Cationic Gemini surfactant as a corrosion inhibitor and a biocide for high salinity sulfidogenic bacteria originating from an oil-field water tank

Article abstract of DOI:10.1007/s11743-013-1551-4

A novel cationic gemini surfactant (NCGS) was synthesized and characterized. The inhibitory effect of NCGS was evaluated on the basis of protecting a metal surface from the salinity (5.49 % NaCl) and the activity of environmental sulfidogenic bacteria which originated from an oil-field water tank. Sulfidogenic bacterial activities were determined based on sulfide production, redox potential, changes in biofilm structures and constituents and metal corrosion rate calculations. At high surfactant concentrations, the sulfide production was completely inhibited as well as a considerable drop in the redox potential was observed in the reactor's bulk phase. A minimum inhibitory concentration of the NCGS was achieved at a concentration of 1 mM. The NCGS showed a high ability to inhibit a biofilm over the metal surface at a concentration of 0.1 mM. The lowest metal corrosion rate was detected at a concentration of 5 mM with a metal corrosion inhibition efficiency of 97 %. In addition the NCGS showed a nonspecific biocidal activity against Gram-positive and Gram-negative bacterial strains.

Full text of DOI:10.1007/s11743-013-1551-4

J Surfact Deterg
DOI 10.1007/s11743-013-1551-4
ORIGINAL ARTICLE
Cationic Gemini Surfactant as a Corrosion Inhibitor
and a Biocide for High Salinity Sulfidogenic Bacteria
Originating from an Oil-Field Water Tank
•
Ahmed Labena Mohamed A. Hegazy
•
•
Harald Horn Elisabeth Mu¨ller
Received: 29 August 2013 / Accepted: 5 November 2013
Ó AOCS 2013
Abstract A novel cationic gemini surfactant (NCGS)
was synthesized and characterized. The inhibitory effect of
NCGS was evaluated on the basis of protecting a metal
surface from the salinity (5.49 % NaCl) and the activity of
environmental sulfidogenic bacteria which originated from
an oil-field water tank. Sulfidogenic bacterial activities
were determined based on sulfide production, redox
potential, changes in biofilm structures and constituents
and metal corrosion rate calculations. At high surfactant
concentrations, the sulfide production was completely
inhibited as well as a considerable drop in the redox
potential was observed in the reactor’s bulk phase. A
minimum inhibitory concentration of the NCGS was
achieved at a concentration of 1 mM. The NCGS showed a
high ability to inhibit a biofilm over the metal surface at a
concentration of 0.1 mM. The lowest metal corrosion rate
was detected at a concentration of 5 mM with a metal
corrosion inhibition efficiency of 97 %. In addition the
NCGS showed a nonspecific biocidal activity against
Gram-positive and Gram-negative bacterial strains.
Keywords Sulfidogenic biofilm ꢀ High salinity
water-tank ꢀ Cationic gemini surfactant ꢀ
Microbiological corrosion ꢀ Antibacterial activity
Introduction
Seawater is widely used in the oil and gas industries for
cooling purposes, fire-fighting, oil-field water injection and
desalination plants [1, 2]. Oil-field seawater is a suitable
medium for sulfidogenic microorganisms as it contains a
high sulfate concentration (*20 mM) and other nutritional
requirements for microbial growth. Sulfidogenic microor-
ganisms grow under anaerobic conditions and gain energy
for growth by oxidizing organic compounds or hydrogen
with sulfate being reduced to hydrogen sulfide [3]. Sul-
fidogenic microorganisms are a big problem in the petro-
leum industries where they cause iron and steel corrosion
[4]. The corrosiveness of these microorganisms is due to
metabolites produced such as hydrogen sulfide (H2S), the
supposed electrochemical effect termed ‘‘cathodic depo-
larization’’, and microbial colonization (biofilm) on the
metal surface. Sulfidogenic biofilms frequently show a
localized attack in the form of a slimy film composed of
multispecies of microbial communities, extracellular
polymeric substances (EPS) and water [5]. Biofilms facil-
itate corrosion by trapping corrosive metabolites products
such as hydrogen sulfide in close proximity to metal sur-
faces and then initialize localized metal corrosion [6].
There are different approaches to achieve corrosion
inhibition. The applications of inhibitors are the most
practical strategy for corrosion mitigation [7]. To be
effective, inhibitors have to provide high microbial inhi-
bition efficiency (biocides), displace water from the metal
surface, interact with anodic or cathodic reaction sites to
A. Labena (&) ꢀ E. Mu¨ller
Chair of Urban Water Systems Engineering, Technical
University of Munich, Am Coulombwall, 85748 Garching,
Germany
e-mail: labena.labena@gmail.com
M. A. Hegazy
Egyptian Petroleum Research Institute (EPRI), Nasr City, Cairo,
Egypt
H. Horn
Chair of Water Chemistry and Water Technology, Karlsruhe
Institute of Technology, Engler-Bunte-Institut,
Engler-Bunte-Ring 1, 76121 Karlsruhe, Germany
123

J Surfact Deterg
retard oxidation and reduction corrosion reactions and to
prevent the transportation of water and corrosive metabo-
lites to the metal surface [8]. A surfactant or surface active
compound is defined as a substance that, at a certain con-
centration, adsorbs partially or completely to the interface
(metal/liquid) in a particular system [9]. A monomeric
surfactant consists of a polar hydrophilic head attached to a
non-polar hydrophobic tail. Dissolved surfactant molecules
escape from water to an available interface due to their
hydrophobic nature. A gemini surfactant represents a new
class of surfactant. It is composed of at least two hydro-
philic heads and two hydrophobic chain tails which are
linked by spacers [9]. A gemini surfactant can protect the
metal surface by adsorption to the metal/liquid interfaces.
Previous studies revealed that gemini surfactants can be
used as corrosion inhibitors and biocides with high metal
corrosion inhibition efficiencies [10, 11].
esterification reaction between 2 mol of N-(2-hydroxy-
ethyl)-N,N-dimethyl-dodecane-1-aminium bromide and
1 mol of phosphoric acid (in the presence of toluene as the
solvent and p-toluene sulfonic acid as the dehydrating
agent). The reaction was completed after the water had
been removed from the reaction system and it was con-
centrated up to 2 mol. Afterward, the reaction mixture was
distilled under a vacuum to completely remove the solvent.
The third step, 1 mol of the resulted products was allowed
to react with 1 mol of potassium hydroxide. Then reflux in
ethanol for 12 h at 70 °C. The reaction mixture was left to
cool for 1 h and then filtered and concentrated by ethanol
evaporation. At the end the pale brown precipitate obtained
was recrystallized twice from ethanol.
The chemical structure of the synthesized surfactant was
confirmed by Fourier transform infrared (FTIR) and
nuclear magnetic resonance (NMR) spectroscopy (Bruker,
Vortex 70 and Bruker, 400 MHz NMR spectrometer,
Avance DRX 400 for FTIR and NMR, respectively). The
synthesized surfactant was more confirmed by 31P NMR
using Jeol ECA 500 NMR spectrometer at 500 MHz.
Although corrosion inhibitors are widely used by the
Egyptian Petroleum Companies, the problem of corrosion
damages is still urgent and considered to be an open
question. To a certain extent this is due to the fact that
sulfidogenic bacteria inducing pitting corrosion are
responsible for all bio-damage cases of high salinity water
tanks. Therefore, a novel cationic gemini surfactant was
synthesized and characterized. Investigation of the activity
and effectiveness of the synthesized surfactant (corrosion
inhibitor and biocide) was carried out to protect the metal
surface from medium salinity and activity of environmental
sulfidogenic bacteria. The sulfidogenic bacteria originated
from a high salinity water tank of the Qarun Petroleum
Company (QPC, Egypt). The activity of the surfactant as a
biocide was discussed on the basis of sulfide production,
redox potential, cultivated biofilm constituent analysis and
metal corrosion rate calculations. The minimal inhibitory
concentration (MIC) of the synthesized surfactant was
determined from a most probable number experiment. In
addition, antibacterial activity of the synthesized surfactant
(against Gram-positive and Gram-negative strains) was
evaluated and the MIC was calculated.
Surface Active Properties of the Synthesized Surfactant
The Surface Tension (c)
The surface tension was analyzed at different concentra-
tions of the synthesized surfactant using a Du Nouy Ten-
siometer (Kru¨ss Type 6). The measurement for the
synthesized surfactant was done in distilled water (with a
surface tension of 72 mN m-1) at 25 °C.
The Effectiveness (pCMC
)
The surface tension value (c) at CCMC point was used to
calculate the surface pressure (effectiveness) value
according to the following equation [13]:
pCMC ¼ co ꢁ cCMC
;
ð1Þ
where co and cCMC are the surface tensions of pure water
and surface tension at CCMC, respectively. The most
effective surfactant is one that gives the greatest lowering
in the surface tension at the CCMC point.
Materials and Methods
Synthesis of the Novel Cationic Gemini Surfactant
(NCGS)
The Surface Excess (Cmax
)
The NCGS in this study was synthesized through three
steps. The first step was a quaternization reaction between
1 mol of 2-(dimethylamino)ethanol and 1 mol of 1-bro-
mododecane in ethanol for 12 h at 70 °C [12]. The mixture
was left to cool and precipitate. Then the white precipitate
obtained was purified by diethyl ether and afterward re-
crystallized from ethanol. The second step was an
The surface excess was calculated according to the Gibbs’
adsorption equation [9]:
ꢀ
ꢁꢀ
ꢁ
ꢁ1
dc
d ln C
Cmax
¼
;
ð2Þ
nRT
where Cmax is the surface excess concentration of surfac-
tant ions, R is the gas constant, T is the absolute
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J Surfact Deterg
temperature, c is the surface tension at a specific concen-
tration, n is the number of species ions in solution and C is
the concentration of surfactant. A surfactant substance that
decreases the surface energy is thus present in excess or
near the surface. That means the surface tension decreases
with increasing activity (concentration) of a surfactant
molecule.
Table 1 Composition of anaerobic modified Postgate’s B (PB) and
Postgate’s C (PC) medium
Composition (g L-1
)
Modified PB
Modified PC
KH2PO4
0.5
1.0
1.0
–
0.5
1.0
4.5
0.06
0.06
4.42
1.0
–
NH4Cl
Na2SO4
CaCl2ꢀ6H2O
MgSO4ꢀ7H2O
Sodium lactate
Yeast extract
Ascorbic acid
2.0
3.5
1.0
0.1
0.1
0.5
–
The Minimum Surface Area per Molecule (Amin
)
The minimum surface area (Amin) is defined as an area
occupied by one molecule in nm2 at the interface. Amin was
calculated according to the following equation [14]:
Sodium thioglycolate
–
FeSO4ꢀ7H2O
Sodium citrate. 2H2O
Salinity (NaCl)
pH
–
1014
Amin
¼
;
ð3Þ
0.3
54.9
5.7
NACmax
54.9
5.7
where NA is Avogadro’s number and Cmax (mol m-2) is the
maximal surface excess of the adsorbed surfactant mole-
cules to the interface.
0.0002 % (w/v) resazurin was used as a redox potential indicator for
anaerobic cultivation
The Conductivity (K)
(modified Postgate’s-B medium see Table 1) according to
Postgate [17]. Modification of Postgate’s-B medium was
done by using the original water salinity (NaCl) and pH
during preparation (see Table 1). The medium was pre-
pared, sparged with nitrogen gas and 0.0002 % (w/v)
resazurin was used as a redox potential indicator for
anaerobic cultivation. The medium was inoculated with
10 % (v/v) water sample and incubated at 37 °C for
14 days. Medium preparation and cultivation were
achieved according to the modified Hungate’s technique
for anaerobes [18]. The appearance of a black precipitate
(Ferrous sulfide) was used as a marker for sulfate
reduction and as an indicator of the activity of sulfido-
genic bacteria in the culture media. The inoculated sam-
ple was enriched three times under anaerobic conditions
and modified Postgate’s-B medium was used as inocula
for inhibition experiments.
The specific conductivity (K) measurement was performed
for the synthesized surfactant using a conductometer (LF
191 WTW) at 25 °C in order to evaluate the CCMC value
and the degree of counter ion dissociation (b). The specific
conductivity is linearly correlated to a surfactant concen-
tration in both a premicellar and a postmicellar region [15].
The intersection point between the two lines gives the
CCMC value. While, the ratio between the two slopes gives
the b value.
The Standard Free Energy of Micellization (DGm0 ic
)
In the charged pseudo-phase model of micellization, the
standard free energy micellization (DG0mic) per mole of the
synthesized surfactant was calculated according to the
following equation [16]:
DGomic ¼ ð2 ꢁ bÞRT ln CCMC
;
ð4Þ
Reactors Setup and Evaluation of Sulfidogenic Activity
where b is the degree of counter ion dissociation, R is the
gas constant, T is the temperature, and CCMC is expresses
the molarity of the surfactant.
In order to investigate the effect of the synthesized sur-
factant on the sulfidogenic activity, batch reactor experi-
ments were carried out using modified Postgate’s-C
medium (see Table 1). The Postgate’s-C medium was
modified by using the original water salinity (NaCl) and pH
during preparation (see Table 1). A mild steel coupon with
the chemical composition reported in Table 2 (CS1018
300 9 1/200 9 11/600 strip, Cormon LTD) was used as a
main iron source for the cultivated bacteria. Inhibition
experiments were evaluated using different concentrations
of the synthesized surfactant. In addition, two control
approaches were carried out (1) blank (medium without
Application of the NCGS as a Biocide and as a Metal
Corrosion Inhibitor
Sulfidogenic Consortia and Cultivation Conditions
A water sample with a salinity of 5.49 % was collected
from the water tank of Qarun Petroleum Company (QPC),
Egypt and labeled Youmna. Onsite inoculation of the
water sample was done in an anaerobic selective media
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J Surfact Deterg
phase. A separate reactor (reactor B) was implemented to
evaluate the biofilm constituents using confocal laser
scanning microscopy (CLSM) and staining procedure.
Moreover, an additional reactor (reactor C) was operated to
examine the biofilm, the metal surface (after removing the
biofilm) and the metal surface inoculated with the enriched
sulfidogenic bacteria and the optimum biocide concentra-
tion using scanning electron microscopy (SEM).
Table 2 Chemical composition of mild steal coupon C1018
Chemical composition (%) (remaining: Fe)
C
0.18
0.035
\0.01
0.02
Mo
Nb
Ni
P
\0.01
\0.01
0.01
Si
Sn
Ti
W
V
0.02
\0.01
\0.01
\0.01
\0.01
\0.0001
Al
Co
Cr
Cu
Mn
0.01
0.02
Pb
S
\0.01
\0.005
0.84
B
Samples from the bulk phase of reactor A were taken
every 5 days for 1 month of cultivation to analyze the
sulfidogenic activity by measuring the sulfide concentra-
tion according to the German Standard Methods [19] and
redox potential using SenTix ORP electrode, WTW. The
biocidal effect of the synthesized surfactants against the
environmental sulfidogenic-bacteria was evaluated by
determine the minimum inhibitory concentration (MIC).
The MIC is defined as the lowest concentration of anti-
microbial agent that inhibits the development of visible
microorganism growth [20]. Samples were taken from the
bulk phase of reactor A after 5 days cultivation and tested
for viable bacterial counts using the most probable number
(MPN) method [21]. At the end of 1 month of incubation
(30 days), the coupons were taken from reactor A and
immersed in Clarke solution (1 L 36 % HCl, 20 g Sb2O3
and 50 g SnCl2) for 10–15 s, washed with deionized water,
ethanol and finally dried in a desiccator. Afterward, the
dried coupons were weighed and the weight loss was
determined by comparison of the weights of the coupons
after and before the inhibition experiments. The corrosion
rate (gm-2 day-1) [22] and metal corrosion inhibition
efficiency [23] were calculated from the weight loss results.
CLSM with staining protocol was used to detect the
change in bacterial cells and EPS distribution within the
sulfidogenic-biofilm matrix in the presence and absence of
the synthesized surfactant. At maximum sulfidogenic-
activity (the highest sulfide value in the bulk phase), the
mild steel coupons with the attached biofilms were taken
from reactor B and immersed first in 0.85 % NaCl to
remove the planktonic cells. For the detection/quantifica-
tion of bacteria, the nucleic acid stain SYTO9 (Invitrogen,
Eugene, USA) was used according to the protocol descri-
bed by Neu [24]. Aleuria aurantia lectin (AAL, LINARIS
Biologische Produkte GmbH, Wertheim-Bettingen, Ger-
many) labeled with AlexaFluor 633 (Invitrogen/Molecular
Probes, Eugene, USA) was applied to stain glycoconju-
gates. Proteins were stained with SYPRO Orange (Invit-
rogen/Molecular Probes, Eugene, USA) according to the
protocol described by Lawrence et al. [25]. A Zeiss
LSM510 META (Carl Zeiss Micro-Imaging GmbH, Jena,
Germany) was used to create image stacks, adjusted by the
AIM software (v. 3.2, Carl Zeiss Micro Imaging GmbH,
Jena, Germany). For excitation, two wavelengths were
used: 488 and 633 nm that are related to the stains used.
Table 3 Reactors operation mode
Reactor A Reactor B
Reactor C
Inoculated
sample
Enriched Youmna-sulfidogenic bacteria
Temperature 37 °C
Shaking 100 rpm
Reactor type Blank-reactor: reactor not inoculated with the
enriched sulfidogenic bacteria
Control-reactor: reactor inoculated with the enriched
sulfidogenic bacteria
Surfactant-reactor: reactor inoculated with the
enriched sulfidogenic bacteria and exposed to
different concentrations of the synthesized
surfactant
Experiment
Analysis
Sulfidogenic Biofilm
activity constituents
Biofilm and
metal surface
analysis
Sulfide
Redox
potential
CLSM with
staining
protocols to
detect nucleic
acids, proteins
and EPS
SEM to detect
the biofilm,
metal surface
(after
removing the
biofilm), and
metal surface
with optimal
biocide
MPN
Corrosion
rate
glycoconjugates
in the cultivated
biofilms
concentration
Examination Every
day 5 days:
At maximum
sulfidogenic
activity (high
sulfide
concentration in
the bulk phase)
After 1 month
cultivation
(30 days)
sulfide and
redox
potential
MPN: after
5 days
Corrosion
rate: after
1 month
(30 days)
bacteria) and (2) control (cultivated sulfidogenic bacteria
without surfactant) (see Table 3).
Three group batch reactors (150 ml working volume)
were inoculated with 3 ml enriched sulfidogenic bacteria
(see Table 3). The first reactor (reactor A) was operated in
order to determine the sulfidogenic activity in the bulk
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J Surfact Deterg
A 63 9 0.95 numerical aperture (NA) water immersion
lens was applied for in-situ observation of biofilms over the
coupons. For each sample, five microscopic fields were
selected randomly and scanned for nucleic acids, EPS
glycoconjugates and proteins. The pinhole was adjusted for
all channels with an identical value to the stack slice
thicknesses of 0.79 lm [26]. For image analysis, the soft-
ware ImageJ (v.1.39i, http://rsb.info.nih.gov/ij/index.html)
was used, image analysis and calculation details were
documented by Wagner et al. [27]. The coverage (C) of
each single image of the image stack was quantified by the
synthesized surfactant was introduced into the wells. The
plates were incubated at 37 °C overnight. The antibacterial
activity was evaluated by measuring the diameter of zones
of inhibition (in mm). In addition, a standard tetracycline
solution was used as a positive control and sterile water
was used as a negative control.
Results and Discussion
Confirmation of Chemical Structure of the Synthesized
NCGS
ꢀ
ImageJ software. The average coverage (Cstack) was cal-
culated for each image stack from the first slice (n = 1)
where the average coverage equal to 0.1 % of the last slice
(n = nmax) where the average coverage was equal to 0.1 %
according to the following equation [27],
The chemical structure of the NCGS was confirmed by
FTIR and NMR (proton and carbon, and phosphorous)
spectroscopy.
n¼nmax
X
1
ꢀ
Cstack
FTIR Spectra
¼
C
ð5Þ
nmax
n¼1
FTIR spectra of the synthesized NCGS demonstrated that
the characteristic bands for the alkyl moiety are 2,922.65,
2,853.03 cm-1 for asymmetric and symmetric stretching
(CH), respectively. While at 1,377.90 cm-1 for symmetric
bending (CH3), at 1,466.93 cm-1 for symmetric bending
(CH2) and at 720.52 cm-1 for –(CH2)n– rock. R4N?
appeared as a band at 1,058.00 cm-1. In addition, there
was a band at 1,097.76 cm-1 for P–O–C aliphatic and a
band at 907.64 cm-1 corresponding to ionic phosphate and
one at 1,150.64 cm-1 for P=O stretching. The FTIR
spectrum confirmed the expected functional groups in the
synthesized NCGS.
Then the mean average coverage values of the five
scanned microscopic fields on the same coupon was
calculated and used to evaluate the change in the nucleic
acids, EPS glycoconjugates and proteins in different
biofilms.
The biofilms, metal surfaces (after removing the bio-
film) and the metal surfaces inoculated with the enriched
sulfidogenic bacteria and the optimum biocide concentra-
tion were examined using scanning electron microscopy
(SEM) model Leica/Cambridge Stereo scan 360 at mag-
nifications ranging from 509 to 10,0009 and operated at
an acceleration voltage of 20 V. After 1 month incubation
time, the metal surface samples were removed from reactor
C, fixed with 3 % glutaraldehyde PBS, pH 7.3–7.4 for 4 h,
washed two times with PBS (5 min each), rinsed with
distilled water for another two times (5 min each) and then
dehydrated using an ethanol gradient (50, 75, 95 and 99 %)
for 10 min before being finally stored in the desiccator.
NMR Spectra
The 1H-NMR (DMSO) spectrum of the synthesized NCGS
showed different bands at d = 0.88 ppm (t, 3H,
NCH2CH2(CH2)9CH3); d = 1.23 ppm (m, 18H, NCH2
CH2–(CH2)9CH3); d = 1.73 ppm (m, 2H, NCH2CH2
(CH2)9CH3); d = 3.35 ppm (t, 2H, NCH2CH2–(CH2)9
CH3); at 3.45 ppm (s, 3H, NCH3); d = 3.70 ppm (t, 3H,
NCH2CH2OP); d = 4.11 ppm (t, 2H, NCH2CH2OP).
The data of the 1H-NMR spectrum confirmed the
expected hydrogen proton distribution in the synthesized
NCGS.
The 13C-NMR (CDCl3) spectrum of the synthesized
NCGS presented different bands at d = 14.07 ppm (NC
H2CH2CH2(CH2)6CH2CH2CH3); d = 18.02 ppm (NCH2
CH2CH2–(CH2)6CH2CH2–CH3); d = 23.19 ppm (NCH2
CH2CH2(CH2)6CH2CH2CH3); d = 29.27 ppm (NCH2CH2
CH2–(CH2)6–CH2CH2CH3); d = 26.55 ppm (NCH2CH2
CH2(CH2)6CH2CH2CH3); d = 23.18 ppm (NCH2–CH2
CH2–(CH2)6CH2CH2–CH3); d = 66.21 ppm (NCH2CH2
CH2–(CH2)6CH2CH2CH3); at d = 51.93 ppm (NCH3);
Antibacterial Activity of the Synthesized Surfactant
The antibacterial (biocidal) activity of the synthesized
surfactant was tested against different bacterial strains
(DSMZ: Deutsche Sammlung von Mikroorganismen und
Zellkulturen) as follows: Gram-positive bacteria (Staphy-
lococcus aureus DSM 3463) and Gram-negative bacteria
(Pseudomonas aeruginosa DSM 50071, Escherichia coli
DSM 30083). The antibacterial activity of the synthesized
surfactant was determined by a modified agar-well diffu-
sion method [28]. In this method, nutrient agar plates were
seeded with the tested microorganism by streaking over the
agar plates. A sterile 10-mm borer was used to cut three
wells of equidistance in each of the plates; 0.2 ml of
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J Surfact Deterg
Fig. 1 Chemical nomenclature
and structure of the synthesized
surfactant according to IUPAC
OH
N
P
OH
N
O
O
HO
O
N,N'-(((hydroxyphosphoryl)bis(oxy))bis(ethane-2,1-diyl))bis(N,N-dimethyldodecan-1-ammonium) hydroxide
concentration increases. The CCMC value (2.3 9 10-3
mol dm-3) was determined from the break points in the
c–log C plots of the synthesized surfactant [14]. This
behavior shown by the surfactant molecule in the solution
is common and is used to determine its purity and CCMC
value (see Table 4).
The Effectiveness (PCMC
)
The effectiveness of the synthesized NCGS is good for
lowering the surface tension of water to the present value
(Table 4). The synthesized NCGS showed higher effec-
tiveness (45 mN m-1) in comparison to other cationic
gemini surfactants (28.2–33.7 mN m-1) at the CCMC points
[29].
Fig. 2 The surface tension of the synthesized surfactant at different
concentrations in water at 25 °C
The Surface Excess (Cmax
)
d = 58.25 ppm (NCH2CH2O); d = 55.95 ppm (NCH2
CH2O).
The surface excess (Cmax) data show that by increasing the
hydrophobic chain length, as in the case of the synthesized
NCGS (see Table 4), the hydrophobicity increases. Thus,
the synthesized surfactant molecules are directed to the
interface and therefore, the surface energy of the solution
decreases. This leads to an increase in the maximum sur-
face access.
The data of 13C-NMR spectra confirmed the expected
carbon distribution in the synthesized NCGS.
The 31P-NMR (DMSO) spectrum of the synthesized
NCGS showed characteristic signal at dp -0.5114 ppm.
The nomenclature and the chemical structure of the
synthesized NCGS are given according to international
union of pure and applied chemistry (IUPAC) (see Fig. 1).
The Minimum Surface Area per Molecule (Amin
)
Surface Active Properties of the Synthesized NCGS
Results reported in Table 4 reveal that the surface pressure
(pCMC) of the synthesized NCGS increases with decreasing
the minimum surface area (Amin) of the adsorbed surfactant
molecules as previously reported [29].
The Surface Tension (c)
The changes in the surface tension (c) values of the syn-
thesized surfactant at different concentrations are presented
in Fig. 2. Significant decreases in the surface tension were
observed with increasing surfactant concentrations. After-
ward the curves break rapidly at relatively low surfactant
concentrations and continue to decrease slowly as the
Conductivity Measurements (K)
The CCMC value calculated from the specific conductivity
figure (Fig. 3) was in agreement with that obtained using
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J Surfact Deterg
Table 4 Critical micelle concentration (CCMC), surface tension at CCMC (cCMC), effectiveness (pCMC), maximum surface excess (Cmax), and
minimum area (Amin), the degree of counter ion dissociation (b) and free energy of micellization (DG0mic) of the synthesized surfactant at 25 °C
Inhibitor CCMC (mol dm-3
)
cCMC (mN m-1
)
pCMC (mN m-1
)
Cmax 9 1010 (mol m-2
)
Amin (nm2)
b
DG0mic (kJ mol-1
)
NCGS
2.3 9 10-3
27
45
6.31
0.26
0.26 -23.18
Fig. 3 The electrical conductivity of the synthesized surfactant at
different concentrations in water at 25 °C
Fig. 4 Sulfidogenic bacterial activities shown on the basis of the
sulfide production in the bulk phase for the different reactors. The
results shown are mean values of duplicates with corresponding
standard deviations
the surface tension result (see Fig. 2). Increasing in the
conductivity of the synthesized NCGS was related to
increases in the cation bulkiness [8].
Deltaproteobacteria). No Archaea with a dsrb-gene were
detected in the DGGE band sequences.
The Standard Free Energy of Micellization (DGm0 ic
)
According to the CCMC value of the synthesized NCGS
(see Table 4), the surfactant concentration-ranges were
selected. The CCMC value of the NCGS was
2.3 9 10-3 mol dm-3 therefore the concentration-ranges
were chosen as 0.01, 0.1, 1 and 5 mM. The enriched
inoculum of the cultivated reactors had initial MPN count
Results listed in Table 4 show that the negative value of
DGo of the synthesized NCGS indicated that there is a
tendency of the synthesized surfactant molecules to be
mic
adsorbed at the interface [30].
of 2.9 9 108 ml-1
.
Inhibition of environmental sulfidogenic bacterial activity
using the novel cationic gemini surfactant (NCGS)
Results presented in Fig. 4 showed that there was a
strong sulfide production in the control reactor and the
sulfidogenic-reactor treated with surfactant at a concen-
tration of 0.01 mM. Sulfide production was gradually
decreased by applying different concentrations of the sur-
factant (biocide) throughout the experiment. The sulfide
result indicates the growth of the sulfidogenic bacteria
which growing by either oxidizing organic compounds or
utilizing hydrogen as an electron donor with sulfate being
reduced to hydrogen sulfide [33]. At a concentration of
0.1 mM NCGS, the Youmna-sulfidogenic activity was
inhibited at the beginning, but after 5 days it started to
resist the biocide effect. Reactor inoculated with the Yo-
umna-sulfidogenic bacteria showed no sulfide production at
high surfactant concentrations of 1, 5 mM. No sulfide
production means that there is no sulfidogenic activity in
the bulk phase or over the metal surface. The inhibition of
sulfide production is an important factor to protect the
The water sample was enriched and used as an inoculum
for biofilm cultivation. The dissimilatory sulfite reductase-
b subunit (dsrb) based on the denaturing gradient gel
electrophoresis (DGGE) was used to identify the sulfido-
genic community’s composition directly from the original
water sample (W) and from its cultivated biofilm (CB). The
DGGE pattern of the Youmna sample (data not shown)
shows a reduction in microbial diversity from nine bands in
W to one band in CB. The change in the DGGE pattern in
W and CB might be attributed to the selective cultivation
conditions which did not reflect the in-situ water properties
such as organic, inorganic contents and cultivation tem-
perature [31, 32].
The most detected sulfidogenic bacteria were Desulf-
ovibrio
genus
(phylum
Proteobacteria,
class
123

J Surfact Deterg
Table 5 Evaluation of the most probable number (MPN) of the
cultivated reactors
Cultivated reactors
MPN (cell ml-1
)
Control
2.6 9 109
2.4 9 109
1.1 9 105
No growth
No growth
0.01 mM
0.10 mM
1.00 mM
5.00 mM
In general, the biocidal activity of the synthesized surfac-
tant depends on the alkyl chain length that is increased in
the synthesized NCGS in comparison to the monomeric
surfactant. The biocidal effect took place at concentrations
of surfactant below the CCMC and is due to the individual
molecules and not to the aggregates [17].
Fig. 5 Sulfidogenic bacterial activities shown on the basis the redox
potential for the different reactors. The results shown are mean values
of duplicates with corresponding standard deviations
The hypothesized biocidal effect of the synthesized
surfactant against bacterial cells can be explained as an
electrostatic interaction and physical disruption. Electro-
static interaction between the negatively charged cell
membrane (lipoprotein) and the positively charged NCGS
two ammonium groups (R4N?). A physical disruption
occurs when hydrophobic chains (alkyl groups) penetrate
into the bacterial cell membrane. That happens because of
similarity of the cell membrane constituents and the
hydrophobic chains of the synthesized surfactants. Pene-
tration of the cell membrane leads to damage of the
selective permeability of the cell, disturbs the metabolic
pathway within the cytoplasm and then death of the
microorganism as has been reported previously [11, 35].
The NCGS contains two hydrophilic groups and two
hydrophobic groups linked by spacer. This spacer contains
one phosphorous and three oxygen atoms. It has been
reported that phosphorus containing compounds are com-
monly used to inhibit metal corrosion in aqueous envi-
ronments [36]. Their use is considered to be risk free due to
their low toxicity [37].
metal surface from microbial metal corrosion. Hydrogen
sulfide can accelerate corrosion of metals by being source
of bound protons and by precipitation of Fe2? as FeS [34].
It has also been proposed that the iron sulfide film that
precipitates over the metal surface plays an important role
in the initiation of pitting corrosion of metal. In this
respect, applying the synthesized surfactant at high con-
centrations of 1, 5 mM protects the metal surface from the
sulfidogenic bacterial activity.
The measured redox potential in the different reactors
(Fig. 5) is in agreement with the sulfidogenic activity
determined by sulfide production. Most sulfidogenic bac-
teria are strictly anaerobic and start to reduce sulfate at a
redox potential below -100 mV [17]. For the control
reactor and the reactors treated with NCGS at concentra-
tions of 0.01 and 0.1 mM the redox potential measured was
optimal for the sulfidogenic bacterial growth. While at high
NCGS concentrations 1, 5 mM the measured redox
potential of the Youmna-sulfidogenic bacteria was
increased up to -100 mV. The sulfide and redox potential
results demonstrate that at high surfactant concentrations,
the sulfate reduction reaction was completely inhibited
causing no sulfide production and a considerable drop in
the redox potential in the reactor’s bulk phase.
The sulfidogenic activities shown were detected in the
bulk phase, not over the metal surface. Therefore, in order
to characterize the possible changes in the biofilm structure
and constituents over the metal surface after surfactant
application, CLSM and staining procedures were used.
Biofilm development was only detected in the control
reactor and reactor inoculated with the synthesized NCGS
at a concentration of 0.01 mM (Fig. 6). At a concentration
of 0.01 mM NCGS, the Youmna-biofilm microbial popu-
lation (nucleic acids) does not reveal significantly changes
in comparison to the control biofilm. When EPS compo-
nents were changed, EPS glycoconjugates were reduced
and the proteins increased. The explanation of EPS com-
ponents changing by applying the biocide is a defensive
behavior of the microbial population. It has been reported
Results presented in Table 5 showed the minimum
inhibitory concentration (MIC) of the synthesized surfac-
tant is detected at a concentration of 1 mM. With
increasing the synthesized surfactant concentrations no
sulfidogenic bacterial growth was detected. At a lower
concentration of 0.01 mM NCGS, there is no significant
biocidal effect against Youmna-sulfidogenic bacteria.
However at a concentration of 0.1 mM NCGS, the Yo-
umna-sulfidogenic bacterial growth was partially inhibited.
123

J Surfact Deterg
Fig. 7 The corrosion rate of sulfidogenic bacteria is demonstrated.
The blank reactor (media without bacteria), the control reactor
(inoculated with the sulfidogenic bacteria without the inhibitor) and
the reactors with different inhibitor concentrations. The results shown
are the mean values of duplicates with corresponding standard
deviations
Fig. 6 The changing of nucleic acids, EPS glycoconjugates and
proteins of the sulfidogenic biofilms control reactor and the biocide
reactor. The figure shows the average coverage of five randomly
selected areas of the cultivated biofilms with corresponding standard
deviations
where the chloride anion penetrates the oxide film of the
metal surface through pores or defects easier than do other
ions. In addition it may colloidally disperse the oxide film
and increase its permeability. On the other hand, according
to the adsorption theory, once the chloride anion is in
contact with the metal surface, it favors hydration of the
metal ions and increases the ease with which metal ions
enter into solution. Then the adsorbed chloride ions lead to
pit and crevice corrosion [41]. The role of chloride in the
metal corrosion process is unique. It can be reacted again
and again and hence even a small number of chloride
anions can sustain the metal corrosion process. Thus the
iron-chloride reaction is self-perpetuating and the free
chloride serves as a reaction catalyst [42].
that, the natural response of microorganisms upon exposure
to a toxic environment is to stimulate the production of
EPS. This natural response has been detected when the
SRB biofilm is exposed to seawater containing toxic heavy
metals and it started to produce more protein than poly-
saccharides [38]. However the biofilm development on the
metal surface at a concentration of 0.1 mM NCGS was
completely inhibited, no complete inhibition was detected
in the bulk phase.
Increasing the NCGS concentrations (0.1, 1, 5 mM)
completely inhibited Youmna-sulfidogenic biofilms devel-
opment over the metal surface. The explanation of this
result, at high concentrations of 1, 5 mMthe synthesized
surfactant, the metal surface was completely protected not
only from the planktonic bacterial cells, as confirmed by
the fact that there was no sulfide production and drop in the
redox potential in the bulk phase, but also from the biofilms
development.
The mechanisms of metal corrosion in the presence of
sulfidogenic bacteria in the bulk phase and over the metal
surface (control reactors) are complex. In an anaerobic envi-
ronment, sulfidogenicbacteriautilizehydrogen(asanelectron
donor) with sulfate (as an electron acceptor) being reduced to
sulfide. In this respect, Von Wolzogen Kuhr and Van der
Vlugt [43] suggested the following corrosion reaction:
At the end of the 1 month cultivation (30 days), the
metal corrosion rate of the coupons was calculated (Fig. 7).
The higher metal corrosion rate of the Youmna blank-
reactor (without biomass) in comparison to the Youmna
control-reactor (inoculated with Youmna-sulfidogenic
consortia), show that the medium salinity (5.49 % NaCl)
caused metal corrosion in the blank-reactor and the sul-
fidogenic biofilm in the control-reactor protected the metal
surface from the harmful chloride anion effect as postu-
lated before [34, 39, 40]. It has been reported that metal
corrosion in an aqueous medium containing NaCl (artificial
seawater) proceeds by chemisorption of chloride ions on
the metal surface. There are two theories of chloride anion/
metal surface interaction. The first theory is the oxide-film,
4Fe þ SO24ꢁ þ 4H2O ! 3FeðOHÞ2þ FeS þ 2OHꢁ
ð6Þ
This reaction is described as a cathodic depolarization
theory. Based on this theory, sulfidogenic bacteria increase
the metal corrosion rate by continuous consume the atomic
hydrogen that accumulated at the cathode by a hydrogenase
enzyme. Therefore it leads to increasing the metal
dissolution. Some researchers [44, 45] suggested that the
metal corrosion rate increases as a result of the cathodic
reduction of H2S:
123

J Surfact Deterg
Fig. 8 Diagram of the
adsorption mechanism of a
cationic gemini surfactant on
the metal surface. Adapted from
Ref. [49]
H2S þ 2eꢁ ! H2 þ S2ꢁ
and the anodic reaction is stimulated by the formation of
iron sulfide which plays an important role in initiatiing the
pitting corrosion of metal:
ð7Þ
can be explained as adsorption of the surfactant molecules and
formation of a tight surfactant layer at the metal/liquid interface
and not due to the micelles [10]. The surfactant layers which are
adsorbed to the metal surface act as an inhibitive shield and
protect the metal surface from the surrounding environment
[48]. Two types of adsorption may take place (physical and
chemical). The procedure of physical adsorption requires the
presence of an electrically charged metal surface and charged
species in the bulk phase. The chemisorption process involves a
charge transfer or charge sharing between the inhibitor mole-
cules and the metal surface. The presence of an inhibitor
molecule that has relatively loosely bound electrons or het-
eroatoms with lone-pair electrons together with a transition
metal have a vacant and low-energy electron orbital, promotes
this adsorption. The NCGS contains two hydrophilic groups
and two hydrophobic groups linked by a spacer. This structure
reflects three different types of adsorption: (1) at low gemini
surfactant concentrations, the adsorption takes place by a hor-
izontal binding of the gemini surfactant to the metal surface
(Fig. 8a). This adsorption attitude is favored by an electrostatic
interaction between the two ammonium groups (R4N?) and the
cathodic sites on the one hand and the hydroxide ion on the
anodic sites on the other hand. (2) At high gemini surfactant
concentration, a perpendicular adsorption occurs as a result of
an inter-hydrophobic chain interaction (Fig. 8b). (3) With
further increase of gemini surfactant concentration, up to the
Fe þ S2ꢁ ! FeS þ 2e
ð8Þ
Directly on the metal surface, the metal corrosion rate
was related to the sulfidogenic bacteria attached to the
surface. It has been hypothesized that EPS have the ability
to entrap metal ions by binding carboxylic groups of the
exopolysaccharides and phosphate groups of the nucleic
acids. This binding influences the electrochemical behavior
of a metal through formation of metal corrosion cells and
galvanic coupling [46, 47]. In addition, the sulfidogenic
biofilm increases corrosion by trapping corrosive sulfide in
close proximity to the metal surfaces [6].
The application of surfactant at different concentrations
gradually decreased the metal corrosion rate. The lowest metal
corrosion rate of the Youmna reactors was achieved at a con-
centration of 5 mM with a metal corrosion inhibition efficiency
of 97 %. At this concentration, the synthesized surfactant
shows a biocidal effect against the environmental sulfidogenic
bacteria in the bulk phase and over the metal surface. In a
medium with high salinity (5.49 % NaCl) with free Youmna-
sulfidogenic bacterial activity, the metal surface corrodes by
the effect of corrosive chloride anions. In this respect the
application of the synthesized surfactant protects the metal
surface from the chloride corrosion effect. Therefore the
hypothesized metal surface corrosion inhibition behavior of the
synthesized surfactant against the cultivated medium salinity
CCMC value, an efficiency plateau appears (Fig. 8c) [49].
The corrosion inhibition efficiency was confirmed by
applying SEM to the metal surface. Figure 9a shows an
SEM image of a carbon steel surface after 1 month incu-
bation with the Youmna-sulfidogenic bacteria which was
123

J Surfact Deterg
Table 6 Antibacterial activity of the synthesized surfactant
Biocide Concentration S. aureus
P. aeruginosa E. coli
(DSM 50071) DSM
(mM)
(DSM
3463)
(mm)
(mm)
30083)
(mm)
NCGS 0.01
0.10
0
0
0
15.8 0.2
0
0
1.00
28
39
40
0
0
0
23
0
15.3 0.5
5.00
24.8 0.2
29.1 0.2
28
31
0
0
10.00
0 mm no effect as a resistance of the cultivated bacteria to the anti-
microbial agent
at high salinity that induces metal corrosion (Fig. 9b).
However Fig. 9c showed the surface of another steel
sample after cultivation for the same period in a medium
containing 5 mM NCGS and inoculated with the enriched
Youmna-sulfidogenic bacteria. The SEM image revealed
that the surfaces are free from pits and completely covered
with the inhibitors. This result indicates a good protective
inhibitor film over the steel surface and confirms the
highest inhibition efficiency of the synthesized NCGS.
Results reported in Table 6 indicate that, the synthesized
surfactant has a nonspecific antibacterial activity. The
synthesized NCGS showed antibacterial (biocidal) activity
not only against environmental sulfidogenic bacteria but
also against Gram-positive (S. aureus) and Gram-negative
(E. coli and P. aeruginosa) species. The antimicrobial
activity depends on the ammonium groups (R4N?) and
hydrophobic alkyl groups of the synthesized surfactant. It
has been reported that increasing the hydrophobicity as in
the gemini surfactant leads to an increase in the biocidal
activity [50]. The hypothesized explanation of the non-
specific antimicrobial (biocidal) effect of the synthesized
surfactant can be attributed to an electrostatic interaction
between the the negatively charged cell membrane (lipo-
protein) and the positively charged surfactant’s ammonium
group (R4N?). Moreover physical disruption of the bacte-
rial cell occurs when the surfactant’s hydrophobic chain
penetrates into the bacterial cell membrane. Penetration of
the cell membrane leads to damage of the selective per-
meability of the cell, and then death of the bacterial cell (as
previously described with the environmental sulfidogenic
bacteria).
Fig. 9 SEM images of, a the sulfidogenic biofilm over the mild steel
coupon, b the metal surface after removing the biofilm and c 5 mM
NCGS with the enriched Youmna-sulfidogenic bacteria. Scale bar
50 lm
Relationship Between the Surface Activity
and Corrosion Inhibition Efficiency
In general, the corrosion inhibition efficiency of synthe-
sized surfactants depends on their ability to adsorb onto the
metal surface. This adsorption leads to the formation of a
protective layer over the metal surface. Therefore the CMC
cultivated at a medium salinity of 5.49 % NaCl. The bio-
film images showed that the metal surface was completely
covered with biofilm. After removing the biofilm, the
surface was damaged as the effect of the cultivated biofilm
123

J Surfact Deterg
plays a key role in determining the effectiveness of the
synthesized surfactant as a corrosion inhibitor. When the
corrosion inhibitor concentration reaches the CCMC, an
adsorbed layer of inhibitor covers the largest possible area
on the mild steel surface according to stereochemistry of a
synthesized surfactant. Adding more surfactant molecules
to the solution combines to form micelles in the bulk
solution [51]. On the basis of this view, the studied sur-
factant NCGS at low CCMC showed high corrosion inhi-
bition efficiency. On the other hand, it shows that the
highest reduction in the surface tension (effectiveness,
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123