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\DOI{10.5802/crchim.227}
\datereceived{2022-11-14}
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\dateposted{2023-04-11}
\begin{document}
%\copyeditor{RK}
\begin{noXML}

%\TopicEF{Materials and Clean Processes for Sustainable Energy and
%Environmental Applications}{Mat\'eriaux et proc\'ed\'es propres pour
%des applications \'energ\'etiques et environnementales}

\title{Influence of sulfate-reducing bacteria on the biocorrosion of mild
steel coated with hybrid polyetherimide-ZnO or CuO bilayer composites}

\alttitle{Influence des bact\'eries sulfato-r\'eductrices sur la
biocorrosion de l'acier doux rev\^etu de composites bicouches hybrides
poly\'etherimide-ZnO ou CuO}

\author{\firstname{Emna} \lastname{Rahali}}
\address{Laboratory for Wastewater and Environment, Water Researches
and Technologies Center (CERTE), Technopark of Borj Cedria, PB 273,
Soliman 8020, Tunisia}
\address{National Institute of Applied Science and Technology (INSAT),
Carthage University, Tunis, Tunisia}
\email[E. Rahali]{rahali93@hotmail.com}

\author{\firstname{Leila} \lastname{El-Bassi}\CDRorcid{0000-0002-3144-4435}\IsCorresp}
\addressSameAs{1}{Laboratory for Wastewater and Environment, Water
Researches and Technologies Center (CERTE), Technopark of Borj Cedria, PB
273, Soliman 8020, Tunisia}
\email[L. El-Bassi]{l.elbassi@gmail.com}

\author{\firstname{Latifa} \lastname{Bousselmi}\CDRorcid{0000-0002-0699-4806}}
\addressSameAs{1}{Laboratory for Wastewater and Environment, Water
Researches and Technologies Center (CERTE), Technopark of Borj Cedria, PB
273, Soliman 8020, Tunisia}
\email[L. Bousselmi]{latifa.bousselmi.certe@gmail.com}

\author{\firstname{Marta M.} \lastname{Alves}\CDRorcid{0000-0002-9679-4022}}
\address{Centro de Qu\'{i}mica Estrutural, Institute of Molecular
Sciences, Departamento de Engenharia Qu\'{i}mica, Instituto Superior
T\'{e}cnico, Universidade de Lisboa, Av.
Rovisco Pais 1049-001, Lisboa, Portugal}
\email[M. M. Alves]{marta4alves@gmail.com}

\author{\firstname{Maria~de~F\'{a}tima} \lastname{Montemor}\CDRorcid{0000-0001-7835-6814}}
\addressSameAs{3}{Centro de Qu\'{i}mica Estrutural, Instituteof
Molecular Sciences, Departamento de Engenharia Qu\'{i}mica, Instituto
Superior T\'{e}cnico, Universidade de Lisboa, Av.
Rovisco Pais 1049-001, Lisboa, Portugal}
\email[M. F. Montemor]{mfmontemor@tecnico.ulisboa.pt}

\author{\firstname{Hanene} \lastname{Akrout}\CDRorcid{0000-0002-4878-6108}}
\addressSameAs{1}{Laboratory for Wastewater and Environment, Water
Researches and Technologies Center (CERTE) Technopark of Borj Cedria PB
273, Soliman 8020, Tunisia}
\email[H. Akrout]{Hanene.akrout@yahoo.com}

\shortrunauthors

\keywords{\kwd{Biocorrosion}
\kwd{\textit{Desulfovibrio} sp}
\kwd{Oxide}
\kwd{EPS}
\kwd{Inhibition}}

\altkeywords{\kwd{Biocorrosion}
\kwd{\textit{Desulfovibrio} sp}
\kwd{Oxyde}
\kwd{EPS}
\kwd{Inhibition}}

\begin{abstract}
Mild steel (MS) coated with protective bilayer hybrid coatings of
polyetherimide, and ZnO/CuO was exposed to artificial treated 
wastewater (ATWW) inoculated with \textit{Desulfovibrio} sp. 
(biotic system). Proteins from the extracellular polymeric substances 
(EPS) matrix of \textit{Desulfovibrio} sp. responded to the coatings. 
PEI-CuO offered the highest performance against biocorrosion. The higher
PEI-CuO resistivity value of $2.17\times
10^{6}~\rmOmega{\cdot}\mathrm{cm}^{2}$ contrasted with the decrease to
$1.5\times 10^{5}$ and $1.6\times 10^{3}~\rmOmega{\cdot}\mathrm{cm}^{2}$
for PEI-ZnO and PEI coatings, respectively. The one order of magnitude
increased resistance of the biotic system, compared to sterilized ATWW
(abiotic system), resulted from the protective nature of the bacterial
biofilm.
\end{abstract}

\begin{altabstract}
L'acier doux (MS) recouvert de rev\^etements hybrides bicouches
protecteurs de poly\'etherimide et de ZnO/CuO a \'et\'e expos\'e \`a
des eaux us\'ees artificielles (ATWW) inocul\'ees par \textit{Desulfovibrio} sp.
(syst\`eme biotique). Les prot\'eines de la matrice des substances
polym\`eres extracellulaires (EPS) de \textit{Desulfovibrio} sp. ont r\'eagi aux
rev\^etements. Le PEI-CuO a offert les meilleures performances contre
la biocorrosion. La valeur de r\'esistivit\'e plus \'elev\'ee de
PEI-CuO de  $2{,}17\times 10^6~\rmOmega{\cdot}\mathrm{cm}^2$ contraste avec la
diminution \`a respectivement  $1{,}5\times 10^5$ et $1{,}6\times
10^3~\rmOmega{\cdot}\mathrm{cm}^2$ pour les rev\^etements PEI-ZnO et PEI.
La r\'esistance accrue d'un ordre de grandeur du syst\`eme biotique, par
rapport \`a l'ATWW st\'erilis\'e (syst\`eme abiotique), r\'esulte de la
nature protectrice du biofilm bact\'erien.
\end{altabstract}


\maketitle
\vspace*{20pt}
\twocolumngrid

\end{noXML}


\section{Introduction}\label{sec1}

Due to its composition, wastewater is the most complex matrix that
favors microbial growth on several metal surfaces under short periods,
particularly on steel commonly used in industrial
equipments~\cite{1}. It comprises two fractions, an organic and an
inorganic one. The former is mainly composed of carbohydrates, proteins,
lipids and industrial residues, where as phosphorus, chlorides,
nitrogen, and limited amounts of heavy metals comprise the latter
fraction~\cite{2}. In fact, microorganisms adhere to the steel surface
where they colonize, reproduce and finally form a biofilm, affecting
the kinetics of the cathodic and anodic corrosion
processes~\cite{3,4,5}. This phenomenon is called biocorrosion or
microbiologically influenced corrosion (MIC)~\cite{6}. 



Well-known microorganisms in steel biocorrosion are the
sulfate-reducing bacteria (SRB)~\cite{7,8,9}. SRB can aerobically
produce sulfide from sulfate and thiosulfate reduction~\cite{10},
which is considered as the highest risk factor in biocorrosion
processes~\cite{7,8,9,10,11}. Besides, SRB can use $\mathrm{O}_{2}$ and
$\mathrm{Fe}^{3+}$ and oxidize hydrogen; they can also utilize
aliphatic and aromatic hydrocarbons, and link sulfate reduction to
intracellular magnetite production~\cite{10}. Furthermore, bacterial
sessile cells within the SRB biofilm have the ability to harvest energy
from $\mathrm{SO}_{4}^{2-}$ reduction for metabolic activities.
Therefore, SRB can reduce $\mathrm{SO}_{4}^{2-}$ to $\mathrm{HS}^{-}$
and the $\mathrm{Fe}^{2+}$ generated by the anodic process then reacts
with $\mathrm{HS}^{-}$ {to form sulfide precipitates (FeS) in the
biofilm. FeS is the typical corrosion product formed when mild steel (MS)
is exposed to an environment containing SRB~\cite{12,13}. Chen
\etal~\cite{14} proved that the presence of SRB in seawater could
generate localized corrosion in carbon steel. The authors reported that
the cathodic and anodic areas present different morphological features
in the presence of SRB strain. In fact, the corrosion products formed
in the cathodic area were more compact than those grown in the anodic
areas. Likewise, Li \etal~\cite{15} studied bacterial distribution in
SRB biofilms. They showed that SRB bacteria could generate both forms
of corrosion: localized and uniform, and that bacterial distribution in
biofilms varied, depending on the type of corrosion. In the presence of
localized corrosion, bacterial cells were distributed at the bottom of
the biofilm, whereas in generalized corrosion, cells gathered outside
the SRB biofilm~\cite{15}.

Apart from $\mathrm{SO}_{4}^{2-}$, chlorides present in the
wastewaters are critical for pitting formation~\cite{16}. El-Bassi
\etal~\cite{16} investigated the effect of chloride in the biocorrosion
of carbon steel immersed in wastewaters. They reported that pitting is
one of the primary forms of corrosion caused by this anion~\cite{16}.
Ferrous ions react with the chlorides to form Fe(OH)$_2$ and
consequently release H$^{+}$ and Cl$^{-}$. The described cycle is
repeated until the protective layer of
$\mathrm{Fe}_{2}{\mathrm{O}}_{3}$ or  $\mathrm{Fe}_{3}\mathrm{O}_{4}$
is entirely abolished. In the presence of biofilm, oxidizing iron
reacts with $\mathrm{Cl}^{-}$, as follows 
(Equations~(\ref{eq1})~and~(\ref{eq2})):
{\begin{eqnarray}
\mathrm{Fe}^{2+}+2\mathrm{Cl}^{-}&\leftrightarrows &
\mathrm{FeCl}_{2} \label{eq1}\Seqnsplit
\mathrm{FeCl}_{2}+2\mathrm{H}_{2}\mathrm{O}&\leftrightarrows &
\mathrm{Fe}(\mathrm{OH})_{2}+2\mathrm{H}^{+}+ 
2\mathrm{Cl}^{-}\label{eq2}
\end{eqnarray}}\unskip

In this context, Ziadi \etal~\cite{12} reported that SRB could induce
pitting corrosion in the first stage of biocorrosion on stainless steel
immersed in wastewater, which produced hydrogen sulfide (H$_{2}$S) or
iron sulfide (FeS$_{2}$) by $\mathrm{SO}_{4}^{2-}$ reduction~\cite{12}.
Rajala \etal~\cite{17} investigated carbon steel biocorrosion in anoxic
groundwater containing SRB and methanogenic archaea (MA)}\cite{17}.
They reported that the corrosion rate in abiotic system ($1~\upmu$m/y)
was the lowest among the studied systems; however, in biotic
environments, the corrosion rates (between $5~\upmu$m/y and
$10~\upmu$m/y) are higher in SRB-enriched environments and lower in SRB
and MA environments~\cite{17}. 

Among the SRB group, several studies investigated the impact
of \textit{Desulfovibrio} sp. on steel in different
environments~\cite{18,19}. Wikie \etal~\cite{20} evaluated the\break 
negative effect of \textit{Desulfovibrio Alaskensis} {sp}{.} biofilms
on the corrosion progress of carbon steel in marine environments. The
biofilm that settled on the MS surface was a complex structure
composed mainly of bacteria, metallic ions, extracellular polymeric
substances (EPS), and biocorrosion products~\cite{21,22}.

Polymeric coatings are relevant in preventing corrosion, particularly to
overcome microbiologically influenced corrosion (MIC). This strategy is 
of primary relevance when the coatings are composed of non-toxic 
substances such as polyetherimide (PEI)~\cite{23}.
Two essential factors should be considered when selecting additives for
coatings' design, which are (i) high resistance to bacterial attack and
(ii) absence of corrosion products formation as a result of the coating
degradation~\cite{24}. To enhance the protectiveness of coatings in
treated wastewaters, several metal oxides, like $\mathrm{TiO}_{2}$,
have been tested as microbial growth inhibitors as possible
alternatives to traditional and toxic biocides~\cite{25}. Ziadi
\etal~\cite{12} demonstrated that incorporating $\mathrm{TiO}_{2}$
nanoparticles in a silane coating can mitigate biocorrosion of
stainless steel in contact with treated urban wastewaters~\cite{12}.

Zinc and copper oxides have shown antibacterial effectiveness in
several applications~\cite{26,27}. Thus, metal oxide additives for
antimicrobial polymers are considered safe solutions with high
antibacterial efficiency, and improved polymer stability and
maintenance of its functional properties~\cite{28}.

Previously, Rahali \etal~\cite{29} studied the impact of hybrid
coatings on the corrosion progress of MS in simplified
abiotic environments (3.5~wt\% NaCl) and reported that the
corrosion resistance of MS was enhanced by ZnO and CuO, leading to a
protective efficiency equal to 70\% and 99\%, respectively. This good
efficiency is probably due to the higher coating thickness and smaller
pores present in the outer layer~\cite{29}. The proven efficiency for
MS corrosion protection in abiotic environments makes these coatings
worthy of exploring in complex biotic environments. 

Thus, the present work aims to understand the biocorrosion progression
of MS coated with hybrid PEI composite bilayers in the
presence of ZnO or CuO. To reach this objective, uncoated and coated MS
samples were exposed to a biotic system simulated by ATWW inoculated with 
\textit{Desulfovibrio} sp. The biofilm and the EPS layers were
characterized, namely the polysaccharide, protein and lipid contents.
The relationship between these layers' compositions and the coatings is
discussed. The protectiveness of the coatings in the biotic system was
compared with an abiotic system (sterilized artificial treated
wastewater, SATWW). This protectiveness was assessed using
electrochemical impedance spectroscopy (EIS) and through a deep
physicochemical analysis of the corrosion layers formed on the bare and
coated MSsurface. 

\section{Materials and methods}\label{sec2}

\subsection{Bacterial strain and cultivation conditions}\label{sec21}

The strain \textit{Desulfovibrio} sp. (MK782593), used as a model of
SRB, was isolated from a previous
study~\cite{30}. Briefly, for cultivation and isolation of
\textit{Desulfovibrio} sp., the biofilm formed on the surface of 304 L
SS immersed in treated urban wastewater was collected after 3 days of
immersion. The biofilm collected was cultivated in SRB selective medium
(consisting of 0.5 g of K$_{2}$HPO$_{4}$, 1.0~g of NH$_{4}$Cl, 0.5~g of
Na$_{2}$SO$_{4}$, 0.1~g of CaCl$_{2}$, 2.0~g of MgSO$_{4}$, 3.5~g of
C$_{3}$H$_{5}$NaO$_{3}$ and 1.0 g of ascorbic acid in 1~L of distilled
water at pH  $7.20 \pm 0.02$).

The inoculated medium was incubated at 30~\textdegree C for 21 days when
black deposits and hydrogen sulphide odour were detected. After this
period, SRB suspensions were cultivated on Petri dishes in the presence
of nutrient agar. Isolation consisted of three successive subcultures
of each strain and then inoculation into liquid media. Before use, the
strain \textit{Desulfovibrio} sp. was re-cultivated in SRB selective
medium. Bacterial inoculation was carried out using a cell suspension
at the exponential growth phase~\cite{12}.
\vspace*{-5pt}

\subsection{Preparation of mild steel (MS) coupons}\label{sec22}

DC01 carbon steel (CS) with chemical composition (wt\%): 
$\mathrm{C} < 0.12$; $\mathrm{Mn} < 0.60$; $\mathrm{P} < 0.045$; 
$\mathrm{S}< 0.045$.
Steel coupons (2.5 cm~$\times$~2.5 cm) were abraded with silicon carbide
(SiC) papers of 230, 320, 600, 1000, and 1200 grit and rinsed with
distilled water. Finally, samples were degreased with ethanol in an
ultrasounds bath and dried with an air stream.
\vspace*{-5pt}

\subsection{Synthesis of sol--gel coating}\label{sec23}
\vspace*{-3pt}

As previously described, the hybrid coating was composed of two layers
applied in separate steps~\cite{29}. Briefly, the first polymeric
layer was synthesized by mixing PEI (Sigma-Aldrich) in
15\% N,N-dimethyl-acetamide (DMAc, Fluka). The coating was applied by
dip-coating, employing a dip coater RSC15 (Bungard Elektronik) controlled
by a SIEMENS SPS unit. The coated samples were cured in an oven for 4~h 
at 160~\textdegree C. The second layer was synthesized precisely as the
previous one, where CuO or ZnO particles (0.123~g/L) were added to the
polymeric solution.
\vspace*{-5pt}

\subsection{Coupons incubations with \textit{Desulfovibrio} sp. strain}
\label{sec24}
\vspace*{-5pt}

Anaerobic vials (250~mL) were filled with 100~mL ATWW (Table~S1). 
Bare and coated steel coupons were
added to each vial before inoculation. Then, each vial was inoculated
with $100~\upmu \mathrm{L}$ of overnight cultivation of SRB suspension
($\mathrm{O.D.}_{660~\mathrm{nm}}\sim1$). The coupons were
sampled after 7~days of immersion, gently washed, and used for
microbiological characterization.
\vspace*{-3pt}

\subsubsection{Extracellular polymeric substances (EPS) extraction and
characterization} \label{sec241}
\vspace*{-2pt}

Bacterial biofilms from the SRB strain were recovered from the MS
surface immersed in artificial urban wastewaters. The recovered biofilm
was washed with 0.9~wt\% NaCl, and suspended in a 5~mL saline
solution (0.9~wt\% NaCl). Biofilm suspensions were then centrifuged
at 12,000 rpm for 20 min at 4~\textdegree C to obtain the EPS
supernatant. EPS extraction was performed according to the method
previously described by Liu \etal~\cite{31}, using EDTA (2\%) as an
extractant. Briefly, 1~mL of EDTA was added to each test tube with
biofilm suspension and rested for 3~h at 4~\textdegree C. The resulting
suspension was filtered through a $0.2~\upmu\mathrm{m}$ membrane,
followed by a dialysis membrane filtration (3500~Da). The obtained
filtrates were lyophilized for 48~h at $-50$~\textdegree C, and the
recovered EPS was quantified using an electronic balance~\cite{31}. 

The polysaccharide content of EPS was determined using the
phenol-sulfuric acid assay with glucose as standard~\cite{32}. The
protein content of EPS was measured by the Bradford assay using casein
as the standard~\cite{33}. The sulfo-phospho-vanillin method was used
to determine the lipid content~\cite{34}. All EPS analysis was
performed in duplicate.
\vspace*{-1pt}

\subsubsection{Surface characterization}\label{sec242}
\vspace*{-2pt}

The morphological observation of the bare and coated samples before and
after immersion in ATWW, with or
without bacteria, was characterized using scanning electron microscopy
(SEM) (PhenomProX G6, JEOL-JSM7001F). The X-ray energy dispersive
spectrometer (EDS) was used to assess the elemental chemical
composition. The conductivity of the samples was improved with a thin
coating of conductive gold/palladium (Polaron E-5100).

An optical camera assessed the water contact angle on the immersed
coated surfaces. In brief, a droplet of water of ${\sim}5~\upmu
\mathrm{L}$ was placed on the coating surface using a tension meter
(AttentionTheta T200).
\vspace*{-5pt}

\subsubsection{Electrochemical measurements}\label{sec243}

The electrochemical impedance spectroscopy (EIS) technique was used to
investigate the interface behaviors of the bare and coated MS 
in both biotic and abiotic systems using an experimental
setup with a three electrodes conventional cell. A potentiostat
Voltalab PGZ301 was used. EIS measurements were undertaken at open
circuit potential ($E_{\mathrm{oc}}$), under a sinusoidal perturbation
of ${\pm}10$~mV for frequencies ranging from 65~kHz to  10~mHz, with an
acquisition frequency of 10 points per decade. The EIS measurements
were performed after 7~days. Each experiment was conducted in
duplicate. The experimental impedance spectra were analyzed and fitted
using the Zsimpwin version 3.30d (EChemSoftware, Ann Arbor, MI, USA)
with suitable equivalent circuit models; to ensure the fitting
reliability, only regressions with a chi-square of ${\leq}10^{-3}$ were
accepted.
\vspace*{-5pt}

\subsubsection{Statistical analyses}\label{sec244}

Both the values of the amount and relative composition of the EPS
content are averaged values of at least three measurements, and the
error bars represent their standard deviations.
\vspace*{-5pt}

\section{Results and discussions}\label{sec3}

\subsection{\textit{Desulfovibrio} sp. biofilms formed on bare and
coated MS immersed in artificial treated wastewater}\label{sec31}

Following the efficacy of PEI-ZnO and PEI-CuO to protect MS under
simplified biotic conditions (3.5~wt\% NaCl)~\cite{29}, the coatings
were tested in quasi-real conditions. Artificial treated wastewater
(ATWW) with bacteria was selected as a biotic system model in this
studyand was compared with an abiotic system of sterilized ATWW (SATWW).

SEM observations were used to characterize the surface of MS, PEI,
PEI-ZnO, and PEI-CuO before and after immersion in ATWW with
or without \textit{Desulfovibrio} sp. (Figure~S1, Figure~\ref{fig1}).

\begin{figure*}[t!]
\includegraphics{fig01}
\vspace*{-3pt}
\caption{\label{fig1}Top view SEM images of (a) MS, (b) PEI, (c) PEI-ZnO
and (d) PEI-CuO immersed in biotic system, (e) MS, (f) PEI, (g) PEI-ZnO
and (h) PEI-CuO immersed in abiotic system; insets are magnified SEM
images.
%\vspace*{-10pt}
}
\end{figure*}

Figure~\ref{fig1}a--d shows the morphological features of the biofilm
formed on bare and coated MS in \textit{Desulfovibrio} sp. inoculated
media. An abiotic assay was performed using the above-indicated coating
but without SRB strain inoculation (Figure~\ref{fig1}e--h). In this
medium, only corrosion products are noticed on the steel surface
(Figure~\ref{fig1}e--h), contrasting with the biotic medium where
corrosion products and individualized SRB cells can be clearly detected
(Figure~\ref{fig1}a--d). Similar results were attained by Rasool
\etal~\cite{35}. In particular, the observation of the bare MS surface 7
days after immersion (Figure~\ref{fig1}a) shows an adherent homogeneous
biofilm, confirming the easiness of SRB to establish and progress to a
biofilm. In PEI-coated MS (Figure~\ref{fig1}b--d), the SEM observation
shows fewer bacterial colonies scattered on the surface. Changes in
bacterial colonization could be related to differences between MS and
MS/PEI surfaces in terms of structure and/or physicochemical properties
of PEI.\looseness=-1

The roughness in the PEI coating can influence the dynamic of biofilm
development and other microbial interactions. Aguirre \etal~\cite{36}
reported that electrostatic adherence between coatings and
microorganisms could lead to leakage of microbial contents and
disruption of the cell wall.

A more heterogeneous structure for the biofilm (Figure~\ref{fig1}c) was
observed on the coated MS after immersion for the PEI-ZnO coating. An
obvious deformation on the bacterial biofilm surface occurred compared
to the non-coated steel (Figure~\ref{fig1}a). This damage could be
attributed to the bactericide effect of PEI-ZnO, which altered the
homogeneity of the deposited bacterial biofilm. Rasheed \etal~\cite{37}
{demonstrated that chitosan-$\mathrm{ZnO}_{\mathrm{NPs}}$ (CZNC-10)
coatings on carbon steel had an inhibitory effect on SRB biofilm
establishment after 7 days of incubation, with a significant biofilm
deformation. This deformation may be due to the inhibition efficiency
of chitosan-$\mathrm{ZnO}_{\mathrm{NPs}}$, which makes the SRB more
susceptible to damage~\cite{37}. The antibacterial activity of ZnO has
been proven by several studies~\cite{38}. Malini \etal~\cite{39}
revealed that the presence of ZnO in membranes blocked biofilm
formation and demonstrated antifouling activity on wastewater
filtration membranes. In addition, Dhillon \etal~\cite{40} noted strong
anti-biofilm activity of chitosan-$\mathrm{ZnO}_{\mathrm{NPs}}$ against
\textit{Staphylococcus aureus} and \textit{Micrococcus luteus even}
when small concentrations of ZnO nanoparticles were added. The
antibacterial activity of ZnO is based on the interaction of the
particles with bacteria.This interaction leads to the release of
$\mathrm{Zn}^{2+}$ and causing to the deterioration of bacterial cell
walls, lipids, proteins and  DNA~\cite{41}.

A pronounced bactericide effect was observed with the PEI-CuO coating
(Figure~\ref{fig1}d), in which the number of SRB individual colonies
was significantly decreased with a notable complicated and porous
structure. The literature demonstrates that CuO had a bactericide
effect on \textit{Desulfovibrio marinisediminis} GSR3 strain and on
\textit{Pseudomonas aureus} by inactivating the catabolic metabolism
and biosynthesis mechanismson the latest~\cite{42,43,44}.\looseness=-1

Fathy \etal~\cite{45} investigated styrene
N-vinylpyrrolidone nanocomposites' efficiency when combined with a metal
oxide (CuO or ZnO) against SRB-induced biocorrosion. Authors
demonstrate that the SRB biocidal activity on coated MS was higher for
the copolymer/ZnO (3\%) and CuO (0.3\%) coatings~\cite{45}. According to the
literature~\cite{29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46},
the abrasive surface shape of ZnO
and CuO particles can destroy bacterial membranes. Also, the presence
of $\mathrm{Zn}^{2+}$ and $\mathrm{Cu}^{2+}$ from particles can be a
possible variant of antibacterial activity, as these cations can
disrupt amino acid metabolisms, enzyme systems and transport of
substances into cells, subsequently inhibiting bacterial cell
growth~\cite{47}.


The altered biofilm structure and corrosion products layer (biolayer),
influenced by the metal oxides in the coatings, affects the surface
properties of both bare and coated metal. If, on the one hand, it is
possible to predict bacterial adhesion by the water contact angle (WCA) 
analysis of the coatings, on the another hand, it is possible to assess the
biocorrosion progression by analysing the WCA of the biolayers formed
over these coatings. WCA measurements were used to
assess such alterations, as presented in (Figure~\ref{fig2})}.

\begin{figure}[t!]
\includegraphics{fig02}
\caption{\label{fig2}Water contact angles of bare and coated steel
before and after immersion in biotic system; the contact angle of
samples before immersion are taken from the authors' previous 
work~\cite{29}.}
\end{figure}

%\break
Based on previous work~\cite{29}, WCA values for PEI and PEI-coatings
are recorded at 84\textdegree~and increased to 92\textdegree~in the
presence of CuO~\cite{29}. According to the literature, the
hydrophobicity of a surface is a major factor governing bacterial
adhesion~\cite{48}. However, the importance of this parameter is
related to the type of bacteria~\cite{49}. In fact, hydrophilic
bacteria like {\textit{Desulfovibrio desulfuricans}} prefer to adhere
to hydrophilic surfaces~\cite{50}. Chen \etal~\cite{50}
reported that an increase in the hydrophobicity of the CS surface
resulted in a remarkable reduction of SRB adhesion. This trend was not
verified as bare MS, with the highest WCA, was the surface with more
bacteria. In contrast, the PEI-CuO coating, with the highest WCA
amongst the coatings, had few bacterial adhesions
(Figure~\ref{fig2}). This was further proved by the hydrophilicity
of the layers formed on bare and coated MS when immersion in the biotic
system. Upon immersion in ATWW with \textit{D. desulfuricans}, it was
noticed that WCA were dramatically decreased (Figure~\ref{fig2}). This
decrease can be due to the type of \unskip\break biolayer form on MS in this
medium~\cite{51}. These biolayers caused a decrease in the WCA of
approximately 90\textdegree~for the bare MS, 72\textdegree~for PEI,
68\textdegree~for PEI-ZnO and 36\textdegree~for the PEI-CuO coatings
(Figure~\ref{fig2}). These findings are in line with the decreased
biofilm and corrosion products formation on bare MS $>$ PEI $>$ PEI-ZnO
$>$ PEI-CuO (Figures~\ref{fig1} and~\ref{fig2}).


\subsection{Impact of \textit{Desulfovibrio} sp. on EPS production and
characterization}\label{sec32}

To further explore SRB settling, the production of EPS, a key 
component in evaluating microbial growth and
bacterial biofilm establishment on surfaces, was studied~\cite{30}.
Thus, further EPS-produced biofilm characterization was made to assess
the coating effect on the bacterial biofilm. It is worth mentioning
that the presence of SRB strain is well recognized to enhance EPS
production, which facilitates biofilm adhesion and shows its ability to
produce EPS, even under small biomass
concentrations~\cite{37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52}.

Figure~\ref{fig3} reports the EPS yields extracted from biofilms grown
on the different coated and bare MS surfaces after 7 days of immersion in
ATWW with \textit{Desulfovibrio} sp. The produced EPS mass was more
relevant in the case of the uncoated MS (Figure~\ref{fig3}). In the
case of the PEI coating, a decrease in EPS mass of about 4\% was
observed compared to the non-coated MS (Figure~\ref{fig3}), indicating
that the PEI coating was not primarily affecting EPS production by
\textit{Desulfovibrio}~sp. Previous studies demonstrated that PEI does
not exhibit bactericide activity~\cite{11,53} and suggest that the
decrease in biofilm production in the presence of PEI maybe somehow
related to anti-adherence mechanisms rather than direct biocidal
effects~\cite{11}.

\begin{figure}[t!]
\includegraphics{fig03}
{\vspace*{-2pt}}
\caption{\label{fig3}EPS weights (mg) obtained of the biofilm grown on
bare and coated mild steel after 7 days of immersion in the biotic
system.}
{\vspace*{-4pt}}
\end{figure}

\begin{figure*}[t!]
\includegraphics{fig04}
\caption{\label{fig4}(a)~Relative composition of the EPS content and
evolution of the EPS, (b) protein content, (c)
polysaccharide and (d) lipid of the extracted EPS from the
biofilm grown on bare and coated MS immersed in ATWW.\vspace*{3pt}}
\end{figure*}

In the case of PEI-ZnO and PEI-CuO coatings, EPS production decreased
compared to the bare MS, about 34\% and 48\%, respectively
(Figure~\ref{fig3}). When added to PEI, Zn or Cu oxides had a notable
decrease rate on the EPS weight (Figure~\ref{fig3}), but it was not
completely inhibited. In fact, the ZnO-PEI composite exhibits high
inhibition rates against \textit{S. aureus} and \textit{E.~coli} even
after being subjected to harsh simulated conditions~\cite{53}. 

Hewayde \etal~\cite{54} investigated the toxicity of CuO to SRB. The
concrete sewer pipes coated with CuO exhibited antimicrobial
characteristics against \textit{Desulfovibrio desulfricans}
isolated from a lab-scale anaerobic digester and showed 99\%
inhibition against bacteria as well as in their EPS production. While
the EPS production observed in our work aligns with Hewayde's
\etal~\cite{54} report, other points in the opposite direction of
EPS enhanced production to preserve the inner microorganisms from harsh
external environmental conditions~\cite{19,20}. Fang
\etal~\cite{55} demonstrated Cu$^{2+}$ as being the most toxic
element to SRB among all tested metals and chemicals. In fact, exposure
of SRB to Cu$^{2+}$ slightly stimulated the production of EPS.
Likewise, Miao \etal~\cite{56} demonstrated that the EPS content
was considerably increased in response to CuO-nanoparticles exposure in
wastewaters. $\mathrm{Zn}^{2+}$ proved less inhibitory to SRB activity,
yet it greatly stimulated EPS production~\cite{55}. Yue \etal~\cite{57}
investigated EPS production by \textit{Desulfovibrio desulfuricans} 
strain when Cu$^{2+}$ and $\mathrm{Zn}^{2+}$  were added to the culture
medium. Authors demonstrated that the EPS extracted from bacteria 
exposed to $\mathrm{Zn}^{2+}$ increased the binding affinity for all
heavy metals, therefore reducing the inhibitory impact of Cu$^{2+}$ on
the SRB~\cite{57}. A similar result was proved by Zhao \etal~\cite{58},
where nano-ZnO's presence accelerated the EPS production from
\textit{Chlorella vulgaris}.


These contradictory data prompt us to investigate further the effect
each coating caused on the EPS content (Figure~\ref{fig4}). The content
distribution of the EPS extracted from the \textit{Desulfovibrio} sp.
biofilm established on the coated MS is reported in Figure~\ref{fig4}.
Protein content represented the highest component among EPS contents
(Figure~\ref{fig4}a). The protein content was about 70 and
$74~\upmu\mathrm{g}/\mathrm{mg}$ for bare MS, and PEI-coated steel,
respectively (Figure~\ref{fig4}a), which underlined that the PEI
coating did not impact protein production. The protein content increased
to 120 and  $139~\upmu\mathrm{g}/\mathrm{mg}$ in the presence of
PEI-ZnO and PEI-CuO coated MS (Figure~\ref{fig4}a). The protein content
increased in the presence of the different coatings to a maximum value
of 34.5\% for PEI-CuO coated steel. According to Figure~\ref{fig4}c,
the polysaccharide content is between 22\% of the EPS content for
PEI-CuO as a minimum value, and 27\% for MS as the maximum value. So
the addition of metal oxides to the coatings significantly increased
protein content but caused a low variation in the polysaccharide
content.

These results agree with P\'{e}rez \etal~\cite{59} that 
\textit{Desulfovibrio} sp. proteins are the main constituents of
biofilms formed on CS when immersed in synthetic seawater. Also, Chen
\etal~\cite{60} demonstrated that \textit{Desulfovibrio vulgaris}
counteracted CuO nanoparticles stress by increasing the protein content
of EPS to protect bacterial biofilm from toxic elements~\cite{56,57}.
Miao \etal~\cite{56} found a dominance of protein contents, compared to
polysaccharides, in response to CuO nanoparticles. Likewise, EPS
containing a higher amount of protein was reported in the presence of
zinc oxide~\cite{58}. On the contrary, Yue \etal~\cite{57} demonstrated
that $\mathrm{Cu}^{2+}$ and $\mathrm{Zn}^{2+}$ added to the culture
medium of \textit{Desulfovibrio desulfuricans} increased
polysaccharides and reduced protein contents. This was explained by a
modification in the carboxyl group of proteins and in the C--O--C group
of polysaccharides contained in the EPS that caused an improved binding
capacity of the metals~\cite{60}.


In our specific case, the particles embedded within the coatings
modified EPS production in such a way that protein production was
favored over polysaccharide production, most probably due to the
combination of PEI with Cu or Zn ions release.

The lipid content in the extracted EPS (Figure~\ref{fig4}a) did not
exceed 6~$\upmu$g/mg for bare and coated steel. Bare MS has the highest
lipid content, with a percentage of 39.5\% (Figure~\ref{fig4}c), while
the lowest was detected for PEI-CuO (1.85~$\upmu$g/mg). Conrad
\etal~\cite{61} indicated that the lipid fraction impacts biofilm
adhesion to the substrate and influences the hydrophobic properties of
EPS. Thus, lipid fractions lead to the maintenance and establishment of
biofilm structures~\cite{62}. Also, lipids may participate membranes
and micelles formation, leading to Van Der Waals interactions in the
biofilm~\cite{63}. The decreased lipid content on PEI-CuO might explain
the damaged structure of biofilm observed by SEM imaging
(Figure~\ref{fig1}). 

Liu and Fang~\cite{63} demonstrated that the hydrophilicity/hydrophobicity 
of EPS highly influences the hydrophobicity of the
bacterial biofilm. The extracted EPS from PEI-CuO coating, in which the
protein represents the main component of the total EPS
(Figure~\ref{fig4}), aligns with the high hydrophobicity observed
over the formed biofilm (Figure~\ref{fig4}).


\subsection{Impact of \textit{Desulfovibrio} sp. biofilms on the
corrosion behavior of bare and coated mild steel}\label{sec33}

To understand the influence of the biofilm on the biocorrosion process
of bare and coated MS, the corrosion products formed under abiotic
conditions were analyzed and compared with those formed under biotic
conditions (Figure~\ref{fig5}). Clearly, in PEI, PEI-ZnO and PEI-CuO,
limited corrosion deposits were observed compared to the non-coated
steel (Figure~\ref{fig5}).

\begin{figure*}[t!]
\vspace*{-3pt}
\includegraphics{fig05}
\vspace*{-3pt}
\caption{\label{fig5}Chemical elements distribution on the coating
surface with (a) MS, (b) PEI, (c) PEI-ZnO, (d)~\mbox{PEI-CuO} immersed in biotic
system, (e) MS, (f) PEI, (g) PEI-ZnO, (h) PEI-CuO immersed 7 days in
abiotic system.}
\vspace*{-4pt}
\end{figure*}

The EDS analysis made over bare and coated MS revealed the presence of
Fe, C, and O. In the abiotic medium, the absence of C element on the MS
(Figure~\ref{fig5}e) is consistent with the absence of bacteria, while
its presence over the coated steel is associated to the polymeric
coatings (Figure~\ref{fig5}f--h). As mentioned above, in the biotic
system (Figure~\ref{fig5}a--d), the C element present in the bare and
coated steel can be associated with SRB biofilm formation
(Figure~\ref{fig1}a--d), and in the coated samples, with the polymeric
matrix from the coating (Figure~\ref{fig1}b--d).

The existence of O can be associated with the organics in the polymeric
coating, with the ZnO and CuO present in the hybrid coatings
(Figure~\ref{fig5}c,d,g,h), (Figure~\ref{fig5}b--d,f--h), and/or with
the existence of corrosion products such as hydroxides or iron oxides
(Figure~\ref{fig5})~\cite{64}. In addition, EDS analysis clearly shows
that Fe is the central element detected as expected considering the
substrate composition and corrosion products formed
(Figure~\ref{fig5}). The overlap of Fe and O elemental distribution with
well-defined bright structures in SEM micrographs (Figure~\ref{fig5})
suggest that the major corrosion products formed are iron compounds
rich in oxygen~\cite{65}.

To further study the influence of the biofilm in the MIC process of MS,
electrochemical impedance spectroscopy (EIS) was used. 

Figure~\ref{fig6} shows the Nyquist and Bode plots of the coated steel
incubated in ATWW with and without \textit{Desulfovibrio} sp. The
increased impedance values compared to the bare MS (Figure~S2 in
Supplementary Data) indicates higher resistances across the
metal/solution electrolyte interface, referring to a higher protection
of the steel by the polymetric matrices~\cite{66}. 

\def\ffigcaption{\label{fig6}}
\xsubfig[t!]{06}{}
\figcnt
\begin{figure*}[t!]
\vspace*{-5pt}
\xincludegraphics{fig06}
\vspace*{-8pt}
\xcaption{\label{fig6}\ffigcaption}{Nyquist and Bode plots of coated MS with
(a) PEI, (b) PEI-ZnO, and (c) PEI-CuO, (d)~the equivalent circuit used
to fit the EIS data for the coated MS immersed in abiotic
system (ATWW) and (e) the equivalent circuit used to fit the EIS data
for the coated MS immersed in biotic system
(ATWW $+$ \textit{Desulfovibrio}).\vspace*{-5pt}}
\end{figure*}

In the case of the coated steel immersed for 7 days in the abiotic
system, a marked slope associated with a diffusion process is evident
in the Nyquist plot, which is associated with the accumulation of the
corrosion products (Figure~\ref{fig6}a--c). Thus, the EIS spectra can
be fitted by the ECC shown in Figure~\ref{fig6}d. In the EEC,
$R_{\mathrm{co}}$ and $Q_{\mathrm{co}}$  are the pores resistance and
the constant-phase element of the coating layer in the presence of
corrosion products, respectively. $R_{\mathrm{ct}}$ and
$Q_{\mathrm{dl}}$ represent the Faradic resistance and double-layer
capacitance, whereas W represents the Warburg resistance due to the
diffusion process of\unskip\break  aggressive ions. This parameter does not exist in
the case of a biotic system. The rationale for this can be
mass-controlled processes' establishment explained by sealing a portion
of pores in the biofilm and the coating. The coating tends to inhibit
the growth of \textit{Desulfovibrio} sp, and the weak biofilm layer
formed on the coated steel surface might be involved with the corrosion
products blocking a part of the pores in the biofilm and
coating~\cite{67}. 

In biotic medium, the coated steel showed two-time constants. The first
one in the high-frequency range is due to the coating, the biofilm and
some corrosion product according to SEM images (Figure~\ref{fig1}), and
the second constant time, at lower frequencies, can be related to the
Faradic responses being at the interface metal/electrolyte
(Figure~\ref{fig6})~\cite{29}. 

Figure~\ref{fig6}e illustrates the EEC used to model the experimental
EIS data for the biotic system to calculate the electrochemical
parameters at the metal/electrolyte interface. In this case, $R_{s}$
represents the solution resistance. $R_{\mathrm{co}}$ and
$Q_{\mathrm{co}}$ are the pores resistance and the constant-phase
element corresponding coating layer with biofilm and 
%{\unskip\break}
corrosion
products, respectively. For the second time constant, $R_{2}$ and
$Q_{2}$ represent the Faradic resistance and double-layer capacitance
at the coating/metal interface, respectively. The evolution of the
impedance parameters obtained by fitting the experimental results are
reported in Table~\ref{tab1}. The chi-squared of the regressions,
around $10^{-3}$ and $10^{-4}$, indicates good fitting 
reliability.


\begin{table*}[t!]
\caption{\label{tab1}Evolution of the impedance parameters extracted by
numerical simulation of the EIS data for the coated mild steel, after 7
days upon immersion in abiotic and biotic systems}
\begin{tabular}{lccccccc}
\thead
Coating & \multicolumn{2}{c}{PEI} & \multicolumn{2}{c}{PEI-ZnO} & 
\multicolumn{2}{c}{PEI-CuO} \\\cline{2-3}\cline{4-5}\cline{6-7}
System incubation & {Abiotic}& Biotic & Abiotic & 
{Biotic} & Abiotic & Biotic \\
\endthead
${R}_{{s}}$ ($\rmOmega{\cdot}\mathrm{cm}^{2}$) & 
900 &  335.8 &  1459 & 2815 & 2616 & 182.5 \\
${R}_{\mathrm{co}}$ ($\rmOmega{\cdot}\mathrm{cm}^{2}$) & 
1143 & 142.3 & 2343 & 4450 & $2.6\times 10^{5}$ & $6\times 10^{5}$ \\
${Q}_{\mathrm{co}}$ ($\rmOmega^{{-1}}{\cdot} \mathrm{cm}^{{2}}{\cdot}\mathrm{s}^{{n}}$) 
& $1.9\times 10^{-5}$ & $1.1\times 10^{-4}$ & $2.3\times 10^{-5}$ & 
$1.7\times 10^{-5}$ & $6.5\times 10^{-8}$ & $1.1\times 10^{-9}$ \\
${n}_{{1}}$ & 0.55 & 0.58  & 0.75 & 0.59 & 0.62 & 0.9 \\
${R}_{{2}}$ ($\rmOmega{\cdot}\mathrm{cm}^{2}$) & 7544 & 1554 & 9754 & 
$1.5\times 10^{5}$ & $6.3\times 10^{5}$ & $2.2\times 10^{6}$ \\
${Q}_{\mathrm{dl}}$ ($\rmOmega ^{-1}{\cdot}\mathrm{cm}^{{2}}{\cdot}\mathrm{s}^{{n}}$) 
& $7.6\times 10^{-5}$ & $3\times 10^{-4}$ & 
$6.5\times 10^{-5}$ & $2.9\times 10^{-5}$ & $8\times 10^{-6}$ 
& $6.5\times 10^{-7}$ \\
${n}_{{2}}$ & 0.68 & 0.59 & 0.86 & 0.56 & 0.77 & 0.48 \\
${W}$ & $3\times 10^{-4}$ & --- & $1.2\times 10^{-8}$ 
& --- & $2.5\times 10^{-6}$ & --- \\
${\upchi}^{{2}}$ & $7.6\times 10^{-3}$ & 
$3\times 10^{-4}$ & $1.5\times 10^{-.3}$ & 
$1.9\times 10^{-3}$ &  $2\times 10^{-4}$ &  $1.5\times 10^{-3}$
\botline 
\end{tabular}
\vspace*{-6pt}
\end{table*}

As observed in EIS plots of PEI (Figure~\ref{fig6}a), a remarkable
decrease in the overall impedance was noticed in the presence of the
SRB strain. The reduction after 7 immersion days can be attributed to
the absence of the protective effect of PEI against the biocorrosion
related to the gradual degradation in the protective film (coating and
biofilm), which leads to an increased corrosion activity compared to
the abiotic system. 

According to the EIS spectra (Figure~\ref{fig6}b,c), middle and
low-frequency time constants observed in PEI-ZnO and PEI-CuO coating
present higher impedance values than those of PEI in both biotic and
abiotic systems. Indicating higher electrical resistances at the
interface of the steels with a solution, and thus decreased corrosion
activity.

\break
When comparing the phase angles values for the bare and coated steels
after 7 days of immersion, the time constant at high and medium-high
frequencies demonstrate that the coatings affect the electrochemical
response of the system. More negative phase angle values and increased
impedance values for PEI-CuO (Figure~\ref{fig6}c) compared to PEI and
PEI-ZnO (Figure~\ref{fig6}a,b) supported the hypothesis of higher
protectiveness of the PEI-CuO coating.

\break
For PEI and PEI-ZnO, the values of log $|Z|$ determined at the lowest
frequency are approximately $2.8\times 10^{3}$ and $9.5 \times 
10^{4}~\rmOmega{\cdot} \mathrm{cm}^{2}$, respectively
(Figure~\ref{fig6}a and b). In contrast, in the PEI-CuO coating, values
were of  $2.5\times 10^{6}~\rmOmega{\cdot} \mathrm{cm}^{2}$
(Figure~\ref{fig6}c). Thus, the PEI-CuO shows higher efficiency as an
anti-corrosive coating in three systems: chloride medium~\cite{29},
%\unskip\break
abiotic system (SATWW) and biotic system (ATWW in the presence of
\textit{Desulfovibrio} sp.), probably due to the higher coating
thickness and decreased porosity in the outer layer~\cite{29}.

As shown in Figure~\ref{fig6}b,c, in the biotic system, the impedance
values are notably higher than abiotic ones demonstrating that
PEI-metal oxides resulted in a biocorrosion inhibition.

The variation of $R_{\mathrm{co}}$ and $R_{2}$ ($R_{\mathrm{ct}}$ in
the case of the abiotic system) in different media for the different
coating is shown in Figure~S3(a,b) and (c,d). $R_{\mathrm{co}}$ and
$R_{2}$ increased with the presence of ZnO and CuO in the abiotic
medium (Figure~S3b--d), which led to the gradual formation of a
protective corrosion product film that decreased the corrosion of 
MS. Besides, the PEI and PEI-ZnO coatings have the lowest resistance
compared to the PEI-CuO, which shows the rapid degradation of the film
and,consequently, the corrosion increases.

In the case of hybrid coatings, the values of $R_{\mathrm{co}}$ and
$R_{2}$ in biotic medium were larger than in the abiotic medium. The
increase in the biolayer thickness and the lower metabolic activity of
SRB can explain these values. The effectiveness of PEI-CuO may also be
related to the higher thickness (51~$\upmu$m) and the limited number of
holes formed before and after immersion in the aggressive medium.
Despite the similar thickness value observed for PEI-ZnO and PEI
(43~$\upmu$m)~\cite{29}, a sharp decrease of $R_{\mathrm{co}}$ and
$R_{2}$ was observed for PEI in the biotic system. This behavior may be
related to a high concentration of corrosive metabolites from bacteria.

Whatever the immersion medium, PEI-CuO had the highest efficiency
(Figure~S3 in Supplementary Data) when compared with the other
coatings. These results may be related to the increased thickness, the
decreased porosity~\cite{29} and/or the composition of the produced EPS
(biotic system). Moreover, as a nobler alloying element, Cu protects
steel by suppressing anodic dissolution~\cite{68}. The percentages of
protection efficiency increased in the presence of
\textit{Desulfovibrio} sp. for PEI-CuO and PEI-ZnO coatings, indicating
that the biofilm formed on the surface of the PEI-metal oxide coatings
has a protective role against biocorrosion. 

Various factors can influence the biocorrosion process, such as biofilm
structure, surface coverage and EPS composition~\cite{69}. For uncoated
and PEI-coated MS, the microbial presence accelerates the
biocorrosion rate by creating different electrochemical potentials
(Figure~S4)~\cite{70}. According to Miranda \etal~\cite{10}, the
existence of \textit{Desulfovibrio} sp on the steel surface results in
a deposition of ferric sulfates. Microbial clusters result in anaerobic
microenvironments beneath the deposits, producing conditions for the
accumulation of Cl$^{-}$ that combine with iron to form acidic ferric
chloride. Generally, these compounds are highly corrosive to steels. In
addition, the presence of sulfides can induce pitting on
steel~\cite{15}. 

According to Mostafaei \etal~\cite{71}, after immersion, ZnO and CuO
reduction within coatings leads to the release of $\mathrm{Zn}^{2+}$
and Cu$^{2+}$. These ions interact with polymers' nitrogen atoms and
form compact clusters that increase the electron-rich benzenoid groups
to facilitate a greater coverage on the metal surface. Consequently, a
delay diffusion of $\mathrm{O}_{2}$, H$_{2}$O, and Cl$^{-}$ occurs on
the steel surface~\cite{29}. In the biotic system, the biofilm formed
on the surface of the composite coating increases the corrosion
resistance of MS. In fact, the protective role of the bacterial
biofilm~\cite{72} is confirmed to be a function of its bacterial
diversity, which influences the biofilm attachment, EPS composition,
biofilm thickness, density, and metal chelation~\cite{26}. In this
context, the corrosion protection by the biofilm seems to be enhanced
by bacterial strains such as \textit{vibrio} and \textit{Serratia
marcescens EF190} are used. The formation of a protective layer on the
steel surface, in the first step of attachment, was demonstrated along
side oxygen consumption by electron transports proteins~\cite{70,23}.

Moreover, biofilm development and its properties are highly influenced
by the composition of extracellular polymeric substances
(EPS)~\cite{17,23,73}. Corrosion inhibition by these proteins have been 
demonstrated earlier~\cite{74,75,76}. Due to their stable adsorption,
proteins interact with metal ions ($\mathrm{Zn}^{2+}$ and Cu$^{2+}$)
and create metal-protein complexes (chelates) on the steel surface.
This reaction helps to decrease the metal release, blocking other
mechanisms such as electrochemical dissolution and, therefore slowing
down corrosion. Moreover, exopolysaccharides were reported to be used
for corrosion protection. Scheerder \etal~\cite{77} reported that when
the first step (Figure~S5) of corrosion takes place, $\mathrm{Fe}^{2+}$
can interact with --C--OH, --CH$_{3}$, --CH$_{2}$-- and --COO$^{-}$ in
the polysaccharide and --C=O-- and --COO-- in the protein molecules. As
such, these ions can no longer participate in the electrochemical
process. Because it is not soluble in the coating matrix, the metal
complex will likely deposit at the coating-metal interface. Thus, such
metal complexes will form a protective layer on the steel surface,
preventing the diffusion of corrosive particles and oxygen dissolved.
Under this layer, the probability of $\mathrm{Fe}^{2+}$ binding with
H$_{2}$O, $\mathrm{O}_{2}$, and polysaccharide/protein molecules is
reduced, the resistance is gradually increased, and corrosion is
inhibited.\vspace*{-5pt}

\section{Conclusion} 

A new approach to reduce biocorrosion induced by \textit{Desulfovibrio}
sp. using new hybrid coatings for MS protection in ATWW was achieved
with PEI-ZnO and PEI-CuO coatings. The bare steel surface had an
adherent homogeneous biofilm, confirming that the biofilm can quickly
be established upon SRB colonization. In PEI-coated MS, fewer
and scattered bacterial colonies developed on the metal surface, either
in separate colonies or small clusters. The PEI coating makes it difficult
for bacteria to colonize and access MS surface under coating and
PEI-ZnO and PEI-CuO coatings led to a more heterogeneous biofilm
structure. A pronounced bactericide effect was depicted for PEI-ZnO and
PEI-CuO coatings, with the number of SRB individual colonies
significantly decreasing with a more intricate porous structure. From
these two coatings, higher protein content, from the EPS
characterization occurred in the presence of the PEI-CuO coating. EIS
measurements in the biotic medium showed that the hybrid coatings,
PEI-ZnO and PEI-CuO, significantly increased the protection of MS
against biocorrosion compared to PEI coatings. The biofilm itself acted
as a protective film, showing increased resistance values for the ZnO
and CuO modified coatings. PEI-CuO seemed more protective, either in the
absence or presence of bacteria, where PEI seemed to act as
anti-adherent agent and CuO as antimicrobial agent. Moreover, EPS
extracted from the biofilm formed on the PEI-CuO surface can be
suggested as a green biocorrosion inhibitor in wastewater.\looseness=-1

\section*{Conflicts of interest}
Authors have no conflict of interest to declare.

\section*{Acknowledgments}
The authors would like to gratefully express their appreciation to the
Tunisian Ministry of Higher Education and Scientific Research and the
Funda\c{c}\~{a}o para a Ci\^{e}ncia e Tecnologia (FCT) through the
project UIDB/00100/2020, IDB/00100/2020, UIDP/00100/2020,
LA/P/0056/2020 for the financial support.

\CDRGrant[FCT]{UIDB/00100/2020}
\CDRGrant[FCT]{IDB/00100/2020}
\CDRGrant[FCT]{UIDP/00100/2020}
\CDRGrant[FCT]{LA/P/0056/2020}

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-227-suppl.pdf}}

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