Bioresource Technology 102 (2011) 3366–3372

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Application of biosurfactant produced from peanut oil cake by Lactobacillus

delbrueckii in biodegradation of crude oil
Rengathavasi Thavasi a,⇑, Singaram Jayalakshmi b, Ibrahim M. Banat c
a
Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 Metrotech Center, Brooklyn, NY 11201, USA
b
CAS in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu, India
c
School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK

a r t i c l e i n f o a b s t r a c t

Article history: Lactobacillus delbrueckii cultured with peanut oil cake as the carbon source yielded 5.35 mg ml1 of
Received 12 October 2010 biosurfactant production. Five sets of microcosm biodegradation experiments were carried out with
Received in revised form 16 November 2010 crude oil as follows: set 1 – bacterial cells + crude oil, set 2 – bacterial cells + crude oil + fertilizer, set 3
Accepted 17 November 2010
– bacterial cells + crude oil + biosurfactant, set 4 – bacterial cells + crude oil + biosurfactant + fertilizer,
Available online 21 November 2010
set 5 – with no bacterial cells, fertilizer and biosurfactant (control). Maximum degradation of crude oil
was observed in set 4 (75%). Interestingly, when biosurfactant and bacterial cells were used (set 3),
Keywords:
significant oil biodegradation activity occurred and the difference between this treatment and that in
Biosurfactant
Peanut oil cake
set 4 was 7% higher degradation level in microcosm experiments. It is evident from the results that bio-
Bioremediation surfactants alone is capable of promoting biodegradation to a large extent without added fertilizers.
Emulsification Ó 2010 Elsevier Ltd. All rights reserved.
Lactobacillus delbrueckii

1. Introduction reaches its peak, nutrient limitation would not be a problem to

Pollution caused by petroleum hydrocarbons in terrestrial and
aquatic environment is a common phenomenon that causes signif-
icant ecological and social problems. Physical and chemical clean-
ing processes used to decontaminate the oil polluted areas have
been limited in their application (Perfumo et al., 2010a). Physical
collection methods such as booms, skimmers, and adsorbents typ-
ically recover no more than 10–15% of the spilled oil and the use of
chemical surfactants as remediating agents is not favorable due to
their toxic effects on the existing biota in the polluted area. There-
fore, despite decades of research, successful bioremediation of oil
contaminated environment still remains a challenge (Perfumo
et al., 2010b).
Biodegradation in aquatic environment is limited by the avail-
ability of nutrients such as nitrogen and phosphorous, which are
necessary for initial microbial cell growth. The use of water-soluble
salts containing nitrogen and phosphorus is effective under labora-
tory conditions, but are readily washed away by surface agitation
and mixing in the aquatic environment. A possible alternate meth-
od could be using biosurfactants along with nutrients in the form
of fertilizers. Because biosurfactants rapidly emulsify the oil and
therefore facilitate fast microbial growth and once the cell number
⇑ Corresponding author. Tel.: +1 718 260 3960; fax: +1 718 260 3075.
E-mail address: hydrobact@gmail.com (R. Thavasi).
precede the biodegradation process.
Biosurfactants are surface active compounds produced by
microorganisms. There are many types of biosurfactants based on
their chemical composition such as glycolipids, lipopolysaccha-
rides, oligosaccharides, and lipopeptides that have been reported
to be produced by diverse bacterial genera (Franzetti et al., 2010;
Banat et al., 2010). Biosurfactants received considerable attention
in the field of environmental remediation processes such as biore-
mediation, soil washing, and soil flushing. Biosurfactants influence
these processes because of their efficacy as dispersion and remedi-
ation agents and their environmentally friendly characteristics
such as low toxicity and high biodegradability (Sivapathasekaran
et al., 2010; Kiran et al., 2010; Satpute et al., 2010).
Although biosurfactants exhibit such important advantages,
they have not yet been employed extensively in industry because
of relatively high production costs. One possible strategy for reduc-
ing costs is the utilization of alternative substrates such as agroin-
dustrial wastes. The main problem related to the use of alternative
substrates as carbon source is to find a waste with the right bal-
ance of nutrients that permits cell growth and product accumula-
tion (Makkar and Cameotra, 1999). Peat hydrolysate (Sheppard and
Mulligan, 1987), molasses (Makkar and Cameotra, 1999), potato
processing effluents (Fox and Bala, 2000), cheese whey and molas-
ses (Rodrigues et al., 2006c), and agriculture residues (Moldes
et al., 2007) are few examples of alternative substrates that have
been used for biosurfactant production. The establishment of

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.11.071
 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372
waste-based medium for biosurfactant production also faces an-
other problem – the kind and the properties of final product are
dependent on the composition of the culture medium. Hence, in
the present study peanut oil cake was tried as a cheaper carbon
source for biosurfactant production. Peanut oil cake is a carbohy-
drate, protein and lipid rich residue generated in large quantities
during the production of peanut oil and the cost of this cake is very
low as compared to other carbon sources like glucose, fructose,
crude oil, and other hydrocarbons.
In this study Lactobacillus delbrueckii was used to investigate its
biosurfactant production and biodegradation potentials. The rea-
son behind selecting this strain for this work is that among many
bacterial genera, genus Lactobacillus is known for its benevolent
uses to humans in many ways such as production of lactic
acid (Wee et al., 2005), yogurt (Omogbai et al., 2005), cheese
(Blaiotta et al., 2001), bacteriocin (Vuyst and Leroy, 2007), and in
some cases the bacteria is used as probiotics, prebiotics
(Teitelbaum and Walker, 2002), and biotherapeutics (Buddington,
2009). Studies on biosurfactant production by Lactobacillus are
scarce and there are only few reports available on biosurfactant
production by the lactic acid bacteria Lactobacillus (Reid et al.,
2002; Rodrigues et al., 2006a; Moldes et al., 2007; Golek et al.,
2007; Gudina et al., 2010) and only two reports on biosurfactant
production using agriculture residues by Lactobacillus pentosus
(Moldes et al., 2007; Portilla-Rivera et al., 2008). So, as an addition
to above reports on beneficial uses of Lactobacillus, L. delbrueckii
used in this study was reported as a strain with biosurfactant pro-
duction and crude oil biodegradation potentials (Thavasi, 2006).
Hence, the present study was undertaken with two objectives: (i)
Evaluation of biosurfactant producing potential of L. delbrueckii
using peanut oil cake, and (ii) Effect of biosurfactant and fertilizer
addition on biodegradation of crude oil by L. delbrueckii.
2. Methods
2.1. Culture conditions and biosurfactant production
L. delbrueckii used in this study was isolated and characterized
as a biosurfactant producing strain and reported earlier (Thavasi,
2006). Optimization of culture conditions such as temperature,
pH, and substrate concentration (carbon source) in shake flask
experiments reported in the above study were used for biosurfac-
tant production and biodegradation experiments in this study.
Biosurfactant production was carried out in a 3 l fermentor with
working volume of 2.1 l (Scigenics India Pvt. Ltd., Chennai).
L. delbrueckii was cultured in mineral salts medium containing
(g l1) 1.0 K2HPO4, 0.2 MgSO47H2O, 0.05 FeSO47H2O, 0.1
CaCl22H2O, 0.001 Na2MoO42H2O, 5.0 NaCl, and peanut oil cake
(2.0%, w/v) as the carbon source. Sterilized culture medium was
inoculated with 1% (v/v) inoculum containing 105 bacterial
cells ml1 and the culture was maintained at 34 °C temperature,
pH 7.5, 350 rpm of agitation and 1.5 l min1 of air flow
(8.5 mg ml1 dissolved oxygen) for 168 h.
Five millilitre samples of culture broth were collected at 24 h
intervals for a period of 168 h. Bacterial cell growth was estimated
using viable cell count method. Briefly, 1 ml of sample drawn from
the culture broth was serially diluted and plated on nutrient agar
plates and incubated for 24 h at 34 °C, colonies developed on the
nutrient agar were counted and expressed as Log CFU ml1.
Concentration of extracellular biosurfactant in the culture broth
was estimated according to the procedure described by Thavasi
et al. (2007) and the biosurfactant concentration was expressed
as mg ml1 (dry weight). Briefly, culture broth was centrifuged at
6000 rpm for 20 min at 4 °C and the cell free culture broth was ex-
tracted twice with chloroform and methanol (2:1, v/v). Solvents in
3367
the extracts were removed by rotary evaporation and the residue
was partially purified in silica gel (60–120 mesh) column eluted
with chloroform and methanol ranging from 20:1 to 2:1 (v/v) in
a gradient manner. The fractions were pooled and solvents were
evaporated. The resulting residue was dialysed against distilled
water and freeze dried. Freeze dried partially purified biosurfactant
was used for further analysis and biodegradation experiments.
2.2. Characterization of biosurfactant
2.2.1. Biochemical composition of biosurfactant
Carbohydrate content of the biosurfactant was determined by
the phenol–sulfuric acid method (Dubois et al., 1956) using D-glu-
cose 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 by following the procedure of
Folch et al. (1956).
2.2.2. Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) is most useful
for identifying types of chemical bonds (functional groups), there-
fore can be used to elucidate some components in an unknown
mixture. Freeze dried biosurfactant (10 mg) was mixed with
100 mg of potassium bromide and pressed with 7500 kg for 30 s
to obtain translucent pellets. Infrared absorption spectra were re-
corded on a Thermo Niocolet, AVATAR 330 FTIR system with a
spectral resolution and wave number accuracy of 4 and
0.01 cm1, respectively. All measurements consisted of 500 scans,
and a potassium bromide pellet was used as background reference.
2.2.3. Mass spectrometric analysis of biosurfactant
Biosurfactant was dissolved in methanol and mixed thoroughly.
The mass spectrometric analysis of the biosurfactant was carried
out in an LCQ™ quadrupole ion-trap mass spectrometer (Finnigan
MAT, San Jose, CA, USA) which utilizes electrospray ionization
(ESI). Standard solutions and samples under investigation were in-
fused into the mass spectrometer at a flow rate of 10 ll min1. In
the ESI, nitrogen and auxiliary gas flows were maintained at 50
and 5 ml min1, respectively and referred to arbitrary values set
by the software. The heated capillary temperature was 250 °C
and the spray voltage was set to 5 kV. Negative ion mode was used
and scanning was performed at 50–2000m/z range.
2.3. Shake flask crude oil biodegradation experiments with different
concentration of fertilizer and biosurfactant
Shake flask biodegradation experiments were carried out in
500 ml Erlenmeyer flasks with 100 ml of mineral salt medium
(same medium used for biosurfactant production) with 2.0%
(w/v) of crude oil. Crude oil used in this study was obtained from
Chennai Refineries Ltd., Chennai, India with a specific gravity of
0.844 at 25 °C. Sterilized culture medium was inoculated with
1% (v/v) inoculum containing 105 bacterial cells ml1 and the cul-
ture flasks were maintained in a shaker at 200 rpm for 168 h. Other
culture conditions used for this experiment were same as
mentioned in biosurfactant production experiments. The effect of
fertilizer and biosurfactant concentration on biodegradation of
crude oil was evaluated with different concentrations of fertilizer
and biosurfactant (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%, w/v). Urea and
K2HPO4 (1:1, w/w) were used as fertilizers. Critical micelle concen-
tration (CMC) obtained for the biosurfactant used in this study was
2 mg ml1 (unpublished data), based on which above concentra-
tions were used to check the effect biosurfactant on biodegrada-
tion. The biosurfactant used in this study for biodegradation
experiments was produced by the same strain that produces the
3368 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372
biosurfactant, i.e., L. delbrueckii. All the experiments were carried
out in duplicate and the mean values were used as results.
2.4. Estimation of growth and crude oil degradation
Crude oil degradation was estimated fluorometrically as de-
scribed in Intergovernmental Oceanographic Commission (IOC)
Manuals and Guide No. 13 (1982). Five millilitres of culture med-
ium was drawn from each above experimental sets and centrifuged
at 6000 rpm to remove the bacterial cells. Crude oil residues from
the cell free culture medium were extracted with n-hexane and to-
tal volume of the extract was made up to 10 ml. Crude oil content
in the n-hexane extract was measured in a Varian, Cary Eclipse
fluorescence spectrophotometer at 310 nm excitation and at
374 nm emission wavelengths. Values were compared with a stan-
dard graph prepared with different concentration of crude oil using
Cary Eclipse software and the degradation levels were expressed as
percentage values. Bacterial cell growth was estimated using viable
cell count method as described in (Section 2.1).
2.5. Laboratory scale microcosm experiment on biodegradation of
crude oil
This experiment was carried out to investigate the influence of
biosurfactant and fertilizers on biodegradation of crude oil in
natural sea water. Seventy five litres plastic tanks were filled with
50 l of filtered and UV treated sea water having 30‰ (‰ – parts per
thousand) salt content, and pH 8.0. Biodegradation experiments
were conducted in five different sets and they are as follows: set
1 – Bacterial cells + mineral salts medium + crude oil (normal),
set 2 – Bacterial cells + mineral salts medium + fertilizer + crude
oil, set 3 – Bacterial cells + mineral salts medium + biosurfactant +
crude oil, set 4 – Bacterial cells + mineral salts medium + fertilizer +
biosurfactant + crude oil, and set 5 – Crude oil + mineral salts med-
ium (control-with no bacterial cells, fertilizer and biosurfactant).
To the above experimental sets, 2.0% (w/v) of crude oil, 0.1%
(w/v) of fertilizer (Urea and K2HPO4), and biosurfactant were
added and inoculated with 1% (v/v) of 24 h old bacterial culture
containing 105 bacterial cells ml1. Continuous aeration was
provided (1.5 l min1) using an oil free aerator and the experimen-
tal sets were maintained at room temperature for a period of 168 h
(Thavasi et al., 2007). Bacterial cell growth and biodegradation of
crude oil was estimated as described above in Sections 2.1 and
2.4, respectively. An uninoculated control (set 5) was maintained
to assess the natural weathering of crude oil and it was estimated
as 6.5% and the value was subtracted from the results obtained in
other experimental sets (sets 1–4).
2.6. Gas chromatographic analysis of degraded residual crude oil
Crude oil extract from culture broth was made up to 10 ml
using n-hexane. A capillary column (30 m Fused Silica column,
Restek Corporation, USA) and GC (Perkin–Elmer 8310) with flame
ionization detector were used for the analysis. The injection and
detector temperature was 250 °C, and column temperature was
programmed as 50 °C/4 min which was then increased at the rate
of 10 °C/min to attain 330 °C and maintained at that temperature
for 20 min. Florida Total Recoverable Petroleum Hydrocarbon
(TRPH) standard (Restek, USA) was used to identify the compounds
in the crude oil (Thavasi et al., 2011).
2.7. Bacterial adhesion to hydrocarbons (BATH assay)
Cell hydrophobicity was measured by bacterial adherence to
hydrocarbons (BATH) as described by Rosenberg et al. (1980). L.
delbrueckii cells obtained from centrifuged culture broth were
washed twice and suspended in a buffer salt solution (g l1 16.9
K2HPO4 and 7.3 KH2PO4) to give an OD of 0.5 at 600 nm. The cell
suspension (2 ml) with 100 ll crude oil was vortex-shaken for
3 min in 5 ml screw capped test tube. After shaking, crude oil
and aqueous phases were allowed to separate for 1 h. The optical
density (OD) of the aqueous phase was then measured at 600 nm
in a spectrophotometer (Varian, Cary Eclipse Spectrophotometer).
For a given sample, three independent determinations were made
and the mean value was calculated.
Cells adhering to oil droplet were visualized by following a
method described by Betts et al. (1989). Briefly, few drops of
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride
(INT) solution was added to the BATH assay culture broth and ob-
served under the microscope. The INT turned red if it was reduced
inside the cells, indicating the viability and visualized the adher-
ence of cells with crude oil droplets. Hydrophobicity was expressed
as the percentage of cell adherence to crude oil and calculated as
follows:
% of bacterial adherence ¼ ð1  ðODshaken with oil =ODoriginal ÞÞ  100
where ODshaken with oil is OD of the mixture containing cells and
crude oil and ODoriginal is the OD of the cell suspension in the buffer
solution (before mixing with crude oil).
2.8. Emulsification assay
Partially purified biosurfactant (1 mg ml1) was dissolved in
5 ml of tris buffer (pH 8.0) in 30 ml test tubes. Hydrocarbons such
as waste motor lubricant oil, crude oil, diesel, kerosene, naphtha-
lene, anthracene, xylene, and peanut oil were tested for emulsifica-
tion activity. Five milligrams of hydrocarbon was added to the
above solution and vortexed for 1 min and the mixture was al-
lowed to stand for 20 min. OD of the emulsified mixture was mea-
sured at 610 nm and the results were expressed as D610. A control
was maintained with above buffer solution and hydrocarbons (no
biosurfactant added), and the values obtained for this control
was subtracted from emulsification values obtained with biosur-
factant. A standard chemical surfactant, Triton X-100 was used to
compare the emulsification of biosurfactant, and the reaction con-
ditions and procedure used were same as described above for
biosurfactant.
3. Results and discussion
3.1. Bacterial cell growth and biosurfactant production
In order to economize the biosurfactant production, the cheaper
carbon source – peanut oil cake was used. Fig. 1 shows the time
course of biosurfactant production by L. delbrueckii with peanut
oil cake as the substrate. Maximum biosurfactant concentration
of 5.35 mg ml1 was observed at 144 h of incubation, when the
cells reached their early stationary phase. Maximum biomass
was observed at 120 h (9.04 Log CFU ml1). Higher biosurfactant
concentration after the offset of growth may be because of the re-
lease of cell-bound biosurfactant at the early stationary phase
(144 h), which leads to an increase in extracellular biosurfactant
concentration in the culture medium. Similar observation was
made by Thavasi et al. (2007, 2008) while culturing Bacillus mega-
terium and Corynebacterium kutscheri with peanut oil cake for bio-
surfactant production. Biosurfactant production reported in this
study for peanut oil cake (5.35 g l1) was higher than earlier re-
ports on use of cheese whey and molasses as carbon sources for
biosurfactant production (Rodrigues et al., 2006a) using four
Lactobacillus strains, in which they obtained 1.45 and 1.7 g l1,
respectively for above carbon sources. This indicated the potential
 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372
6 Biosurfactant
Biosurfactant concentration (mg/ml)
5
4
3 5
2
1
0 0
0 24
Fig. 1. Growth and biosurfactant production by L. delbrueckii using peanut oil cake in fermentor.
use of peanut oil cake as a renewable cheaper carbon source for
biosurfactant production.
3.2. Characterization of biosurfactant
Biosurfactant produced by L. delbrueckii in this study was clas-
sified as a glycolipid with carbohydrate and lipid combination of
30%:70% (w/w). The molecular composition of the biosurfactant
evaluated by FTIR revealed that the most important bands were
located at 2962, 2924, and 2854 cm1 (for the CH aliphatic
stretching), 1793 cm1 (for the C@O ester bond), 1061 cm1 (PII
band:polysaccharides) and 766, 700 cm1 (for the CH2 group)
and 3388 and 3696 cm1 (for OAH bonds) confirming the presence
of glycolipid moieties (figure not shown here, see Supplementary
data) (Thavasi et al., 2008; Rodrigues et al. 2006b). In addition,
the mass spectrometric analysis of the biosurfactant (figure not
shown here, see Supplementary data) also confirmed the above
FTIR results with peaks observed at m/z = 326.5 and at 663.4 for li-
pid and glycolipid moieties, respectively (Thavasi et al., 2008).
However, a detailed structural analysis of the biosurfactant pro-
duced by L. delbrueckii is needed.
3.3. Growth and biodegradation of crude oil with different
concentration of fertilizer and biosurfactant
Experiments conducted with different concentrations of
fertilizer revealed that, 0.1% of fertilizer concentration resulted
maximum degradation of crude oil (61.25%), and cell growth
(9.04 Log CFU ml1) (Fig.2a and c). Beyond 0.1% there was no signif-
icant change observed in bacterial cell growth and biodegradation
rate. As observations made in this experiment, Vyas and Dave
(2010) reported that addition of excess nutrients beyond certain
limit in bioremediation would have no impact on cell growth and
biodegradation process and excess nutrient content can be toxic
to cell growth. Thus, 0.1% fertilizer concentration was used as the
optimum level in microcosm experiments carried out in this study.
Among the different biosurfactant concentrations used in bio-
degradation experiments, maximum crude oil degradation and cell
growth was observed with 0.1% biosurfactant concentration (Fig.2b
and c). There was no significant increase in degradation activity ob-
served at concentrations above 0.1%, which indicates that at this
concentration sufficient emulsification of the crude oil occurred
making it bioavailable for degradation. Biosurfactants are known
for their potential emulsification activity even at very low concen-
tration (Raza et al., 2009). Bacterial strain used in this study is a
3369
Log CFU/ml 10
8
7
Log CFU/ml
6
4
3
2
1
48 72 96 120 144 168
Incubation time (h)
biosurfactant producer and the additional initial biosurfactant
added to the culture medium could be enough to facilitate the cell
access to the crude oil followed with more biosurfactant produc-
tion. In this study, we used the biosurfactant produced by the same
strain in its biodegradation experiments; however a detailed inves-
tigation is needed to determine the effect of biosurfactant pro-
duced by one bacterium on other bacteria to select the potential
biosurfactant, most useful for all biodegrading microbes.
Bacterial cell growth in above experiments conducted with dif-
ferent concentrations of fertilizer and biosurfactant revealed that
cell growth was in its maximum at 120 h of incubation (Fig 2a
and b), which is similar to the observation made in this study for
this strain during biosurfactant production (Fig. 1). Similar growth
trend was reported by Thavasi et al. (2011) for three marine bacte-
rial isolates, B. megaterium, C. kutscheri and Pseudomonas aeruginosa
while culturing with different concentration fertilizer and biosur-
factant in biodegradation experiments.
3.4. Biodegradation of crude oil in laboratory scale microcosm
experiments
Biodegradation of crude oil in laboratory scale microcosm
experiment (Fig. 3a and b) showed maximum growth and biodeg-
radation in set 4 (bacterial cells + crude oil + biosurfactant + fertil-
izer). The difference in biodegradation levels between sets 1
(bacterial cells + crude oil) and 4 was 20.25%. However there was
not much difference between sets 3 (bacterial cells + crude
oil + biosurfactant) and 4 (7%). The difference between sets 4 and
2 (bacterial cells + crude oil + fertilizer) was 12.5%. Interestingly,
the difference in biodegradation rate between experiments with
sets 1 (normal, bacterial cells + crude oil) and 3 (bacterial cells +
crude oil + biosurfactant) was 13.25%, which is higher than the
values obtained for sets 1 and 2 (7.15%). This indicated that the
main factor promoting oil degradation was the presence of biosur-
factant rather than the fertilizer. The improved biodegradation
levels obtained with biosurfactant indicated that they represent
the most efficient accelerators for hydrocarbon biodegradation
through increasing the bioavailability of oil (Perfumo et al.,
2010a, b). The use of biosurfactant in combination with fertilizer
could reduce the actual amount of fertilizer to be added to polluted
sites. In some studies, water soluble fertilizers encountered
problem such as washing away and rapid dilution in aquatic
environment. Maki et al. (2003) reported that fertilizers only stim-
ulate the early stage degradation rate of the oil and that the final
degradation efficiencies with fertilizers were not significantly
3370 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372

12
0.05% 0.10% 0.50% 1% 1.50% 2%
10

8
Log CFU/ml

0
0 24 48 72 96 120 144 168
Incubation time (h)

12
0.05% 0.10% 0.50% 1% 1.50% 2%

10

8
Log CFU/ml

0
0 24 48 72 96 120 144 168
Incubation time (h)

100
Biosurfactant Fertilizer
90

80
% of crude oil degradation

70

60

50

40

30

20

10

0
0.05 0.1 0.5 1 1.5 2
Biosurfactant & Fertilizer concentration (%)

Fig. 2. Growth with different concentrations of (a) fertilizer, (b) biosurfactant, and (c) biodegradation of crude oil by L. delbrueckii in shake flask experiments. Biosurfactant
and fertilizer concentrations are represented in % (w/v).

different from those where no fertilizers were used. It is important
however to keep in mind that nutrients or fertilizer use may be
essential in some environments with insufficient nutrient levels.
Gas chromatographic analysis of the degraded crude oil ex-
tracted from the culture medium commensurate with the degrada-
tion values obtained by quantitative fluorometric analysis.
Bacterium used in this study was able to break the compounds
present in crude oil. Preferential degradation of compounds by
L. delbrueckii revealed that compounds with carbon numbers C22
and C23 were completely degraded (data not shown). Similar
selective degradation of compounds was reported by Thavasi
et al. (2011) for three marine bacterial isolates – B. megaterium,
 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372 3371

12
Normal Fertilizer Biosurfactant Biosurfactant & Fertilizer

10

8
Log CFU/ml
6

0
0 24 48 72 96 120 144 168
Incubation time (h)

100

90

80
% of crude oil degradation

70

60

50

40

30

20

10

0
Normal Fertilizer Biosurfactant Biosurfactant &
Fertilizer

Fig. 3. (a) Growth and (b) biodegradation of crude oil by L. delbrueckii in lab scale microcosm experiments – normal (without biosurfactant and fertilizer) (set 1), fertilizer (set
2), biosurfactant (set 3), and biosurfactant + fertilizer (set 4).

Table 1
C. kutscheri and P. aeruginosa, while culturing them for crude oil Emulsification of hydrocarbons by biosurfactant isolated from L. delbrueckii.
degradation.
Hydrocarbons Emulsification activity (D610)
Biosurfactanta Triton X-100
3.5. BATH assay
Waste motor lubricant oil 1.85 ± 0.14 1.93 ± 0.08
Crude oil 1.75 ± 0.07 1.85 ± 0.07
BATH assay results revealed that a high cell adherence of
Peanut oil 1.45 ± 0.08 1.56 ± 0.04
93.2 ± 1.2% was found for L. delbrueckii cells with crude oil, which Kerosene 1.05 1.12 ± 0.10
directly correlated with the biodegradation potential observed in Diesel 0.85 ± 0.05 0.94 ± 0.02
this study for this strain. Similar high cell hydrophobicity and deg- Xylene 0.55 ± 0.04 0.78 ± 0.12
radation reported by Thavasi et al. (2011) for P. aeruginosa support Anthracene 0.45 ± 0.11 0.58 ± 0.01
Naphthalene 0.40 ± 0.01 0.63
the results obtained in this study. Cell surface properties are
a
important factors that determine the rate of degradation of hydro- Biosurfactant isolated from L. delbrueckii.
phobic substrates. Therefore, isolates with high hydrophobicity are
likely to be more efficient degraders, as reported in this study for L.
delbrueckii. Cell hydrophobicity is also an indication of biosurfac- lower toxicity, biodegradability and ecological acceptability, their
tant production (Franzetti et al., 2009). use in bioremediation activity becomes more favorable. Emulsifi-
cation of different hydrocarbons by the biosurfactant was in the
3.6. Emulsification assay order of waste motor lubricant oil > crude oil > peanut oil >
kerosene > diesel > xylene > anthracene > naphthalene. Crude oil
Emulsification activity of biosurfactant isolated from L. del- therefore was the second compound that has been highly emulsi-
brueckii in this study was comparatively less than the emulsifica- fied by biosurfactant used in this study, which coincided with
tion activity recorded with the standard chemical surfactant the enhanced biodegradation of crude oil obtained in sets 3 and
Triton X-100 (Table 1). However, when considering the advantages 4 in this study. It is evident from the above results that emulsifica-
of biosurfactants over chemically synthesized surfactants such as tion is an essential process in biodegradation.
3372 R. Thavasi et al. / Bioresource Technology 102 (2011) 3366–3372
Influence on biodegradation and emulsification activity of the
biosurfactant isolated in this study was comparatively less than
earlier reports on emulsification and biodegradation of crude oil
with biosurfactants isolated from marine bacterial isolates (Thav-
asi et al., 2010). But the ability to improve the degradation process
and emulsification of hydrocarbons are still comparable with
above report. A detailed study on influence of biosurfactant iso-
lated from L. delbrueckii on biodegradation of crude oil with other
potential oil degrading bacteria and its toxicity on those bacteria
could make this biosurfactant more economically and environ-
mentally viable.
4. Conclusion
Biosurfactant produced by L. delbrueckii using peanut oil cake in
this study showed its potential to be used in bioremediation pro-
cess. Unlike medicinal applications, environmental application of
biosurfactants needs comparatively less purity and high activity.
In this study peanut oil cake was used as the carbon source for bio-
surfactant production. Even though the biosurfactant was not puri-
fied to its purest form and structurally not well characterized but
the results on emulsification and biodegradation experiments re-
vealed the potential use of this biosurfactant in bioremediation
of hydrocarbon pollution. Which emphasize that for environmen-
tal applications the biosurfactants need not be pure and could be
synthesized from a mixed cheaper carbon source like peanut oil
cake used in this study. All these approaches will make the biore-
mediation process an economically and environmentally viable
mitigation technology.
Acknowledgements
We thank the authorities of Annamalai University for providing
the facilities and Department of Ocean Development (DOD) and
Council of Scientific and Industrial Research (CSIR), Government
of India for providing financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2010.11.071.
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