Study of biofilm influenced corrosion on cast iron pipes in reclaimed water

Accepted Manuscript Title: Study of biofilm influenced corrosion on cast iron pipes in reclaimed water Author: Haiya Zhang Yimei Tian Jianmei Wan Peng Zhao PII: DOI: Reference:

S0169-4332(15)02112-1 http://dx.doi.org/doi:10.1016/j.apsusc.2015.09.021 APSUSC 31235

To appear in:

APSUSC

Received date: Revised date: Accepted date:

15-4-2015 31-8-2015 1-9-2015

Please cite this article as: H. Zhang, Study of biofilm influenced corrosion on cast iron pipes in reclaimed water, Applied Surface Science (2015), http://dx.doi.org/10.1016/j.apsusc.2015.09.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights

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Compared to sterile water, biofilm in reclaimed water promoted corrosion process

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significantly.

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Corrosion rate was accelerated by the biofilm in the first 7 days but was inhibited

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afterward.

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There was an inverse correlation between the biofilm thickness and general

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corrosion rate.

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Corrosion process was influenced by bacteria, EPS and corrosion products

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comprehensively.

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The corrosion process can be divided into three different stages in our study.

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Study of biofilm influenced corrosion on cast iron pipes in reclaimed water

Haiya Zhang, Yimei Tian, Jianmei Wan, Peng Zhao*

Department of Environmental Engineering, School of Environmental Science and Engineering, Tianjin University, Tianjin, China

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*Correspondence author:

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Peng Zhao, Department of Environmental Engineering, School of Environmental

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Science and Engineering, Tianjin University, Tianjin, 300072, China; Tel. +86 22

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27408298; Fax: +86 22 27408298; E-mail: [email protected]

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Other e-mail addresses in order: [email protected]; [email protected];

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[email protected]

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Abstract: Biofilm influenced corrosion on cast iron pipes in reclaimed water was

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systemically studied using the weight loss method and electrochemical impedance

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spectroscopy (EIS). The results demonstrated that compared to sterile water, the

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existence of the biofilm in reclaimed water promoted the corrosion process

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significantly. The characteristics of biofilm on cast iron coupons were examined by

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the surface profiler, scanning electron microscopy (SEM) and energy dispersive

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spectroscopy (EDS). The bacterial counts in the biofilm were determined using the

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standard plate count method and the most probable number (MPN). The results

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demonstrated that the corrosion process was influenced by the settled bacteria, EPS,

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and corrosion products in the biofilm comprehensively. But, the corrosion

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mechanisms were different with respect to time and could be divided into three stages

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in our study. Furthermore, several corresponding corrosion mechanisms were

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proposed for different immersion times.

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Keywords: biofilm influenced corrosion; settled bacteria; EPS; corrosion products;

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cast iron pipes; reclaimed water

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1. Introduction Population growth, industrialization and urbanization all increase the water

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demand and aggravate water resource crisis, thus the reclaimed water emerge at the

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right moment to alleviate the dilemma [1]. One problem facing reclaiming water

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compared to potable water is its complexity, which causes the growth of

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microorganisms on the pipe surface over a short period of time [2,3]. Generally,

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microorganisms attach themselves to the surface of pipes, colonize, proliferate and

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finally form a biofilm, which tends to affect the kinetics of the cathodic and anodic

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corrosion process [4-6]. Consequently, although the final reclaimed water meets

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regulatory standards at treatment plants, biofilm influenced corrosion in reclaimed

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water distribution systems may cause a deterioration of water quality [7,8], especially

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when iron pipes are used [9,10].

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A large number of studies have reported microbiologically influenced corrosion

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(MIC) on iron, copper, aluminum and their alloys in the presence of pure or mixed

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culture bacteria [11]. Several types of bacteria have been found to accelerate the

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corrosion process [12-16], while others can inhibit corrosion [17,18]. However, MIC

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is not a distinct type of corrosion form, rather, MIC is the synergistic interaction

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between the metal surface, abiotic corrosion products, and bacterial cells and their

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metabolities [11,19]. X. X. Sheng et al. [20] reported that the biofilm induced by

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different sulfate-reducing bacteria (SRB) strains showed a different morphology and

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polarization resistance, which had a direct role on the corrosion process. In addition,

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extracellular polymeric substance (EPS) was also considered to be one of the factors

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causing the metal corrosion process. J. T. Jin et al. [9] and Q. Bao et al. [21] also

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reported that to a certain extent, EPS is able to protect the substrate from corrosion by

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forming a protective film and minimize the cathodic reduction of oxygen. Meanwhile,

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corrosion products, which were essentially a type of extracellular biomineralization,

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can also change the corrosion potential in either a positive or a negative direction

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[22,23]. Based on these results, several mechanisms involving sulfate-reducing

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bacteria biofilm induced corrosion have been proposed, which include but are not

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limited to the cathodic depolarization theory, formation of iron sulfides in corrosion

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products, the existence of inorganic and organic sulfides, the presence of a densely

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packed EPS film, but mainly in sea water [15,20,24,25,26]. Only a small number of

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studies have been conducted to investigate aerobic biofilm influenced corrosion

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[18,27,28]. However, to the best of our knowledge, most of these studies provide no

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insight into the mixed species biofilm influenced corrosion with the combination of

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aerobic and anaerobic bacteria, especially in reclaimed water.

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The present study was designed under laboratory conditions to elucidate the

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mixed species biofilm (IOB and SRB) influenced corrosion on cast iron pipes in

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reclaimed water. As a control experiment, corrosion analyses were also performed on

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samples in sterile water. The major objectives of this work were to investigate (1) the

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interfacial evolution of the biofilm characteristics with respect to time, as analyzed by

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the surface profiler and SEM/EDS; (2) the corrosion process by comparing reclaimed

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and sterile water using the weight loss method and EIS; (3) whether the biofilm in

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reclaimed water promoted or inhibited the corrosion process for cast iron pipes. In

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addition, the bacterial counts in the biofilm were determined using the standard plate

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count method and the most probable number (MPN). Furthermore, several corrosion

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mechanisms were proposed between the corrosion process and the biofilm in

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reclaimed water respect to time.

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2. Materials and Methods

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2.1 Materials and preparation

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The cast iron coupons used for our study were purchased from Yangzhou

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Xiangwei machinery manufacturing Co. Ltd. The coupons were manufactured

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according to HG/T 3523-2008 [29]. The dimensions for each coupon were length ×

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width × thickness= (50.0±0.1) mm × (25.0±0.1) mm × (2.0±0.1) mm and with an

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exposed surface area 28.00 cm2. The composition of the cast iron coupons were C

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19.08%, O 6.09%, Si 2.06%, Ca 0.58%, P 0.65%, S 1.60%, Fe 65%, Cu 1.98%, Mn

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0.92%, and Zn 2.04%. Prior to the experiment, the coupons were cut into square

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shapes with a length of 0.8 cm. To remove the uncertainty of nanometer-scale surface

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roughness [30], the coupons were sequentially ground using a series of waterproof

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abrasive papers (200 #, 400 #, 600 #, 800 #, 1000 #, 1200 # and 1500 #). The polished

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coupons were degreased in acetone, dehydrated in absolute ethanol, and dried in a

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laminar flow cabinet. Subsequently, the coupons were stored in a drying cabinet and

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exposed to UV light for 30 min before use.

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2.2 Water quality analysis and bacteria preparation The influent water was taken from the effluent of the wastewater treatment plant

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in northern China. The water quality parameters are shown in (Supporting

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Information 1). The main technological processes (Supporting Information 2) in the

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reclaimed water treatment plant were Coagulation and Sedimentation, CMF

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(Continuous Micro-Filtration) and RO (Reverse Osmosis). The effluent water quality

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parameters are summarized in Table 1. No significant changes occurred in the

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reclaimed water quality during the entire experiment.

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effluent water

pH

6.77

BOD5 (mg/L)

0.31

Turbidity (NTU)

0.244

effluent water

Total hardness (mg/L)

241.5

TP (mg/L)

0.8

NH3-N (mg/L)

5.00

<1

TDS (mg/L)

866

77.1

Chloride (mg/L)

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Total alkalinity (mg/L)

water quality parameters

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Table 1 Average water quality parameter of the effluent water

SS (mg/L)

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Due to the high concentration of chlorine in the disinfection tank, bacteria in the

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effluent water were barely detected. Therefore, a simulated flowing reclaimed water

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distribution system was established in the laboratory to enrich the bacterial content in

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reclaimed water.

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Unsterilized effluent water from the simulated reclaimed water distribution

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system was used on one group of samples to investigate the development and effect of

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biofilm on the coupon corrosion. The effluent water exposed to ultraviolet light (180

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w/cm2) for 30 min was used in a second group as the control sterile water experiment.

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After sterilization, the total bacterial count was <1 cfu/ml in the control sterile water.

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Other water quality parameters remained the same, as shown in Table 1. The sterile

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water was regularly replaced to maintain the aseptic condition.

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2.3 Bacteria cultivation and corrosion tests

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The cast iron coupons were immersed into the two types of water: (1) the control

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sterile water and (2) the reclaimed water rich in microorganisms (in which biofilm can

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grow on the cast iron coupons). All tests were conducted using a nutrient-rich

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medium. The medium had the following composition per liter of reclaimed water

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(sterile water): 5.0 g NaCl, 3.0 g beef extract and 10.0 g peptone. The pH was

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adjusted to 7.0 using 1 M NaOH and the solution was autoclaved at 121℃ for 20

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min.

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Next, 250 ml ground glass stoppered bottles containing the two types of water

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were incubated at 30℃ in a biochemical incubator to start the corrosion test. The

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bottles used in our study were not transparent to natural light, which simulated a dark

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environment in the reclaimed water distribution system. In all batch experiments, the

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cast iron coupons were sampled at predetermined cultivation times of 3, 7, 10, 15, 20,

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25 and 30 days. Each sample was examined for general corrosion rate test. The

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sampling process was kept under a strict aseptic environment such that the other

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bacteria would remain isolated from the experiment.

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2.4 Biofilm characteristics analysis by surface profiler and SEM/EDS

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The biofilm thickness and roughness of the cast iron coupons were measured by

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the D-100 surface profiler (KLA-Tencor Company, USA). The surface morphology

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and elemental composition of the biofilm were examined by the S4800 field emission

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scanning electron microscopy (Hitachi Company, Japan), which was equipped with

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the Genesis XM2 Electron Energy Disperse Spectroscopy (EDAX Company, USA).

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Surface analysis was performed on the representative 3, 7, 10, and 15 day samples.

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The coupons with biofilm were prepared for surface analysis using the following

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procedure: the coupons were gently washed twice with sterile deionized water; fixed

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with a 2.5 vol % Glutaradehyde fixation fluid for 2 h at 4

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washed for the second time with the Phosphate Buffer solution (PBS) and aseptic

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water; separately dehydrated for 10 min using grade ethanol (25%, 50%, 75%, 100%).

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Finally, the coupons were dried overnight in a vacuum freeze drier and coated with

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platinum before observed by SEM/EDS.

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in a refrigerator; then

The phosphate buffer solution was prepared by dissolving NaCl 7.9 g, KCl 0.2 g,

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KH2PO4 0.24 g and K2HPO4 1.8 g into 800 ml distilled water. The pH of solution was

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adjusted to 7.4 with hydrochloric acid solution. The solution was subsequently settled

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to 1 L with distilled water and stored in a refrigerator at 4 ℃ for later use.

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2.5 Bacterial counts in the biofilm

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The cast iron coupons obtained in section 2.3 were washed gently with the

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aseptic water. Then, the coupons were washed for 20 min in an Ultrasonic Cleaning

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Systems with 0.01 M phosphate buffer solution (PBS) to obtain the biofilm bacteria

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solution. The frequency of the Ultrasonic Cleaning Systems was 40 KHz. The 1 mL 8

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sample of the biofilm bacteria solution was used to statistically determine the total

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number of bacteria by the plate count method [31]. The number of iron bacteria and

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sulfate reducing bacteria was determined by the Maximum Probable Number (MPN)

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[32,33].

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The term “iron bacteria” refers to the iron oxidizing bacteria, which can survive

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through the iron oxidation process. The Maximum Probable Number on the iron

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bacteria was determined using the following steps: MgSO4 0.5 g, (NH4)2SO4 0.5 g,

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Na2HPO4 0.5 g, CaCl2 0.2 g, NaNO3 0.5 g, and FeC6H5O7.NH4OH;C6H10FeNO8 10.0

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g were dissolved into 1000 ml distilled water to prepare the iron bacteria culture

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medium. The pH of the solution was adjusted to 6.8±0.2 with hydrochloric acid or

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sodium hydroxide solutions. Next, 5 ml of solution was transferred to a sterilized

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glass tube, wrapped in craft paper and sterilized at (121±1)℃ for 15 min. The 1 mL

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biofilm bacteria solution was diluted with distilled water to different multiples from

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10-1 to 10-7. Each sample was added to a glass tube with culture medium and then

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incubated at (29±1)℃ for 14 days to enumerate counts when the original medium

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disappear into a transparent color with the presence of brown and black deposits.

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The Maximum Probable Number on the sulfate reducing bacteria are as follows:

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K2HPO4 0.5 g, NH4Cl 1.0 g, Na2SO4 0.5 g, CaCl2 0.1 g, MgSO4 2.0 g, C3H5NaO3 3.5 g

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and Lebedev juice 1.0 g were dissolved into 1000 ml distilled water to prepare the

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sulfate-reducing bacteria culture medium. The pH of solution was adjusted to 7.2±0.2

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with hydrochloric acid or sodium hydroxide solution. Next, 350 ml solution was

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transferred to a sterilized conical flask (500 ml) with a cotton stopper, wrapped in

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craft paper and sterilized at (121±1)℃ for 15 min. The 1 mL biofilm bacteria solution

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was diluted with distilled water to different multiples from 10-1 to 10-7. Each sample

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was added into a conical flask with the culture medium and incubated at (29±1) ℃ for

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21 days to enumerate counts when black deposits and hydrogen sulfide odor was

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detected.

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2.6 General corrosion rate measurement

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The weight loss method was utilized to determine the general corrosion rate of

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the coupons in the reclaimed and control sterile water respectively. The weight of the

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cast iron coupons was measured at fixed days (as explained in section 2.3) using a

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high accuracy mass balance (Sartorius, Germany, readability 0.01 mg). The coupons

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were washed with the Ultrasonic Cleaning Systems to remove the biofilm and

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corrosion products on the coupon surface with the cleaning liquid of 10%

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hydrochloric acid and 0.5% Hexamethylenetetramine. After that, the coupons were

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briefly dehydrated twice with absolute ethylalcohol, and then passivated for a moment

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with 5N (200 g/L) NaOH solution, dried with filter paper, wrapped and stored in a

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drying cabinet. The samples were weighed after 24 h and the weight loss could be got.

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The corresponding corrosion rate was calculated using the formula below [34]:

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corrosion rate（mm/year）= 87600 ÷×△W / ( A × ρ × T )

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where △W is the weight loss of the cast iron coupons in mg, A is the surface area of

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the cast iron coupons in cm2 (the value in this study was 28 cm2), ρ is the density of

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the test coupons in kg/m3 (the value in this study was 7.30 kg/m3), and T is the

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corrosion time in hours.

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2.7 Electrochemical impedance spectroscopy (EIS) measurements

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Electrochemical impedance spectroscopy measurements were conducted in a

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conventional three-electrode glass corrosion cell with a capacity of 250 mL. A

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platinum gauze electrode was used as the counter electrode (CE), an Ag/ (AgCl) / KCl

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(0.1 mol/L) as the reference electrode (RE), and a cast iron coupon was used as the

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working electrode (WE).

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The procedure for preparing the working electrodes was as follows: the cast iron

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coupons were cut into cylinders with a diameter of 8 mm, thickness of 6 mm and

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working area of 0.5 cm2. The cylinder was subsequently polished to remove oil and

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rust, welded with a metal wire (the wire was inserted into the prepared PVC pipe) and

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finally, sealed with epoxy resin. The electrode was successively sanded with abrasive

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water in 200 #, 400 #, 600 #, 800 #, 1000 #,1200 # and 1500 #, buffed by polishing

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powder, steeped in acetone to remove oil, hydrated in absolute ethanol, and dried for

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use. The working electrodes were exposed to UV light for 30 min before being used.

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A CS350 electrochemical working station (KeSiTe, WuHan, China) was used to

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measure the electrochemical impedance spectroscopy at 30 . The EIS measurements 11

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were conducted by applying a 5 mV sinusoidal perturbation ranging from 0.01 Hz to

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100000 Hz. The experimental data were analyzed by the Origin8.1 software. The

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Zsimpwin software was employed to fit the EIS data using the equivalent circuit

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model. All experiments were performed in duplicate.

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3. Results and Discussion

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3.1 Biofilm thickness and roughness analysis

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Biofilm thickness on the cast iron coupons Biofilm roughness on the cast iron coupons

40000

20000

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biofilm roughness

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30000

0

5

10

20000

15000 (A)

Biofilm thickness

10000 20000

10000

15000 20000

30000

40000

50000

5000

10000

5000

0

0

0

15

25000

20

0

25

Biofilm roughness on the cast iron coupons( A)

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Biofilm thickness on the cast iron coupons( A)

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30

Immersion time(days)

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Fig. 1 Biofilm thickness and roughness formed on the cast iron coupons at different times

In our study, the biofilm on the cast iron coupons was composed of settled

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bacterial cells (including iron oxidizing bacteria and sulfate reducing bacteria),

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extracellular polymeric substance (EPS) and corrosion products [35]. The thickness

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and roughness of the biofilm attached to the cast iron coupons were found to increase

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with immersion time for the whole experiment process as shown in Fig. 1. The two 12

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variations were almost the same, which exhibited faster growth at an earlier stage and

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slower growth at a later period. The thickness of the biofilm increased significantly due to bacterial propagation

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and the accumulation of corrosion products and metabolic products on the cast iron

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surface. On average, the biofilm thickness increased from 266 A to 42570 A during its

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development from 1 to 30 days, which corresponded to a drastic increase during the

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first 15 days. It is known that, in an undisturbed environment, autogenic biofilm

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adhesion process appeared relatively slow in the end [36, 37], which can explain the

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lower biofilm thichkness growth as observed after 15 days of colonization.

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The initial biofilm roughness was 108 A, and it increased gradually to 23062 A

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by 20 day. The roughness later decreased rapidly to 17148 A by 30 day. Initially, the

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individual bacterial cells and bacterial colonies gave rise to the biofilm roughness on

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the cast iron surface (Fig. 3a, 3b). Subsequently, as the bacteria colonized, the

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individual bacterial cells would have most likely embedded with the metabolic

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products and smooth EPS matrix (Fig. 3d) [38], resulting in a sharp decline in biofilm

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roughness after 20 days.

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In our experiments, a significant linear relationship between biofilm thickness

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and biofilm roughness was found during the first 15 days (r=0.992, p=0.001), but the

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relationship between them was more tenuous after 15 days. This finding demonstrated

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that the biofilm roughness was not necessarily associated with the biofilm thickness

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and that the biofilm absorbed onto the cast iron coupons was random and

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heterogeneous. Many studies have also observed the heterogeneous biofilm structure

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in the artificial seawater with the sulphate-reducing bacteria [20, 26] Here, we report

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the biofilm for such phenomenon under the combination of iron-oxidizing bacteria

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(IOB) and sulfate reducing bacteria (SRB). SEM analysis also validated this

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phenomenon.

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3.2 Bacterial counts in the biofilm

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Total bacteria Iron oxidizing bacteria Sulfate reducing bacteria

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40000

20000

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Bacterial counts(cfu/mL.cm2)

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266 267 268

5

10

15

20

25

30

Immersion time (days)

Fig. 2 Bacterial counts of the biofilm on the cast iron coupons at different times

Fig. 2 shows the total bacteria (TB), iron-oxidizing bacteria (IOB), and sulfate

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reducing bacteria (SRB) counts of the biofilm on the cast iron coupons at different

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culturing times. However, since our study was not carried out with pure culture

271

medium for IOB or SRB, the other heterotrophic bacteria might grow as well. As a

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result, the total bacteria counts were always higher than the sum of the IOB counts

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and SRB counts at any time.

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The initial counts of the total bacteria settled on the cast iron surface was 26980 14

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(cfu/mL·cm2) at 3 day, and it increased gradually to a maximum of 115000

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(cfu/mL·cm2) by 15 day. A significant reduction was observed after 15 days, and

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afterward, and the total bacterial counts decreased gradually to 33800 (cfu/mL·cm2)

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by 30 day due to the limition of nutrient. Typically, as observed in most experiments

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performed in limited culture medium at laboratory scale, bacterial counts colonize

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with a high biomass logarithmic phase, followed by an aging phase later [39,40].

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Iron-oxidizing bacteria (IOB) is a type of aerobic bacteria, and its growth

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depends strongly on dissolved oxygen and the matrix material [4], which can obtain

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life activities for growth through the iron oxidation process. The initial IOB counts

284

was 13600 (cfu/mL·cm2) at 3 day, which was 15 times higher than that of SRB (896

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cfu/mL·cm2). This means that at the start of the test, the iron donor supplied from the

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oxidation of iron is more likely to help stimulate the growth of IOB than that of SRB

287

in reclaimed water [41]. The relatively higher DO concentration (>0.3 mg/L) before 7

288

day (Supporting Information 3) also provided a favorable condition for the growth of

289

IOB. The IOB counts increased to the maximum 16800 (cfu/mL·cm2) at 7 day. The

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respiration of iron-oxidizing bacteria on cast iron coupons was shown by Eq.(1),

291

Eq.(2) and Eq.(3) [2].

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O2 + 2H2O + 4e- → 4OH-

(1)

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2Fe → 4e- + 2Fe2+

(2)

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4Fe + 6H2O + 3O2 → 4Fe(OH)3

(3)

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The response also indicates that as soon as the iron is oxidized to iron ion by the

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IOB, it might be converted to the fixed forms of dense precipitates (Fe(OH)3) on the

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coupon surface. Subsequently, the ferric hydroxide deposited in the fibrin sheath of

298

IOB, form the dense oxide layers and corrosion tubercles on the cast iron surface [42]

299

(Fig. 3b), which restrict the access of fresh oxygen to the metal surface. Meanwhile,

300

with the activity of IOB, the available DO in the electrolyte became depleted to as low

301

as 0.2mg/L after 7 days (Supporting Information 3), almost reaching anaerobic

302

conditions. All these inhibited the aerobic activity of IOB, but provided better

303

conditions for the anaerobic SRB metabolism [43], leading to the decrease of the

304

aerobic IOB counts and the increase of anaerobic SRB counts [44] after 7 days. At

305

this time, SRB obtains energy for growth and cell synthesis by oxidizing molecular

306

hydrogen (anode) and reducing sulfate to sulfide (cathode) beneath the biofilm [45],

307

as shown by Eq.(4), Eq.(5) and Eq.(6) [20]. Also, with the increase of the biofilm

308

thickness (Fig.1), the inside of the dense biofilm layers become more anaerobic and

309

more beneficial for the growth of SRB. Then, the SRB counts showed a faster upward

310

trend and reached a maximum of 13500 (cfu/mL·cm2) by 15 day. Afterward, with the

311

excessive consumption of sulfate in reclaimed water [46], the SRB counts decreased

312

dramatically to 10100 (cfu/mL·cm2) after 30 days of incubation.

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Fe → 2e- + Fe2+

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SO42-+8H→S2-+4H2O

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Fe2+ + S2- → FeS

(4) (5) (6)

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3.3.Morphology structure of the biofilm (SEM Result)

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Fig. 3a SEM results of the biofilm on the cast iron coupons at 3 days

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Fig. 3b SEM results of the biofilm on the cast iron coupons at 7 days

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Fig. 3c SEM results of the biofilm on the cast iron coupons at 10 days

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Fig. 3d SEM results of the biofilm on the cast iron coupons at 15 days

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Fig. 3 Scanning electron microscopy (SEM) micrographs of the biofilm formed on the

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cast iron coupons at 3a: 3 days, 3b: 7 days, 3c: 10 days, 3d: 15 days

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Fig. 3a-3d show the representative SEM micrographs of the biofilm on the cast

332

iron coupons at different times. The scanning ranges of the electron microscope were

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20 µm, 10 µm and 5µm. The results revealed that the development of the biofilm on

334

the cast iron surface was a dynamic process. Samples taken from the reclaimed water

335

after an immersion period of 3 days still exhibited uncovered surface areas that were

336

clearly distinguishable from the biofilm-covered areas (Fig. 3a). In addition to the

337

bare cast iron substrate, we could see that the typical individual ellipsoidal iron-

338

oxidizing bacteria cells (about 1μm) randomly aggregated on the cast iron surface

339

with a coverage nearly 70%, which finally resulted in the formation of small corrosion 19

Page 19 of 43

tubercles by Eq.(3) [42], but without continuous biofilm. The typical rod-shaped SRB

341

cells were hardly been found, indicating that SRB were not jet significantly growth on

342

the surface at this time. The whole biofilm structure was heterogeneous and porous,

343

which may not be protective to the metal.

ip t

340

Then, with the colonization and propagation of iron bacteria (Fig. 2), metabolic

345

products and corrosion tubercles accumulated gradually on the cast iron surface. After

346

being immersed in reclaimed water for 7 days (Fig. 3b), deposits covering the cast

347

iron surfaces, had a layered structure. In addition to the typical ellipsoidal IOB cells,

348

the ferric hydroxide deposited in the fibrin sheath of IOB, formed the dense upper

349

oxide layers. Among the sedimentation of the upper layer, accumulation of rod-shaped

350

SRB cells deposited in the plain, thick surface were observed in the inner layer. The

351

inner deposits were further identified by EDS as ferric sulfide. Iron sulfide deposits

352

are indeed often found inside the iron-rich corrosion products by IOB [47]. Finally,

353

the substrate of the cast iron could hardly be seen and the whole surfaces were

354

covered completely by the dense, nonuniform layer of deposits.

us

an

M

d

Ac ce pt e

355

cr

344

After being immersed in reclaimed water for 10 days, as displayed in Fig. 3c, the

356

individual bacterial cells and corrosion products could not clearly visible on the

357

surface, which were enclosed with the biofilm matrix of bacteria metabolism. The

358

sheaths of IOB assist in the formation of biofim matrix that is relatively impervious

359

[42]. And, the large downy globular cluster with exopolysaccharide fibers became the

360

main morphology on the cast iron surface. The biofilm structure presented a

20

Page 20 of 43

361

snowflake shape. Many studies by SEM of the iron corrosion products confirmed the

362

principal structure with globular floccules morphologies [3]. Furthermore, in the rapid growth phase (1-15 day) as shown in Fig. 2, an

364

abundance of EPS was produced with the vigorously metabolizing of bacteria, which

365

adhered onto the metal surface strongly and formed a densely packed film [21]. As a

366

result, the bacterial cells and corrosion products were hardly be seen, which were

367

wrapped with this thick, slimy, like a jelly glue EPS. The EPS film presented a

368

double-layered structure. The tightly bound EPS located in the inner layer, which

369

combined closely with the cast iron surface, and, the loosely bound EPS located in the

370

outer layer, which was amorphous and has a relatively unconfined structure [48].

371

3.4 Elemental composition of the biofilm

cr

us

an

M

d

Table 2 Elemental composition ratio of the biofilm on cast iron coupon surface

Ac ce pt e

372

ip t

363

Weight %

Immersion times (days)

373

C

O

Na

Al

S

Cl

Ca

Fe

Au

0

5.41

2.33

-

0.67

0.32

-

-

86.86

6.74

3

5.48

17.79

1.84

0.29

0.00

0.00

0.12

47.57

22.94

7

12.08

17.44

1.73

0.29

3.08

0.00

-

32.17

26.50

10

12.50

26.82

2.86

0.00

3.29

0.26

0.27

37.15

-

15

38.24

11.52

1.50

-

11.63

0.00

0.30

36.80

-

EDS was used to obtain the elemental composition of the biofilm, which

374

deposited on the cast iron coupon surfaces. The results at the 3, 7, 10 and 15 day are

375

shown in Table 2. It is important to point out that the small peaks of Na and Cl were

376

stemed from the culture medium and the Au peaks was related with the pretreatment

377

process before SEM/EDS. 21

Page 21 of 43

In the scrapings of the cast iron coupon, the relative iron amounting to 86.86

379

wt%, carbon content to 5.41 wt% and oxygen content to 2.33 wt% are the major

380

constituents at 0 day. However, the deposits collected from the cast iron surface has

381

undergone great changes in our study. As the increase of total bacterial counts at 3

382

day, the accumulation of bacterial cells and the metabolic by-products [22] on the

383

surface gave rise to the relative content of carbon (5.48 wt%) and oxygen. Also, the

384

respiratory activity of iron bacteria during their proliferation promoted the formation

385

of ferric hydroxide. As a result, the relative oxygen content of the biofilm increased

386

sharply to 17.19 wt% at 3 day. Thus, compared to the cast iron matrix, the relative

387

iron content decreased rapidly to 47.57 wt% at 3 day due to the deposits of bacterial

388

cells and corrosion oxides.

M

an

us

cr

ip t

378

Afterwards, as the gradually accumulation of ferric hydroxide, together with the

390

bacterial cells (Fig. 2) and the metabolic by-products (Fig. 3c), the relative oxygen

391

content increased to a maximum of 26.82 wt% by the 10 day. Meanwhile, the

392

elemental sulfur of the biofilm began to appear at the 7 day with a relative content of

393

3.08 wt%, which could be an evidence of the formation of FeS in the biofilm [15,26].

394

It also indicated that the relatively high SRB activity (Fig. 2) in the anaerobic

395

conditions beneath the biofilm may significantly participate in the deposit formation

396

[49], producing large amount of ferric sulfide at 7 day. Subsequently, with the

397

proliferation of SRB, the relative sulfur content increased to 3.29 wt% at 10 day.

398

Further, as the progressively increased SRB counts at 15 day, the excess generation of

Ac ce pt e

d

389

22

Page 22 of 43

H2S can migrate to the edges of ferric hydroxide where the original corrosion

400

products (Fe(OH)3) may be reduced to ferrous iron compounds as shown by Eq.(7)

401

[44]. This can be used to explain the decrease of oxygen content in the biofilm after

402

10 days. And, in the environment containing large amounts of H2S, the composition of

403

the corrosion products can transformed from FeS to other polysulfides as shown by

404

Eq.(8). The ratio of S/Fe increased from the 8.86 % at the 10 day to the maximum

405

(31.6 %) at the 15 day also confirmed the formation of the polysulfides. All these

406

revealed that mineral species of corrosion products can transform from one form to

407

another either abiogenically or biogenically [22].

an

us

cr

ip t

399

408

3H2S+2Fe(OH)3=Fe2S3+6H2O

409

FeS+ H2S=FeS2+2H++2e-

410

Finally, the relative carbon content of the biofilm increased to 38.24 wt% by 15

411

day, which was a result of the accumulation of abundant bacterial cells and

412

extracellular polymeric substance (EPS) on the cast iron surface (Fig. 3d). EPS are

413

composed dominantly of exopolysaccharides and proteins [50], the primary

414

component of them was C [51].

(8)

Ac ce pt e

d

M

(7)

23

Page 23 of 43

415

3.5 General corrosion rate analysis

0.25 cast iron coupons in sterile water cast iron coupons in reclaimed water

ip t

0.15 0.10

cr

v (mm/a)

0.20

0.00

us

0.05

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

an

Immersion times (days)

Fig. 4 General corrosion rate of the cast iron coupons comparing reclaimed water and sterile water

419

Fig. 4 shows the general corrosion rate trends of the cast iron coupons in

420

reclaimed water and sterile water. The general corrosion rate in reclaimed water was

421

higher than that of the sterile water at every time period. This result indicated that the

422

existence of the biofilm (containing IOB and SRB) in reclaimed water promoted the

423

general corrosion rate significantly. The increase of corrosion rate at the biofilm/metal

424

interface had been observed in the presence of sulfate reducing bacteria in several

425

studies [19,25]. About the co-existence of IOB and SRB, C. M. Xu et al. [38] reported

426

they can promoted the pitting corrosion by reducing the corrosion potential. In the

427

present study, the whole biofilm on the cast iron coupons, which was composed of

428

absorbed bacterial cells (containing IOB and SRB), extracellular polymeric substance

429

(EPS) and corrosion products also promoted the general corrosion rate.

Ac ce pt e

d

M

416 417 418

24

Page 24 of 43

As seen in Fig. 1, the biofilm thickness increased from 0 to 30 days, which was

431

less than 5μm during the whole experiment. However, the curve of the general

432

corrosion rate in reclaimed water exhibited three stages. During the period of 0-7 days

433

(Stage I), the corrosion rate increased sharply from 0.07 mm/a to 0.24 mm/a. After 7

434

days, which was Stage II (7-15 days), the corrosion rate decreased sharply from 0.24

435

mm/a to 0.06 mm/a before smoothing out. After 15 days, the corrosion rate continued

436

to decrease to 0.03 mm/a until 30 day at Stage III (15-30 days). Through the

437

above analysis, it was determined that there was an inverse correlation between the

438

biofilm thickness and general corrosion rate in our study (r=-0.891, p=0.049).

439

Nevertheless, other biofilm characteristics, such as biofilm structure, elemental

440

composition and aerobic or anaerobic bacteria might also influenced the corrosion

441

rate significantly [38]. To determine how these factors contribute to the corrosion

442

process, a systematic discussion would be necessary.

cr

us

an

M

d

Ac ce pt e

443

ip t

430

The high concentration of dissolved oxygen in reclaimed water before 7 days

444

(Supporting Information 3) can form a large cathodic area, which could activate the

445

corrosion process on the cast iron surface initially [52]. Also, the discontinuity biofilm

446

at 3 day made some of the cast iron substrate bare to the reclaimed water (Fig. 3a),

447

which create environments that are conducive to sustaining the growth of IOB and

448

subsequently promote corrosion through turning the atomic iron into ionic iron during

449

the initial stage [53]. Meanwhile, under these conditions, there will be a pronounced

450

growth of small corrosion tubercles produced from the metabolic activity of IOB at 3

25

Page 25 of 43

day (Fig. 3a). The small corrosion tubercles also plays a very important role in the

452

corrosion process [4]. Because, the difference in the potential between the iron surface

453

underneath and outside the small corrosion tubercles increase, and thus corrosion get

454

accelerated further [54]. Therefore, the general corrosion rate increased sharply to

455

0.24 mm/a at 7 day (Stage I). Another significant observation is that the peaks of

456

corrosion rate in reclaimed water coincide with the peaks of IOB counts on the cast

457

iron coupons, which occurred at 7 day in our study. Consequently, all these analysis

458

demonstrated that the increment of the settled IOB counts and the discontinuous,

459

porous biofilm structure contributed to the increased corrosion rate in 7 days together.

an

us

cr

ip t

451

Subsequently, with the accumulation of bacterial cells (IOB and SRB), metabolic

461

products and corrosion tubercles, the whole surfaces were covered completely by the

462

dense, nonuniform layer of deposits at 7 day, which could alter the microenvironment

463

at the mental surface [55]. Such changes also have an obvious consequence for the

464

corrosion process. Firstly, SRB have been documented to show aggressive corrosion

465

with iron under anaerobic conditions [15]. As the increase of SRB counts between 7

466

day to 15 day, the ferric sulfide came under observation on the inner surface at 7 day,

467

which was reported has a poor protective effect on the metal surface [16]. Even, it has

468

reported that more the ferric sulfide formation, the higher is the corrosion rate [56].

469

While, in our study, the general corrosion rate decreased from 0.24 mm/a to 0.06

470

mm/a between 7 day and 15 day (Stage II). Thus, the corrosion rate of cast iron

471

coupons can not correlate directly with the settled SRB counts (r=-0.544, p=0.634)

Ac ce pt e

d

M

460

26

Page 26 of 43

472

and the ferric sulfide, which was associated with the oxidiation of H2S induced by the

473

SRB [57]. P. Angell et al. [58] also reported that there was no direct correlation

474

between numbers of SRB and corrosion rates. In contrast to SRB, we detected that the decreased corrosion rate was positively

476

correlated with the reduced number of settled IOB counts after 7 days (r=0.952,

477

p=0.013). In addition, the dense upper oxide layers (Fig. 3b) composed of ferric

478

hydroxide and fibrin sheath of IOB at 7 day could also prevent the transport of metal

479

ions away from the surface [45], thereby inhibiting the development of corrosion

480

process further. Thus, the biofilm at this moment were in the passive state. J. Lin et al.

481

[25] and X. D. Zhao et al. [59] also reported the protective effect of the corrosion

482

products film on the steel in sea water. Furthermore, bacterial cells and corrosion

483

products enclosed with exopolysaccharide fibers (Fig. 3c) were observed on the cast

484

iron surface due to the bacterial multiplication at 10 day (Fig. 2). The

485

exopolysaccharide fibers was relatively impervious [42], and, inhibited the corrosion

486

process more. A significant negative correlation relationship was also obtained

487

between the corrosion rate and the carbon content in the biofilm here (r=-1, p<0.01).

488

Therefore, the general corrosion rate continued to decrease after 10 days. As a result,

489

we concluded that the corrosion process between 7 day and 15 day (Stage II) could be

490

dominated by the declining IOB counts as well as the dense biofilm composed of

491

corrosion products and exopolysaccharide fibers.

492

Ac ce pt e

d

M

an

us

cr

ip t

475

Later on, as the vigorously metabolize of bacteria, bacterial cells and corrosion

27

Page 27 of 43

products were covered with double-layered slimy EPS at 15 day. The tightly bound

494

EPS located in the inner layer are organized in well defined capsules around the

495

bacterial cells and corrosion products. Although specific chemical components of EPS

496

could not be obtained during the corrosion process in our study, many studies reported

497

the EPS mainly consist of exopolysaccharides and proteins with plenty of hydroxyl

498

and carboxyl groups [21,48]. J. T. Jin et al. [9] suggested a positive correlation

499

between the corrosion inhibition efficiency and the carboxyl groups on cast iron pipes

500

in reclaimed water. These analysis can demonstrate that the densely packed EPS film

501

at 15 day protected the cast iron from corrosion to a certain degree afterwards. The

502

inhibition of EPS was also reported by some previous studies [60,61]. Keeping in

503

mind that was mentioned above concerning EPS, the existence of corrosion products

504

and bacterial cells inside the biofilm on corrosion also must be considered. For long

505

immersion period (15-30 days), the metabolic activity of IOB and SRB declined a lot

506

because of the lack of nutrients (DO and sulfate ion) for life activity [62], as a result,

507

the corrosion rate decreased smoothly and tend to be a constant 0.03 mm/a until 30

508

day (Stage III). According to the analysis above, the decreased corrosion rate after 15

509

day is attributed to the synergies between the declined metabolic activity of settled

510

bacteria (IOB and SRB) and the stabilization of corrosion products beneath the

511

densely packed EPS film.

Ac ce pt e

d

M

an

us

cr

ip t

493

28

Page 28 of 43

3.6 Electrochemical impedance spectra (EIS) analysis 4000

1800

3500

1200

2

2000 1500 1000

1000 800 600 400

500 0

200 0

500

0

1000 1500 2000 2500 3000 3500 4000

200

400

600

2

Fig. 5a Nyquist plots in sterile water

Fig. 6a Nyquist plots in reclaimed water

4500

4500 3 days 7 days 9 days 15 days 20 days 23 days

3500

3500 3000

2

2

|Z|(Ohm﹒cm )

3000

4000

|Z| Ohm﹒cm )

4000

2500 2000

(

1500 1000

2500 2000 1500 1000

500

500 0.1

1

10

100

1000

an

0 0.01

800 1000 1200 1400 1600 1800

Z' (Ohm﹒ cm )

cr

513 514

0 0.01

10000 100000

f (Hz)

Fig. 5b Bode magnitude plots in sterile water

0.1

1

10

100

1000

3 days 7 days 9 days 15 days 20 days 23 days

10000 100000

f (Hz)

Fig. 6b Bode magnitude plots in reclaimed water

M

515 516

0

2

Z' (Ohm﹒ cm )

ip t

2

2500

3 days 7 days 9 days 15 days 20 days 23 days

1400 -Z''(Ohm﹒cm )

3000 -Z''(Ohm﹒cm )

1600

3 days 7 days 9 days 15 days 20 days 23 days

us

512

1.60

1.570

1.55

1.565

o

d

1.550 1.545

3 days 7 days 9 days 15 days 20 days 23 days

1.540 1.535 1.530

0.01

517 518 519 520

phase angle( )

1.50

1.555

Ac ce pt e

o

phase angle( )

1.560

0.1

1

10

100

1000

1.40

3 days 7 days 9 days 15 days 20 days 23 days

1.35 1.30

10000 100000

0.01

f (Hz)

Fig. 5c Bode phase angle plots in sterile water

1.45

0.1

1

10

100

1000

10000 100000

f (Hz)

Fig. 6c Bode phase angle plots in reclaimed water

Fig. 5 and Fig. 6 respectively show the Nyquist plots and Bode plots of the cast

521

iron coupons immersed in sterile and reclaimed water for different time periods. From

522

the Bode plots, it can be observed that the total impedance and phase angle magnitude

523

were higher at the same frequency in sterile water than that in the reclaimed water at

524

the same corresponding period. These results demonstrated that the corrosion process

525

was promoted by the biofilm in reclaimed water, confirming the results of the general

29

Page 29 of 43

526

corrosion rate test. This have also validated that the biofilm can interfere with the

527

electrochemical reactions at the time that the mixed microorganisms colonize on the

528

metal surface [63]. While carrying out the impedance study in reclaimed water, the irregular semi-

530

circle capacitive resistance arc in the Nyquist plots suggested that the formation of the

531

biofilm on the cast iron surface, due to which the corrosion may be affected by the

532

deposition of corrosion products beneath the biofilm [64, 65]. Meanwhile, the

533

increase in the capacitive loop in terms of size with the extension of immersion time,

534

demonstrated that the biofilm was thickened and the protection afforded by the

535

corrosion biofilm layer was improved [66]. The smallest diameter of the impedance

536

loops at 3 day shows the least reaction resistance through the biofilm on the surface,

537

indicating that the corrosion processes are activated by the presence of DO and IOB in

538

reclaimed water. This behavior can be used to explain the increased corrosion rate

539

before 7 day. Subsequently, the diameter of the impedance loops increased sharply

540

and reached a maximum at 7 day, which was inconsistent with the general corrosion

541

result. Thus, it is important to mention that the cast iron seems to suffer a surface

542

modification as a consequence of the biofilm accumulation at this time. Since the cast

543

iron surface was covered with the dense, passive biofilm consisted a layered structure

544

composed of an upper ferric hydroxide layer and an inner ferric sulfide layer at 7 day

545

(Fig. 3b). This modification in the biofilm structure and the chemical composition

546

(Table 2) could have a great impact on the electrode surface, therefore, resulting the

Ac ce pt e

d

M

an

us

cr

ip t

529

30

Page 30 of 43

deviation of impedance, which also indicating the distinct variation of corrosion

548

mechanisms at 7 day. Afterwards, the diameter of the impedance loop returned to

549

normal at 15 day, later, increased by exposure time. This fact suggests that this stage

550

is more impeded probably due to the accumulation of corrosion products and the EPS

551

film [67] together with the reduced metabolic activity of IOB and SRB, which also

552

implied the decrement of corrosion rate in reclaimed water after 15 days. These trends

553

was in accordance with the general corrosion test.

us

cr

ip t

547

From the bode phase angle plots (Fig. 6c), two peaks can be distinguished at high

555

frequencies and low frequencies respectively. The phase angle magnitude, which

556

corresponds to the low frequencies peak, increased sharply from 1.52° at 3 day to a

557

maximum of 1.56° by 7 day, thereby demonstrating the increased biofilm density as

558

shown in Fig. 3b. Next, the phase angle magnitude decreased to 1.51° at 15 day,

559

which indicated the relatively loose biofilm structure due to loosely bound EPS

560

located in the outer layer (Fig. 3d).

M

d

Ac ce pt e

561

an

554

Furthermore, the equivalent circuit Rs(Qbf (Rbf(QdlRct) ) ) as shown in Fig. 7 was

562

used to describe the corrosion process where biofilm adhesion occurs [68-70], which

563

was consistent with the measurement conditions made in this work. The results of the

564

equivalent circuit model parameters are shown in Table 3. The constant phase element

565

(CPE) was used to substitute the pure capacitance component (C) due to the diffusion

566

process on the electrode surface [71].

31

Page 31 of 43

Qbf

RS Q dl

ip t

Rbf Rct

Table 3 Equivalent circuit model parameters of cast iron pipe in reclaimed water RS

Rct 2

Qdl 2

-2 n-1

ndl

Rbf

nbf

(Ω·cm )

(µF·cm-2·sn-1)

0.74

13.51

0.0034

0.95

0.1045

0.68

43.78

0.0098

0.90

0.0143

0.64

60.57

0.0147

0.89

375.76

0.0128

0.65

72.37

0.0238

0.87

10.26

396.30

0.0098

0.57

94.32

0.0224

0.86

12.64

399.17

0.0096

0.58

104.56

0.0328

0.85

(Ω·cm )

(Ω·cm )

(µF·cm ·s )

3

1.78

364.81

0.0124

7

4.574

526.39

9

6.36

375.24

15

9.64

20 23

an

(days)

2

Qbf

M

Immersion times

Ac ce pt e

570

us

Fig. 7 Equivalent circuit of the cast iron pipe in reclaimed water

d

569

cr

567 568

Rs denotes the resistance of solution between the working electrode (WE) and

571

reference electrode (RE). Rbf is the resistance of biofilm formed on the cast iron

572

surface and Rct is the charge transfer resistance. Qdl is the constant phase element

573

(CPE) of the electric double layer and Qbf is the CPE of the surface film, n is depicted

574

as the dispersion parameter and its value being less than the one indicating an

575

imperfect capacitor. Increase of the biofilm roughness means the decrease of the n

576

value [38].

577

The value of Rs changed slightly in the whole reaction process and was all below

578

13 Ω·cm2, indicating that the solution conductivity of the electrolyte was not 32

Page 32 of 43

obviously affected by the bacterial adhesion. The biofilm resistance Rbf increased

580

from 13.51 Ω·cm2 at 3 day to 104.56 Ω·cm2 by 23 day, probably due to the growth of

581

the biofilm thickness. Therefore, it can be concluded that the accumulation of

582

metabolic by-products and corrosion products on the electrode could hinder the

583

reaction between the biofilm and the electrode surface. However, the charge transfer

584

resistance Rct was higher than solution resistance (Rs) and biofilm resistance (Rbf) for

585

1 to 2 orders of magnitude, indicating that the Rct was the primary factor that

586

controlled the biofilm influenced corrosion in reclaimed water. The variation tendency

587

of Rct was consistent with the diameter of the impedance loops.

an

us

cr

ip t

579

The fact that the morphology and chemical composition of the biofilm may be

589

correlated to the capacitance magnitudes has been reported in the literature [72]. Qdl

590

represents the double-layer capacitance between the biofilm and the iron surface,

591

which increased from 0.0124 µF·cm-2·sn-1 at 3 day to a maximum of 0.1045 µF·cm-

592

2 n-1

593

is worth mentioning that there may be not only an ferric hydroxide on the cast iron

594

surface, but mainly high-conductive layer of FeS formation due to the metabolic

595

activity of SRB in the biofilm [73]. These modifications could also be confirmed by

596

the SEM and EDS result. Subsequently, as the accumulation of exopolysaccharide

597

fibers and EPS on the cast iron surface, Qdl declined rapidly to 0.0143 µF·cm-2·sn-1 at 9

598

day, later it decreased slightly, and stabilized at 0.0096 µF·cm-2·sn-1 at 23 day.

·s

Ac ce pt e

d

M

588

by 7 day, most likely reflecting the formation of relatively conductive biofilm. It

33

Page 33 of 43

599

4. Conclusions In this laboratory study, the mixed species biofim induced by the synergy of IOB

601

and SRB was found presenting a random and heterogeneous structure on the cast iron

602

surface, which was composed of the settled bacterial cells, extracellular polymeric

603

substance (EPS) and corrosion products. The effects of this biofilm on the corrosion

604

of cast iron coupons in reclaimed water versus sterile water were investigated. The

605

results demonstrated that compared to sterile water, the existence of the biofilm in

606

reclaimed water promoted the corrosion process significantly.

us

cr

ip t

600

The evolution of impedance spectra in terms of size at different times in

608

reclaimed water demonstrated that the corrosion process was affected by the biofilm.

609

Due to the existence of the biofilm in reclaimed water, the general corrosion rate was

610

accelerated before 7 day but was inhibited afterward. Though the biofilm thickness

611

was less than 5μm during the whole experiment, it was determined that there was an

612

inverse correlation between the biofilm thickness and general corrosion rate in our

613

study (r=-0.891, p=0.049). Thus, the corrosion process was influenced by the settled

614

bacteria imbibed in EPS and corrosion products in the biofilm comprehensively.

M

d

Ac ce pt e

615

an

607

But, the corrosion mechanisms were different with respect to time and could be

616

divided into three stages in our study. Firstly, the increment of settled IOB counts and

617

the discontinuity, porous biofilm structure contributed to the increased corrosion rate

618

in 7 days together at stage I (0-7 days). While at stage II (7-15 days), the decreased

619

corrosion rate could be dominated by the declining IOB counts as well as the dense

34

Page 34 of 43

biofilm composed of corrosion products, SRB and exopolysaccharide fibers on the

621

cast iron surface. Finally, the continue decreased corrosion rate at stage III (15-30

622

days) is attributed to the synergies between the declined metabolic activity of settled

623

bacteria (IOB and SRB) and the densely packed corrosion products in the EPS film.

624

Electrochemical impedance spectra (EIS) analysis also confirmed the results of the

625

general corrosion rate test.

cr

ip t

620

With the greater widespread application of cast iron pipes used in reclaimed

627

water distribution system, biofilm influenced corrosion is predicted to be an ever-

628

present problem in need of further understanding.

an

Acknowledgments

M

629 630

us

626

This research was supported by National Natural Science Foundation of China

632

(No.51478307) and Specialized Research Fund for the Doctoral Program of Higher

633

Education of China (No.20130032110032).

Ac ce pt e

634

d

631

635

References

636

[1]M. Meneses, J. C. Pasqualino, F. Castells, Environmental assessment of urban

637

wastewater reuse: Treatment alternatives and applications, Chemosphere. 81 (2010)

638

266-272.

639

[2]]F. Teng, Y. T. Guan, W. P. Zhu, Effect of biofilm on cast iron pipe corrosion in

640

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Fig. 1 Biofilm thickness and roughness formed on the cast iron coupons at different

878

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Fig. 2 Bacterial counts of the biofilm on the cast iron coupons at different times

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Fig. 7 Equivalent circuit of the cast iron pipe in reclaimed water

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