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Corrosion Science 114 (2017) 102–111

Contents lists available at ScienceDirect

Corrosion Science

j ourna l h o mepa ge: www.elsev ier .com/ locate /corsc i

he corrosion behavior and mechanism of carbon steel induced byxtracellular polymeric substances of iron-oxidizing bacteria

ongwei Liua, Tingyue Gub, Muhammad Asif a, Guoan Zhanga, Hongfang Liua,∗

Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, Hubei Key Laboratory of Materials Chemistry and Serviceailure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR ChinaDepartment of Chemical and Biomolecular Engineering, Institute for Corrosion and Multiphase Technology, Ohio University, Athens, OH 45701, USA

r t i c l e i n f o

rticle history:eceived 18 June 2016eceived in revised form4 September 2016ccepted 23 October 2016

a b s t r a c t

In this work, the corrosion behavior and mechanism of carbon steel induced by extracellular polymericsubstances (EPS) extracted from an iron-oxidizing bacterium culture were studied using surface analysisand electrochemical measurements in 3.5 wt% NaCl solutions. Results of electrochemical measurementsshowed that 7 day-old EPS at 240 mg L−1 inhibited the corrosion of carbon steel, especially after the EPSsolution was heat treated to deactivate the enzymes in the EPS. However, 7 day-old EPS at much higher

vailable online 24 October 2016

eywords:: Mild steel: EIS: SEM

and much lower concentrations accelerated corrosion. Furthermore, 14 day-old EPS and 28 day-old EPSat 240 mg L−1 also accelerated corrosion.

© 2016 Elsevier Ltd. All rights reserved.

: Microbiological corrosion

. Introduction

Microbiologically influenced corrosion (MIC) is present in vari-us environments, including fresh water, seawater, cooling waternd oil-field produced water, etc [1–4]. MIC is closely associatedith the formation and attachment of a biofilm on a metal surface.ith the formation of the biofilm, the probability of metal corro-

ion is enhanced considerably [5,6]. A biofilm is mainly composedf microbial cells, extracellular polymeric substances (EPS), tracesf inorganic minerals and some organics adsorbed from the bulkuid or culture medium [7–9]. Sessile cells, rather than planktonicells, are the main culprit of MIC [8].

In a biofilm, EPS are composed of polysaccharides, proteins,ipids and a small amount of nucleic acids [10]. EPS can be foundoth in the biofilm and the bulk medium. EPS play a key role

n the corrosion process of metals [11]. In addition to promotinghe growth of sessile bacteria, EPS indirectly influence the metalorrosion process [12]. The components, structures and electricharge distribution of EPS are closely related to the growth cycles of

icroorganisms and thus influencing the metal corrosion process

13]. Some electroactive EPS molecules in a biofilm can act as elec-ron “shuttles” to transport electrons from a metal surface to the

∗ Corresponding author.E-mail address: liuhf@hust.edu.cn (H. Liu).

ttp://dx.doi.org/10.1016/j.corsci.2016.10.025010-938X/© 2016 Elsevier Ltd. All rights reserved.

terminal electron acceptor (e.g., O2), which accelerates corrosion[14]. On the other hand, EPS may also inhibit corrosion by acting asan oxygen barrier [10,15].

Some effects of EPS on the corrosion behavior of carbon steelhave been reported in the literature. Ghafari et al. [16] isolated avariety of bacterial strains from garden soil and hot spring waterto investigate the corrosion inhibition by EPS, and found thatcarboxylic acids in the EPS were responsible for steel corrosionresistance. Jin et al. [8] also found that EPS could inhibit cast ironcorrosion, and the formation of protective EPS film could inhibit thecathodic reaction. Moreover, the effect of EPS on metal corrosiondepends on the microbial species, growth stages and the enzymeactivities in the EPS [17]. In the presence of dissolved oxygen, someorganic functional groups in EPS containing lone pair electrons thatcan be easily transferred to the d-unoccupied-orbital of the Fe atom,thus strengthening the chemisorption of EPS on the steel surface.It is the adsorbed EPS film that offers corrosion inhibition.

Some recent studies paid more attention to the effects of EPSfrom sulfate reducing bacteria (SRB) on metal corrosion [18,19].SRB are corrosive anaerobes and need an anaerobic environmentto grow. In an open-air system, SRB can grow underneath an aerobicbiofilm that provides a locally anaerobic environment. Dong et al.[10] found that low concentrations of EPS secreted by SRB could

inhibit, while the high concentration of EPS enhanced the corrosionof carbon steel. Chanet et al. [20] found that EPS secreted by SRB

Science 114 (2017) 102–111 103

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sIaep

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Table 1Polysaccharide and protein contents of EPS after incubation times. The protein andpolysaccharide content of EPS were measured using Bradfordassay and phenol-sulfuric acid method.

Protein content (mg g−1) Polysaccharide content (mg g−1)

7 day-old EPS 2.16 11.7914 day-old EPS 7.61 14.0928 day-old EPS 4.03 4.54

Table 2The pH measurement results of the 3.5 wt% NaCl corrosion medium with differentEPS concentration and species after immersion for 24 h.

CEPS (mg L−1) pH values

Control (EPS-free) 0 7.07 day-old EPS 24 6.5

240 5.91200 4.5

H. Liu et al. / Corrosion

ould enhance steel corrosion mainly due to the oxidizing powerf EPS.

Iron oxidizing bacteria (IOB) are another major type of corro-ive bacteria, causing serious corrosion damages in the field [21].owever, the exact role of EPS from IOB on the corrosion of car-on steel is not clear. The study of EPS from IOB on the metalorrosion is needed to reveal the MIC mechanism of carbon steely IOB. EPS secreted by IOB are different from the EPS secretedy other microorganisms. For example, the EPS of IOB contain Fexidases [21–23], and the corrosion process influenced by IOB islosely related with Fe oxidases.

In this work, the effects of EPS secreted by IOB on the corro-ion behavior of mild carbon steel are studied for the first time.t aimed to determine the effects of EPS on the corrosion processnd electrochemical behavior of carbon steel. Surface analyses andlectrochemical measurements were used to probe the corrosionrocess and the corrosion inhibition by EPS.

. Experimental

.1. Coupon preparation

Q235 mild carbon steel were used for corrosion coupons withhe chemical composition (wt%) of C 0.3, Si 0.01, Mn 0.42, S 0.029,

0.01, and Fe balance. Cylindrical shaped coupons with a diame-er of 10 mm and height of 10 mm were used. Only one end face0.785 cm2) was exposed and the rest was sealed with an epoxyesin. A copper wire was soldered to each coupon for electro-hemical measurements. Coin-shaped coupons with a diameter of5 mm and thickness of 2 mm were used for surface analyses. Allhe coupons were abraded through 600, 800 and 1200-grit siliconarbide metallurgical papers, degreased in acetone, washed withnhydrous ethanol, and finally dried under nitrogen gas. They weretored in a desiccator until use.

.2. Microbial inoculation and cultivation

IOB were isolated from the sludge of a Sinopec oilfield in China.he bacteria were identified in a previous study [1]. The IOB cul-ure medium contained (g L−1): K2HPO4 0.5, NaNO3 0.5, CaCl2 0.2,

gSO4·7H2O 0.5, (NH4)2SO4 0.5 and ammonium iron citrate 10.0pH 6.5). Before inoculation, the IOB culture medium was auto-laved at 121 ◦C for 20 min. The IOB inoculum size was 1% (v/v).

.3. Extraction and characterization of EPS

EPS were isolated from 1 L IOB culture medium after 7, 14 and8 d of incubation, respectively. Initially, the culture medium wasentrifuged at 12,000 rpm at 4 ◦C for 20 min using centrifuge tubef 10 mL to obtain an EPS supernatant. Next, the EPS supernatantas filtered 3 times using a 0.22 �m membrane filter to remove

acterial cells. Then the filtrate containing EPS was lyophilisedt −50 ◦C. The protein content of the EPS was determined byhe Bradfordassay with bovineserum albumin as the standard24]. The polysaccharide content of EPS was quantified using thehenol-sulfuric acid method with glucose as the standard [25]. Thextracted EPS were used for corrosion tests in the absence of IOB.

.4. Surface analysis

In this work, a 3.5 wt% NaCl solution was used as the corrosion

est medium. Before scanning electron microscopy (SEM) obser-ation and energy dispersive X-ray spectrum (EDS) analysis oforrosion product-EPS films, coupons were taken out from the testedium after 24 h of immersion. Prior to SEM observation, the

14 day-old EPS 240 5.628 day-old EPS 240 5.4

coupons were coated with a thin gold film by means of a vac-uum sputter to provide electrical conductivity. X-ray photoelectronspectroscopy (XPS) was used to analyze the composition of cor-rosion products on the coupon surfaces. Atomic force microscopy(AFM) was used to measure the force curves of EPS adsorption onthe coupon surfaces.

2.5. Electrochemical measurements

All the electrochemical measurements were conducted usinga CS350 electrochemical workstation (CS350, Corrtest, WuhanChina) with a saturated calomel electrode (SCE) as the referenceelectrode and platinum plate as the counter electrode. Electro-chemical impedance spectroscopy (EIS) were obtained at thesteady-state open circuit potential (OCP) by applying a sinusoidalvoltage signal of 10 mV in the frequency range of 10−2–105 Hz.The EIS data were analyzed using the Zview2 software (Scribner,Inc.) with a suitable equivalent circuit model. Potentiodynamicpolarization curves were measured by scanning the potential from−250 mV to +300 mV versus OCP after the working electrodereached a stable state at a scan rate of 0.5 mV s−1. All polariza-tion curves were analyzed using the Cview2 software (Scribner,Inc.). Tests were repeated at least 3 times at 30 ◦C unless otherwisestated.

3. Results

3.1. The chemical component analysis of EPS

The EPS quantities varied slightly for different IOB incubationtimes. The outputs of 7 day-old EPS, 14 day-old EPS and 28 day-old EPS were 1.39 g L−1, 1.70 g L−1 and 1.47 g L−1, respectively. Theoutput of 14 day-old EPS was the highest. The polysaccharide andprotein contents of the EPS sample with different incubation timesare shown in Table 1. Table 1 indicates that the polysaccharide andprotein contents of 14 day-old EPS were also the highest. After 28d incubation, the contents of polysaccharide and protein declinedgreatly.

Steel corrosion process is related to the pH of the test corrosionmedium. The pH values of the test corrosion medium with differentEPS concentration of and species after immersion for 24 are shown

in Table 2. The pH values decreased with the decrease of the 7 day-old EPS concentration with the pH value of 28 day-old EPS beingthe lowest.

104 H. Liu et al. / Corrosion Science 114 (2017) 102–111

Fig. 1. SEM images of corrosion products on coupon surfaces exposed to an 3.5 wt% NaCl corrosion medium after immersion for 24 h: a and b, control; c and d, 7 day-oldEPS; e and f, 14 day- old EPS; g and h, 28 day-old EPS (b, d, f, and h are enlarged views of a, c, e, and g, respectively.). The EPS concentration was 240 mg L−1 and the controlcorrosion medium was an 3.5 wt% NaCl solution without any EPS.

H. Liu et al. / Corrosion Science 114 (2017) 102–111 105

0 2 4 6 8 10

O

Na Cl

Fe

Fe

FeInte

nsity

(a.u

.)

Energy (ke V)

(a)

0 2 4 6 8 10

(b) O

ClC

Fe

Fe

Fe

Inte

nsity

(a.u

.)

Energy (keV)

0 2 4 6 8 10

Na ClC

O

Fe

Fe

Fe(c)

Inte

nsity

(a.u

.)

Energy (keV)0 2 4 6 8 10

ClC

OFe

Fe

Fe(d)

Inte

nsity

(a.u

.)

Energy (keV)

Fig. 2. EDS analysis of corrosion product films on coupons exposed to an 3.5 wt% NaCl cora, control; b, 7 day-old EPS; c 14 day-old EPS; d, 28 day-old EPS. The control corrosion me

Table 3EDS quantitative analysis results of corrosion product or EPS adsorbed films oncoupons in an 3.5 wt% NaCl corrosion medium with EPS concentration of 240 mg L−1

after immersion for 24 h.

Elements (wt%) C O Cl Na Fe

Control 15.88 1.10 3.23 79.787 day-old EPS 4.46 26.59 0.45 68.50

3

cep2cc(i(EgtrfrasaEfiEp

carbon steel

14 day-old EPS 4.23 17.32 0.79 3.24 74.4228 day-old EPS 4.13 13.67 0.65 81.55

.2. SEM analysis of corrosion products

SEM and EDS were conducted to analyze the morphology andomposition of corrosion products on a coupon surface in the pres-nce and absence of EPS. Fig. 1 shows the SEM images of corrosionroducts on the surface of coupon after immersion for 24 h with40 mg L−1 EPS. For the control coupon (in the absence of EPS), theorrosion product film was loose and agglomerates of inorganicorrosion products could be seen in Fig. 1b. EDS analysis resultsFig. 2a and Table 3) show that the corrosion products were mainlyron oxides. In the corrosion test medium containing 7 day-old EPSFig. 1c and d), the corrosion product film, which was mixed withPS, was compact and rather dense. Some amorphous film and inor-anic corrosion products could be seen in Fig. 1d, which suggestedhat EPS could absorb on the carbon steel surface. The EDS analysisesults (Fig. 2b and Table 3) show the presence of C element, whichurther verified the adsorption of EPS. In the 14 day-old EPS cor-osion test medium (Fig. 1e and f), the corrosion product film waslso compact and with some granular corrosion products on theurface (Fig. 1f). Fig. 1f shows that the corrosion product film was

little porous, thus prone to further corrosion. In the 28 day-oldPS corrosion test medium (Fig. 1g and h), the corrosion product

lm showed some cracks and pores could be seen in Fig. 1h. TheDS analysis results of 14 day-old EPS and 28 day-old EPS corrosionroducts also verified the adsorption of EPS (presence of C element

rosion medium after immersion for 24 h with the EPS concentration of 240 mg L−1:dium was only an 3.5 wt% NaCl solution without any EPS.

detected) on the carbon steel surfaces and the formation of ironoxides.

3.3. XPS analysis of corrosion products

Fig. 3 shows high-resolution XPS spectra of N 1s, S 2p, O 1s andFe 2p for coupons after immersion for 24 h with 240 mg L−1 EPS. Inthe presence of 7 day-old EPS, 14 day-old EPS and 28 day-old EPS,in each case, N 1s peak at about 400 eV corresponded to C N, C Nand NH [26], which were assigned to the structures of protein in theEPS. The fitting results of S 2p data in the presence of 7 day-old EPS,14 day-old EPS and 28 day-old EPS all showed the presence of C S.The elements N and S were characteristic of proteins. In the con-trol, N and S elements were not detected. The presence of proteinsfurther verified the adsorption of EPS on the coupon surfaces. Thefitting results of O 1s data both in the control and in the presenceof EPS indicated that Fe2O3 and FeOOH were the main corrosionproducts [27–29]. In the presence of 7 day-old EPS, The presenceof organic O in Fig. 3 once again supported the argument of EPSadsorption [30]. In the control, and in the presence of 7 day-old EPSand 28 day-old EPS, all the Fe 2p spectra were curve-fitted withthree peaks. The peaks at 709 eV and 710.8 eV both correspondedto Fe2O3, while the peak at 711.8 eV was attributed to FeOOH [17].In the presence of 14 day-old EPS, the Fe 2p spectra were curve-fitted with three peaks. The peak at 709.6 eV corresponded to FeO[27], and the peaks at 710.4 eV and 710.8 eV corresponded to Fe2O3and FeOOH, respectively [17].

3.4. Electrochemical measurements

3.4.1. The effect of EPS concentration on the corrosion behavior of

The batch growth cycle of IOB was usually 14 days in lab tests,and the activity of 7 day-old EPS was the highest [22]. Thus, 7 day-old EPS were chosen to investigate the effects of EPS concentration

106 H. Liu et al. / Corrosion Science 114 (2017) 102–111

408 406 404 402 40 0 39 8 39 6 394 392

NHC=NC-N

28 day-old EP S

7 day -old EP S

14 day -old EPS

Inte

nsity

(cps

)

Bind ing Energy (eV)

NHC=NC-N

NH

C=NC-N

(a) N 1s

174 172 170 168 16 6 16 4 162 160 158

(b)

C=S

C=S

C=S

28 day -old EP S

14 day -old EP S

7 day-old EP SInte

nsity

(cps

)

Bind ing Energy (eV)

S 2p

538 53 6 534 532 530 528 526

Organic O

Control

7 day-old EP S

FeOO H

FeOOH

Fe2O3

Fe2O3

Inte

nsity

(cps

)

Bind ing Energy (eV)

O 1s(c)

538 536 534 532 530 52 8 526

28 day-old EP S

14 day -old EP S

O 1s

FeOOH

FeOOH

Fe2O3

Fe2O3(d)

Inte

nsity

(cps

)

Binding Energy (eV)720 716 71 2 708 70 4

Control

7 day -old EP S

Fe2O3

Fe2O3

FeOOH

FeOOH

Inte

nsity

(cps

)

Binding Energy (eV)

Fe 2p(e)

720 716 712 708 70 4

FeO

Fe2O3

Fe2O3FeOOH

FeOO H

28 day-old EP S

14 day-old EP S

Inte

nsity

(cps

)

Binding Energy (eV)

(f) Fe 2p

Fig. 3. High resolution XPS spectra of N 1s (a), S 2p (b), O 1s (c and d) and Fe 2p (e and f14 day-old and 28 day-old EPS with the concentration of 240 mg L−1 after immersion for 2EPS.

0 5 10 15 20 25-0.76

-0.74

-0.72

-0.70

-0.68

-0.66

-0.64

OC

P (V

vs.

SC

E)

t (h)

Con trol (EPS-free ) 24 mg L-1 EPS 240 mg L-1 EP S 1200 mg L-1 EPS

Fm

owTdtvc

ccsdiwfidtWmc

−1

ig. 4. Change of open circuit potential with time in the 3.5 wt% NaCl corrosionedium containing different concentration of 7 day-old EPS.

n the corrosion behavior of carbon steel. The 3.5 wt% NaCl solutionas used as the corrosion test medium for the control (EPS-free).

he change of OCP with time in the corrosion medium containingifferent concentrations of 7 day-old EPS was shown in Fig. 4. Inhe control medium, the OCP decreased gradually with time. Thealues of OCP was lowest in the presence of 7 day-old EPS at theoncentration of 240 mg L−1.

Fig. 6 shows the Nyquist and Bode plots for the coupons afterontinuous immersion up to 24 h in the presence of different con-entrations of 7 day-old EPS. All the EIS were carried out under thetable OCP (Fig. 5). For the control coupon (Fig. 6a and b), the smalliameter of the Nyquist plots suggested serious corrosion. With an

ncreased immersion time, the diameter of Nyquist plot increased,hich indicated the formation of a protective corrosion productlm on the coupon surface [31]. As shown in Fig. 6c, the smalliameter of Nyquist plot indicated that EPS with a low concentra-

−1

ion (24 mg L ) increased corrosion. Furthermore, the presence ofarburg impedance in the low frequency range suggested the for-ation of a more compact corrosion products film. When the EPS

oncentration reached 240 mg L−1, the diameters of Nyquist plots

) for coupons exposed to an 3.5 wt% NaCl corrosion medium containing 7 day-old,4 h. The control corrosion medium was only an 3.5 wt% NaCl solution without any

(Fig. 6e) were bigger than those of the control, indicating better pro-tection against corrosion by the EPS. Two obviously different timeconstants can be seen in the Bode plots (Fig. 6f), which suggestedthe formation of a protective corrosion product film on the couponsurface. In the presence of a high EPS concentration (1200 mg L−1)in the corrosion test medium, the diameters of Nyquist plot (Fig. 6g)were very small after immersion for 1 h, indicating that the highconcentration of EPS promoted corrosion. With a prolonged cor-rosion time, the formation of a protective corrosion product filmresulted in an increase of impedance (Fig. 6g).

The experimental impedance spectra were curve-fitted satisfac-torily (errors <10%) with suitable equivalent circuits (Fig. 7). It wasnecessary to consider distributed capacitance through constant-phase element (Q) instead of capacitance. Impedance of Q wascalculated from the equation below,

ZQ = Y−10 (jw)−� (1)

where � is angular frequency in rad s−1, Y0 and � the Q parame-ters, and � the deviation from ideal behavior [32]. In the electricalanalog circuits (Fig. 7), Rs represents solution resistance, Rf and Qfthe resistance and the capacitance of the corrosion product film,respectively. Rct and Qdl correspond to a charge transfer resistanceand a double layer capacitance, respectively. W is the Warburgimpedance.

The EIS fitting results of Rp (Rf + Rct) with time are shown in Fig. 8.The Rp values were associated with corrosion rates, with a lowervalue for a higher corrosion rate [21]. It could be seen that the val-ues of Rp with the EPS concentration of 240 mg L−1 were biggest,suggesting the protection of EPS for the coupon. However, the pres-ence of the low (24 mg L−1) or high (1200 mg L−1) concentrationof EPS, the values of Rp on the whole were smaller than those ofthe control. Thus, both the low (24 mg L−1) and high (1200 mg L−1)concentrations of EPS promoted the corrosion, and the corrosion ofcoupon was more serious in presence of the low concentration EPS

(24 mg L ) corrosion test medium.

Fig. 9 shows the potentiodynamic polarization curves ofcoupons in corrosion test media containing different concentra-tions of 7 day-old EPS. The corresponding electrochemical fitting

H. Liu et al. / Corrosion Science 114 (2017) 102–111 107

0 200 40 0 60 0 80 0 1000 12 00-0.74

-0.72

-0.70

-0.68

-0.66

8 h 14 h 24 h

OC

P (V

vs.

SC

E)

t (s)

1 h 4 h

(a)

0 200 40 0 60 0 80 0 1000 12 00

-0.72 0

-0.71 2

-0.70 4

-0.69 6

-0.68 8

OC

P (V

vs.

SC

E)

t (s)

1 h 4 h 8 h 14 h 24 h

(b)

0 200 40 0 600 800 1000 12 00-0.748

-0.744

-0.740

-0.736 8 h 14 h 24 h

OC

P (V

vs.

SC

E)

t (s)

1 h 4 h

(c)

0 200 400 60 0 80 0 100 0 1200

-0.728

-0.724

-0.720

-0.716

OC

P (V

vs.

SC

E)

t (s)

1 h 4 h 8 h 14 h 24 h

(d)

Fig. 5. The change of OCP with time before EIS measurements in the 3.5 wt% NaCl corrosion medium containing with different concentrations of 7 day-old EPS: control (a),24 mg L−1 EPS (b), 240 mg L−1 EPS (c) and 1200 mg L−1 EPS (d). The OCP was in the steady-state before EIS measurements.

Table 4Electrochemical parameters fitted from the potentiodynamic polarization data ofcoupons in 3.5 wt% NaCl corrosion mediums containing different concentration of7 day-old EPS (Errors represent the standard deviations from 3 measurements).

CEPS (mg L−1) ba (V dec−1) bc (V dec−1) Ecorr (V vs. SCE) icorr (�A cm−2)

0 0.07 ± 0.01 −0.43 ± 0.13 −0.761 ± 0.02 12.6 ± 1.824 0.06 ± 0.01 −0.36 ± 0.08 −0.745 ± 0.01 20.0 ± 2.5

pccb7(cw

3c

sd[itacEpswso

Table 5Electrochemical parameters fitted from the potentiodynamic polarization data inFig. 10 in the 3.5 wt% NaCl corrosion medium containing 240 mg L−1 7 day-old EPSwith deactivated enzymes after immersion for 24 h (Errors represent the standarddeviations from 3 measurements).

CEPS (mg L−1) ba (V dec−1) bc (V dec−1) Ecorr (V vs. SCE) icorr (�A cm−2)

0 0.07 ± 0.01 −0.43 ± 0.13 −0.761 ± 0.02 12.6 ± 1.8240 0.06 ± 0.01 −0.09 ± 0.02 −0.772 ± 0.05 1.10 ± 0.35

Table 6Electrochemical parameters fitted from the potentiodynamic polarization data inFig. 11 in the 3.5 wt% NaCl corrosion solution containing 14 day-old EPS and 28 day-old EPS both with EPS concentration of 240 mg L−1 after immersion for 24 h (Errorsrepresent the standard deviations from 3 measurements).

ba (V dec−1) bc (V dec−1) Ecorr (V vs. SCE) icorr (�A cm−2)

240 0.06 ± 0.02 −0.17 ± 0.06 −0.759 ± 0.01 9.50 ± 0.701200 0.06 ± 0.01 −0.38 ± 0.06 −0.736 ± 0.02 19.9 ± 2.1

arameters are listed in Table 4. As shown in Fig. 9, in the EPSorrosion media with lower (24 mg L−1) and higher (1200 mg L−1)oncentrations, the cathodic reaction processes were promotedy EPS. Table 4 shows that icorr was lowest with 240 mg L−1 of

day-old EPS, indicating its inhibition of corrosion, while at low24 mg L−1) and high (1200 mg L−1) concentrations, EPS enhancedorrosion. The analysis of potentiodynamic polarization curvesere consistent with the EIS data above.

.4.2. The effect of enzymatic activity of EPS on the carbon steelorrosion

EPS were composed of polysaccharide, proteins, lipids and amall amount of nucleic acids and DNA. In addition, some Fe oxi-ases produced by IOB could be transferred to the bulk solution33]. Enzymes would lose their bioactivity at a higher temperaturerreversibly. Thus, the effects of enzymes with or without bioac-ivity on the coupon corrosion should be different. Based on thisrgument, the corrosion test medium with 7 day-old EPS at theoncentration of 240 mg L−1 was first pretreated by heating thePS solution for 20 min at 90 ◦C to denature the EPS enzymes. Theotentiodynamic polarization curves of the coupons and the corre-

ponding electrochemical fitting parameters in the presence of EPSith deactivated enzymes are shown in Fig. 10 and Table 5. Fig. 10

hows that the corrosion current density (icorr) decreased obvi-usly compared with the control. Compared with the EPS without

14 day-old EPS 0.07 ± 0.01 −0.24 ± 0.06 −0.761 ± 0.03 15.5 ± 1.528 day-old EPS 0.22 ± 0.05 −0.10 ± 0.03 −0.809 ± 0.02 18.6 ± 2.3

enzyme deactivation (Fig. 9), both the cathodic and anodic reac-tion processes were inhibited due to enzyme deactivation. Table 5shows that the value of icorr decreased by one order of magnitude,further indicating that EPS without enzyme activities provided bet-ter corrosion inhibition for the coupons. This meant that metalsurface binding EPS with enzymes deactivated could potentiallybe used as a corrosion inhibitor.

3.4.3. The effect of EPS types on carbon steel corrosionThe study above has indicated that the corrosion of carbon steel

coupons could be protected by 7 day-old EPS at the concentrationof 240 mg L−1 (Table 4). Thus, this concentration were chosen toinvestigate the effect of EPS types on the corrosion behavior of car-

bon steel coupons. Fig. 11 shows the potentiodynamic polarizationcurves of coupons in the presence of 14 day-old EPS and 28 day-oldEPS at 240 mg L−1 after immersion for 24 h. The corresponding elec-trochemical fitting parameters are listed in Table 6. Fig. 11 shows

108 H. Liu et al. / Corrosion Science 114 (2017) 102–111

0 100 200 300 400 500 600 7000

-100

-200

-300

-400

-500

-600

-700

0.08 Hz

0.13 Hz

Z'' (

Ω c

m2 )

Z' (Ω cm2)

1 h4 h8 h14 h24 hFitted li ne

(a)

-2 -1 0 1 2 3 4 50.5

1.0

1.5

2.0

2.5

3.01 h4 h8 h14 h24 h

logf (Hz)

0

-10

-20

-30

-40

-50

-60(b)

log|Z|

(Ω c

m2 )

Fitt ed li ne

Phas

e (d

egre

e)

0 10 0 200 300 400 5000

-100

-200

-300

-400

-500

Z'' (

Ω c

m2 )

Z' (Ω cm2)

1 h4 h8 h14 h24 hFitt ed li ne

(c)

0.42 Hz

0.13 Hz

0.08 Hz-2 -1 0 1 2 3 4 5

0.0

0.5

1.0

1.5

2.0

2.5

3.0(d)

Phas

e (d

egre

e)

1 h4 h8 h14 h24 h

log|Z|

( Ω c

m2 )

logf (Hz)

Fitted li ne

0

-10

-20

-30

-40

-50

-60

0 100 200 30 0 400 500 600 70 0 800 9000

-100-200-300-400-500-600-700-800-900

(e)

0.06 Hz

0.08 Hz

1 h4 h8 h14 h24 h

Z'' (

Ω c

m2 )

Z' (Ω cm2)

Fitt ed li ne

-2 -1 0 1 2 3 4 50.5

1.0

1.5

2.0

2.5

3.0(f)

log|Z|

(Ω c

m2 )

logf (Hz)

1 h4 h8 h14 h24 hFitted line

10

0

-10

-20

-30

-40

-50

-60

Phas

e (d

egre

e)

0 100 200 300 400 500 600 70 0 800 9000

-100-200-300-400-500-600-700-800-900

(g)1 h4 h8 h14 h24 h

Z'' (

Ω c

m2 )

Z' (Ω cm2)

Fitted li ne 0 50 100 15 0 20 00

-50

-10 0

-15 0

-20 0

Z'' (

Ω c

m2 )

Z' (Ω cm2)

0.85 Hz

1.4 Hz

-2 -1 0 1 2 3 4 50.5

1.0

1.5

2.0

2.5

3.0

log|Z|

(Ω c

m2 )

1 h4 h8 h14 h24 h

logf (Hz)

0

-10

-20

-30

-40

-50

-60(h)

Phas

e (d

egre

e)

Fitt ed li ne

F mediuu EPS (g

taittcc

ig. 6. Nyquist and Bode plots for the coupons exposed to an 3.5 wt% NaCl corrosionp to 24 h: control (a, b), 24 mg L−1 EPS (c, d), 240 mg L−1 EPS (e, f) and 1200 mg L−1

hat 28 day-old EPS promoted the cathodic reaction process, thusccelerating corrosion. Table 6 shows that both the icorr of couponsn the presence of 14 day-old EPS and 28 day-old EPS were largerhan those of the EPS-free control (Table 4), again showing that

he 14 day-old EPS and 28 day-old EPS at 240 mg L−1 acceleratedorrosion. Between them, the 28 day-old EPS was slightly moreorrosive.

m containing different concentrations of 7 day-old EPS after continuous immersion, h). The control corrosion medium was an 3.5 wt% NaCl solution without any EPS.

3.5. AFM force curves

AFM force curves were measured to understand the adhesionof EPS on the coupon surfaces [34], and the results are shown in

Fig. 12. Fig. 12a and b indicates that the force curves in the differentcorrosion test media were different. Fig. 12c shows that the viscousforces increased with the increase of the 7 day-old EPS concentra-tion. The change of viscous force followed closely the trend of the

H. Liu et al. / Corrosion Science 114 (2017) 102–111 109

Fig. 7. Equivalent circuits simulating experimental impedance diagrams in Fig. 6: (a)coupons immersed in the 3.5 wt% NaCl solution with the 7 day-old EPS concentrationof 0, 240, and 1200 mg L−1 and (b) other coupon.

0 5 10 15 20 250

200

400

600

800

1000

1200

240 mg L-1EPS 120 0 mg L-1EPS

Rp

(Ω c

m2 )

t (h)

Con trol (EPS-f ree) 24 mg L-1 EPS

Fig. 8. Changes of Rp with time fitted from EIS of coupons in Fig. 6 after continuousimmersion up to 24 h in the 3.5 wt% NaCl corrosion medium containing differentconcentrations of 7 day-old EPS (Rp = Rf + Rct). The control corrosion medium was an3.5 wt% NaCl solution without any EPS.

-8 -7 -6 -5 -4 -3 -2 -1-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

E (V

vs.

SC

E)

log(i, A cm-2)

Con trol (EPS -free) 24 mg L-1 EPS 240 mg L-1 EPS 1200 mg L-1 EPS

Fig. 9. Potentiodynamic polarization curves of coupons exposed to an 3.5 wt% NaClc7w

cccsfifiohccf

-8 -7 -6 -5 -4 -3 -2-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

E (V

vs.

SC

E)

log(i, A cm-2)

Con trol (EP S-free)240 mg L-1 EP S

Fig. 10. Potentiodynamic polarization curves of coupons in the 3.5 wt% NaCl corro-sion medium containing 240 mg L−1 7 day-old EPS with deactivated enzymes afterimmersion for 24 h. The control corrosion medium was only an 3.5 wt% NaCl solutionwithout any EPS.

-7 -6 -5 -4 -3 -2 -1-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

E (V

vs.

SC

E)

log(i, A cm-2)

14 day-old EP S28 day-old EP S

orrosion medium after immersion for 24 h containing different concentration of day-old EPS. The control corrosion medium was only an 3.5 wt% NaCl solutionithout any EPS.

hange in the total organics on the coupon surfaces. Higher EPS con-entrations led to higher viscous forces. The viscous force was alsolosely related to the compactness of adsorbed EPS on the couponurfaces, higher viscous forces corresponding to more compact EPSlms. It was mentioned in the discussion above that a compact EPSlm could provide corrosion protection. After enzymes in the 7 day-ld EPS were heat deactivated, the viscous force was highest. The

igher viscose force was likely a factor that the EPS provided betterorrosion protection (Fig. 10 and Table 5). At the same EPS con-entration (240 mg L−1) without enzyme deactivation, the viscousorce of the 14 day-old EPS was bigger than that of the 28 day-old

Fig. 11. Potentiodynamic polarization curves of coupons in the 3.5 wt% NaCl cor-rosion solution containing 14 day-old EPS and 28 day-old EPS both with EPSconcentration of 240 mg L−1 after immersion for 24 h.

EPS. Thus, the 14 day-old EPS was less corrosive than 28 day-oldEPS (Fig. 11 and Table 6).

4. Discussion

4.1. The effect of IOB culture time on the EPS components

The growth cycle of IOB is usually 14 days in lab cultures [21].After 7 d incubation, the activity of IOB is usually very high at thattime. With abundant nutrients, the metabolic rate of IOB is highand copious amounts of EPS can be produced [23,35,36]. After 14 dincubation, IOB enters the decline phase. This leads to less secretionof EPS, while the cumulative EPS production increases. Thus, thepolysaccharide and protein contents of 14 day-old EPS in this workwere more than those of 7 day-old EPS (Table 1). After 28 d of IOBincubation, nutrient starvation occurred, which could lead to EPSscavenging by the IOB. Table 1 indicates that the polysaccharideand protein contents of the 28 day-old EPS in this work decreaseddramatically compared with those of the 14 day-old EPS due to theprolonged incubation time.

4.2. The corrosion mechanism of carbon steel induced by EPS

The corrosion of carbon steel is an electrochemical process inthe aerobic solution, and the electrochemical mechanism can beexpressed as follows:

Anodic reaction:

Fe → Fe2++2e− (2)

Fe2+ → Fe3+ + e− (3)

110 H. Liu et al. / Corrosion Science 114 (2017) 102–111

-2000 -1000 0 1000 2000 3000-6-4-20246810

7 day -old EPS (12 00 mg L-1)

14 day-old EP S (240 mg L-1) 28 day-old EPS (240 mg L-1)

7 day-old EPS (90 °C+240 mg L-1)

Forc

e (n

N)

Distance (nm)

Control

7 day-old EPS (24 mg L-1) 7 day-old EPS (240 mg L-1)

(a)

500 10 00 1500-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5(b)

Forc

e (n

N)

Distance (nm)

0

-1

-2

-3

-4

-5

-6

-7

28day-EPS

(c)

240 mg L-1

240 mg L-1

90 °C+240 mg L-1

1200 mg L-1

240 mg L-1

24 mg L-1

14day-EPS

7day-EPS

7day-EPS

7day-EPS

7day-EPS

Visc

osity

forc

e (n

N)

Control

F be andd rrespom

C

mFR

F

F

F

2

fpaoeabi

iocEtw[amop

hfaEoptBotc[

ig. 12. Typical AFM force–distance curves: (a) for the interaction between the proifferent EPS concentration and species, (b) a part of enlarged image of (a), and (c) coedium was an 3.5 wt% NaCl solution without any EPS.

athodicreaction : 1/2O2 + H2O + 2e− → 2OH− (4)

XPS and EDS analysis results (Figs. 2 and 3) indicated that theain corrosion products in the presence of EPS are FeOOH and

e2O3. So, The supply of OH− from the O2 reduction reaction ineaction (4) pushes Reactions (5) and (6) forward:

e2+ + 2OH− → Fe(OH)2 (5)

e3+ + 3OH− → Fe(OH)3 (6)

Fe(OH)2 can be oxidized to FeOOH by O2, and the unstableeOOH can decompose into Fe2O3.

Fe(OH)2 → Fe2O3 + H2O (7)

The effect of EPS on the corrosion of carbon steel is related to theormation of adsorbed EPS film on the coupon surface and the com-lexation of EPS and Fe ions [32,37,38]. Fig. 9 shows that both thenodic reaction and cathodic reaction are inhibited in the presencef 7 day-old EPS at the concentration of 240 mg L−1. The protectiveffect of EPS for the coupon came from the formation of a compactdsorbed EPS film. The compact EPS adsorbed film served as an O2arrier and inhibited the dissolution of Fe0, leading to corrosion

nhibition.Fig. 9 also shows that only the cathodic reaction was accelerated

n the presence of 7 day-old EPS at the concentration of 24 mg L−1

r 1200 mg L−1. It indicates that 7 day-old EPS with this low or highoncentration promoted the dissolution of Fe0. Some electroactivePS molecules (Fe oxidases) in EPS can act as electron “shuttles”o transport electrons from Fe0 to O2 in an aerobic environment,hich promotes the cathodic reaction and accelerates corrosion

14]. The weak electrostatics action between EPS and metal ions canccelerate the corrosion of carbon steel [39,40]. The EPS can pro-ote the nucleation of iron minerals, thus promoting the growth

f corrosion products [41]. In addition, once the compact corrosionroduct film is partly damaged, the corrosion will go on [42].

In the test using 7 day-old EPS of 1200 mg L−1, the test solutionad a lower pH of 4.5 (Table 2). Thus, acid corrosion was one of the

actors promoting corrosion at this high EPS concentration [43]. Thecidity was due to the organic acids such as gluconic acids in thePS. However, at lower EPS concentrations listed in Table 2, the pHf the corrosion test medium was 5.4 and above. At this kind ofH, acid corrosion was not a key factor in carbon steel corrosion inhis work. The main corrosion process should be oxygen corrosion.oth the 14 day-old EPS and 28 day-old EPS with the concentration

f 240 mg L−1 promote the corrosion of carbon steel also throughhe acceleration of cathodic reaction (Table 4 and Table 6). The weakorrosion product film could be a reason for corrosion acceleration44,45].

coupons after immersion for 24 h in the 3.5 wt% NaCl corrosion test medium withnding viscose forces on different coupons calculated from (a). The control corrosion

4.3. The effect of EPS enzyme inactivation on corrosion

The 7 day-old EPS at the concentration of 240 mg L−1 inhibitedcorrosion (Table 4) due to the formation of a compact corrosionproduct film (Fig. 1). Seven day-old EPS at the concentration of240 mg L−1 was first pretreated for 20 min under 90 ◦C to deac-tivate enzymes. After this treatment, 7 day-old EPS significantlyinhibited the corrosion (Table 5) and both the anodic and cathodicreactions were inhibited. Based on these results, it can be said thatthe enzymes in EPS played key roles in the corrosion process. Thiswas not a surprise because enzymes can act as electron “shut-tles” to transport electrons from Fe0 to O2 [14]. The enzymes inthe EPS of IOB are mainly composed of some Fe oxidases [46].After the heat treatment, 7 day-old EPS at the concentration of240 mg L−1 adsorbed onto the coupon surface to form an EPS film.This EPS film was the most compact among all tested cases (Fig. 12).With enzyme deactivation, electron transport was inhibited, thusenhancing corrosion inhibition greatly. Thus, 7 day-old EPS at theconcentration of 240 mg L−1 after enzyme deactivation offered bet-ter corrosion inhibition.

5. Conclusions

(1) SEM analysis results indicated that the corrosion product filmwas loose for the EPS-free control coupon, while the corrosionproduct film were more compact with some cracks and poresin the presence of EPS.

(2) EIS and potentiodynamic polarization results indicated that acorrosion product film could form on the steel surface. Sevenday-old EPS at 240 mg L−1 inhibited corrosion while at muchlower and much higher concentrations, they accelerated cor-rosion. Furthermore, 14 day-old EPS and 28 day-old EPS at240 mg L−1 both promoted corrosion. It was found that the cor-rosion promotion was due to accelerated cathodic reaction.

(3) XPS analysis results confirmed that the corrosion products weremainly FeOOH and Fe2O3 with the oxygen as the electron accep-tor in the test using 7 day-old EPS at 240 mg L−1.

(4) After heat deactivation of EPS enzymes, 7 day-old EPS at240 mg L−1 inhibited corrosion more effectively.

Acknowledgements

This research was supported by Shenzhen strategic emerg-ing industry development special fund project (Project No.JCYJ20130401144744190), the Innovation Foundation of Huazhong

University of Science and Technology (Project No. 2015TS150,2015ZZGH010). We also acknowledge the support of the Analyt-ical and Testing Center of the Huazhong University of Science andTechnology for SEM and XPS measurements.

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