Production and stability studies of the biosurfactant isolated from ...

Desalination 285 (2012) 198–204
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Desalination
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Production and stability studies of the biosurfactant isolated from marine
Nocardiopsis sp. B4
A. Khopade a , R. Biao b , X. Liu b , K. Mahadik a , L. Zhang b , C. Kokare a,c, ⁎
a Department of Pharmaceutical Biotechnology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, 411 038, India
b Chinese Academy of Science Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
c Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune, 411 041, India
article
info
abstract
Article history:
Received 7 June 2011
Received in revised form 21 September 2011
Accepted 4 October 2011
Available online 9 November 2011
Keywords:
Biosurfactant
Nocardiopsis
Optimization
Stability
Actinomycetes
A potential biosurfactant producing strain, marine Nocardiopsis B4 was isolated from the West coast of India.
Culture conditions involving variations in carbon and nitrogen sources were examined at constant pH, temperature
and revolutions per min (rpm), with the aim of increasing productivity in the process. The biosurfactant
production was followed by measuring the surface tension, emulsification assay and emulsifying
index E24. Enhanced biosurfactant production was carried out using olive oil as the carbon source and phenyl
alanine as the nitrogen source. The maximum production of the biosurfactant by Nocardiopsis occurred at a C/
N ratio of 2:1 and the optimized bioprocess condition was pH 7.0, temperature 30°C and salt concentration
3%. The production of the biosurfactant was growth dependent. The surface tension was reduced up to
29 mN/m as well as the emulsification index E24 was 80% in 6 to 9 days. Properties of the biosurfactant
that was separated by acid precipitation were investigated. The biosurfactant activity was stable at high temperature,
a wide range of pH and salt concentrations thus, indicating its application in bioremediation, food,
pharmaceutical and cosmetics industries.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Research in the area of biosurfactants has expanded in recent
years due to its potential use in different areas, such as the food industry,
agriculture, pharmaceuticals, oil industry, petro chemistry,
paper and pulp industry. The development of this line of research is
of paramount importance, mainly in view of the present concern regarding
the protection of the environment. They are not only useful
as antibacterial, antifungal and antiviral agents, but also have potential
for use as major immunomodulatory molecules, anti-adhesive
agents and even in vaccines and gene therapy [1–4]. Involvement of
biosurfactants in microbial adhesion and desorption has been widely
described. For example, inhibition of uropathogen biofilm formation
on silicone rubber by protein-like biosurfactants obtained from
Lactobacillus fermentum RC-14 was reported [1]. Also, exposure to
suspensions of active probiotics and the consumption of buttermilk
containing Lactococcus lactis 53 were reported to influence the
biofilm formation on silicone rubber voice prostheses [1], possibly
due to the release of biosurfactants. Biosurfactants, are produced by
bacteria or yeast from various substrates including sugars, glycerol,
oils, hydrocarbons and agricultural wastes. Biosurfactants are
⁎ Corresponding author at: Department of Pharmaceutics, STES, Sinhgad Institute of
Pharmacy, Narhe, Pune, 411 041, India. Tel.: +91 20 66831806; fax: +91 20 24699051.
E-mail address: kokare71@rediffmail.com (C. Kokare).
classified as glycolipids, lipopeptides, phospholipids, fatty acids, neutral
lipids, and polymeric or particulate compounds [2,5]. The hydrophobic
portion of the molecule is long-chain fatty acids, hydroxyl
fatty acids or a-alkyl-b-hydroxyl fatty acids. The hydrophilic moiety
can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic
acid or alcohol. Biosurfactants have been receiving increasing attention
as a result of their unique properties, i.e. mild production
conditions, lower toxicity and higher biodegradability, compared to
their synthetic chemical counterparts [3]. Even though interest in biosurfactants
is increasing, these compounds do not compete economically
with synthetic surfactants. To reduce production costs, different
routes could be investigated such as the increase of yields and product
accumulation; the development of economical engineering
processes and the use of cost-free or cost credit feedstock for microorganism
growth and surfactant production. The choice of inexpensive
raw materials is important to overall economy of the process because
they account for 50% of the final product cost and also reduce the
expenses with wastes treatment [1,6,7]. Glycolipid from
Rhodococcus erythropolis, R. aurantiacus and surface active lipid from
Nocardia erythropolis were studied in the literature survey. There
are very few reports on biosurfactants production from marine
actinomycetes [6].
The objective of present study was to isolate marine actinomycetes
and the identification of strain for potent biosurfactant
production. The cultural conditions for maximum production of
biosurfactant and the stability study of the product were investigated.
0011-9164/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.desal.2011.10.002

A. Khopade et al. / Desalination 285 (2012) 198–204
199
2. Material and methods
2.1. Isolation of actinomycetes strain B4
Marine sediment sample was collected from Mumbai coastal region
of India at the time of low tide [8]. Collected sediment sample
was suspended in sterile water and mixed on rotary incubator shaker
(New Brunswick Scientific, Model Excella E24, USA), at 150 rpm for
20 min. Different marine actinomycete species were isolated by
using selective media such as glycerol yeast extract agar, starch casein
agar, maltose yeast extract agar and glucose aspargine agar (Himedia,
India). The isolated strains were screened for biosurfactant production
by using different techniques [9–14]. Maximum biosurfactant
producing marine sp. B4 was maintained on glycerol yeast extract
agar medium [15].
2.2. Identification of strain B4
Identification of strain was done by scanning electron microscopy
(SEM) (Pune, India), 16S r-DNA sequencing, biochemical and cultural
characterizations. The method adopted for preparation of slide culture
for SEM analysis was used, as described by Williams and Davies
[12,15–17].
2.3. Inoculum preparation and culture condition
The glycerol yeast extract medium prepared in artificial seawater
(ASW) was used for development of inoculum. The seed culture
was prepared in 100 ml conical flasks containing 50 ml of medium
by inoculating 2.0 ml of spore suspension containing 2.5 to
3.0×10 6 CFU ml −1 and cultivated with agitation (150 rpm) at 28 °C
for 4 days. The seed culture (50 ml) was inoculated in the 1 l fermentation
medium prepared in artificial sea water supplemented with
0.1 ml trace element solution. The pH of the medium was adjusted
to 7.0. Fermentation was carried out in 2 l bench scale fermenter
(New Brunswick Scientific, USA) for 12 days. The operating conditions
during batch fermentation were temperature 28 °C, agitation
rate 150 rpm and the aeration rate 1.0 VVM [8].
2.4. Medium optimization
The medium optimization was conducted in a series of experiments
changing one variable at a time, keeping other factors
unchanged. The production of biosurfactant was growth dependent.
Cell growth and the accumulation of metabolic products were strongly
influenced by medium composition such as carbon sources, nitrogen
sources. Three factors were chosen aiming to obtain higher
productivity of the biosurfactant: carbon source (C), nitrogen source
(N) and C/N ratio. The carbon sources used were n-hexadecane (2%
w/v) (Himedia, India), olive oil (2% w/v) (commercial type), sucrose
(Himedia, India), trehalose (Himedia, India), maltose (Himedia,
India), dextrose and glucose (Himedia, India) (20 g/l), with ammonium
chloride (NH 4 Cl) (Himedia, India) as nitrogen source. For evaluation
of the most appropriate nitrogen sources for the production of
biosurfactants, phenyl alanine (Himedia, India), urea (Himedia,
India), ammonium sulfate (Merck, India), NH 4 Cl and sodium nitrate
(NaNO 3 )(Merck, India), were employed at a concentration of 1 g/l
with the optimum carbon source. The C/N ratio (with optimized
carbon and nitrogen sources) was varied from 10 to 40 by keeping a
constant nitrogen source concentration 1 g/l [18–21].
2.5. Biosurfactant production kinetics
The kinetics of biosurfactant production was followed in batch
cultures during 12 days at optimum conditions by measuring surface
tension and emulsification assay of supernatant samples obtained
after cell separation [18,19].
2.6. Effect of pH, temperature, sodium chloride and aeration on
biosurfactant production and activity
In order to evaluate the effect of pH and temperature on the biosurfactant
production, the pH of medium was adjusted in the range
between 4 and 12 and the temperature was set at 4, 15, 25, 30, 35
40, 45 and 60 °C. The pH of the medium was measured with a digital
pH-meter (Systronics, India). To examine the effect of sodium chloride
on biosurfactant production in optimized medium, the sodium
chloride was added in medium to achieve final concentrations of
1–10% (w/v). Effect of aeration on production of biosurfactant was
detected by incubating inoculated fermentation media at different
aeration conditions such as 50, 75, 100, 125, 150, 175, 200, 225 and
300 rpm. Biosurfactant production was measured by emulsification
assay and absorbance was measured at 400 nm [21].
2.7. Effect of oils, surfactants and hydrocarbon on biosurfactant
production
The effect of crude oil and surfactant was evaluated for biosurfactant
production. The different oils were used such as castor oil, codliver
oil, eucalyptus oil, sesame oil, mustard oil and surfactants such
as ethylene diamine tetra acetic acid (EDTA) (Himedia, India), cetyl
trimethyl ammonium bromide (CTAB) (Himedia, India), sodium
dodicyl sulfate (SDS) (Loba Chemie, India), tweens 20, 40, 80 (Loba
Chemie, India) were added separately in 1% (v/v) and emulsification
activity of medium was measured. The hydrocarbons such as diesel,
petrol, toluene, xylene, n-hexane and kerosene (commercial grade,
India) were added [1% (v/v)] separately in optimized medium and
their effect was observed. The surface tension measurement was carried
out using the du Nouy ring method [6,8].
2.8. Surface tension measurement
The surface tension measurement of cell free supernatant was
determined in a K6 tensiometer, using the du Nouy ring method.
The values reported were the mean of three measurements. All measurements
were made on cell-free broth obtained by centrifuging the
cultures at 10,000 rpm for 20 min [18].
2.9. Bioemulsifier production assay
Actinomycetes species were grown for 12 to 15 days in glycerol
yeast extract (GYE) broth. The microbial cells were separated by centrifugation
(Eppendorff, model 5810R, Germany) at 10,000 rpm for
15 min at 30 °C. Cell free culture broth (3 ml) was added in 0.5 ml
test oil, mixed vigorously for 2 min and incubated at 30 °C for 1 h
for phase separation. Aqueous phase was removed carefully and absorbance
of aqueous phase was recorded at 400 nm. The absorbance
maxima arrived after scanning at entire visible light spectrum. The
blank was prepared with sterile medium. An absorbance of
0.01 units at 400 nm multiplied by dilution factor, if any, was considered
as one unit of emulsification activity per ml (EU/ml) [6].
2.10. Emulsification index (E24)
Emulsification index of culture samples was determined by adding
2 ml of a hydrocarbon to the same amount of culture, mixing with a
vortex for 2 min, and left standing for 24 h. The E24 index is given
as percentage of height of emulsified layer (mm) divided by total
height of the liquid column (mm) [9–11].

200 A. Khopade et al. / Desalination 285 (2012) 198–204
2.11. Biosurfactant recovery
Culture broth was centrifuged at 10,000 rpm for 20 min to get cell
free broth. Biosurfactant was precipitated by adjusting the pH of the
cell free broth to 2.0 using 6 N hydrochloric acid (HCl) (Merck,
India) and keeping it overnight at 4 °C. The precipitate thus formed
was collected by centrifugation (10,000 rpm, 20 min; 4 °C) and dissolved
in distilled water. Its pH was adjusted to 8.0 with 1 N sodium
hydroxide (NaOH) (Qualigens, India), and the solution was lyophilized
[18].
2.12. Stability studies
To determine the thermal stability of the biosurfactant, cell-free
broth was maintained at a constant temperature range of 20–100 °C
for 15 min, then cooled to room temperature and activity of the biosurfactant
was investigated. To determine the effect of pH on activity,
the pH of the cell free broth was adjusted to different values using 1 N
NaOH or 1 N HCl. The effect of addition of different concentration of
NaCl on the activity of the biosurfactant was investigated. The biosurfactant
was re-dissolved after purification with distilled water containing
the specific concentration of NaCl (0–9%, w/v) [22,23].
3. Results and discussions
The advantages of biosurfactants over synthetic ones include
lower toxicity, biodegradability, selectivity, specific activity at extreme
temperatures, pH and salinity. In our laboratory, we have
isolated the strain of marine Nocardiopsis from sediments.
3.1. Characterization of strain B4
The strain B4 showed good growth in the temperature range
25–45 °C in 7 days on glycerol yeast extract agar medium. Outer surface
of colonies was perfectly round initially, but later they developed
aerial mycelium that may appear velvety and spore formation started
after the 4th day of incubation. Spore chain was long and sporulating
hyphae were straight. Spores were oval and warty, appeared like
hairy and were 1–2.5 mm in size (Fig. 1A and 1B). Good growth was
observed at neutral pH. By morphology, SEM and 16S DNA sequencing
(Fig. 2), the isolated strain was found to be a member of
Nocardiopsis genus [8].
3.2. Growth characteristics and biosurfactant production from strain B4
Most of the actinomycetes species are slow-growing. Biosurfactant
production started in early log phase but there was drastic
increase in production at late growth phase and early stationary
phase; and biosurfactant production continued up to late stationary
phase and after that it declined (Fig. 3). It clearly indicates that biosurfactant
production was dependent on the growth phase [8].
3.3. Optimization of cultivation medium
3.3.1. Effect of carbon source
The production of biosurfactant was studied by using carbon
sources such as n-hexadecane, olive oil, trehalose, sucrose, fructose,
maltose and glucose (Fig. 4A). The use of vegetable oil as carbon
sources to produce biosurfactants seems to be an interesting and
low cost alternative [18]. Screening of nutrient substrates showed
that Nocardiopsis supported growth on all substrates although the
yield was limited with galactose or starch as carbon sources because
of inhibition due to the decrease in pH is probably caused by the
production of secondary acid such as uronic acid [18]. The maximum
biosurfactant production was occurring only with trehalose, hexadecane
and olive oil. The strain grew on starch but did not produce
Fig. 1. Scanning electron microscopy of strain B4 [A] ×3000 magnification, and
[B] ×6000 magnification.
the surfactant under these conditions. Olive oil was the best carbon
source for biosurfactant synthesis. The isolated biosurfactant decreased
the surface tension to 30 mN/m and the emulsifying activity
was 80%. Similar results were found with biosurfactant production
form P. aeruginosa 44T1 [24,25].
3.3.2. Effect of nitrogen source
The effect of nitrogen source affects the biosurfactant production
as shown in Fig. 4B. Nocardiopsis sp. was able to use nitrogen sources
such as ammonia and urea for biosurfactant production. However, in
order to obtain high concentrations of biosurfactant it is necessary to
have restrained conditions of these macro-nutrients. Phenyl alanine
was the best source of nitrogen for growth and biosurfactant synthesis.
Ammonium salts in the form of ammonium chloride were used
for growth but not for biosurfactant production and caused a significant
decrease in pH (4.03) [24,26,27]. The maximum emulsifying
activity and minimal surface tension (30 mN/m) were reached in
media with phenyl alanine. No significant change in pH was observed
in this case. A similar result was reported in biosurfactant isolated
from Pseudomonas fluorescens by Abouseoud et al. [18].
3.3.3. Effect of carbon/nitrogen ratio
The fundamental aspect to the improvement of biosurfactant productivity
was the ratio of C/N. These results were obtained using olive
oil and phenyl alanine as carbon and nitrogen source respectively
since the best results were attained with lower values of this parameter
(C/N=20 ) (ST=30; EA=265 U/ml) (Fig. 4C). There were no
significant differences between C/N ratios of 30, 35 and 40 in relation
to emulsification index, but a C/N ratio of 20 presented a significant
difference in relation to the emulsification index. These results are
similar with those found using waste frying oil and sodium nitrate
as carbon and nitrogen sources respectively [25–28]. Guerra-Santos
et al. [29] observed that biosurfactant production was poor with

A. Khopade et al. / Desalination 285 (2012) 198–204
201
76
76
95
99 Nocardiopsis exhalans ES10.1 T (AY036000)
Nocardiopsis valliformis 20028 T (AY336503)
Nocardiopsis metallicus KBS6 T (AJ420769)
Nocardiopsis ganjiahuensis HBUM 20038 T (AY336513)
Nocardiopsis prasina DSM 43845 T (X97884)
Nocardiopsis listeri DSM40297 T (X97887)
76
Nocardiopsis alkaliphila DSM 44657 T (AY230848)
B4
Nocardiopsis alba DSM 43377 T (X97883)
Nocardiopsis umidischolae 66/93 T (AY036001)
100
Nocardiopsis tropica VKM Ac -1457 T (AF105971)
Marinactinospora thermotoleransSCSIO 00652 T (EU698029)
0.005
Fig. 2. Neighbor-joining phylogenetic tree of strain B4 made by MEGA 4.0. Numbers at nodes indicate levels of bootstrap support (%) based on a neighbor-joining analysis of 1000
resampled datasets; only values >50% are given. NCBI accession numbers are given in parentheses. Bar, 0.005 nucleotide substitutions per site.
both yeast extract and nitrate as nitrogen sources. When the yeast extract
was omitted, the biomass concentration decreased, rhamnolipid
increased and a moderate accumulation of glucose occurred, indicating
a nitrogen-limiting medium.
3.4. Kinetics of biosurfactant production
The biosurfactant production and surface tension were dependent
on growth of culture in the fermentation medium. The surface tension
dropped rapidly after inoculation, reaching its lowest value
(29 mN/m) during exponential phase after about 9 days of growth
(Fig. 3A and Fig. 3B). The emulsification activity plot, a measure of
biosurfactant concentration, showed that insufficient surfactant was
initially present to form micelles. At about 6th day of growth, the surfactant
concentration started to increase, reaching its maximum after
about 9th day. The increase in surface tension and the decrease in E24
after 12th of incubation showed that biosurfactant biosynthesis
stopped and is probably due to the production of secondary metabolites
which could interfere with emulsion formation and the adsorption
of surfactant molecules at the oil–water interface [30]. These
results indicate that the biosurfactant biosynthesis from olive oil occurred
predominantly during the exponential growth phase, suggesting
that the biosurfactant is produced as primary metabolite
accompanying cellular biomass formation (growth-associated kinetics)
[31]. This property suggests that biosurfactant could be effectively
produced under chemostat conditions or by immobilized cells
[18,19,32,33].
3.5. Effect of sodium chloride, pH, temperature and aeration on
biosurfactant production
The strain B4 was found to be moderately halophilic in nature as
maximum biosurfactant production was obtained in presence of 3%
Fig. 3. Growth kinetics and biosurfactant production of Nocardiopsis B4 sp. (OD600).
(w/v) of NaCl and it retained almost 80% of its activity in presence
of 12% (w/v) of NaCl. (Fig. 5A). The strain B4 showed gradual increase
in biosurfactant production and optimum pH for biosurfactant production
was found to be 7 (Fig. 5B). The research was focused on
the isolation of alkaline biosurfactant from microbes because there
is tremendous potentiality of biosurfactant in detergent industry.
The strain B4 showed good growth in the temperature range of
25–45 °C but optimum growth was observed at 30 °C (Fig. 5C). This
clearly indicates the moderately thermostable nature of the biosurfactant.
The maximum biosurfactant production was obtained at
150 rpm. Until today, bacteria belonging to genus Bacillus have been
exploited for commercial production of biosurfactant [21,24]. There
is no such report on isolation of moderately thermostable surfactant
from marine Nocardiopsis sp.
3.6. Effect of oils, surfactants and hydrocarbons on production of
biosurfactant
Fermentation was carried out with addition of different concentrations
of oils, surfactant and hydrocarbons in the fermentation medium.
It was observed that olive oil, tween 80 and hexane as a
substrate showed maximum activity against all test oils, surfactants
and hydrocarbons respectively. Olive oil and tween 80 showed emulsification
activity at 198 EU/ml and 225 EU/ml respectively (Fig. 6A
and 6B). Hexane was used [6] as a substrate for biosurfactant production
and it was observed that 1% v/v showed maximum biosurfactant
production activity (Fig. 6C).
3.7. Stability study
3.7.1. Temperature stability
The applicability of biosurfactants in several fields depends on
their stability at different temperatures and pH values. The stability
of biosurfactant was tested over a wide range of temperature. The
biosurfactant produced by Nocardiopsis sp. was shown to be thermostable
(Fig. 7A). Heating of the biosurfactant to 100 °C caused no significant
effect on the biosurfactant performance. The emulsification
activity was quite stable at the temperatures used (E24=66%) in
comparison with synthetic surfactants such as SDS which exhibits a
significant loss of emulsification activity beginning at 70 °C [31].
Therefore, it can be concluded that this biosurfactant maintains its
surface properties unaffected in the range of temperatures between
30 and 100 °C. This activity was discovered indicating the usefulness
of the biosurfactant in food, pharmaceutical and cosmetics industries
where heating to achieve sterility is of paramount importance
[18,25].

202 A. Khopade et al. / Desalination 285 (2012) 198–204
Surface tension (mN/m)
AEmulsification activity (EU/ml)
E24 (%)
B
Surface tension (mN/m)
C
100
80
60
40
20
0
80
60
40
20
0
Olive oil
Galato se
Ammo. sulphate
300
200
100
0
Phenyl alanine
Dextrose
Strac h
Mannitol
Sucrose
Trehalose
Carbon sources
Alanine
Ammo. chloride
Urea
Nitrogen source
Hexadecne
Fructo se
Maltose
Aspargine
Sodium nitrate
Emlusification activity
Surface tension
10 15 20 25 30 35 40
C:N ratio
A
Surface tension (mN/m)
0 20 40 60 80
B
Surface tension (mN/m)
C
Surface tension (mN/m)
80
60
40
20
0
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10
NaCl (% w/v)
4
5
6
7
4
15
25
30
8
pH
35
9
10
11
12
40
Temperature (°C)
45
60
Fig. 4. [A] Effect of carbon source on biosurfactant production, [B] effect of nitrogen
source on biosurfactant production, and [C] effect of C:N ratio on biosurfactant
production.
Fig. 5. [A] Effect of NaCl on biosurfactant production, [B] effect of pH on biosurfactant
production, and [C] effect of temperature on biosurfactant production.
3.7.2. pH stability
The surface activity of the crude biosurfactant remained relatively
stable to pH changes between pH 8 and 12, showing higher stability
at alkaline pH 9 than acidic conditions. At pH 12, the value in emulsification
activity (E24) showed almost 66% activity, whereas below pH
7 activity was decreased up to 55%. In addition, for pH values lower
than 6, the samples become turbid, due to partial precipitation of
the biosurfactant. Fig. 7B shows the effect of pH on the biosurfactant
properties. These results indicate that increase pH has a positive effect
on emulsification activity and emulsion stability. This could be
caused by a better stability of fatty acid surfactant micelles in the
presence of NaOH and the precipitation of secondary metabolites at
higher pH values. The effect of pH on surface activity has been
reported for biosurfactants for different microorganisms [18,34].
3.7.3. Effect of salinity
The effect of sodium chloride addition on biosurfactant produced
from Nocardiopsis was studied. Optimum stability of biosurfactant
was observed at 3% NaCl concentration. Little changes were observed
in increased concentration of NaCl up to 8% (w/v) (Fig. 7C). At higher
concentration of NaCl the biosurfactant retains 50% of the emulsification
activity. The biosurfactant has stability at alkaline pH and high
salinity; such a biosurfactant may be useful for bioremediation of
spills in marine environment because of its stability in alkaline condition
and in the presence of salt. Stability of emulsion in the presence

A. Khopade et al. / Desalination 285 (2012) 198–204
203
A
250
A
150
Emulsification activity
(EU/ml)
200
150
100
50
E24 (%)
100
50
0
Caster oil
Olive oil
Ecualyptus oil
Clove oil
Coconut oil
Oils
Cod liver oil
Senamom oil
B
0
150
30
40
50
60
70
80
Temperature (°C)
90
100
B
Emulsification activity (EU/ml)
300
200
100
0
EDTA
CTAB
SDS
Tween 20
Tween 40
Surfactants
Tween 80
Triton X-100
E24 (%)
C
100
50
0
150
4
5
6
7
8
pH
9
10
11
12
C
Surface tension (mN/m)
80
60
40
20
E24 (%)
100
50
0
1
2
3
4
5
6
NaCl (% w/v)
7
8
9
of salt has been reported as one of the properties of the biosurfactant
produced by Bacillus licheniformis strain JF-2 [21].
4. Conclusions
0
Tolune
n Hexane
Xylene
Kerosens
Hydrocarbons
Petrol
Diesel
Fig. 6. [A] Effect of oils on biosurfactant production, [B] effect of surfactants on biosurfactant
production, and [C] effect of hydrocarbons on biosurfactant production.
In the present study the biosurfactant isolated from marine
Nocardiopsis sp. showed good stability at high temperature, a wide
range of pH and salt concentrations and the maximum biosurfactant
production was observed with olive oil as a carbon source and phenyl
alanine as the nitrogen source. The important finding was thermo
stability of biosurfactant; isolated biosurfactant was extreme stability
Fig. 7. Effect of [A] temperature, [B] pH and [C] salinity on biosurfactant stability.
at high temperature (100°). The thermal stability of the biosurfactants
increases its scope of application in a broader perspective
including at conditions where high temperatures prevail as in microbial
enhanced oil recovery. Considering the potential need of halotolerant
strains and biosurfactants for the bioremediation of oil
contaminated sites (oil spills), it is mandatory to screen and develop
potential biosurfactant producers from the marine environment.
It was found that the biosurfactant produced by the marine
Nocardiopsis was stable up to 8% NaCl; however the chemical surfactants
are deactivated by 2–3% salt concentration. The biosurfactant
was isolated from natural sources thus, indicating the application of
the biosurfactant in food, pharmaceutical and cosmetics industries.
Acknowledgments
The authors would like to acknowledge All India Council for Technical
Education (AICTE), HRD Ministry, New Delhi, Govt. of India for

204 A. Khopade et al. / Desalination 285 (2012) 198–204
financial support to this research project under National Doctoral
Fellowship (NDF), 2010–2013.
References
[1] L.R. Rodrigues, J.A. Teixeira, H.C. Vander Mei, R. Oliveira, Physicochemical and
functional characterization of a biosurfactant produced by Lactococcus lactis 53,
Colloids Surf., B 49 (2006) 79–86.
[2] J.D. Desai, I.M. Banat, Microbial production of surfactants and their commercial
potential, Microbiol. Mol. Biol. Rev. 61 (1997) 47–64.
[3] E.Z. Ron, E. Rosenberg, A review of natural roles of biosurfactants, Environ. Microbiol.
3 (2001) 229–236.
[4] I.M. Banat, R.S. Markkar, S.S. Cameotra, Potential commercial applications of microbial
surfactants, Appl. Microbiol. Biotechnol. 53 (2000) 495–508.
[5] S. Mukherjee, P. Das, R. Sen, Towards commercial production of microbial surfactants,
Trends Biotechnol. 24 (2006) 509–515.
[6] C.R. Kokare, S.S. Kadam, K.R. Mahadik, B.A. Chopade, Studies on bioemulsifier production
from marine Streptomyces sp. S1, Ind. J Biotech. 6 (2007) 78–84.
[7] S. Maneerat, K. Phetrong, K. Song, Isolation of biosurfactant-producing marine
bacteria and characteristics of selected biosurfactant, J. Sci. Technol. 29 (2007)
781–791.
[8] S. Chakraborty, A. Khopade, C. Kokare, K. Mahadik, B. Chopade, Isolation and characterization
of novel α-amylase from marine Streptomyces sp. D1, J. Mol. Catal. B:
Enzym. 58 (2009) 17–23.
[9] T.A.A. Moussa, G.M. Ahmed, S.M.S. Abdel-hamid, Optimization of cultural conditions
for biosurfactant production from Nocardia amarae, J. Appl. Sci. Res. 2 (11)
(2006) 844–850.
[10] A.S. Kumar, K. Mody, B. Jha, Evaluation of biosurfactant/bioemulsifier production
by a marine bacterium, Bull. Environ. Contam. Toxicol. 79 (2007) 617–621.
[11] C.D. Cunha, M. do Rosário, A.S. Rosado, S.G.F. Leite, Serratia sp. SVGG16: a promising
biosurfactant producer isolated from tropical soil during growth with
ethanol-blended gasoline, Process. Biochem. 39 (2004) 2277–2282.
[12] F.M. Bento, F.A. Oliveira Camargo, B.C. Okekeb, W.T. Frankenberger, Diversity of
biosurfactant producing microorganisms isolated from soils contaminated with
diesel oil, Microbiol. Res. 160 (2005) 249–255.
[13] S. Joshi, C. Bharucha, S. Jha, S. Yadav, A. Nerurkar, A.J. Desai, Biosurfactant production
using molasses and whey under thermophilic conditions, Bioresour. Technol.
99 (2008) 195–199.
[14] G.S. Kiran, T.A. Thomas, J. Selvin, Production of a new glycolipid biosurfactant
from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation, Colloids
Surf., B 78 (2010) 8–16.
[15] S. Chakraborty, A. Khopade, R. Biao, W. Jian, X.Y. Liub, K. Mahadik, B. Chopade, L.
Zhang, C. Kokare, Characterization and stability studies on surfactant, detergent
and oxidant stable α-amylase from marine haloalkaliphilic Saccharopolyspora
sp. A9, J. Mol. Catal. B: Enzym. 68 (2011) 52–58.
[16] A. Gnanamania, V. Kavithaa, N. Radhakrishnana, G.S. Rajakumara, G. Sekaranb, A.B.
Mandala, Microbial products (biosurfactant and extracellular chromate reductase)
of marine microorganism are the potential agents reduce the oxidative stress
induced by toxic heavy metals, Colloids Surf., B 79 (2010) 334–339.
[17] S.T. Williams, F.L. Davies, Use of scanning electron microscope for the examination
of actinomycetes, J. Gen. Microbiol. 48 (1964) 171–177.
[18] M. Abouseoud, R. Maachi, A. Amrane, S. Boudergua, A. Nabi, Evaluation of different
carbon and nitrogen sources in production of biosurfactant by Pseudomonas
fluorescens, Desalination 223 (2008) 143–151.
[19] T.B. Lotfabada, M. Shourianc, R. Roostaazada, A.R. Najafabadi, M.R. Adelzadeha, K.A.
Noghabic, An efficient biosurfactant-producing bacterium Pseudomonas
aeruginosa MR01, isolated from oil excavation areas in south of Iran, Colloids Surf.,
B 69 (2009) 183–193.
[20] G. Seghal Kiran, T.A. Hema, R. Gandhimathi, J. Selvin, T. Anto Thomas, T.R. Ravji, K.
Natarajaseenivasan, Optimization and production of a biosurfactant from the
sponge-associated marine fungus Aspergillus ustus MSF3, Colloids Surf., B 73
(2009) 250–256.
[21] M.O. Ilori, C.J. Amobi, A.C. Odocha, Factors affecting biosurfactant production by
oil degrading Aeromonas spp. isolated from a tropical environment, Chemosphere
61 (2005) 985–992.
[22] H. Ghojavand, F. Vahabzadeh, E. Roayaei, A.K. Shahraki, Production and properties
of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC
1696), J. Colloid Interface Sci. 324 (2008) 172–176.
[23] E.J. Gudi˜na, J.A. Teixeira, L.R. Rodrigues, Isolation and functional characterization
of a biosurfactant produced by Lactobacillus paracasei, Colloids Surf., B 76 (2010)
298–304.
[24] L.M. Prieto, M. Michelon, J.F.M. Burkert, S.J. Kalil, C.A.V. Burkert, The production of
rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal
zone in Brazil, Chemosphere 71 (2008) 1781–1785.
[25] C.N. Mulligan, B.F. Gibbs, Correlation of nitrogen metabolism with biosurfactant
production by Pseudomonas aeruginosa, Appl. Environ. Microbiol. 55 (1989)
3016–3019.
[26] L.M. Santa Anna, G.V. Sebastian, E.P. Menezes, T.L.M. Alves, A.S. Santos, J.N.
Pereira, D.M.G. Freire, Production of biosurfactants from Pseudomonas aeruginosa
PA1 isolated in oil environments, Braz. J. Chem. Eng. 19 (2002) 159–166.
[27] H. Rashedi, E. Jamshidi, M.M. Assadi, B. Bonakdaepour, Isolation and production of
biosurfactant from Pseudomonas aeruginosa isolated from Iranian southern wells
oil, Int. J. Environ. Sci. Technol. 2 (2005) 121–127.
[28] C.C. Chen, L. Riadi, S.J. Suh, D.E. Ohman, L.K. Ju, Degradation and synthesis kinetics
of quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation, J. Biotechnol.
117 (2005) 1–10.
[29] L. Guerra-Santos, O. Käppeli, A. Fiechter, Pseudomonas aeruginosa biosurfactant
production in continuous culture with glucose as carbon source, Appl. Environ.
Microbiol. 48 (1984) 301–305.
[30] M. Bonilla, C. Olivaro, M. Corona, A. Vazquez, M. Soubes, Production and characterization
of a new bioemulsifier from Pseudomonas putida ML2, J. Appl. Microbiol.
98 (2005) 456–463.
[31] A. Persson, E. Österberg, M. Dostalek, Biosurfactant production by Pseudomonas
fluorescens 378: growth and product characteristics, Appl. Microbiol. Biotechnol.
29 (1) (1988) 1–4.
[32] J. Klein, F. Wagner, Different strategies to optimize the production phase of
immobilized cells, Ann. N.Y. Acad. Sci. 501 (1987) 306–316.
[33] H. Kim, B. Yoon, C. Lee, H. Suh, H. Oh, T. Katsuragi, Y. Tani, Production and properties
of a lipopeptide biosurfactant from Bacillus subtilis C9, J. Ferment. Bioeng.
84 (1) (1997) 41–46.
[34] A.S. Abu-Ruwaida, I.M. Banat, S. Haditirto, A. Salem, M. Kadri, Isolation of biosurfactant
producing bacteria, product characterization, and evaluation, Acta Biotechnol.
11 (1991) 315–324.