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Colloids and Surfaces B: Biointerfaces 67 (2008) 192–198

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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esicle formation in hydrocarbons assisted with microbialydrolases and biosurfactants

. Gnanamania,∗, V. Kavithaa, G. Sekaranb, G. Suseela Rajakumara

Microbiology Division, Central Leather Research Institute (Council of Scientific and Industrial Research, New Delhi), Adyar, Chennai, Tamil Nadu, IndiaDepartment of Environmental Technology, Central Leather Research Institute (Council of Scientific and Industrial Research, New Delhi), Adyar, Chennai, Tamil Nadu, India

r t i c l e i n f o

rticle history:eceived 23 June 2008eceived in revised form 7 August 2008ccepted 16 August 2008

a b s t r a c t

The present study demonstrates the role of microbial hydrolases in the transformation of hydrocarbons(soybean, sunflower, groundnut and gingelly oil, etc.) to vesicles. The combined effect of lipolytic enzymegeneration and biosurfactants production during microbial growth at optimized media and environmentalconditions mediates this transformation. Among the microbial species, Candida albicans exhibit complete

vailable online 26 August 2008

eywords:esiclesydrocarbonsiscoelasticity

transformation compared to Pseudomonads and Bacillus sps. Within hydrocarbons, only soybean and sun-flower oils transformed to solid mass and no change with the remaining oils. Characterization of thevesicles revealed an increase in total weight by 160–180% compared to the original weight of hydrocar-bon taken for the study and more than 73% increases in viscosity. Acid value and saponification valuealso showed an increase, respectively, by 78 and 84%. The bound water content estimated was 26%. Light

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. Introduction

The present advancement in nanomaterials invites new prod-cts for wide applications in various fields of studies and in specific,anomaterials for biology, electronics, transport and informationechnology are also in demand because of their electrical, opticalnd magnetic properties. However, development of these nanoma-erials depends on number of factors. Nanomaterials from organicr inorganic sources require surfactants of biological or syntheticrigin [1]. Further, most of the nanoparticles are for the humanse, the biocompatibility parameter is a prerequisite [2]. Nano-aterials in the form of vesicles, in general, are self-assembled

tructures with bilayers of amphiphilic molecules able to accom-odate micro and macromolecules [3]. Fatty acid vesicles (FAV)

re an excellent choice due to its biocompatibility properties. Com-ared to liposome, FAVs are distinctive, however, their formationnd the stability varied with the concentration of ingredients3,4] and the environmental factors like, pH and temperature5–7]. According to Morigaki and Walde [7], the formation of vesi-

les involves formation of disk-like micelles followed by its slowrowth and finally converted to vesicles. Further, compared toicelles, thermal stability of vesicle is still vague. Though, stud-

es on the dynamic formation of these fatty acid vesicles provide

∗ Corresponding author.E-mail address: agmani 2000@yahoo.com (A. Gnanamani).

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927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2008.08.016

esence of unilamellar and bi-lamellar structures.© 2008 Elsevier B.V. All rights reserved.

he unknown truth in the ‘origin of life’, however, still continuousesearch is in need to have solution for the unanswered ques-ions.

Further, gellation or vesicle formation is an attractive phe-omenon in which, the self-assembly of molecules occurs atefinite environmental conditions. Gellation of oils resolves num-er of problems associated with oil spills and their relative toxicffects. Bhattacharya et al. [8] reported fatty acid amides of l-lanine can able to gellate the oil in the oil–water mixture. Further,he cavities inside the gel structures can able to entangle the

olecules of either organic or inorganic and thus finds immensepplication [1]. With respect to gel formation and liphopilicity,nly the long chain carbon compounds able to transform into vesi-les or solid form [6,9]. Gebicki and Hicks [10,11] established theesicular formation in oleic acid. This discovery indent quantumf research in the field of monolayer, bilayer forming potentialf fatty acids. The length and the degree of unsaturation of theydrocarbon chains also play an important role in determininghe layering property and the van der Waal’s interactions betweenhe hydrocarbon chains and the deprotonated and protonated acid

olecules, induces bilayer system. In addition, other than fattycids, polypropanol derivatives also form vesicles, which can able

o capture macromolecules like DNA [12]. Compared to dendrimers,esicles carry higher load because of their capacious interiors.oreover, the formation of reverse micelle in the mixture of organic

olvent and surfactants is quite interesting, in which, due to theon-polar nature of the solvents/oils, the hydrophilic head of the

A. Gnanamani et al. / Colloids and Surfaces B: Biointerfaces 67 (2008) 192–198 193

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ig. 1. Visualization of vesicle formation in soybean oil. (a) Control sample containpon gellation of soybean oil.

urfactants are concealed in the core of the micellar particles andhe hydrophobic tails are outward and contact the solvent/oil.

The present study, exemplify, in-situ vesicle formation in hydro-arbons and in detail, it demonstrates the role of in-situ generationf lipolytic enzymes and biosurfactants during the growth oficroorganisms in the formation of vesicles of hydrocarbons when

rovided along with the growth medium. Further, the article revealshe physico-chemical and microscopical characteristics of vesiclesormed in hydrocarbon, soybean oil.

. Experimental

.1. Organisms

Candida albicans (MTTC 3017), Pseudomonad sp. (MTTC 1942)nd Bacillus sp. (MTTC 2731) obtained from IMTECH, Chandigarh,ndia, was sub-cultured in the suitable medium and stored as glyc-rol stock at 4 ◦C.

.2. Media composition and development of vesicles

The growth medium used in the present study contains (periter distilled water) 100 g Hydrocarbons (oil); 1 g NH4NO3; 2.55 gaH2PO4; 0.5 g MgSO4·7H2O; 0.1 g CaCl2·H2O; 0.02 g MnSO4·H2O;g Peptone; 0.5 g Glucose; pH 7.8. All the ingredients were mixednd autoclaved at 121 ◦C for 15 min. Hydrocarbon at the concen-ration of 2, 4, 6, 8 and 10% was added to sterile medium andncubated at 38 ◦C at 200-rpm for 168–240 h. Growth of the organ-sm (OD measured at 600 nm in UV-Visible spectrophotometer) wasbserved up to 4 days and lipase activity [13] phospholipid [14] andlycerol [15] content of the broth were estimated at 24 h intervalsor the period of 240 h.

Observations on formation of micelle, followed by aggregationf micelles and the transformation of oil to vesicles were madehroughout the experimental period. The experiments were contin-ed till the complete transformation of oil to vesicles occurs. On theay of completion, the mass obtained was removed by decanting

2

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rowth medium, soybean oil and inoculated microorganism; (b) vesicle developed

he aqueous medium and stored at 4 ◦C and used for experimentalnalysis.

.3. Characterization of vesicles

.3.1. Physical characterization of vesiclesNature, odour, solubility, viscosity, pH, effect of temperature and

ound water content are the parameters analyzed for the vesi-le formed. Both nature and odour of the vesicle was assessed byhysical observations. Solubility was checked with both polar andon-polar solvents. Viscosity of the vesicles was measured as perhe standard procedure using Oswald viscometer [16]. pH of theesicle was measured using Elico pH meter. Bound water contentas measured using Karl Fischer titration method.

.4. Chemical characterization of vesicle

UV-Visible spectral analysis of the vesicles was made usingary 100 UV-Visible spectrophotometer with the spectral range of00–900 nm. The samples were dissolved in ethyl acetate (w/v) andhe clear solution was subjected to scanning. With respect to FT-R analysis, the vesicles were directly mixed with the KBr and theesultant pellet was analyzed using Spectrum one Perkin–Elmero., USA model. CHN analysis of the sample was carried out usingHN analyzer. Molecular weight estimations of the polymers wereerformed on a JASCO GPC chromatograph model MX-2080-31tted with PL gel 5Ìm Mixed-C columns, 300 × 7.5 mm, in tetrahy-rofuran (THF) with a flow rate at 1 ml/min at 30 ◦C using aefractive index (RI) detector. Thermogravimetric analysis (TGA)as carried out using TGA Q50 V20.6 Build 31 and differential scan-ing calorimetery (DSC) was made with DSC Q200 V23.10 Build9.

.5. Detection of lipids

The nature of lipid content in the vesicle was assessed byhin layer chromatography (TLC) using chloroform, methanol and

194 A. Gnanamani et al. / Colloids and Surfaces B: Biointerfaces 67 (2008) 192–198

regatio

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TitwpowtaavdtvsSstrm4raaan increase in mass of 4197 for the vesicle compared to 3162 for

Fig. 2. Microscopical analysis of vesicles obtained from soybean oil. (a) Agg

ater (65:25:4) as mobile phase. Detection of phospholipids wasade with ammonium molybdate [17], and diphenylamine was

mployed for the detection of glycolipids.

. Results

Liquid culturing using mineral medium exhibited vigorousrowth of C. albicans at 38 ◦C under shaking conditions and growthas not affected by the incorporation of the hydrocarbons (soybeanil, sunflower oil, gingelly oil, groundnut oil). The stationary phasef the growth was observed 34 h. Analysis on Lipase activity exhib-ted an increase from (3.5 U/ml) 12th hour onwards and stabilized2.5–3.0 U/ml) after 48 h declined (1.2 U/ml) when the incubationeriod increases to 72 h. With respect to biosurfactant activity,esults obtained from drops collapse test revealed existence of bio-urfactant activity encountered from 22nd hour and increases upo 6th day and decreases afterwards. A thin layer chromatographytudy further confirms the presence of both phospholipids and gly-olipids (results not shown). pH of the growth medium decreasesinitial pH 7.8) slowly and on the final day of the experiment itas estimated as 3.3. Glycerol content of the cell free extract ana-

yzed on different days of incubation revealed an initial increasen glycerol content up to 28 h and marginal decrease up to 96 hnd on the day of completion approximately 650 mg/ml of glycerolas estimated. The micelle formation and development of vesicle

nd transformation of oil to vesicles is illustrated in Fig. 1(a and

) after day 7. Further optical analysis of the vesicles (Fig. 2(a–d))xhibit different nature of vesicles and the micelles formed. Inter-stingly, the unilamellar as well as the bi-lamellar structures werelso observed.

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n of vesicles; (b) multilamellar structure; (c) and (d) bi-lamellar structure.

With regard to physico-chemical characterization of the vesicle,able 1 illustrates the physical features, viz., colour, odour, viscos-ty, solubility, heating, freezing, pH, ash and bound water content ofhe vesicles obtained. Mass of the vesicle increases proportionatelyith the volume of the hydrocarbons provided and the maximumercentage yield of 160–180% was obtained with 10% of soybeanil. With respect to viscosity, more than 73% increase in viscosityas realized. When tested for solubility, complete dissolution of

he vesicle was observed with ethyl acetate. When these vesiclesre subjected to higher temperatures (100 ◦C) liquefaction occursnd when the temperature decreases again solidification starts pro-ides the gel like structure with high viscosity. pH of the vesicle wasetermined as 3.5 and about 26% of bound water was estimatedhrough Karl Fischer titration. Acid and saponification value of theesicle varied at significant level compared to oil. The acid value ofoybean oil was estimated as 1.1, whereas, it was 87.5 for vesicles.imilarly, the saponification value of 1761 was realized with theolid mass, whereas it was only 280 for soybean oil. With regardo the unsaponifiable value, there was no change and it was in theange of 180–188 for both the samples. CHN analysis of the solidass revealed about 2% increase in nitrogen content and about

0% decrease in carbon content was noticed when compared toaw soybean oil. FT-IR analysis showed the presence of amide link-ge at 3448/cm, C O at 1682 and 1740/cm, C C at 1243/cm, CH3symmetric stretch at 2927/cm. Gel permeation analysis showed

he raw soybean oil. Figs. 3 and 4(a and b) illustrate the thermalnalysis of the vesicle and soybean oil. Soybean oil showed hightability up to 385 ◦C and further increase in temperature slowlyeduces the mass percentage weight and at 470 ◦C the mass of the

A. Gnanamani et al. / Colloids and Surfaces B: Biointerfaces 67 (2008) 192–198 195

Table 1Characterization of vesicles obtained from hydrocarbon (soybean oil) in the presence of hydrolytic enzyme and biosurfactants of C. albicans

S. No. Characteristics Substrate Sample

1 Colour Light yellow Creamy white2 Odour Odourless Irritating odour3 Appearance Liquid Thick clumpy mass4 Viscosity Viscous [22.79 × 103 cps] Highly viscous [77.20 × 103 cps]5 Freezing under −20 ◦C No change Solidify immediately6 Thawing No change Semisolid state7 Heating Boiling Liquefy

8 SolubilityEthyl acetate Dissolved Completely dissolvedTHF Precipitate Precipitate

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scontain carbon chain more than C12; (ii) ionic concentrations(low/high pH); (iii) surfactants (bio or synthetic); (iv) environ-mental conditions (static/stirring); (v) trace elements; (vi) fattymolecules, etc. The chirality nature of the molecules also play an

Methanol

9 pH10 Weight

ompound reached zero percentage. However, TGA analysis of vesi-le showed a different pattern with immediate weight percentageoss when the temperature reaches 80 ◦C and a further increase inemperature reduce the mass slowly and at 480 ◦C the mass per-entage reaches zero (Fig. 3). The immediate loss in the percentageeight of the vesicle might be due to the presence of bound water

ontent, which starts evaporating when the temperature increases.ith regard to DSC, vesicles showed a clear strong absorbance peak

t 141.36 ◦C (Fig. 4(a)), whereas, soybean oil alone doesn’t exhibitny absorption peak (Fig. 4(b)).

. Discussion

Self-organization is an important property and the early lifeeneration is based on the self-organization behavior of variousolecules. Recently, studies on self-organization of molecules or

tilization of self-organized molecules are in the lime light, since,hese molecules can able to encapsulate the compounds and releasehem through their layer opening system. For example in the field ofrug delivery, identification of suitable matrix is the current prob-

em in the field of medicine. However, exploitation of self-organizedolecules having supramolecular structure, in turn satisfy the

hysicians, since most of the self-organized molecules are highlyiocompatible, in addition, most of them are from lipids.

Recently, Dey and Roy [6] studied the self-organizing behav-or of N-acyl amino acid surfactants (NAAS). NAAS have been toelf-organize in nature and also inorganic solvents to form variousypes of supramolecular structure. These supramolecular struc-

ig. 3. TGA thermogram of soybean oil and vesicle under in vivo condition in theresence of hydrolase enzyme and biosurfactants of C. albicans.

Fc

cipitate Precipitate

–5.0 3.0–4.0/100 ml 17–18 g/100 ml

ures assemble to generate bilayer in nature with shapes such aslanar membrane, tubules, helices, ribbons and rods. Among these,he tubules and helical ribbons are the most interlay as far as tech-ological application of nano structured materials are concerned.heir compatibility with the body system, either topically or orallyr invasive or non-invasive provide high soothing effect and alsoithout any side effect.

Research on developing supramolecular structure by inducingelf-organization behavior of molecules, requires, (i) molecules

ig. 4. (a) and (b) DSC thermogram of soybean oil and vesicle obtained under in vivoondition in the presence of hydrolase enzyme and biosurfactants of C. albicans.

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96 A. Gnanamani et al. / Colloids and Su

mportant role in developing supramolecular structure [6,18] andormation of vesicular structure are mostly depends on the stere-chemistry of the chiral amphiphiles. Further, charge in the headroup and the hydrocarbon tail affects the morphology of the vesi-les. Infact, the hydrophobic interaction between long hydrocarbonhains is essential during the formation of supramolecular aggre-ates, and the interaction between head groups is a possible drivingorce for the formation of helical and or cylindrical aggregates.

Rallison [19] and Stone [20] reported the interface between twoimple fluid is governed by the surface tension, which is isotropichen there is no surfactant, but in the presence of surfactant, the

nterfacial area is minimized and it becomes anisotropic and thehape has been transformed to spherical and increases the micel-ar compactness which results with an increase in viscosity at fiveold level. Further, the external aqueous medium facilitates thencreased level of bound water content in the vesicles [21].

TGA analyses of both raw sample (soy bean oil) and the vesiclesevealed stability of the vesicles reduced with an increase in tem-erature and the percentage of weight loss of about 13, 23 and 36%bserved between 110 and 263 ◦C, respectively, might be due to theoss in the bound water present in the vesicles [22]. However, oilhowed stability up to 362 ◦C. With respect to DSC, heating scanso sub zero temperature provides a sharp peak which indicates thexistence of water molecules bound to the lipid head groups [21].

Further, to have stable vesicle, the hydrophobic tail havingength equal to that of a C16 chain is suitable [6]. In the presenttudy, though four different oils have been chosen, however, onlyoybean oil and sunflower oil provided expected results and thisrticle exemplify only the results obtained with soybean oil. Theatty acid composition of soybean oil showed both unsaturatednd saturated acids in the ratio of 5:7, viz., C16:0 = 7%; C18:0 = 4%;18:1 = 24%; C18:2 = 54% and C18:3 = 7%. With respect to three dif-

erent organisms chosen, only C. albicans exhibit the gellationroperty and which could be due to lipase production and biosur-

actant activity during the growth. Haba et al. [23] and Kim et al. [24]eported the existence of lipase activity and biosurfactant activity in. albians. Results obtained with Pseudomonad and Bacillus sps. didot exhibit appreciable levels of lipase and biosurfactant activity,hich is reflected in the transformation of hydrocarbons, where,o conversion was encountered. Fernandez and Jose [25] reported

ipase activity of Pseudomonad species depends on medium com-osition and type of the species exploited. Similarly, biosurfactantctivity was not at appreciable level in Bacillus species [26]. Thencorporation of soybean oil in the growth medium of C. albicanshowed three transition phases, viz., CMC, micellar growth, andicellar entanglement results with gellation. The amide linkage

ear the surfactant head group [8] as evidenced through FT-IR spec-ral analysis must reasons the micellar aggregation.

Further, the vesicles formation was confirmed by conductiv-ty measurements KCl (results not shown). With respect to pH,s a result of protonation of the –COO− group, the ionic repulsionre reduced which promote the intermolecular hydrogen bondingetween the –COOH and –COO− or between –COOH and –CONHroups. The intermolecular hydrogen bonding as a result of proto-ation of the –COO− group increases the curvature of the bilayerggregates to produce closed vesicle [27]. Moreover, the pKa valueas found to be higher than pKa value of test sample. Similar

ncrease of pKa values upon aggregation have been also suggestedor other fatty acids by Walde et al. [28] and Bachmann et al. [29].

With respect to intramolecular hydrogen bonding, there may be

ntermolecular hydrogen bonding between the secondary amideroups at the end of the hydrocarbon tails and thus leads to theormation of bilayer structures. The two layer arrays of intermolec-lar hydrogen bonding interactions through the amide bonds of theeighboring surfactant molecules results in the formation of a par-

tmthr

B: Biointerfaces 67 (2008) 192–198

llel arrangement of the corresponding hydrophobic tails such thathe surfactant molecules can self-organize into bilayer structure inater. The secondary amide hydrogen bond chains act as a stability

actor to over hydration energies.Microscopical evaluation of the aggregates reveals existence of

pherical as well as cylindrical vesicles has an internal diameterf 1–2 �m. The population of small size vesicle is large comparedo the large size vesicles. The vesicles appear to be multilamellarype and it was clear that there is an entrapped aqueous phasend the hydrophobic lamella of the vesicle. The thickness of theamella is high and suggests that the vesicles are multilamellar intructure. The conclusion on lamellar size was made based on theeport of Lasic [5]. Kumar and Katare [30] in their review discussedhe lecithin organogels as a potential phospholipids structuredystem for tropical drug delivery. Lecithin organogels are jelly-ike phases, consists of three-dimensional networks of entangledeverse micelles, within immobilizes the continuous or macro-copic external organic phase thus turns a liquid into a gel. Thepherical reverse micellar state of lipid aggregate turns into elon-ated tubular micelles with the addition of water and subsequentlyntangles and immobilizes the liquid phase, thus providing a gelorm or the jelly-like state of the initial non-viscous solution toiscous solution.

Gelling formation depends on the nature of fatty acids. Onlynsaturated fatty acids can able to form a gel and the saturated orydrogenated fatty acids never form a gel. With reference to theffect of unsaturation in phospholipids on gelling, no such system-tic investigative studies have been conducted so far. However it haseen established that unsaturation in phospholipids molecules is aesired property. The unsaturation contributes to the value factor ofhe non-polar region of phospholipids molecules and may alter thealue of critical packing parameter (CPP) favouring the formationf reverse micelle structures. In contrast to the saturated hydro-enated phospholipids, unsaturation in phospholipid moleculesould result better hydration of the polar head group, thereby

ncreasing the area per lipid polar head group. Consequently, largerea and relatively smaller volume would favourably alter the spon-aneous curvature of lipid monomers for the formation of micellesnd subsequently their self-assembly to form the micellar network.

Comparing the phosphatidyes of soybean oil, sunflower oil, gin-elly oil and ground nut oil, the percentage of �6 (linoleic acid)s 54% in soybean oil, 68% in sunflower oil, 45% in gingelly oil and0% in groundnut oil. While �3 (linolenic acid) was present only

n soybean oil (7%) and sunflower oil (1%). This implies that theormation of gel like structure may depends on the phospholipidsontent of the oil chosen and the presence of biosurfactant inducehe vesicle formation, which increases the viscoelastic propertiesf the vesicles.

As mentioned, three components are actually in need of toonvert free flowing oil to highly viscous semi solid matter. Thechematic representation of the involvement of these componentsas been illustrated as Scheme 1. The first component may be theurfactant. In the present study, the organism chosen was able toecrete surfactants, i.e. biosurfactants. Active biosurfactant pres-nce has been evidenced through a drop collapse test. Further,LC analysis also provides the presence of glycolipids surfac-ants. Sequentially, hydrolysis of soybean oil by lipolytic enzymeseleased by C. albicans provides fatty acids of both phospholipidsnd glycolipids and the fatty acids released by hydrolysis in turnelf-aggregate in the presence of biosurfactant and with the help of

he third component, the aqueous medium, intensifies the reverse

icelle formation. As reported by Kumar and Katare [30], evenhe glycerol can able to induce the self-aggregation because of itsigh surface tension. In the present study, the quantity of glyceroleleased is directly proportional to the incubation period, and the

A. Gnanamani et al. / Colloids and Surfaces B: Biointerfaces 67 (2008) 192–198 197

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Scheme 1. Schematic representation of in-situ transformatio

aximum release was observed till 7 days of incubation. Further,hese results exemplifies, the resultant vesicles are of fatty acidsesicles, generated during hydrolysis of soybean oil.

. Conclusions

Self-assembly in hydrocarbons finds immense applications inarious fields of research including drug delivery and enzymeatalysis. In the present study self-assembly and vesicle forma-ion in soybean oil has been mediated by the in-situ generation ofydrolytic enzymes and biosurfactants production by C. albicans.n terms of molecular shape approach [31], aggregate trans-ormations and the reverse micelle formation followed by thection of hydrolytic enzymes of the organism on hydrocarbons,urther the micelle begin to overlap, entangle themselves. This

akes a crossover to a system characterized by increased viscositythree fold increases) and viscoelastic properties. The vesicles thusbtained contain a considerable amount of (i.e. water) entrappedn the spaces between the entangled reverse micelle. The hydrogenonding network built up by molecules of polar additive and phos-

hate groups is also accompanied by stiffness of the phospholipidsolecule in the region of phosphate group and glycerol residuehich stabilizes the micellar aggregates that even the impure orixed components can able to form vesicles in the presence of

n-situ secretion of surfactants and other hydrolytic enzymes.

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oybean oil to vesicles during the growth of microorganisms.

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98 A. Gnanamani et al. / Colloids and Su

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