International Biodeterioration & Biodegradation 132 (2018) 66–73

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International Biodeterioration & Biodegradation

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

Allium sativum (garlic extract) as a green corrosion inhibitor with biocidal T

properties for the control of MIC in carbon steel and stainless steel in oilfield
environments
Punniyakotti Parthipana,b,∗∗, Punniyakotti Elumalaic, Jayaraman Narenkumara,
Laura L. Machucad, Kadarkarai Murugane,f, Obuli P. Karthikeyang,h,i, Aruliah Rajasekara,∗
a
Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, Tamilnadu, 632 115, India
b
Electrochemical Energy Research Lab, Centre for Nanoscience and Technology, Pondicherry University, Puducherry, 605 014, India
c
Division of Biotechnology, Advanced Institute of Environment and Biosciences, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan,
Jeonbuk, 54596, South Korea
d
Curtin Corrosion Centre, WA School of Mines: Minerals, Energy and Chemical Engineering, Curtin University, Bentley, WA, 6102, Australia
e
Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu, 641 046, India
f
Thiruvalluvar University, Serkkadu, Vellore, Tamilnadu, 632 115, India
g
Institute of Subtropical Agriculture, Chinese Academy of Science, Changsha, China
h
Queens University of Belfast, Belfast, United Kingdom
i
ProLog Biologicals Pvt. Ltd., Hyderabad, India

A R T I C LE I N FO A B S T R A C T

Keywords: In the present study, the effectiveness of garlic extract to inhibit the bio-corrosion of carbon steel API 5LX (CS)
Allium sativum and stainless steel 316 (SS) in the presence of Bacillus subtilis A1 and Streptomyces parvus B7 was estimated. The
Bio-corrosion antibacterial activity of the garlic extract (GAE) was tested; 100 ppm of the GAE was identified as the minimal
Biofilm inhibitory concentration for bacterial growth. Weight loss and electrochemical studies including linear polar-
Green inhibitor
ization and AC impedance along with surface analysis were used to examine the corrosion inhibition efficiency
Weight loss
Electrochemical impedance spectroscopy
(IE) for both metals in the presence of GAE. The strains A1, B7 and their mixed consortium caused severe
corrosion to both metals. In the presence of GAE the IE for abiotic system was about 81 ± 3% and 75 ± 3%,
while in the presence of the mixed consortium the IE was 72 ± 3% and 69 ± 3% for CS and SS, respectively.
Gas chromatography mass spectrum analysis of GAE indicated that GAE contains a sulphur rich compound
which plays a key role in the inhibition of both bacterial development and corrosion. This is the first time garlic
extract is proposed as a green corrosion inhibitor with biocidal activity to control biocorrosion in hypersaline
corrosive environment containing microorganisms.

1. Introduction
Microbiologically induced corrosion (MIC) is an electrochemical
process where microorganisms enhance metal deterioration (Rajasekar
et al., 2007a; Dheilly et al., 2008; Machuca et al., 2016; Parthipan et al.,
2018). Microbial metabolism in oil reservoir leads to fuel turbidity,
contamination, and deterioration of storage tanks and pipelines
(Hamilton, 1985; Rajasekar et al., 2010). The presence of micro-
organisms are key factors responsible for corrosion issues associated
with the oil industries (Rajasekar et al., 2010; Stevenson et al., 2011;
Lenhart et al., 2014; Lyles et al., 2014). Moreover, water can also
stratify at the substructure of crude oil transporting pipeline, if the oil
velocity is not enough to entrain water and sweep it through the
transporting pipeline system (Rajasekar et al., 2007b).
Microbial corrosion can lead to pipeline failure and has been shown
to increase the operation and maintenance costs of the crude oil in-
dustry (Lee et al., 2010; Suflita et al., 2012; Elumalai et al., 2017).
Generally, 40% of the pipeline metal corrosion in the gas and oil in-
dustries is caused by numerous microorganisms (Rajasekar et al.,
2007b). The microbial strains present near to metal surface might re-
lease metabolic products which are highly corrosive to the metals
(Swaroop et al., 2016). Extracellular polymeric substances (EPS)

∗
Corresponding author.
∗∗
Corresponding author. Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, Tamilnadu, 632
115, India.
E-mail addresses: pparthibiotech@gmail.com (P. Parthipan), rajasekargood@gmail.com, rajasekargood@tvu.edu.in (A. Rajasekar).

https://doi.org/10.1016/j.ibiod.2018.05.005
Received 8 March 2017; Received in revised form 3 April 2018; Accepted 9 May 2018
Available online 26 May 2018
0964-8305/ © 2018 Elsevier Ltd. All rights reserved.
P. Parthipan et al.
participate as an important biological factors in the biofilm develop-
ment on metallic surfaces (Javed et al., 2015). Biofilm formation in-
itiates with the early attachment of microorganisms on the solid sur-
face, and further secretion of EPS leads to the development of a thicker
biofilm and further dispersal of cells which yet again begin with new
biofilms on nearby metal surfaces.
Carbon steel is a commonly used engineering material for the
transportation and storage of the oil products, due to their higher me-
chanical strength and known performance in diverse environments. It
has been widely studied by many researchers in regards to biocorrosion
(Rajasekar et al., 2007a, 2010; Bhola et al., 2014). Stainless steel has
high mechanical strength and enhanced corrosion resistance potential
compared to carbon steel. Corrosion of stainless steel represents a
crucial research area due to their broad use and exceptional applica-
tions in chemical and mechanized industries under aggressive operating
circumstances (Psyllaki et al., 2013; Vazdirvanidis et al., 2017; Xu
et al., 2017). Several biocorrosion studies have also been conducted to
study the corrosion performance of these materials in the presence of
microorganisms (Machuca et al., 2013, 2014a, b; Machuca, 2017).
Prevention of biocorrosion may be achieved by minimizing biofilm
development on metal surfaces. Chemical treatments applied to control
biofilm formation include the use of biocides and other products such as
inhibitors or dispersive agents (Guiamet and Gomez De Saravia, 2005).
Several factors including spectrum of antimicrobial activity, compat-
ibility with other chemicals, cost, and environmental impacts must be
assessed when selecting corrosion inhibitors/biocides (Gaylarde and
Videla, 1992).
Many chemical inhibitors like formaldehyde, ethylene glycol, glu-
taraldehyde, sodium molybdate and quaternary ammonium salts have
been shown to restrict microbial activity effectively and also limit
biofilm formation. However, environmental concerns regarding the
application of these chemical inhibitors represent an ongoing challenge
to the industry (Guiamet and Gomez De Saravia, 2005; Queiroz et al.,
2005; Starosvetsky et al., 2007; Narenkumar et al., 2017,2018). Natural
biocides/inhibitors such as plant materials have received increased
attention lately. Plant derivatives are economical, easily available and
environmentally friendly which make them ideal candidates for MIC
control.
Different biocides include plant extracts and antimicrobial peptides
which are eco-friendly and have been used to control bio-corrosion
(Jayaraman et al., 1999). Recently neem extract was used as green
corrosion inhibitor to control copper and carbon steel corrosion in the
presence of Arthrobacter sulfureus, Bacillus sp., Pseudomonas sp., and
Acinetobacter sp. (Swaroop et al., 2016; Parthipan et al., 2017a). Bhola
et al. (2014) applied neem extracts to manage sulphate reducing bac-
teria (SRB) mediated bio-corrosion; neem extracts controlled microbial
growth as well as biofilm development. Many plant derivatives have
been used to control chemical corrosion, for instance Ervatamia cor-
onaria, Phyllanthus amarus, garlic peel extract, Aloe vera, Lemon verbena,
Gossipium hirsutum, bamboo leaves, Artemisia pallens, orange peel, Musa
Paradisica, Tagetes erecta, among others (Abiola et al., 2009; Abiola and
James, 2010; de Assuncao Araujo Pereira et al., 2012; Garai et al.,
2012; Sethuraman et al., 2017; Li et al., 2014; Ji et al., 2015; Mourya
et al., 2014; Mhiri et al., 2016; Anupama et al., 2016; Fattah-alhosseini
and Noori, 2016). Garlic has many advantages over other plant extracts
including availability and low cost. It contains a mixture of organo-
sulphur compounds which have been shown to display inhibition
properties against microorganisms (de Assuncao Araujo Pereira et al.,
2012). Many researchers have used garlic extract as corrosion inhibitors
for the control of acid corrosion. These studies have demonstrated the
presence of sulphur-containing compounds, which play a key role in the
corrosion inhibition activity (de Assuncao Araujo Pereira et al., 2012;
Rajam et al., 2013; Al-Mhyawi, 2014; Rodriguez-Clemente et al., 2014,
2015). The corrosion inhibition of copper and carbon steel in acidic
medium was over 70–96% in the presence of garlic extract at 400 ppm
concentration. To our knowledge, there are no reports available on the
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International Biodeterioration & Biodegradation 132 (2018) 66–73
inhibitory actions of garlic extract against microbial corrosion in hy-
persaline conditions.
The current investigation focused on the application of garlic extract
(GAE) as a green inhibitor to control MIC of carbon steel API 5LX (CS)
and stainless steel 316 (SS). Weight loss experiments, electrochemical
studies including polarization and AC impedance, X-ray diffraction
(XRD) and gas-chromatography mass spectroscopy (GCMS) were ap-
plied to assess the role of GAE in the different corrosion systems.
2. Material and methods
2.1. Microbial strains and culture conditions
Two bacterial strains were used in this study namely Bacillus subtilis
A1 and Streptomyces parvus B7 which were isolated from an Indian
crude oil reservoir. These strains were identified by 16S rRNA se-
quencing and deposited under NCBI Genbank accession numbers
KP895564 and KP895570 respectively (Parthipan et al., 2017b). Both
bacterial strains were retrieved from glycerol stocks 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
incubated at 37 °C for 24 h. Bacterial inocula were prepared using single
colony in LB broth (pH 7.0) and kept in an orbital shaker (150 rpm) for
24 h at 37 °C.
2.2. Biocide preparation
Aqueous garlic extracts were obtained by weighing 10 g of peeled
garlic cloves and mashed with 100 mL of deionized water. This extract
was filtered using Whatman filter paper to remove the residue and
stored at 4 °C until further use (Arunachalam, 2011).
2.3. Chemical analysis of garlic extract
2.3.1. Gas chromatography analysis
GAE was analyzed using gas chromatograph mass spectrometry to
identify the chemical nature of the inhibitor. 1 μL of sample was in-
jected into a gas chromatography (Shimadzu QP2010 Ultra, Rtx-5Sil MS
(30 m × 0.25 mm ID × 0.25 μm)). The carrier gas was He, the flow rate
was set as 1.5 mL min−1 and the working temperature of the GC in-
jector was 260 °C. The temperature was set between 60 and 260 °C, at a
speed of 5 °C min−1, through an isothermal phase of 10 min at the end
of the analysis. The electron impact ion source was sustained at 200 °C.
Mass spectra were recorded at 70 keV. The mass spectra were obtained
with an m/z range: 40–600 ultra-high resolution modes with an ac-
quisition speed of 6 spectra/second. The identification of components
was done in scan mode by using NIST11 and Wiley8 library and the
target mass spectra obtained from sample are compared with the mass
spectra obtained from the library.
2.3.2. Fourier transform infra-red spectroscopy (FT-IR)
FT-IR analysis was carried to identify the chemical groups present in
the garlic extract. For FT-IR analysis, a few drops of garlic extract were
dried over glass plate and further dried sample was crushed with the
addition of potassium bromide in 1:100 ratio and the pellet was fixed in
the sample container, and analyzed using FT-IR (model Jasco) in the
mid IR region 400-4000 cm−1.
2.4. Antibacterial properties of garlic extract
2.4.1. Agar-well diffusion assay
To assess the antibacterial activity of the GAE, Mueller Hinton Agar
(MH) (Himedia-India) plates were prepared as per the instructions of
the manufacturer. Five different concentrations; 20 ppm, 50 ppm,
100 ppm, 150 ppm and 200 ppm of the GAE were selected for evalua-
tion of their bactericidal activity. Both bacterial strains A1 and B7 were
P. Parthipan et al.
inoculated separately and uniformly spread over the surface of MH agar
plates. Wells (6 mm dia with 5 mm depth) were prepared on the solid
MH agar using a sterile glass borer. GAE at the different concentrations
were dispensed to each respective well. 10 μg of Gentamycin was used
as a positive control because of their broad range of bactericidal ac-
tivity. Sterile distilled water was used as negative control and incubated
at 37 °C for 24 h. Zones of inhibition were measured and expressed in
millimetres (mm). Each experiment was done in triplicates.
2.4.2. Biofilm inhibition assay
Overnight cultures of A1 and B7 in Luria Bertani (LB) medium were
diluted in 1:20 ratio with fresh LB medium with different concentration
of GAE such as (50 ppm, 100 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 the same culture
broth was preferred without GAE, which was served as control. The
suspended culture medium was removed and the 96-well plate was
washed with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl,
10 mM NaHPO4, 2 mM KHPO4, pH:7.2), 120 μL of crystal violet was
added to the each well and left for 20 min. Finally, 125 μL of acetic acid
was added over the crystal violet and incubated for additional 15 min at
37 °C. Visible changes in emergence were noticed to confirm the biofilm
inhibition capability of the GAE at the lowest concentration (O'Tool
et al., 1999).
2.5. Weight loss experiments
MIC behavior of carbon steel and stainless steel were investigated as
described by Rajasekar et al. (2010) with the modification in the sys-
tems design as follows: produced water collected from an oil reservoir
was used as corrosive medium. Collected produced water contained
higher concentrations of inorganic chemical substances such as chloride
(36 g/L), iron (32 mg/L), sulphate (354 mg/L), magnesium (529 mg/L)
and calcium (1800 mg/L) as major components. Carbon steel API 5LX
coupons (metals components in (%): 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) and 316 stainless
steel (metals components in (%): Cr 14.0, Mo 1.0, Ni 2.0 and C 1.0)
were cut into 25 × 25 × 0.4 mm and 10 mm × 10 mm x 1 mm size
coupons for weight loss studies and electrochemical measurements
(impedance and potentiodynamic polarization studies) respectively.
Coupons were smoothened with different grades of silicon carbide pa-
pers (180, 500, 800, 1200, and 1500) and finally polished with alumina
powder (0.3 μM) (Rajasekar et al., 2010). The polished working elec-
trodes (WE) with an exposed area of 1.0 cm2 were used for the EIS
studies. The electrodes were sterilized under UV light prior to the ex-
periment. For weight loss measurements, initial weights of the each
coupon were recorded before immersion into the corrosion systems.
The coupons were first dipped into the deionized water and degreased
with trichloroethylene. System I was set as coupons (in triplicates)
immersed in a 500 mL Erlenmeyer flask with 300 mL sterile corrosive
medium (produced water) which acted as abiotic control systems.
System II –V are similar to that of system I. The system II was added
with 100 ppm of GAE, system III was inoculated with bacterial strain A1
(106 CFU/mL), system IV was inoculated with bacterial strain B7 (about
106 CFU/mL). System V was inoculated with both A1 and B7 (mixed
consortia) and system VI was similar to system V with the addition of
GAE (100 ppm), similar systems were used for the SS. Triplicate tests
were carried out for each system. These systems were left undisturbed
for 20 days at 37 °C. At the end of the 20th day, the coupons were
studied using EIS and x-ray powder diffraction (XRD). To examine the
antibacterial activity of the GAE on the corrosion studies, the total vi-
able count (TVC) of the bacterial biofilm on the metal surface was
scrapped at two day intervals during the biocorrosion studies and TVC
was calculated using plate count method.
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International Biodeterioration & Biodegradation 132 (2018) 66–73
2.6. Electrochemical studies
EIS studies were conducted using CH Instruments Inc., USA (Model
CHI-608E) with a three electrode system, a standard calomel electrode
(SCE) employed as reference electrode, a platinum wire was used as
counter electrode and the metal coupons (both CS and SS), recovered at
the end of the each weight loss study, acted as the working electrode.
The corrosive media from respective systems (weight loss experiments)
were used as the electrolyte solution for the EIS analysis. The im-
pedance measurements were conducted under steady state conditions
in the frequency range of 0.1–105 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 corrosion potential (Ecorr). Zsim Demo
3.20d software was used to analyse electrochemical parameters. All the
measurements were recorded in triplicate.
2.7. Surface analysis
At the end of the weight loss experiment, the coupons were re-
covered and the corrosion products were detached carefully. The
weight loss coupons were surface cleaned with Clark's solution
(Rajasekar et al., 2011). Corrosion rate was calculated as previously
described by Rajasekar et al. (2017) and Parthipan et al. (2017a). The
inhibition efficiency (IE%) was measured using the following formula
(de Assuncao Araujo Pereira et al., 2012):
W0−W
IE %= x100
W0
Where W0 and W are the weight loss in absence and presence of the
GAE, respectively. Rust products collected from each systems were
dried and firmed as powder and characterized using XRD to validate the
presence of the oxides using X-ray diffractometer (JEOL JDX-8030) and
scanned using CuKα radiation (Ni filter) in the range between 10 and
85° at 40 kV, 20 mA.
3. Results and discussion
3.1. Antimicrobial assay
Different concentrations of the garlic extract were checked for their
antibacterial activity using Agar-Well diffusion method as shown in the
Fig. 1. 20 ppm of the GAE showed no inhibition activity on the MH
agar. The remaining concentrations showed inhibition activities on the
Fig. 1. Antibacterial activity of the garlic extracts on the MH agar with strain
A1 and B7. All of the bar graphs represent the mean of triplicate tests. The error
bars represent standard deviations of the triplicate tests.
P. Parthipan et al. International Biodeterioration & Biodegradation 132 (2018) 66–73

Table 1
Corrosion rate and inhibition efficiency for the carbon steel and stainless steel in presence and absence of Bacillus subtilis A1 and Streptomyces parvus B7 and garlic
extract. All values are means of triplicate tests.
Systems Carbon steel API 5LX Stainless steel 316

Weight loss (mg) Corrosion rate (mm/y) Inhibition efficiency Weight loss (mg) Corrosion rate (mm/y) Inhibition efficiency
(%) (%)

I - Abiotic Control 11.4 ± 2 0.042 ± 0.001 – 1.6 ± 2 0.006 ± 0.0007 –

II- Abiotic control with 100 ppm of 2.1 ± 0.5 0.0078 ± 0.0016 81 ± 3 0.4 ± 0.5 0.0015 ± 0.0001 75 ± 3
garlic extract
III – B. subtilis A1 19.5 ± 2 0.072 ± 0.001 – 3.5 ± 2 0.013 ± 0.001 –
IV – S. parvus B7 22.2 ± 2 0.082 ± 0.001 – 2.5 ± 2 0.009 ± 0.001 –
V - Mixed consortia 28.7 ± 1 0.107 ± 0.001 – 6.5 ± 2 0.024 ± 0.001 –
VI - Mixed consortia with 100 ppm of 3.2 ± 3 0.012 ± 0.001 72 ± 3 0.6 ± 2 0.0022 ± 0.0007 69 ± 3
garlic extract

mm/y - millimeter per year.

agar surfaces. Zone of the inhibition increased with increasing con-

centration. Further 50 ppm, 100 ppm, 150 ppm and 200 ppm were se-
lected for the biofilm inhibition assay. In biofilm inhibition assays, both
bacterial strains A1 and B7 were checked by microtiter plate with
crystal violet (O'Tool et al., 1999). This analysis confirmed the cap-
abilities of the bacterial strains to form biofilm; at the same time in the
presence of GAE at different concentrations (100 ppm, 150 ppm and
200 ppm), biofilm development was greatly inhibited. A lower GAE
concentration (50 ppm) showed moderate inhibition on both strains.
Hence, 100 ppm of the GAE was identified as the minimum inhibitory
concentration for both bacterial strains and this concentration was used
for biocorrosion experiments.

3.2. Weight loss experiment

The outcomes of the weight loss experiments for carbon steel API
5LX and 316 stainless steel in the presence and absence of bacterial
strains and GAE after 20 days of immersion were presented in Table 1.
In the absence of bacterial strains (abiotic control) the mean corrosion
rate (CR) was 0.042 ± 0.001 mm/y for CS and 0.006 ± 0.0007 mm/y
for SS respectively. However, in the presence of individual bacterial
strains A1 and B7, the CR were doubled for both metals
(0.072 ± 0.001 mm/y, 0.082 ± 0.001 mm/y for CS and
0.013 ± 0.001 mm/y, 0.009 ± 0.001 mm/y for SS, respectively) as
compared to abiotic control system (Table 1). In the presence of mixed
consortium (system V) the CR was slightly higher (0.107 ± 0.001 mm/
y and 0.024 ± 0.001 mm/y for CS and SS, respectively) compared to
individual strains in systems III and IV (Table 1). Abiotic control system
with GAE (system II) showed tremendous IE for both (CS and SS) metals
as 81 ± 3% and 75 ± 3%, respectively. The CR and IE for the mixed
consortia with GAE system-VI for both (CS and SS) metals were ob-
served as 0.012 ± 0.001 mm/y (72 ± 3%) and
0.0022 ± 0.0007 mm/y (69 ± 3%), respectively. Therefore, the ad-
dition of GAE resulted in the lowest CR for both CS and SS.
The growth curve of the biofilm in the different corrosion systems,
i.e. abiotic control, A1, B7, mixed consortia with and without GAE on
Fig. 2. Bacterial growth curve of the different corrosion system in presence of
CS and SS metal surfaces, are shown in Fig. 2A–B. The growth curve the strain A1, B7, mixed consortia with and without GAE biocide. A. Carbon
showed that biofilms of A1, B7 and mixed consortia on carbon steel steel API 5LX and B. Stainless steel 316. All values are mean of triplicate test
were observed in the range of 4.7–6.5 × 106 (CFU/cm2) whereas in the and error bars stand for standard deviation. All of the bar graphs represent the
presence of GAE biofilm cell counts were reduced to below 6 × 101 mean of triplicate tests. The error bars represent standard deviations of the
(CFU/cm2) for CS. Similarly, biofilm cells on SS were measured as triplicate tests.
4.2–5.5 × 105 (CFU/cm2) for A1, B7 and mixed consortia and 5 × 101
(CFU/cm2) in the presence of GAE. No colony was found in the abiotic Assuncao Araujo Pereira et al. (2012) used garlic peel extract as green
control system with GAE inhibitor. It reveals that GAE had an inhibiting inhibitor for control of the chemical corrosion in acidic corrosive
effect on biofilm development on both CS and SS metal surfaces. medium and reported that the 100 ppm of the garlic peel extract was
Weight loss measurements used for the analysis of corrosion rate required to achieve a maximum inhibition efficiency of about 84%.
and inhibition efficiency usually offer more consistent results than Inhibition efficiency slightly increased with increasing the concentra-
electrochemical methods since the experimental settings more strongly tion of the garlic peel extract. In this study, GAE exhibited biostatic
imitate real conditions (de Souza and Spinelli, 2009). Recently, de

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P. Parthipan et al. International Biodeterioration & Biodegradation 132 (2018) 66–73

Table 2
Polarization and impedance parameters for carbon steel API 5LX in the presence/absence Bacillus subtilis A1 and Streptomyces parvus B7 and garlic extract. All values
are means of triplicate tests.
Systems polarization data impedance data

Icorr (A/cm2) Ecorr (V) βa (mV/dec) βc (mV/dec) Rct (Ω cm2) Rs (Ω cm2)

I- Abiotic control 3.5 × 10−5 ± 0.1 × 10−5 −509 ± 2 7.31 ± 0.1 −1.75 ± 0.01 99.5 ± 1 5.8 ± 0.1
II- Abiotic control with 100 ppm GAE 4.9 × 10−5 ± 0.1 × 10−5 −477 ± 2 7.02 ± 0.1 −1.30 ± 0.01 135.2 ± 2 5.9 ± 0.1
III- B. subtilis A1 4.3 × 10−5 ± 0.1 × 10−5 −562 ± 3 8.09 ± 0.1 −2.25 ± 0.1 68.2 ± 1 5.6 ± 0.1
IV- S. parvus B7 3.7 × 10−5 ± 0.01 × 10−5 −556 ± 3 7.92 ± 0.1 −1.91 ± 0.01 72.6 ± 1 5.5 ± 0.1
V- Mixed consortia 6.1 × 10−5 ± 0.1 × 10−5 −568 ± 2 8.24 ± 0.1 −2.69 ± 0.1 91.7 ± 2 5.2 ± 0.1
VI- Mixed consortia with 100 ppm GAE 4.8 × 10−5 ± 0.1 × 10−5 −487 ± 2 7.10 ± 0.1 −1.41 ± 0.01 126.7 ± 2 5.7 ± 0.1

Icorr-Corrosion current, Ecorr - Corrosion potential, βa - anodic tafel slope, βc - cathodic tafel slope, Rct - Charge transfer resistance, Rs- Solution resistance.

properties resulting in higher inhibition efficiency when compared to
the other natural products like neem extract used as corrosion in-
hibitor/biocide against SRB (about 50% of IE only) (Bhola et al., 2014).
3.3. Electrochemical studies
3.3.1. Potentiodynamic polarization
The potentiodynamic polarization curves of the CS in the presence
and absence of the bacterial strains and GAE after 20 days immersion
are presented in Fig. S1 (see supplementary information for details).
The electrochemical parameters such as corrosion potential (Ecorr), the
corrosion current density (Icorr), anodic (βa) and cathodic (βc) Tafel
values were presented in Table 2, which were collected from Tafel
plots. Similarly, polarization curves and electrochemical parameters for
the SS are shown in Fig. S2 (see supplementary information for details)
and Table 3, respectively. Tafel parameters of the A1, B7 and mixed
consortia confirmed that bacterial strains have the highest capabilities
to cause metal deterioration in the chloride rich environment. From the
Tafel values, it can be seen that the Icorr decreased in presence of the
GAE as compared with mixed consortia for both the metals. Both βc and
βa of the systems with GAE were decreased in comparison with the
other evaluated systems in both metals. Similar observation was noticed
by Faustin et al. (2015) and this finding implies that the active com-
pounds present in garlic extract adsorbed on the metal surface and
hindered the corrosion process.
3.3.2. Electrochemical impedance spectroscopy (EIS)
Fig. S3 and S4 (see supplementary information for details) present
the electrochemical impedance for the carbon steel and stainless steel in
the presence and absence of A1 and B7 and GAE, respectively. The
impedance parameters such as charge transfer values (Rct) and solution
resistance (Rs) values of the different systems for both CS and SS are
shown in Tables 2 and 3, respectively. The Rct values were maximum
recorded in the abiotic system with GAE (135.2 ± 2 and
3.8 ± 0.1 Ω cm2 for CS and SS respectively) followed by mixed con-
sortia with GAE (126.7 ± 2 and 3.2 ± 0.3 Ω cm2 for CS and SS re-
spectively) compared to the abiotic system (99.5 ± 1 and
2.9 ± 0.2 Ω cm2 for CS and SS respectively). The increases in Rct va-
lues were associated with the development of protective films at the
metal/solution interface (de Assuncao Araujo Pereira et al., 2012;
Mourya et al., 2014). Therefore, this observation might be ascribed to
the adsorption of the compounds of GAE on the metal/solution inter-
face (Kamal and Sethuraman, 2012). This hypothesis is supported by
both anodic/cathodic polarization curves and the corrosion potential
effects (de Assuncao Araujo Pereira et al., 2012).
3.4. Surface analysis using X-ray diffractometer
Fig. 3 shows the XRD patterns of the corrosion products on the
carbon steel in the presence/absence of the bacterial strains and the
GAE. XRD confirmed that the presence of ferric oxide, manganese oxide
and iron oxide-hydroxide as corrosion products at the end of the bio-
corrosion studies. When compared to the abiotic control, higher intense
peaks were observed in the A1, B7 and mixed consortia system. Lower
intensity of the peaks was noticed in the mixed consortia with GAE.
Similarly Fig. 4 shows the XRD patterns of the stainless steel in the
presence and absence of the A1 and B7 and the GAE. From the XRD
data, the corrosion products collected from the SS metal surface was
included with Fe2O3, MnO3, FeOOH, α-FeOOH and Fe3O4. Similar to
the CS, SS also showed higher intensity peaks of these phases in the
presence of A1 and B7 as compared to the abiotic and mixed consortia
with GAE systems. The presence of these iron particles in the corrosion
products of bacterial systems indicates that both bacterial strains A1
and B7 has the ability to oxidize inorganic compounds present in the
both metals and utilize them as inorganic source for their growth
(Rajasekar et al., 2005; Suflita et al., 2012).
The antibacterial activities of the GAE against biofilms on the metal
surfaces were confirmed from the biofilm inhibition assay. XRD results
suggest that GAE not only suppresses growth of the bacteria but also
minimises corrosion in the corrosive environment likely via adsorption
on the metal surface. This observation was supported by few studies
(Swaroop et al., 2016) which showed that garlic extract act as corrosion
inhibitor in different corrosive media including acidic and aqueous
system (de Assuncao Araujo Pereira et al., 2012).

Table 3
Polarization and impedance parameters for stainless steel 316 in the presence/absence Bacillus subtilis A1 and Streptomyces parvus B7 and garlic extract. All values are
means of triplicate tests.
Systems polarization data impedance data

Icorr (A/cm2) Ecorr (V) βa (mV/dec) βc (mV/dec) Rct (Ω cm2) Rs (Ω cm2)

I- Abiotic control 1.9 × 10−5 ± 0.1 × 10−5 −257 ± 2 6.03 ± 0.1 −3.72 ± 0.1 2.9 ± 0.2 5.6 ± 0.02
II- Abiotic control with 100 ppm GAE 1.6 × 10−5 ± 0.1 × 10−5 −217 ± 2 5.86 ± 0.1 −2.27 ± 0.1 3.8 ± 0.1 5.9 ± 0.02
III- B. subtilis A1 1.8 × 10−5 ± 0.1 × 10−5 −322 ± 3 6.76 ± 0.1 −4.87 ± 0.1 0.8 ± 0.2 5.7 ± 0.03
IV- S. parvus B7 1.8 × 10−5 ± 0.1 × 10−5 −320 ± 3 5.73 ± 0.1 −4.76 ± 0.1 1.6 ± 0.1 5.1 ± 0.03
V- Mixed consortia 2.6 × 10−5 ± 0.1 × 10−5 −295 ± 3 6.92 ± 0.1 −3.72 ± 0.1 0.6 ± 0.2 5.2 ± 0.02
VI- Mixed consortia with 100 ppm GAE 1.7 × 10−5 ± 0.1 × 10−5 −227 ± 2 5.98 ± 0.1 −2.32 ± 0.1 3.2 ± 0.3 5.7 ± 0.02

Icorr-Corrosion current, Ecorr - Corrosion potential, βa - anodic tafel slope, βc - cathodic tafel slope, Rct - Charge transfer resistance, Rs- Solution resistance.

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P. Parthipan et al. International Biodeterioration & Biodegradation 132 (2018) 66–73

Fig. 3. XRD patterns of the corrosion products of the carbon steel API 5LX in
presence/absence of the bacterial strains and the GAE, A-abiotic control, B-
Fig. 4. XRD patterns of the corrosion products of the stainless steel 316 in
strain A1, C- strain B7, D-mixed consortia and E-mixed consortia with 100 ppm
presence/absence of the bacterial strains and the GAE, A-abiotic control, B-
GAE.
strain A1, C- strain B7, D-mixed consortia and E-mixed consortia with 100 ppm
GAE.
3.5. Gas chromatographic analysis of the garlic extract
Table 4
Gas chromatographic characterization of the garlic extracts con- GCMS characterization of the garlic extract.
firmed that presence of the organosulphur compounds in the GAE.
RT Compound Chemical MW Chemical structure
Important compounds found in the GAE were diallyl disulphide
formula
(C6H10S2) (de Assuncao Araujo Pereira et al., 2012) and 2-fur-
ancarboxaldehyde, 5-(hydroxymethyl) - (C6H6O3), also other sulphur 7.50 Diallyl Disulphide C6H10S2 146
containing components such as trisulfide, di-2-propenyl and di-n-de-
10.67 Trisulfide, Di-2-Propenyl C6H10S3 178
cylsulfone were confirmed in the GAE extracts (Table 4). These com-
pounds have been identified in GAE previously (de Assuncao Araujo
Pereira et al., 2012).
Fig. 5 shows the FT-IR spectra of garlic extract. It was found that in
11.30 2-Furancarboxaldehyde, C6H6O3 126
the garlic extract, the –OH stretch at 3410 cm−1, the C-H stretch at 5-(Hydroxymethyl)-
2930 cm−1, band at 1630 cm−1 is assigned to stretching of amides
(C=O). The band found at 1407 cm−1 could be due to the presence of
–O–H bend of carboxylates. Bands at the 1128 cm−1 and 1028 cm−1
can be attributed to SO2 (sulfones) and C–N stretching vibrations due to
28.83 Di-N-Decylsulfone C20H42O2S 346
presence of amines respectively. 926 cm−1 is assigned to -C–H,
818 cm−1 could be attributed to N–H bend of primary amines and
602 cm−1 for C–H bend of alkynes. The predicted peaks were mainly
endorsed to the sulphur and nitrogen based components present in the
garlic extract (Arunachalam, 2011).

3.6. Corrosion inhibition mechanism by garlic extract Note: RT- Retention time and MW- Molecular weight.

The biofilm inhibition assay revealed that both bacterial strains

71
P. Parthipan et al.
Fig. 5. FTIR spectra of garlic extract.
were very sensitive to the GAE. It was also evident from the potentio-
dynamic polarization, AC impedance and XRD analysis with bacterial
strains and GAE systems. The electrochemical parameters indicate that
bacterial activity was different in the presence and absence of the GAE.
The garlic extract had been identified as metal protector against cor-
rosion by adsorption of their organic compounds on the metal surface
(de Assuncao Araujo Pereira et al., 2012).
The bactericidal compounds present in the Allium sativum are diallyl
disulphide, trisulfide and other compounds as presented in Table 4 (de
Assuncao Araujo Pereira et al., 2012). It is known that biofilm is formed
on the metal surface due to the electrostatic forces, contact between the
metal surface/bacterial cells and extracellular polymeric substances.
Metal surface roughness and accretion of charges also are involved in
biofilm development (Swaroop et al., 2016). Once biofilms are formed,
bacterial metabolites and EPS produced across the metal surface can
lead to increase corrosion rates in different metals (Zhai et al., 2015).
Electron transfer mechanisms are also believed to be liable for MIC
(Zhai et al., 2015; Zhang et al., 2015). Results from the present study
shows that garlic extract exhibits both corrosion inhibition properties
and bactericidal properties which affected biofilm activity and resulted
in the lower biocorrosion rates in a hypersaline environment. This in-
hibition resulted from the adsorbed phytochemical components of the
GAE, which could have controlled the biofilm formation and protected
the carbon steel surface from corrosion (de Assuncao Araujo Pereira
et al., 2012). Increasing GAE concentration is expected to lead higher
inhibition efficiency.
4. Conclusions
This study revealed that the GAE can act as an efficient, eco-friendly
and natural green corrosion inhibitor with biocidal properties for
carbon steel API 5LX and stainless steel 316 in hypersaline produced
water environment which was enriched with corrosive bacteria. Weight
loss and electrochemical analysis showed that B. subtilis A1 and S.
parvus B7 increased the corrosion in both metals. At the same time,
these two bacterial strains were very sensitive to the GAE at a con-
centration of 100 ppm. Viable cell counts in biofilms of the mixed
consortia formed on surface of both metals were greatly reduced in the
presence of GAE. In addition, GAE was shown to inhibit corrosion in
both metals in the presence and absence of bacteria; inhibition effi-
ciencies were about 81 ± 3% and 75 ± 3% for abiotic system while
72 ± 3% and 69 ± 3% in the presence of mixed bacterial consortia
for CS and SS, respectively. The presence of sulphur rich compounds in
GAE plays a key role in the corrosion inhibitory mechanism. Based on
these findings garlic extract is hereby proposed as a green corrosion
72
International Biodeterioration & Biodegradation 132 (2018) 66–73
inhibitor with biocidal properties for control of the MIC. Further studies
are needed to extend the use of the garlic extract as efficient green
inhibitor for a broader range of microorganisms, as stand-alone ap-
proach or in combination with other inhibitor/biocidal compounds.
Acknowledgments
P. Parthipan, gratefully acknowledge the Science and Engineering
Research Board, Department of Science and Technology, for providing
research fellowship under National Postdoctoral Fellowship (PDF/
2017/001134). Dr. A. Rajasekar, acknowledge Department of
Biotechnology, Government of India (BT/RLF/Re-entry/17/2012),
DST-SERB, Government of India (EEQ/2016/000449) and University
Grants Commission-MRP (MRP-MAJOR-MICRO-2013-31825).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.ibiod.2018.05.005.
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