(2) ARTICLE IN PRESS 136. A. Reyes et al. / International Biodeterioration & Biodegradation 61 (2008) 135–141. copper by EPS, and the generation of a weakly acidic environment (Geesey et al., 1986). MIC causes high release of copper by-products to drinking water and pitting attack (Geesey et al., 1994; Arens et al., 1995; Webster et al., 1996; Edwards and Jacobs, 2000; Critchley et al., 2004). MIC may be promoted when the copper pipes are in contact with drinking water during long stagnation times (Geesey et al., 1994). Pseudomonas paucimobilis and Pseudomonas solanacearum (Angell et al., 1990; Campbell et al., 1993); Sphingomonas sp. and Pseudomonas fluorescens (Arens et al., 1995); P. paucimobilis, Rhodotorula sp., and Flavobacterium sp. (Webster et al., 1996); Acidovorax delafieldii, Cytophaga johnsonae, and Micrococcus kristinae (Critchley and Fallowﬁeld, 2001); Acidovorax sp. and Sphingomonas sp. (Critchley et al., 2004) have been identiﬁed in bioﬁlms that cause MIC in copper pipes. Cases of MIC have been associated with blue water phenomenon (Webster et al., 2000; Geesey et al., 2002), and reported mainly in large buildings (Angell et al., 1990; Fischer et al., 1992, 1994; Campbell et al., 1993; Geesey et al., 1994; Bremer et al., 2001). The aim of this work was to determine if the high release of copper by-products in low-pH water measured in the ﬁeld was associated with the presence of microbial bioﬁlms. 2. Materials and methods 2.1. Study sites This study was carried out in ﬁve houses in Talca, a city of approximately 200,000 inhabitants located 256 km south of Santiago, Chile. Three houses were located in the rural area and two in the urban area. All houses included in this study used well water for drinking. In the rural houses the water was obtained from private wells and was not chlorinated, whereas in the urban houses the water was both chlorinated and ﬂuorinated before use.. quality according to APHA, AWWA, WEF (1998). Parameters such as residual chlorine, phosphate, sulﬁde, nitrite, ammonia, aluminum, manganese, silicon, sodium, tin, zinc, potassium, magnesium, calcium, sulfate, chloride, and nitrate were also measured. Temperature, pH, and DO were measured immediately after sampling. Water samples for determination of concentrations of soluble and particulate copper were taken on site and immediately ﬁltered (0.45 mm cellulose nitrate) according to APHA, AWWA, WEF (1998) protocols. The copper concentration was measured using a Perkin-Elmer Analyst 100 atomic absorption spectrophotometer (Shelton, CT, USA). DOC concentrations were measured using a Shimadzu VCPH total organic carbon analyser (Kyoto, Japan). DO, pH, residual chlorine, conductivity and temperature measurements were made on site using portable instruments from WTW (Weilheim, Germany).. 2.4. Copper pipe samples preparation for scanning electron microscopy (SEM) Copper samples (1 cm2) cut from the copper pipes were used for bioﬁlm SEM analysis according to methods previously described (Angell et al., 1990). The bioﬁlm present on the internal copper pipe surfaces was ﬁxed by soaking in 3% glutaraldehyde prepared in sodium cacodylate buffer (0.1 M, pH 7.2) for 2 h at 48 1C, rinsed three times in sodium cacodylate buffer, and ﬁve times in double distilled water. The samples were then dehydrated in graded acetone baths (30%, 50%, 70%, and 100%), and dried in a critical point drying apparatus using CO2. The copper pipe samples obtained were ﬁxed on metal supports, sputter-coated with gold (10 nm) (MED 010, Balzers Union Limited, Liechtenstein), and observed with a Jeol JSM 25 S SEM (Tokyo, Japan). Micrographs were taken on Kodak t-max 100 ﬁlm with exposure times of 8–30 s. The presence of pitting was determined by SEM examination of copper 1 cm2 sections after removing the corrosion products, by pickling in 10% (w/w) citric acid (Fischer et al., 1992).. 2.5. X-ray diffraction (XRD) Sections of copper pipe (1 cm2) inner surfaces were analysed by XRD. The analysis was carried out in a Siemens D-5000 diffractometer (Karlsruhe, Germany).. 2.6. Microbial methods 2.2. Water and pipe sampling Samples of well water (1 L), 8 h stagnant water (125 mL), and running water or 2-min ﬂushed water (1 L) were taken from each house studied. The sampling was conducted according to the protocol described by US Environmental Protection Agency (1991). Sampling of water for dissolved organic carbon (DOC) measurement was carried out according to APHA, AWWA, WEF (1998). Chemical, physical, and microbiological analyses were carried out on these samples. All water samples were collected in high-density polyethylene container (APHA, AWWA, WEF, 1998), except those that were taken for DOC analysis, and immediately stored at 4 1C. Samples for DOC analysis were collected in glass bottles according to APHA, AWWA, WEF (1998). Pipe sampling was carried out using gloves. Pipes were sawed with a sterilized saw down to 30-cm length pieces. Pipes were then transferred into sterile glass bottles and ﬁlled with drinking water taken from the same site, and stored at 4 1C for 12 h for subsequent surface analysis.. 2.3. Water analysis Well, stagnant, and running water were analysed for copper concentration (total, soluble, and particulate), pH, alkalinity, dissolved oxygen (DO), DOC, temperature, conductivity, and microbiological. In order to obtain information about the types of microorganisms present in microbial bioﬁlms, a protocol was developed to isolate bacteria by culturing samples from the inner surface of the copper pipes. Cells were released from 5-cm copper pipe sections by sonication in an ultrasound bath (Tru-SweepTM, 50/60 Hz, Crest, NJ, USA) for 30 s, three times, on ice. Cells were collected by centrifugation at 13,000 rpm for 10 min to obtain a cellular pellet. Colony-forming units were detected by plating 100 mL of concentrated cell suspension on R2A agar media, followed by incubation for 5 d at 30 1C. Bacterial isolates were ﬁrst grouped by colony morphology and then by restriction fragment length polymorphism (RFLP) analysis of the respective 16S rRNA genes (see below). One isolate from each group was selected for partial sequencing of its 16S rRNA gene. Oligonucleotide primers designed for detection of bacterial 16S rRNA genes [8F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1392R (50 -ACG GGC GGT GTG TAC-30 )] (Amann et al., 1995) were used for the ampliﬁcation of rRNA genes. Sequencing was carried out in a Perkin-Elmer ABI Prism 310 sequencer (Foster City, CA, USA) using the same primers. Polymerase chain reactions (PCR) and electrophoresis conditions were described by Jordan et al. (2002). For RFLP analysis, the PCR products were digested with 20 U of HhaI or HaeIII restriction enzymes in a total volume of 20 mL at 37 1C for 3 h. Digested PCR products were visualized after electrophoresis on 3% agarose TAE gels at 70 mV for 3 h, and subsequently stained in ethidium bromide for 10 min..
(3) ARTICLE IN PRESS A. Reyes et al. / International Biodeterioration & Biodegradation 61 (2008) 135–141. 2.7. Thermodynamic calculations Thermodynamic calculations were made using MINEQL+ software (Schecher and Mcavoy, 2001). Chemical parameters of drinking water measured in the ﬁve houses were entered and the copper solubility values obtained were compared with concentrations of soluble copper determined experimentally after ﬁltrations of stagnant water samples.. 137. composition of running and standing water is shown in Table 2. It is observed that the DOC and the particulate copper concentrations increased markedly during water stagnation in the rural houses and did not increase during water stagnation in the urban houses. 3.2. Biofilms, porous scales, and high copper by-product release. 3. Results 3.1. Changes observed during water stagnation Chemical and microbiological water quality parameters of sampled well waters are shown in Table 1 and the Table 1 Composition of well waters sampled Parametera. Rural housesb,c,d. Urban housesd. pH Alkalinity (mg/L as CaCO3) TOC DO Conductivity (mS/cm) Total copper Soluble copper Particulate copper Iron Lead Bacterial counts (E. coli cells per 100 mL). 6.3 60 o0.5 5.10 271 0.024 0.019 0.004 0.004 o0.002 Not detectable. 6.9 95 6.9 1.70 341 0.073 0.059 0.002 0.007 o0.002 Not detectable. The SEM examination of the rural house copper pipes showed extensive bioﬁlms along with corrosion scales (Figs. 1a–c). The surface of the copper pipes in two rural houses was completely and uniformly covered with bluish green corrosion products, which were identiﬁed as malachite by XRD analysis (Table 3). The copper pipe sampled from one of the rural houses did not contain bluish green corrosion products and the surface had a reddish brown colour. In this case, only cuprite was detected by XRD (Table 3). In addition, the SEM images show that the corrosion scales covering the surfaces of copper pipe samples of the three rural houses were porous. The visual inspection of these samples showed non-adherent and easily sloughed corrosion scales. The SEM analysis of copper pipe sections from the urban houses showed no presence of bioﬁlms (Figs. 1d and e) and XRD analysis showed that the inner surface was covered solely with cuprite and tenorite (Table 3). Visual inspection of these samples showed more uniform and compact corrosion scales in comparison with the rural houses.. a. Units in mg/L unless otherwise stated. Non-chlorinated water. c Average values of three houses. d Other parameters: phosphate o0.01, sulﬁde o0.5, nitrite o0.05, ammonia o0.05, aluminum o0.01, manganese o0.01, silicon 21, sodium 11.2, tin o0.01, zinc 0.01, potassium 2.96, magnesium 5.6, calcium 3.7, sulfate o10, chloride 12, nitrate 2.6, and residual chlorine 0.5. b. 3.3. Variovorax sp. was isolated from bacterial biofilms of copper pipes in rural houses Two main colony morphotypes were obtained from the copper pipe samples in the rural houses. We did not detect. Table 2 Composition of running and stagnant waters in rural and urban houses Parametera. pH Alkalinity (mg/L as CaCO3) TOC DO Conductivity (mS/cm) Total copper Soluble copper Particulate copper Particulate stagnant copper (%) Iron Lead a. Rural housesb,c,d,e. Urban housesf. Running water. Stagnant water. Running water. Stagnant water. 6.2 63 o0.5 5.10 271 0.094 0.084 0.004 3.9 0.005 o0.002. 6.2 72 2.9 3.10 285 5.258 4.326 0.87 17.0 0.023 0.044. 7.2 98 1.7 6.90 341 0.076 0.067 0.002 2.7 0.007 o0.002. 6.9 100 1.5 5.51 360 0.377 0.552 0.039 6.0 0.014 0.023. Units in mg/L unless otherwise stated. Non-chlorinated water. c Average values of three houses. d Parameters without changes: residual chlorine 0.5, phosphate o0.01, sulﬁde o0.5, nitrite o0.05, ammonia o0.05, aluminum o0.01, manganese o0.01, silicon 19, sodium 11.1, tin o0.01, zinc 0.01, potassium 3.7, magnesium 10, calcium 12.7, sulfate 14, chloride 7.9, and nitrate 3.8. e,f Not detectable E. coli cells in 100 mL water sample. b.
(4) ARTICLE IN PRESS A. Reyes et al. / International Biodeterioration & Biodegradation 61 (2008) 135–141. 138. Fig. 1. SEM images of cold-water pipes. Rural area: (a) rod-shaped bacteria and EPS in House 1. (b) Rod-shaped bacteria in association with corrosion products and EPS in House 2. (c) Copious layer of EPS and rod-shaped bacteria in House 3. Urban area: (d) corrosion products in House 4. (e) Corrosion products in House 5.. Table 3 Other characteristics of copper pipes Parametersa. Location (cold-water tap)/T (1C) Inner diameter (in) Pipe age (years) Pipe length (m), from well to tap Compounds. Rural houses. Urban houses. House 1. House 2. House 3. House 4. House 5. Bathroom/16.5. Kitchen/18.7. Bathroom/15.6. Bathroom/17.0. Bathroom/ 17.7. 1 2. 3 8. 3 8. 3 8. 3 8. 6 31. 4 42. 4 23. 4 8. 6 9. Malachite (Cu2(CO3)(OH)2) Cuprite (Cu2O). Cuprite (Cu2O). Cuprite (Cu2O). Cuprite (Cu2O). Cuprite (Cu2O). Malachite (Cu2(CO3)(OH)2). Tenorite (CuO). Melanothallite (Cu2OCl2) Cu8SiS6. Microorganisms a. Cu15Si4 Variovorax sp.. Units in mg/L unless otherwise stated.. Not identiﬁed Variovorax sp.. Belloit (Cu(OH)Cl) Copper nitrate (Cu(NO3)2 6H2O) Manganese ferrite (MnFe2O4) None detected. None detected.
(5) ARTICLE IN PRESS A. Reyes et al. / International Biodeterioration & Biodegradation 61 (2008) 135–141. colony formation on plates from the urban house. This data were correlated with the RFLP analysis, because two ribotypes were observed in copper pipe samples from rural houses. One of these ribotypes was common to both house samples. A sequencing analysis of a fragment encoding the 16S rRNA (1200 bp) gene was performed for each ribotype. After comparison of the sequence with the Ribosomal Database Project and NCBI databases, the analysis showed that all the ribotypes matched the group of the b-proteobacteria, particularly the Variovorax genus (95% identity). It is known that bioﬁlms contain a number of microorganisms which can be detected by modern molecular techniques. However, in the present case we could not amplify 16S rRNA genes from the metagenomic DNA of the samples, possibly due to a low yield of DNA and, even more signiﬁcant, a high amount of copper and calcium ions that could inhibit the PCR reaction. The isolation of individual species from bioﬁlms will be considered in a future work to test the effect of bioﬁlms on copper release in laboratory controlled experiments.. 139. 4. Discussion The increase of DOC observed during water stagnation in rural houses was possibly caused by sloughing of organic matter from the bacterial bioﬁlms because of the porous and low-adherence nature of scales, similar to that reported by several authors when MIC occurs on copper pipes (Geesey et al., 1986; Bremer and Geesey, 1991; Davidson et al., 1996; Webster et al., 1996, 2000). It is possible that, under these conditions, the release of copper by-product increases because the protective scale formation is inhibited by the bioﬁlms (Webster et al., 2000). According to Davidson et al. (1996) the production of acidic metabolites by bacterial bioﬁlms may be associated with high copper by-product release, especially in the form of cellular material bound to copper particulates. A similar mechanism could explain the high particulate copper concentrations generated in water of rural houses (Table 2). However, it is important to emphasize the pH inﬂuences on copper corrosion with or without bioﬁlms. At pH values. Fig. 2. SEM images of cold-water pipes after pickling with 10% citric acid. Rural area: (a) copper pipe from House 1 showing pitting. (b) Uniform attack on copper pipe from House 2. (c) Copper pipe from House 3 showing pitting. Urban area: (d) copper pipe from House 4 showing uniform attack. (e) Copper pipe from House 5 showing uniform attack..
(6) ARTICLE IN PRESS 140. A. Reyes et al. / International Biodeterioration & Biodegradation 61 (2008) 135–141. less than 6.0 the corrosion rates can be very high (Edwards et al., 1994), which support the uniform corrosion (Mattsson and Fredrikksson, 1968; Cruse et al., 1985). Equilibrium calculations (MINEQL+) using the water composition measured in 4 year old copper pipes of rural houses, and considering malachite as the solid that controls the solubility, predicted just about one-fourth of the copper concentration measured after stagnation. These results can be explained because the simulation includes only the abiotic component, without considering that a fraction of copper could be originated by effect of bacterial bioﬁlms. After removing the microbial bioﬁlms and inorganic deposits on copper pipes, the SEM micrographs showed numerous pits. The pits had spherical shape, whose diameter did not exceed 20–30 mm and were located under the bluish green scales (Figs. 2a and c). No pits were found in pipes which did not contain bioﬁlms (Figs. 2d and e). Geesey et al. (1987) suggested that pits are caused when metallic copper is oxidized by acidic groups of EPS contained in bioﬁlms. Variovorax strains have been mainly described in soils. However, Variovorax strains have been also reported in groundwater (Rooney-Varga et al., 1999; Humphries et al., 2005). The ability to form bioﬁlms has been recently described (Steinberger and Holden, 2005). Factors observed in rural houses, such as infrequently used pipes, giving rise to very long stagnation periods (472 h) and the length of the pipes (Table 3) contributed to the bioﬁlm growth such as that which has been reported (Bremer et al., 2001; Geesey et al., 1994). Due to the multiple evidences shown before, we concluded that the microbial bioﬁlms along with the aggressive water quality could be the main factors involved in the high levels of copper by-product liberation in rural houses. 5. Conclusions MIC probably caused the increase of the copper byproduct release to water and the presence of pits on copper pipes in water of a pH range of 6.1–6.3. A bacterial strain identiﬁed as Variovorax sp. was isolated from the bioﬁlms present on the inner surfaces of the copper pipes. Acknowledgements This research was supported by a grant provided by the International Copper Association, the Grant FONDAPFONDECYT (Grant 1501-0001, program 7), the Millennium Nucleus Grant P04/P-007-F, and the Grant FONDECYT (no. 1040607). References Amann, R., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identiﬁcation and in situ detection of individual microbial cells without cultivation. Microbiology and Molecular Biology Reviews 59, 143–169.. Angell, P., Campbell, H.S., Chamberlain, A.H.L., 1990. Microbial involvement in corrosion of copper in fresh water. Interim Report, International Copper Association (ICA Project 405), New York, pp. 1–63. APHA, AWWA, WEF, 1998. 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