Glycerol as substrate for the production of biosurfactant by Pseudomonas aeruginosa UCP0992

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Colloids and Surfaces B: Biointerfaces 79 (2010) 174–183

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Glycerol as substrate for the production of biosurfactant by Pseudomonasaeruginosa UCP0992

S.N.R.L. Silvaa,b, C.B.B. Fariasb,c, R.D. Rufinob,d, J.M. Lunab,d, L.A. Sarubbob,e,∗

a Catholic University of Pernambuco, Rua do Príncipe, n. 526, Boa Vista, CEP 50050-900, Recife-Pernambuco, Brazilb Centre of Research in Environmental Sciences, Catholic University of Pernambuco, Rua Nunes Machado, n. 42, Bl J, Térreo, Boa Vista, CEP 50050-590, Recife-Pernambuco, Brazilc State University of Campinas, Campinas, SP, Brazild Centre of Biological Sciences, Federal University of Pernambuco, Av. Prof. Moraes Rego, Cidade Universitária, CEP 50670-901, Recife-Pernambuco, Brazile Centre of Science and Technology, Catholic University of Pernambuco, Rua do Príncipe, n. 526, Boa Vista, CEP 50050-900, Recife-Pernambuco, Brazil

a r t i c l e i n f o

Article history:Received 31 December 2009Received in revised form 19 February 2010Accepted 30 March 2010Available online 8 April 2010

Keywords:BiosurfactantEmulsificationGlycerolPseudomonas aeruginosaSurface tensionToxicity

a b s t r a c t

In this work the influence of the carbon source concentration, of the type and concentration of the nitrogensource and of the cultivation conditions (temperature, aeration and agitation speed) in mineral mediumformulated with glycerol was studied for biosurfactant production by Pseudomonas aeruginosa UCP0992.The kinetics of microorganism growth and biosurfactant production have been described for the mediumsupplemented with 3% glycerol and 0.6% NaNO3, at 28 ◦C during 120 h under 200 rpm, showing a growth-associated production. The isolated biosurfactant corresponded to a concentration of 8.0 g/l after 96 h. Themedium surface tension was reduced to 27.4 mN/m and the emulsification index of hexadecane reached75–80% after 72 h. A CMC of 700 mg/l and an interfacial tension against hexadecane of 2 mN/m wereobtained. The biosurfactant showed stability when submitted at 90 ◦C during 2 h, and thermal (4–120 ◦C)and pH (4–12) stability regarding the surface tension reduction and the emulsification capacity of veg-etable oils and hydrocarbons, and tolerance under high salt concentrations (2–10%). The biosurfactantwas characterized as a group of rhamnolipids with anionic nature. The crude biosurfactant did not showtoxicity against the microcrustacean Artemia salina and the cabbage (Brassica oleracea), while the isolatedbiosurfactant showed toxicity against the microcrustacean at the CMC. The application of the biosur-factant in diesel recovery from sand was demonstrated by removal percentiles above 85%. The resultsobtained in this work are noteworthy for possible biosurfactant production from glycerol with potentialof application in the environment.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Oil spill accidents result in significant contamination of theocean and shoreline environments [1]. It is estimated that0.08–0.4% of the total worldwide production of petroleum even-tually reaches the oceans. In Brazil, many accidents involvingpetroleum hydrocarbons, such as gasoline and fuel oil has causedserious environmental problems. In 1998, 1200 m3 of fuel oil werereleased because of pipeline corrosion in Cubatão city, São Paulo.The corrosion of pipelines was also responsible for 1300 m3 of fueloil discharged in Baía de Guanabara, Rio de Janeiro, which had beenstrongly contaminated before by other oil spills [2].

∗ Corresponding author at: Centre of Research in Environmental Sciences, CatholicUniversity of Pernambuco, Rua Nunes Machado, n. 42, Bl J, Térreo, Boa Vista, CEP50050-590, Recife-Pernambuco, Brazil. Tel.: +55 81 21194048;fax: +55 81 21194043.

E-mail addresses: [email protected], [email protected] (L.A. Sarubbo).

Such incidents have intensified attempts to develop proceduresand technologies for combating oil pollution in the environment.Soil that is accidentally contaminated with petroleum hydro-carbons can be remediated by physical, chemical, or biologicalmethods. However, new trends in soil and water restoration avoidintroducing synthetic chemicals. Among the remediation tech-niques available for contaminated sites, bioremediation is regardedas environmentally friendly because it preserves the soil structure,requires little energy input, and involves the complete destructionor immobilization of the contaminants, although the efficiency ofbiodegradation of oil pollutants is often limited by their poor watersolubility [2,3]. One of the approaches to enhance biodegradationof oils is to use biosurfactants, which could increase solubility ofoils in water to enhance the bioavailability of the hydrophobicsubstrates, leading to higher oil degradation rates. Biosurfactantsare natural surface-active products from a variety of microorgan-isms and can be categorized as glycolipids, lipopetides, fatty acids,polysaccharide-protein complexes, peptides, phospholipids, andneutral lipids. Being amphiphilic molecules, they generally absorb

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S.N.R.L. Silva et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 174–183 175

of at interfaces and reduce the surface tension between two liq-uid phases, enabling the uptake of hydrophobic substrates by themicroorganism. In addition to environmental applications, biosur-factants also find their applications in pharmaceutical, cosmetic,fine chemical, and food industries [3]. Biosurfactants also offer sev-eral advantages over chemical surfactants as biodegradability andlow toxicity, and can be produced from renewable substrates [2,4].

Many biosurfactants have been produced and applied to hydro-carbons degradation, specially the rhamnolipids produced by thegram-negative bacterium Pseudomonas aeruginosa [4]. Rhamno-lipids are a group of biosurfactants of glycolipid nature, composedof a hydrophilic head formed by one or two rhamnose molecules,known respectively as monorhamnolipid and dirhamnolipid, anda hydrophobic tail which contains one or two fatty acids [5]. Thetypes of rhamnolipids produced will depend on the strain, thecarbon source used and the culture conditions. Rhamnolipids con-stitute one of the most interesting classes of biosurfactants becauseof their advantageous characteristics. With respect to their pro-duction, they show high concentrations as compared to otherbiosurfactants, and several renewable materials, like used oils orwastes from the food industry, can be used as carbon sources[6]. Rhamnolipids are able to reduce the water/air surface tensionfrom 72 mN/m to values close to 30 mN/m, as well as the water/oilinterface tension from 43 mN/m to values around 1 mN/m [7]. Thecritical micelle concentration of pure rhamnolipids and their mix-tures depends to a great extent on the chemical composition of thevarious species, and varies from 50 to 200 mg/l [1].

Despite the advantages and potential applicability of these bio-logical compounds, the success of biosurfactants depends on theeconomy of process and the use of low cost raw materials whichaccount for the 10–30% of the overall costs [6,7].

The industrial conversion of renewable resources into usefulcompounds has been receiving much attention from the environ-mental point of view. The currently available resources are oils,sugar, cellulose and lignocellulose from plants, and a variety oforganic wastes from agriculture and related industries [8]. Amongthese bioresources, the utilization of waste glycerol is becomingvery important, because the amount of waste has been increasingyear by year through the increasing production of biodiesel andother oleochemicals [9]. On the other hand, glycerol is success-fully used as the water-soluble carbon source for different microbialproductions [10,11].

Thus, the aim of this work was to evaluate the production of abiosurfactant from P. aeruginosa UCP0992 cultivated in glycerol asthe carbon source, and to study the surface and emulsifier activity ofthis biosurfactant mixture in order to verify its potential industrialapplication in the environment.

2. Materials and methods

2.1. Materials

All the reagents were of the highest purity available.

2.2. Bacterial strain and preparation of seed culture

A strain of P. aeruginosa UCP0992 was provided from the cul-ture collection of the Centre of Research in Environmental Sciences,Catholic University of Pernambuco, Brazil, registered with theWorld Federation of Culture Collections (WFCC). The cultures weremaintained in nutrient agar slants at 4 ◦C. For pre-culture, the strainfrom a 24-h culture on nutrient agar was transferred into 50 mlnutrient broth to prepare the seed culture. The cultivation conditionfor the seed culture was 28 ◦C, 150 rpm, and 10–14 h of incubationtime.

2.3. Fermentation media and conditions

For liquid fermentation, a 2% cell suspension of 0.7 OD(optical density) at 600 nm, corresponding to an inoculum of107 CFU/ml, was inoculated into 500-ml flask containing 100 mlmineral medium consisting of 0.1% KH2PO4, 0.1% K2HPO4, 0.02%MgSO4·7H2O, 0.002% CaCl2·2H2O, 0.005% FeCl3·6H2O and 0.2%NaNO3, according to Patel and Desai [12], amended with glycerol asa sole carbon substrate. The pH of the medium was initially adjustedto 6.8 by 1.0 M HCl. The culture temperature and agitation rate was28 ◦C and 150 rpm, respectively, otherwise indicated. At the end offermentation (120 h), samples were taken from the liquid cultureto determine the surface tension and surfactant concentration.

For the experiments exploiting the effect of the carbon sourceconcentration on biosurfactant production, the concentration ofglycerol was adjusted to 2, 3, 5 and 7%. Using the most appropriateglycerol concentration, the following nitrogen sources were evalu-ated at 0.2%: sodium nitrate, ammonium nitrate, urea, ammoniumsulphate, peptone, yeast extract and corn steep liquor, the latestobtained from the factory Corn Products do Brasil, Cabo de SantoAgostinho-PE, Brazil.

Using the selected glycerol concentration and a variable con-centration of the selected nitrogen source (0.05, 0.1, 0.2, 0.4 and0.6%), different C/N ratios were tested: 150, 75, 46, 37.5 and 18,respectively.

After the establishment of the medium, the effects of differ-ent fermentation conditions on biosurfactant production wereevaluated. The factors studied included: effect of incubation tem-perature (28 and 37 ◦C), aeration (studied by varying the volume ofheadspace in 500 ml conical flasks created by variation of the vol-ume of the medium in the flask which will be reflected in variationof the level of aeration) and agitation speeds (150 and 200 rpm).

After selection of the best medium composition and conditionsof cultivation, kinetics of microorganism growth and biosurfac-tant production were monitored during 120 h. At regular intervals,different process parameters such as growth, pH, surface tension,biosurfactant concentration, glycerol consumption and emulsifica-tion activity against hexadecane were evaluated.

2.4. Biomass determination

For biomass determination, 10 ml samples were centrifuged at5000 × g during 30 min and the cell pellet dried in an oven at 105 ◦Cfor 24 h.

2.5. Glycerol concentration

The glycerol concentration was determined by the enzymatic-colorimetric assay using a triglycerides kit (Bioclin-Quibasa, Brazil).

2.6. Rhamnolipid quantification

Rhamnolipids expressed as rhamnose (g/l) were measured inthe cell-free culture medium using the phenol-sulphuric method[13]. One millilitre of the cell-free culture broth was mixed with0.5 ml of 80% phenol and 2.5 ml of concentrate sulphuric acid. Afterincubation during 10 min at room temperature, the absorbancewas measured at 490 nm and the rhamnolipid concentration wascalculated using a standard curve prepared using different concen-trations of rhamnose [1].

2.7. Emulsifying activity with different hydrophobic compounds

Emulsification index (EI) was measured using the methoddescribed by Cooper and Goldenberg [14] whereby 2 ml of a liquidhydrophobic compound (n-hexadecane, benzene, diesel, motor oil,


176 S.N.R.L. Silva et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 174–183

petroleum, cotton seed corn rice oil and soybean vegetable oils) wasadded to 2 ml of the culture broth free of cells in a graduated screw-cap test tube, and vortexed at high speed for 2 min. The emulsionstability was determined after 24 h and the emulsification indexwas calculated by dividing the measured height of the emulsionlayer by the mixture’s total height and multiplying by 100.

2.8. Surface tension and CMC determination

Surface tension and interfacial tension (against hexadecane)changes were carried out on the cell-free broth obtained by cen-trifuging the cultures at 5000 × g for 20 min by the ring methodusing a Sigma 70 Tensiometer (KSV Instruments Ltd., Finland) atroom temperature. Tensiometers determine the surface tensionwith the help of an optimally wettable ring suspended from aprecision balance. In the Ring method the liquid is raised untilcontact with the surface is registered. The sample is then low-ered again so that the liquid film produced beneath the liquidis stretched. As the film is stretched a maximum force is expe-rienced, the force is measured and used to calculate the surfacetension. The instrument was calibrated against Mill-Q-4 ultrapuredistilled water (Millipore, Ilhinois, USA). Prior to use the platinumplate and all the glassware were sequentially washed with chromicacid, deionised water, acetone and finally flamed with a Bunsenburner.

The critical micelle concentration (CMC) was determined bymeasuring the surface tensions of dilutions of isolated biosurfac-tant in distilled water up to a constant value of surface tension.Stabilization was allowed to occur until standard deviation of 10successive measurements was less than 0.4 mN/m. Each result wasthe average of 10 determinations after stabilization. The value ofCMC was obtained from the plot of surface tension against sur-factant concentration. The CMC value was determined to be g/l ofbiosurfactant.

2.9. Effect of environmental factors on biosurfactant activity

The effect of addition of different concentrations of NaCl onthe activity of the biosurfactant was investigated in the cell-freebroth. A specific concentration of NaCl (2–10%, w/v) was addedand surface tension and emulsification activity were determined aspreviously stated. The cell-free broth was also maintained at a con-stant temperature (4, 28, 70, 100 and 120 ◦C) for 60 min and usedfor surface tension and emulsification measurements. To evaluatethe effect of time of heating on the biosurfactant activity, the cul-ture broth was maintained at 90 ◦C for 10, 20, 40 and 60 min. Theeffect of pH on surface tension and emulsification was evaluatedafter adjustment of the broth pH to 2, 4, 6, 8, 10, 12 and 14 with6.0 M NaOH or HCl.

2.10. Biosurfactant isolation

The rhamnolipid mixtures were extracted from culture mediaafter cell removal by centrifugation at 5000 × g for 30 min. Thesupernatant pH was adjusted to 2.0 with 6.0 M HCl, and anequal volume of CHCl3/CH3OH (2:1) was added. The mixture wasvigorously shaken for 15 min and allowed to set until phase sep-aration. The organic phase was removed and the operation wasrepeated twice again. The rhamnolipid product was concentratedfrom the pooled organic phases using a rotary evaporator. Theviscous yellowish product obtained was dissolved in methanoland concentrated again by evaporation of the solvent at 45 ◦C[15].

2.11. Biosurfactant characterization by thin-layerchromatography

After isolating the biosurfactant, a sample of 0.1 g was dis-solved in methanol and analyzed by thin-layer chromatography(TLC) on silica gel plates (G60; Merck, Germany). Chromatogramswere developed with chloroform:methanol:acetic acid (65:15:2,v/v) and the detection was done by the following methods: (1)exposure to iodine vapours for lipid stains, (2) exposure to the Mol-ish reagent for sugar detection and (3) exposure to 1% ninhydrinsolution for free amino groups. The reagents were sprayed, and theplates were heated for 30–40 min at 110 ◦C until the appearance ofthe respective colour [16,17].

2.12. Determination of biosurfactant fatty acids composition

The fatty acids composition of crude biosurfactant extract wereanalyzed by GC-FID (CP-3380, Varian Inc., California, USA) of fattyacids methyl ethers, as described by Durham and Kloos [18].

The analyses were carried out in a gas chromatograph equippedwith capillary column HR-SS-10. The carrier gas was helium at24 ml/min. The injector and detector (FID) temperature was 250 ◦C,oven temperature at 130 ◦C, starting and increasing to 170 ◦C at1 ◦C/min, to 180 ◦C at 3 ◦C/min, kept isothermically for 10 min. Thesample volume was 10 �l. The chromatographic peaks were iden-tified by comparing with the chromatogram of the standard fattyacid methyl mixture (Sigma Chemical Co., St. Louis, MO, USA).

2.13. Determination of biosurfactant ionic character

The ionic charge of the biosurfactant was determined using theagar double diffusion technique [19]. Two regularly spaced rowsof wells were made in an agar of low hardness (1% agar). Wells ofone row were filled with the biosurfactant solution and wells of theother were filled a pure compound of known ionic charge.

The anionic substance chosen was sodium dodecyl sulphate(SDS) 20 Mm and the cationic one was barium chloride, 50 mM.The appearance of precipitation lines between the wells, indicativeof the ionic character of the biosurfactant, was monitored over a48-h period at ambient temperature.

2.14. Phytotoxicity assay

The phytotoxicity of the biosurfactant was evaluated in statictest by seed germination and root elongation of cabbage (Brassicaoleracea) according to Tiquia et al. [20]. Solutions of the isolatedbiosurfactant were prepared with distilled water in concentrationsof 175, 350, 525 and 700 mg/l. The toxicity was determined in ster-ilized Petri dishes (1 cm × 10 cm) containing Whatman N◦ 1 filterpaper. The seeds were pre-treated with sodium hypochlorite and10 seeds were inoculated in each Petri dish which was inoculatedwith 5 ml of the test solution at 27 ◦C. After 5 days of incubationin the dark, the seed germination, root elongation (≥5 mm) andgermination index (GI, a factor of relative seed germination andrelative root elongation) were determined as follows:

relative seed germination (%)

=(

number of seeds germinated in the extractnumber of seeds germinated in control

)× 100

relative root length (%) =(

mean root length in the extractmean root length in control

)× 100

GI = (% seed germination) × (% root growth)100%



Controls were prepared with distilled water to replace the biosur-factant solutions. The mean and standard deviation of triplicatesamples from each concentration were calculated.

2.15. Artemia assay

The toxicity assay was performed with the isolated biosurfac-tant using brine shrimp (the microcrustacean Artemia salina) as thetoxicity indicator. Brine shrimp eggs were obtained in a local story.Larvae were used within 1 day of hatching.

Following dilutions of a biosurfactant solution at the CMC(700 mg/l) with saline water (33 mg/l) to give concentrations of175, 350, 525 and 700 mg/l, the assays were conducted in penicillintubes of 10 ml capacity containing 10 brine shrimp larvae in 5 mlof saline water per tube. The cell-free broth containing the biosur-factant was also tested. The brine shrimp larvae in each tube weretested using 5 ml per concentration level of biosurfactant solution.They were observed for 24 h to calculate mortality [21]. The toxicitythreshold concentration, expressed as biosurfactant concentrationper 100 ml of saline water, was defined as the lowest concentrationthat kills all tested brine shrimp within 24 h. Each test was run intriplicate, and saline water was used as the control.

2.16. Application of the biosurfactant in hydrophobiccontaminants removal from sand

Biosurfactant suitability for enhanced oil recovery was carriedout using artificially contaminated sand with 10% of petroleum ordiesel. Samples of 20 g of 40/50 mesh (0.3–0.42 mm) and 20/30mesh (0.6–0.85 mm) fractions of the contaminated Brazilian stan-dard sand NBR 7214 [22] were transferred to 250-ml Erlenmeyerflasks, which were submitted to the following treatments: additionof 40 ml distilled water (control) or 40 ml of the cell-free broth or40 ml of a solution of the isolated biosurfactant at the CMC and fivetimes superior its CMC. The samples were incubated on a rotaryshaker (150 rpm) for 24 h at 27 ◦C and then were centrifuged at5000 × g for 20 min for separation of the laundering solution andthe sand. The pH of the samples was also measured before andafter the treatment. The amount of oil residing in the sand afterthe impact of biosurfactant was gravimetrically determined as theamount of material extracted from the sand by hexane [23].

2.17. Statistical analysis

All surface tensions, biosurfactant concentrations and emulsi-fication activities determinations were performed at least threetimes. Means and standard errors were calculated using theMicrosoft Office Excel 2003 (Version 7).

3. Results and discussion

Studies in the field of biosurfactants have indicated that themedium composition and growth cultivation conditions play acritical role on the type and concentration of the biosurfactantsproduced, being of fundamental importance the investigation ofthese parameters [24].

In this sense, the effect of the carbon source concentration, ofthe nitrogen source and concentration and of the cultivation con-ditions including agitation velocity, aeration and temperature wereconsecutively evaluated, in single-factor experiments, to establishthe best conditions for the production of a biosurfactant from P.aeruginosa UCP0992.

Table 1Effect of glycerol concentration on the biosurfactant production by P. aeruginosaUCP0992 cultivated in mineral medium during 120 h in terms of surface tensionreduction and product concentration.

Glycerol (%) Surface tension reduction (mN/m) Concentration (g/l)

2 28.50 ± 0.20 2.05 ± 0.093 28.30 ± 0.10 3.40 ± 0.055 28.00 ± 0.31 3.00 ± 0.067 30.00 ± 0.25 3.50 ± 0.04

3.1. Effect of glycerol concentration on biosurfactant production

The concentrations of glycerol tested were estimated accordingto the data from our preliminary studies as well as the results indi-cated in the literature [24]. It was observed that the concentrationof 3% was the most favourable in terms of both surface tension andbiosurfactant concentration (Table 1).

3.2. Effect of nitrogen sources on biosurfactant production

There is evidence that nitrogen plays an important role in theproduction of surface-active compounds by microorganisms [24].

From the best glycerol concentration, the effect of the nitro-gen source was examined for the most commonly used organicand inorganic nitrogen sources reported in the literature. It wasobserved that NaNO3 and corn steep liquor were the most efficientnitrogen sources. Thus, sodium nitrate was selected for furtherexperiments, considering its availability and low cost. The cornsteep liquor, on the other hand, due to its variable composition,i.e., its residual origin, could influence the composition and concen-trations of the biosurfactants produced (Table 2). Ammonium saltsand urea were not utilized for the production of the biosurfactant.

Santa Anna et al. [25] also studied the effect of the nitrogensource on the production of a biosurfactant by P. aeruginosa PA1 cul-tivated in mineral medium containing 3% glycerol. The NaNO3 wasmost effective than (NH4)2SO4, for a C/N ratio of 60, correspond-ing to a concentration of 0.145. % NaNO3, observing that nutritionallimitations direct cellular metabolism to product formation.

According to Mulligan and Gibbs [26], P. aeruginosa uses nitrates,ammonia and amino acids as nitrogen sources. The nitrates mustbe reduced to nitrite and then to ammonia. The ammonia canbe assimilated either by glutamate dehydrogenase (EC 1.4.1.4) toform glutamate or, with glutamine, by glutamine synthetase (EC6.3.1.2) to form glutamine. Glutamine and �-ketoglutarate are thenconverted to glutamine by l-glutamine 2-oxoglutarate aminotrans-ferase (EC 1.4.1.13). On the other hand, during the biosynthesis ofrhamnolipid, lipid formation, but not sugar, is the rate-determiningfactor, and nitrogen limitation may promote lipid accumulation.Therefore, in comparison with ammonia, the assimilation of nitrateas a nitrogen source is slower, simulating a nitrogen-limiting con-dition that is favourable to rhamnolipid production.

Table 2Effect of nitrogen sources on the biosurfactant production by P. aeruginosa UCP0992cultivated in mineral medium during 120 h in terms of surface tension reductionand product concentration.

Nitrogen source (0.2%) Surface tensionreduction (mN/m)

Concentration (g/l)

NaNO3 28.30 ± 0.01 3.30 ± 0.03Urea 43.29 ± 0.51 ND(NH4)2SO4 50.50 ± 0.73 NDPeptone 36.50 ± 0.42 ND(NH4)NO3 39.20 ± 0.44 NDCorn steep liquor 28.20 ± 0.23 2.50 ± 0.05Yeast extract 32.00 ± 0.18 ND

ND: not determined.



It has been reported that the organic nitrogen sources could helpcell growth but were unfavourable to the production of glycolipidbiosurfactants [24,27].

Guerra-Santos et al. [28] observed that biosurfactant productionwas poor with both yeast extract and nitrate as nitrogen sources.When the yeast extract was omitted, the biomass concentrationdecreased, rhamnolipid increased and a moderate accumulation ofglucose occurred, indicating a nitrogen-limiting medium.

The P. aeruginosa 44T1 cultivated in olive oil [29] and in glu-cose [27] produced higher quantities of biosurfactant, measured assurface tension reduction, when utilizing NaNO3 in preference tothe ammonium salts (NH4)2SO4, NH4NO3 and NH4Cl. On the otherhand, Cha et al. [30], studying different nitrogen sources, observedmaximum biosurfactant production by P. aeruginosa EMS1 culti-vated in 2% soybean oil with 0.3% NH4NO3. The concentration ofisolate product was 5 g/l. Prieto et al. [31], studying the productionof a biosurfactant from P. aeruginosa LBM10, observed, however,that, although there was no a significant difference between ammo-nium nitrate (0.4%) and sodium nitrate (0.85%) in relation to therhamnose concentration, a significant difference was observed inrelation to the emulsification index. Sodium nitrate was consideredto be the best nitrogen source for rhamnolipid production, reach-ing a rhamnose concentration of 0.94 g/l. Urea and ammoniumsulphate, as the results obtained in this work, were not adequatenitrogen sources for biosurfactant production.

3.3. Effect of nitrogen concentration on biosurfactant production

In many fermentative processes, the C/N ration is an extremelysensitive parameter, thus affecting the metabolites accumulation.High C/N ratios, i.e., reduced levels of nitrogen, limit bacterialgrowth, favouring the cellular metabolism towards the productionof metabolites. On the other hand, an excess of the nitrogen sourcedirects the substrate to the synthesis of cellular material, limitingrelatively the accumulation of products [27].

Table 3Effect of NaNO3 concentration on the biosurfactant production by P. aeruginosaUCP0992 cultivated in mineral medium during 120 h in terms of surface tensionreduction and product concentration.

NaNO3 (%) Surface tension reduction (mN/m) Concentration (g/l)

0.05 30.00 ± 0.10 2.0 ± 0.070.10 30.00 ± 0.50 2.3 ± 0.010.20 28.30 ± 0.09 3.3 ± 0.030.40 27.80 ± 0.12 3.5 ± 0.050.60 27.40 ± 0.35 5.5 ± 0.02

After selecting the nitrogen source, the effect of NaNO3 con-centration was examined. The best results were obtained at aconcentration of 0.6%, corresponding to a C/N ratio of 12.24(Table 3). The increase of the C/N ratio to 18, 46, 37,5, 75 and 150did not favour the production of the biosurfactant.

The results obtained in this work are consistent with the findingsof Haba et al. [32] who varied the concentration of NaNO3 at 0.3, 0.4,0.5 and 0.7%, observing that the highest biosurfactant concentrationfrom P. aeruginosa was obtained with 0.5% NaNO3, correspondingto a C/N ratio of 8.

Wu et al. [24], for P. aeruginosa cultivated in 4% glycerol, obtained7.5 g/l rhamnolipids when NaNO3 was used as the nitrogen sourceat 0.2%, corresponding to a C/N ratio of 52. In contrast, the produc-tivity was similar for a C/N ratio of 6.5 to 52, but markedly decreasedas the C/N ratio was increased to 130. On the other hand, Lima et al.[33] obtained 2.2 g/l as rhamnose when NH4NO3 was used as thenitrogen source at 0.56% for P. aeruginosa cultivated in 2.2% residualsoybean oil.

3.4. Effect of cultivation conditions on biosurfactant production

The effect of rotation velocity (agitation) on the biosurfactantconcentration and surface tension reduction was tested at 150 and200 rpm.

Fig. 1. Growth, pH, surface tension, biosurfactant concentration and hexadecane emulsification profiles of P. aeruginosa UCP0992 grown in mineral medium supplementedwith 3% glycerol and 0.6% NaNO3 during 120 h at 200 rpm and 28 ◦C.



The results obtained showed that the velocity of 200 rpmfavoured the accumulation of biosurfactant (6.5 g/l) against theconcentration of 5.0 g/l obtained at 150 rpm, although the surfacetension (27.5 mN/m) remained unaltered for both velocities tested.

The effect of the variation in the agitation speed from culturesof 50–200 rpm has been studied by Oliveira et al. [34] for P. alcali-genes cultivated in 0.5% palm oil. They observed that the increase ofthe rotation velocity from 260 to 300 favoured the reduction of thesurface tension of the cell-free broth to 27.6 mN/m. In a previouswork, however, Oliveira et al. [35] observed, for P. aeruginosa cul-tivated in palm oil, the decrease of the surface tension to 35 mN/mat 150 rpm, while the velocities of 50, 100 and 200 rpm increasedthe surface tension to 55, 39 and 51 mN/m, respectively.

Cunha et al. [36] observed that agitation had a negative effect onsurface tension reduction by the biosurfactant produced by Serratiasp. SVGG16 cultivated in hydrocarbons, and that best results wereobtained with the lowest value, of 100 rpm, when compared to 200and 300 rpm.

P. aeruginosa is a facultative microorganism that may presentgrowth in environments with low oxygen concentration; the pro-duction of surface-active compounds, however, involves stages ofoxidation of the substrate [35]. The agitation speed of the mediumis a determining factor in the mixture of the aqueous and oily phaseas well as in the oxygen mass transfer into the cultures using agi-tated flasks. Thus, it is possible that, in the case of this work, whereno an oil phase is involved, the agitation speed may have influ-enced the surfactant production by the applied strain, promoting aphase mixture and/or an adequate oxygen transfer rate for a mosteffective biosurfactant production at 200 rpm.

The effect of aeration was evaluated for 60, 80 and 90%, asdescribed earlier. It was observed that the surface tension and con-centration in isolate biosurfactant remained unaltered for thesevalues tested. On the other hand, Abdel-Mawgoud et al. [37]observed maximum surfactin production at an aeration percent-age of 90% and a sharp decline in the production upon decrease ofaeration for Bacillus subtilis BS5.

It has been described the importance of intense aeration is toinhibit the bacterium anaerobic mechanism, which can be utilizedthroughout the use of nitrates as final acceptors of electrons for thecellular respiration [27].

The effect of the culture temperature was also verified in thiswork at 28 and 37 ◦C. There was no differences between bothtemperatures regarded the surface tension values and the biosur-factant concentration, suggesting that the metabolism of the strainP. aeruginosa UCP0992 was not affected by the temperatures tested.

Considering that elevates temperatures contribute to a majorproduction cost, the temperature of 28 ◦C was selected for furtherexperiments.

Guerra-Santos et al. [38], observed that the temperature range of32–34 ◦C resulted in high rhamnolipids production by P. aeruginosa,while Subasioglu and Cansunar [39] obtained maximum rhamno-lipids production by P. aeruginosa cultivated in manitol at 34.5 ◦C,with a great reduction for temperatures above 36 ◦C. On the otherhand, Robert et al. [27] observed that the temperature of 37 ◦Cfavoured the accumulation of biosurfactant by P. aeruginosa 44T1cultivated in glucose as substrate, when compared with the tem-perature of 27 ◦C.

3.5. Growth kinetics and biosurfactant production

After the establishment of the best medium and conditions ofcultivation, the growth kinetics and biosurfactant production weredescribed for P. aeruginosa UCP0992 cultivated in 500-ml Erlen-meyer flasks containing 100 ml mineral medium (aeration of 80%)supplemented with 3% glycerol and 0.6% NaNO3, as the nitrogensource, at 28 ◦C during 120 h under agitation speed of 200 rpm.

The kinetics curves showed that there was an almost paral-lel relationship between biosurfactant production, cell growth,consume of glycerol, surface tension reduction, emulsificationof hexadecane and substrate utilization, suggesting a growth-associated production (Fig. 1).

Biosurfactant production started soon after inoculation, show-ing a biphasic profile. The first phase extended up to 24 h, remainingconstant until 48 h, whereas in the second phase, production wasincreasing at a slower rate and a slight increase was obtained until96 h. At this point, it was observed not only the maximum biosur-factant accumulation (8.0 g/l), but also the minor surface tension,which was reduced from 56 mN/m to 27.4 mN/m, and the highestbiomass concentration (4.0 g/l).

The glycerol consumption curve showed a similar profile to thesurface tension reduction curve, while the hexadecane emulsifica-tion followed the biosurfactant production, confirming the possibleuse of surface tension and emulsification as indicative parame-ters of the presence of surface-active products in the medium. Theemulsification increased along the time, reaching its maximumafter 72 h and remaining constant until the end of cultivation, with80–75% of hexadecane emulsification.

The literature describes the occurrence of different kinetic pro-files for rhamnolipids. P. aeruginosa GS3 had been reported toproduce biosurfactant when the cell reached the stationary phase[12]. However, the growth-associated production of biosurfactantshad also been reported from several researches [30].

The fermentative pH exerts great influence over the productsmetabolism velocity. For the cultivation of P. aeruginosa UCP0992,the pH decreased to 6.0 during the first 24 h, corresponding tothe minor biomass and to the biphasic phase of biosurfactant pro-duction and hexadecane emulsification. After that, it increasedgradually, reaching the highest values, around 8.5, after 120 ofcultivation.

In fermentation without pH control, its value is the result ofthe microorganism metabolism. For the experiments conducted inpresence of nitrate, the bacterium promoted the medium alcalin-ization.

The culture pH had also increased along the time when using 2%buriti or cupuacu brazilian vegetable oil as substrates for P. aerug-inosa LB1, reaching values over 8.0 after 120 h of cultivation [15].Robert et al. [27], studying the production of rhamnolipids from P.aeruginosa 44T1 cultivated in glucose, had also observed a reduc-tion in the pH to 6.0 in the first 20 h of cultivation, and after that itstarted to increase until values around 9.0 at the end of fermenta-tion.

The quantification of rhamnolipids in the cell-free broth, deter-mined throughout rhamnose concentration gave a result of 6.8 g/l.Fermentations for rhamnolipids production using frying oil as sub-strate [32], refinery residue [40], milk whey [41] and molasses [12]gave 2.7, 1.85, 1.78 and 0.24 g/l, respectively.

3.6. Biosurfactant emulsification capacity

In addition to surface and interfacial tension, stabilization ofan oil and water emulsion is commonly used as a surface activ-ity indicator, although the ability of a molecule in forming a stableemulsion is not always associated with the capacity in reducing thesurface tension [42].

Most microbial surfactants are substrate specific, solubilizing oremulsifying different hydrocarbons at different rates. Poor emulsi-fication of some of the hydrocarbons might be due to the inabilityof the biosurfactant to stabilize the microscopic droplets [42].

Different oil-degrading microorganisms produce surface-activesubstances and some of them make stable oil in water emulsions.These microorganisms can be divided in two categories, one thatproduces low molecular weight surfactant but usually do not make



Table 4Emulsification index (EI) of hydrophobic substrates bythe cell-free broth containing the biosurfactant from P.aeruginosa UCP0992 cultivated in mineral medium sup-plemented with 3% glycerol and 0.6% NaNO3 during 96 h.

Substrate EI (%)

Cotton seed oil 60.0 ± 5.12Corn oil 58.1 ± 3.42Rice oil 57.2 ± 3.00Soybean oil 40.5 ± 2.50Hexadecane 75.2 ± 4.52Benzene 56.3 ± 5.02Motor oil 50.2 ± 5.70Diesel 53.7 ± 3.24Petroleum 100 ± 5.32

stable emulsions and another group that produces polymers thatprimarily act as emulsion stabilisers but usually do not affect thesurface tension. In some cases, few bacteria and yeasts have bothtype of properties [43]. The data obtained in this work suggestthat the P. aeruginosa UCP0992 can be included in the last cate-gory of microorganisms once the bacterium was able to reduce thesurface tension and also make stable emulsions with different sub-strates (Table 4). High degrees of emulsification (above 50%) wereobserved. The cell-free broth containing the biosurfactant obtainedafter 96 h of cultivation was able to emulsify 100% petroleum. Thewater-oil emulsions showed to be compact and remained stable formore than 6 months at room temperature, suggesting that the addi-tion of such biosurfactant into a remediation process may enhancethe availability of the recalcitrant hydrocarbon. O the other hand,the ability to emulsify the vegetable oils suggests a potential appli-cation in the pharmaceutical and cosmetic industries.

3.7. Biosurfactant stability related to surface tension andemulsification

The application of biosurfactants in the industrial sector willrequire, undoubtly, stable products to the operational and envi-ronmental conditions. In the case of the petrochemical industry,specifically, the use of biosurfactants as coadjutants in the Micro-bial Enhanced Oil Recovery processes is promising, although theconditions found in the oil reservoirs such as high temperature,pressure and salinity will require the utilization of efficient andstable agents [44].

Purification accounts for up to 60% of the total production cost ofbiosurfactants. Because of economic considerations in the oil indus-try, most biosurfactants would require either whole-cell culturebroths or crude preparations. Therefore, the application of the bio-surfactant from P. aeruginosa UCP0992 in its crude form withoutprior costly extraction steps was investigated.

Firstly, the resistance of the cell-free broth containing thebiosurfactant to the presence of NaCl was investigated. The bio-surfactant maintained the capacity of reducing the surface tensionup to 10% NaCl, showing a slight tendency of reducing the surfacetension proportional to the increase of the salt concentration, whilethe capacity of emulsifying the hydrophobic compounds tested alsoremained practically unaltered in the presence of the salt (Table 5).This phenomenon was interpreted by the findings of Helvaci et al.[45]. They stated that electrolytes directly affect the carboxylategroups of the rhamnolipids. The solution/air interface has a net neg-ative charge due to the ionized carboxylic acid groups at alkalinepH with strong repulsive electrostatic forces between the rhamno-lipid molecules. This negative charge is shielded by the Na+ ions inthe electrical double layer in the presence of NaCl, causing the for-mation of a close-packed monolayer and consequently a decreasein surface tension values.

The effect of salinities on the activity of the biosurfactant wasevaluated to investigate its applicability in the bioremediation ofcontaminated marines. Considering that the highest sea salinity inthe world is of 3%, the biosurfactant from P. aeruginosa UCP0992could be applied in saline environments.

The stability of the biosurfactant was also tested over a widetemperature range. The biosurfactant showed to be stable dur-ing incubation for 1 h at temperatures ranging from 4 to 120 ◦C(Table 5). The surface tension reducing activity and emulsificationactivity were quite stable at these used temperatures, indicatingthe usefulness of the biosurfactant in industries where heating toachieve sterility is of paramount importance.

The stability of the biosurfactant at different pH values is alsoan important issue that can affect its application spectrum. Studieson reduction of surface tension and capacity of emulsification bythe cell-free broth containing the biosurfactant showed that it wasstable over a wide range of pH, while a reduction in the surface ten-sion has been observed at extremes pH of 2.0 and 14.0, suggestingthe occurrence of some alteration in the surfactant structure underthese pHs. A similar behaviour was observed for the emulsifica-tion capacity of the vegetable oils tested, although with a contraryeffect, i.e., an elevate reduction in the emulsion percentages. Theemulsification of diesel, however, remained unchanged along pH,with exception of pH 2.0 (Table 5).

The rhamnolipids have their optimum aqueous solubility atneutral to alkaline pHs. This phenomenon is attributed to theiracidic nature, once the reported pKa is 5.6. Rhamnolipids contain asingle free carboxylic acid group corresponding to the �-hydroxyfatty acid moiety, and this confers the anionic nature of rhamno-lipids. It has been reported that as the pH increases from 5 to 8, thenegative charge of the polar head increases, and this is reflected byincreased aqueous solubility. If the surface-active molecules are athigh concentrations, the increase of pH can change the morphologyof the micelle structure formed above the CMC, which change fromlamellar to vesicles, and finally to micelles [37].

The effect of the time of heating over the cell-free broth fromP. aeruginosa UCP0992 at 90 ◦C is also shown in Table 5. It couldbe observed the stability of the emulsification capacity and of thesurface tension reduction along the time range tested, althougha slightly tendency to increasing the surface tension had beenobserved.

Promising results related to the stability under extreme condi-tions have also been demonstrated for other surfactants producedfrom bacteria [31,37].

3.8. Biosurfactant characterization

Rhamnolipids comprise a mixture of homologous species R1, R2,R3 and R4, although a great variation of other homologues had beendetected. It is known that the properties of rhamnolipids depend onthe distribution of these homologues, i.e., on their composition anddistribution that vary according to bacterial strain, culture condi-tion, and medium composition Another possibility to explain thesedifferences can be the presence of impurities such as extracted non-metabolized fatty acids from the culture broth that could influencethe surface-active properties. Most of the reported research on bio-surfactant production by Pseudomonas strains has focused on theobtainment of the glycolipids rhamnolipids [46].

The crude extract form P. aeruginosa UCP0992 appeared as vis-cous sticky oily residue with ambar to yellowish brown colour.The biosurfactant was soluble in aqueous solution and in organicsolvents like methanol, chloroform ethylether and ethylacetate.Similar physical properties had been described for other rhamno-lipids surfactants [37].

Agar double diffusion tests revealed the appearance of precip-itation lines between the biosurfactant produced by P. aeruginosa



Table 5Influences of salt concentration, temperature, pH and time of heating at 90 ◦C on the surface tension reducing activity and on the emulsifying activity of the cell-free brothcontaining the biosurfactant from P. aeruginosa UCP0992 cultivated in mineral medium supplemented with 3% glycerol and 0.6% NaNO3 during 96 h.

Surface tension reduction (mN/m) EI (%)a EI (%)b EI (%)c

NaCl (%)2.0 27.9 ± 0.15 62.5 ± 5.10 59.3 ± 5.10 56.1 ± 3.004.0 27.4 ± 0.10 58.5 ± 5.02 62.5 ± 2.09 60.0 ± 3.076.0 27.0 ± 0.12 59.3 ± 3.17 62.5 ± 4.47 60.2 ± 2.528.0 27.0 ± 0.20 56.2 ± 4.28 51.4 ± 3.52 60.1 ± 4.0210.0 26.5 ± 0.30 57.0 ± 4.30 55.1 ± 3.07 58.3 ± 5.34

Temperature (◦C)4 28.0 ± 0.34 58.0 ± 5.11 54.3 ± 4.38 50.2 ± 3.0028 28.5 ± 0.42 60.2 ± 4.02 58.0 ± 2.85 53.4 ± 2.8970 29.0 ± 0.39 60.1 ± 5.00 58.1 ± 3.27 53.2 ± 3.49100 29.5 ± 0.35 55.5 ± 3.03 56.5 ± 4.15 55.4 ± 2.31120 30.0 ± 0.28 53.5 ± 4.30 55.1 ± 5.02 56.8 ± 3.02

pH2 24.0 ± 0.35 22.0 ± 2.97 17.0 ± 5.12 30.0 ± 3.244 26.1 ± 0.12 46.1 ± 3.08 46.0 ± 3.80 50.0 ± 3.026 26.5 ± 0.41 60.0 ± 4.09 60.1 ± 2.35 50.0 ± 2.958 28.5 ± 0.32 60.0 ± 4.03 58.2 ± 3.21 53.0 ± 3.4510 29.0 ± 0.42 58.2 ± 2.51 51.0 ± 4.57 60.0 ± 3.0112 28.5 ± 0.22 60.0 ± 3.15 60.0 ± 3.43 53.0 ± 3.3214 24.3 ± 0.52 20.0 ± 4.00 11.1 ± 5.13 53.0 ± 4.00

Time (min)10 29.3 ± 0.12 54.0 ± 3.30 57.1 ± 2.80 60.0 ± 3.8120 29.3 ± 0.10 53.1 ± 2.81 55.0 ± 3.02 60.0 ± 4.1040 29.6 ± 0.08 60.0 ± 3.09 55.0 ± 4.09 58.3 ± 4.2460 29.7 ± 0.09 60.0 ± 3.31 55.0 ± 3.32 60.0 ± 2.51120 29.9 ± 0.05 54.2 ± 2.13 55.0 ± 2.55 58.0 ± 4.27

a Emulsification index of cotton seed oil.b Emulsification index of corn oil.c Emulsification index of diesel.

UCP0992 and the cationic compound selected (barium chloride),while no lines had formed between the biosurfactant and theanionic compound (SDS). Under the experimental conditions of thiswork, this very simple test confirmed the anionic character of thebiosurfactant produced. A similar result had been observed for thebiosurfactant from P. fluorescens 495 submitted to the same test[19].

The fatty acid composition of the isolated biosurfactant analyzedby Gas Chromatography revealed the presence of C16:0 (42.3%) andC18:1 (39.8%) as the major components, and C18:0 (6.5%), C16:1(5.5%), C18:2 (3.6%) and C12:0 (2.3%) in minor quantities.

The biosurfactant extracted from the cell-free broth was devel-oped by thin-layer chromatography (TLC) and visualized withspecific reagents and produced spots with different Rf (retentionfactor), as shown in Fig. 2. Five spot with different Rf values, includ-ing more and less mobile fractions, showed positive reactions forsugars with Molish reagents and for lipids with iodine vapours,but negative reactions for amino groups with ninhydrin. The pres-ence of both glycosyl units and lipid moieties on the same spotsindicated that the sample was a glycolipid. These results suggestthat P. aeruginosa UCP0992 produced glycolipids in medium sup-plemented with glycerol as the sole carbon source.

The results obtained in this work exhibited similarities in theseparation profile with previously reported TLC results of commer-cially available purified rhamnolipids and produced rhamnolipidsfrom P. aeruginosa strains, as described below.

George and Jayachandran [46], studying the production of bio-surfactant by P. aeruginosa MTCC2297 cultivated in orange fruitpeelings detected spots with Rf values of 0.19 (dirhamnolipids),0.36 (monorhamnolipids), 0.59 and 0.71 (various rhamnolipidforms), 0.82 and 0.98 in silica plates eluted with the same systemused in his work. Haba et al. [32], on the other hand, observed thatPseudomonas sp. cultivated in medium supplemented with usedvegetable oils produced a mixture of two rhamnolipids with a Rf

of 0.7 and 0.45. Arino et al. [47] characterized the rhamnolipidmixture produced by P. aeruginosa GL1. The Rf values for differentspots were calculated and it corresponds to R1 0.72 (Rha-C10C10,),R2 0.40 (Rha-C10), R3 0.32 (Rha-Rha-C10C10) and R4 0.13 (Rha-Rha-C10). Monteiro et al. [5], studying the production of a biosurfactantby P. aeruginosa DAAUPE614 cultivated in glycerol and ammoniumnitrate, detected two orcinol-positive spots, with Rf values of 0.55and 0.36 in chloroform:methanol:water (65:15:2), representing

Fig. 2. Rhamnolipid components detected by thin-layer chromatography. Thesamples were applied in 20 �l volumes in TLC plates and developed withCHCl3:MeOH:H3COOH (65:15:2, v/v) solvent system. TLC plate was sprayed withMolisch reagent, followed by heating at 100 ◦C for 30–40 min.



Fig. 3. Surface tension versus concentration of the isolated biosurfactant from P.aeruginosa UCP0992 cultivated in mineral medium supplemented with 3% glyceroland 0.6% NaNO3 during 96 h.

the monorhamnolipids and dirhamnolipids, respectively, whereasZhang et al. [48] for the rhaminolipis from P. aeruginosa cultivatedin glycerol and sodium nitrate had also detected two spots with Rfof 0.52 and 0.32. Parra et al. [7] detected the presence of two spotswith Rf of 0.45 and 0.70, also identified as R1 (Rha-C10C10) and R2(Rha-Rha-C10) for P. aeruginosa 44T1 cultivated in kerosene.

3.9. Surface and interfacial tensions and critical micelleconcentration (CMC) of the biosurfactant

The surface-active properties of biosurfactant mainly dependon its ability to lower surface and interfacial tensions, CMCvalue and formation of stable emulsion. The ability to reducethe surface tension depends on the specific concentration ofsurface-active compound, i.e., the CMC which is defined as theminimum concentration of biosurfactant required to give max-imum surface tension reduction of water and initiate micelleformation. Efficient surfactants have very low CMC values,i.e., less surfactant is required to decrease surface tension[43,46].

Surface tensions versus the isolated biosurfactant concen-trations were plotted (Fig. 3). The surface tension of waterdecreased gradually with increasing biosurfactant concentra-tion from 70 to 29 mN/m, with a biosurfactant concentrationof 700 mg/l, and then remained constant, while the solutionof the isolated biosurfactant at the CMC reduced the inter-facial tension of the mixture water/hexadecane from 40 to1 mN/m.

This CMC value differs greatly from that of 53 mg/l reported forthe rhamnolipids mixture produced by P. aeruginosa UG2 on corn oil[49], or 230 mg/l found for a mixture of seven homologues [50], or120 mg/l found for a mixture of six homologues from P. aeruginosaLB1 cultivated in soapstock [1].

The different CMC values may have resulted from differencesin purity and composition of rhamnolipid. In this study, the lowpurity of the test rhamnolpids mixture may be responsible for thedifference in CMC values between the test and other rhamnolipiddescribed in the literature [37]. Due to the intrinsic variability of therhamnolipids accumulated and the complexity of its composition,number and proportions of homologues, presence of unsaturatedbonds, branching and length of the aliphatic chain of the rhamno-lipid can all affect the CMC and surface tension values between therhamnolipids produced [43]. The proportions of the homologuesproduced can also differ with the bacterium, medium and cultiva-tion conditions [24].

3.10. Biosurfactant toxicity

The biosurfactant from P. aeruginosa was first tested for its toxic-ity in a short term bioassay using brine shrimp. The biosurfactant at700 and 525 mg/l caused 100 and 50% brine shrimp lethality after24 h, respectively, while no lethality was observed at the minorconcentrations tested and with the cell-free broth. On the otherhand, Wang et al. [51] showed that the rhamnolipid biosurfactantsproduced by P. aeruginosa had potential algicidal effects on theharmful algal bloom (HAB) species, Heterosigma akashiwo, above0.4 mg/l. These results indicate the low toxicity of the biosurfactantfrom P. aeruginosa UCP0992. The acute toxicity tests of the surfac-tant JE1058BS produced by the bacterium Gordonia sp. against twospecies of marine larvae, Mysidopsis bahia (shrimp) and Menidiaberyllina (fish), also showed the low toxicity of this biosurfactant[52].

The germination index (GI), which combines measures of rela-tive seed germination and relative root elongation, has been used toevaluate the toxicity of the biosurfactant on cabbage (B. oleracea).Considering that a GI value of 80% has been used as an indicator ofthe disappearance of phytotoxicity [20], the results obtained indi-cated that the solutions tested did not show inhibitory effects onthe seed germination and root elongation of cabbage, since GI of245, 187, 132 and 86% were found for biosurfactant solutions of175, 350, 525 and 700 mg/l, respectively. It was also possible tovisualize the leaves and the elongation of secondary roots for allthe conditions tested.

3.11. Application of the biosurfactant in hydrophobiccontaminants removal

Apart from the industrial applications of biosurfactants envis-age, their application in the oil industry is one of the potential useswhich requires lower purity specifications so that whole-cell brothcould be used, eliminating the purification steps that representalmost 60% of the total production costs [37].

In order to investigate the application of the biosurfactantproduced by P. aeruginosa UCP0992 in contaminant removal, a

Table 6Removal of petroleum and diesel adsorbed in standard sand samples by the biosurfactant produced by P. aeruginosa UCP0992 cultivated in mineral medium supplementedwith 3% glycerol during 96 h and by distilled water (as the control).

Removal agent Pollutant removal from sand (%)

40/50 mesh (0.3–0.42 mm) 20/30 mesh (0.6–0.85 mm)

Diesel Petroleum Diesel Petroleum

Biosurfactant (cell-free broth) 87.5 ± 0.5 10.0 ± 0.6 85.4 ± 0.3 0Solution of the isolated biosurfactant at theCMC (700 mg/l)

95.0 ± 0.4 20 ± 0.9 95 ± 0.5 10 ± 0.9

Solution of the isolated biosurfactant at aconcentration five times superior its CMC(3500 mg/l)

91.0 ± 0.5 10 ± 0.7 90.0 ± 0.4 7 ± 0.8

Control (distilled water) 50.0 ± 0.1 0 45.2 ± 0.2 0



preliminary experiment using the cell-free broth containing thesurfactant and solutions of the isolated surfactant at and above theCMC were performed to verify the removal of hydrocarbons fromsand samples (Table 6).

High percentage removals of diesel were observed for all solu-tions, although the biosurfactant has not proven to be very effectivein removing petroleum under the conditions tested in this work.The particle size of the soil did not exercised great influence onthe percentage removal of the pollutants, neither the biosurfactantconcentration. It could be observed that the cell-free broth con-taining the crude biosurfactant was practically as effective as theisolated biosurfactant when removing diesel oil, thus indicating thepossible use of the biosurfactant without purification steps, whichwould increase the production costs.

We can also suggest that the biosurfactant from P. aeruginosaUCP0992 utilized the mechanism of displacement to release the oilsdroplets once the increase of the concentration did not improve theremoval of the pollutants.

4. Conclusions

The use of glycerol is a promising and cheap alternative forthe production of biosurfactants due the increasing demand ofbiodiesel in the world. The rhamnolipids produced by P. aerugi-nosa UCP0992 show a stable surface tension reduction capacityand ability to emulsify different hydrophobic substrates underhigh temperatures, different pHs and saline conditions. The cruderhamnolipid-containing preparations (cell-free broth) gave highemulsifying activities against different oils, capacity to removehydrophobic contaminants and did not show toxicity. This com-plies with the nowadays need for the use of biosurfactants in theircrude form for the purpose of cost reduction. Thus, the biosur-factant will be of interest for potential use in a wide variety ofindustrial, environmental and biotechnology applications. Emul-sifying biosurfactants that are stable in environments with high pHand salinity would find application for bioremediation of spills atseas. The biosurfactant may also be useful for bioremediation worksin hot and alkaline environments.

Acknowledgements

This work was financially supported by National Council forScientific and Technological Development (CNPq), and CatholicUniversity of Pernambuco (UNICAP), Brazil.

References

[1] M. Benincasa, A. Abalos, I. Oliveira, A. Manresa, Antonie Van Leewenhoek 85(2004) 1.

[2] M. Benincasa, Curr. Microbiol. 54 (2007) 445.[3] J. Sabaté, M. Vinas, A.M. Solanas, Int. Biodeter. Biodegr. 54 (2004) 19.[4] M. Sánchez, F.J. Aranda, M.J. Espuny, A. Marqués, J.A. Teruel, A. Manresa, A. Ortiz,

J. Colloid Interf. Sci. 307 (2007) 246.[5] S.A. Monteiro, G.L. Sassaki, L.M. Souza, J.A. Meira, J.M. Araújo, D.A. Mitchell, L.P.

Ramos, N. Krieger, Chem. Phys. Lipids 147 (2007) 1.[6] S. Lang, D. Wullbrandt, Appl. Microbiol. Biotechnol. 51 (1999) 22.[7] J.L. Parra, J. Guinea, M.A. Manresa, M. Robert, M.E. Mercadè, F. Comelles, M.P.

Bosch, JAOCS 66 (1989) 141.[8] T. Morita, M. Konishi, T. Fukuoka, T. Imura, D. Kitamoto, J. Biosci. Bioeng. 104

(2007) 78.

[9] E.R. Easterling, W.T. French, R. Hernandez, M. Licha, Bioresour. Technol. 100(2009) 356.

[10] Y.C. Sharma, B. Singh, S.N. Upadhyay, Fuel 87 (2008) 2355.[11] G.P. Silva, M. Mack, J. Contiero, Biotechnol. Adv. 27 (2009) 30.[12] R.M. Patel, A.J. Desai, Lett. Appl. Microbiol. 25 (1997) 91.[13] M. Dubois, K.A. Gilles, J.K. Hamilton, Anal. Chem. 28 (1956) 350.[14] D.G. Cooper, B.G. Goldenberg, Appl. Environ. Microbiol. 53 (1987) 224.[15] S.G.V.A.O. Costa, M. Nitschke, R. Haddad, M.N. Eberlin, J. Contiero, Process

Biochem. 21 (2005) 1593.[16] M. Deshpande, L. Daniels, Bioresour. Technol. 54 (1995) 143.[17] A.S. Santos, A.P.W. Sampaio, G.S. Vasquez, L.M.S. Anna, N. Pereira Jr., D.M.G.

Freire, Appl. Biochem. Biotechnol. 98 (2002) 1025.[18] D.R. Durham, W.E. Kloos, Int. J. Syst. Bacteriol. 28 (1978) 228.[19] T. Meylheuc, C.J. Van Oss, M.N. Bellon-Fontaine, J. Appl. Microbiol. 91 (2001)

822.[20] S.M. Tiquia, N.F.Y. Tam, I.J. Hodgkiss, Environ. Pollut. 93 (1996) 249.[21] B.N.N.R. Meyer, J.E. Ferrigni, L.B. Putnam, D.E. Jacobsen, D.E. Nichols, J.L.

McLaughlin, J. Med. Plant Res. 45 (1982) 31.[22] Associacão Brasileira de Normas Técnicas (ABNT), NBR8492: tijolo macico de

solo-cimento: determinacão da resistência à compressão e da absorcão de água,método de ensaio, Rio de Janeiro, 1982.

[23] J.M. Luna, L.A. Sarubbo, G.M. Campos-Takaki, Br. Arch. Biol. Technol. 52 (2009)785.

[24] J.-Y. Wu, K.-L. Yeh, W.-B. Lu, C.-L. Lin, J.-S. Chang, Bioresour. Technol. 99 (2008)1157.

[25] I.M. Santa Anna, G.V. Sebastian, N. Pereira Jr., T.L.M. Alves, E.P. Menezes, D.M.G.Freire, Appl. Biochem. Biotechnol. 91 (2002) 459.

[26] C.N. Mulligan, B.F. Gibbs, Appl. Environ. Microbiol. 55 (1989) 3016.[27] M. Robert, E. Mercadé, C. Andrés, M.J. Espuny, M.A. Manresa, Grasas Aceites 42

(1991) 1.[28] L.H. Guerra-Santos, O. Kãppeli, A. Fiechter, Appl. Environ. Microbiol. 48 (1984)

301.[29] M. Robert, M.E. Mercadé, M.P. Bosch, J.L. Parra, M.J. Espuny, M.A. Manresa, J.

Guinea, Biotechnol. Lett. 11 (1989) 871.[30] M. Cha, N. Lee, M. Kim, M. Kim, S. Lee, Bioresour. Technol. 99 (2008) 2192.[31] L.M. Prieto, M. Michelon, J.F.M. Burkert, S.J. Kalil, C.A.V. Burkert, Chemosphere

71 (2008) 1781.[32] E. Haba, M.J. Espuny, M. Busquets, A. Manresa, J. Appl. Microbiol. 88 (2000)

379–387.[33] C.J.B. Lima, F.P. Franca, E.F.C. Sérvulo, M.M. Resende, V.L. Cardoso, Appl.

Biochem. Biotechnol. 7 (2007) 136.[34] F.J.S. Oliveira, L. Vazquez, N.P. Campos, F.P. Franca, Process Biochem. 44 (2009)

383.[35] F.J.S. Oliveira, L. Vazquez, N.P. Campos, F.P. Franca, Appl. Biochem. Biotechnol.

6 (2006) 727.[36] C.D. Cunha, M. Rosário, A.S. Rosado, S.G.F. Leite, Process Biochem. 39 (2004)

2277.[37] A.M. Abdel-Mawgoud, M.M. Aboulwafa, N.A-H. Hassouna, Appl. Biochem.

Biotechnol. 157 (2009) 329.[38] L. Guerra-Santos, O. Kappeli, A. Fiechter, Appl. Microbiol. Biotechnol. 24 (1986)

443.[39] T. Subasioglu, E. Cansunar, J. Appl. Biol. Sci. 2 (2008) 31.[40] A.K. Koch, J. Reiser, O. Kappeli, A. Fiechter, Biotechnology 51 (1988) 1335.[41] P.S. Babu, A.N. Vaidya, A.S. Bal, R. Kapur, A. Juwarkar, P. Khanna, Biotechnol.

Lett. 18 (1996) 263.[42] N.H. Youssef, K.E. Duncan, D.P. Nagle, K.N. Savage, R.M. Hnapp, M.J. Mcinerney,

J. Microbiol. Meth. 56 (2004) 339.[43] L.A. Sarubbo, J.M. Luna, G.M. Campos-Takaki, Eletronic J. Biotechnol. 9 (2006)

400.[44] S. Joshi, C. Bharuch, S. Jha, S. Yadav, A. Nerurkar, A.J. Desai, Bioresour. Technol.

99 (2008) 195.[45] S.S. Helvaci, S. Peker, G. Ozdemir, Colloid Surf. B: Biointerface 35 (2004) 225.[46] S. George, K. Jayachandran, Appl. Biochem. Biotechnol. (2009), doi:10.1007/

s12010-008-8337-6.[47] S. Arino, R. Marchal, J.P. Vandecasteele, Appl. Environ. Microbiol. 45 (1996)

162.[48] G.-L. Zhang, Y.-T. Wu, X.-P. Qian, Q. Meng, J. Zhejiang, Univ. Sci. 6B (2005)

725.[49] J.C. Mata-Sandoval, J. Karns, A. Torrents, J. Chromatogr. A 864 (1999) 211.[50] A. Abalos, A. Pinazo, M.R. Infante, M. Casals, F. García, A. Manresa, Langmuir 17

(2001) 1367.[51] X. Wang, L. Gong, S. Liang, X. Han, C. Zhu, Y. Li, Harmful Algae 4 (2005) 433.[52] H. Saeki, K.M. Sasaki, O. Komatsu, A. Miura, H. Matsuda, Bioresour. Technol. 100

(2009) 572.

Glycerol as substrate for the production of biosurfactant by Pseudomonas aeruginosa UCP0992

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