Real-time Online Monitoring for Assessing Bacteria Removal by

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Real-time Online Monitoring for Assessing Bacteria Removal by Reverse Osmosis Takahiro Fujioka, Anh Tram Hoang, Hidenobu Aizawa, Hiroki Ashiba, Makoto Fujimaki, and Menu Leddy Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00200 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Real-time Online Monitoring for Assessing Bacteria Removal

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by Reverse Osmosis

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Takahiro Fujioka,†,* Anh T. Hoang,† Hidenobu Aizawa,‡

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Hiroki Ashiba,§ Makoto Fujimaki,§ Menu Leddy,ǁ



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Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan ‡

Environment Management Research Institute, National Institute of Advanced Industrial Science

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Water and Environmental Engineering, Graduate School of Engineering,

and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569 Japan §

Electronics and Photonics Research Institute, National Institute of Advanced Industrial Science

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and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

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ǁ

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Orange County Water District, 18700 Ward Street, Fountain Valley, CA 92708, USA

_______________________ * Corresponding author: Takahiro Fujioka, Email: [email protected], Tel: +81 095 819 2695, Fax: +81 95 819 2620

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Abstract

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Rigorous monitoring of microbial water quality is essential to ensure the safety of recycled water

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after advanced treatment for indirect and direct potable reuse. This study evaluated real-time

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bacterial monitoring for assessing reverse osmosis (RO) treatment for removal of bacteria. A

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strategy was employed to monitor bacterial counts on-line and in real time in RO feed and

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permeate water using a real-time continuous bacteriological counter. Over the course of 68-hours

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pilot-scale testing, bacterial counts were monitored in real-time at an approximate range of

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1×103–4×104 and 4–342 counts/mL in RO feed (ultrafiltration-treated wastewater) and permeate,

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respectively. The results indicate that the bacteriological counter can track the variations in

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bacterial counts in RO feed and permeate. Bacterial concentrations were confirmed by Epi-

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fluorescence microscopy for total bacterial counts. A high correlation (R2 = 0.83) was identified

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between the online bacterial counts and Epi-fluorescence counts in RO feed; a negligible

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correlation was observed for RO permeate. In this study we evaluated a real-time bacteriological

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counter (i.e. counts/mL every second) to ensure continuous removal of bacterial contaminants by

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RO treatment.

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INTRODUCTION

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Potable reuse (PR) has been increasingly used to augment potable water supplies in arid regions.1,

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2

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and reliability in recycled water quality, particularly microbiological quality, is a critical

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component for public health protection. This is especially important for direct potable reuse

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(DPR), where highly treated wastewater at advanced water treatment plants (AWTPs) is directly

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used as a potable water source without going through an environmental buffer.3 At AWTPs,

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reverse osmosis (RO) membrane process can have an important role in removing most of the

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dissolved ions, trace organic chemicals and microorganisms in treated wastewater.4 However, the

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credibility of RO membranes for removing microorganisms and pathogens has been undervalued.

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For example, current RO membrane integrity monitoring methods are mostly based on the

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removal of surrogate substances: total organic carbon (TOC) and electrical conductivity; these

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surrogate indicators provide up to a 2-log reduction (i.e. 99% removal) for viruses and protozoa.5

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Bacterial water quality has also attracted much attention in DPR to minimize health risk of

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infection from enteric bacterial pathogens such as Salmonella spp.3, 5, 6 For example, a 9-log

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reduction of total coliform bacteria through the treatment processes has been suggested for

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DPR.7

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Considering RO membrane deterioration over time and unforeseen spikes of bacteria in raw

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sewage, the implementation of continuous monitoring of bacterial contaminants at low

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concentrations after RO treatment or in RO permeate will considerably enhance monitoring for

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bacterial contaminants in recycled water for water quality disruptions. For this purpose, the

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analytical instruments must be fast, reliable, sensitive and accurate. Many commercial devices

Potable reuse turns treated wastewater effluent into potable water; thus, the assurance of safety

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are capable of detecting bacteriological cells within a short analysis time (every 5 min to several

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hours).8 Among them, flow cytometric bacterial cell counters combined with general nucleic acid

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staining is an emerging technology capable of rapidly counting total bacterial cells.9, 10 In recent

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years, several near real-time or real-time bacteriological sensing technologies (e.g. BioSentry

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sensor)11 have been evaluated for relatively clean waters including drinking water.12

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Nevertheless, to date, real-time monitoring techniques have not been fully established in potable

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reuse due to limitations with availability and adaptability in analyzing treated wastewater.

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A real-time continuous bacterial counting technique that is capable of monitoring bacterial

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counts as low as one count per second in ultrapure water at a flow rate of 0.16 mL/s was

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evaluated with RO feed and permeate of ultrafiltration (UF)-treated secondary wastewater in this

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study. The real-time bacteriological counter can differentiate bacterial and non-bacterial particles

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using auto-fluorescence light emitted from riboflavin and nicotinamide adenine dinucleotide -

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hydrogen (NADH) and their scattered light.13 This technique has an advantage of speed, no

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additional chemicals and sensitivity over other bacteriological monitoring technologies,

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including flow cytrometry that has a range of approximately 20–100 cells/mL.14, 15 In addition to

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continuous online monitoring for bacteria, this technique, when applied to RO feed and permeate,

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has a potential application for real-time membrane integrity monitoring. However, big challenges

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for real-time RO monitoring in RO feed are interferences by humic-like substances that can mask

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the detection of bacterial counts.16 To enable online monitoring of RO feed, a new strategy was

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adopted for the RO feed that is likely to contain higher concentrations of bacteria than RO

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permeate was continuously diluted on line in real-time.

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This study aimed to evaluate the ability of real-time bacteriological counters to ensure that

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microbial contaminants are being removed by RO treatment. The study was performed by 3

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tracking the variation in bacterial counts in RO feed and permeate at the pilot scale. The

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reduction in bacterial counts by RO treatment was confirmed by determining the total bacterial

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counts using Epi-fluorescence microscopy.

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MATERIALS AND METHODS

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Analytical techniques

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Two real-time bacteriological counters (IMD-WTM, Azbil Corporation, Tokyo, Japan) were used

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to monitor bacterial counts in the RO feed and RO permeate in real-time. The real-time counter

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is capable of detecting the number of bacterial particles at one count per second by introducing

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part of the sample flow into the counter at a sampling flow rate of 0.16 mL/s. In other words, it

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can provide bacterial counts at as low as 1 counts/mL every second. The real-time

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bacteriological counter first irradiates the excitation light (wavelength = 405 nm) to the running

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sample solution, which primes the system to identify particles with scattered light (Fig. S1). If

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the particle is a bacteria, intrinsic fluorescence emission is induced due to their auto-fluorescence

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property from riboflavin and NADH. The intensity of faint fluorescent light is received by two

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fluorescence detectors with different wavelength bands (wavelength = about 415–450 and 490–

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530 nm) (Fig. S1). Particles holding a certain level of auto-fluorescence light are recognised as

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bacteria and counted as biological particle. The real-time instrument counts all particles similar

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in size to bacteria in the sample and determines whether they are bacterial or non-bacterial

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particles. To confirm the real-time bacteriological counts, this study also analysed total bacterial

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counts using a fluorescence microscopy method with 4’-6-diamidino-2-phenylindole (DAPI) dye

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(Text S1). Excitation emission matrix (EEM) fluorescence spectra were obtained using Aqualog

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(Horiba, Kyoto, Japan). Details for the analytical conditions can be found elsewhere.17

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Validation protocol

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A pilot-scale cross-flow RO filtration system comprised of a 4-in. spiral wound RO membrane

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element with the surface area of 7.43 m2 (ESPA2-LD-4040, Hydranautics/Nitto, Oceanside, CA,

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USA) was used (Fig. S2 and Text S2). The operation was performed by recirculating RO

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permeate and concentrate into the feed reservoir and maintained at a permeate flux of 20 L/m2h,

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RO feed temperature of 14–16 °C and a recovery of 20% (permeate and concentrate flow rate =

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2.5 and 10 L/min, respectively). To stabilize the process condition, the RO system was first

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operated using a drinking water disinfected with chlorine (