Elsevier

Water Research

Volume 183, 15 September 2020, 115951
Water Research

Corroding copper and steel exposed to intermittently flowing tap water promote biofilm formation and growth of Legionella pneumophila

https://doi.org/10.1016/j.watres.2020.115951Get rights and content

Highlights

  • Copper and steel can promote biofilm formation and growth of Legionella pneumophila.

  • Copper released less corrosion by-products and biomass than mild steel.

  • Biofilm formation likely is enhanced by low-molecular-weight carboxylic acids and H2.

  • Water stagnation time may affect growth of L. pneumophila in copper pipes.

Abstract

The information about the impact of copper pipes on the growth of Legionella pneumophila in premise plumbing is controversial. For this reason, pipe segments of copper, stainless steel (SS), mild steel (MS), polyethylene, chlorinated polyvinylchloride (CPVC) and glass (controls) were exposed to intermittently flowing (20 min stagnation time) nonchlorinated tap water of 37 °C or 16 °C (ambient temperature) during six months to study the impact of metals on biofilm formation and growth of L. pneumophila. Biofilm concentrations (BfC, measured as ATP) on copper were 3 (at 37 °C) to 6 (at 16 °C) times higher than on SS. The maximum colony counts of L. pneumophila on the materials tested at 37 °C showed a quadratic relationship with the associated BfCs, with highest values on copper and MS. The average Cu concentration on the glass control of copper (glass-copper) was more than two log units lower than the Fe concentration on glass-MS, suggesting that copper released less corrosion by-products than MS. The release of corrosion by-products with attached biomass from MS most likely enhanced biofilm formation on glass-MS. Cloning and 16S RNA gene sequence analysis of the predominating biofilm bacteria revealed that an uncultured Xanthobacteraceae bacterium and Reyranella accounted for 75% of the bacterial community on copper at 37 °C. The nitrite-oxidizing Nitrospira moscoviensis, which can also utilize hydrogen (H2) and formate, accounted for >50% of the bacterial abundance in the biofilms on MS and glass-MS at 37 °C. The predominating presence of the strictly anaerobic non-fermentative Fe(III)-reducing Geobacter and the Fe(II)-oxidizing Gallionella on MS exposed to tap water of 16 °C indicated anoxic niches and the availability of H2, low molecular weight carboxylic acids (LMWCAs) and Fe(II) at the MS surface. LMWCAs likely also promoted bacterial growth on copper, but the release mechanisms from natural organic matter at the surface of corroding metals are unclear. The effects of water stagnation time and flow dynamics on biofilm formation on copper requires further investigation.

Introduction

The number of reported cases of Legionnaires’ Disease (LD) in Europe increases continuously (ECDC, 2019) and the disease burden of LD is the highest of all waterborne illnesses (Cassini et al., 2018). Moreover, in recent years most drinking-water-related cases of disease and death in the United States are caused by Legionella pneumophila in premise-plumbing systems (Benedict et al., 2017). At temperatures of 25–45 °C this organism can multiply within host amoebae that graze on the biofilms on water-exposed surfaces (Fields et al., 2002). Biofilm formation in premise plumbing is affected by a variety of factors, including water composition (presence of disinfectant, growth-promoting compounds), temperature, hydraulics, and also the nature of plumbing materials. In early studies rubber tap washers that enhanced biofilm formation were identified as a cause of growth for L. pneumophila (Colbourne et al., 1984). Batch tests confirmed that a variety of polymeric materials, including natural rubber, synthetic rubber, silicone rubber, plasticized PVC, (cross-linked) polyethylene, polypropylene and polybutylene in contact with tap water enhanced bacterial growth and proliferation of L. pneumophila, as compared to glass, stainless steel (SS) and copper (Niedeveld et al., 1986; van der Kooij et al., 2002). A number of synthetic materials also promoted biofilm formation and growth of L. pneumophila under dynamic flow conditions in model systems (Schoenen et al., 1988; Rogers et al., 1994a; Flemming et al., 2014). The information about the impact of copper, a major pipe material in tap water installations, on the growth of Legionella is controversial. Pringler et al. (2002) reported that colony counts of L. pneumophila in hot water systems with copper pipes in large buildings were lower than in systems with galvanized-iron pipes. L. pneumophila did not multiply in small-diameter copper pipes with warm tap water, fed for 10 days with tap water containing L. pneumophila and subsequently flushed two times each week with autoclaved tap water during 250 days (Schoenen et al., 1988) nor on copper coupons in a two-stage batch model system (Rogers et al., 1994b). Moreover, electrolytically-generated Cu ions are effective in the control of L. pneumophila in plumbing systems (Lin et al., 2011). However, in household plumbing systems with copper pipes significantly higher L. pneumophila colony counts were observed than in systems of galvanized steel or synthetic pipes (Tiefenbrunner et al., 1993; Mathys et al., 2008). Furthermore, copper pipes of a model plumbing system simulating domestic water use supported biofilm formation and growth of L. pneumophila although the colony counts initially were below those in pipes of PEX and SS (van der Kooij et al., 2005). Also Flemming et al. (2014) and Buse et al. (2017) reported that L. pneumophila multiplied in biofilms in copper pipes after inoculation of the test system.

Studies with the boiler biofilm monitor (BBM) system showed that biofilm formation with growth of L. pneumophila on surfaces of glass and CPVC exposed to intermittently flowing warm tap water without disinfectant was related with the AOC concentration of the water (van der Kooij et al. 2017, 2018). However, copper pipe segments exposed to warm tap water in the BBM showed significantly more biofilm formation and growth of L. pneumophila than the glass control (unpublished observation, Fig. S1). This effect contrasted with the observations in the model plumbing system (van der Kooij et al., 2005) and results of batch tests where concentrations of biofilm and L. pneumophila on copper were not or only slightly higher than on glass, SS and CPVC (van der Kooij et al., 2002; van der Kooij and Veenendaal, 2014). Therefore, biofilm formation and growth of L. pneumophila on copper under dynamic flow conditions was further studied in the BBM in comparison with SS and mild (corrosive) steel (MS). The objectives of the study were to measure (i) the biofilm formation on these metallic materials in comparison with glass and CPVC exposed to tap water of 37 °C or the ambient temperature (16 °C); (ii) the growth of L. pneumophila in the biofilms at 37 °C; (iii) the concentration of corrosion by-products (Cu and Fe) in the biofilms, and (iv) to obtain information about growth-affecting environmental conditions from the ecophysiological characteristics of the bacterial species predominating on copper and MS.

Section snippets

Tap water

Tap water used in the investigation originated from a municipal supply (annual production 5 × 106 m3) treating anaerobic groundwater by perforated-plate aeration and rapid sand filtration. The finished water was distributed without disinfectant. Selected water quality characteristics are shown in Table S1 (Supplemental Material). The used tap water has a low AOC concentration (≤5 μg acetate-C equivalents L−1; van der Kooij et al., 2017).

Boiler biofilm monitor (BBM) and discontinuous biofilm monitor (DBM)

The BBM has been described in detail in a previous

Comparison of copper with SS, HDPE and CPVC

To confirm the enhanced growth observed on the copper pipe segments (Fig. S1), copper was tested in the BBM in comparison with SS, HDPE, a growth-promoting material, CPVC, a commonly used material in warm water systems, and glass (control). All materials supported biofilm formation (Fig. S2) and growth of L. pneumophila (Fig. S3). The L. pneumophila colony count increased with the biofilm concentration (BfC) (Fig. S4). The BfC after ≥100 days of exposure, defined as Biofilm Formation Potential

Biofilm formation potential of materials

Quantification of the biomass production potential (BPP) of materials in contact with drinking water by measuring the concentration of attached and suspended biomass by ATP analysis in batch tests using tap water with a low bacterial growth potential showed BPP levels ranging from <100 pg of ATP cm−2 for glass, SS and CPVC to values > 10,000 pg ATP cm−2 for plasticized PVC and natural rubber (van der Kooij et al., 2002; Hambsch et al., 2014). The elevated biomass production in comparison with

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The study was conducted as part of the Joint Research Program of the Water Supply Companies in the Netherlands. The authors thank the staff of the Laboratory for Microbiology of KWR Water Research Institute for assistance in sampling and analyses and Gertjan Medema for comments on the manuscript.

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