Journal of Molecular Liquids 240 (2017) 121–127

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Neem extract as a green inhibitor for microbiologically influenced

corrosion of carbon steel API 5LX in a hypersaline environments
Punniyakotti Parthipan a, Jayaraman Narenkumar a, Punniyakotti Elumalai a, Parameswaran Sujatha Preethi b,
Ayyakkannu Usha Raja Nanthini b, Akhil Agrawal c, Aruliah Rajasekar a,⁎
a
Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore 632 115, Tamilnadu, India
b
Department of Biotechnology, Mother Teresa Women's University, Kodaikanal 624 101, Tamilnadu India
c
Department of Microbiology, Central University of Rajasthan, Kishangarh, Ajmer 305 801, India

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the efficiency of neem extract to inhibit the corrosion of carbon steel API 5LX in the presence of dif-
Received 7 February 2017 ferent bacterial strains (Bacillus subtilis A1, Streptomyces parvus B7, Pseudomonas stutzeri NA3 and Acinetobacter
Received in revised form 3 May 2017 baumannii MN3) were evaluated. 150 ppm of Azadirachta indica leave extract (AILE) was identified as minimal
Accepted 14 May 2017
inhibitory concentration for the bacterial strains B. subtilis A1, S. parvus B7, P. stutzeri NA3 and A. baumannii
Available online 16 May 2017
MN3. Weight loss, electrochemical studies including potential measurements, potentiodynamic polarization
Keywords:
and impedance and surface analysis includes Fourier transform infrared spectroscopy (FT-IR) were used to exam-
Azadirachta indica ine the corrosion inhibition efficiency (IE) of AILE on carbon steel API 5LX. The individual bacterial strains and
Green inhibitor mixed consortium had showed severe corrosion of carbon steel API 5LX in hypersaline environment. In contrast
Biocorrosion with AILE, the IE of abiotic control and mixed consortia were about 81% and 72% respectively. The corrosion rate
EIS was observed as increased trends in presence of all bacterial strains (0.179 mm/y) when compared to abiotic sys-
Weight loss tem with AILE system (0.012 mm/y). X-ray diffraction spectroscopy (XRD) confirms that all bacterial strains has
Biofilm accelerates the corrosion by the production of higher intensity peaks of corrosion products when compared to
AILE systems. FT-IR data reveals that AILE extracts consists of azadirachtin and other phytochemical compound
which was play a key role in control of bacterial biofilm on metal surface and thus leads to inhibit corrosion pro-
cess. This is the first report on the application of AILE as a natural, eco-friendly corrosion inhibitor with antibac-
terial activities to control microbiologically influenced corrosion in hypersaline environment of oil reservoir.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Microbiologically influenced corrosion (MIC) is an electrochemical
process where microbes accelerate/cause metal deterioration by their
action of metabolism on the metal surface [1–4]. As a result of microbi-
ological action in reservoir fuel contamination, fuel turbidity, metal de-
terioration in storage tanks and pipelines, and souring of oil products
occurs [5,6]. The presence of microorganisms is one of the key factors re-
sponsible for the corrosion issues associated with the oil industries [6–
9]. MIC is one of the most important features for pipeline failure, and
one of the key factor involved in operation and maintenance cost issues
of crude oil industry [10,11]. Overall, about 40% of internal corrosion in
gas and oil industries is originated from microorganisms [12–14]. Car-
bon steel is a widely used engineering material for the transportation
⁎ Corresponding author.
E-mail address: rajasekargood@gmail.com (A. Rajasekar).
and storage of the oil products and many researchers have reported
the biocorrosion of carbon steel [1,6,15].
Control and anticipation of microbial corrosion have potential im-
portance in at the moment in sight of the harshness of the problems
caused. The electrostatic charge accumulation on metal surface, which
are irregular and positive discrimination for biofilm development. Bio-
film is a formed by the mixture of microbial cells, spores and their me-
tabolites along with grime materials which are harden to a greasy film
by the action of cellular secretions [4]. The microbial activity leads to
formation of products which are very corrosive to the metal surfaces.
Different microbial communities (aerobic/anaerobic) in a system acts
on surface of metals in different behavior [3,16].
Using inhibitors is an important method of protecting materials
against deterioration due to corrosion [7,17]. The influence of heterocy-
clic compounds containing oxygen, sulfur and nitrogen on the corrosion
of different steel in acidic environments has been investigated by some
researchers [17–20]. Many of the synthetic compounds give high anti-
corrosive performance, but most of them are extremely toxic to envi-
ronment [21–23]. The protection and ecological concerns of synthetic

http://dx.doi.org/10.1016/j.molliq.2017.05.059
0167-7322/© 2017 Elsevier B.V. All rights reserved.
122 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127
corrosion inhibitors in industries have constantly exist as global appre-
hension [24]. The known hazardous effect of most synthetic corrosion
inhibitors has motivated scientists to use natural products as corrosion
inhibitors as they are inexpensive, readily available and renewable
sources of materials, environmentally friendly, and ecologically accept-
able [7,25–27]. Up to date, many corrosion inhibitors have been synthe-
sised, and they sort from rare earth elements [28] to organic material
[29,30].
Recently many of the plant derivatives were used as green corrosion
inhibitor for the control of chemical corrosion in steels, such as Areca
catechu, Syzygium cumini, Tragia plukenetii, Azadirachta indica, Calotropis
gigantea, Gossypium hirsutum, Artemisia pallens, garlic peel extract, Pa-
paya seeds and etc., [3,24,26,31–39]. Among these natural products
neem (Azadirachta indica) only used as inhibitor with biocidel activities
for control of the microbial corrosion in API 5L X80 linepipe steel and
copper [3,15].
Neem tree is distinguished for its extraordinary biological properties
around the world. Neem oil, leaves and bark are acknowledged to ac-
quire diverse medical uses for the healing of many diseases [40]. Ex-
tracts of neem tree contains several compounds, such as not limited
to: azadirachtin, gedunin, nimbin, nimbandiol, azadirone, nimbolide,
nimbinene, nimbolin, nimonol, margolone, vilasanin, flavonoids and
etc. [15,31,40–42]. Among identified neem compounds azadirachtin
has highest antimicrobial activity against both Gram positive and
Gram negative bacteria [15,40]. Previously neem was used as potential
inhibitor for acid corrosion of the different metals such as mild steel,
copper, zinc, X80 carbon steel [31,32,40,43,].
The present research focused on the application of Azadirachta indica
leaves extract (AILE) as green inhibitor to control MIC of carbon steel
API 5LX (CS) in the hypersaline environment. Weight loss test, electro-
chemical studies (polarization and impedance spectroscopy), X-ray dif-
fraction spectroscopy (XRD) and Fourier transform infrared spectrum
(FT-IR) were utilized to characterize the role of AILE on control of MIC
of carbon steel API 5LX.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Four bacterial strains were exploited in this study namely Bacillus
subtilis A1, Streptomyces parvus B7, Pseudomonas stutzeri NA3 and
Acinetobacter baumannii MN3 which were isolated and identified from
an Indian crude oil reservoir. These strains were identified by 16S
rDNA sequencing and deposited under NCBI Genbank accession num-
bers KP895564, KP895570, KU708859 and KU708860 respectively. All
the strains were retrieved from glycerol stock and sub-cultured in
Luria–Bertani (LB) agar plates (g/L: 10.0 tryptone, 5.0 yeast extract,
10.0 sodium chloride with 15.0 agar (Himedia, Mumbai, India)) and in-
cubated at 37 °C for 24 h. Further sub cultured by single colony inocula-
tion method using LB broth (pH: 7.0) and incubated in an orbital shaker
(150 rpm) for 24 h at 37 °C.
2.2. Corrosion inhibitor preparation
Fresh Azadirachta indica leaves were collected from Thiruvalluvar
University campus and surface cleaned with deionised water and
grained with ethanol (99.9%). The liquid thus acquired was sieved via
Whatman filter paper to eliminate the dregs. The filtered solution was
rotary evaporated and sterilized using autoclave prior to the further
uses and stored at 4 °C [3]. The functional groups present in Azadirachta
indica were qualitatively characterized by Fourier transform infrared
spectrum (FT-IR). The samples were pulverized with the addition of po-
tassium bromide in a 1:100 ratio and the pellet was fixed in the sample
container, and analyzed in the mid IR region 400–4000 cm−1.
2.3. Antibacterial properties of AILE
2.3.1. Agar-well diffusion assay
To assess the antibacterial activity of the AILE, Mueller Hinton Agar
(MH) (Himedia-India) plates was prepared as per manufacturer's in-
structions. Six different concentrations at 20 ppm, 50 ppm, 100 ppm,
150 ppm, 200 ppm and 250 ppm of the AILE were selected to evaluate
their bactericidal activity of the four bacterial strains (B. subtilis A1, S.
parvus B7, P. stutzeri NA3 and A. baumannii MN3). All strains were inoc-
ulated separately on the MH agar plates and evenly spread over the en-
tire surface of MH agar. Wells of 6 mm diameter with 5 mm deepness
were made on the solid MH agar using a sterile glass borer. 50 μL of
AILE at the different concentrations were loaded to each respective
well. 10 μg gentamycin was used as a positive control because of its
broad range bactericidal activity. Sterile ethanol was used as negative
control and each experiment was done in triplicates and incubated at
37 °C for 24 h. Zone of the inhibition was measured and expressed in
millimetres (mm).
2.3.2. Biofilm inhibition assay
Overnight bacterial cultures of B. subtilis A1, S. parvus B7, P. stutzeri
NA3 and A. baumannii MN3 in Luria Bertani (LB) medium were diluted
in 1:20 ratio with fresh LB medium with different concentration of AILE
such as (100 ppm, 150 ppm, 200 ppm and 250 ppm) and further these
diluted cultures (100 μL) were transferred into 96-well microtiter plate
and incubated at 37 °C for 24 h. For comparison same culture broth was
preferred without AILE and served as control. The suspended culture
medium were removed and the 96-well plate was washed with phos-
phate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO4,
2 mM KHPO4, pH:7.2), 120 μL of crystal violet were added to each well
and left for 20 min. Finally, 125 μL of acetic acid was added over the crys-
tal violet and incubated for additional 15 min at 37 °C. Visible changes in
appearance were observed to confirm the biofilm inhibition capability
of the AILE at the lowest concentration [44].
2.4. Corrosion studies
2.4.1. Carbon steel coupon preparation
MIC behavior of carbon steel API 5LX was investigated as described
by Rajasekar et al. [6]. with the modification in the systems design as fol-
lows: produced water collected from an oil reservoir was served as cor-
rosive medium. Collected produced water contained higher amount of
the inorganic corrosive substances such as chloride (36 g/L), sulphate
(354 mg/L), iron (32 mg/L) as major components and other chemicals
as trace amounts like magnesium (529 mg/L), calcium (1800 mg/L)
among others. Carbon steel API 5LX coupons (metals components in
(wt%): C 0.070, Si 0.195, Cr 0.03, Ni 0.02, Mn 1.05, Cu 0.05 and Al
0.029, balanced with Fe) was cut into 25 × 25 × 0.4 mm and 10 × 10
× 1 mm size coupons for weight loss studies and electrochemical mea-
surements (impedance and potentiodynamic polarization studies) re-
spectively. All the coupons were sequentially smoothened with
different grade silicon carbide papers (180, 500, 800, 1200, and 1500)
and finally polished with 0.3 μM alumina powder [6]. The polished car-
bon steel API 5LX which are acted as working electrodes (WE) with an
exposed area of 1.0 cm2 were used for EIS studies. The electrodes
were sterilized by UV light prior to the experiment.
2.4.2. Weight loss experiments
For weight loss measurements, initial weights of the each coupon (in
triplicate) were recorded before immersion into the corrosion systems.
The coupons were first dipped into the deionised water and degreased
with trichloroethylene. System I consisted of coupons (in triplicates)
immersed in a 500 mL Erlenmeyer flask with 300 mL sterile corrosive
medium (produced water) which acted as abiotic control systems. Sys-
tem II –XII are similar to that of system of I. The system II was added
with 150 ppm of ethanolic extract of AILE, system III, IV, VII, IX were
 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127
inoculated separately with each bacterial strain (B. subtilis A1, S. parvus
B7, P. stutzeri NA3 and A. baumannii MN3 respectively) (about
104 CFU/mL). Similarly system IV, VI, VIII and X were inoculated sepa-
rately with each bacterial strain (about 104 CFU/mL), in addition of
150 ppm ethanolic extract of AILE also added. System XI was inoculated
with all bacterial strains as mixed consortium (B. subtilis A1, S. parvus
B7, P. stutzeri NA3 and A. baumannii MN3) and system XII was similar
to system XI with the addition of AILE (150 ppm). Triplicate tests
were carried out for each weight loss system. These systems were left
undisturbed for 20 days at 37 °C. At the end of the 20th day, the coupons
were studied using electrochemical impedance spectroscopy (EIS) and
x-ray powder diffraction (XRD). For examine the antibacterial activity
of the AILE on the corrosion studies, the total viable count (TVC) of the
bacterial biofilm on metal surface of each systems were done by every
two days interval during the biocorrosion studies. TVC of the biofilms
were enumerated by the standard plate count method.
2.5. Electrochemical studies
2.5.1. 2.6.1. Potential measurement
The open circuit potential (EOCP) of carbon steel API 5LX samples in
different corrosion experiments in presence/absence of bacterial cul-
tures and neem extract were monitored using CH Instruments Inc.,
USA (Model CHI-608E) for every day up to end of the experiments. Sat-
urated calomel electrode (SCE) was employed as a reference electrode.
EOCP was observed at frequency of a one reading per 30 s.
2.5.2. 2.6.2. Impedance and potentiodynamic polarization measurement
The electrochemical impedance spectroscopy (EIS) studies were
conducted with CH Instruments Inc., USA (Model CHI-608E). The stud-
ies were performed with a three electrode system, a SCE employed as
reference electrode, a platinum wire was used as counter electrode
and the metal coupons (carbon steel), recovered at the end of the
each weight loss study, acted as the working electrode. The used medi-
um from each of the relevant systems (weight loss experiments) were
used as the electrolyte solution. Impedance measurements were con-
ducted under steady state conditions in the frequency range of 0.1 to
105 Hz at a scan rate 10 mV/min. The potentiodynamic polarization
analysis was conducted by polarizing towards + 200 mV anodically
and −200 mV cathodically at scan rate of 0.002 V/s with respect to cor-
rosion potential (Ecorr). Zsim Demo 3.20d software was used to analysis
electrochemical parameters. All the measurements were recorded in
triplicate.
2.6. Surface analysis
At the end of the each weight loss experiment, the coupons were re-
moved and the corrosion products were detached carefully. The cou-
pons were then pickled with Clark solution as described by Rajasekar
et al. [45] briefly 2% antimony trioxide with 5% stannous chloride
suspended in con. HCl at room temperature with regular rousing
about 5–10 min) further washed with deionised water and air dried.
The corrosion inhibition efficiency (IE%) of AILE on the carbon steel
API 5LX was obtained using the following Eq. [34].
IE% ¼ W0 −W  100
W0
where W0 and W are the weight loss in absence and presence of the
AILE respectively. Corrosion products collected from each biocorrosion
systems were dried and crushed to a fine powder and analyzed using
X-ray diffractometer (XRD) to confirm the nature of the oxides. XRD
was analyzed using Bruker D8 and scanned with CuKα radiation (Ni fil-
ter) in the range 10–85° at the rating of 40 kV, 20 mA.
123
3. Results and discussion
3.1. Antibacterial assay
The ethanolic extracts of the Azadirachta indica leave compounds
was tested for their antibacterial activities using agar-well diffusion
method. Different concentration of the AILE was utilized and primary
selection observation was shown in the Fig. 1. Overall ethanolic extract
of the AILE had showed the effective biofilm inhibition activities, specif-
ically both 20 and 50 ppm of the ethanol extracts shows very minute
zones on the MH agar. While in higher concentration (100–250 ppm)
ethanolic extract of AILE showed the good biofilm inhibition effect on
all the bacterial strains and also in mixed consortium. It indicates that,
biofilm inhibition activity was directly proportional to the concentra-
tion of the AILE.
Further 100 ppm, 150 ppm, 200 ppm and 250 ppm were selected for
the biofilm inhibition assay. In biofilm assays, all four strains were
checked by microtiter plate with crystal violet method [44]. This analy-
sis confirmed that the capability of the strains to form biofilm on metal
surface. In presence of AILE at different concentration 150 ppm,
200 ppm, 250 ppm, the biofilm development on metal surface was high-
ly inhibited. The AILE concentration of 150 ppm identified as minimum
inhibitory concentration against all the selected bacterial strains which
was further chosen for the biocorrosion experiments.
3.2. Biocorrosion studies
3.2.1. Weight loss study
The results of the weight loss experiments for the carbon steel API
5LX immersed in the presence and absence of bacterial strains and
AILE after 20 days were summarized and presented in Table 1. It is no-
ticed from Table 1, the produced water contributes to the corrosion of
carbon steel to a lesser extent. The mean weight loss of the carbon
steel coupons in the abiotic system was 16 mg. Besides, abiotic system
with AILE inhibitor showed very less weight loss (3.0 mg). The mean
weight losses of carbon steel in presence of individual bacterial strains
were ranged between 33 and 40 mg. Among the individual bacterial
strain S. parvus B7 showed that the highest weight loss which was
about 40 mg. Individual bacterial strains with AILE showed the lower
weight loss which was ranged from 3 to 4 mg. Mixed bacterial consortia
showed the maximum weight loss about 48 mg, but in the mixed con-
sortia with AILE showed the considerably lesser weight loss (4 mg).
The weight loss information of the abiotic and biotic system with and
without AILE confirm that's the Azadirachta indica extract has both the
activities of bactericidal and corrosion inhibition, since the weight loss
Fig. 1. Antibacterial activities of the neem extract on the Mueller Hinton agar with
different bacterial strains.
124 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127

of the abiotic control with AILE inhibitor showed the lower level of cor-
rosion carbon steel metal [3,15].
The corrosion rate (CR) of the different biocorrosion system was pre-
sented in the Table 1. In the absence of bacteria (abiotic system) the
mean CR was 0.060 mm/y for carbon steel. However, in the presence
of individual bacterial strains CR was doubled (0.13 mm/y), at the
same time very lower level of CR (0.015 mm/y) was recorded for indi-
vidual bacterial strains with AILE. The highest CR (0.179 mm/y) was no-
ticed in the mixed consortia system. The doubled amount of the
corrosion rate in the bacterial systems indicates that each bacterial
strains were utilized the inorganic metal from the metal surface and
it's very prone to metal deteriorations. It was evidenced from corrosion
rate, AILE extract contains biocidel activities with corrosion inhibition
properties.
The corrosion inhibition efficiency (IE %) of the AILE in presence and
absence of the bacterial strains were presented in Table 1. Highest IE
was observed in abiotic system with AILE (81%), and also bacterial
strains with AILE showed considerable inhibition activities such as 78%
(B7), 75% (NA3) and mixed bacterial system (72%). This rate of inhibi-
tion efficiency was very higher as compared to other available reports
[32,40].
The growth curves of the bacterial culture of different corrosion sys-
tems, i.e. bacterial systems with and without AILE were shown in Fig. 2.
The growth curve reveals that biofilm of each bacterial systems were
observed in the range of 105–106 (CFU/cm2) whereas in presence of
AILE, the biofilm cells were highly reduced up to 100–101 (CFU/cm2).
Too low to count was found in the abiotic control system with AILE in-
hibitor. It reveals that AILE had antibacterial activity on inhibition of
bacterial biofilms on carbon steel metal surface.
Weight loss measurements used for the analysis of corrosion rates
and inhibitor efficiency usually extend more reliable results than elec-
trochemical methods since the experimental systems more robustly im-
itate real conditions [46]. In this study, AILE showed the biostatic
properties resulting in higher corrosion inhibition efficiency when com-
pared to inhibit SRB about 50% of IE which was reported by Bhola et al.
[15].
3.3. Electrochemical studies
3.3.1. Potential changes
The changes in OCP towards the incubation period in presence and
absence of bacterial cultures and neem extracts have been shown in
Table 1
Corrosion rate and inhibition efficiency for the carbon steel API 5LX in the presence and
absence of bacterial strains and neem extract.
Systems Carbon steel API 5LX
Fig. 2. Bacterial growth curve of the different corrosion systems with and without of
bacterial strains and neem extract. Note: A1 - B. subtilis, B7 - S. parvus, NA3 - P. stutzeri,
MN3 - A. baumannii, AC - abiotic control, AILE- Azadirachta indica leave extract.
Fig. 3. Potential changes was measured immediately after the addition
of the bacterial cultures and neem extract, and found that initial values
about −490 to −580 mV vs SCE. After the one day of incubation nega-
tive shift was observed for each corrosion systems. Afterwards the 2nd
day of incubation very small changes was observed as low potential
values was measured in the abiotic systems − 610 mV vs SCE at 6th
day of incubation, besides highest potential was recorded in the mixed
consortia with neem extracts about −800 mV vs SCE. Further recorded
values confirms that potential was stable until the end of the exposure
period. Compared to respective bacterial and inhibitor systems positive
shift was observed in the bacterial systems, it reveals that biofilm play
important role in the corrosion of carbon steel. The negative shift in
the inhibitor systems indicates that corrosion inhibition was occurred
in the presence of neem extract [15].
3.3.2. Potentiodynamic polarization
The potentiodynamic tafel polarization curves of the carbon steel API
5LX in presence and absence of bacterial strains and AILE after immer-
sion of 20 days were shown in Fig. 4. The electrochemical parameters
such as corrosion potential (Ecorr), the corrosion current density (Icorr),
and the anodic (βa) and cathodic (βc) tafel values were presented in
Table 2, which were collected from tafel plots. Tafel parameters of the
bacterial strains B. subtilis A1, S. parvus B7, P. stutzeri NA3 and A.
baumannii MN3 confirmed that bacterial strains have the highest capa-
bility to cause metal deterioration in the hypersaline environment.
From the tafel values it can be noticed that the Icorr decreased in pres-
ence of AILE as compared with each bacterial strains and mixed consor-
tia. Both βc and βa of the systems with AILE were decreased in

Weight loss Corrosion rate Corrosion inhibition

(mg) (mm/y) efficiency (%)

I - Abiotic control 16.0 ± 1 0.060 –

II - Abiotic control 3.1 ± 0.5 0.012 81
with AILE
III - S. parvus B7 40.2 ± 2 0.150 –
IV - S. parvus B7 with 3.5 ± 0.5 0.013 78
AILE
V - B. subtilis A1 38.6 ± 2 0.114 –
VI - B. subtilis A1 with 4.4 ± 0.5 0.017 72
AILE
VII - P. stutzeri NA3 36.4 ± 1 0.136 –
VIII - P. stutzeri NA3 4.0 ± 0.5 0.015 75
with AILE
IX - A. baumannii MN3 32.8 ± 1 0.122 –
X - A. baumannii MN3 4.2 ± 0.5 0.016 74
with AILE
XI - Mixed consortia 48.2 ± 2 0.179 –
XII - Mixed consortia 4.5 ± 0.5 0.017 72
Fig. 3. Change of open circuit potential of the carbon steel API 5LX in presence and absence
with AILE
of bacterial strains and neem extract. Note: A1 - B. subtilis, B7 - S. parvus, NA3 - P. stutzeri,
Note: AILE- Azadirachta indica leave extract. MN3 - A. baumannii, AC - Abiotic control, AILE- Azadirachta indica leave extract.
 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127 125

Fig. 4. The potentiodynamic polarization curves of the carbon steel API 5LX in presence Fig. 5. Electrochemical impedance illustration of the carbon steel API 5LX in the presence
and absence of bacterial strains and neem extract. Note: A1 - B. subtilis, B7 - S. parvus, and absence of bacterial strain and neem extract. Note: A1 - B. subtilis, B7 - S. parvus, NA3 -
NA3 - P. stutzeri, MN3 - A. baumannii, AC - Abiotic control, AILE - Azadirachta indica leave P. stutzeri, MN3 - A. baumannii, AC - Abiotic control, AILE- Azadirachta indica leave extract.
extract.

comparison with the other evaluated systems in carbon steel metals.
Similar observation was noticed by Faustin et al. [47] for the garlic ex-
tracts as corrosion inhibitor. This finding implies that the active com-
pounds present in AILE, adsorbed on the metal surface and hindered
the corrosion process. The positive shift of the corrosion potential are
key phenomenon and explained to occur as a effect of the biofilm for-
mation due to the growth of bacterial cells and their metabolites. The
development of biofilm and towards a positive shift in corrosion poten-
tial could be credited to the enlarged biological cathodic electron uptake
effect and thus development in the reduction capability of the system to
hurry up anodic dissolution [3,48]. The Ecorr values showed a positive
shift in presence of AILE which could be owing to the adsorption of in-
hibitor compounds, biofilm and the electrolyte components. Recently,
Swaroop et al. [3] also described that neem extract has the capabilities
to positive shift of the corrosion potential which are reduces the corro-
sion reactions.
3.3.3. Impedance spectroscopy
Fig. 5 represents the electrochemical impedance diagram for the car-
bon steel API 5LX in presence and absence of bacterial strains and AILE.
The impedance parameters, charge transfer values (Rct) and solution re-
sistance (Rs) values of the different system were presented in Table 2.
The Rct values were maximum recorded in the abiotic system with
AILE (20.2 Ω·cm2) followed by mixed consortia with AILE (19.53
Ω·cm2) compared to the abiotic system (16.13 Ω·cm2).The increase
in Rct values was connected with the development of protective layer
at the metal/solution interface [34,49]. Hence, this finding might be at-
tributed to the adsorption of the compounds of AILE on the metal/solu-
tion interface [50]. This theory was well supported by both anodic/
cathodic polarization curves and the corrosion potential effects [34].
3.4. X-ray diffraction analysis
Fig. 6 shows the XRD patterns of the corrosion products on the car-
bon steel API 5LX in presence/absence of the different bacterial strains
and AILE. XRD confirmed the presence of ferric oxide, manganese
oxide and iron oxide-hydroxide as corrosion product at the end of the
biocorrosion studies. When compared to abiotic control, higher intense
peaks were observed in all the bacterial systems. Lower intensity of
peaks was noticed in the bacterial systems with AILE. The presence of
these iron particles as corrosion products in bacterial systems indicates
that all the bacterial strains has the ability to oxidize the inorganic com-
pounds present in the carbon steel composition [11,51]. XRD results
suggest that AILE not only control the development of bacteria but
also it minimises the corrosion reaction in the corrosive medium prob-
ably via adsorption over the metal surface. This observation was sup-
ported by recent report of Swaroop et al. [3] that showed that neem
extract act as corrosion inhibitor in different corrosive medium includ-
ing acidic and aqueous system [3]. The antibacterial activity of the
AILE against biofilms on metal surfaces was confirmed from the biofilm
inhibition assay.

Table 2
Polarization and impedance parameters for carbon steel API 5LX in the presence/absence bacterial strains and neem extract.

Systems Polarization data Impedance data

Icorr Ecorr (V) βa βc Rct Rs

(A/cm2) (mV/dec) (mV/dec) (Ω·cm2) (ohm)

I - Abiotic control 1.9 × 10−4 −596 7.53 2.29 16.13 9.65

II- Abiotic control with AILE 1.0 × 10−4 −570 7.22 1.82 20.2 9.98
III - S. parvus B7 2.0 × 10−4 −647 9.45 2.84 11.15 8.69
IV - S. parvus B7 with AILE 1.3 × 10−4 −610 7.56 2.20 15.95 9.21
V - B. subtilis A1 2.3 × 10−4 −632 8.49 2.87 12.01 7.53
VI - B. subtilis A1 with AILE 2.0 × 10−4 −594 7.95 2.38 17.83 9.45
VII - P. stutzeri NA3 2.6 × 10−4 −640 8.93 2.97 10.99 7.95
VIII - P. stutzeri NA3 with AILE 2.2 × 10−4 −615 8.09 2.18 16.42 9.36
IX - A. baumannii MN3 1.7 × 10−4 −649 9.55 3.09 11.54 7.49
X - A. baumannii MN3 with AILE 1.2 × 10−4 −603 7.87 2.48 17.56 9.22
XI - Mixed consortia 3.3 × 10−4 −656 10.18 3.53 11.1 7.01
XII - Mixed consortia with AILE 1.6 × 10−4 −586 7.36 2.20 19.53 9.74

Note: AILE - Azadirachta indica leave extract, Icorr - Corrosion current, Ecorr - Corrosion potential, βa - Anodic tafel slope, βa - Cathodic tafel slope, Rct - Charge transfer resistance, Rs - Solution
resistance.
126 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127
Fig. 6. XRD patterns of the corrosion products of the carbon steel API 5LX in presence/
absence of the bacterial strains and the neem extract. Note: A1 - B. subtilis, B7 - S. parvus,
NA3 - P. stutzeri, MN3 - A. baumannii, AC - Abiotic control, AILE - Azadirachta indica leave
extract.
3.5. Corrosion inhibition mechanism by Azadirachta indica
The biofilm inhibition assay reveals that all bacterial strains had very
sensitive to the AILE. It was also evidenced from potentiodynamic polar-
ization, electrochemical impedance spectroscopy and XRD analysis with
bacterial and AILE systems. The electrochemical parameters strongly
confirmed that nature of the bacterial activities was different in pres-
ence and absence of the AILE. The Azadirachta indica had been identified
as metal protector against corrosion by adsorption of their organic com-
pounds on the metal surface [15,52]. Fig. 7 show the IR spectra of the
ethanolic extracts of the neem extract, were used as inhibitor. From
the results, it was found that for the leaves extract, the\\OH stretch at
3321 cm− 1, the C\\H stretch at 2926.16 cm− 1, the N\\H bend at
1622 cm−1, the C\\O stretch at 1028 cm−1. The predicted peaks are
mainly endorsed to limonoid (specifically azadirachtin) flavonoids and
terpenoids exceptionally present in AILE extract [53,54].
Fig. 7. FT-IR spectrum of the Azadirachta indica leave extract.
The primary bactericidal compound present in Azadirachta indica
was azadirachtin [3,15]. The bacterial metabolites produced over the
metal surface from biofilm are corrosive in nature for different metals
[55]. Electron transfer reactions are believed to be liable for MIC [55,
56]. The possible ways of the biocorrosion are oxidation of metal togeth-
er with intracellular reduction under biocatalysis, here MIC was formed
by electron transport commencing outside medium into the cell. Anoth-
er way is by corrosive metabolites produced from bacterial cells which
are act as oxidants to the most of the metal and lead to biocorrosion
[3,15]. Based on these theories, in the present study biocorrosion was
supposed to take place with the bacterial development as biofilm and
their metabolites. The acquaintance of the biofilm on carbon steel API
5LX surface plays a key task in the biocorrosion of hypersaline environ-
ment. Biofilm sticks on the metal surface due to the electrostatic forces,
contact between the metal surface/bacterial cells and extracellular poly-
meric substances. Metal surface roughness and accretion of charges also
involved in the biofilm development [3,57]. The AILE has the properties
of corrosion inhibition and the antibacterial effects on control of corro-
sion caused by bacterial strains (B. subtilis A1, S. parvus B7, P. stutzeri
NA3 and A. baumannii MN3) in hypersaline environment (rich chloride
contents). The bactericidal activities of the AILE terminate the bacterial
growth and the adsorbed phytochemical components of the AILE pro-
tect the carbon steel surface from the adsorption corrosion and biofilm
[15]. Many researchers used synthetic chemicals such as ethylene gly-
col, 8-hydroxyl-N′-(2–quinolin-8-yloxy)acetyl)quinolone-5-
sulfonohydrazide (HQS), bishydrazone derivatives as corrosion inhibi-
tors. These synthetic chemical inhibitors have anticorrosive and antimi-
crobial properties, at the same time they are toxic in nature and
therefore not preferred. Distant from such synthetic inhibitors which
are destructive to the environment, neem extract act as natural green
biocides with higher inhibition efficiency, which are of plant derivation
are of enormous importance nowadays. They are inexpensive, easily
available and environmentally secure which make them favoured con-
testant [3].
4. Conclusions
This study revealed that Azadirachta indica can act as an efficient,
eco-friendly green corrosion inhibitor with biocidal properties for con-
trol of MIC on carbon steel API 5LX in hypersaline environment. From
the growth curve, the total viable counts of the bacterial cells in corro-
sion systems with AILE were highly reduced to 100. It reveals that,
AILE has an antibacterial effect against all the bacterial biofilm on carbon
steel surfaces. Weight loss and electrochemical analysis showed that
bacterial strains S. parvus B7, B. subtilis A1, P. stutzeri NA3 and A.
baumannii MN3 increased the corrosion rate of carbon steel. At the
same time these bacterial strains were very sensitive to the AILE at the
concentration of 150 ppm. AILE suppressed the corrosion mechanisms
in carbon steel and the inhibition efficiency were ranged as 72–81%. In-
creased concentration of the AILE components will expect to lead to
higher corrosion inhibition efficiency. The presence of biocidel com-
pounds azadirachtin and other phytochemical in the AILE could be
playing an important role in the inhibitory mechanism of biocorrosion.
The active compounds Azadirachta co-ordinates with iron on metal sur-
face and form a protective film as Az-Fe complex. Based on these find-
ings Azadirachta indica can be suggested as a green inhibitor with
biocidal properties for control of MIC in crude oil reservoir environment.
Acknowledgments
This study was funded by Department of Biotechnology, Govern-
ment of India (BT/RLF/Re-entry/17/2012), Science and Engineering Re-
search Board, Department of Science and Technology, Government of
India (EEQ/2016/000449 & SB/YS/LS-40/2013) and University Grants
Commission (MRP-MAJOR-MICRO-2013-31825). P. Parthipan acknowl-
edge the DBT, Government of India for financial support through project
 P. Parthipan et al. / Journal of Molecular Liquids 240 (2017) 121–127
scheme (DBT-RLF). Authors also acknowledge the help received from
Dr. J. Madhavan and Dr. R.A. Senthil, Department of Chemistry,
Thiruvalluvar University regarding electrochemical studies and related
discussions.
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