Accepted Manuscript

Title: THERMODYNAMIC PROPERTIES OFRHAMNOLIPID MICELLIZATION AND ADSORPTION

Author: DIANA Manko ANNA Zdziennicka BRONISŁAWJanczuk

PII: S0927-7765(14)00218-5DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.020Reference: COLSUB 6397

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 15-2-2014Revised date: 4-4-2014Accepted date: 23-4-2014

Please cite this article as: D.I.A.N.A. Manko, A.N.N.A. Zdziennicka,B.R.O.N.I.S.L.A.W. Janczuk, THERMODYNAMIC PROPERTIES OFRHAMNOLIPID MICELLIZATION AND ADSORPTION, Colloids and Surfaces B:Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.020

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THERMODYNAMIC PROPERTIES OF RHAMNOLIPID MICELLIZATION AND ADSORPTION

DIANA MAŃKO, ANNA ZDZIENNICKA*, AND BRONISŁAW JAŃCZUK

Department of Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland

Running title: Thermodynamic properties of rhamnolipid

Total number of words: 6697

Total number of figures: 7

Total number of tables: 0

*To whom correspondence should be addressed

phone (48-81) 537-56-70

fax (48-81) 533-3348

e-mail aniaz@hektor.umcs.lublin.pl

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Abstract

Measurements of the surface tension, density, viscosity and conductivity of aqueous solutions

of rhamnolipid at natural and controlled pH were made at 293 K. On the basis of the obtained

results the critical micelle concentration of rhamnolipid and its Gibbs surface excess

concentration at the water-air interface were determined. The maximal surface excess

concentration was considered in the light of the size of rhamnolipid molecule. Next the Gibbs

standard free energy of rhamnolipid adsorption at this interface was determined on the basis

of the different approaches to this energy. The standard free energy of adsorption was also

deduced on the basis of the surface tension of n-hexane and water-n-hexane interface tension.

Standard free energy obtained in this way was close to those determined by using the

Langmuir, Szyszkowski, Aronson and Rosen, Gu and Zhu as well as modified Gamboa and

Olea equations. The standard free energy of rhamnolipid adsorption at the water-air interface

was compared to its standard free energy of micellization which was determined from the

Philips equation taking into account the degree of rhamnolipid dissociation in the micelles.

Key words: Biosurfactant, rhamnolipid, micellization, adsorption, standard free energy of

micellization and adsorption.

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Introduction

Surfactants are amphihilic molecules which have two different parts: hydrophobic chain (tail)

and hydrophilic group (head) [1]. These compounds influence the surface and interface

tension through the formation of the aggregates co-called micelles and adsorption on different

interfaces. Surfactants can be divided into two main groups: synthetic surfactants and

biosurfactants. Synthetic surfactants are produced by organic chemical reactions and

biosurfactans are produced by a number of microorganisms, including bacteria, yeats and

fungi [2]. Biosurfactants have very interesting properties such as: good biodegradability, low

toxicity or effectiveness at extreme temperature, pH and salinity. Because of their properties

biosurfactants can be treated as potential substitutes of chemical compounds obtained by

classical synthesis [3-6]. Among biosurfactants rhamnolipids have very interesting properties

from theoretical and practical points of view. Rhamnolipids which are mainly produced by

Pseudomonas aeruginosa during cultivation on glucose, glycerol or triglycerides [7-13]

represents of glycolipid. There are many types of rhamnolipids, however, they possess similar

chemical structures [14]. Generally rhamnolipids contain a hydrophilic head formed by one or

two rhamnose molecules and a hydrophobic group composed of one or two fatty acid chains

[14-16]. The form of rhamnolipids produced by bacteria and the proportion between them

depend on their strain, the carbon source and the culture conditions [2,17]. Rhamnolipids have

various applications such as pharmaceuticals [18,19], cosmetic products, food items,

detergents [20] and bioremediation enhancers [21,22]. Additionally, rhamnolipids possess

anti-proliferative activity against a human breast cancer cell [16,23] and anti-microbial

against bacteria and phytopathogenic fungi species [11,24].

Though there are the many studies dealing with the adsorption and volumetric properties of

rhamnolipids, the problem of their surface activity and tendency to form micelles in aqueous

solutions is not quite clear. In the literature it is even possible to find different values of

efficiency and effectiveness of rhamnolipid adsorption as well as of critical micelle

concentration [25-27]. On one hand, it can result from the fact that rhamnolipids produced by

Pseudomonas sp. can be a mixture of various 4 – 28 homologues where monorhamnolipid is a

dominant form [14]. One the other hand, the studies dealing with the adsorption and

aggregation properties of rhamnolipid are based mainly on the measurements of the surface

tension. Moreover, the data connected with these properties are different and incoherent in

many cases [25-27].

We must also remember that in practical application of rhamnolipid the knowledge of its

tendency to adsorb at the water-air interface and to aggregate in the bulk phase should be very

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helpful. These tendencies can be expressed and predicted by the thermodynamic parameters

of the rhamnolipid adsorption and micellization process. In the literature it is difficult to find a

complex analysis of these processes based on the data obtained from the measurements of

different physicochemical properties of aqueous solutions of rhamnolipid. Thus, the purpose

of our studies was to determine the critical micelle concentration of the studied rhamnolipid

and its surface excess concentration at the water-air interface and standard free energy of

adsorption and micellization by using different approaches to these processes. This purpose

was achieved by measurements of the surface tension, density, dynamic viscosity and

conductivity of aqueous solutions of rhamnolipid at natural pH in a wide range of its

concentration as well as their theoretical consideration.

2. Material and methods

2.1. Materials

R-95 Rhamnolipid obtained from SIGMA-ALDRICH (95%) was used without further

purification. The aqueous solutions of rhamnolipid were prepared using doubly distilled and

deionized water (Destamat Bi18E) which had an internal specific resistance of 18.2 MΩ. The

purity of water was additionally controlled by the surface tension measurements before

preparing the solutions. The concentration of rhamnolipid was changed in the range from 2 x

10-4 to 40 mg/dm3.

2.2. Measurements

The equilibrium surface tension ( LV ) of the aqueous solution of rhamnolipid was measured

by the Krüss K9 tensiometer according to the platinum ring detachment method (du Nouy’s

method). Before the surface tension measurements, the tensiometer was calibrated using water

( LV = 72.8 mN/m) and methanol ( LV = 22.5 mN/m) according to the procedure of C. Huh

and S. G. Mason [28]. The ring was cleaned with distilled water and heated to red colour with

a Bunsen burner before each measurement. In all cases more than 10 successive

measurements were carried out. The standard deviation depending on the surfactant

concentration was in the range from ± 0.1 to ± 0.25 mN/m. The measurement temperature

was controlled by a jacketed vessel joined to a thermostatic water bath with the accuracy ± 0.1

K. The uncertainty of the surface tension measurements was equal from 0.3 to 0.7 %

depending on the range of surfactant concentration. All the experiments were done at 293 K

within ± 0.1K.

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The density of the aqueous solutions of rhamnolipid was measured with a U-tube

densitometer (DMA 5000 Anton Paar) at the constant temperature 293 K. The precision of the

density and temperature measurements given by the manufacturer is ±0.000001 g/m3 and

±0.001K. Uncertainty was calculated to be equal to 0.01%.

All the viscosity measurements of the aqueous solutions of rhamnolipid were performed with

the Anton Paar viscosimeter (AMVn) at 293K ±0.01K with the precision of 0.0001

smPa and uncertainty 0.3%. The densitometer and viscosimeter were calibrated regularly

with distilled and deionized water.

The specific conductivity ( ) measurements of the aqueous solutions of rhamnolipid were

made by the conductometer, Mettler Toledo, joined with the thermostat LAUDA RE 415S

with the temperature precision equal to ±0.1K. The relative uncertainty of the conductivity

measurements did not exceed 0.5%.

All these physicochemical properties were determined at natural pH of aqueous solutions of

rhamnolipids.

3. Results and discussion

According to the studies of many authors [9,17,22, 29-32] rhamnolipids produced by

Pseudomonas aeruginosa, grown with different carbon sources can be mixtures of 4 – 28

different homologues. Among them the mono- and dirhamnolipids are present

[8,10,11,33,34]. Therefore, for determination of the molar concentration of the rhamnolipid

studied (C) by us the molecular weight of mono- (504) and dirhamnolipid (650) was taken

into account. Therefore , for the calculations of all quantities as well as expression of some

quantities as a function of molar concentration of rhamnolipid double values of this

concentration were used.

3.1. Surface excess concentration of rhamnolipid at water-air interface

The shape of the isotherm of surface tension ( LV ) of aqueous solutions of rhamnolipid (Fig.

1) depends on the density and orientation of biosurfactant molecules in the adsorption

monolayer at the water-air interface. The density of surfactants in this layer can be

determined, among other things, by using the Gibbs adsorption equation. For the ionic

surfactant of the type AB electrolyte (AB↔A++B-) if f ≈1 ( f is the surfactant activity

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coefficient) and C

X (where X is the mole fraction of the surface active agent, C is the

molar concentration of the surface active agent and ω is the number of molecules of water in

1 dm3) the Gibbs adsorption equation assumes the following form [1,35,36]:

Cd

d

RTCd

d

RTdC

d

RT

C LVLVLV

log606.4

1

ln2

1

2

(1)

where is the Gibbs surface excess concentration of the ionic surfactant of the type AB

electrolyte.

It appeared that the changes of the surface tension of the aqueous solutions of rhamnolipid as

a function of its concentration (C) (Fig. 1) can be described by the first order exponential

function. Thus, it was possible to calculate dCLV / and then from Eq. (1). However, the

maximal Gibbs surface excess concentration of rhamnolipid was determined from the

relationship between the surface tension of solutions and logC in this concentration range. It

appeared that the isotherm of rhamnolipid surface excess concentration (Fig. 2) has a shape

typical of classical surfactants [37]. It is difficult to compare the shape of the isotherm to

those in the literature because it is difficult to find such data. However, the value of the

maximal surface excess concentration of rhamnolipid obtained by us equal to 2.01 x 10-6

mol/m2 (Fig. 2) is close to that obtained by Chen et al. [25]. It should be noted that in the

literature it is possible to find different values of the maximal rhamnolipid surface excess

concentration [26,27]. It is a question whether the maximal Gibbs surface excess

concentration of rhamnolipid at the water-air interface is reasonable taking into account the

size of rhamnolipid molecule. The value of 2.01 x 10-6 mol/m2 (Fig. 2) corresponds to the

area occupied by a molecule of rhamnolipid at the water-air interface which is equal to 82.6

Å2. If we assume that the Gibbs plane at the solution-air interface is chosen in such a way

that the hydrophilic part of rhamnolipid molecule is in the liquid phase and the hydrophobic

one in the air, the hydrophilic group is oriented parallel to the interface and the hydrophobic

one perpendicular to the interface, respectively, then the minimal length of a molecule

calculated on the basis of the length of bonds between particular atoms and the contact angle

between bonds [38], at the first approximation, is equal to 15.31 Å and the width 4.512 Å,

respectively. In the calculations it was assumed that the average minimal distance between

molecules is equal to 1.58 Å [39] and that the heterocyclic ring is oriented perpendicular to

the –O-CH-CH2-CO-CH-CH2—COOH group. Expressing, at the first approximation, the

minimal area occupied by a rhamnolipid molecule by the rectangle [40], it was found equal to

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69.08 Å2. This area corresponds to 2.403 x 10-6 mol/m2 of the Gibbs surface excess

concentration and can be treated as the limiting area for a representative monorhamnolipid

molecule in the surface layer at the water-air interface (Ao). This value is higher than the

maximal surface excess concentration calculated from Eq. (1). On the other hand, it is more

probable that the average minimal distance between the rhamnolipid molecule can be higher

than 2 Å [40] and the minimal area occupied by the representative monorhamnolipid

corresponds to the saturated adsorbed monolayer should be higher than 77.55 Å2 ( = 2.14 x

10-6 mol/m2). Taking this fact into account, it seems that the minimal area occupied by one

molecule of rhamnolipid determined by us (82.6 Å2) is reasonable and that the studied

rhamnolipid contains mainly the representative monorhamnolipid. It is interesting that the

most literature data dealing with the maximal surface excess concentration of rhamnolipid at

the water-air interface are close to this value [25,27]. If we assume that the ratio of the

calculated limiting area to the minimal area obtained from the Gibbs equation is equal to the

fraction surface occupied by rhamnolipid at the solution-air interface, then it is possible, at the

first approximation, to establish the minimal surface tension of the aqueous solutions of

rhamnolipid. The decrease of the water surface tension depends on the surface tension of the

compounds from which the hydrophobic part of surfactant was formed. The representative

monorhamnolipid can be treated as possessing the hexyl group as a hydrophobic one. The

surface tension of n-hexane at 293 K is equal to 18.49 mN/m [41] and that of water to 72.8

mN/m. Taking into account the values of surface tension and mole fraction of the area

occupied by rhamnolipid at the surface-air interface, we obtained the minimal surface tension

of the representative monorhamnolipid as equal to 27.38 mN/m [42]. This value is close to the

measured minimal values of the rhamnolipid surface tension which is equal to 27.89 mN/m

and which is in the accordance with the literature data [15].

2. Critical micelle concentration

Another specific property of surfactants is the tendency to form aggregates in the bulk phase

at a concentration called the critical micelle concentration (CMC). It should be stressed that

the CMC values determined for rhamnolipid by various researchers were obtained mainly

from the changes of the surface tension of solutions as a function of their concentration.

However, there is a lack of confirmation of this data of CMC by the measurements of other

physicochemical properties. Therefore, apart from the surface tension, the changes of density,

viscosity and conductivity as a function of rhamnolipid concentration for its CMC

determination were taken into account (Figs. 3 – 5).

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From Figs. 1 and 3 – 5 it appeared that the inflection point was observed on the surface

tension, density, viscosity and conductivity isotherms and the CMC of rhamnolipid obtained

on the basis of these isotherms is equal to 26.24; 24.06; 25.72 and 25.43 mg/dm3,

respectively. The CMC values obtained by us are in the range of rhamnolipid CMC

determined by other authors (1 – 200 mg/dm3) [10,11,16,24,43,44]. The wide range of the

CMC rhamnolipid whch can be find in the literature [10,11,16,24,43,44] probably results

from the fact that rhamnolipid is a mixture of various 4 – 28 homologues where

monorhamnolipid is a dominant form [9]. It should be also noticed that the discrepancy in the

CMC values can suggest that CMC should be treated rather as a range of concentration in

which aggregates can be formed but not as one pinpoint or that each method of CMC

determination is sensitive to different sizes of aggregates.

3.3. Apparent and partial molar volume of rhamnolipid

The micelle formation is correlated with the changes of solution structure and it should be

reflected in the apparent ( V ) and partial ( MV ) molar volumes of surfactant.

The apparent molar volume was determined from the following equation [45]:

0

0

0

1000

S

SV C

M (2)

where MS is the molecular weight of surfactant, CS is its concentration mol/cm3 and 0 and

are the density of a “pure” solvent and the solution, respectively.

In our calculations according to the rhamnolipid form [26] there was taken into account the

molecular weight equal to 650 and 504 g/mol for di- and monorhamnolipids, respectively, as

two main and representative forms of this biosurfactant.

The partial molar volume MV was calculated from equation [46]:

p

pSM

dC

dCMV

1001 (3)

where Cp is the percentage weight of the solute.

It appeared that the data fit a polynomial of Cp given by:

2PP dCbCa (4)

where a, b and d are the constants.

It proved that both apparent and partial molar volumes of rhamnolipid changed very slightly

as a function of rhamnolipid concentration (Fig. 6). However, on the CV curves a very

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small infection point can be found. It is interesting that this point corresponds to the

rhamnolipd CMC. On the contrary, the dependence between MV and the rhamnolipid

concentration is linear (Fig. 6). Unfortunately, the values of apparent and partial molar

volumes of rhamnolipid obtained by us are difficult to compare with the literature data

because of the lack of this type data.

3.4. Standard free energy of micellization and adsorption at the water-air interface

The presence of surfactant in water causes the increase of the surface free energy which can

be minimalized by the adsorption and micellization process of surfactants. It is reflected in the

values of the standard Gibbs free energy of adsorption and micellization.

3.4.1. Standard free energy of rhamnolipid micellization

For the ionic surfactants of the type AB electrolyte (1:1) the Gibbs standard free energy of

micellization ( omicG ) can be calculated, among other things, from the Philips equation [47]

which has the following form:

CMC

RTn

pGmic ln20

(5)

where n is the number of surfactant ions forming a micelle, p is the number of counterions

bound to the micelle and p/n is equal to (1-) where is the degree of the surfactant

dissociation in the micelle.

Because the rhamnolipids are typical anionic surfactants due to the presence of the carboxylic

group in their molecule, it was possible to determine the degree of H+ bonding to the micelle

(p/n) from the conductivity changes as a function of its concentration [1,48]. This degree was

calculated on the basis of the slope of linear part of the C curve after and before CMC and

is equal to 0.11.

Taking into account the obtained by us values of p/n and rhamnolipid CMC, the values of

omicG were determined from Eq. (5). They are in the range from -61.66 to -60.17 kJ/mol.

Thus, independently of the method of CMC determination and the molecular weight of di-

and monorhamolipids the obtained values of omicG are close to each other.

It should be stressed that omicG of rhamnolipid is considerably lower than for the classical

anionic surfactants and even nonionic one [49]. It means that rhamnolipid tendency to form

micelle is very high.

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3.4.2. Standard free energy of rhamnolipid adsorption at the water-air interface

In the literature there are many approaches for determination of the standard free energy of

adsorption ( oadsG ). The most commonly used is the Langmuir equation modified by de Boer

which has the form [50]:

RT

GC

AA

A

AA

A ads0

0

0

0

0 expexp

(6)

where A is the area occupied per molecule at the water-air interface and Ao is the “excluded

area”, i.e., the area of the interface unavailable to one molecule due to the presence of another.

Assuming that this excluded area is equal to the limiting area occupied by one molecule of the

representative monorhamnolipid at the water-air interface determined by us, from the size of

this rhamnolipid we calculated oadsG from Eq. (6) taking into account 2RT instead RT. Of

course, the oadsG values corresponding to the range of rhamnolipid concentration (0 - 0.02

mg/dm3) in which the unsaturated monolayer at the water-air interface is formed are stable

(Figs.1,7) and then rapidly decrease to the concentration of rhamnolipid equal to 5 mg/dm3.

This value is lower than that of rhamnolipid CMC obtained by us but it is in the range of the

literature data [44] . Above this concentration the increase of oadsG is observed. It is known

that the Langmuir equation can be applied for oadsG determination by using the area occupied

per rhamnolipid molecules in the unsaturated monolayer in which there are no mutual

interactions between the adsorbed molecules. The stable values of oadsG calculated from

Eq.(6) corresponding to the concentration in the range from 0 to 0.02 mg/dm3 suggest that in

this concentration range, there are no interactions between rhamnolipid molecules in the

monolayer at the water-air interface and oadsG determined for this range of rhamnolipid

concentration can be treated as a standard free energy of rhamnolipid adsorption. The

oadsG values determined in this way are equal to -85.04 or -86.28 kJ/mol depending on the

molecular weight taken into account for the determination of rhamnolipid concentration (for

mono- or dirhamnolipid). It is known that it is difficult to obtain good quality values of the

surface tension of aqueous solutions of surfactant at their low concentration. Therefore it is

sometimes more reasonable to determine oadsG on the basis of the surface tension values

corresponding to the saturated monolayer at the water-air interface by using the Rosen and

Aronson equation which for the ionic surfactants of type AB electrolyte can be expressed in

the following form [1,51]:

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1ln2 w

CRTG o

ads (7)

where C and correspond to the saturated monolayer of surfactants ( is the difference

between the surface tension of solvent ( oLV ) and solution ( LV )) , 1 is the area occupied by

one mole of the surfactant at the water-air interface.

The values of oadsG calculated from Eq. (7) are equal to -83.64 and -84.88 kJ/mol depending

on the rhamnolipid molecular weight taken into account for its concentration determination

(for mono- or dirhamnolipid).

From the analysis of the Gibbs surface excess concentration of surfactant at the water-air

interface, it results that the monolayer for most surfactants is saturated already at the

concentration of surfactant in the bulk phase corresponding to that at which the reduction of

water surface tension by 20 mN/m is obtained [1]. As results from Fig.1 the studied

rhamnolipid fulfilled this condition. In such case it is possible to calculate oadsG on the basis

of the rhamnolipid concentration corresponding to the surface tension of aqueous solutions of

rhamnolipid equal to 52.8 mN/m ( = 20 mN/m) by using the modified Gamboa and Olea

equation which for the ionic surfactants type AB electrolyte (1:1) has the following form

[52,53]:

)(606.4 120 KpCRTG oads (8)

where K1 is the constant.

The constant K1 was calculated on the basis of the limiting area of rhamnolipid molecule at

the water-air interface equal to 69.08 Å2 (K1 = 2.49). The value of oadsG calculated from Eq.

(8) is equal to -88.22 kJ/mol.

As mentioned above, Eq. (6) is fulfilled for the low concentration of surfactant and Eq. (7) for

the concentration corresponding to the saturated monolayer at the water-air interface,

however, the Szyszkowski equation gives the possibility of oadsG determination on the basis

of surface tension of aqueous solution of rhamnolipid in the range from 0 to CMC because the

constant b in this equation is correlated with the standard Gibbs free energy of adsorption and

for rhamnolipid fulfills the following expression [49]:

RT

Gb

oads

2exp

(9)

The value of oadsG calculated in this way is equal to -84.4 kJ/mol.

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In our previous study [37] we found that the Gu and Zhu isotherm adsorption equation [54-

56] derived for the solid-solution interface can be satisfactorily applied for the solution-air

interface.

The general equation of the Gu and Zhu adsorption isotherm has the form [55]:

121

121

11

1

a

a

n

n

a

CkCk

Ckn

Ck

(10)

where k1 and k2 are the equilibrium constants of the surface monolayer and micelle formation,

respectively (there is the equilibrium between the adsorbed and free species in the bulk

phase), and an is the aggregation number of the surface micelles.

If 1an and 122 Ck from Eq. (10) we obtain [55]:

a

a

n

n

KC

KC

1 (11)

where 21kkK .

Eq. (11) can be transformed to the logarithmic form:

CnK a logloglog

(12)

If a plot of

log versus Clog is a straight line, then the K and an constants can be

determined from Eq. (12). In the case when an = 1, then aK /1 where a is the constant in

the Langmuir equation which at 293 K for anionic surfactants type AB electrolyte (1:1) fulfills

the condition [1]:

RT

Ga ads

2exp4.55

0 (13)

It appeared that using equal to 2.403 x 10-6 mol/m2 the plot of

log versus

Clog is linear in the low range of rhamnolipid concentration whose slope is equal to 1.01.

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Thus, it was possible to calculate of oadsG from Eq. (13) and it is equal to -86.04 and -87.3

kJ/mol depending on the rhamnolipd molecular weight taken into account in its concentration

determination (for mono- or dirhamnolipid).

Comparing the oadsG values obtained from the Langmuir [1,50], Szyszkowski [1] and Gu and

Zhu equations [54-56] it can be stated that there is good agreement between them and these

values are close to those determined from the Rosen and Aronson [1,41], and modified

Gamboa and Olea equations [52]. This agreement indicates that the limiting area occupied by

the representative monorhamnolipid at the water-air interface calculated on the basis of the

size and proper orientation of the hydrophilic group at the water-air interface is quite

reasonable because this value was used for the calculation of oadsG in Eqs. (6), (8) and (9).

The additional confirmation of our statement are the oadsG values determined from the

following equation [1,57]:

max CMCo

micoads GG

(14)

where CMC is the surface pressure in CMC and CMCLV

oLVCMC ( CMC

LV is the surface

tension at CMC and CMC is the surface excess concentration at CMC).

The calculated values of oadsG are in the range from -82.83 to -82.45 and from -83.94 to -

82.69 kJ/mol depending on the rhamnolipid molecular weight taken into account for

determination (for mono- or dhirhamnolipid) of its concentration in mol/dm3.

The adsorption process of the surfactant at the interface is connected with work of the

hydrophilic (head) and hydrophobic (tail) parts of its molecules transfer from the bulk phase

to the interface. However, the contribution of this work to the standard free energy of

surfactant adsorption is different. Therefore, these two works should be taken into account

considering the adsorption of a given surfactant at the water-air interface [1].

According to van Oss and Constanzo [39], the surface free energy of a surfactant can be

divided into the surface free energies of the hydrocarbon tail (the state when the surfactant

molecules are oriented by the hydrophobic groups toward the air phase) and head (the state

when the surfactant molecules are oriented by the hydrophilic groups toward the air). The

surface free energy of the hydrocarbon tail results from the Lifshitz-van der Waals

intermolecular interactions, and that of the hydrophilic head from the Lifshitz-van der Waals,

Lewis acid-base and electrostatic interactions.

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If it is assumed that after adsorption at the aqueous solution of the surfactant-air interface, the

hydrophobic tail or its part is in the air phase and the hydrophilic head or head with a part of

tail is in the solution phase, the transfer of surfactant molecules from the bulk aqueous phase

to the surface monolayer is associated with changes of the interfacial free energy of the water-

tail ( WT ) to the surface free energy of tail ( T ) and the interfacial free energy water-head

( WH ) from WH to 1WH because of the dehydration of the head during the adsorption

process [40].

Thus, the standard free energy of adsorption at the aqueous solution of surfactant-air interface

should fulfill the condition [40]:

WHWHHWTTToads NANAG 1 (15)

where TA is the contactable area of the surfactant tail or its part, HA is the contactable area of

the surfactant head or head with a part of tail.

If during the transport of the surfactant molecule from the bulk phase of solution to the

surface monolayer its head does not dehydrate then Eq. (15) can be expressed in the form

[40]:

WTTToads NAG (16)

It was shown earlier [34] that the contactable area of n-alkane molecule (A) can be calculated

from the simple expression:

2)(24 dwdwdlA (17)

In the case of the hydrocarbon surfactant having the alkyl group as a hydrophobic one the

contactable area of such group is found by the following expression:

2)())(2/(4 dwdwdlAT (18)

If it is assumed that the representative monorhamnolipid has two hexyl groups as a

hydrophobic one (tail) and after adsorption in the aqueous solution of surfactant-air interface,

this group is in the air phase and the hydrophilic group (head) in the solution phase the

transfer of rhamnolipid molecules from the bulk aqueous phase to the surface monolayer is

associated with the change the interfacial free energy of water-n-hexane to the surface free

energy of n-hexane. Taking into account that the 2/dl is equal to 10.82 Å and dw = 4.6

Å the contactable area of the hexyl group was calculated and it was equal to 220.25 Å2.

Because we assumed that the representative monorhamnolipid has two hexyl groups therefore

the total contactable area was equal to 440.5 Å2. Knowing that the surface free energy of n-

hexane is equal to 18.49 mJ/m2 [41] at 293K and the water-n-hexane interfacial free energy

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51.1 mJ/m2 [41], the calculated value of oadsG from Eq. (16) is equal to -86.52 kJ/mol. Of

course, for our calculation we assumed that the hexyl groups are oriented perpendicular to the

solution-air interface. This value is in good accordance with that obtained from the Langmuir

equation.

The obtained values of oadsG for rhamnolipid determined from on the basis of all equations

used in our calculations are almost twice as low as the standard free energy of adsorption of

nonionic Tritons and lower than that of the classical anionic and cationic synthetic surfactants

[43]. It is not clear that the standard free energy of rhamnolipid adsorption being the measure

of its efficiency to adsrob at the water-air interface is twice as low as that of Tritons. It is

connected with application of 2RT instead of RT in all equations used for oadsG calculation.

However, the obtained oadsG value by using Eq. (16) is close to those obtained from the other

equations in which 2RT was applied. On the other hand, oadsG calculated from the Langmuir

equation deals with the dilute solution of surfactant for which the unsaturated monolayer is

formed at the water-air interface. For such case the parallel orientation of the hydrophobic

group at the water-air interface is more probable than the perpendicular one. Taking this into

account and the fact that even two groups of –CH2– can be present in the water phase [49], the

calculated value of oadsG from Eq. (16) is equal to -47.78 kJ/mol. This value is close to that

obtained from the Aronson and Rosen equation [1,51] if RT instead of 2RT is used in this

equation and somewhat lower than that determined from the Langmuir equation [44] under

the same assumption. The values calculated in such way are only slightly lower than oadsG

for Tritons [49].

4. Conclusions

From the measurements and thermodynamic considerations it results that:

The area occupied by rhamnolipid in the saturated monolayer at the water- air interface is

equal to 82.6 Å2. This area is somewhat higher than the “excluded area” of monorhamnolipid

(which is equal to 69.08 Å2) determined on the basis of the cross sectional area of the

hydrophilic and hydrophobic groups being in the monolayer at the water-air interface oriented

perpendicularly to this interface.

The tendency of rhamnolipid to adsorb at the water -air interface and form the micelles is

higher than for the classical synthetic surfactants because the standard Gibbs free energy of

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adsorption and micellization of rhamnolipid determined by using different models is

considerably lower than those of the synthetic ones.

It is possible to predict the standard Gibbs free energy of adsorption on the basis of the

surface tension of n-hexane and the n-hexane-water interface tension.

The rhamnolipid molar volume changes only slightly during the micelles formation and CMC

determined by surface tension, density, viscosity and specific conductivity is considerably

lower even than such nonionic surfactant as Triton TX-100.

The dissociation of the rhamnolipid molecules in the micelles is only slightly lower than in

the monomeric form.

References

[1] J.M. Rosen, Surfactants and Interfacial Phenomena, 3rd Ed., Wiley Interscience, New

York, 2004.

[2] M. Sánchez, F.J. Aranda, M.J. Espuny, A. Marqués, J.A. Teruel, Á. Manresa, A. Ortiz,

Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonos

aeruginosa in aqueous media, J. Colloid Interface Sci. 307 (2007) 246-253.

[3] J.D. Desai, I.M. Banat, Microbial production of surfactants and their commercial potential

Microbiol. Mol. Rev. 61 (1997) 47-64.

[4] S.S. Cameotra, R.S. Makkar, Synthesis of biosurfactants in extreme conditions

Appl. Microbiol. Biotechnol. 50 (1998) 520-529.

[5] P. Singh. S.S. Cameotra, Potential applications of microbial surfactants in biomedical

sciences, Trends Biotechnol. 22 (2004) 142-146.

[6] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveria, Biosurfactants: potential applications in

medicine, J. Animicrob. Chemother. 57 (2006) 609-618.

[7] Y.H. Wei , Ch.L. Cheng, Ch.Ch. Chien, H.M. Wan, Enhanced di-rhamnolipid production

with an indigenous isolate Pseudomonas aeruginosa J16, Process Biochem. 43 (2008)

769-774.

[8] M.I. Van Dyke, P. Couture, M. Brauer, H. Lee, J.T. Trevors, Pseudomonas aeruginosa

UG2 rhamnolipid biosurfactants: structural characterization and their use in removing

hydrophobic compounds from soil, Can. J. Microbiol. 39 (1993) 1071-1078.

[9] T.B. Lotfabad, H. Abassi, R. Ahmadkhaniha, R. Roostaazad, R. Masooma, H S. Zahiri, G.

Ahamdian, H. Vali, K.A. Noghabi, Structural characterization of a rhamnolipid-type

biosurfactant produced by Pseudomonas aeruginosa MR01: Enhancement of di-

Page 17 of 27

Accep

ted

Man

uscr

ipt

rhamnolipid proportion using gamma irradiation, Colloids Surfaces B: Biointerfaces 81

(2010) 397-405.

[10] J.C. Mata-Sandoval, J. Karns, A. Torrents, High-performance liquid chromatography

method for the characterization of rhamnolipid mixtures produced by Pseudomonas

aeruginosa UG2 on corn oil, J. Chromatogr. A. 864 (1999) 211-220.

[11] A. Abalos, A. Pinazo, M.R. Casals, F. Garcia, A. Manresa, Physicochemical and

antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa

AT10 from soybean oil refinery wastes, Langmuir, 17 (2001) 1367-1371.

[12] A.M. Abbdel-Mawgoud, F. Lépine, E. Déziel. Rhamnolipids: diversity of structures,

microbial origins and roles, Appl. Microbiol. Biotechnol. 86 (2010) 1323-1336.

[13] C. Chayabutra. J. Wu, L.K. Ju, Rhamnolipid production by Pseudomonas

aeruginosa under denitrification: Effects of limiting nutrients and carbon substrates,

Biotechnol. Bioeng. 72 (2001) 25-33.

[14] J.L. Torrens, D.C. Herman, R.M. Miller-Maier, Biosurfactant (Rhamnolipid) sorption

and the impact on Rhamnolipid-facilitated removal of cadmium from various soils under

saturated flow conditions, Environmental Sci. Technol. 32 (1998) 776-781.

[15] H. Abbasi, K. A. Noghabi, M.M. Harmedi, H.S. Zahiri, A.A. Moosavi-Movahedi, M.

Amanlou, J. A. Teruel, A. Ortiz, Physicochemical characterization of a monorhamnolipid

secreted by Pseudomonas aeruginosa MA01 in aqueous media. An experimental and

molecular dynamics study, Colloids Surfaces B, 101 (2013) 256-265.

[16] O. Pornsunthorntwee, S. Chavadej, R. Rujiravanit, Solution properties and vesicle

formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4,

Colloids Surfaces B: Biointerfaces, 72 (2009) 6-15

[17] G. Soberón-Chávez, F. Lépine, E. Déziel. Production of rhamnolipids by Pseudomonas

aeruginosa, Appl. Microbiol. Biotechnol. 68 (2005) 705-717.

[18] T. Stipcevic, A. Piljac, G. Piljac, Enhanced healing of full-thickness burn wounds using

di-rhamnolipid, Burns, 32 (2006) 24-34.

[19] T. Stipcevic, T. Piljac, R. R. Isseroff, Di-rhamnolipid from Pseudomonas aeruginosa

displays differential effects on human keratinocyte and fibroblast cultures, J. Dertmatol.

Sci. 40 (2005) 141-143.

[20] R. Makkar, S. Cameotra, An update on the use of unconventional substrates for

biosurfactant production and their new applications, Appl. Microbiol. Biotechnol. 58

(2002) 428-434.

Page 18 of 27

Accep

ted

Man

uscr

ipt

[21] C.C. Lai, Y.C. Huang, Y.H. Wei, j. S. Chang, Biosurfactant-enhanced removal of total

petroleum hydrocarbons from contaminated soil, J. Hazardous Mater. 167 (2009) 609-

614.

[22] C.N. Mulligan, Environmental applications for biosurfactants,Environ. Pollut. 133 (2005)

183-198.

[23] B. Thanomsub, W. Pumeechockchai, A. Limtrakul, P. Arunrattiyakorn, W. Petchleelaha,

T. Nitoda, Chemical structures and biological activities of rhamnolipids produced

by Pseudomonas aeruginosa B189 isolated from milk factory waste, Bioresource

Technology, 97 (2006) 2457-2461.

[24] M. Benincasa, A. Abalos, I. Oliveira, A. Manresa, Chemical structure, surface properties

and biological activities of the biosurfactant Chemical structures and biological activities

of rhamnolipids produced by Pseudomonas aeruginosa LBI from soapstock, Antonie van

Leeuwenhoec 85 (2004) 1-8.

[25] M.L. Chen, J. Penfold, R.K. Thomas, T.J.P. Smyth, A. Perfumo, R. Marchant, I. M.

Banat, P. Stevenson, A. Parry, I. Tucker, I. Grillo, Solution self-assembly and adsorption

at the air-water interface of the monorhamnose and dirhamnose rhamnolipids and their

mixtures, Langmuir, 26 (2010) 18281-18292.

[26] S.S. Helvaci, S. Peker, G. Özdemir, Effect of electrolytes on the surface behaviour of

rhamnoipids R1 and R2, Colloids Surf. B, 35 (2004) 225-233.

[27] J. Penfold, R. K. Thomas, Hsin-Hui Shen, Adsorption and self-assembly of

biosurfactants studied by neutron reflectivity and small neutron scattering:glycolipids,

lipopetides and proteins, Soft Matter, 8 (2012) 578-591.

[28] C. Huh, A. Mason, Rigorous Theory of Ring Tensiometry; Colloid Polym. Sci. 253

(1975) 566-580.

[29] E. Dézíel, F. Lépine, S. Milot, R. Villemur, Mass spectrometry monitoring of

rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP,

Biochemica at Biophysica Acta 1485 (2000) 143-152.

[30] S.A. Monteiro, G.L. Sassaki, L.M de Souza, J. A. Meira, J. M. de Araújo, D.A. Mitchell,

L.P. Ramos, N. Krieger, Molecular and structural characterization of the biosurfactant

produced by Pseudomonas aeruginosa DAUPE 614, Chem. Phys. Lipids 147 (2007) 1-

13.

[31] M. Nitschke, S.G.V.A. Costa, J. Contiero, Rhamnolipid surfactants: An update on the

general aspects of these remarkable biomolecules, Biotechnol. Prog. 21 (2005) 1593-

1600.

Page 19 of 27

Accep

ted

Man

uscr

ipt

[32] M. Nitschke, S.G.V.A. Costa, R. Haddad, L.A.G. Goncalves, M.N. Eberlin, J. Contiero,

Biotechnol. Prog. 21 (2005) 1562-1566.

[33] S. Lang, D. Wulibrandt, Rhamnose lipids – biosynthesis, microbial production and

application potential, Appl. Microbiol. Biotechnol. 51 (1999) 22-32.

[34] C. Syldatk, S. Lang, F. Wagner, V. Wray, L. Witte, Chemical and physical

characterization of four interfacial-active rhamnolipids from Pseudomonas spec. DSM

2874 grown on n-alkanes, Z. Naturforsch, 40 (1985) 51-60.

[35] A.W. Adamson, A.P. Gast, Physical Chemistry of Surfaces, 6th ed., Wiley-Interscience,

New York, 1997.

[36] D.K. Chattoraj, K.S. Birdi, Adsorption and Gibbs Surface Excess, Plenum Press, New

York, 1984, p.83.

[37] K. Szymczyk, A. Zdziennicka, J. Krawczyk, B. Jańczuk, Behaviour of

cetyltrimethylammonium bromide, Triton X-100 and Triton X-114 in mixed monolayer

at the water-air interface, J. Chemical Thermodynamics, 69 (2014) 85-92.

[38] L. Pauling, "The nature of the Chemical Bond" Cornell Univ. Press, Ithaca, Ny, 1945

[39] C.J. van Oss, P.M. Constanzo, Adhesion of anionic surfactants to polymer surfaces and

low-energy materials, J. Adhesion Sci. Technol., 4 (1992) 477-487.

[40] B. Jańczuk, J.A. Méndez-Sierra, M.L. Gonzaléz-Martín, J.M. Bruque, W. Wójcik,

Properties of decylammonium chloride and cesium perfluorooctanoate at interfaces and

standard free energy of their adsorption, J. Colloid Interface Sci., 192 (1997) 408-414.

[41] B. Jańczuk, W. Wójcik, A. Zdziennicka, Determiantion of the components of the surface

tension of some liquids from interfacial liquid-liquid tension measurements, J. Colloid

Interface Sci., 157 (1993) 384-393.

[42] F.M. Fowkes, Attractive forces at interfaces. Ind. Eng Chem 1964;56(12):40-56.

[43] Y. Zhang, R.M. Miller, Enhanced octadecane dispersion and biodegradation by a

Pseudomonas rhamnolipid surfactant (biosurfactant), Appl. Environ. Microbiol., 58

(1992) 3276-3282.

[44] S.G.V.A.O. Costa, M. Nitschke, F. Lépine, E. Déziel, J. Contiero, Structure, properties

and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from

cassava wastewater, Process Biochem. 45 (2010) 1511-1516.

[45] K.M. Kale, R. Zana, Effect of the nature of the counterion on the volume change upon

micellization of ionic detergents in aqueous solutions , J. Colloid Interface Sci. 61 (1977)

312-322.

Page 20 of 27

Accep

ted

Man

uscr

ipt

[46] L. Benjamin, Partial molal volume changes during micellization and solution of

nonionic surfactants and perfluorocarboxylates using a magnetic density balance, J. Phys.

Chem.70 (1966) 3790-3797.

[47] J.N. Philips, Energetics of micelle formation, Trans Faraday Soc. 51(1955) 561-569.

[48] S. Yiv, R. Zana, Chemical relaxation and equilibrium studies of association in aqueous-

solutions of bolaform detergents .2. Hexadecane-1,16-bis(trimethylammonium bromide)

and dodecane-1,12-bis(tributylammonium bromide), J. Colloid interface sci., 77 (1980)

449-455.

[49] K. Szymczyk, A. Zdziennicka, J. Krawczyk, B. Jańczuk, Activity and thermodynamic

parameters of some surfactants adsorption at the water- air interface. Fluid Phase

equilibria, 318 (2012) 25-33.

[50] J. H. de Boer, The Dynamic Character of Adsorption, Oxford University, Oxford, 1953.

[51] J.M. Rosen, S. Aronson, Standard free energies of adsorption of surfactants at the

aqueous solution/air interface from surface tension data in the vicinity of the critical

micelle concentration, Colloids Surf. A, 3 (1981) 201-208.

[52] C. Gamboa, A.F. Olea, Association of cationic surfactants to humic acid: effect on the

surface activity, Colloids Surf. A, 278 (2006) 241-245.

[53] A. Zdziennicka, B. Jańczuk, Behaviour of anionic surfactansts and short chain alcohols

mixtures in the monolayer at the water-air interface, J. Surfact. Detergent. 14 (2011) 257-

267.

[54] T. Gu, B-Y. Zhu, The s-type isotherm equation for adsorption of nonionic surfactants at

the silica gel–water interface, Colloids and Surfaces, 44 (1990) 81-87.

[55] T. Gu, B-Y. Zhu, H. Rupprecht, Surfactant adsorption and surface micellization, Prog.

Colloid Polym. Sci., 88 (1992) 74-85.

[56] B-Y. Zhu, T. Gu, T. Reverse Hemimicelle Formation of 1-Decanol from Heptane at the

Solution/Graphite Interface; Colloid Surf. 46 (1990) 339-345.

[57] T.F. Tadros, Surfactants in Agrochemicals, Marcel Dekker, New York, 1994.

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Figure legends

Fig. 1. A plot of the surface tension ( LV ) of the aqueous solutions of rhamnolipid vs. the

logarithm of concentration (C). Curves 1 and 2 correspond to the molar concentration

of representative monorhamnolipid and dirhamnolipid, respectively.

Fig. 2. A plot of the Gibbs surface excess concentration ( ) of rhamnolipid vs. the logarithm

of concentration (C). Curves 1 and 2 correspond to the molar concentration of

representative monorhamnolipid and dirhamnolipid, respectively.

Fig. 3. A plot of the density of the aqueous solutions of rhamnolipid vs. the concentration

(C). Curves 1 and 2 correspond to the molar concentration of representative

monorhamnolipid and dirhamnolipid, respectively.

Fig. 4. A plot of the viscosity of the aqueous solutions of rhamnolipid) vs. the

concentration (C). Curves 1 and 2 correspond to the molar concentration of

representative monorhamnolipid and dirhamnolipid, respectively.

Fig.5. A plot of the conductivity (κ) of the aqueous solutions of rhamnolipid vs. the

concentration (C). Curves 1 and 2 correspond to the molar concentration of

representative monorhamnolipid and dirhamnolipid, respectively.

Fig.6. A plot of the apparent ( V ) (curves 1,1' and 1'') and the partial molar volumes ( MV )

(curves 2 and 2') of rhamnolipid vs. the logarithm concentration (C). Curves 1, 1'' and

2 correspond to the molar concentration of representative monorhamnolipid and 1' and

2' dirhamnolipid, respectively.

Fig. 7. A plot of the standard Gibbs free energy of rhamnolipid adsorption ( oadsG ) calculated

from Eq.(6) vs. the logarithm of concentration (C). Curves 1 and 2 correspond to the

molar concentration of representative monorhamnolipid and dirhamnolipid,

respectively.

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THERMODYNAMIC PROPERTIES OF RHAMNOLIPID MICELLIZATION AND ADSORPTION

DIANA MAŃKO, ANNA ZDZIENNICKA*, AND BRONISŁAW JAŃCZUK

Department of Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20-031 Lublin, Poland

The representative monorhamnolipid at the water-air interface.

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Highlights

The Gibbs surface excess concentration and CMC of rhamnolipid were determined.

The limiting surface area of rhamnolipid molecule at interface was established.

The oadsG was determined by using different methods.

The correlation between oadsG and o

micG was shown.

The oadsG of rhamnolipid was predicted from tail-air and tail-water tensions.