Mar Biotechnol (2009) 11:551–556
DOI 10.1007/s10126-008-9162-1
SHORT COMMUNICATION
Biosurfactant Production by Azotobacter chroococcum
Isolated 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 marine
environment showed maximum biomass and biosurfactant
production at 120 and 132 h, respectively, at pH 8.0, 38°C,
and 30‰ salinity utilizing a 2% carbon substrate. It grew
and produced biosurfactant on crude oil, waste motor
lubricant oil, and peanut oil cake. Peanut oil cake gave
the highest biosurfactant production (4.6 mg/mL) under
fermentation 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, Fourier
transform infrared spectroscopy, and mass spectral analysis
indicated that the biosurfactant was a lipopeptide with
percentage lipid and protein proportion of 31.3:68.7.
R. Thavasi (*)
Department of Chemical and Biological Sciences,
Polytechnic Institute of New York University,
Six Metrotech Center,
Brooklyn, NY 11201,, USA
e-mail: hydrobact@rediffmail.com
V. R. M. Subramanyam Nambaru : S. Jayalakshmi :
T. Balasubramanian
CAS in Marine Biology, Annamalai University,
Parangipettai 608 502, Tamil Nadu, India
V. R. M. Subramanyam Nambaru
e-mail: nvrmsubash@rediffmail.com
S. Jayalakshmi
e-mail: hydrobact@gmail.com
T. Balasubramanian
e-mail: stbcas@nic.in
I. M. Banat
School of Biomedical Sciences, University of Ulster,
Coleraine BT52 1SA, Northern Ireland, UK
e-mail: im.banat@uslter.ac.uk
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 of
reducing surface tension and facilitating hydrocarbon
uptake and emulsification/dispersion. They can improve
the bioavailability of hydrocarbons to the microbial cells by
increasing the area of contact at the aqueous–hydrocarbon
interface. This increases the rate of hydrocarbon dissolution
and their utilization by microorganisms (Rahaman et al.
2002a; Vasileva-Tonkova and Gesheva 2005; Perfumo et al.
2006). Biosurfactants can be used in many processes
involving emulsification, foaming, detergency, wetting,
dispersion, and solubilization of hydrophobic compounds
(Desai and Banat 1997). They have several advantages
over the chemical surfactants, such as lower toxicity,
higher biodegradability (Zajic et al. 1977), environmental
compatibility (Georgiou et al. 1992), higher foaming
(Razafindralambo et al. 1996), selectivity and specific
activity at extreme temperatures, pH, and salinity (Velikonja
and Kosaric 1993), and the ability to be synthesized from
renewable feedstock (Desai and Banat 1997; Nitschke and
Pastore 2004).
Although biosurfactants have many interesting properties,
their industrial importance depends on the cost and ease of
production (Banat et al. 2000). Low yields are a major
limitation for profitable industrial production and commer-
cialization. Hence, in the present study, peanut oil cake and
waste motor lubricant oil were selected as cheaper carbon
sources for production. Peanut oil cake is a cheap carbohy-
drate, protein, and lipid-rich residue generated in large
552
amounts during the production of peanut oil. Waste motor
lubricant oil is waste oil drained from geared motor vehicles
containing weathered hydrocarbon fractions. The aims of this
study were to optimize biosurfactant production under
fermentation condition using cheaper carbon sources and
characterize the biosurfactant using biochemical, Fourier
transform infrared spectroscopy (FTIR) and mass spectral
analysis.
Materials and Methods
Microorganism
Azotobacter chroococcum was isolated from water sample
collected 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,
Fig. 1 Growth and biosurfactant production of A. chroococcum at different pH (a, b), temperatures (c, d), and salinities (e, f)
Mar Biotechnol (2009) 11:551–556
Na2MoO4·2H2O 0.001, and NaCl 5.0 (Benson 1990)
supplemented with crude oil or waste motor lubricant oil
or peanut oil cake was used for optimization and production
in a fermentor. The strain was cultured at different temper-
atures (30°C to 46°C), substrate concentrations (0.5% to
2.5%, w/v of crude oil, waste motor lubricant oil, and
peanut oil cake), pH (5.0 to 9.0), and salinities (NaCl 0‰ to
40‰). All the experiments were carried out in 500-mL
conical flasks containing 100 mL mineral salt medium. The
culture was maintained in a water bath shaker at 150 rpm.
Statistical analysis (analysis of variance, ANOVA) was
carried 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. The
culture conditions were: pH 8.0, temperature 38°C,
salinity 30‰ and 2.0% substrate (crude oil, peanut oil
cake, and waste motor lubricant oil), stirring at 350 rpm,
8.0 mg/L dissolved oxygen concentration, and 0.5-kg/cm2
pressure.
Mar Biotechnol (2009) 11:551–556
Growth, Biochemical Analysis, and Emulsification
Measurements
Five-milliliter culture broth samples were collected at
12-h intervals for a period of 168 h for gravimetrical
biomass measurements in milligrams per milliliter dry
weight 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 was
determined by the method of Lowry et al. (1951) using
bovine serum albumin as a standard, and lipid content was
estimated 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) in
a 30-mL screw-capped test tube. Five milligrams of
hydrocarbon (waste motor lubricant oil, crude oil, diesel,
kerosene, naphthalene, anthracene, or xylene) was added to
the above solution which was shaken well for 20 min and
allowed to stand for 20 min. The optical density of the
mixture was then measured at 610 nm, and the results were
expressed as OD610 (Rosenberg et al. 1979).
Fig. 2 Growth and biosurfac-
tant production by A. chroococ-
cum at different concentrations
of crude oil (a, b), waste motor
lubricant oil (c, d), and peanut
oil cake (e, f)
553
Purification of Biosurfactant
The culture broth was centrifuged at 6,000 rpm for 20 min
and extracted with chloroform and methanol (2:1, v/v). The
solvents were removed by rotary evaporation and the
residue purified on a silica gel (60–120 mesh) column
eluting with a chloroform/methanol gradient ranging from
20:1 to 2:1 (v/v), collecting ten fractions. The fractions were
pooled and the solvents evaporated; the resulting residue
was dialyzed against distilled water and lyophilized as
reported by Li et al. (1984). Weight of the biosurfactant was
expressed in terms of milligrams per milliliter (dry weight).
Fourier Transform Infrared Spectroscopy and Mass
Spectrometric Analysis
FTIR is most useful for identifying types of chemical bonds
(functional groups) and therefore can be used to elucidate
some components of an unknown mixture. Ten milligrams
of freeze-dried pure biosurfactant was ground with 100 mg
of KBr and pressed with 7,500 kg for 30 s to obtain
translucent pellets. Infrared absorption spectra were
554
Fig. 3 Biosurfactant production
(a) and growth (b) of A.
chrooroccum in a fermentor
recorded on a Thermo Niocolet, AVATAR 330 FTIR system
with a spectral resolution and wavenumber accuracy of 4
and 0.01 cm−1, respectively. All measurements consisted of
500 scans, and a KBr pellet was used as background
reference.
Mass spectrometric analysis was carried out as described
by Rahaman et al. (2002b). The purified biosurfactant was
dissolved in methanol and mixed thoroughly. The mass
spectrometric analysis of the biosurfactant was carried out
in a LCQTM quadrupole ion trap mass spectrometer
(Finnigan MAT, San Jose, CA, USA) utilizing electrospray
ionization (ESI). Standard solutions and samples under
investigation were infused into the mass spectrometer at a
flow rate of 10 μL/min. In the ESI, nitrogen and auxiliary
gas 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 spray
voltage set to 5 kV. Negative ion mode was used and
scanning was done at 50–2,000 m/z range.
Fig. 4 FTIR spectrum of
biosurfactant produced by
A. chroococcum
Mar Biotechnol (2009) 11:551–556
Results and Discussion
Growth and biosurfactant production followed similar
patterns on crude oil with maximum detected at pH 8.0
(Fig. 1a,b), at a temperature of 38°C (Fig. 1c,d), and a
salinity of 30‰ (Fig. 1e,f). Biomass and biosurfactant
production were in the range of 1.26 to 3.12 and 0.98 to
2.97 mg/mL, respectively. The strain tolerated 30‰ NaCl,
which is higher than the results reported by Page (1986) at
23‰ for such a strain.
Growth and biosurfactant production on crude oil
(Fig. 2a,b), waste motor lubricant oil (Fig. 2c,d), and
peanut oil cake (Fig. 2e,f) showed a maximum at 2.0%
substrate concentration for both the substrates tested (1.9
and 3.6 mg/mL, respectively). Biosurfactant production on
peanut oil cake both in flask and in fermentor conditions
was higher than on crude oil and waste motor lubricant oil,
which indicated its suitability as a cheaper substrate for
large-scale biosurfactant production. In all culture condi-
Mar Biotechnol (2009) 11:551–556
tions tested, biosurfactant concentration was highest at the
early stationary phase, 120–132 h. Higher concentration of
biosurfactant at the early stationary phase may be due to the
release of cell-bound biosurfactant into the culture broth
which led to a rise in extracellular biosurfactant concentra-
tion (Goldman et al. 1982). Statistical analysis (ANOVA) of
the influence of pH, temperature, salinity, and substrate
concentration on biosurfactant production showed a high
significance (p=0.05).
Biomass and biosurfactant production were higher under
fermentation conditions ranging from 4.86 to 2.15 mg/mL
for crude oil, 5.12 to 2.82 mg/mL for waste motor lubricant
oil, and 8.7 to 4.6 mg/mL for peanut oil cake, respectively
(Fig. 3a,b). These results indicated the possibility of
biosurfactant production at industrial scale using a cheaper
cost substrate such as peanut oil cake. In other studies,
vegetable oils had been used as carbon sources for
biosurfactant production (Rahaman et al. 2002b; Bednarski
et al. 2004), whereas in the present study, peanut oil cake is
used, 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 > crude
oil > 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 results
by two marine strains, Pseudomonas nautical and Mar-
inobacter hydrocarboclasticus, and concluded that emulsi-
fication is a major essential process in alkane
biodegradation. Emulsification of various hydrocarbons by
the biosurfactant used in the present study indicated the
possibility of their application in the remediation of
different types of hydrocarbon pollution either as a means
of their direct removal or as a promoter of biodegradation
(Thavasi et al. 2007).
The biosurfactant component of A. chroococcum is a
lipopeptide with a lipid and protein combination of
31.30:68.69%. The FTIR analysis of biosurfactant revealed
that wavenumbers 2,852, 2,923, 1,421, and 1,465 cm−1
resulting from the C–H stretching mode suggested the
presence of aliphatic chain. The presence of N–H and CO–
N bonds was indicated by wavenumbers 3,383 and
1,647 cm−1, respectively. The C–O bonds were observed
at 1,058 cm−1. The obtained wavenumbers are consistent
with the lipopeptide moieties of the biosurfactant (Fig. 4).
The FTIR analysis of the biosurfactant was similar to the
results obtained by earlier workers (Onwurah 1999; Kuiper
et al. 2004; Das and Mukherjee 2007) who used FTIR as an
analytical tool for the characterization of biosurfactants.
The mass spectral analysis of the biosurfactant showed
ionized compounds with molecular weights of m/z=326.5,
663.4, and 1,347.3 which may represent the lipid and
555
protein molecules, respectively. Similar results were
obtained by Kuiper et al. (2004) using Pseudomonas putida
with a lipopeptide biosurfactant and by Kalinovskaya et al.
(1995) for surfactin, a lipopeptide biosurfactant produced
by Bacillus pumilus.
In conclusion, biosurfactants can be produced growing
A. chroococcum on economically cheaper carbon source
such as peanut oil cake or waste motor lubricant oil for
application in oil pollution removal or bioremediation.
Acknowledgments We thank the authorities of Annamalai Univer-
sity for providing the facilities and DOD and CSIR, Government of
India for financial support.
References
Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial
applications of microbial surfactants. Appl Microbiol Biotechnol
53:495–508
Bednarski W, Adamczak M, Tomasik J, Plaszczyk M (2004)
Application of oil refinery waste in the biosynthesis of
glycolipids by yeast. Bioresour Technol 95:15–18
Benson JH (1990) Microbial applications: a laboratory manual in
general microbiology. WM.C. Brown, Dubuque
Das K, Mukherjee AK (2007) Comparison of lipopeptide biosurfac-
tants production by Bacillus subtilis strains in submerged and
solid state fermentation systems using a cheap carbon source:
some industrial applications of biosurfactants. Process Biochem
42:1191–1199
Desai JD, Banat IM (1997) Microbial production of surfactants and
their commercial potential. Microbiol Mol Biol Rev 61:47–64
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)
Colorimetric method for determination of sugars and related
substances. Anal Chem 28:350–356
Fernandez-Linares L, Acquaviva M, Bertrand J-C, Gauthier M (1996)
Effect of sodium chloride concentration on growth and degrada-
tion of eicosane by marine halotolerent bacterium Marinobacter
hydrocarbonoclastieus. Appl Microbiol 19:113–121
Folch JM, Lees M, Stanly HS (1956) A simple method for the
isolation and quantification of total lipids from animal tissues. J
Biol Chem 226:497–509
Georgiou G, Lin SC, Sharma MM (1992) Surface-active compounds
from microorganism. Biotechnology 10:60–65
Goldman S, Shabtai Y, Rubinovitz C, Rosenberg E, Gutnick DL
(1982) Emulsan in Acinetobacter calcoaceticus RAG-I: distribu-
tion of cell-free and cell associated cross-reacting materials. Appl
Environ Microbiol 44:165–170
Kalinovskaya N, Kuznetsova T, Rashkes Ya, Mil’grom Yu, Mil’grom
E, Willis R, Wood A, Kurtz H, Carabedian C, Murphy P, Elyakov
G (1995) Surfactin-like structures of five cyclic despsipeptises
from the marine isolates of Bacillus pumilus. Russ Chem Bull
44:951–955 (English translation)
Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GEM,
Thomas-Oates JE, Lugtenberg BJJ, Bloemberg GV (2004)
Characterization of two Pseudomonas putida lipopeptide
biosurfactants, putisolvin I and II, which inhibit biofilm
formation and break down existing biofilms. Mol Microbiol
51:97–113
Li Z-Y, Lang S, Wagner F, Witte L, Wary V (1984) Formation and
identification of interfacial-active glycolipids from resting mi-
crobial cells. Appl Environ Microbiol 48:610–617
556
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the Folin phenol reagent. J Biol Chem
193:265–275
Nitschke M, Pastore GM (2004) Biosurfactant production by Bacillus
subtilis using cassava processing effluent. Appl Biochem
Biotechnol 112:163–172
Onwurah INE (1999) Role of diazotropic bacteria in the bioremedi-
ation of crude oil-polluted soil. J Chem Technol Biotechnol
74:957–964
Page WJ (1986) Sodium-dependent growth of Azotobactor chroococ-
cum. Appl Environ Microbiol 51:510–514
Perfumo A, Banat IM, Canganella F, Marchant R (2006) Rhamnolipid
production by a novel thermotolerant hydrocarbon-degrading
Pseudomonas aeruginosa AP02-1. Appl Microbiol Biotechnol
72:132–138
Rahaman KSM, Banat IM, Thahira J, Thayumanavan T, Lakshmana-
perumalsamy P (2002a) Bioremediation of gasoline contaminated
soil by bacterial consortium with poultry litter, coir pith and
rhamnolipid biosurfactant. Bioresour Technol 81:25–32
Rahaman KSM, Rahman TJ, McClean S, Marchant R, Banat IM
(2002b) Rhamnolipid biosurfactant production by strains of
Pseudomonas aeruginosa using low-cost raw materials. Bio-
technol Prog 18:1277–1281
Mar Biotechnol (2009) 11:551–556
Razafindralambo H, Paquot M, Baniel A, Popineau Y, Hbid C,
Jacques P, Thonart P (1996) Foaming properties of surfactin, a
lipopeptide biosurfactant from Bacillus subtilis. J Am Oil Chem
Soc 73:149–151
Rosenberg E, Zuckerberg A, Rubinovitz C, Gutnick DL (1979)
Emulsifier of Arthrobacter RAG-I: isolation and emulsifying
properties. Appl Environ Microbiol 37:402–408
Thavasi R, Jayalakshmi S, Balasubramanian T, Banat IM (2006)
Biodegradation of crude oil by nitrogen fixing marine bacteria
Azotobacter chroococcum. Res J Microbiol 1:401–408
Thavasi R, Jayalakshmi S, Balasubramanian T, Banat IM (2007)
Biosurfactant production by Corynebacterium kutscheri from
waste motor lubricant oil and peanut oil cake. Lett Appl
Microbiol 45:686–691
Vasileva-Tonkova E, Gesheva V (2005) Glycolipids produced by
Antarctic Nocardioides sp. during growth on n-paraffin. Process
Biochem 40:2387–2391
Velikonja J, Kosaric N (1993) Biosurfactant in food application. In:
Kosaric N (ed) Biosurfactants: production, properties, applica-
tions. Marcel Dekker, New York
Zajic JE, Gignard H, Gerson DF (1977) Properties and biodegradation
of a bioemulsifier from Corynebacterium hydrocarbonoclasticus.
Biotechnol Bioeng 91:1303–1320