Bioelectrochemistry 97 (2014) 15–22

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Multi-technique approach to assess the effects of microbial biofilms involved in copper plumbing corrosion Ignacio T. Vargas a,b,⁎, Marco A. Alsina b, Juan P. Pavissich b, Gustavo A. Jeria b, Pablo A. Pastén a,b, Magdalena Walczak c, Gonzalo E. Pizarro a,b a b c

Centro de Desarrollo Urbano Sustentable (CEDEUS), Pontificia Universidad Católica de Chile, Santiago, Chile Departamento de Ingeniería Hidráulica y Ambiental, Pontificia Universidad Católica de Chile, Santiago, Chile Departamento de Ingeniería Mecánica y Metalúrgica, Pontificia Universidad Católica de Chile, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 15 December 2012 Received in revised form 19 November 2013 Accepted 26 November 2013 Available online 4 December 2013 Keywords: Copper MIC Biofilm Electron microscopy GI-XRD

a b s t r a c t Microbially influenced corrosion (MIC) is recognized as an unusual and severe type of corrosion that causes costly failures around the world. A microbial biofilm could enhance the copper release from copper plumbing into the water by forming a reactive interface. The biofilm increases the corrosion rate, the mobility of labile copper from its matrix and the detachment of particles enriched with copper under variable shear stress due to flow conditions. MIC is currently considered as a series of interdependent processes occurring at the metal–liquid interface. The presence of a biofilm results in the following effects: (a) the formation of localized microenvironments with distinct pH, dissolved oxygen concentrations, and redox conditions; (b) sorption and desorption of labile copper bonded to organic compounds under changing water chemistry conditions; (c) change in morphology by deposition of solid corrosion by-products; (d) diffusive transport of reactive chemical species from or towards the metal surface; and (e) detachment of scale particles under flow conditions. Using a multi-technique approach that combines pipe and coupon experiments this paper reviews the effects of microbial biofilms on the corrosion of copper plumbing systems, and proposes an integrated conceptual model for this phenomenon supported by new experimental data. © 2013 Elsevier B.V. All rights reserved.

1. Introduction During the last decades, corrosion of metallic piping systems for drinking water has been considered relevant because of health risks related to the release of metals into drinking water, general failure of the piping system, and the costs associated with repairing infrastructure damaged by corrosion [1]. Due to its relatively low price and durability, copper is the plumbing material of choice for household drinking water systems [2]. Corrosion of copper pipes proceeds as an electrochemical reaction, where metallic copper is oxidized to copper ions (anodic half-reaction), while an oxidizing agent is reduced, typically dissolved oxygen (DO) (cathodic half-reaction) [3]. The water chemistry and operational conditions of the piping system control the formation/ dissolution of scales and the release of copper into the water [2]. Microbially influenced corrosion (MIC) has been identified as an unusual and severe type of corrosion that appears in the form of pitting (known as Type 1½), and occurs mainly in soft water supplies in the presence of microorganisms [1,4]. Microbial colonization and the subsequent biofilm development induce drastic changes in the vicinity of the inorganic environment formed on the metallic surface [1,5]. This type of ⁎ Corresponding author at: Av. Vicuña Mackenna 4860, 7820436 Macul, Santiago, Chile. Tel.: +56 2 26864218; fax: +56 2 2354 5876. E-mail address: [email protected] (I.T. Vargas). 1567-5394/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bioelechem.2013.11.005

corrosion has caused failures in institutional buildings in several countries around the world, including the United Kingdom, Germany, Saudi Arabia, the USA and Chile [1]. The cost of replacing the damaged piping can be as high as 100 million, as estimated for a Scottish hospital where the entire piping system collapsed [1]. Relevant advances in the understanding of MIC were accomplished during the 1980s as a response to strong industrial demand to mitigate related failures. Several research groups worked on unraveling MIC mechanisms, including the effect of microorganisms on the corrosion processes. By the end of the 1990s and the beginning of the 21st century, the development of new techniques for surface analysis and electrochemical characterization have helped to elucidate the function of biofilms as reactive interfaces that control the mobility of metallic species between the metal and the liquid [6]. Recent advances in molecular biology, including culture-independent techniques constitute a powerful analytical tool and a next step for understanding MIC. These advances allow the determination of microbial communities, and the formulation of a conceptual model that includes metal–microbe interactions [6–9]. A microbial biofilm is basically composed of microorganisms (mainly bacteria) embedded in a heterogeneous matrix of extracellular polymeric substances (EPS) that facilitate the attachment of the microbial community growing on the aggressive environment existing at the metal– liquid interface. EPS are a complex mixture of various macromolecules

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(i.e. proteins, polysaccharides, lipids, nucleic acids and amphiphilic polymers), whose nature depends on the specific microbial species in the biofilm, and relevant environmental factors such as nutrient availability and environmental stress [10]. The reactivity of the biofilm to copper is determined by the capability of EPS and the bacterial cells itself to bind copper ions (sorption) and retain copper particles produced as a consequence of the precipitation of corrosion by-products [11,12]. This patchwork of bacteria, EPS, and solid corrosion byproducts form a complex reactive surface that induces metallic copper oxidation, resulting in the release of ions and/or particulate corrosion by-products into the water during the operational flow–stagnation cycle. The study of MIC requires the integration of several analytical and modeling techniques contributed by a number of study fields, including microbiology, surface and interface science, electrochemistry, water chemistry, and fluid mechanics. The complexity of the processes involved in MIC poses considerable difficulty for a robust simulation and, consequently, the existing models of MIC are not yet complete to thoroughly describe the interactions between the biofilm and the corrosion by-products under flow–stagnation cycles. Current models consider MIC as a series of simultaneous or successive processes. The proposed mechanisms include the following effects of the biofilm matrix: (a) formation of localized microenvironments with different pH, DO concentrations, and redox conditions [6,13,14], (b) sorption and desorption of labile metal bonded to organic compounds under changing water chemistry conditions [13,15], (c) change in morphology of the solid corrosion by-products [6], (d) diffusive transport of chemical species at the metal surface [6], and (e) detachment of scale particles from the pipe surface under flow conditions [6,13]. While abundant information related to the first two mechanisms (a, b) is available in the literature [6,13–15], we consider that the experimental evidence to support the remaining mechanisms (c–e) is still insufficient. The study of the biofilm effects should consider the local properties of the material where the biofilm is growing. Copper is a scale-forming material that, under abiotic conditions, shows moderate corrosion rates [2]. Dissolved copper initially released from the metallic surface leads to precipitation of corrosion by-products on the inner surface of the pipe, thus passivating the surface and decreasing the concentration of dissolved copper in about an order of magnitude compared to the initial dissolved copper released from the metallic surface [2]. In the presence of a biofilm, passivation is prevented and EPS can accumulate the released copper. Moreover, the biofilm heterogeneity could also induce particle detachment that, in case of a scaling forming material, may highly increase the mass of copper released into the bulk water [13]. Under certain conditions, particle detachment increases the copper concentration of the bulk water to concentration up to 20 mg/L, giving the water a blue–green coloration [16]. Such episodes are known as “blue water” which is associated with microbial activity [5,16–18]. Non-scale forming materials (i.e. stainless steel) also form passive films, but in those cases corrosion is so slow that high concentrations of metallic ions never accumulate and the presence of a biofilm may result in different effects. Due to its antimicrobial properties, copper has currently been used to replace stainless steel, aluminum and plastic touch surfaces designed for human contact (e.g. door handles, bathroom fixtures, or bed rails) and reduce hospital microbial contamination [19]. Thus, a conceptual model of MIC in copper should include the interaction between corrosion by-products and the biofilm, the EPS matrix reactivity, and the effect of copper toxicity on the microbial ecology. In this paper, we review the MIC mechanisms related to copper by a combination of traditional tests (e.g. electrochemistry) and new approaches used by our group (e.g. in-situ DO measurements) to account for the effect of microorganisms on corrosion. Here, we present new experimental evidence based on results obtained in laboratory tests (e.g. pipes and metal coupons) and field campaigns performed in a model household drinking water distribution system affected by MIC.

We finally propose a new conceptualization of MIC that may be used as a more thorough framework for the development of mathematical models of copper corrosion. 2. Materials and methods 2.1. Pipe experiments Pipe experiments were conducted on copper pipes with an internal diameter of 1.95 cm (3/4″) (type L, manufactured by MADECO-Chile under ASTM-B88 standard). The pipes were extracted from a household system affected by MIC [13] and tested under laboratory conditions using tap water from the same location. Additionally, abiotic experiments were prepared using synthetic MilliQ water, sodium bicarbonate (ACS grade, 99.7% pure, Merck KGaA, Darmstadt, Germany) and nitric acid (p.a. EMSURE® ISO, 65% pure, Merck KGaA, Darmstadt, Germany) to adjust the pH (tap water conditions). 2.1.1. DO consumption experiments DO consumption experiments were carried out under biotic (pH = 6.0, HCO− 3 = 1.6 mM) and abiotic conditions (pH adjusted to 6.0 ± 0.1, [HCO− 3 ] of 0, 1.6, 3.0, and 7 mM). DO concentration was continuously measured during stagnation according to the methodology developed in our previous works using both a 2 mm diameter Fiber Optic Oxygen Sensor (FOXY, Ocean Optics, Inc.) [20] and a Luminescence Dissolved Oxygen (LDO) sensor connected to a HQ40d multimeter (HACH Company, Loveland, CO, USA) [21]. Fluorescence sensors were inserted in the bulk water at one end of the tested pipe, while the opposite end was closed with a rubber stopper. 2.1.2. Flushing tests Flushing experiments were carried out in accordance to the methodology presented elsewhere [13,22]. The laboratory tests were conducted on 1 m long copper pipes of 0.3 L volume. Pipes under biotic and abiotic conditions were previously aged through water changes three times per week (Monday, Wednesday and Friday) [23] using synthetic water with 3.2 mM [HCO− 3 ], and water obtained from the field [13], respectively. After 4 weeks of aging and 8 h of stagnation, pipes were flushed at a flow rate of 0.24 L/min at 25 °C, to simulate the behavior of domestic pipe systems under laminar conditions (Re = 280.44). During flushing, samples of 15 mL for the first 0.6 L, and 100 mL for the remaining volume, were taken to determine copper concentration until approximately 6 L of water was flushed through the pipe. An ICP/OES spectrometer (Thermo Electron iCAP 6300) was used to measure the concentration of copper released into the bulk water. The mass of copper released was estimated by integrating the copper concentration versus the volume of water extracted from the pipe. 2.1.3. Surface analysis Scanning electron microscopy (SEM, LEO 1420VP) with elemental analysis (EDS, Oxford 7424 solid-state detector) was used to study the morphology and composition of the biofilm matrix and the corrosion scales formed on the inner surface of the pipes. Several 1 × 1 cm coupons were cut from (i) new pipes tested in the laboratory under biotic and abiotic conditions, and (ii) used pipes extracted from the model household system affected by MIC. The samples were kept hydrated with the field water before preparation for microscopic examination. The coupons were treated with critical point drying [24] and coated with a thin gold film. Transmission electron microscopy (TEM, Phillips Tecnai 12 Bio Twin) was used to determine the presence of colloidal material (b 0.45 μm). The particles were captured through a three step procedure, adapted from the methodology developed by Lienemann et al. [25] for the examination of colloids. First, the water sample (50 mL) was centrifuged at 500 rpm (RCF = 27 ×g) for 5 min to exclude particles above the colloidal size. After this preliminary centrifugation, the supernatant was carefully withdrawn and 5 mL of the

I.T. Vargas et al. / Bioelectrochemistry 97 (2014) 15–22 Table 1 Composition of water extracted from the studied location affected by microbial influenced corrosion [13]. Parameter

Unit

Concentration

Copper Iron Chloride Total alkalinity Total hardness Sulfate Nitrate Phosphate pH Dissolved oxygen Conductivity Dissolved inorganic carbon (DOC) Magnesium

mg Cu/L mg Fe/L mg Cl2/L mg CaCO3/L mg CaCO3/L mg SO−2 4 /L mg NO− 3 –N/L mg PO−3 4 /L – mg O2/L μS/cm mg/L mg Mg/L

0.07 0.045 15.6 80 160 30.2 3.2 1.66 6 8.11 235 1.5 6.44

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CH Instruments Inc.) as reference electrode. Autoclaved water extracted from the field location was used as electrolyte. Epifluorescence microscopy (Olympus CX31) was used to detect the presence of a biofilm on the tested coupons. Micrographs were taken using an attached digital camera. Sample preparation consisted in cell fixation with ethanol and subsequent staining of nucleic acids with 1% v/v Acridine Orange for 20 min. 3. Results and discussion Results below are organized to discuss the biofilm effects on the corrosion processes (mechanisms (a) through (e)) of drinking water copper pipes. 3.1. Microenvironments

sample was extracted from the bottom of the centrifugation tube, resuspended with MilliQ water, and centrifuged at 6000 rpm (RCF = 3823 ×g) for 30 min. After centrifugation, the sample was filtered using a 0.45 μm pore-size acetate cellulose membrane. Finally, an aliquot of the filtered water was deposited over a hydrophilic resin (Nanoplast). 2.2. Coupon experiments Electrochemical experiments to estimate corrosion rates under biotic and abiotic conditions were conducted using pretreated copper coupons (99.99% purity) of 2.5 cm2 surface area. Pretreatment included a 10 minute immersion in a 1:4 HCl/distilled water solution followed by several washes with distilled water until reaching circumneutral pH. After pretreatment, coupons were aged immediately in 100 mL glass bottles filled with water extracted from the study location (Table 1). Bottles were incubated in triplicate for 150 days in a shaking incubator at 25 °C and 25 rpm. The abiotic controls were aged using the same water after autoclaving. Grazing incidence X-ray diffraction (GI-XRD) [26] was used to analyze the solid phase formed on the tested copper coupons. GI-XRD measurements were performed using a Bruker D2 Phaser diffractometer with a Lynxeye detector and a Cu Kα radiation. The scans were measured from 10° to 80° with a step size of 0.02°. Electrochemical measurements were conducted using a potentiostat (Gamry, Reference 600). Corrosion rates of copper coupons were estimated by linear polarization using the Echem Analyst Software (Gamry, Philadelphia, PA, USA). Experiments were performed using a 50 mL homemade electrochemical cell. A platinum wire (CHI115, CH Instruments Inc.) was used as counter electrode and Ag/AgCl (CHI111,

SEM micrographs in Fig. 1 show the inner surface of a copper pipe affected by MIC. The presence of a biofilm together with corrosion scales forms a heterogeneous matrix that may result in the formation of microenvironments on the surface due to spatial chemical gradients. Fig. 1a–b shows botryoidal scales, observed by light microscopy as greenish solid phases and identified through X-ray absorption spectroscopy as mainly being malachite (Cu2CO3(OH)2) [13] a carbonate hydroxide corrosion by-product with passivating properties. Within the malachite, well defined pits were observed. The presence of pits surrounding the corrosion by-products strongly suggests the formation of anodic and cathodic zones where the metallic copper oxidation and oxygen reduction take place, respectively. The diffusional resistance and metabolic activity of a mature biofilm matrix (microorganisms, EPS, and inorganic solid phases) limit the diffusion of oxygen to cathodic areas and diffusion of ions from anodic areas [6]. This kind of patchwork arrangement was observed by Geesey et al. [4], and described as an unusual type of pitting corrosion named corrosion type ½. In a study assessing the interactions between microorganisms and minerals, Little et al. [26] suggested that the presence of a biofilm maintains diverse chemical microenvironments at the metal–liquid interface, which may be completely different from the bulk liquid in terms of pH, DO, and organic and inorganic species [27]. Therefore, thermodynamic calculations based on the bulk chemistry may not be adequate to predict the precipitation and solid phase replacement reactions (e.g., cupric hydroxide (Cu(OH)2) to tenorite (CuO) or malachite) that occur at the metal–liquid interface [27]. It is likely then that a complex matrix of biological and inorganic phases, like the one observed in Fig. 1c, increases the physical heterogeneity and chemical heterogeneity of the surface producing microenvironments that facilitate the formation of

Fig. 1. SEM micrographs of a copper pipe surface extracted from a 2 year old system affected by MIC. Images reveal physical heterogeneity of the surface attributed to the biofilm–mineral matrix. Large (a) and small (b) pits (2500× of magnification). (c) Bacteria and minerals near the pit (10,000× of magnification).

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isolates obtained in this previous work [7], we performed soluble copper sorption tests. Preliminary results have shown sorption capacities of as much as 18 mg Cu/mg of dry biomass (unpublished data). These initial results suggest that biofilms in copper pipes may have a very high copper accumulation capacity. 3.3. Morphology of scales

Fig. 2. SEM micrographs of the biofilm growing on a 2 year old copper pipe affected by MIC. Summary of EDS analysis for the EPS structures (a): C = 63 wt.%, O = 16 wt.%, Cu = 21 wt.%; the bacteria (b): C = 65 wt.%, O = 6 wt.%, Cu = 30 wt.%; and beneath the biofilm (c): C = 27 wt.%, O = 2 wt.%, Cu = 71 wt.%.

electrochemical cells, enhancing copper oxidation and the consequent increase of copper released into the bulk water. 3.2. Sorption of soluble copper As a reactive barrier, biofilms can sequester and accumulate labile copper. This process has the potential to disturb the chemical equilibrium and to induce high copper concentrations into the bulk water during flushing events. In general, the biofilm matrix accumulates copper by two mechanisms: copper ions are bound to EPS and cells during stagnation periods (sorption), and copper phases are enriched in the biofilm matrix through the formation of corrosion by-products. Furthermore, corrosion by-products may also sorb copper ions, although this could be more difficult to observe and advanced synchrotron spectroscopic techniques would be needed to quantify this process. Fig. 2 shows a biofilm formed on the inner surface of a 2 year old copper pipe. EDS analysis suggested that both microorganisms and EPS matrix accumulate soluble copper by mineralization and sorption of labile copper. Analysis of the EPS structures indicated an elemental composition of 63% C, 16% O and 21% Cu, while on the bacterial cells showed a composition of 65% C, 6% O and 30% Cu. The surface beneath the biofilm seemed to be formed basically by corrosion by-products and metallic copper (27% C, 2% O, 71% Cu). In a previous study [7] we performed a bacterial community analysis of the biofilm growing on copper pipes affected by MIC and concluded that the microbial ability, linked to biofilm growth, is a key factor related to the observed increase in surface heterogeneity, corrosion and the occurrence of microenvironments and bacterial survival. Using bacterial

Malachite mineralization produced during corrosion events commonly displays a botryoidal morphology; although observed texture features seem to vary according to the formation conditions, including the presence of a biofilm. Malachite formed under abiotic conditions tends to show a plumose texture, resembling “spines” [21,28]. Malachite formed under biofilm presence tends to show an acicular texture, with “hair-like” filaments [27]. SEM micrographs of copper pipes affected by biotic corrosion and abiotic corrosion (Fig. 3) indicate that the presence of the microbial–EPS matrix alters the morphology of malachite precipitates [13,20]. This is consistent with our findings in copper corroded pipes, while “hair-like” filaments were observed under biotic conditions (Fig. 3a), the formation of morphologies with columnar crystal habits was observed under abiotic conditions (Fig. 3b). Interestingly, under biotic conditions malachite maintains a botryoidal configuration (Fig. 3c) found also for abiotic conditions. Thus, this “hair-like” morphology of malachite together with the observed patchwork between the biofilm and precipitates form a film that, under the shear stress associated with the pipe flow, could increase the release of particles into the bulk water. 3.4. Transport of chemical species Passivation of the metallic surface by accumulation of corrosion by-products controls the transport of ions to the bulk water, limiting the rate of copper oxidation [29]. However, microbial local acidification induces chemical weathering and dissolution of solid phases such as tenorite or malachite, thus forcing mineral replacement reactions that, in turn, enhance copper release into the bulk [27]. GI-XRD analysis of coupons exposed for 20 days to water extracted from the study field suggested solid phase replacement due to microbial activity at the metal surface. X-ray diffraction data identifies cuprite (Cu2O) and tenorite on the surface in the biotic experiments, but only tenorite in the abiotic ones (data not shown). This result suggested changes in the local chemistry at the solid–liquid interface due to the presence of the biofilm. Presence and absence of bacteria were checked using epifluorescence microscopy. In a previous work we detected cupric hydroxide, a non-expected metastable amorphous phase, coexisting with malachite in an old piping system affected by MIC [13]. Using ellipsometric measurements on Al-brass and Ti surface tested under biotic and abiotic conditions Busalmen et al. [30] concluded that

Fig. 3. SEM micrographs (10,000× of magnification) of malachite structures formed under (a) biotic conditions, (b) abiotic conditions, and (c) larger field of (a).

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Fig. 4. SEM micrographs of a copper pipe surface under stagnation. (a) Biotic conditions (t = 3 months; 700× of magnification), (b) zoom of a (5000× of magnification), (c) abiotic conditions (t = 1 week; 700× of magnification), and (d) biotic conditions (t = 8 h; 5000× of magnification). Circles indicate individual cells.

bacteria inhibit the kinetics of oxide growth resulting in decreased oxide film thickness. The thinning occurs probably due to a local drop of pH, along with restricted oxygen diffusion through the biofilm. SEM micrographs of the inner surface of the pipes tested under laboratory conditions showed the development of a biofilm after 3 months of stagnation (Fig. 4a–b), where it is possible to observe that the bacteria–EPS–scales matrix compromises film homogeneity and thus inhibits the passivation of copper. The presence of a biofilm increased the heterogeneity and overall surface roughness compared with a surface tested under abiotic conditions (Fig. 4c). According to our previous studies [20,21] abiotic conditions at longer aging times lead to passivation of the metallic surface by precipitation of malachite. Fig. 4c shows that, in the absence of a biological matrix, corrosion by-products are formed using the surface features forming the pipe manufacturing process as template. That is, parallel to the direction of the flow and consistent with the direction of the pipe extrusion. The aggregation pattern observed in Fig. 4a was not observed in the biotic experiments (Fig. 4a), probably due to the heterogeneity imposed by the biofilm. In addition, Fig. 4d shows initial microbial colonization (open circles) on

Fig. 5. Dissolved oxygen measurements during water stagnation on copper pipes under biotic (pH = 6.0, [HCO− 3 ] = 1.6 mM) and abiotic conditions (pH adjusted to 6.0 ± 0.1, [HCO− 3 ] of 0, 1.6, 3.0, and 7 mM, respectively).

a new copper pipe after 8 h of stagnation. The evidence of microorganisms attached to initial corrosion films without a supportive organic matrix suggested that microbes and EPS are intimately entrained with the inorganic compounds, preventing homogeneous passivation of the surface. Previous analysis of bacterial isolates extracted from the field site revealed the presence of copper resistance genes and high copper tolerance [31] in some of the most abundant bacterial groups. These findings are consistent with the observation of microbial colonization without a supportive organic matrix (Fig. 4d). In-situ DO measurements under biotic and abiotic conditions (Fig. 5) suggested that a biofilm in the inner surface of the copper pipe induces metallic copper oxidation. In previous works [20,21] we used in-situ DO measurements in drinking water copper pipes to estimate rates of metal oxidation during stagnation in water under or without passivating conditions due to precipitation of malachite. Notorious differences have been observed in DO consumption kinetics between passivated and non-passivated surfaces where electron transfer and subsequent corrosion take place [20,21]. Normalized DO measurements tests show that DO consumption rates are similar in biotic and abiotic tests with negligible dissolved carbonate concentration, in which no surface passivation is expected. Indeed in abiotic tests with 0 mM HCO− 3 and the biotic test with 1.6 mM HCO− 3 , where a biofilm–mineral matrix is formed (Fig. 2) instead of passivation, DO is depleted between 40 and 50 h, following a zero-order kinetic rate law [20]. On the other hand, in abiotic tests with higher carbonate concentrations (where a passive film is expected to form), DO is depleted after 70 h following a firstorder kinetic rate law [20]. According to our estimations the consumption of DO due to microbial respiration is negligible in biotic tests with 1.5 mg/L of dissolved organic carbon (DOC). Hence, the physical heterogeneity and increase in surface roughness that a biofilm imposes on the inner surface of the pipe may prevent passivation (Fig. 4a–b). We propose that the measurement of DO consumption is a helpful nondestructive test to evaluate changes on surface passivation due to the establishment of a microbial biofilm in copper pipes. This test could be used as an alternative or complementary tool to electrochemical experiments in drinking water piping systems. Corrosion rate experiments conducted on the coupons suggested that the age of the biofilm is related with its effect on corrosion. In short-term experiments new coupons (NC) showed a corrosion rate of

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(a) Short-term experiments (20 days) Corosion rate (mpy)

433

246 157

New coupon

After 20 days of treatment

After 20 days of treatment (sterilized)

on corrosion rates for both tested conditions (NC = 420 ± 20 mpy, BC = 147 ± 47 mpy, AC = 29 ± 0 mpy) as shown in Fig. 6b. However, differences in corrosion rates after 150 days suggested that the presence of a microbial biofilm decreased surface passivation. Hence, the age of the biofilm determines its effects on copper corrosion. Observed differences in the initial corrosion rates of NC between short and long-term experiments could be explained due to atmospheric corrosion during storage time. Differences between the biofilm community structures of field pipes with an operation of more than 1 year and pipes tested in the laboratory for 8 weeks (using same water as inoculum and feed) suggested microbial changes and probably ecological succession over time [7]. These microbial community dynamics probably explain the different corrosion rates observed in the coupons. While colonization and early biofilm formation may prevent corrosion, a mature biofilm could actually induce it.

(b) 420

Long-term experiments (150 days) Corosion rate (mpy)

147

29

New coupon

After 150 days of treatment

After 150 days of treatment (sterilized)

Fig. 6. Corrosion rates estimated by linear polarization in pretreated copper coupons (99.99% purity, 2.5 cm2 surface area) aged in water extracted from the study location. (a) Short-term experiments (t = 20 days of aging). (b) Long-term experiments (t = 150 days of aging). Experimental condition: agitation = 25 rpm; temperature = 25 °C.

246 ± 7 mpy (miles per year), after 20 days of aging while under biotic conditions (BC) the corrosion rate decreased to 157 ± 18 mpy, and under (AC) abiotic conditions (autoclaved water) the corrosion rate reaches 433 ± 73 mpy (Fig. 6a). These results are in agreement with our previous observations of copper release in drinking water copper pipes [7], where, after 3 weeks, passivation seems to control copper release. In contrast, the long-term experiments showed a decrease

3.5. Particle detachment Under flow conditions the bulk water velocity field imposes mechanical stress on the pipe surface, mainly shear, due to the viscosity of the water [13]. When shear forces exceed the mechanical resistance of the material, detachment of biofilm and corrosion by-products from the surface can occur. Sloughing of corrosion scales enhances copper release into the bulk water. Although studies have reported a correlation between hydrodynamics and metal release from metallic surfaces, this has focused mostly on mechanical abrasion under flow conditions [32]. Recent abiotic laboratory tests in copper pipes [22] showed the detachment of nano and micro-particles of malachite (0.05–0.2 μm), induced by the flow shear stress, increasing the mass of copper released into the water. Under abiotic conditions, the mass of copper related to nano-particles was estimated in 0.93 mg (for 4.5 L of flushed water), explaining the difference between measurements of total and dissolved copper. For biotic conditions, we expect the physical heterogeneity of the biofilm to facilitate the detachment of protective corrosion by-products films. Fig. 7 shows micrographs of a 1 year old pipe affected by MIC (Fig. 7a) [33] and of detached particles released from the pipe surface, including micro-particles (N0.45 μm) (Fig. 7b) and nano-particles (Fig. 7c) captured from the bulk water. Laboratory flushing experiments using biotic and abiotic conditions showed that the mass of copper released into the water due to flowing water is higher when biofilms are present (Fig. 8). Experiments were done with a flow rate of 0.24 L/min, and a total flushing volume of 6 L. The mass of copper released with the first 600 mL of flushed water is

Fig. 7. Electron microscopy micrographs of solid by-products from a 1 year old copper pipe affected by MIC. (a) SEM of the pipe surface (500× of magnification), (b) SEM of a corrosion by-product particle (10,000× of magnification), (c) TEM of corrosion by-product particles N0.45 μm.

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directly transfer electrons to and from electrodes respectively. Hence, the mechanisms and the proposed conceptual model presented here are required to model MIC in copper drinking plumbing systems under flow–stagnation cycles.

4. Conclusions

Fig. 8. Mass of total copper released from the pipe during flushing. Solid line: biotic experiments (field water), dashed line: abiotic experiments (synthetic water). Conditions: aging time = 4 weeks; stagnation time (previous to the flow) = 8 h; flow rate = 0.24 L/min; temperature = 25 °C.

similar under biotic (0.062 mg of Cu) and abiotic (0.058 mg of Cu) conditions. However, after this initial flushing volume (i.e., from 0.6 L to 6 L) there is a significant difference in the mass of copper released under biotic (0.0361 mg of Cu) and abiotic (0.0191 mg of Cu) conditions. Although the mass of copper released in the flushing experiments cannot be directly linked to particle detachment, it is reasonable to assume that during the flushing test (approximately 25 min) the copper release due to other mechanisms, such as metallic copper oxidation or scale dissolution, is negligible. Thus, our results indicated that biofilm growth on the surface enhances release of copper due to detachment of corrosion by-products. 3.6. Conceptual model The integration of the revised mechanisms related with MIC of copper pipes and the release of copper into water are presented as a conceptual model in Fig. 9. This conceptual model includes (i) the development of microenvironments and the formation of anodes and cathodes across the corroding pipe inner surface, and (ii) sorption of copper ions on the biofilm and/or mineralization on the EPS–microbial matrix, forming a heterogeneous surface that prevents passivation, and facilitates detachment of copper particles under flow conditions. The presented conceptual model does not include microbial mechanisms of direct electron up-take from solid and dissolved metal phases, which have been observed for iron and carbon but not for copper. For example, iron-respiring [34], and iron-oxidizing [35] bacteria

This study points out that the conventional MIC theoretical concept of a microorganisms–EPS matrix spatially separated from the corrosion by-products is not adequate to understand the effect of biofilms on the corrosion processes and release of copper into the bulk water in plumbing systems under flow–stagnation cycles. Microscopic observations, and analyses of samples extracted from an actual copper piping system affected by MIC suggested that the presence of a biofilm plays an important role on the morphology and reactivity of the surface (i.e. solid phase replacement, sorption, transport of chemical species). Our experimental results confirmed that biofilms increase the release of copper into the water, mainly due to corrosion and particle detachment. The evidence presented in this work emphasizes the idea that biofilm reactivity is not only associated with a single mechanism (e.g. sorption and desorption of labile copper). The overall effect of a biofilm involved in MIC events, with the consequent enhancement of copper concentration in drinking water, is the sum of several mechanisms that require an interdisciplinary view, integrating aspects of analytical chemistry, water chemistry, electrochemistry, microbiology, hydrodynamics, and mathematical modeling. Therefore, in order to study such complex phenomena and elaborate a model for practical uses, it is necessary to combine a number of experimental techniques that allow verifying the individual mechanisms and interaction between them. Some of the techniques are established instruments of the above-mentioned disciplines but new techniques are also developed, as is the case of the here presented monitoring of DO consumption that may serve as a non-destructive alternative to the conventional electrochemical approach.

Acknowledgments This research was funded by FONDECYT projects 1110440/2011 and 11110112/2011. This paper was presented under project CONICYT/ FONDAP/15110020. Special thanks to Katherine Lizama for helping with the TEM sample preparation, José Tomás Corthon, Max Felis, and Sara Acevedo for laboratory work and conducting electrochemical analysis. The authors wish to acknowledge Dr. Bernardo Gonzalez for his valuable advice on microbiological analysis.

Fig. 9. MIC conceptual model including the five corrosion mechanisms discussed in this article.

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