SHORT COMMUNICATION

Biosurfactant Production by Azotobacter chroococcumIsolated from the Marine Environment

R. Thavasi & V. R. M. Subramanyam Nambaru &

S. Jayalakshmi & T. Balasubramanian &

Ibrahim M. Banat

Received: 19 October 2007 /Accepted: 29 October 2008 /Published online: 26 November 2008# Springer Science + Business Media, LLC 2008

Abstract Preliminary characterization of a biosurfactant-producing Azotobacter chroococcum isolated from marineenvironment showed maximum biomass and biosurfactantproduction at 120 and 132 h, respectively, at pH 8.0, 38°C,and 30‰ salinity utilizing a 2% carbon substrate. It grewand produced biosurfactant on crude oil, waste motorlubricant oil, and peanut oil cake. Peanut oil cake gavethe highest biosurfactant production (4.6 mg/mL) underfermentation conditions. The biosurfactant product emulsi-fied waste motor lubricant oil, crude oil, diesel, kerosene,naphthalene, anthracene, and xylene. Preliminary charac-terization of the biosurfactant using biochemical, Fouriertransform infrared spectroscopy, and mass spectral analysisindicated that the biosurfactant was a lipopeptide withpercentage lipid and protein proportion of 31.3:68.7.

Keywords Biosurfactant . Emulsification . Hydrocarbon .

Crude oil .Waste motor lubricant oil . Peanut oil cake

Introduction

Microbial biosurfactants are extracellular products contain-ing both hydrophilic and hydrophobic moieties capable ofreducing surface tension and facilitating hydrocarbonuptake and emulsification/dispersion. They can improvethe bioavailability of hydrocarbons to the microbial cells byincreasing the area of contact at the aqueous–hydrocarboninterface. This increases the rate of hydrocarbon dissolutionand their utilization by microorganisms (Rahaman et al.2002a; Vasileva-Tonkova and Gesheva 2005; Perfumo et al.2006). Biosurfactants can be used in many processesinvolving emulsification, foaming, detergency, wetting,dispersion, and solubilization of hydrophobic compounds(Desai and Banat 1997). They have several advantagesover the chemical surfactants, such as lower toxicity,higher biodegradability (Zajic et al. 1977), environmentalcompatibility (Georgiou et al. 1992), higher foaming(Razafindralambo et al. 1996), selectivity and specificactivity at extreme temperatures, pH, and salinity (Velikonjaand Kosaric 1993), and the ability to be synthesized fromrenewable feedstock (Desai and Banat 1997; Nitschke andPastore 2004).

Although biosurfactants have many interesting properties,their industrial importance depends on the cost and ease ofproduction (Banat et al. 2000). Low yields are a majorlimitation for profitable industrial production and commer-cialization. Hence, in the present study, peanut oil cake andwaste motor lubricant oil were selected as cheaper carbonsources for production. Peanut oil cake is a cheap carbohy-drate, protein, and lipid-rich residue generated in large

Mar Biotechnol (2009) 11:551–556DOI 10.1007/s10126-008-9162-1

R. Thavasi (*)Department of Chemical and Biological Sciences,Polytechnic Institute of New York University,Six Metrotech Center,Brooklyn, NY 11201,, USAe-mail: hydrobact@rediffmail.com

V. R. M. Subramanyam Nambaru : S. Jayalakshmi :T. BalasubramanianCAS in Marine Biology, Annamalai University,Parangipettai 608 502, Tamil Nadu, India

V. R. M. Subramanyam Nambarue-mail: nvrmsubash@rediffmail.com

S. Jayalakshmie-mail: hydrobact@gmail.com

T. Balasubramaniane-mail: stbcas@nic.in

I. M. BanatSchool of Biomedical Sciences, University of Ulster,Coleraine BT52 1SA, Northern Ireland, UKe-mail: im.banat@uslter.ac.uk

amounts during the production of peanut oil. Waste motorlubricant oil is waste oil drained from geared motor vehiclescontaining weathered hydrocarbon fractions. The aims of thisstudy were to optimize biosurfactant production underfermentation condition using cheaper carbon sources andcharacterize the biosurfactant using biochemical, Fouriertransform infrared spectroscopy (FTIR) and mass spectralanalysis.

Materials and Methods

Microorganism

Azotobacter chroococcum was isolated from water samplecollected at Tuticorin harbor, Tamil Nadu, India (08°45′ N,78°13′ E) and characterized by Thavasi et al. (2006).

Optimization of Biosurfactant Production in Shake Flasks

Mineral salt medium containing (g/L) K2HPO4 1.0,MgSO4·7H2O 0.2, FeSO4·7H2O 0.05, CaCl2·2H2O 0.1,

Na2MoO4·2H2O 0.001, and NaCl 5.0 (Benson 1990)supplemented with crude oil or waste motor lubricant oilor peanut oil cake was used for optimization and productionin a fermentor. The strain was cultured at different temper-atures (30°C to 46°C), substrate concentrations (0.5% to2.5%, w/v of crude oil, waste motor lubricant oil, andpeanut oil cake), pH (5.0 to 9.0), and salinities (NaCl 0‰ to40‰). All the experiments were carried out in 500-mLconical flasks containing 100 mL mineral salt medium. Theculture was maintained in a water bath shaker at 150 rpm.Statistical analysis (analysis of variance, ANOVA) wascarried out for all experiments.

Biosurfactant Production in Fermentor

A 3-L laboratory fermentor (Scigenics, India Pvt. Ltd.,Chennai) with a 2.1-L working volume was used. Theculture conditions were: pH 8.0, temperature 38°C,salinity 30‰ and 2.0% substrate (crude oil, peanut oilcake, and waste motor lubricant oil), stirring at 350 rpm,8.0 mg/L dissolved oxygen concentration, and 0.5-kg/cm2

pressure.

Fig. 1 Growth and biosurfactant production of A. chroococcum at different pH (a, b), temperatures (c, d), and salinities (e, f)

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Growth, Biochemical Analysis, and EmulsificationMeasurements

Five-milliliter culture broth samples were collected at12-h intervals for a period of 168 h for gravimetricalbiomass measurements in milligrams per milliliter dryweight as described by Thavasi et al. (2006).

Carbohydrate content of the biosurfactant was deter-mined by the phenol sulfuric acid method (Dubois et al.1956) using D-glucose as a standard. Protein content wasdetermined by the method of Lowry et al. (1951) usingbovine serum albumin as a standard, and lipid content wasestimated adopting the procedure of Folch et al. (1956).

To estimate emulsification activity, purified biosurfactant(1 mg/mL) was dissolved in 5 mL of Tris buffer (pH 8.0) ina 30-mL screw-capped test tube. Five milligrams ofhydrocarbon (waste motor lubricant oil, crude oil, diesel,kerosene, naphthalene, anthracene, or xylene) was added tothe above solution which was shaken well for 20 min andallowed to stand for 20 min. The optical density of themixture was then measured at 610 nm, and the results wereexpressed as OD610 (Rosenberg et al. 1979).

Purification of Biosurfactant

The culture broth was centrifuged at 6,000 rpm for 20 minand extracted with chloroform and methanol (2:1, v/v). Thesolvents were removed by rotary evaporation and theresidue purified on a silica gel (60–120 mesh) columneluting with a chloroform/methanol gradient ranging from20:1 to 2:1 (v/v), collecting ten fractions. The fractions werepooled and the solvents evaporated; the resulting residuewas dialyzed against distilled water and lyophilized asreported by Li et al. (1984). Weight of the biosurfactant wasexpressed in terms of milligrams per milliliter (dry weight).

Fourier Transform Infrared Spectroscopy and MassSpectrometric Analysis

FTIR is most useful for identifying types of chemical bonds(functional groups) and therefore can be used to elucidatesome components of an unknown mixture. Ten milligramsof freeze-dried pure biosurfactant was ground with 100 mgof KBr and pressed with 7,500 kg for 30 s to obtaintranslucent pellets. Infrared absorption spectra were

Fig. 2 Growth and biosurfac-tant production by A. chroococ-cum at different concentrationsof crude oil (a, b), waste motorlubricant oil (c, d), and peanutoil cake (e, f)

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recorded on a Thermo Niocolet, AVATAR 330 FTIR systemwith a spectral resolution and wavenumber accuracy of 4and 0.01 cm−1, respectively. All measurements consisted of500 scans, and a KBr pellet was used as backgroundreference.

Mass spectrometric analysis was carried out as describedby Rahaman et al. (2002b). The purified biosurfactant wasdissolved in methanol and mixed thoroughly. The massspectrometric analysis of the biosurfactant was carried outin a LCQTM quadrupole ion trap mass spectrometer(Finnigan MAT, San Jose, CA, USA) utilizing electrosprayionization (ESI). Standard solutions and samples underinvestigation were infused into the mass spectrometer at aflow rate of 10 μL/min. In the ESI, nitrogen and auxiliarygas flows were maintained at 50 and 5 mL/min, respec-tively, and referred to arbitrary values set by the software.The heated capillary temperature was 250°C and the sprayvoltage set to 5 kV. Negative ion mode was used andscanning was done at 50–2,000 m/z range.

Results and Discussion

Growth and biosurfactant production followed similarpatterns on crude oil with maximum detected at pH 8.0(Fig. 1a,b), at a temperature of 38°C (Fig. 1c,d), and asalinity of 30‰ (Fig. 1e,f). Biomass and biosurfactantproduction were in the range of 1.26 to 3.12 and 0.98 to2.97 mg/mL, respectively. The strain tolerated 30‰ NaCl,which is higher than the results reported by Page (1986) at23‰ for such a strain.

Growth and biosurfactant production on crude oil(Fig. 2a,b), waste motor lubricant oil (Fig. 2c,d), andpeanut oil cake (Fig. 2e,f) showed a maximum at 2.0%substrate concentration for both the substrates tested (1.9and 3.6 mg/mL, respectively). Biosurfactant production onpeanut oil cake both in flask and in fermentor conditionswas higher than on crude oil and waste motor lubricant oil,which indicated its suitability as a cheaper substrate forlarge-scale biosurfactant production. In all culture condi-

Fig. 4 FTIR spectrum ofbiosurfactant produced byA. chroococcum

Fig. 3 Biosurfactant production(a) and growth (b) of A.chrooroccum in a fermentor

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tions tested, biosurfactant concentration was highest at theearly stationary phase, 120–132 h. Higher concentration ofbiosurfactant at the early stationary phase may be due to therelease of cell-bound biosurfactant into the culture brothwhich led to a rise in extracellular biosurfactant concentra-tion (Goldman et al. 1982). Statistical analysis (ANOVA) ofthe influence of pH, temperature, salinity, and substrateconcentration on biosurfactant production showed a highsignificance (p=0.05).

Biomass and biosurfactant production were higher underfermentation conditions ranging from 4.86 to 2.15 mg/mLfor crude oil, 5.12 to 2.82 mg/mL for waste motor lubricantoil, and 8.7 to 4.6 mg/mL for peanut oil cake, respectively(Fig. 3a,b). These results indicated the possibility ofbiosurfactant production at industrial scale using a cheapercost substrate such as peanut oil cake. In other studies,vegetable oils had been used as carbon sources forbiosurfactant production (Rahaman et al. 2002b; Bednarskiet al. 2004), whereas in the present study, peanut oil cake isused, which may be more economically viable for large-scale production.

Emulsification of different hydrocarbons by the biosur-factant was in the order of waste motor lubricant oil > crudeoil > kerosene > diesel > naphthalene > anthracene >xylene, and the emulsification activity (OD610) was 1.51,1.43, 0.67, 0.36, 0.33, and 0.42, respectively. Fernandez-Linares et al. (1996) reported similar emulsification resultsby two marine strains, Pseudomonas nautical and Mar-inobacter hydrocarboclasticus, and concluded that emulsi-fication is a major essential process in alkanebiodegradation. Emulsification of various hydrocarbons bythe biosurfactant used in the present study indicated thepossibility of their application in the remediation ofdifferent types of hydrocarbon pollution either as a meansof their direct removal or as a promoter of biodegradation(Thavasi et al. 2007).

The biosurfactant component of A. chroococcum is alipopeptide with a lipid and protein combination of31.30:68.69%. The FTIR analysis of biosurfactant revealedthat wavenumbers 2,852, 2,923, 1,421, and 1,465 cm−1

resulting from the C–H stretching mode suggested thepresence of aliphatic chain. The presence of N–H and CO–N bonds was indicated by wavenumbers 3,383 and1,647 cm−1, respectively. The C–O bonds were observedat 1,058 cm−1. The obtained wavenumbers are consistentwith the lipopeptide moieties of the biosurfactant (Fig. 4).The FTIR analysis of the biosurfactant was similar to theresults obtained by earlier workers (Onwurah 1999; Kuiperet al. 2004; Das and Mukherjee 2007) who used FTIR as ananalytical tool for the characterization of biosurfactants.

The mass spectral analysis of the biosurfactant showedionized compounds with molecular weights of m/z=326.5,663.4, and 1,347.3 which may represent the lipid and

protein molecules, respectively. Similar results wereobtained by Kuiper et al. (2004) using Pseudomonas putidawith a lipopeptide biosurfactant and by Kalinovskaya et al.(1995) for surfactin, a lipopeptide biosurfactant producedby Bacillus pumilus.

In conclusion, biosurfactants can be produced growingA. chroococcum on economically cheaper carbon sourcesuch as peanut oil cake or waste motor lubricant oil forapplication in oil pollution removal or bioremediation.

Acknowledgments We thank the authorities of Annamalai Univer-sity for providing the facilities and DOD and CSIR, Government ofIndia for financial support.

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