Improved biostability assessment of drinking water with a suite of test methods at a water supply treating eutrophic lake water

Water Research 87 (2015) 347e355

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Improved biostability assessment of drinking water with a suite of test methods at a water supply treating eutrophic lake water Dick van der Kooij a, Bram Martijn b, Peter G. Schaap c, Wim Hoogenboezem d, Harm R. Veenendaal a, Paul W.J.J. van der Wielen a, * a

KWR Watercycle Research Institute, Post Box 1072, 3430 BB Nieuwegein, The Netherlands PWN Technologies, PO Box 2046, 1990 AA Velserbroek, The Netherlands Water Supply Company Noord-Holland PWN, Rijksweg 501, Velserbroek, The Netherlands d Het Waterlaboratorium, J.W. Lucasweg 2, 2031 BE Haarlem, The Netherlands b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 March 2015 Received in revised form 24 September 2015 Accepted 25 September 2015 Available online 30 September 2015

Assessment of drinking-water biostability is generally based on measuring bacterial growth in shortterm batch tests. However, microbial growth in the distribution system is affected by multiple interactions between water, biofilms and sediments. Therefore a diversity of test methods was applied to characterize the biostability of drinking water distributed without disinfectant residual at a surfacewater supply. This drinking water complied with the standards for the heterotrophic plate count and coliforms, but aeromonads periodically exceeded the regulatory limit (1000 CFU 100 mL
1). Compounds promoting growth of the biopolymer-utilizing Flavobacterium johnsoniae strain A3 accounted for c. 21% of the easily assimilable organic carbon (AOC) concentration (17 ± 2 mg C L
1) determined by growth of pure cultures in the water after granular activated-carbon filtration (GACF). Growth of the indigenous bacteria measured as adenosine tri-phosphate in water samples incubated at 25  C confirmed the low AOC in the GACF but revealed the presence of compounds promoting growth after more than one week of incubation. Furthermore, the concentration of particulate organic carbon in the GACF (83 ± 42 mg C L
1, including 65% carbohydrates) exceeded the AOC concentration. The increased biomass accumulation rate in the continuous biofouling monitor (CBM) at the distribution system reservoir demonstrated the presence of easily biodegradable by-products related to ClO2 dosage to the GACF and in the CBM at 42 km from the treatment plant an iron-associated biomass accumulation was observed. The various methods applied thus distinguished between easily assimilable compounds, biopolymers, slowly biodegradable compounds and biomass-accumulation potential, providing an improved assessment of the biostability of the water. Regrowth of aeromonads may be related to biomass-turnover processes in the distribution system, but establishment of quantitative relationships is needed for confirmation. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Biostability Easily assimilable organic carbon Slowly-biodegradable organic compounds Particulate organic carbon Biomass-accumulation rate Aeromonas

1. Introduction About one-third of the drinking water distributed by the water supply companies in the Netherlands is produced from surfacewater sources and two-thirds from groundwater (Geudens, 2012). Treated water is distributed without a disinfectant residual, although a low concentration of chlorine dioxide (<0.2 mg ClO2

* Corresponding author. E-mail addresses: [email protected] (D. van der Kooij), bram. [email protected] (B. Martijn), [email protected] (P.G. Schaap), [email protected] (W. Hoogenboezem), paul.van.der. [email protected] (P.W.J.J. van der Wielen). http://dx.doi.org/10.1016/j.watres.2015.09.043 0043-1354/© 2015 Elsevier Ltd. All rights reserved.

L
1) is added to the water directly after treatment at a few surfacewater supplies. The ClO2 residual in the water leaving the treatment plant is < 0.05 mg L
1 and ClO2 is not detectable in the distribution system. Microbial growth in the distribution system can lead to water quality deterioration, e.g. elevated heterotrophic plate counts, presence of coliforms, opportunistic pathogens or invertebrates (van der Kooij and van der Wielen, 2014). Therefore, the water supply companies in the Netherlands aim at distributing drinking water with a high level of biostability (van der Kooij et al., 1999). Assessment of drinking-water biostability is generally based on the concentration of easily assimilable organic carbon (AOC) derived from growth of either Pseudomonas fluorescens strain P17 and Spirillum sp. strain NOX (van der Kooij, 1992; Volk and

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LeChevallier, 2002) or a natural microbial consortium (Hammes et al., 2010) in short-term batch tests. However, microbial growth in distribution systems is affected by water quality variations and multiple interactions between water, biofilms and sediments. Therefore, additional methods have been developed recently to improve the assessment and monitoring of the biostability of drinking water. These methods include: (i) the use of an additional bacterial culture in the AOC test, Flavobacterium johnsoniae strain A3, with the ability to utilize polysaccharides and proteins at the microgram-per-litre level (Sack et al., 2010, 2011), (ii) the biomass production potential (BPP) test for determining the effect of incubation of a water sample with the indigenous microbial community on the active biomass concentration measured as adenosine tri-phosphate (ATP) (van der Kooij and Veenendaal, 2014), (iii) determination of the concentration of particulate organic carbon (POC) and particulate carbohydrate carbon (PCHC), (iv) the continuous biofouling monitor (CBM) for determining the accumulation rates of biomass, iron and manganese on glass beads (van der Kooij and Veenendaal, 2014). These methods were applied to the water at various treatment stages and in the distribution system of a surface-water supply using eutrophic lake water as the source and distributing drinking water without a disinfectant residual. The colony count of Aeromonas, which is used as an indicator for regrowth, periodically exceed the legislative limit value of 1000 CFU (100 mL)
1 in the distribution system of this supply. Moreover, at a few locations in the distribution system, consumers complained about discoloured water. The objectives of our study were to (i) evaluate the usefulness of the recently developed methods for an improved assessment of the biostability of the water and (ii) elucidate the cause(s) of the regrowth in the distribution system. 2. Materials and methods

Table 1 Physical and chemical quality characteristics of water from the open storage reservoir (intake) and finished water in 2010a. Parameter

Intake (n)b

Finished water (n)

Temperature (minemax) ( C) Chlorophyll (mg L
1) Suspended solids (mg L
1) Turbidity (NTU) Ammonium-N (mg L
1) Nitrate-N (mg L
1) Total phosphate-P (mg L
1) Iron (mg L
1) Manganese (mg L
1) DOC (mg L
1) UVA (m
1)g Coliforms (n 100 mL
1) Escherichia coli (n 100 mL
1) Aeromonas (CFU 100 mL
1) HPC22 (CFU mL
1)

0.3e21.0 (52) 18 ± 12 (50)c 9.9 ± 2.6 (42) Naf 0.13 ± 0.09 (52) 0.98 ± 0.6 (4) 0.03 ± 0.02 (4) 191 ± 99 (4) 38 ± 21 (4) 6.7 ± 0.7 (52) 11.5 ± 1.4 (52) 84 ± 198 (52) 81 ± 193 (52) 10,670 ± 12,740 (13) Naf

0.9e21.8 (52) <0.1d (DMF, 52)e <0.2d (DMF, 52)e 0.02 ± 0.01 (52) <0.02d (52) 1.2 ± 0.55 (52) 0.01d (8) <10d (52)a <5d (52)a 1.5 ± 0.2 (52) 1.6 ± 0.2 (52) <1 (365) <1 (365) <1 (13) 2 ± 3 (52)

a Concentration of iron and manganese in finished water in 2011 (water treatment as in 2010). b n, number of samples. c Maximum in research period: 74 mg L
1. d Detection limit. e In dual-media filtrate. f na, not analysed. g UV absorbance at 254 nm.

distribution-system reservoir (DR) at 17 km pipe length from the treatment facility, and (v) three locations in the distribution system, viz. proximal (D8) at 7.6 km from the treatment facility, central (D35) at 17.5 km from DR, with 42% of pipes <500 mm, and distal (D42) at 25 km from DR, with 98% of the pipes >500 mm. The sampling locations are shown in Fig. S1 (Supplementary material). Transportation time (T, h) of the water and the average surface-tovolume ratio (S/V, m
1) of the pipes connecting the location to the treatment plant (DR, D8) or to DR (D35, D42) are given in the Supplementary material Table S1, together with the water-tosurface contact intensity (T S V
1, h m
1). Samples were stored at 5 ± 3  C and processed within 24 h.

2.1. Water treatment and distribution Water treatment includes: storage of Lake IJssel water (2e4 weeks) in an open reservoir with NaOH dosage for softening, CO2 dosage at the intake from the reservoir, followed by microsieving (35 mm), dosage of FeClSO4 (20 mg L
1), coagulation, flocculation (upflow sludge-blanket filtration), dual-media filtration [empty bed contact time (EBCT) 2 min], H2O2 dosage (6 mg L
1), UV irradiation (560 mJ cm
2), GAC filtration (EBCT 25 min), microsieves (35 mm), ClO2 dosage (0.08e0.12 mg L
1) and clear-well storage (avg. residence time of 1.9 h). A scheme of the treatment chain is depicted in the Supplementary material (Fig. S1). Table 1 shows a number of physical, chemical and microbiological quality characteristics of raw and finished water, based on routine monitoring using standard methods. The maximum residence time of treated water in the distribution system, with one reservoir (7.5 h residence time) and a maximum distance of 45 km to the treatment facility, is estimated at about 150 h. The major pipe materials of the distribution system include asbestos cement (45%), unplasticized PVC (27%), polyethylene (21%) and mortar-lined nodular-graphite cast iron (c. 1%), with 73.7% of the distribution pipes <150 mm diameter. 2.2. Sampling locations From March to December 2010, samples were collected from (i) dual-media filtrate (DMF), (ii) water after UV-H2O2 treatment, GAC filtrate (GACF), (iii) finished water from the clear well (FW), (iv) the

2.3. Microbiological water quality parameters The heterotrophic plate count (HPC22) was determined with glucose-yeast-extract-agar, incubated at 22 ± 2  C according to standard NEN-EN-ISO- 6222 (NEN, 1999). The number of coliform bacteria was determined with the membrane-filter method according to standard NEN-EN-ISO 9308-1 (NEN, 2000). The colony count of Aeromonas was determined on ampicillin dextrin agar (Havelaar et al., 1987). Sample volumes of 100 mL were membrane filtered (0.45 mm) and the membrane was placed on the medium. Typical colonies were counted after 24 ± 2 h of incubation at 30 ± 1  C. These parameters were part of the routine monitoring programme and were not tested at the described sampling locations. The ATP concentration in water was determined by measuring the light production in the luciferine-luciferase assay as described previously (van der Wielen and van der Kooij, 2010). The detection limit of the analysis is 1 ng ATP L
1. The total cell count (TCC) and the number of membrane-intact cells in water samples were determined with a flowcytometer (BD FACS Calibur) (van der Wielen and van der Kooij, 2013). TCC in biomass suspensions from the CBM was determined after acridine-orange staining of the cells collected by membrane filtration, followed by enumeration using epifluorescence microscopy (Hobbie et al., 1977).

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2.4. Particulate organic carbon (POC) and carbohydrate carbon (PCHC) The concentrations of POC and PCHC were obtained by applying cross-flow ultrafiltration to the water at the selected locations, followed by analysis of the collected concentrate. The hemoflow membrane HF80S (Fresenius Medical Care), with a molecularweight-cut off of 30 kD, was used for the collection of particles and colloids >0.01 mm. The membrane element was placed in a pressure vessel and supplied with the water at a flow of approx. 0.9 L min
1, during about 24 h (Heijnen et al., 2009). The collected concentrate (0.5e0.8 L) was stored at 5 ± 3  C and analysed within 24 h for the total concentration of organic carbon (Shimadzu TOC-V analyser) and carbohydrates (Dubois et al., 1956). The concentrations of POC and PCHC in the water were calculated from the concentration in the concentrate and the concentration factor (usually 2000e2500), with a detection limit of 1 mg L
1. 2.5. Assimilable organic carbon (AOC) and biomass production potential (BPP) The concentration of easily assimilable organic carbon (AOC-P17/ NOX) was determined with the AOC method using P. fluorescens strain P17 and Spirillum sp. strain NOX (van der Kooij, 1992). The introduction of a few mg ortho-phosphate-P L
1 with the inoculum of these strains ensured the presence of sufficient P for the utilization of more than 100 mg of C L
1. Sodium sulphite (37.5 mg L
1) was added to UV-H2O2 treated water prior to AOC analysis to inactivate residual H2O2. The concentration of biopolymers available to F. johnsoniae strain A3 [AOC(A3)] was measured by incubation (at 15  C) of pasteurised water inoculated with strains P17 and A3 as previously described (Sack et al., 2010). The AOC(A3) concentration was calculated from the maximum colony count (Nmax, CFU mL
1) by using the average yield (1.43  107 CFU mg
1 C) of strain A3 on polysaccharides and proteins (Sack et al., 2011). The biomassproduction potential (BPP) was determined by incubation of water (600 mL) sampled in a glass-stoppered Pyrex-glass Erlenmeyer flask of 1 L that had been thoroughly cleaned following the procedure applied for the AOC test. One mL of membrane-filtered (1.2 mm) river water was added to the water sample with the indigenous bacteria to ensure the presence of a large diversity of bacteria. Furthermore, phosphate-P (0.33 mg L
1) and nitrate-N (0.8 mg L
1) were added to prevent growth limitation by these nutrients. The concentration of active bacteria in the water samples incubated in duplicate at 25  C was monitored by periodic ATP analysis. The maximal ATP concentration within 7 days of incubation (BP7, ng ATP L
1) is used as a measure of the concentration of easily biodegradable compounds. The maximal concentration attained between 7 and 28 days of incubation (BP28), is used as a measure of the concentration of slowly biodegradable compounds. 2.6. Continuous biofouling monitor (CBM) The CBM was used for the semi-continuous determination of the biomass accumulation rate (BAR, pg ATP cm
2 d
1) of the water (van der Kooij and Veenendaal, 2014). In brief, four parallel columns with a glass cylinder (id 1 cm, length 2 cm) containing glass beads (2 mm) representing an external surface of approx. 20 cm2, were each supplied with the water under investigation at a flow rate of 10 L h
1. Every two weeks two cylinders were removed from the system and replaced by cylinders with clean beads. The glass beads from the removed cylinders were transferred to 10 mL of autoclaved drinking water and treated during 2 min with low-energy ultrasound in a water bath (Branson Sonication Unit 5050). This procedure was repeated twice with replaced water. The obtained

349

suspension (30 mL) was analysed for ATP, TCC, Fe and Mn. Fe and Mn were measured in samples acidified (pH < 2) with HNO3, according to standard NEN-EN-ISO 17294-2 (NEN, 2003). The average concentration of these parameters on the surface of the glass beads was calculated and from these concentrations and the period of exposure (28 days; 14 days for the first 2 cylinders) the BAR value (pg ATP cm
2 d
1) and the accumulation rates of Fe (FeAR, mg Fe m
2 d
1) and Mn (MnAR, mg Mn m
2 d
1) were obtained. Replacement of the cylinders enabled a continuing periodic assessment of the BAR, the FeAR and the MnAR. CBMs were placed at GACF, DR (not at FW because of the ClO2 residual), D8 and D42. 2.7. Statistics For determining significant differences between the concentrations of the water quality parameters at the different treatment stages and in the distribution system the analysis of variance (ANOVA) with the Bonferroni post hoc test was used on parameters with normally-distributed results, affirmed with the KolmogoroveSmirnov test. The KruskaeWallis test with pair-wise comparison was used on parameters with not normally distributed results. Quantitative relationships between water quality parameters were identified by linear regression analysis. 3. Results 3.1. Heterotrophic plate count (HPC22) and Aeromonas The HPC22 ranged from <1 CFU mL
1 to 8260 CFU mL
1 in the distribution system (441 samples) with 8% >100 CFU mL
1, in most cases at a water temperature <15  C. The geometric average (8 CFU mL
1) was clearly below the legislative limit (100 CFU mL
1). The colony count of aeromonads in drinking water in the distribution system ranged from <1 (100 mL)
1 in 11% of 253 samples to 9800 CFU (100 mL)
1, with a concentration >1000 CFU (100 mL)
1 in 8% of the samples. Aeromonas counts >1000 CFU (100 mL)
1 were more frequently (p < 0.01) observed at a water temperature >15  C. Furthermore, the Aeromonas count was <1000 CFU (100 mL)
1 in all combined samples (n ¼ 24) with HPC22 >100 CFU mL
1. Apparently, HPC22 bacteria and Aeromonas behave differently in the distribution system. 3.2. Total cell counts (TCC), ATP and particulate organic carbon (POC) TCC values exceeded 5  105 cells mL
1 in most DMF samples and ranged from 1e2  105 cells mL
1 in most samples from the GACF and the distribution system (Fig. 1A; Table 2). The average percentage of intact cells ranged from more than 90% (DMF) to 23% (DR) indicating the impact of ClO2 dosage. The TCC values at D8, D35 and D42 did not differ significantly (p > 0.01) from those in the GACF and at DR and the intact-cells concentration in the distribution system remained below the level in the GACF. The ATP concentration was >10 ng L
1 in all DMF samples at a water temperature >15  C (Fig. 1B) and in a few samples collected in April at an elevated chlorophyll concentration (about 20 mg L
1) in the intake water (Fig. S2, Supplementary material). The ATP concentration in the GACF was significantly lower (p < 0.01) than in the DMF (Fig. 2). The ATP concentration at DR, D8, D35 and D42 did not differ significantly from the concentration in the GACF (p > 0.05). Overall, the collected TCC and ATP data did not indicate regrowth in the distribution system. The POC concentration in water collected from different treatment stages and the distribution system varied from 55 to 160 mg C L
1 in most samples (Table 2). The PCHC accounted for

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Fig. 1. Total cell count (TCC) and ATP concentration in water during treatment and distribution. Broken line shows water temperature. DMF, dual-media filtrate; GACF, granular activated-carbon filtrate; DR, water from distribution-system reservoir; D8, distribution system, proximal location; D35, distribution system, central location and D42, distal location.

Table 2 Water quality characteristics at the test locations in water treatment and the distribution system. Parametera

Concentration of bacteria TCC (cells mL
1) (n ¼ 12) Intact cells (mL
1) (n ¼ 12) % Intact cells (n ¼ 12) ATP (ng L
1) (n ¼ 12) ATP/intact cell (fg) (n ¼ 12) Particulate organic carbon POC (mg C L
1) (n ¼ 4) PCHC (mg C L
1) (n ¼ 4) PCHC/POC (%) (n ¼ 4) Assimilable organic carbon (AOC) AOC(P17/NOX) (mg ac-C eq L
1) (n ¼ 4) AOC(P17) (n ¼ 4) AOC(A3) (mg C L
1) (n ¼ 3) AOC(T) (mg C L
1) (n ¼ 3) Biomass production potential BP7 (ng ATP L
1) (n ¼ 3e5) BP28 (ng ATP L
1) (n ¼ 3e5) Continuous biofouling monitor BAR (pg ATP cm
2 d
1) (n ¼ 20) TCC (cells cm
2)e (n ¼ 16) ATP/cell (fg) (n ¼ 16) FeAR (mg m
2 d
1) (n ¼ 16) MnAR (mg m
2 d
1) (n ¼ 16)

Treatment stage

Distribution system

Dual-media filtrate (DMF)

After H2O2- GAC filtrate UV (GACF)

Distribution-system reservoir (DR)

Proximal location Distal location (D8) (D42)

Central location (D35)

5.1 (±1.2)  105c 4.9 (±1.0)  105c 91 ± 10 12.8 ± 3.2c 0.027 ± 0.01

ndb nd nd nd nd

1.9 (±0.4)  105 1.5 (±0.3)  105 82.4 ± 11.5 5.9 ± 2.8 0.037 ± 0.013

1.7 (±0.4)  105 3.6 (±1.7)  104 23.3 ± 7.0 3.2 ± 1.6 0.079 ± 0.04

2.0 (±0.5)  105 9.5 (±4.4)  104 50.1 ± 12.8 4.8 ± 2.3 0.047 ± 0.013

1.7 (±0.4)  105 8.3 (±4.4)  104 46.3 ± 6.9 4.0 ± 1.1 0.057 ± 0.019

1.8 (±0.3)  105 8.4 (±2.4)  104 54.6 ± 14 4.9 ± 1.9 0.045 ± 0.01

164 (n ¼ 1) 102 (n ¼ 1) 62 (n ¼ 1)

134 ± 28 80 ± 21 59 ± 6

83 ± 42 53 ± 25 65 ± 12

71 ± 14 39 ± 12 53 ± 7

88 ± 44 51 ± 29 57 ± 5

106 ± 80 74 ± 46 55 ± 6

nd nd nd

5.3 ± 1.4c

52 ± 15c

11.3 ± 4.0

12.6 ± 2.2d

13.1 ± 2.5d

9.2 ± 3.0d

nd

d

3.0 ± 0.8 8.4 ± 0.9c 13.9 ± 2.5

28 ± 13 nd nd

0.8 ± 0.6 3.5 ± 0.4 16.6 ± 2.2

ng 4.4 ± 1.7 17.9 ± 2.2

ng 0.7 ± 0.4c 13.8 ± 2.9

ng 2.9 ± 4.0 13.2 ± 3.6

nd nd nd

9.2 ± 2.2 31 ± 7.3c

36 ± 7.1c 21.3 ± 5.0

9.1 ± 2.1 14.5 ± 3.1

7.0 ± 3.5 11.8 ± 3.3

6.2 ± 3.7 13.4 ± 8.8

4.1 ± 1.0 9.1 ± 5.5

6.0 ± 1.8 24.3 ± 5.1

nd nd nd nd nd

nd nd nd nd nd

10 ± 6.7 1.8 (±1.2)  106 0.25 ± 0.22 0.11 ± 0.5 0.03 ± 0.02

39 ± 16c 8.9 (±7.0)  106c 0.16 ± 0.06 0.8 ± 0.6c 0.18 ± 0.19c

16 ± 11 5.7 (±4.7)  106 0.09 ± 0.02c 0.6 ± 0.4 0.05 ± 0.04

26 ± 20 7.7 (±5.7)  106c 0.09 ± 0.03 1.9 ± 0.7c 0.10 ± 0.10

nd nd nd nd nd

a TCC, total cell count, ATP, adenosine triphosphate, AOC, assimilable organic carbon; AOC(T) ¼ AOC(P17/NOX) þ AOC(A3); BP7, maximal biomass concentration within 7 days of incubation; BP28, maximal biomass concentration between 7 and 28 days of incubation; Hemoflow, ultrafiltration; POC, particulate organic carbon; PCHC, particulate carbohydrate carbon; BAR, biomass accumulation rate; FeAR, iron accumulation rate; MnAR, manganese accumulation rate. b nd, not determined. c Concentration significantly (p < 0.01) different from concentration in GACF. d No growth (ng) of strain P17. e After 28 days exposure.

53e65% of the POC (avg. 57 ± 10%). Temperature significantly affected the POC in the GACF with lowest concentrations at temperatures >15  C (Fig. 2). At 3  C (day 347), the concentration in the GACF was only slightly lower (14%) than in the DMF, indicating that UV-H2O2 treatment and GAC filtration hardly affected the POC at this temperature. 3.3. Assimilable organic carbon (AOC) and biomass production potential (BPP) Coagulation and upflow sludge-blanket filtration followed by dual-media filtration reduced the high AOC(P17/NOX)

concentration in the water from the open intake reservoir (1800 ± 1300 mg ac-C eq L
1; n ¼ 5) to <10 mg ac-C eq L
1, but the AOC(A3) concentration in the DMF revealed the presence of biopolymers exceeding the AOC(P17/NOX) concentration (Table 2). The increased AOC (P17/NOX) in water after UV-H2O2 treatment demonstrated the formation of growth-promoting compounds by this process, with 54% used by strain P17. GAC filtration caused an 80% reduction of the AOC(P17/NOX), with more than 95% removal of the AOC(P17) fraction, but the concentration in the GACF was higher (p < 0.01) than in the DMF. The AOC(A3) was also reduced and accounted for 21% of the average total AOC concentration in the GACF (Table 2). The AOC(P17/NOX) at DR, D8 and D42 did not differ

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3.4. Continuous biofouling monitor (CBM)

Fig. 2. Particulate organic carbon (POC) in water sampled from different treatment stages and the distribution system in relation to the water temperature during treatment. Relationship between POC and temperature for GACF: POC (mg L
1) ¼ (
5.3 ± 0.9) Temp ( C) þ (150 ± 13) (r2 ¼ 0.94; p ¼ 0.03). UV-H2O2: water after the UV-H2O2 process; for other abbreviations see Fig. 1.

significantly from the concentration in the GACF (p > 0.01), but strain P17 failed to grow and even disappeared in the samples from these locations, despite repeated inoculation with this organism. In June and September, the AOC(A3) concentration at D8 and D42 was clearly below the concentrations in GACF and DR on these days, indicating a decrease during distribution in summer. Incubation of water in the BPP test affected the active-biomass (ATP) concentration, depending on water type and incubation time (Fig. 3). The increase of the ATP concentration in the DMF after about 14 days of incubation demonstrated the presence of slowly biodegradable compounds whereas growth within five days in water after UV-H2O2 treatment confirmed the presence of easily assimilable compounds. GAC filtration reduced the BP7 to <10 ng ATP L
1 and the average of BP28 in the GACF was significantly lower (p < 0.01) than in the DMF (Table 2), but still showed the presence of slowly biodegradable compounds (Fig. 3B). The growth within five days in the finished water collected on day 327 confirmed that ClO2 dosage had increased the concentration of easily assimilable compounds, but the utilization of slowly biodegradable compounds remained below the level observed in the GACF (Fig. 3). The BP7 in water collected from the distribution system was <10 ng ATP l
1 in most samples including location DR (Table 2). The low and stable concentrations of AOC(P17/NOX) and BP7 in the distribution system are consistent with the stability of the TCC and ATP concentrations. However, the elevated BP28 levels at D35 after about 3 weeks of incubation (Fig. 3C), revealed the local presence of slowly biodegradable compounds.

On day 166 the CBM was installed at the GACF and the locations DR, D8 and D42. The BAR varied during the research period and was highest at DR, D8 and D42 at a water temperature >20  C (Fig. 4A). An exponential relationship was observed between the BAR and water temperature at DR, corresponding with a 3.8 times BAR decrease at a 10  C temperature decline (Supplementary material, Fig. S3). TCC values in the accumulated biomass were strongly related to the ATP concentration (Supplementary material, Fig. S4). The average ATP amount per cell in the CBM was 2e6 times higher than the average ATP amount of the intact bacteria in the water at the test locations (Table 2), indicating attachment-enhanced substrate uptake. In the CBMs also a temperature-depending accumulation of iron and manganese was observed (Fig. 4). At location D42 the FeAR attained 3 mg Fe m
2 d
1 and was significantly higher (p < 0.01) than at the other locations, with the lowest FeAR in the GACF (Table 2). Also the Fe to biomass ratio in the CBM at location D42 was clearly higher than at the other locations (Fig. 4C). The MnAR also differed between the test locations (Fig. 4D) and was significantly (p < 0.01) higher at DR than in the GACF, indicating an elevated MnO2 concentration at DR. 4. Discussion The methods applied in this study showed the presence of easily assimilable organic compounds, growth-promoting biopolymers and slowly biodegradable compounds, included in the POC in water at different treatment stages and in distributed drinking water. The significance of the methods for biostability assessment is discussed below. 4.1. Regrowth The concentrations of ATP and TCC in drinking water were within the ranges observed in other water supplies operated without disinfectant residual (van der Wielen and van der Kooij, 2010; Hammes et al., 2010; Vital et al., 2012) and did not increase at the selected locations in the distribution system. Furthermore, the low HPC22 values and absence of coliforms in association with a low AOC(P17/NOX) concentration are consistent with earlier observations in drinking water supplies in the Netherlands (van der Kooij, 1992). However, the Aeromonas count exceeded the limit value of 1000 CFU (100 mL)
1 in 8% of the samples, in most cases at a water temperature >15  C. Regrowth of Aeromonas in water supplies has been reported in earlier studies in the Netherlands (Havelaar et al., 1990) and elsewhere (Burke et al., 1984; Gavriel et al., 1998; Kühn et al., 1997). In the Netherlands the Aeromonas count at 30  C was introduced in the drinking water regulations as

Fig. 3. Effect of water treatment and distribution on the biomass production in water incubated at 25  C (BPP-test). FW, finished water; for other abbreviations, see Fig. 1. Lines show average of observations in duplicate flasks.

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Fig. 4. A: Biomass accumulation rate (BAR) in the continuous biofouling monitor (CBM) supplied with the GAC filtrate (GACF), water from the distribution system reservoir (DR) and water in the distribution system at a proximal (D8) and a distal (D42) location, respectively. Broken line indicates water temperature; B: iron-accumulation rate (FeAR); C: relationships between iron and biomass accumulated in the CBMs (best fitting curves shown, see Supplementary Material, Table S2); D: manganese-accumulation rate (MnAR).

an additional regrowth indicator (VROM, 2001). Aeromonads contribute to HPC22, but the Aeromonas/HPC22 ratio shows a large variation and the relationship between these parameters is very weak (Supplementary material, Fig. S5). Aeromonads can multiply at low HPC22 values and low and stable concentrations of TCC and ATP. The defined limit for Aeromonas therefore is a more stringent regrowth criterion than the standards for HPC22 and coliforms. 4.2. Microbial-growth-potential tests The original AOC test is based on the growth of the nutritionally versatile P. fluorescens strain P17 (van der Kooij et al., 1982) and Spirillum sp. strain NOX specialized in the utilization of lowmolecular-weight (hydroxy)carboxylic acids in a batch test (van der Kooij and Hijnen, 1984). However, strains P17 and NOX do not utilize polysaccharides and proteins and the AOC test therefore was extended by including F. johnsoniae strain A3 that can grow at microgram-per-litre levels of these biopolymers (Sack et al., 2010, 2011). The BPP test with the indigenous bacteria and bacteria originating from river water was used to assess the presence of all kinds of compounds, both dissolved and suspended, serving as a source of energy and carbon for bacterial growth. This test initially was developed for assessing the enhancement of microbial growth by construction materials in contact with drinking water (van der Kooij and Veenendaal, 2001). Addition of an easily biodegradable compound, e.g. acetate, glucose, or maltose to tap water caused an increase of the ATP concentration within a few days (van der Kooij and Veenendaal, 2014). Growth within a few days in a sample of treated water implies uptake within the residence time of the water

in the distribution system. The incubation period in the BPP test was extended to four weeks to obtain information about slowly biodegradable compounds that may contribute to regrowth when accumulating in the distribution system. The POC includes microbial biomass and biomass-associated high-molecular-weight compounds (>30 kD). This parameter is not a direct measure of the microbial growth potential of water, but compounds deposited in the distribution system may become available for growth of (micro)organisms. For seawater a 50% POC recovery efficiency of tangential ultrafiltration has been reported (Benner et al., 1997). The actual concentrations of POC and PCHC therefore may be higher than determined with the hemoflow method, but the well-controlled sampling procedure (Heijnen et al., 2009) enables comparison between water types. The continuous biofouling monitor (CBM) enables a semicontinuous monitoring of the (variations in the) rate of accumulation of active biomass, Fe and Mn on water-exposed glass beads. The data thus provide information about the kinetics of the microbial-growth potential and interactions between fouling characteristics. 4.3. Effects of treatment on easily and slowly biodegradable compounds The raw water contained a high and fluctuating concentration of algae (Supplementary material Fig. S1), but in the DMF the concentrations of chlorophyll and suspended solids were below the detection limit, demonstrating an effective removal (Table 1). The AOC(P17/NOX) in the DMF was also low (Table 2), but growth of strain A3 and the increased growth in the BPP test after prolonged

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incubation demonstrated the presence biopolymers and slowlybiodegradable compounds (Fig. 3A; Table 2). The elevated BP7 in water after UV-H2O2 treatment corresponds with the increased AOC(P17/NOX) concentration (Table 2) and confirmed earlier reports about the formation of easily biodegradable compounds by UV-H2O2 treatment (Martijn et al., 2007). GAC filtration effectively removed the compounds contributing to AOC(P17/NOX), AOC(A3) and BP7, but the increase of the ATP concentration in the GACF at prolonged incubation revealed the presence of slowly biodegradable compounds (Fig. 3) and also POC removal was limited. The PCHC fraction of the POC in the GACF (65 ± 12%) and also in the DMF and in water after H2O2-UV is high as compared to the carbohydrate fraction in the biomass of bacteria (5%; Neidhardt, 1987) and algae (15e20%; Becker, 1994). The production of large amounts of heteropolysaccharides by algae (Biersmith and Benner, 1998) suggests that algal EPS is a major fraction of the PCHC. The POC in the DMF and the GACF therefore most likely originates from the algae in the eutrophic lake water (Supplementary material, Fig. S2) but insufficient data are available for establishing quantitative relationships. EPS consists of a large variety of proteins and polysaccharides with different sugar moieties and functional groups, e.g. uronic acids, in branched and linear polymers (Sutherland, 2001; Flemming and Wingender, 2010). Utilization of these compounds requires an array of highly specific exoenzymes produced by a diverse bacterial assemblage (Arnosti et al., 1994; Sutherland, 1999). Certain compounds included in the POC and PCHC may have contributed to the AOC(A3) and also to the BP28, but the POC concentrations in the DMF and GACF were more than 10 times higher than the AOC(A3) concentrations (Table 2). The POC concentration in the GACF also is more than 10 times higher as compared to treated groundwater (<10 mg C in 9 of 11 supplies) in the Netherlands (unpublished results). The low BAR value (10 ± 6.7 pg ATP cm
2 d
1) in the CBM supplied with the GACF is consistent with the low biofilm formation rate (BFR) of the GACF in the biofilm monitor (Martijn et al., 2007). Much higher BFR values were observed in drinking water supplemented with 10 mg of acetate-C L
1 (635e825 pg ATP cm
2 d
1) or maltose-C L
1 (267 pg ATP cm
2 d
1) (Sack et al., 2014). Obviously, the concentration of compounds promoting rapid biofilm formation was low in the GACF. Compounds included in PCHC and other POC fractions apparently did not enhance the BAR, which is consistent with their GAC passage at an EBCT of 25 min. The elevated average ATP content of the cells in the biomass accumulated in the CBMs (0.09e0.25 fg cell
1), as compared to the average ATP content of the membrane-intact bacteria in the water (0.04e0.08 fg cell
1) (Table 2), shows uptake of energy source(s) from the GACF and at the other locations. 4.4. Effects of ClO2 and distribution BP7 in finished water collected from the clear well exceeded 10 ng ATP L
1 (Fig. 3B) and indicated the presence of easily biodegradable compounds, which are typical by-products of the ClO2 dosage (Rav-Acha, 1984; Ramseier et al., 2011). Their presence was confirmed by the elevated BAR value in water from the distribution system reservoir (DR). The BAR decline with decreasing temperature (Fig. 4A) can be attributed to the operationally lowering of the ClO2 dosage, aiming at a residual of 0.03e0.04 mg l
1 in the water leaving the clear well, and probably also to the effect of temperature on biofilm formation. The lower BAR values at location D8 with an average residence time of 37 h indicate that the growth-promoting by-products were utilized during transportation as were compounds contributing to the AOC(A3) (Table 2). The low AOC(P17/NOX) in the distributed water

353

did not differ significantly (p > 0.05) between DR, D8 and D42 and the BP7 was below 10 ng ATP L
1 and only slightly above the ATP concentration in the distributed drinking water (Table 2). The limited utilization of compounds contributing to AOC(P17/NOX) and BP7 is consistent with the stable TCC and ATP concentrations at the sampled locations. The growth inhibition of strain P17 in the AOC test in all samples from the distribution system may be related to the ClO2 dosage, but was not eliminated by thiosulfate addition (data not shown) and remains unexplained. The POC at the locations in the distribution system did not differ significantly (p > 0.05) from those in the GACF, but the elevated BP28 values in drinking water at location D35 (Fig. 3; Table 2), suggest a local accumulation of slowly biodegradable compounds. The relatively high BAR value at location D42, at 25 km from location DR with 99% of the pipes 500 mm, is associated with a significantly elevated FeAR (p < 0.01) as compared to the GACF, DR and D8 (Table 2), and a high iron to biomass ratio (Fig. 4C). The BAR and FeAR of the GACF (Table 2, Fig. 4) were low at the low iron concentration (<10 mg L
1 in treated water; Table 1). Mortar-lined cast-iron pipes accounted for <0.05% of the pipe length between location DR and location D42. Fe concentrations >10 mg L
1 (range: 10.2e65 mg L
1) were observed in 11 of 208 samples collected from the distribution system, indicating the local presence of iron deposits. The elevated BAR and FeAR in the CBM at location D42 therefore is attributed to the deposition of iron-associated biomass originating from the biofilm and sediments in the transportation pipes. The MnAR was highest at DR (Fig. 4D), most likely as the result of the oxidation of dissolved Mn2þ to insoluble Mn3þ by ClO2
and Bader, 1994). Hence, the CBM can be used to trace local (Hoigne fouling (conditions) that may lead to discoloured-water complaints associated with elevated concentrations of Fe3þ and/or Mn3þ (Sly et al., 1990; Vreeburg and Boxall, 2007). Overall, the observations with the CBM revealed local differences and temporal changes in water quality affecting biomass accumulation in the distribution system that were not observed with the other test methods. 4.5. Regrowth-promoting conditions Coagulation, flocculation, UV-H2O2 and GAC filtration of surface water from the open storage reservoir with high concentrations of algae and DOC yielded low values of the AOC(P17/NOX), BP7 and BAR. However, the GACF contained biodegradable biopolymers [AOC(A3)], slowly biodegradable compounds (BP28) and elevated concentrations of POC and PCHC. Furthermore, by-products of ClO2 dosage increased the BAR of the drinking water. These water quality characteristics did not cause noncompliance for HPC22 and coliforms, nor induced an increase of the total cell count and the ATP concentration in the drinking water during distribution. However, the temperature-related growth of Aeromonas, with a wide range in colony counts at specific sampling dates, indicates that multiplication of this organism depends on local conditions in the distribution system. Sloughing of the by-products-induced biofilm and adsorption, coagulation and sedimentation of particulates and colloids, including POC, Fe and Mn, may lead to a distance-related deposition, in particular at locations with a low flow rate or stagnation (Vreeburg et al., 2009). Local fouling in the distribution system with loose deposits can enhance microbial growth (Lehtola et al., 2004; Gauthier et al., 1999). A rise in water temperature increases the activity of free-living protozoa and invertebrates that feed on biofilms and sediments (van Lieverloo et al., 2012; Christensen et al., 2011). Bacteria-consuming protists release nutrients into the environment, e.g. ammonia and amino acids (Pernthaler, 2005), thereby enhancing secondary growth of bacteria. These biomass-turnover processes may favour growth of aeromonads, which can utilize amino acids and long-chain fatty acids

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at the microgram-per-litre level (van der Kooij and Hijnen, 1988). Also regrowth of coliforms has been associated with elevated fouling levels, e.g. algal organic matter, and sediments, at a temperature >15  C (LeChevallier et al., 1996; Bouteleux et al., 2005), but quantitative data about growth in association with deposits seem to be lacking. Further application of the BPP-test, AOC(A3), POC/PCHC analysis and the CBM may lead to the establishment of relationships between the concentration of slowly biodegradable compounds in drinking water, as well as deposits formation, and multiplication of Aeromonas and other (micro)organisms in drinking water distribution systems. 5. Conclusions  The presence of slowly biodegradable compounds and an elevated concentration of particulate organic carbon at a low AOC(P17/NOX) concentration in the GAC filtrate of a surfacewater supply indicates that biostability assessment of drinking water only based on the concentration of easily assimilable organic carbon may not be correct. Growth of F. johnsoniae strain A3, the ATP-based biomass production potential test, and particulate organic carbon analysis provide additional information about the biostability.  The use of the continuous biofouling monitor for measuring the accumulation rates of active biomass, iron and manganese facilitates the assessment of the effects of operational and seasonal variations in water treatment processes and distribution on water quality.  Regrowth of Aeromonas may be related to temperaturedepending biomass-turnover processes in biofilms and sediments in the distribution system, but establishment of quantitative relationships is needed for confirmation. Acknowledgements The biostability assessment of drinking water at the full-scale water treatment plant was financed by Water Supply Company Noord Holland (PWN). The methods for determining the biostability of drinking water have been developed within the framework of the Joint Research Programme (BTO) of the water supply companies in the Netherlands. The authors are much indebted to the staff of the Laboratory of Microbiology at KWR for punctual analysis of the microbial growth characteristics of water, to Meindert de Graaf for collecting samples with the CBM and the hemoflow system and to Peter Hendriks (Het Waterlaboratorium, Haarlem) for collecting the routine-monitoring data. Appendix A. Supplementary material Supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.watres.2015.09.043. References Arnosti, C., Repeta, D.J., Blough, N.V., 1994. Rapid bacterial degradation of polysaccharides in anoxic marine sediments. Geochim. Cosmochim. Acta 58, 26-392652. Becker, E.W., 1994. Microalgae: Biotechnology and Microbiology. Cambridge University Press. ISBN 13:978-0521350204. Benner, R., Biddanda, B., Black, B., McCarthy, M., 1997. Abundance, distribution and stable carbon and nitrogen isotope compositions of organic matter isolated by tangential-flow ultrafiltration. Mar. Chem. 57, 243e263. Biersmith, A., Benner, R., 1998. Carbohydrates in phytoplankton and freshly produced dissolved organic matter. Mar. Chem. 63, 131e144. Bouteleux, C., Saby, S., Tozza, D., Cavard, J., Lahoussine, V., Hartemann, P., Mathieu, L., 2005. Escherichia coli behaviour in the presence of organic matter released by algae exposed to water treatment chemicals. Appl. Environ. Microbiol. 71 (2), 734e740.

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