Surface and Antimicrobial Activity of Sulfobetaines

Article abstract of DOI:10.1007/s11743-016-1838-3

Sulfobetaines belong to the group of zwitterionic surfactants. They are electroneutral salts, which have in the same molecule, two ionic centers with different charge. Due to the specific structure they exhibit excellent properties such as good solubility in water and detergency. In this paper we present surface properties and adsorption parameters of sulfobetaines in water/air systems. From the adsorption isotherms the CMC value, the surface tension and surface pressure at the CMC as well as the efficiency of adsorption were determined. Physicochemical analyses of the data allowed for the further description of adsorption process. Results showed that sulfobetaines exhibit good surface properties especially low CMC and p20 values. Additionally the antimicrobial activity of sulfobetaines solutions against gram-positive and gram-negative bacteria were tested by the well-diffusion method. MIC values and growth kinetics were determined by microdilution method. Antimicrobial assays demonstrated that sulfobetaines can be good antibacterial agents, but the activity of surfactants strongly depends on alkyl chain length.

Full text of DOI:10.1007/s11743-016-1838-3

J Surfact Deterg
DOI 10.1007/s11743-016-1838-3
ORIGINAL ARTICLE
Surface and Antimicrobial Activity of Sulfobetaines
1
2
3
4
•
Daria Wieczorek
Tang-Long Shen
•
Daniela Gwiazdowska
•
Katarzyna Staszak
•
Ying-Lien Chen
4
Received: 1 December 2015 / Accepted: 19 May 2016
Ó AOCS 2016
Abstract Sulfobetaines belong to the group of zwitterionic
surfactants. They are electroneutral salts, which have in the
same molecule, two ionic centers with different charge.
Due to the specific structure they exhibit excellent prop-
erties such as good solubility in water and detergency. In
this paper we present surface properties and adsorption
parameters of sulfobetaines in water/air systems. From the
adsorption isotherms the CMC value, the surface tension
and surface pressure at the CMC as well as the efficiency of
adsorption were determined. Physicochemical analyses of
the data allowed for the further description of adsorption
process. Results showed that sulfobetaines exhibit good
surface properties especially low CMC and p20 values.
Additionally the antimicrobial activity of sulfobetaines
solutions against gram-positive and gram-negative bacteria
were tested by the well-diffusion method. MIC values and
growth kinetics were determined by microdilution method.
Antimicrobial assays demonstrated that sulfobetaines can
be good antibacterial agents, but the activity of surfactants
strongly depends on alkyl chain length.
Keywords Sulfobetaine Á Antimicrobial activity Á Surface
activity Á Surfactant Á MIC
Introduction
Sulfobetaine surfactants are electroneutral salts possessing,
in the same molecule, two ionic centers of different charge
[1]. Sulfobetaines are compounds structurally similar to
alkylbetaines, however the carboxylic acid has been
replace by a sulfonate group. Compounds belonging to this
group can be divided into several subgroups differing from
each other with the length and presence of a hydroxyl
group spacer separating the quaternary ammonium center
from the sulfonate group [2, 3].
Sulfobetaine surfactants have no net charge [4]. They
are pH-insensitive, in neutral alkaline and cationic solu-
tions they exist in the form of internal salts [5]. Together
with the pH–dependent alkylbetaines, they are classified as
zwitterionic surfactants [6].
&
Daria Wieczorek
d.wieczorek@ue.poznan.pl
1
Sulfobetaines, whose hydrophilic polar headgroup
carries both a positive and a negative charge in the
molecule are interesting in several aspects. As reported in
the literature they can be mild to skin and eyes. Moreover
they are not toxic and can produce copious amounts of
stable foam. Additionally sulfobetaines are hard water
tolerant and more resistant to degradation by oxidizing
and reducing agents resistance compounds compared to
many ionic surfactants [7, 8]. Sulfobetaines are insensi-
tive to pH changes. The effects of temperature changes
and addition of electrolyte on sulfobetaines have been
found to be minimal [6, 8].
Department of Technology and Instrumental Analysis,
Faculty of Commodity Science, Poznan University of
Economics and Business, al. Niepodległo s´ ci 10,
6
1-875 Poznan, Poland
2
3
4
Department of Natural Science and Quality Assurance,
Faculty of Commodity Science, Poznan University of
Economics, al. Niepodległo s´ ci 10, 61-875 Poznan, Poland
Institute of Technology and Chemical Engineering, Poznan
University of Technology, ul. Berdychowo 4, 60-965 Poznan,
Poland
Department of Plant Pathology and Microbiology, National
Taiwan University, No.1, Sec. 4, Roosevelt Road,
Taipei 10617, Taiwan
1
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J Surfact Deterg
Because they contain both cationic and anionic groups
in the same molecule, sulfobetaines exhibit behavior
intermediate between ionic and nonionic surfactants [1].
Due to their specific structure they exhibit excellent per-
formance properties including detergency, foaming, emul-
sification, biodegradability and good solubility in water.
Sulfobetaines are widely used ingredients not only in
cosmetic products like shampoos and shower gels, but also
in domestic detergents [1, 9–11] for two reasons: (1) sul-
fobetaines do not transport substances across the skin
barrier, (2) they are often combined with anionic or
cationic surfactants and exhibit higher surface activity than
typical betaines.
propanesultone or 1,4-butanesultone with corresponding
N,N-dimethylalkylamine in ethyl acetate at room temper-
ature. The resulting crystalline products were separated
from the reaction mixture by filtration and further purified
by recrystallization. The structure of the obtained com-
pounds was confirmed by spectroscopic methods and ele-
mental analysis. All surfactants synthesized are listed in
Table 1.
The surface tension of the aqueous solutions of surfac-
tants was measured with a Tracker (I.T.Concept, France)
tensiometer at 21 °C by the drop shape method. The optical
tensiometer records the drop shape as a function of time on
the basis of image analysis of an air bubble immersed in the
aqueous surfactant solution. Measurements were made
using a 50 mM stock solution. A series of 19 aqueous
solutions were obtained by serial dilution. All the surfac-
tants solutions were prepared using water from the
PURELAB Classic, Elga with resistivity = 18.2 MX cm.
The adsorption parameters and CMC were determined
from the surface tension isotherms.
Sulfobetaines are known to solubilize membrane
receptors and proteins and therefore could be used to
investigate structural biology [12]. The possibility of
using these compounds to prepare ultrafiltration mem-
branes via the phase inversion method is described in the
literature. The advantage to using these substances is
effective reduction of membrane fouling over a wide pH
range and longer operational life-times. The
hydrophilicity and the antifouling properties of the
membrane could be improved by increasing the number of
sulfobetaine groups in the membrane. For example,
membranes obtained from polyurethane modified by
cross-linked sulfobetaine methacrylate (SBMA) can
effectively resist nonspecific protein adsorption, espe-
cially if the surface density of the zwitterionic groups is
controlled at a high level [13, 14].
Microorganisms and Culture Conditions
Eight microorganisms were used in the experiments. This
group included gram-positive bacteria: Staphylococcus
aureus ATCC 33862, Staphylococcus epidermidis ATCC
12228, Bacillus megaterium PCM 1854, Bacillus subtilis
PCM 2021 and gram-negative bacteria: Aeromonas
hydrophila ATCC 7966, Escherichia coli ATCC 8739,
Proteus vulgaris PCM 549, Pseudomonas aeruginosa
ATCC 9027. Nutrient agar or broth (Biocorp Polska Ltd.)
was used for bacterial cultivation. Before the experiments,
all microorganisms were subcultured on fresh media and
then incubated for 24 h at 30 °C (A. hydrophila) and 37 °C
(remaining bacteria). Subsequently, the microorganisms
were suspended in saline and the cell density was adjusted
to 0.5 according to McFarland Standard.
The aim of the work was to determine the surface ten-
sion and micellization properties of sulfobetaines as well as
the determination of their antimicrobial activity.
Experimental Section
Materials and Methods
The laboratory synthesis of sulfobetaines that belong to the
group of N-alkyl-N,N-dimethyl-3-ammonio-1-propanesul-
fonate and N-alkyl-N,N-dimethyl-4-ammonio-1-butanesul-
fonate (Fig. 1) was carried out by the reaction of 1,3-
Determination of Antimicrobial Activity
of Sulfobetaines
Antimicrobial activity of aqueous sulfobetaine solutions of
was determined by a well-diffusion assay. Suspensions of
microorganisms were overlaid with agar media and after
medium solidification, the wells (10 mm in diameter) were
cut with a sterile cork borer, to which 100 ll of surfactant
solution was introduced. All the aqueous surfactant solu-
tions were prepared at the concentration of 1.0 and
-
3
0
.5 g cm . The plates were incubated for 24 h at 30 or
37 °C depending on the indicator microorganism. After
incubation the sizes of the inhibition zones were measured
in millimeters. Tests were performed in triplicate and the
mean values are presented.
Fig. 1 Structure of sulfobetaines
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J Surfact Deterg
Table 1 Surfactants
synthesized in the study and
abbreviations used in this work
No.
Surfactant name
Abbreviation
1
2
3
4
5
6
7
8
N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
N-Decyl-N,N-dimethyl-4-ammonio-1-butanesulfonate
N-Dodecyl-N,N-dimethyl-4-ammonio-1-butanesulfonate
N-Tetradecyl-N,N-dimethyl-4-ammonio-1-butanesulfonate
N-Hexadecyl-N,N-dimethyl-4-ammonio-1-butanesulfonate
SB3C10
SB3C12
SB3C14
SB3C16
SB4C10
SB4C12
SB4C14
SB4C16
Minimal Inhibition Concentration Determination
determined from the inflection point on the surface tension
isotherms. Moreover the value of surface tension at the
CMC (cCMC), value of surface pressure at CMC (PCMC),
and the efficiency of adsorption (pC ) was calculated. The
The Minimal Inhibition Concentration (MIC) of sulfobe-
taines was determined by the microdilution method with
Mueller-Hinton medium. First, serial twofold dilutions of
tested sulfobetaines were made in Mueller–Hinton broth.
The final concentration of samples ranged from 19 to
2
0
0
standard free energy of micellization (DG ) was calculated
m
from the following equation:
0
m
DG ¼ RTlnX
ð1Þ
-
3
CMC
2
500 lg cm . Next, solutions of microorganisms in saline
with 0.5 turbidity on the McFarland Scale were added at
0–180 ll broth with sulfobetaines. As a negative control
where R is the gas constant, T absolute temperature, XCMC
molar fraction of surfactant [15].
2
Mueller–Hinton medium was applied and as a positive
control suspension of microorganism with an addition of
water was used. After 24 h of plate incubation at 30 or 37 °C
the optical density at a wavelength 600 nm through 24 h was
measured. Results were presented as a minimal inhibitory
concentration, which is necessary to cause at least 80 % of
inhibition growth of indicator microorganism.
The CMC deceases with increasing alkyl chain length.
The increasing molecular hydrophobic moiety could be an
explanation of this phenomenon. The dependence of the
logarithm CMC values on the number of carbon atoms in
the alkyl chain for the investigated compounds is linear for
both series of sulfobetaine synthesized. The relationship
between CMC values and hydrocarbon chain length is
described
by
the
well-known
equation:
log
Growth Kinetics of Indicator Microorganisms
with the Addition of Sulfobetaines
CMC = A - Bn, where n is the number of carbon atoms in
the alkyl chain, A and B are constants [6]. At a given
temperature, A is a constant characteristic for a ionic head
and B takes the value of 0.3 and 0.5 at 25 °C for the
conventional anionic and cationic and for nonionic and
zwitterionic surfactants respectively [16]. Studies suggest
that this parameter B is proportional to the hydrophobic
interaction energy of micelle formation. The values of
A and B for SB3 and SB4 homologues series are: 1.63,
-0.40 and 2.76, -0.46 respectively. The values are similar
to those for the other betaine-surfactants: 0.39 for alkyl-
betaine zwitterionic gemini surfactants, 0.49 for zwitteri-
onic monomeric surfactants N-alkylbetaine [16] and 0.37
for heterogemini sulfobetaines [4]. Comparing both
homologous series of surfactants, it can be seen that a
decrease in the CMC values with increasing chain length is
larger for the butane derivatives.
The kinetics of growth of chosen indicator microorgan-
isms: S. aureus and P. aeruginosa in the presence of dif-
ferent concentrations of sulfobetaines was evaluated on
microtiter plates. Samples were prepared by the method of
serial dilutions in the same way as in the determination of
MIC. In temperature of 37 °C plates were incubated and
the optical density at 600 nm wavelength was recorded
every hour for 24 h. Results were presented as growth
curves. The curves were created by plotting OD values
versus incubation time.
Results
Surface Tension and Critical Micelle Concentration
of Sulfobetaines
The adsorption and micellization of surfactants can be
expressed by pC20 and PCMC parameters. The pC20 value
describes the adsorption efficiency of surfactant molecules at
the air/water interface. The larger the pC20 value, the higher
the adsorption efficiency of surfactant molecules. To esti-
mate the effectiveness in surface tension reduction, the value
Results of surface tension measurements for aqueous sul-
fobetaine solutions of surfactants are presented in Table 2.
Values of critical micellar concentrations (CMC) were
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J Surfact Deterg
of PCMC can be used. Both parameters are included in
Table 2. For sulfobetaines the value of pC20 increases with
increasing alkyl chain length, but the nominal values are
lower for propane derivatives than for corresponding butane.
0
The DG values are negative and become more negative
m
with increasing hydrocarbon chain length. This suggests
that the surfactants show great ability to form micelles in
aqueous solution and this ability increases for longer alkyl
chain in the sulfobetaine molecule, suggesting that the
stronger hydrophobic interaction favors micellar
aggregation.
The analyses of physicochemical parameters were based
on the surface tension data. The surface tension isotherms
at concentrations below the CMC point were fit to the
Szyszkowski equation. The adsorption coefficients values
of the Szyszkowski isotherm (A and B ) were determined
sz
sz
and used to calculate the surface excess at the saturated
interface (C ). Additionally the minimum molecular area
?
in the adsorption layer at the saturated interface (Amin) and
the Gibbs free energy of adsorption (DGads) for the systems
considered are given in Table 2. All these parameters were
calculated according to the equations [17]:
BSzc0
C
1
¼
ð2Þ
RT
1
Amin ¼
ð3Þ
ð4Þ
C1NA
Sz
DG ¼ RTlnðAmin
Þ
ads
where c , N , R, T stand for interfacial tension for the
0
A
solvent, here water, the Avogadro constant, the gas con-
stant and temperature, respectively.
Values of the Gibbs free energy of adsorption are,
similarly to the standard free energy of micellization,
negative and become more negative with increasing num-
ber of carbon atoms in alkyl chain. However, the absolute
0
values of DGads are higher than corresponding DG , indi-
m
cating that the adsorption process is preferred over
micellization.
From Table 2, it is clear that the C values decrease
?
-
6
-6
-6
from 1.93910
to 1.84910
and 2.39910
-
.17910 mol m for propane and butane derivatives
to
6
-2
1
respectively, with increase of the hydrophobic chain length
from 10 to 16 with a local maximum for chain length of 12.
In consequence, the statistical area at the air/water interface
that the adsorbed surfactant molecules occupy increases
with hydrophobic chain length. Moreover, the area occu-
pied by 12-carbon surfactants is extremely small. The
relationship between the values of C? and the number of
carbon atoms in the alkyl chain is not linearly dependent.
The highest value of C? is observed for surfactants with 12
carbon atoms in both homologous series. Similar situation
can be observed for the Amin parameter; however the value
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J Surfact Deterg
of the minimum area occupied by a single adsorbed
molecule is the smallest for the 12 carbon derivatives.
quantitatively by determining the MIC for the tested
microorganisms (Table 4). Twofold dilutions of surfactants
containing 12, 14 and 16 carbon atoms were prepared
-
3
Antimicrobial Properties
(from 5000 to 19 lg cm ). It was found that the MIC
varied for the tested bacteria and reached a level from 19 to
-
3
To evaluate antimicrobial efficiency of sulfobetaines,
activity spectra and the minimal inhibitory concentrations
were determined. Results of these experiments are pre-
sented in Tables 3 and 4. Results show that antimicrobial
activity of tested sulfobetaines was differentiated and
depends on both the surfactant structure and indicator
microorganism. Results presented in the Table 3 indicate
that surfactants with 10 carbon atoms did not exhibit
antimicrobial activity towards indicator strains, while sur-
factants with 12, 14 and 16 carbon atoms showed broad
spectrum activity. Gram-negative bacteria were less sen-
sitive than Gram-positive bacteria. Among tested Gram-
negative bacteria, E. coli was almost completely insensitive
to tested compounds, while P. aeruginosa and A. hydro-
phila were the most susceptible. All examined Gram-pos-
itive bacteria were inhibited by surfactants, however the
susceptibility varied. The strongest inhibition was observed
in the case of S. aureus, B. subtilis and B. megaterium
treated with compounds with 12, 14 and 16 carbon atoms.
S. epidermidis was weakly inhibited only by surfactants
containing 14 carbon atoms as well as SB3C12 at a con-
5000 lg cm , depending on the bacteria and sulfobetaine.
In the case of Gram-positive bacteria, MIC decrease with
increasing surfactant chain length. The surfactants with 16
carbon atoms had the lowest MIC value against A. hydro-
phila and B. megaterium or P. vulgaris for the propane or
butane derivatives respectively; although in the well-dif-
fusion assay the zones of inhibition of some microorgan-
isms were lower than zones caused by surfactants with 14
carbon atoms. This could be due to weaker diffusion of
surfactants with a longer chain in the agar medium.
Kinetics of Microbial Growth Depending
on the Concentration of Sulfobetaines
Figures 2, 3 presents growth curves of S. aureus and P.
aeruginosa after treatment with sulfobetaines containing
14 and 16 carbon atoms at different concentrations. These
species were chosen as being representatives of Gram-
positive and Gram-negative bacteria, often used as indi-
cator microorganisms in studying the microbiological
quality of cosmetics or household chemicals. According to
the data, growth of tested microorganisms was dependent
on the surfactant concentration. S. aureus (Fig. 2) was
completely inhibited by all examined sulfobetaines in the
-
3
centration of 1.0 g cm
.
Minimal Inhibitory Concentration
-
3
range 78–1250 lg cm . The compound SB3C16 totally
inhibited growth of S. aureus at a concentration of
THe tested surfactants, which showed antimicrobial
activity by the well-diffusion method, were assayed
-
3
39 lg cm , while at the lowest level studied the growth
Table 3 The size of inhibition zones of growth of tested microorganisms determined by the well-diffusion method [mm]
-
3
)
Surfactant
Concentration (g cm
Gram-positive bacteria
Gram-negative bacteria
S.
aureus
S.
epidermidis
B.
subtilis
B.
megaterium
A.
hydrophila
E. coli
P.
aeruginosa
P.
vulgaris
SB3C10
SB3C12
SB3C14
0.5
.0
0.5
.0
0.5
.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1
0.0
0.0
0.0
12.0
16.0
16.0
20.0
22.0
18.0
0.0
0.0
0.0
0.0
0.0
17.0
20.0
26.0
26.0
24.0
0.0
0.0
18.0
18.0
26.0
28.0
24.0
0.0
16.0
20.0
20.0
22.0
20.0
0.0
0.0
16.0
20.0
20.0
22.0
20.0
0.0
15.0
16.0
20.0
22.0
17.0
0.0
1
16.0
18.0
18.0
0.0
14.0
12.0
12.0
0.0
1
SB3C16
SB4C10
0.5
0.5
0.0
0.0
1
.0
0.5
.0
0.5
.0
0.5
12.0
20.0
20.0
26.0
26.0
22.0
0.0
0.0
12.0
16.0
18.0
22.0
18.0
18.0
0.0
0.0
12.0
18.0
20.0
20.0
22.0
18.0
0.0
SB4C12
SB4C14
SB4C16
0.0
14.0
18.0
22.0
28.0
22.0
16.0
18.0
18.0
20.0
18.0
0.0
16.0
18.0
22.0
22.0
18.0
1
0.0
14.0
12.0
12.0
0.0
14.0
18.0
0.0
1
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J Surfact Deterg
Table 4 MIC value for the
sensitive microorganisms
-3
Minimal inhibitory concentration (lg cm )
Microorganism
SB3C12
SB3C14
SB3C16
SB4C12
SB4C14
SB4C16
S. aureus
625
1250
1250
312
78
1250
625
39
39
1250
1250
1250
625
156
1250
625
78
S. epidermidis
B. subtilis
–
–
625
19
625
156
78
B. megaterium
A. hydrophila
E. coli
156
312
78
19
625
156
1250
1250
625
625
1250
312
625
5000
156
1250
2500
625
1250
2500
156
–
P. aeruginosa
P. vulgaris
2500
39
Fig. 2 Kinetics of S. aureus growth
curve decreased in comparison to the control curve.
P. aeruginosa was inhibited only at the high concen-
trations of the tested compounds. The sulfobetaines with a
longer chain had weaker activity towards these bacteria and
inhibitory activity was observed in the concentration
Moreover, growth of S. aureus in the presence of
3
-
1
9 lg cm of SB3C16 was delayed by 2 h. Sulfobetaines
with 14 carbon atoms influenced the growth of S. aureus in
-
3
-3
a similar way. At a concentration of 39 lg cm , inhibition
and delayed cell growth was observed. The compound
1250–5000 lgÁcm , however even high concentrations
did not completely inhibit the growth of P. aeruginosa. In
the case of surfactants with 14 carbon atoms, the effect was
more varied. Better results were observed at the higher
-
3
SB4C16 in the concentration 19 and 39 lg cm did not
show antibacterial activity against S. aureus.
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J Surfact Deterg
Fig. 3 Kinetics of P. aeruginosa growth
-
3
concentration, but even at the 156 lgÁcm concentration,
the alkyl chain. The opposite dependence was observed by
Staszak et al. where the butane-sulfobetaines exhibited
lower CMC values than the corresponding propane
derivatives [4]. This suggests that the CMC values depend
not only on the length of hydrocarbon groups, but also the
structure of the hydrophilic head group.
sulfobetaines decreased growth curves.
Discussion
Surfactant adsorption at the water/air interface depends on
the molecular structure. The adsorption of surfactants
increases with increasing concentration up to the CMC
where the interface is saturated [18]. Analyzing the surface
tension isotherms, it is clear that the CMC decreases with
increasing elongation of the alkyl chain. Such a phe-
nomenon is observed for most surfactants irrespective of
the charge class to which they are classified and can be
explained by the fact that micelle formation is favored
when stabilizing hydrophobic interactions are accentuated.
The values of CMC are comparable to those presented by
Cheng et al., however the nominal values are slightly dif-
ferent [19]. Butane derivatives of sulfobetaines examined
exhibited higher CMC values than the corresponding pro-
pane ones with elongation of alkyl chain, but the differ-
ences decreased with increasing number of carbon atoms in
Two parameters which described the ability of surfac-
tants to reduce the surface tension, efficiency and effec-
tiveness, are sometimes inversely related [20]. The
surfactant effectiveness is the maximum reduction of the
surface tension, whereas the efficiency is the surfactant
concentration required to reduce of surface tension to the
significant amount.
The spreading pressure at the CMC (PCMC) is a measure
of surfactant effectiveness. PCMC values were calculated
by subtracting the surface tension at the CMC from the
surface tension of pure water. Our measurements show no
dependence between PCMC and elongation of the alkyl
chain. The greater surface pressure was measured for
propane-sulfobetaines. The surfactant molecules can be
loosely or densely packed at the interface as an effect of
interfacial properties and their structure. Depending on
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J Surfact Deterg
which is greater––the hydrophobic chain cross-sectional
area or the area required for close packing of head groups,
determines the area occupied by molecule. Straight
hydrocarbon chains and big head groups (relative to the tail
cross section) favor close effective packing, while bran-
ched, bulky, or multiple hydrophobic chains give rise to
steric hindrance at the interface. On the other hand, within
a series of single straight-chain surfactants, increasing the
alkyl chain length could be that the long hydrophobic
chains exhibit the tendency to bend and thus make the
value of Amin larger. A similar relationship was observed
by Xue et al. in their studies of synthesis and properties of
alkylbetaine zwitterionic gemini surfactants [23].
Surfactants are compounds known for their ability to
inhibit the growth of bacteria and fungi [24–28] as well as
increase the efficacy of some antimicrobial agents [29, 30].
Generally, ionic surfactants present higher toxicity than
nonionic. It is also known that among ionic surfactants,
cationic compounds show better antimicrobial properties
[31].
hydrocarbon chain length from C to C20 will have little
8
effect of adsorption effectiveness [21].
The efficiency of surfactant adsorption (p20) is defined
as the negative logarithm of the surfactant concentration
-
1
required to reduce the surface tension by 20 m Nm . In
general, efficiency of adsorption increases with increasing
hydrophobic character of the surfactant. For the sulfobe-
taine systems, the p20 values decrease with increasing
alkyl chain length. Consequently the adsorption efficiency
increases with increasing carbon atoms in the alkyl chain.
When considering the thermodynamics of adsorption
and micellization of sulfobetaines in aqueous solutions it
must be noted that both parameters increases with
increasing alkyl chain length. That indicates that the
hydrophobic effect observed in the systems where the
dissolved surfactants molecules carrying over a dozen
hydrocarbon groups causes distortion of the solvent struc-
ture thus the free energy of the system increase. Moreover
more negative values of free energy of micellization for
surfactants with longer alkyl chain could be explained by
the favoring of micellar aggregation due to larger
hydrophobic interaction. A similar relationship was
observed for alkyl sulfobetaines [22]. The smaller (more
negative) contribution assigned to the polar group propane
sulfobetaine derivative indicates that this group is more
conducive to the formation of the micelles than derivative
with butane sulfobetaine polar group. The same relation-
ship was observed for 3-(N-alkylmorpholinium)-1-propane
Recently, several betaines have also been described as
compounds presenting good antibacterial activity. Ward
et al. obtained a polymethacrylic sulfopropylbetaine
copolymer exhibiting antimicrobial activity against some
common pathogens such as E. coli and S. aureus [31]. The
authors stated that activity was related to the molecular
characteristics including molar composition and structure
of the hydrophobic co-monomer. Cheng et al. described
zwitterionic poly(carboxybetaine methacrylate) (pCBMA)
grafted surfaces resistant to long-term bacterial biofilm
formation by P. aeruginosa and P. putida [32]. Similar
properties were ascribed to surfaces treated with long-chain
zwitterionic poly(sulfobetaine methacrylate), which
reduced adhesion and biofilm formation by S. epidermidis
and P. aeruginosa [33]. Liu et al. described stronger
antibacterial activity and a broader inhibitory spectrum of
chitosan/betaine derivative complex in comparison to chi-
tosan alone [34].
Sulfobetaines tested in our work showed antibacterial
activity against Gram-positive and Gram-negative bacteria
with higher activity against Gram-positive bacteria.
According to the literature, Gram-negative bacteria are
often more resistant to different biocides than Gram-posi-
tive bacteria. Some reports indicate that non-ionic surfac-
tants may interact with proteins and membrane
phospholipids, increasing permeability of the membrane
and thereby causing leakage of compounds with low
molecular mass. Due to loss of ions, amino acids and other
small molecules, cell damage or cell death may occur [35].
The bacterial cell wall differs between Gram-positive and
Gram-negative bacteria. In Gram-positive bacteria there is
a thick layer of peptidoglycan, teichoic and lipoteichoic
acids, which may serve as chelating agents. According to
literature data the lipoteichoic acid layer may participate in
the adsorption of positively charged molecules [36]. The
higher resistance displayed by Gram-negative bacteria
could be explained by the presence of an external mem-
brane in the cell wall structure, composed of lipopolysac-
charides and proteins. This structure generally limits the
penetration of different biocides and amphiphilic com-
pounds into the cell [37, 38]. Moreover, some reports
or butane-sulfonate [4]. However the nominal values of
0
DG and DGads show that the adsorption process is
m
promoted.
A fundamental physical quantity associated with the
formation of an oriented surfactant monolayer is the sur-
face excess. For sulfobetaines solutions, the surface excess
at the saturated interface has no relationships with the
length of the alkyl chain. The highest value of C? are
obtained for SB3C12 from propane-sulfobetaines and
SB4C12 from butane-sulfobetaines. In addition to the
above, the same situation is observed when considering the
values of the minimum molecular area occupied by
molecules at the saturated interface. The value of Amin is
the smallest for both 12 hydrocarbon group derivatives.
The explanation of this phenomenon may be that the
hydrocarbon chains form a film at the interface. The reason
of increasing of the values of Amin with the increase of
1
23

J Surfact Deterg
indicate that perturbation of the outer membrane requires
an appropriate hydrophile-lipophile balance (HLB) and
differences in HLB may cause diverse effect of surfactants
with a similar structure on bacterial species [39]. It is worth
noting that sulfobetaines examined in the present work
exhibited antibacterial activity against two types of bacte-
ria. Similar, Zaky et al. tested germinate non-ionic sur-
factants against pathogenic bacteria and stated that activity
of synthesized compounds towards Gram-positive and
Gram-negative bacteria was not too much different from
each other [36]. The authors explained this high efficiency
against two types of bacteria by the presence of the ethy-
lene oxide units in the surfactant structure which increased
the adsorption and penetration of these compounds into the
cell membrane. Hamouda et al. described non-ionic sur-
factant nanoemulsion designed as 8N8 exhibiting biocidal
activity against some Gram-positive and Gram-negative
bacteria [40]. However, they observed that P. aeruginosa
and most enteric Gram-negative bacteria such as E. coli, S.
typhimurium or S. dysentariae were resistant to the exam-
ined compound. The authors explained that resistance of
some bacteria may be connected with the cell wall struc-
ture which contains negatively charge lipopolysaccharides.
Taking into account that 8N8 also had a negative charge;
repulsive forces could prevent attachment of nanoemulsion
droplets to the bacterial cell wall [25]. However, it is also
possible that more than one mechanism is responsible for
interaction of the tested nanoemulsion and bacterial cells
sulfobetaines is unknown, however it is currently under
investigation.
Acknowledgments This research was supported with 03/32/DS-PB/
0
501 grant.
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