction of GO seems to create a greater number of

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adsorption sites available to EPS as shown by the increased specific surface area (Table S1).

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The higher isotherm nonlinearity suggests that the adsorbing EPS molecules could compete

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more strongly for the additional sites and/or for the modified rGO surface. The higher

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adsorption capacity of rGO versus GO agreed well with a recent report of Yan et al. 35, in

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which oxidized GO exhibited a lower extent of adsorption for aromatic organic compounds

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(i.e., aniline, nitrobenzene, and chlorobenzene). In addition, oxidizing GO surface (i.e., more

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oxygen presence) can make the adsorption of water molecules more favorable than EPS

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adsorption and/or it may localize the π electrons, reducing the adsorption of EPS through π-π

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interaction. 36 Considering that a lower 1/n value typically reflects more heterogeneous

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surfaces with respect to adsorption sites,37 the higher nonlinear isotherms for rGO versus GO

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observed for this study may be associated with a greater degree of the defected sp2 structures

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(Figure S1c) and the reduced presence of C-OH (or more equal abundance of C-OH to those

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of other carbon components) for rGO (Figures S1a and S2). This is because the defected sp2

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structures and the equal abundances of different oxygen-containing carbon components can

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represent more heterogeneous properties of rGO with respect to its carbon structure and

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functional groups, respectively.

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3.2. PARAFAC components and their adsorption behaviors Four different fluorescent components, as presented in Figure 2, were identified based on

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a core consistency test (80.7%) and split-half validation. Component 1 (C1), assigned as

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microbial humic-like fluorophores, had two peaks at the excitation/emission wavelengths

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(Ex/Em) of 220 nm/430 nm and 310 nm/430 nm. Component 2 (C2), which can be assigned

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to an aromatic protein-like component, exhibited two peaks at 220 nm/355 nm and 280 10



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nm/355 nm (Ex/Em). Component 3 (C3), denoted as a humic-like component, presented its

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peaks at longer wavelengths (Em) than other components (Ex/Em = 250 nm/460 nm, 360

268

nm/460 nm). Component 4 (C4) appears to be associated with protein-like substances. All the

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assignments of the PARAFAC components were based on the EEM peak locations

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previously reported for EPS.38,39

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For this study, compositional changes in EPS upon adsorption were tracked by using two

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PARAFAC ratios (i.e., C2/C1 and C4/C3) in Fmax values of the residual EPS after adsorption,

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which were plotted against percent adsorption in Figure 3. For all the graphene materials, the

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two ratios of the residual EPS were much lower than the original values before adsorption

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(12.7 for C2/C1 and 3.5 for C4/C3), ranging from 0.5 to 5.0 and 0.0 to 1.5 for C2/C1 and

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C4/C3, respectively. The lower ratios of the residual EPS after adsorption indicate

277

preferential adsorption of protein-like components (C2 and C4) onto graphene surfaces over

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humic-like components (C1 and C3) in EPS. Such a preferential adsorption behavior was also

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confirmed by comparing the Freundlich model parameters of different PARAFAC

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components individually fitted to the isotherms (Table 2 and Figure S7), in which the

281

adsorption affinities (i.e., KF values) were much higher for the protein-like components than

282

for the humic-like components regardless of graphene type.

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Interestingly, the C2/C1 and C4/C3 ratios showed a decreasing trend with increasing

284

percent of adsorption (Figure 3), suggesting that the degree of the preferential adsorption

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became greater for the situation in which the adsorption sites are more available and thus less

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competition is expected among different EPS molecules. The deviations of the two ratios

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from the original values were even more pronounced for GO versus rGO as shown by the

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lower ratios for GO at the same percent adsorption rates (Figure 3). The difference in the

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degree of the preferential adsorption between GO and rGO may be attributed to relatively 11


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more hydrophilic nature of GO surfaces, which favors the adsorption of protein-like EPS

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substances (e.g., C2 and C4 components for this study) rather than humic-like components.

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Our results can also be explained by electrostatic interaction of nitrogen-containing

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functional groups with oxygen functional group of GO. 35 Importance of electrostatic

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interaction between proteins (i.e., amide groups) and GO surfaces is well described in a

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review article.40 It is notable from our study that the preferential adsorption of protein-like

296

versus humic-like components was more obvious for GO, which has relatively more

297

hydrophilic surface and more acidic functional groups, than for rGO.

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For the same graphene materials, a higher isotherm nonlinearity (or a lower 1/n) was

299

exhibited for protein-like components (C2 and C4) than for humic-like components (C1 and

300

C3) (Table 2), suggesting that the adsorption of protein-related EPS molecules may be driven

301

by site-limiting mechanisms (e.g., electrostatic interaction) to a greater extent compared to

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humic substances. It is notable that the presence of simple aromatic amino acids is a major

303

contributor to the protein-like fluorophores, with their peaks at shorter wavelengths (Em).

304 305

3.3. Changes in the ATR-FTIR spectra of graphene materials after EPS adsorption

306

The ATR-FTIR spectra of graphene materials (GO and rGO-8h) were compared before

307

and after EPS adsorption in Figure 4. The EPS-adsorbed graphene showed additional peaks

308

of amide I (near 1640 cm-1) and amide II (near 1545 cm-1), which originated from the EPS

309

only (Figure 1 and Table S5). Although it is easily inferred that adsorption of aromatic carbon

310

structures of EPS predominantly occurred, the related peaks were not directly confirmed from

311

the ATR-FTIR spectra probably because of the overwhelming presence of sp2 structures in

312

GO. The bands at 1380 cm-1, 1236 cm-1, 1081 cm-1 and 980 cm-1 were also found for both

313

GO and rGO after EPS adsorption. However, the origins were not clear although the 12



314

possibility cannot be ruled out that the bands of EPS could be shifted upon adsorption to the

315

GO and rGO-8h. It is notable that those bands were not observed for the FTIR spectrum of

316

the EPS only.

317

In contrast to the EPS-adsorbed GO, the amide I band of EPS-adsorbed rGO-8h was

318

shifted toward a longer wavenumber from 1635 cm-1 to 1655 cm-1 (Figure 4b), implying

319

strong binding between the corresponding EPS structures and rGO surfaces, which was also

320

reported in Xu and Jiang.41 Considering that the amide I band originates from the peptide

321

backbone C=O stretching vibration, the difference between GO and rGO indicates that the

322

interaction between graphene surfaces and peptide backbone structures can be intensified by

323

chemical reduction of GO. Enhancement of both amide II and aromatic C-C bands (1450 cm-

324

1

325

(i.e., restoration of sp2 structures) could lead to greater adsorption of some aromatic and/or

326

nitrogenous EPS molecules. This is consistent with our previous observation of a higher

327

adsorption affinity of aromatic protein-like component (i.e., C2) on rGO-8h versus GO

328

(Table 2). Our results are also supported by Chen and Chen 37, who demonstrated that the

329

adsorption sites of GO through π-π interaction could be expanded by the restoration of sp2

330

structures upon chemical reduction.

) with no shifts was also found for the EPS-adsorbed rGO-8h, suggesting that GO reduction

331 332 333

3.4. 2D correlation ATR-FTIR spectroscopy To obtain more detailed information on the structural changes of EPS upon adsorption,

334

2D-COS was conducted using a series of the FTIR spectra of graphene materials with

335

increasing EPS adsorption amounts (Figure 5 and Figure S8), which were based on the

336

previous isotherm data. From the synchronous maps, two main auto peaks were observed at

337

the amide I (1600-1700 cm-1) and amide II (1500-1560 cm-1) bands for both GO and rGO-8h 13


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(Figures 5a and 5c). The band corresponding to the aromatic C-C band (1450 cm-1) seemed to

339

overlap with the amide II band in the synchronous maps of both graphene materials. There

340

was a notable difference in the synchronous maps between GO and rGO-8h for this study.

341

For GO, the amide I band was split into two separated peaks, while only one broad peak was

342

observed at the same location for rGO-8h. In general, the amide I band of FTIR spectra can

343

be further separated into two different bands of 1650-1700 cm-1 and 1600-1630 cm-1, each of

344

which is known to be turn and α-helix structure and β-sheet structure of peptides,

345

respectively.42 No polysaccharide-related peak (e.g., 1040 cm-1) was observed in the

346

synchronous maps although carbohydrate was one major constitute of EPS (Table S5). This

347

suggests that adsorption of polysaccharide in EPS may not be significant compared to those

348

of other EPS structures.

349

The intensities of the auto peaks can represent the extent of adsorption of the

350

corresponding structures with increasing adsorption amounts. For this study, higher

351

intensities of amide I and aromatic C-C auto peaks were found for rGO-8h than for GO,

352

while those of amide II band were similar for both graphene materials (Table S6). Our results

353

confirmed the previous observation (Figure 4) that the adsorption of the EPS molecules

354

relating to amide I and aromatic C-C bands might be greater for rGO-8h versus GO.

355

For the cross peaks (i.e., the peaks located off the diagonal line) of the synchronous map

356

of GO (Figure 5a), it was found that the two separated amide I bands at x-axis/y-axis of 1672

357

cm-1 / 1615 cm-1, each of which corresponds to α-helix and β-sheet structures, had positive

358

and weak negative signs, respectively. This suggests that the adsorption behavior might be

359

different between α-helix and β-sheet structures in responding to the site limitation of GO

360

(i.e., more surface coverage). The positive sign may reflect more favorable adsorption upon

361

the site limitation. For rGO-8h, unfortunately, only a broad positive peak appeared at the 14



362

same location of the synchronous map, which makes the interpretation of the adsorption

363

behavior of the two secondary protein structures difficult. The other cross peaks at 1380 cm-1,

364

1236 cm-1, 1081 cm-1 and 980 cm-1 along the y-axis shared the locations of the x-axis

365

corresponding to the band of either amide I or amide II (Figures 5a and 5c), suggesting that

366

the associated structures were also involved in the adsorption of protein EPS structures.

367

Notably, such a concurrent adsorption of different EPS structures could be only confirmed by

368

2D-COS, not by the original FTIR spectra.

369

Asynchronous maps are useful in explaining the sequence of variable changes (or

370

different responses to an external perturbation) when the locations and signs of cross peaks

371

were compared between synchronous and asynchronous maps.43 For the asynchronous map

372

of GO, α-helix/β-sheet and α-helix/amide II (x-axis/y-axis) peaks displayed a negative and a

373

positive signs, respectively (Figure 5b and Table S7), which were the same as the signs in the

374

synchronous map. However, β-sheet/amide II peak showed a weak positive sign, which was

375

opposite to the sign in the synchronous map (Figure 5b and Table S7). For rGO-8h, the two

376

cross peaks of α-helix/β-sheet and α-helix/amide II in the asynchronous map showed the

377

same signs as those of the synchronous maps, while β-sheet/amide II peak had the opposite

378

signs between the two maps. Interestingly, secondary protein structures (i.e., α-helix and β-

379

sheet structures) within the amide I band, which were not identified from the previous

380

synchronous map of rGO-8h, could be distinguished by the asynchronous map (Figure 5).

381

This suggests that asynchronous maps could capture subtle responses of the associated EPS

382

structures in adsorption to increasing adsorption amount (or greater site limitation).

383

Following Noda’s rule, the adsorption of such protein-related structures seems to occur

384

on the order of α-helix → amide II → β-sheet structures for both GO and rGO-8h with

385

increasing adsorption amounts. Although no direct evidence was available in the literature to 15


Page 16 of 27

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386

support our findings, recent studies have demonstrated the possibilities of the sequential

387

adsorption of secondary protein structures onto graphene materials. For example, Katoch et al.

388

(2012) found using FTIR spectra that α-helix structures of peptides bound to graphene

389

surfaces might undergo a transition to a complex reticular form by the interaction with the

390

surface.42 Park et al. (2014) demonstrated different adsorption affinities between single- and

391

double-stranded DNA for GO surface.16

392 393

Supporting Information

394

17 pages including text, 9 figures, 7 tables, and a reference list. This material is available free

395

of charge via the Internet at http://pubs.acs.org.

396 397

Acknowledgements

398

This work was supported by a National Research Foundation of Korea (NRF) grant funded

399

by the Korean government (MSIP) (No.2014R1A2A2A09049496) and also by Korea

400

Ministry of Environment (MOE) through Waste to energy recycling Human resource

401

development Project

402 403

References

404

(1)

405 406 407 408 409 410 411 412 413

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527 528 529 530 531 532 533 534 535 536 537 538 19


Page 20 of 27

Page 21 of 27


539 540 541 542

Tables

543 544

Table 1. Freundlich Isotherm model parameters of EPS adsorption onto graphene materials. Adsorbent type

K F*

1/n**

R2

GO

3.49 ± 0.34

0.77 ± 0.03

0.97

rGO-2h

6.59 ± 0.74

0.71 ± 0.04

0.95

rGO-8h (mg C/g)/(mg C/L)(1/n) ** Unitless

25.09 ± 1.64

0.37 ± 0.02

0.94

545 546 547 548 549 550

*

551

C2, C3 and C4) for EPS adsorption onto graphene materials.

Table 2. Freundlich isotherm model parameters of the individual PARAFAC components (C1,

Components C1

C2

C3

C4

Adsorbent type GO

KF (QSE/mg)/(QSE)(1/n) 4.0×10-5 ± 2.10-5

1/n (Unitless) 2.10 ± 0.09

0.98

rGO-2h

0.03 ± 0.01

0.83 ± 0.09

0.86

rGO-8h

0.08 ± 0.01

0.66 ± 0.04

0.95

GO

0.11 ± 0.01

0.79 ± 0.03

0.99

rGO-2h

0.43 ± 0.12

0.62 ± 0.06

0.91

rGO-8h

2.61 ± 0.14

0.35 ± 0.01

0.99

GO

0.01 ± 0.002

0.80 ± 0.03

0.99

rGO-2h

0.05 ± 0.003

0.50 ± 0.03

0.96

rGO-8h

0.12 ± 0.002

0.39 ± 0.01

0.98

GO

0.26 ± 0.05

0.38 ± 0.08

0.69

rGO-2h

0.29 ± 0.03

0.36 ± 0.04

0.87

rGO-8h

0.72 ± 0.04

0.25 ± 0.02

0.95

552 553 554 555 20


R2


Page 22 of 27

556 557 558 559 560

Figures

(a) Absorbance (arb. unit)

1635 1540

1000 1040

1400 1454 1236

2000

1750

561

1500 1250 1000 -1 Wavenumber (cm )

750

500

45

Proportion of fractions (%)

40

(b)

35 30 25 20 15 10 5 0

562 563

HPO-A

HPO-N

TPI-A

TPI-N

HPI

Figure 1. Characteristics of EPS (a) FTIR spectra, and (b) proportion of resin fractions. 21


Page 23 of 27

564 565 566 567 568 569 570 571 572


Figure 2. Fluorescent components identified by EEM-PARAFAC (a) C1, (b) C2, (c) C3, and (d) C4.

573

22



Page 24 of 27

6 GO

(a)

rGO-2h

5

rGO-8h EPS initial = 12.7

C2/C1

4 3 2

1 Increasing adsorption amount (More surface coverage) 0 20

30

574

40 50 Percent adsorption (%)

60

70

1.6 Increasing adsorption amount (More surface coverage)

(b)

1.4 1.2

C4/C3

1 GO 0.8

rGO-2h rGO-8h

0.6

EPS initial = 3.50 0.4 0.2 0 20 575 576 577 578 579 580

30

40 50 Percent adsorption (%)

60

70

Figure 3. Changes in the ratios of protein-like to humic-like PARAFAC components for the residual EPS after adsorption with increasing percent adsorption or with less surface coverage. (a) C2/C1 and (b) C4/C3.

23


Page 25 of 27


Amide I

(a)

Amide II Polysaccharides ester PO2-

Absorbance (arb. unit)

Aromatic C-C Carboxylate uronic acid

EPS

epoxy C-O

Aromatic C-C

Carboxy COO

C=O 2000

1700

(b)

GO

Alkoxy C-O

1400 1100 Wavenumber (cm-1)

581

EPS-adsorbed GO

800

500

Amide I Amide II

Absorbance (arb. unit)

Polysaccharides Aromatic C-C ester PO2Carboxylate uronic acid

EPS

epoxy C-O

EPS-adsorbed rGO-8h

Aromatic C-C Carboxy COO

C=O 2000 582 583 584

1700

rGO-8h

Alkoxy C-O

1400 1100 Wavenumber (cm-1)

800

500

Figure 4. Comparison of FTIR spectra of GO (a) and rGO-8h (b) with and without adsorption of EPS in the band regions from 750 to 1860 cm-1.

24



585 586 587 588

Figure 5. Synchronous and asynchronous maps of 2D FTIR correlation spectra of GO (a, b) and rGO-8h (c, d) with different EPS amounts in the region of 750-1860 cm-1.

589 590 591 592 593 594 595

25


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Adsorption Behavior of Extracellular Polymeric ... - ACS Publications

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