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Acute Responses of Microorganisms from MBR in the Presence of NaOCl: Protective Mechanisms of Extracellular Polymeric Substances Xiaomeng Han, Zhiwei Wang, Mei Chen, Xingran Zhang, Chuyang Y. Tang, and Zhichao Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05475 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017
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Acute Responses of Microorganisms from MBR in the Presence of
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NaOCl:
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Substances
Protective
Mechanisms
of
Extracellular
Polymeric
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Xiaomeng Han1,3, Zhiwei Wang*,1, Mei Chen1, Xingran Zhang1, Chuyang Y. Tang2, Zhichao
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Wu1
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1
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Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental
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2
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China
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3
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Center Co. Ltd., Shanghai 200082, China
Department of Civil Engineering, The University of Hong Kong, Pokfulam, Hong Kong,
Shanghai Urban Water Resources Development and Utilization National Engineering
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Revised Manuscript Submitted to: Environmental Science & Technology (clean version)
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March 3, 2017
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ABSTRACT
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Extracellular polymeric substances (EPS) are key foulants in membrane bioreactors (MBRs).
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On the other hand, their positive functions of protecting microorganisms from
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environmental stresses, e.g., during in-situ hypochlorite chemical cleaning of membranes,
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have not been adequately elucidated. In this work, we investigated the response of
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microorganisms in an MBR to various dosages of NaOCl, with a particular emphasis on the
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mechanistic roles of EPS. Results showed that functional groups in EPS such as the
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hydroxyl and amino groups were attacked by NaOCl, causing the oxidation of
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polysaccharides, denaturation of amino acids, damage to protein secondary structure, and
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transformation of tryptophan protein-like substances to condensed aromatic ring substances.
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The presence of EPS alleviated the negative impacts on catalase and superoxide dismutase,
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which in turn reduced the concentration of reactive oxygen species (ROS) in microbial cells.
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The direct extracellular reaction and the mitigated intracellular oxidative responses
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facilitated the maintenance of microbial metabolism, as indicated by the quantity of
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adenosine triphosphate and the activity of dehydrogenase. The reaction with NaOCl also led
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to the changes of cell integrity and adhesion properties of EPS, which promoted the release
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of organic matter into bulk solution. Our results systematically demonstrate the protective
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roles of EPS and the underlying mechanisms in resisting the environmental stress caused by
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NaOCl, which provides important implications for in-situ chemical cleaning in MBRs.
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INTRODUCTION
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In membrane bioreactors (MBRs), fouling due to the adsorption/accumulation of
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extracellular polymeric substances (EPS) on membranes is a critical issue affecting MBR
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performance.1 Controlling EPS-related biofouling in MBRs has been a hot topic in past
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decades.2,3 Meanwhile, EPS are important for microorganisms in activated sludge processes
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as they can mediate cell-cell interactions, promote structural development of flocs, and
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serve as carbon/energy sources in starvation.4-6 It has been reported that EPS also play an
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important role in protecting cells against environmental stresses, e.g., desiccation and in the
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presence of heavy metals and antibiotics.7-10
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In the context of MBRs, sodium hypochlorite (NaOCl) is a widely used reagent for
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in-situ chemical cleaning of membranes to restore their permeability11-13. Microbial cells are
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inevitably exposed to NaOCl during this cleaning process. We hypothesize that EPS
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function as a protective layer to alleviate the adverse impacts of NaOCl on microbial
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viability. Furthermore, the mechanisms involved in EPS protection might be related to the
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interaction of EPS with NaOCl since EPS have abundant hydroxyls, carbonyls, and amine
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groups14,15 that can be attacked by NaOCl.5 However, there is an obvious lack of studies
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investigating the role of EPS in protecting microorganisms during membrane cleaning in
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MBRs.
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Previous studies on biofilm formation show that EPS produced by microorganisms
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significantly increase the tolerance of biofilm or pure bacteria strains to antibiotics and
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disinfectants.16-18 Mechanisms of EPS protection for biofilm include transport limitation of
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biocides through EPS matrix, sacrificial reaction of EPS with biocides, formation of a 3
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nutrient gradient within biofilm leading to complex phenotypes of cells, and change of
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biofilm structure.5 Xue et al. further reported that capsular EPS of Pseudomonas aeruginosa
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either acted as a disinfectant consumer (for chlorine inactivation) or limited access to
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reactive sites on cell membrane (for monochloramine inactivation) via deformation of key
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functional groups in EPS and cell membrane.19 Similarly, EPS content was observed to be
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reduced in biofilm in the presence of ozone due to its reaction with EPS. 20 Ozone can attack
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amino acid residue on polypeptide chain of proteins and decrease the molecular weight of
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polysaccharides and proteins. 21 It has been also reported that EPS can bind heavy metals
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(e.g., Cu2+, Pb2+, and Zn2+) or reduce high-oxidation-state metals (e.g., U6+ and Cr6+),
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hindering their intracellular penetration.22-24
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Although the recent research16-19,22-24 on the role of EPS in biofilm tolerance and heavy
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metal binding/remediation has laid the groundwork for understanding EPS-NaOCl
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interactions, the detailed protective mechanisms of EPS for microorganisms from MBRs in
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the presence of NaOCl remain to be established. Currently, no systematic study has been
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conducted to illustrate the role of EPS in protecting microorganisms during membrane
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cleaning. In addition, the acute responses of microorganisms may lead to the change of
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metabolism behaviors and release of organic matters into mixed liquor, causing the
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deterioration of MBR performance and increase of membrane fouling rate. Investigation on
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the microbial stress responses and protective mechanisms of EPS is essential to recognizing
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the impacts of chemical cleaning on microorganisms and optimizing MBR performance
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during/after cleaning.
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Therefore, in the present work, we aim to elucidate the role of EPS in microbial stress 4
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responses to NaOCl application in an MBR. Specifically, this study investigated (i) the
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effect of NaOCl stress on microbial metabolism behaviors using microbial cells with and
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without EPS; (ii) the protective role of EPS in microbial stress responses by examining
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deformation of EPS’s functional groups and generation of intracellular reactive oxygen
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species (ROS); and (iii) the adhesion and release behaviors of EPS after exposure to NaOCl.
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MATERIALS AND METHODS
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Reagents. All chemicals used in this study were of analytical grade unless stated
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otherwise. Sodium sulfite, sodium chloride, sodium hydroxide, chloride acid and sodium
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hypochlorite (~6%) were received from Aladdin (China). Calcein-AM (CAM) and
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propidium iodide (PI) were purchased from AAT Bioquest, USA. All solutions were
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prepared using deionized (DI) water. Solution pH was adjusted by adding 1 M NaOH or 1
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M HCl where applicable. Catalase (CAT) and superoxide dismutase (SOD) assay kits were
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purchased from Cayman Chemical (MI, USA).
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Microorganism samples. Microorganism samples were collected from a lab-scale
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anoxic/oxic MBR treating real municipal wastewater. Detailed information about the MBR
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can be found in Supporting Information (SI) Section S1. The microbial community was
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analyzed during the period of sampling, which is shown in Figure S1 with the analytical
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procedure documented in SI Section S2.
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Microorganisms collected from the MBR were washed and centrifuged twice (4000 g,
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10 min) to remove the supernatant. The remaining microorganisms were re-suspended in DI
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water. The prepared microorganisms were denoted as original ones, i.e., ORI. ORI 5
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microorganisms with pre-determined volume were subject to EPS extraction25 with detailed
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procedures described in SI Section S3. The quantity and compositions of extracted EPS are
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listed in Table S1. The microorganisms after the removal of EPS were named as removed
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ones, i.e., REM. Both ORI and REM microorganisms were exposed to NaOCl solutions.
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The NaOCl dosages of 0-0.67 mmol/g-SS were chosen based on the typical dosages applied
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during chemical cleaning in MBRs.26 An exposure duration of 2 h was used to simulate a
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shock load of NaOCl and to facilitate the evaluation of the acute responses of
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microorganisms.
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Microbial metabolism behaviors. Dehydrogenase (DHA) activity and adenosine
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triphosphate (ATP) quantity were measured for ORI and REM since DHA and ATP can well
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reflect heterotrophic metabolism behaviors.11,27,28 Detailed measurement procedures for
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DHA and ATP can be found in SI Section S3.
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For confirming the heterotrophic metabolism behaviors affected by NaOCl,
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biodegradation of sodium acetate (NaAc) by ORI and REM was performed. Briefly, ORI
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and REM samples were transferred into magnetically agitated reaction vessels, and
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pre-weighted amount of NaAc was dosed to make an initial NaAc concentration of 850
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mg/L. Dissolved oxygen concentration was maintained at about 5 mg/L during the
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experiment. Mixed liquor samples, collected from the vessels at pre-determined durations (0,
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20, 40, 60, 90, 120 min), were centrifuged (8000g, 5 min) and filtered (0.45-µm PTFE
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membrane) prior to TOC analyses (TOC-LCPH, Shimadzu, Japan). Nitrification and
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denitrification tests were also performed for ORI and REM based on the protocols reported
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elsewhere.11 6
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Evaluation of the deformation of EPS’s functional groups and the generation of
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intracellular reactive oxygen species. The extracted EPS were subject to reaction with the
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same dosages of NaOCl as described above. The ionic strength of the solutions was adjusted
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to 15.4 mM to eliminate the impacts of ionic strength. After reaction at pre-determined
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durations (0, 30, 60, 90, 120 min), samples were collected and subject to combined
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spectroscopy
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fluorescence spectroscopy and Fourier-transform infrared spectroscopy (FTIR).29,30 The
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analytical procedures are described in SI Section S4. The degree of acetylation due to the
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replacement of hydroxyl by acetyl group is characterized by 100×A1665/(1.33×A3450).31,32
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Dynamic light scattering was employed to identify the changes in the hydrodynamic
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diameter of EPS.33
analyses,
i.e.,
three-dimensional
excitation-emission
matrix
(EEM)
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ROS are normally generated from sequential univalent reductions of oxygen during
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oxidative phosphorylation, resulting in generation of ROS including superoxide anion (O2●-),
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hydrogen peroxide (H2O2) and hydroxyl radicals (●OH).34 Cells are equipped with enzymes
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including catalase (CAT), superoxide dismutase (SOD) and other antioxidants to maintain a
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balance between the production and elimination of ROS.35 However, the presence/diffusion
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of NaOCl into microbial cells might cause damage to key enzymes, leading to deleterious
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oxidative stress. Production of ROS was detected by a detection kit36 (H2DCF-DA,
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Life-Technologies, USA). Briefly, H2DCF-DA was added into ORI and REM and incubated
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for 20 min at 37oC. The residual probes were removed by centrifugation and washing using
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DI water. NaOCl was then added into ORI and REM, and the intracellular ROS was
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measured by a multi-mode microplate reader (Excitation/Emission wavelength = 488 7
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nm/525 nm, Synergy-4, Bio-Tek, USA). CAT and SOD, capable of mitigating ROS, were
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measured by CAT and SOD assay kits, respectively.
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Cell integrity and organic matter release upon exposure to NaOCl. Cell integrity
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tests were performed by a double staining method37 using calcein-AM (CAM) (for live cells)
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and propidium iodide (PI) (for dead cells). The analytical procedures are documented in SI
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Section S5.
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EPS release was evaluated for ORI during the 2-h exposure to NaOCl. Samples were
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periodically collected and pre-weighted amount of sodium sulfite was added to quench the
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residue NaOCl. The samples were then centrifuged (8000g, 5 min) and the supernatant was
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filtered by 0.45-µm PTFE membrane before TOC analyses. DNA was also monitored for
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verifying whether the release of intracellular substances occurred, since a dramatic increase
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of DNA can indicate cell lysis and intracellular component release.38
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QCM-D analysis33 was performed to elucidate the adhesion behaviors of EPS after
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exposure to NaOCl. Deionized water was initially injected into QCM-D (E4, Q-sense,
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Sweden) for stabilization (frequency drift
