Nanometer growth of marine bio-films of different metallic alloys in seawater by electrochemical impedance spectroscopy

The main purpose of this study was to monitor the growth of the marine bio-film, of micro/macro-organisms, on the surface of different metallic alloys in seawater by electrochemical impedance spectroscopy (EIS). The alloys used in this study were; UNS 1020 carbon steel, stainless steel 304, stainless steel 316L, Sanicro 28, Cu–Ni 70–30, Hastelloy G-30, and titanium. The EIS was used to measure the A.C. Impedance (Z) and the double layer capacitance (Cdl) of the formed bio-film in seawater on a frequent basis. The total exposure time of the tests ranged between 90 days to 180 days. The visual inspection of the tested samples showed a bio-film formation on the surface of these samples. The microbiologically influenced (induced) corrosion (MIC) was observed only on the carbon steel. Monitoring the growth of the bio-film formation was accomplished by the EIS during the 90-180 days exposure of the tested samples. A gradual monitoring of the growth of the bio-film formation was achieved by mathematically correlating the obtained the A.C. Impedance (Z) and the double layer capacitance (Cdl) of the bio-film to the thickness of the bio-film formation. The advantage of EIS is a non-invasive technique with a sensing (spatial) resolution in a nanometer scale in a comparison to other techniques of monitoring the growth of bio-films on metallic alloys in aqueous solutions. © 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5026305


INTRODUCTION
It has been widely known that marine bio-fouling of metal surfaces in contact with seawater is the main cause of several technical problems and economic loss in industry.Marine bio-fouling is defined as the community of organisms found growing on permanently submerged surfaces of objects in seawater.It has been reported that a total of 84 species of fouling organisms belonging to 69 genera, 49 families, and 10 phyla were recorded over the entire year dependent on the temperature of the seawater. 1The growth of this community usually interferes with the efficient use of the surfaces.For example, the organism is known to reduce the flowing of the fluids through the pipelines, to reduce the heat transfer in the heat exchanger systems, to reduce the ship's speed and encrusting of the support legs of oil rigs. 2,3Marine micro fouling is defined as the growth of micro-organisms at metal-solution interfaces.Also, marine micro fouling can be even up to a few micrometers thick.It is known that marine micro fouling reduces heat transfer coefficient in heat exchange systems by as much as 40% besides causing what is known as microbiologically induced corrosion (MIC). 4,5Different types of bacteria cause corrosion by various mechanisms ranging from formation of differential aeration cells to production of aggressive environments through chemical changes. 6It is expected that the seasonal changes in the Gulf seawater will have an effect on the MIC, due to the growth of the marine biofouling film at metal-solution interfaces.In other words, the growth of the marine biofouling film at metal-solution interfaces would control the corrosivity of the submerged metals in seawater.
Therefore, in this study, roles of seasonal changes on the growth of the marine biofouling film at metal-solution interfaces will be investigated on different alloys such as UNS 1020 carbon steel, stainless steel 304, stainless steel 316L, Sanicro 28, Cu-Ni 70-30, Hastelloy G-30, and titanium.Measurements of the marine biofouling film of the seven alloys will be carried out by electrochemical impedance spectroscopy (EIS) for a period of 6 months under a continuous flowing seawater condition.

THEORETICAL ANALYSIS
In literature, the relationship between the A.C. Impedance (Z) and the double layer capacitance (C dl ) of a developed film on a metal sample to the thickness of the formed film is given as the following 7,8 |Z = 1 C dl = L/(e e o A) Where: Z is the A.C. impedance of the formed film, Ohm.cm 2 .C dl is the Double layer capacitance of the formed bio-film, µF. e o is the permittivity of the free space, 8.85 X 10 -14 Farad/cm.A is the area of the sample that exposed to the seawater, 1cm 2 .L is the thickness of the formed biofilm, nm, which can be obtained here by EIS, via equ.(1).
e is the static dielectric constant of the material under the investigation, of the biofilm.
In this investigation, the dielectric constant of the bio-film has been extended to account for the volume fraction of the seawater in the film, as biological membrane. 9,10So; e= (e bf + vsw esw), where e bf is the dielectric constant of the formed biofilm; e bf =5, when the pore fraction is equal to 0, an adhered thin layer. 9,10sw is the dielectric constant of the seawater, esw =70.2 at 18 o C 11 and esw =67.4,at 33 o C. 11 vsw is the volume fraction of the seawater in the film.The value of vsw =50% and 75% will be considered in the film as the film grows outward of the surface of the alloys.The value of esw =70.2 at 18 o C 11 will be considered at an exposure time of 0, 1, 5, 10, 20, 30, 60, 90 days, from the November to February season.Furthermore, the value of esw =67.4 at 33 o C 11 will be considered at an exposure time of 130, 150, 180 days, from the March to May season.
Equation (1) describes the relationship between the A.C. impedance and the double layer capacitance of the thickness of the biofilm.In other words, one can measure the thickness of the biofilm by knowing the A.C. impedance as well as the double layer capacitance of the biofilm from EIS measurements.

EXPERIMENTAL WORK
Metallic samples of UNS 1020 carbon steel (0.18-.23% C, 0.3-0.6%Mn, and balanced of Fe), stainless steel 304, UNS S304, (18-20% Cr, 8-12% Ni, 2% Mn, 1% Si, 0.08% C, and balance of Fe) stainless steel 316L, UNS S31603, (16-18% Cr, 10-14% Ni, 2-3% Mo, 2% Mn,1% Si, 0.03% C, and balance of Fe), Sanicro 28, UNS S 62800, (27% Cr, 31% Ni, 3.5% Mo, 0.002% C, and balance of Fe), Cu-Ni 70-30 (69% Cu, 30% Ni, 0.5% Fe, and 0.6% Mn), Hastelloy G-30 (44% Ni, 22% Cr, 7% Mo, 20% Fe, 2% Cu, and 0.015% C) and commercial pure titanium, UNS R50250, (0.2% Fe, 0.18% O, and balance of Ti) were used in this investigation.Those seven alloys have been selected in this study because of their common applications in seawater. 12All samples were mounted on a Teflon (Trade name) sample holder.The sample holder was especially designed to seal the samples from the surroundings, except the exposed area of 1 cm 2 .The exposed surface of each material was first cleaned with 5% HCl, and then mechanically polished with silicon carbide paper up to the finest grade, 1200 grit finish.The electrochemical test cell was designed to support the continuous flow of fresh seawater of the Gulf seawater and to facilitate the EIS measurements.The dimensions of the cell were 100 cm in length x 60 cm in height x 40 cm in width.The seawater was pumped from a depth of several meters, of Doha Desalination plant, in Kuwait, to storage tanks.Then the seawater was filtered prior to the electrochemical tests.The seawater was filtered in a way to allow microorganisms to pass through and to shield all unnecessarily items, i.e., sea-shells, barnacles, and so on, to the storage tank.This process was carried out on a frequent basis during the 180 days of exposure to seawater.More details on the experimental set up is given elsewhere. 13Also, the cell is equipped with a reference electrode which was a saturated calomel electrode (SCE), 241 mV versus SHE, and a counter electrode which was a graphite rod.Electrochemical impedance measurements [14][15][16] were performed using an EG&G impedance analyzer system model 6310, and the software used for analyzing the outputs was EG&G, 398 model (Trade names).The frequency range used was between 100 kHz to 10 mHz, and the voltage amplitude was ±5 mV root mean square (rms).Each EIS test was obtained after monitoring the opencircuit potential for 1 h.The output data of EIS were the Nyquist and Bode plots.From these plots, the A.C. Impedance (Z) and the double layer capacitance C dl were obtained.A simple Randles circuit was used for interpreting the EIS data.The Randles semi circle was used for data fitting of the experimental results.The acquired electrochemical data (Z & C dl ) were obtained from 11 tests in 180 days of exposure to seawater (0, 1, 5, 10, 20, 30, 60, 90, 120, 150, and 180 days) for each alloy.The temperature of the seawater during the tests was varied from 18 ○ C in winter days to 33 ○ C in the summer days.Also, the resistance of the seawater has an average value of 8 Ohms cm 2 .

RESULTS AND DISCUSSION
An example of an electrochemical impedance spectra of the stainless steel 304 in seawater is given in Figure 1 at different periods of time.The obtained data of the C dl and the calculated of L of the bio-film, from equ. (1), for the UNS 1020 carbon steel, stainless steel 304, stainless steel 316L, Sanicro 28, Cu-Ni 70-30, Hastelloy G-30, and titanium are given in Tables I-III.The value of L was calculated as a function of the volume fraction of seawater in the biofilm, vsw =0, 50, 75%, and the dielectric constant of the seawater at different temperature, esw =70.2 at 18 o C (November to February) & esw =67.4 at 33 o C (March to May).Tables I-III contain the calculated L of the bio-film of all alloys in seawater, when vsw =0%, vsw =50%, vsw =75%, respectively.
Figures 2-8 illustrate the growth of the bio-film (L) as a function of exposure time for the UNS 1020 carbon steel, stainless steel 304, stainless steel 316L, Sanicro 28, Cu-Ni 70-30, Hastelloy G-30, and titanium, respectively.In general, biofilms were visually observed on the seven alloys during the 0-180 days exposure.The L of the biofilm was visually estimated in a range of 20-50µm 13 .On the contrary, the L value of the biofilms, which corresponds to the obtained values of C dl , was calculated in the range of nanometer to fraction of micrometer, see Tables I-III In other words, the predicted L was much smaller than the visually estimated one.Also, it is obvious from the Figs.2-8 that as the volume fraction of the seawater (vsw) was observed to increase in the bio-film, as a biological membrane, the L value of the biofilm increased as well, in the outward direction from the surface of alloys.This implies that the closer the bio-film layer to the surface of the alloys, the more adhered the bio-film to the surface in a comparison to the outer layers of the biofilm.Consequently, the obtained C dl corresponds to the adhered biofilm layer, blue line in Figs.2-8, rather than the loosely biofilm layers, orange & gray lines in Figs.2-8.
In addition, it seems that the growth of the adhered biofilms has maintained a constant stable thickness during the 180 days of exposure of the alloys to seawater, Figs.2-8.In fact, there is no particular feature that one can tell on the effect of the seasonal temperature on the growth of the adhered bio-film, blue line in Figs.2-8.In contrast, the growth of the loosely layer of the biofilms, orange & gray lines in Figs.2-8, had different feature than the adhered layer of the bio-film.For instance, in the case of the carbon steel sample, Fig. 2, the layer has initially increased up to a range of 224.7nm-323 nm in the first 10 days of exposure.Then, the layer attained a steady thickness range between 150-250nm in the rest of the 90 days exposure time of the sample to seawater.Likewise, in the case of the stainless steel 316L (Fig. 4) and the Sanicro 28 (Fig. 5) similar behavior to the growth of the biofilm of the carbon steel was recorded.The test of the carbon steel samples was terminated after 90 days of exposure to the seawater due to the susceptibility of the samples to microbiological induced corrosion (MIC). 13n the contrary, the stainless steel 316L and the Sanicro 28 were observed free of any corrosion during the 180 days of exposure to  the seawater.In contrast, the bio-films on the surface of stainless steel 304 (Fig. 3), Cu-Ni 70-30 (Fig. 6), Hastelloy G-30 (Fig. 7) and the titanium (Fig. 8) were observed to grow in a random fashion, independent of the seasonal temperature.Furthermore, the stainless steel 304, Cu-Ni 70-30, Hastelloy G-30, and the titanium were found free of any corrosion during the 180 days of exposure to the seawater.
It is worth noting that there are other real-time techniques for monitoring MIC due to the growth of the bio-film on metallic alloys in seawater.For instance, the BioGeorge probe 17 (Trade name) and ALVIM 18,19 (Trade name) are electrochemical sensors of bio-film formation on metallic alloys in aqueous solutions.Both sensors work based on a signal acquisition of an applied current or a potential to two different electrodes in aqueous solutions, in which one of the electrode is susceptible to form a bio-film under the steady applied current or the steady potential.Consequently, a potential signal or a current signal would be detected under the Galvanostatic (constant current) or potentialstatic (constant potential), respectively, as the biofilm forms on the metallic sample.In other words, the acquisition of the electrochemical signal by both sensors corresponds to the depolarization of the polarized sample due to the gradual formation of the bio-film.Furthermore, both sensors are capable to detect bio-film in a micrometer scale.On the contrary, the disadvantages of both sensors are not only invasive methods to the electrochemical system but also have a lower sensing (spatial) resolution, in a comparison to the EIS.

CONCLUSIONS
The following conclusions have been drawn from the present investigation: 1.The EIS has been successfully applied to real-time monitoring the growth (L) of the biofilm of several metallic alloys in sweater, leading to the MIC in solely the carbon steel samples during 90 days of exposure.

FIG. 1 .
FIG. 1.The electrochemical impedance spectra of the stainless steel 304 samples in seawater at different periods of time.

2 .
EIS was found a non-invasive technique of monitoring the MIC due to the growth of the bio-film of metallic alloys in seawater in a comparison to other techniques.3. The sensing (spatial) resolution of EIS was found in a nanometer scale as compared to the other techniques, in a micrometer scale.4. The L value of the biofilms, which corresponds to the obtained values of C dl , was calculated in the range of nanometer to fraction of micrometer, as a function of the fraction of seawater in the biofilm, vsw =0,50,75%, and the dielectric constant of the seawater at different temperature, esw =70.2 at 18 o C (November to February) & esw =67.4 at 33 o C (March to May). 5.The growth of the adhered bio-film layer was observed to maintain a constant thickness during the 180 days of of alloys to seawater, blue line Figs.2-8. 6.The loosely biofilm layer was observed to grow in a random fashion during the 180 days of exposure of the alloys to seawater, orange and gray lines in Figs.2-8, independent of the seasonal temperature.

TABLE I .
The EIS data of C dl & calculated L of the bio-film of all alloys in seawater, vsw =0%.

TABLE III .
The EIS data of C dl & calculated L of the bio-film of all alloys in seawater, vsw =75% and esw =70.2 at 18 o C (0, 1, 5, 10, 20, 30, 60, 90 days) & esw =67.4 at 33 o C (120, 150, 180 days).The growth of the bio-film as a function of exposure time of the carbon steel to seawater.The lines represent vsw =0% (blue), vsw =50% (orange), and vsw =75% (gray) in the bio-film.The average temperature of the seawater was 18 o C during the 90 days, November-February.The growth of the bio-film as a function of exposure time of the Sanicro 28 in seawater.The lines represent vsw =0% (blue), vsw =50% (orange), and vsw =75% (gray) in the bio-film.The average temperature of the seawater was 18 o C during the 1 st 90 days, November-February and was 33 o C during the 2 nd 90 days March-May.The growth of the bio-film as a function of exposure time of the Titanium in seawater.The lines represent vsw =0% (blue), vsw =50% (orange), and vsw =75% (gray) in the bio-film.The average temperature of the seawater was 18 o C during the 1 st 90 days, November-February and was 33 o C during the 2 nd 90 days March-May.