Assessing bacterial growth potential in a model distribution system receiving nanofiltration membrane treated water

Desalination 296 (2012) 7–15

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Assessing bacterial growth potential in a model distribution system receiving nanofiltration membrane treated water Se-Keun Park a,⁎, Jae-Hoon Choi b, Jiang Yong Hu c a b c

Waterworks Research Institute, Seoul Metropolitan Government, 716-10 Cheonhodaero, Gwangjin-gu, Seoul 143-820, Republic of Korea R&D Center, Samsung Engineering Co. Ltd., 415-10 Woncheon-dong, Youngtong-gu, Suwon, Gyeonggi-do 443-823, Republic of Korea Division of Environmental Science and Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore

a r t i c l e

i n f o

Article history: Received 2 November 2011 Received in revised form 26 March 2012 Accepted 28 March 2012 Available online 25 April 2012 Keywords: Nanofiltration (NF) Drinking water Biostablility Bacterial growth Biofilm

a b s t r a c t This study was designed to assess the extent of bacterial growth in a model distribution system fed with the water treated by nanofiltration (NF) membranes under controlled conditions. The three NF membranes were used in improving the quality of tap water in a lab-scale process. The changes in total cell counts and culturable heterotrophic bacteria in both bulk water and biofilm were monitored during transport of NF permeate water through the model distribution system. The NF membranes were capable of removing the assimilable organic carbon (AOC) contents, but not low enough to restrict bacterial growth. Bacterial adhesion and cellular proliferation were observed in the NF permeate-fed systems, which were apparently characterized by the increase in the fraction of culturable heterotrophic bacteria and the relative abundance of α-Proteobacteria in bacterial community of biofilms. The overall specific cell growth rate which indicates the growth potential in the whole system was calculated as 0.07–0.08 day − 1. Results indicate that the NF permeate water has some potential to support bacterial growth in a distribution system, although the NF membrane is able to improve the quality of drinking water. Consequently, proper care should be taken to secure biostability to acceptable levels. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In drinking water distribution systems, the undesirable growth of bacteria and the vigorous biofilm buildup at the water-pipe material interface can lead to the deterioration of hygienic water quality and, in some cases, give rise to a public health concern and an issue about service quality [1,2]. Concerns about bacteriologically safe drinking water are encouraging the application of advanced water treatment technologies to remove low concentrations of nutrients available for microbial growth from the water and to provide effective barriers against microorganisms in drinking water treatment, which can minimize the use of disinfectants [3–5]. A promising technology for the enhanced removal of the contaminants can be the membrane separation, particularly nanofiltration (NF) that has a high rejection of inorganic and organic compounds and microorganisms in feed waters [6–8]. NF membranes, mainly because of their small pore size and charge property, can remove organic substances with molecular weights of higher than 200–300 Da and multivalent ions [8]. Research findings showed that NF significantly enhanced drinking water quality by

⁎ Corresponding author. Tel.: + 82 2 3146 1781; fax: + 82 2 3146 1876. E-mail address: [email protected] (S.-K. Park). 0011-9164/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2012.03.022

decreasing concentrations of organic matter and bacterial counts, resulting in more efficient disinfection and lowering disinfection by-product formation potential [3,4,7–10]. It was also reported that NF was more successful in removing a large fraction of biodegradable dissolved organic carbon (BDOC) than assimilable organic carbon (AOC) depending on feed water qualities such as pH, hardness, and ionic strength [5,6,11]. Biostability refers to the ability of water to support the growth of microorganisms. Previous studies on the biostability of NF permeate water mainly focused on changes in AOC and BDOC concentrations in treated water. The AOC and BDOC measurements may be useful for assessing the biostability or bacterial regrowth potential of a specific water, but cannot provide a quantitative analysis on the amount of bacterial biomass supported by the water. There is still little information available with regard to the bacterial biomassbased biostability of the water treated by NF membranes, and thus further investigation is required. This study was conducted to provide further information on the biostability of NF membrane treated water by means of quantitative measurements of bacterial growth. Three flat-sheet NF membranes were selected for additional treatment of tap water produced by a conventional treatment system. Experiments were designed to assess the growth of biofilm and bulk bacteria in a model distribution system fed with NF permeate waters in the absence of a disinfectant.

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2. Materials and methods

2.3. Operational conditions of membrane filtration test unit

2.1. NF membranes

Prior to membrane filtration tests, the membrane unit was cleaned with NaOCl (0.01% available chlorine), and followed by Milli-Q water for 30 min in order to prevent the microbial contamination. For the membrane pretreatment, fresh membrane sheets were soaked in Milli-Q water overnight. The membrane sheets were again rinsed with Milli-Q before being transferred to the test cell. Each filtration test was conducted in three steps: (1) the membranes were compacted at their operating pressures for at least 2 h using Milli-Q water; (2) the pure water flux of each membrane was determined at the end of the compacting process; (3) the feed tank was emptied and filled with feed water for the filtration test. In this study, the feed water was filtered through the unit at an operating pressure of 7.9–9.0, 4.1–4.8, and 3.8–4.1 bar for NF1, NF2 and NF3 membranes, respectively. The permeate fluxes for NF1, NF2 and NF3 membranes were 3.4–3.9, 9.6–11.1 and 11.6–12.5 L/m 2 h bar at 20 °C, respectively, which allowed the flow rate for each membrane to be 1% of the retentate flow rate. During the filtration experiments, the retentate was circulated back to the feed tank and the permeate was collected in a 2-L Pyrex storage bottle. On a daily basis for 3 weeks, each NF membrane sheet produced 1-L permeate from 5-L feed water and in total 20-L permeate from 100-L feed water over the course of the experiments. At operational intervals, the permeate and feed samples were collected for analysis. The rejection efficiency (R) of any membrane is defined as:

Three commercial flat-sheet NF membranes, denoted as NF1 (CK, GE Osmonics, Minneetonka, MN, USA), NF2 (HL, GE Osmonics, Minneetonka, MN, USA) and NF3 (NF270, Dow Filmtec, Minneapolis, MN, USA), were used in this study. The membrane properties given by the manufacturers in addition to some values determined in this study are shown in Table 1. NF1 and NF2 membranes are composed of cellulose acetate and thin film composite polyamide, respectively. NF3 has a very thin semi-aromatic piperazine-based polyamide active layer. The molecular weight cut-off (MWCO) of membranes estimated in this study is quite similar (Table 1). NF2 and NF3 are relatively hydrophilic (represented by a lower contact angle; Table 1) compared with NF1. All the three membranes are negatively charged at the pH 7.6±0.2 of feed water used in this study. This suggests the possibility of electrostatic repulsion of negatively charged components by the membrane [12].

2.2. Membrane filtration test unit and feed water A laboratory-scale membrane filtration test unit (Sepa Cell System, GE Osmonics, Minneetonka, MN, USA) was used in this study. A schematic diagram of the experimental setup is illustrated in Fig. 1a, which includes the membrane test cell, pump (370 W, 1725 rpm), pressure gage, flow meter, 10-L feed tank, and refrigeration unit (Model Recirculating Chiller B-740/14, Büchi Co., New Castle, DE, USA). A test cell houses a round-shaped membrane sheet that has an effective area of 78.5 cm 2 (10 cm in diameter). In the test unit, the water in the feed tank was fed to the cell through the side inlet by a pump, and the retentate and the permeate were collected from the center of the top and the bottom of the cell, respectively. Tap water from a local waterworks (Choa Chu Kang Waterworks, Singapore), which was treated by the combination of coagulation, sedimentation, rapid sand filtration, chloramination and pH adjustment, was used as feed water. Detailed characteristics of the tap water during the filtration are presented in Table 2. Prior to the filtration test, 5 mL of 3% (w/v) sodium thiosulfate solution was spiked into the feed water to remove free and combined chlorine residuals. The temperature of the feed water was kept at 20 °C throughout the experiment by means of a thermostatically controlled cooling coil connected to a refrigeration unit.

Table 1 Characteristics of the NF membranes used in the study. NF1 Manufacturer/ designation Material Molecular weight cut offa Contact angle(°)a Zeta potential (mV)a Operating pH rangec Salt rejection (%)c Operating pressure (bar)a Permeate flux (L/m2 h bar)a

NF2

NF3

GE Osmonics/CK GE Osmonics/HL Dow Filmtec/NF270 CA ~ 300

TFC ~ 350

PAp TFC ~ 350

62.3 ± 1.2 − 16.7 ± 1.8b 2–8 92d 7.9–9.0

32.5 ± 0.5 − 21.8 ± 0.6b 3–9 98e 4.1–4.8

30.2 ± 1.3 − 27.9 ± 0.8b 3–10 97e 3.8–4.1

3.4–3.9

9.6–11.1

11.6–12.5

CA: cellulose acetate, TFC: thin film composite, PAp: semi-aromatic piperazine-based plolyamide. a Determined with feed water in this study. b Determined in the feed (tap) water (pH 7.6 ± 0.2). c Based on the manufacturer's specifications. d Based on Na2SO4.

Rð% Þ ¼

1−

Cp Cf

!  100

ð1Þ

where Cp and Cf are the permeate and feed concentrations for each of the constituents, respectively. 2.4. Model distribution system and operational conditions Biofilm reactor (Model CBR 90-1 CDC, Biosurface Technologies, Bozeman, MT, USA) was used as a model distribution system. A scheme of the biofilm reactor is shown in Fig. 1b. Basically, the reactor consists of eight polypropylene coupon holders supported by an ultra-high-molecular-mass polyethylene ported lid. Each coupon holder accommodates three removable coupons. Each coupon has an exposed surface area of 2.53 cm 2 (1.27 cm diameter and 0.3 cm thickness). A total of 24 polyvinyl chloride (PVC) coupons which support biofilm growth are held in a biofilm reactor. The lid with coupon holders and coupons is mounted in a 1-L Pyrex glass vessel with side-arm discharge port. The glass vessel is placed on a controlled stir plate to provide constant rotation of the baffled stir bar at a designated speed. Rotation of the baffle provides constant mixing and consistent shear to the coupon surface. The reactor has a 350 mL working volume. Prior to the beginning of each run, the reactors were prepared as recommended by the manufacturer (Biosurface Technologies, Bozeman, MT, USA); reactor components (reactor top/vessel, coupons, rods, baffled stir bar) were soaked for 2 h in 2 M HCl, rinsed abundantly with Milli-Q water, air-dried, and then assembled. The assembled reactors were then autoclaved at 121 °C for 15 min, and air vent and tubing were attached to the rigid tubes in the reactor top. At the beginning of the runs, the three biofilm reactors (denoted as NFR1, NFR2 and NFR3) were fed with dechlorinated/dechloraminated tap water for 1 day so that they can be colonized by indigenous bacteria present in tap water ahead of a supply of NF-treated water. After then, the biofilm reactors were supplied with NF-treated water (permeate) and run at a flow rate of 14.5 mL/h to provide a hydraulic retention time of 1 day throughout the course of this study. Water retention times within a distribution system commonly range from

S.-K. Park et al. / Desalination 296 (2012) 7–15

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Fig. 1. Schematics of (a) the membrane filtration unit and (b) the biofilm reactor.

several hours to days as water is delivered to households, with typical lengths of 1 to 3 days [13]. The reactor was stirred at a defined baffle speed (rpm) of 50 rpm, with rotation of the baffle consistently mixing the contents of the reactor and applying approximately 0.01 N/m 2 of shear force to the coupon surfaces. Each reactor run lasted 20 days at room temperature (25–29 °C) with periodic bulk water and biofilm sampling. All reactors were covered with opaque material to prevent the potential for phototrophic growth in the reactor system. For comparison purposes, a control reactor (denoted as CR) was run by feeding the dechlorinated/dechloraminated tap water into the biofilm reactor under the same operating conditions.

2.5. Biofilm sampling and analysis The biofilm was sampled by removing PVC coupons and placing them in a tube containing 10 mL of sterile saline solution (0.85% (w/v) NaCl). The coupons were scraped manually with a sterile disposable cell lifter (Fisherbrand, Pittsburgh, PA, USA). The tube was vigorously vortexed for 3 min, sonicated for 3 min in an ultrasonic cleaning bath (37 kHz and 25 °C) (Elmasonic S60, Elma Hans Schmidbauer GmbH & Co. KG, Singen, Germany), and vortexed again for 3 min. Subsequently the coupons were removed from the biofilm suspension.

The total cell counts (TCC) in the biofilm suspension sample were filtered through a 0.22 μm black polycarbonate membrane (GTBP 02500, Millipore, Bedford, MA, USA) and enumerated using an epifluorescence microscope (Olympus BX51, Olympus America, Melville, NY, USA) following 4′,6-diamidino-2-phenylindole (DAPI) staining [14]. Biofilm TCC was expressed as cells/cm2. Heterotrophic plate counts (HPC) in the biofilm sample was analyzed by the spread plate method (9215 C) or the membrane filter method (9215 D) of Standard Methods [15] on R2A agar (Difco Laboratories, Detroit, MI, USA) and incubation at 25 °C for 7 days. Biofilm HPC was expressed as CFU/cm 2. The composition of biofilm populations taken at the end of the operation was investigated using fluorescently labeled oligonucleotides specific for the α-, β- and γ-subclasses of Proteobacteria called fluorescence in situ hybridization (FISH) assay. Biofilm suspensions were collected by filtration onto a 25-mm diameter and 0.22-μm white polycarbonate membrane (GTTP02500, Millipore, Bedford, MA, USA). Fixation of the sample was done by a 4% paraformaldehydephosphate buffered saline solution [16]. Samples were dehydrated and hybridized with the gene probes (3 h, 46 °C) using a formamide concentration of 20% or 35% in the hybridization solution. The following fluorescently labeled gene probes were used (synthesized by Research Biolabs, Singapore): EUB338 (kingdom-specific for bacteria, 5′-GCTGCCTCCCGTAGGAGT-3′), ALF1b (specific for the α-subclass of

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Table 2 Permeate quality and rejection efficiency (R) of the NF membranes. Parameters

Feed water

NF1

NF2

NF3

pH Conductivity (μS/cm) R (%) TDS (mg/L) R (%) Alkalinity (mg/L as CaCO3) R (%) Hardness (mg/L as CaCO3) R (%) Turbidity (NTU) R (%) NPOC (mg/L) R (%) UV254 (cm− 1) R (%) AOC (μg/L) R (%) TCC (cells/mL) NH4+ (mg/L) R (%) NO3− (mg/L) R (%) PO43 − (mg/L) R (%) Na+ (mg/L) R (%) K+ (mg/L) R (%) Mg2 + (mg/L) R (%) Ca2 + (mg/L) R (%) Cl− (mg/L) R (%) SO42 − (mg/L) R (%)

7.95 (7.72–8.12) 139.0 (136.3–144.6)

6.97 ± 0.24 21.3 ± 1.5 (86.0 ± 1.1) 8.5 ± 0.9 (85.7 ± 1.1) 4.8 ± 1.9 (73.9 ± 9.1) 9.6 ± 0.6 (79.9 ± 1.1) 0.13 ± 0.03 (66.8 ± 7.4) 0.08 ± 0.02 (91.5 ± 2.7) 0.005 ± 0.002 (88.1 ± 3.5) 10.8 ± 5.5 (69.0 ± 11.4) – 0.08 ± 0.02 (66.1 ± 7.0) 2.15 ± 0.56 (44.0 ± 11.4) 0.04 ± 0.02 (73.9 ± 5.8) 2.10 ± 0.31 (73.4 ± 3.4) 0.92 ± 0.20 (80.9 ± 3.6) 0.07 ± 0.03 (92.3 ± 2.8) 1.19 ± 0.48 (92.2 ± 2.5) 2.74 ± 1.01 (74.5 ± 8.2) 0.19 ± 0.19 (99.0 ± 0.7)

7.54 ± 0.30 57.1 ± 4.5 (58.9 ± 4.6) 22.8 ± 1.9 (60.0 ± 1.1) 8.6 ± 1.8 (47.2 ± 4.2) 16.8 ± 0.9 (64.8 ± 1.8) 0.11 ± 0.01 (60.8 ± 7.4) 0.10 ± 0.04 (87.6 ± 5.4) 0.005 ± 0.000 (87.8 ± 5.4) 11.6 ± 6.5 (66.7 ± 16.7) – 0.10 ± 0.04 (62.2 ± 17.3) 1.92 ± 0.43 (44.6 ± 8.0) 0.04 ± 0.03 (73.1 ± 12.9) 4.61 ± 0.16 (37.9 ± 2.4) 2.26 ± 0.29 (47.7 ± 4.5) 0.26 ± 0.02 (74.4 ± 3.6) 5.48 ± 0.23 (67.5 ± 1.8) 7.75 ± 0.17 (26.8 ± 5.7) 1.01 ± 0.07 (94.8 ± 0.4)

7.52 ± 0.18 65.6 ± 2.6 (56.8 ± 1.6) 24.8 ± 0.8 (57.7 ± 1.2) 9.8 ± 1.5 (41.6 ± 5.2) 17.0 ± 0.4 (63.7 ± 1.1) 0.13 ± 0.04 (56.7 ± 7.9) 0.14 ± 0.03 (85.0 ± 3.2) 0.006 ± 0.001 (85.3 ± 2.7) 12.5 ± 7.5 (66.2 ± 20.4) – 0.13 ± 0.03 (50.2 ± 11.8) 1.77 ± 0.21 (52.1 ± 5.4) 0.05 ± 0.01 (73.5 ± 6.3) 5.36 ± 1.43 (32.5 ± 19.0) 2.25 ± 0.16 (49.8 ± 0.2) 0.31 ± 0.02 (65.9 ± 2.2) 5.32 ± 0.10 (65.6 ± 0.5) 7.55 ± 0.30 (27.5 ± 1.9) 0.93 ± 0.43 (93.5 ± 2.6)

54 (47–57) 17 (10–22) 45.9 (45.4–46.4) 0.31 (0.21–0.44) 0.80 (0.63–0.91) 0.036 (0.021–0.046) 30.60 (12.32–58.20) 6.4 × 103 (1.9–8.8 × 103) 0.24 (0.19–0.28) 3.13 (2.31–3.69) 0.10 (0.04–0.20) 7.68 (7.22–8.29) 4.52 (4.17–4.83) 0.97 (0.89–1.07) 15.82 (14.26–17.60) 10.55 (10.17–11.51) 16.38 (11.89–21.86)

the Proteobacteria, 5′-CGTTCGYTCTGAGCCAG-3′), BET42a (specific for the β-subclass of the Proteobacteria, 5′-GCCTTCCCACTTCGTTT-3′) in combination with the unlabelled probe GAM42a, and GAM42a (specific for the γ-subclass of the Proteobacteria, 5′-GCCTTCCCACATCGTTT-3′) in combination with the unlabelled probe BET42a. After a washing step with hybridization buffer without formamide at 48°C for 20 min [16], the sample was incubated with DAPI (1 mL of 0.05 μg/mL DAPI solution) for 10 min for the staining of all cells.

2.6. Membrane and water quality analysis The zeta potential values of the tested NF membranes were measured using a SurPASS electro kinetic analyzer (Anton Paar, Graz, Austria) following the streaming current methodology described by Childress and Elimelech [17]. The contact angle measurement for membrane hydrophobicity was performed according to the sessile drop technique using a goniometer (VCA Optima Systems, AST Product Inc., Billerica, MA, USA). A drop (0.5–1 μL) of deionized water was placed on the membrane surface using a micro-syringe. The contact angle of each membrane was calculated from the average of the contact angles at both left and right sides of the drop. To measure the MWCO of each membrane, non-ionic organic compounds were chosen with a molecular weight range of 46 to 594 Daltons (Da) (ethanol, ethylene glycol, butanol, glucose, sucrose and raffinose). The total organic carbon (TOC) concentration of each solution was prepared at 50 mg/L. Feed and filtrate streams were analyzed for TOC concentrations to obtain the molecular weight distribution curves. The molecular weight of the solute for which the rejection by the membrane is 90% was taken as the MWCO of the NF membranes [18].

The pH, conductivity and total dissolved solid (TDS) were measured using Orion 4-Star Plus Portable pH/Conductivity Meter (Thermo Fisher Scientific Inc., Beverly, MA, USA). Alkalinity and hardness were analyzed with the titration method using mixed bromocresol green-methyl red indicator (2320 B) and the ethylenediaminetetraacetic acid titrimetric method (2340 C) of Standard Methods [15], respectively. Turbidity and UV absorption at 254 nm were measured with 2100N Laboratory Turbidimeter (Hach Co., Loveland, CO, USA) and UV–VIS spectrophotometer (Model UV1700, Shimadzu, Tokyo, Japan) with a 1 cm quartz cell, respectively. TOC and non-purgeable organic carbon (NPOC) were measured using a TOC analyzer (TOC-VCSH, Shimadzu, Tokyo, Japan). AOC concentrations were determined by Standard Methods [15] utilizing Pseudomonas fluorescence strain P17 and Spirillum sp. strain NOX. Cations and anions were analyzed by ionic chromatography (Model DX 500, Dionex, Sunnyvale, CA, USA) using CS12A cation column and AS9-HC anion column. For NF permeate water, grab samples (100 mL) were centrifuged at 5000 ×g for 10 min. The cell pellet was resuspended in 10 mL sterile saline solution. The suspension was then subjected to a similar treatment as the biofilm suspension. TCC and HPC levels present in the bulk water samples of the biofilm reactor were determined by the same method used for biofilm TCC and HPC enumeration. Results were expressed as cells/mL and CFU/mL for TCC and HPC, respectively. 2.7. Growth model for biofilm and bulk bacteria Using the TCC data obtained from influent water, bulk water and biofilm samples, the bacterial growth rates were determined using the method described by Manuel et al. [19].

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An overall model for biofilm can be expressed by the following equation [20–22]:

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3. Results 3.1. NF permeate quality

dX biof ilm ¼ P−β X biof ilm dt

ð2Þ

where is the biofilm cell number per unit surface area (cells/cm 2), is the biofilm production rate per unit surface area (cells/ cm 2/day), which includes the attachment of new cells and cell growth within the biofilm, and represents the average residence time of cells within the biofilm.

Xbiofilm P

β− 1

Assuming that P is constant, as demonstrated by Melo and Vieira [22] and Pereira et al. [23], the integration of Eq. (2) then leads to   −βt X biof ilm ¼ X biof ilm; max 1−e

ð3Þ

and X biof ilm; max ¼

P β

ð4Þ

where Xbiofilm, max is the maximum density of biofilm at steady-state (cells/ cm 2). By the definition of Manuel et al. [19], the specific growth rate of the biofilm at steady state, μbiofilm (day− 1), can be expressed as a function of the steady-state maximum density of the biofilm (Xbiofilm, max) and the biofilm production rate (P) by the following equation: μ biof ilm ¼

P X biof ilm; max

ð5Þ

which means that the cell growth in the biofilm at steady state is equal to the cell detachment from the biofilm. From the specific growth rate of the biofilm cell (μbiofilm), the specific growth rate of bulk cell at steady state, μbulk (day− 1), can be calculated:

μ bulk ¼

Q ðX bulk −X in Þ−μ biof ilm X biof ilm; max A X bulk V

The permeate qualities and related rejection efficiencies for the three membranes are listed in Table 2. As expected, the NF membranes improved the quality of tap water. The levels of pH, conductivity, TDS, alkalinity, hardness and turbidity in the permeate water were within a range of 6.74–7.87, 18.8–67.6 μS/cm, 7–26 mg/L, 2–12 mg/L as CaCO3, 9–18 mg/L as CaCO3 and 0.1–0.2 NTU, respectively. The three NF membranes resulted in a significant removal of organic carbon from the tap water, as measured by NPOC, UV254 and AOC (Table 2). During the entire operation, they produced water with NPOC concentration below 0.17 mg/L and showed more than 80% removal efficiency. In the case of UV254, removal efficiencies by NF membranes were similar to those of NPOC (Table 2). The measurements of AOC showed that the NF membranes are capable of removing AOC present in water. These results are consistent with some other studies [5,8,11]. The NF permeate water had AOC concentrations ranging between 6.1 and 23.8 μg/L and the removal rates of AOC were relatively lower than those of NPOC and UV254 (Table 2). AOC compounds are typically composed of small and low molecular weight organic compounds with negatively charged functionality [25]. Charge repulsion has been suggested as the main mechanism for AOC removal by negatively charged NF membranes [11]. At conditions of low hardness and ionic strength (TDS) like the tap water used in this study, AOC removals could result from charge repulsion between AOC compounds and membrane surfaces. The fluctuations in AOC removals were observed near the end of the operation. This can be attributed to a decrease in the net negative charge on the membrane. Accumulation of counter-ions which have a charge opposite to that of the membrane is able to screen the membrane charge, thus lessening the charge exclusion of the charged organic compounds from the membrane [26]. Another possibility for the fluctuations in AOC removals is that the presence of background anions (e.g. SO42 −, Cl−) would hinder electrostatic interactions between the membranes and charged organic compounds [25], leading to a reduction in the membranes' AOC removal efficiency. The average for the rejection of some inorganic ions is presented in Table 3. The NF membranes removed inorganic ions from the feed water, as reported previously in other studies [5,8,27,28]. The concentrations of ammonium, nitrate and phosphate ions in the permeate water were in the range of 0.05–0.16, 1.42–2.80, and 0.02–0.07 mg/L, respectively. The driving mechanisms responsible for the rejection of inorganics by NF membrane, such as Donnan (charge) and steric (size) exclusion, have been reported elsewhere [27–29].

ð6Þ 3.2. Biofilm and bulk cell growth in NF permeate-fed reactors

where Q V Xbulk Xin A

is the inlet flow rate (mL/day), is the volume of the system (mL), is the bulk cell concentration at steady state (cells/mL), is the cell concentration at the inlet water (cells/mL), and is the internal surface area (cm 2).

At steady state, an overall specific cell growth rate, μoverall (day − 1), defined as a weighted average of the cell growth rates in the biofilm and in the bulk water [24], can be expressed by μ overall ¼

Q ðX bulk −X in Þ X bulk V þ X biof ilm; max A

ð7Þ

The derivation of the above equations is described in detail by Manuel et al. [19].

In this study, it was assumed that there is no permeation of bacterial cells through the NF membrane. The experiments were designed to allow indigenous bacteria to colonize the biofilm reactor for 1 day while feeding with dechlorinated/dechloraminated tap water. The biofilm reactors were colonized by bacteria at a concentration of 1.0 × 103 ± 5.2 × 102 CFU/mL and 2.2 × 104 ± 3.2 × 103 cells/mL in the bulk water phase, and 9.3 × 101 ± 2.2 × 10 1 CFU/cm2 and 1.2 × 10 4 ± 4.5 × 103 cells/ cm2 in the biofilm phase. These levels were excluded from the assessment of bacterial growth in NF permeate-fed reactors. After the bacterial colonization of the biofilm reactors, the NF permeate water processed by the three NF membranes were continuously fed into the biofilm reactors (NFR1, NFR2 and NFR3). Post-disinfection of NF permeate water was not employed in this study, thereby all reactors were operated without any disinfectant addition. In such a case, bacterial growth may be mainly influenced by the content of substrate available in the permeate water.

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Under given conditions, biofilms grown on PVC coupons in the biofilm reactors were measured by TCC and HPC. The biofilm TCC and HPC densities for each of the reactors are shown in Fig. 2. The cell numbers for biofilms grown in NFR1, NFR2 and NFR3 were in reasonably similar magnitude, but lower than in CR. Biofilm TCC at a quasi-steady state averaged 9.1 × 10 3 ± 2.6 × 10 3, 8.7 × 10 3 ± 3.3 × 10 3 and 1.2 × 10 4 ± 2.6 × 10 3 cells/cm 2 for NFR1, NFR2 and NFR3, respectively, while 6.7 × 10 5 ± 3.0 × 10 5 cells/cm 2 for CR (Fig. 2a). At a quasi-steady state, biofilm HPC represented 23.2 ± 2.4, 26.8 ± 3.3 and 19.6 ± 4.0% of biofilm TCC for NFR1, NFR2 and NFR3, respectively, while 14.9 ± 1.4% for CR (Fig. 2b). Bulk water samples from the biofilm reactors were periodically collected for 20 days and analyzed by TCC and HPC. The densities of cells in the bulk water of the biofilm reactors are shown in Fig. 3. The cell numbers in the bulk water showed limited variation after 5 to 7 days of operation. TCC numbers in the bulk water at a quasi-steady state averaged 1.6× 103 ± 4.5 × 10 2, 1.6 × 10 3 ± 4.5 × 102 and 1.5 × 103 ± 3.6 × 102 cells/mL for NFR1, NFR2 and NFR3, respectively, while 2.1 × 105 ± 2.7× 104 cells/mL for CR (Fig. 3a). These TCC levels observed in the bulk water of NFR1, NFR2 and NFR3 were about two orders of magnitude lower than in CR. At a quasi-steady state, HPC numbers in the bulk water represented 33.2± 11.2%, 35.5 ± 6.4% and 33.2 ± 11.1% of bulk TCC for NFR1, NFR2 and NFR3, respectively, while 18.4 ± 5.0% for CR (Fig. 3b). The ratio of HPC to TCC numbers is linked to the culturability in the bacterial populations, which is indicative of the ability of bacteria to form colonies on commonly used media like R2A. In quasi-steady state, the range of bacterial culturability observed in CR fed with the tap water was 13.6–16.4% for the biofilm and 14.4–27.1% for the bulk water, respectively (Figs. 2 and 3). These ranges are similar with

those reported previously in other studies [19,30,31] on drinking water. Of particular interest is that at quasi-steady state the range of culturability (14.4–30.3% for biofilm, 16.8–48.0% for bulk water) observed in the NFRs (NFR1, NFR2 and NFR3) was considerably above the range of values in CR (Figs. 2 and 3). It is generally accepted that poorly culturable and identified microbes can be present in drinking water distribution systems. The tap water conveyed through a pipe network could include a diverse group of poorly culturable or nonculturable bacteria. The recruitment of diverse species affecting bacterial community development is more facile in CR than in NFRs. It would therefore cause a noticeable difference in the fraction of culturable HPC bacteria between CR and NFRs. Considering that the source water used in this study was conveyed through the chloraminated drinking water distribution pipe, for instance, in the case of CR a large fraction of bacteria with recalcitrance to HPC culture may be attributed to the presence of nitrifying bacteria such as ammonia-oxidizing bacteria (AOB) often occurring in chloraminated drinking water distribution system.

3.3. Bacterial growth rates in NF permeate-fed reactors The bacterial growth rates were calculated on the basis of the results from TCC numbers (Figs. 2a and 3a). The model used in this study assumes that at steady state the net biofilm growth is balanced by the biofilm detachment. The steady-state maximum density of the biofilm (Xbiofilm, max) and the biofilm production rate (P) were determined by Eqs. (3) and (4). The specific growth rate of biofilm bacteria (μbiofilm) was calculated with Eqs. (3) and (5). The specific growth rate of bulk bacteria (μbulk) and the overall specific growth

(a) Bulk TCC (cells/mL)

105

104

103

102 CR

NFR1

NFR2

NFR3

101

(b) Bulk HPC (CFU/mL)

105

104

103

102

101 0

5

10

15

20

Time (days) Fig. 2. (a) TCC and (b) HPC levels in the biofilms observed in model distribution systems fed with NF permeate and tap water.

Fig. 3. (a) TCC and (b) HPC levels in the bulk waters observed in model distribution systems fed with NF permeate and tap water.

S.-K. Park et al. / Desalination 296 (2012) 7–15

13

Table 3 Bacterial growth rates in biofilm and bulk water.

Xbiofilm, max (cells/cm2) P (cells/cm2/day) μbiofilm (day− 1) μbulk (day− 1) μoverall (day− 1)

CR

NFR1

NFR2

NFR3

9.8 × 105 ± 1.8 × 105 9.5 × 104 ± 6.7 × 103 0.10 ± 0.04 0.51 ± 0.06 0.17 ± 0.02

1.8 × 104 ± 4.8 × 103 9.0 × 102 ± 1.0 × 102 0.05 ± 0.02 0.38 ± 0.19 0.08 ± 0.02

1.7 × 104 ± 6.4 × 103 9.2 × 102 ± 2.2 × 102 0.06 ± 0.03 0.40 ± 0.18 0.08 ± 0.02

2.0 × 104 ± 2.0 × 103 1.2 × 103 ± 1.9 × 101 0.06 ± 0.01 0.36 ± 0.17 0.07 ± 0.02

rate (μoverall) were calculated using Eqs. (6) and (7), respectively. The estimated values are summarized in Table 3. The estimation of the growth rate parameters yielded lower values for the three NF permeate-fed reactors (NFR1, NFR2 and NFR3) than for CR, suggesting that biofilm and bulk phase bacteria in the NF permeate-fed reactors are slow growing under the given conditions. The values were not significantly different in the three NF permeatefed reactors (NFR1, NFR2 and NFR3) (Table 3). The Xbiofilm, max and P values determined in the three NF permeate-fed reactors were on the order of 10 4 cells/cm 2 and 10 2–10 3 cells/cm 2/day, respectively. The P values determined in the three NF permeate-fed reactors are about 2 orders of magnitude lower than in CR. The specific growth rates for biofilm (μbiofilm) and bulk phase bacteria (μbulk) were within the range of 0.05–0.06 day − 1 and 0.32–0.36 day − 1, respectively, which corresponded to a doubling time of 11.2–13.7 days and 1.7–1.9 days, respectively. The overall growth rate (μoverall) which indicates the growth potential including biofilm and bulk bacteria in the whole system was calculated as 0.07–0.08 day − 1 for the NF permeate-fed reactors, representing a doubling time of 8.6–10.5 days. This is comparable to the overall growth rate of 0.17 day − 1 (a doubling time of 4.0 days) obtained from CR.

3.4. Bacterial biofilm composition In an attempt to investigate bacterial community composition in biofilm, FISH analysis using gene probes which are specific for different subclasses of the Proteobacteria was performed on biofilm samples taken at the end of each run. The percentage of EUB-detected bacteria relative to DAPI-stained bacteria (TCC) is presented in Table 4. The bacterial cells not being detected with rRNA-targeted oligonucleotide probes might not contain enough intracellular rRNA to be observed by epifluorescent microscopy [32]. The biofilm growth in CR was represented by an abundance of bacteria belonging to the α and β subclasses of Proteobacteria; the relative abundance of the αProteobacteria (11.3%) was not as high as the β-Proteobacteria (18.5%) (Table 4). The γ subclass of Proteobacteria was presented with minor proportion (5.3%). The α and β subclasses of Proteobacteria have been found to be typical bacterial groups in biofilms of drinking water networks [16,33]. The prevalence of these bacterial divisions seems to be a common feature of most drinking water systems. FISH showed that the α and β subclasses of Proteobacteria were more abundant than the γ-subclass of Proteobacteria in the biofilms of NFRs (Table 4). It is interestingly observed that the proportions of the α- and β-Proteobacteria in NFRs differed from those of the α- and

β-Proteobacteria in CR. For the biofilms of NFRs, the α-Proteobacteria were detected in a higher proportion than the β-Proteobacteria. Some studies have demonstrated that the β-Proteobacteria involve AOB responsible for nitrification occurrence in a chloraminated drinking water distribution system [34,35]. Considering that the source water used in this study was conveyed through the chloraminated drinking water distribution pipe, AOB affiliated with the β-Proteobacteria can constitute a part of the biofilm community. As shown in Table 4, a large fraction of the β-Proteobacteria in the biofilms of CR may be attributed to the presence of AOB inhabiting the biofilms. Unlike CR, the introduction of AOB into the reactor could be restricted, since the membranes are capable of rejecting bacteria by size exclusion, electrostatic repulsion, etc. [36]. The NF-driven nutrient scarcity could enable AOB growth to be situated in a harsh environment. These might be possible reasons for a reduction in the proportion of the β-Proteobacteria in the biofilms of NFRs. 4. Discussion The uniqueness of NF membranes is highlighted by their ability to selectively reject dissolved salts and ions, and low molecular weight organic compounds. The biostability of the water is determined, in part, by the concentration of AOC consisting of a broad range of low molecular weight organic compounds. Although NF membranes have been shown to be able to remove AOC compounds [5,8,11], the permeability of AOC through NF membranes should not be overlooked. Meylan et al. [5] have shown that AOC compounds can permeate through NF membranes. The 10 μg/L of AOC has been proposed as the threshold value of AOC for achieving biostability in the absence of a disinfectant [37]. According to the AOC threshold value (10 μg/L), in this study the AOC contents measured in the NF permeates were not low enough to restrict bacterial growth over the entire concentration range, although there had been the case where the AOC concentration was as low as b10 μg/L. With such AOC levels above 10 μg/L (Table 2), the NF permeate waters are not likely to be regarded as biologically stable. Several studies have determined different values for specific growth rate of bacteria in drinking water biofilms under the different experimental conditions; the values ranging between 0.03 and 0.72 day − 1 have been reported in the literature [24,38–42]. Of particular, the specific growth rates of biofilm bacteria (0.04– 0.06 day − 1) observed in chlorinated drinking water systems [39,40] are quite similar to what were observed in the three NF permeate-fed reactors, despite the fact that no disinfectant residual was maintained in this study. This emphasizes the importance of a limited availability of nutrients on bacterial growth.

Table 4 Bacterial composition of three-week old biofilms grown in model distribution systems fed with tap water (CR) and NF permeates (NFR1, NFR2, and NFR3). CR 2

TCC (cells/cm ) EUB338/TCC (%) ALF1b/TCC (%) BET42a/TCC (%) GAM42a/TCC (%)

NFR1 5

8.2 × 10 ± 1.6 × 10 49.2 (± 3.7) 11.3 ± 0.04 18.5 ± 0.06 5.3 ± 0.05

5

NFR2 4

2.0 × 10 ± 3.2 × 10 40.9 (± 2.3) 15.7 ± 0.02 10.2 ± 0.19 2.5 ± 0.02

3

NFR3 4

2.9 × 10 ± 3.8 × 10 42.5 (± 3.6) 20.2 ± 0.18 8.5 ± 0.11 3.3 ± 0.02

3

2.6 × 104 ± 5.6 × 103 42.0 (± 2.5) 17.5 ± 0.11 8.7 ± 0.07 2.8 ± 0.03

14

S.-K. Park et al. / Desalination 296 (2012) 7–15

µbiofilm (day-1)

Although the average specific growth rates of bulk phase bacteria observed in the three NF permeate-fed reactors were lower than those in CR, large standard deviation values were observed (Table 3). An analysis of variance proved that the differences in the specific growth rates of bulk bacteria between the three NF permeate-fed reactors and CR were not statistically significant at a significance level of 0.05. It should be pointed out that bulk bacteria in the NF permeate-fed reactors were less abundant, but not necessarily consistent with the lower growth rates. The specific growth rates of bacteria in the bulk water were much higher than those in the biofilms (Table 3), suggesting that the growth of bulk phase bacteria is a significant contributor to the total bacterial production. This is in agreement with some other studies [19,24,31]. The results of Manuel et al. [19] for specific growth rates in bulk water are around 10 times higher than in biofilm. Thus these results suggest that biofilm bacteria may not always dominate the distribution system as is commonly believed [31]. It is generally accepted that the bulk phase bacteria are susceptible to environmental stress (e.g. nutrient starvation, disinfectant), while the biofilm bacteria are less. In the present study, specific conditions such as the absence of disinfectant and long residence time in the system would be conducive to an environment more favorable to bulk phase bacteria than biofilm bacteria. However, it should not be overlooked that the biofilms are able to contribute to microbial load in bulk water as a consequence of their detachment. Besides, the existence of biofilms can significantly reduce the time required for an increase of microbial levels in the bulk water [41]. Results from this study clearly showed that the NF permeate waters did promote bacterial adhesion and cellular proliferation in the distribution system. Therefore, proper care should be taken to secure biostability to acceptable levels. Since the smallest and most easily biodegradable nutrient compounds such as AOC are able to pass through the NF membrane [5,8], they would probably favor microbial growth in the distribution system. Meylan et al. [5] recommended the NF process combined with biological filtration using granular active carbon media in order to remove the AOC compounds effectively. Apart from the AOC removal performance by biofiltration/NF process in particular, it doesn't seem that the bacterial growth potential in distribution systems can be assessed solely by using the empirical values for AOC concentrations. Some studies have reported that even in the low amount of AOC below 10 μg/L a significant biofilm growth could occur [24,38]. The relationship between specific growth rate of biofilm cells and AOC concentrations from the literature and our study is summarized in

1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.12 0.10 0.08 This study Boe-Hansen et al. [24,38] Van der Kooij et al. [42] Kasahara et al. [43]

0.06 0.04 0.02 0.00 0

20

40

60

80

AOC (µg/L) Fig. 4. Relationship between specific growth rate of biofilm cells and AOC concentrations observed in drinking water distribution systems.

Fig. 4. Van der Kooij et al. [42] reported that the 10 μg/L of AOC caused a strong increase of HPC in the biofilm (1.13 day − 1 of μbiofilm), which is higher than the growth rate of the biofilm cells (0.72 day − 1) at a concentration of 70 μg/L [43]. Moreover, even in very pure water a group of bacteria known as oligotrophs is likely to utilize whatever energy resources become available, or face starvation [44]. In addition to remove the AOC compounds available for microbial growth, postdisinfection using disinfectant like chlorine is recommended as a safety barrier for suppressing microbial growth in the distribution system [8]. It can be expected that a high-quality NF permeate water enables an improved stability of chlorine residual in the network, as shown by Peltier et al. [4]. Hence, introduction of a biological filtration step followed by NF process to limit easily biodegradable nutrient compounds during the treatment process may significantly improve the biostability of the water and allow utilities to use a minor dosage of disinfectant, and consequently increase the microbiological quality of water in the network.

5. Conclusions The biostability of NF treated water was systematically investigated in this study. Three commercially available NF membranes were able to improve the quality of drinking water in terms of inorganic and organic contents. Bacterial growth in the NF permeatefed systems was characterized by low bacterial levels and slow growth rates. It was also characterized by the increase in the fraction of culturable heterotrophic bacteria, the relative abundance of αProteobacteria in the biofilm community, and the lower specific growth rate of the biofilm cells compared to the bulk phase cells. The results from AOC measurements and bacterial growth assessments indicate that NF permeate water still has some potential to support bacterial adhesion and cellular proliferation in a distribution system. Proper care, such as biofiltration/NF process and post-disinfection, should be taken to secure biostability to acceptable levels.

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