Journal of Colloid and Interface Science 367 (2012) 311–318

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Journal of Colloid and Interface Science

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Production and characterization of biosurfactant from marine

Streptomyces species B3
Abhijit Khopade a, Biao Ren b, Xiang-Yang Liu b, Kakasaheb Mahadik a, Lixin Zhang b, Chandrakant 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, PR China
c
Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune 411 041, India

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

Article history: The present study demonstrates the production and properties of a biosurfactant isolated from marine
Received 12 July 2011 Streptomyces species B3. The production of the biosurfactant was found to be higher in medium contain-
Accepted 3 November 2011 ing sucrose and lower in the medium containing glycerol. Yeast extract was the best nitrogen source for
Available online 15 November 2011
the production of the biosurfactant. The isolated biosurfactant reduced the surface tension of water to
29 mN/m. The purified biosurfactant was shown critical micelle concentrations of 110 mg/l. The emulsi-
Keywords: fying activity and stability of the biosurfactant was investigated at different salinities, pH, and tempera-
Biosurfactant
ture. The biosurfactant was effective at very low concentrations over a wide range of temperature, pH,
Streptomyces
Surface tension
and salt concentration. The purified biosurfactant was shown strong antimicrobial activity. The biosurfac-
Stability tant was produced from the marine Streptomyces sp. using non-hydrocarbon substrates such as sucrose
Critical micelle concentration that was readily available and not required extensive purification procedure. Streptomyces species B3 can
Antimicrobial activity be used for microbially enhanced oil recovery process.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction soil and sand or in the cleanup of hydrocarbon contamination in

Biosurfactants are a structurally diverse group of surface active
molecules synthesized by microorganisms. Virtually, all the surfac-
tants are chemically synthesized [1]. Nevertheless, in recent years,
much attention has been directed toward biosurfactants owing to
their advantages such as low toxicity, high biodegradability, better
environmental compatibility, high foaming capability, higher
selectivity, specific activity at extreme temperature, pH, salinity,
and the ability to synthesize them from renewable food stocks
[2–5]. Surfactants are amphiphilic agents which, by accumulating
at interface between immiscible phases, can reduce surface and
interfacial tension. The significant reduction of interfacial tension
caused by the biosurfactant increases the solubility and emulsifica-
tion of the immiscible phases and bioavailability of the insoluble
substrate for microorganism [3–6]. Biosurfactants with such sur-
face properties make good candidates for enhanced oil recovery
(EOR). The most effective biosurfactants reduce the surface tension
(ST) of water from 72 dynes/cm to value range of 25–30 dynes/cm
[6–9]. Some biosurfactants are known to have therapeutic applica-
tions [10,11]. Biosurfactants can also be used in bioremediation of
⇑ Corresponding author at: Department of Pharmaceutics, STES, Sinhgad Institute
of Pharmacy, Narhe, Pune 411 041, India. Fax: +91 20 24699051.
E-mail address: kokare71@rediffmail.com (C. Kokare).
groundwater [12–14].
Recently, biosurfactants have been widely used in environmen-
tal protection, including EOR, oil spills control, biodegradation, and
detoxification of oil contaminated industrial effluents and soil. An-
other most important applications of a bioemulsifier is the stimu-
lation of oil production in marginal wells that have approached
their economic limit of operation [6,12,15]. Biosurfactant produc-
tion by actinomycetes has been reported in very few cases. Glyco-
lipid from Rhodococcus erythropolis and Rhodococcus aurantiacus
and surface active lipid from Nocardia erythropolis were studied
in the literature search [16]. Among the many classes of biosurfac-
tants, lipopeptides represent a class of microbial surfactants with
remarkable surface properties and biological activities, such as sur-
plus crude oil recovery, food-processing, de-emulsification, antimi-
crobial, antitumor, antiviral, and antiadhesive activities. Surfactin,
produced by various Bacillus subtilis strains, is one of the most
powerful and effective lipopeptide-type biosurfactant, which is
composed of seven amino acids, linked via a lactone ring to a-hy-
droxy fatty acid with 13, 14, or 15 carbon atoms. Apart from many
characteristic functional activities of biosurfactants, surfactin can
also inhibits fibrin clot formation, induces formation of ion chan-
nels in lipid bilayer membranes, and inhibits cyclic adenosine
monophosphate (cAMP). In addition, a several authors have docu-
mented the antiviral action of surfactin, which is primarily due to a
physiochemical interaction between the membrane active

0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2011.11.009
312 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318
surfactant and the virus lipid membrane [17,18]. The objective of
the present study was production and characterizes the main
functional properties of the biosurfactant from marine Streptomy-
ces. Characterization included the determination of minimum sur-
face tension, critical micelle concentration, effect of different
hydrocarbon and oil on production, compositional analysis, func-
tional group detection, and stability to different factors such as
pH and temperature. Further, the antimicrobial activity of this bio-
surfactant was assayed against different microorganisms.
2. Materials and methods
2.1. Isolation and identification of marine actinomycetes strain
Marine sediment samples were collected from the West coast of
India. Different marine actinomycetes species were isolated by
using selective media such as glycerol yeast extract agar, starch
casein agar, and maltose yeast extract agar [19–22]. The isolated
strains were screened for biosurfactant production by using differ-
ent techniques. Identification of biosurfactant producing strain B3
was done by Scanning Electron Microscopy (SEM), 16S rDNA tech-
nology, biochemical and cultural characterization. The slide culture
preparation for SEM was done as described by Williams and Davies
[23,24].
2.2. Screening methods for potential biosurfactant producers
The potential biosurfactant producer was screened by different
method such as hemolytic assay, drop collapsing test, oil displace-
ment test, and lipase activity [16,25–27]. Maximum biosurfactant
producing marine actinomycetes sp. B3 was maintained on glyc-
erol yeast extract agar medium for further study.
2.3. Inoculum preparation and culture conditions
The glycerol yeast extract medium prepared in artificial seawa-
ter (ASW) was used for development of inoculum. The seed culture
was prepared in 100 ml Erlenmeyer flasks containing 50 ml of
medium by inoculating 2.0 ml of spore suspension containing
2.5–3.0  106 CFU/ml and cultivated under agitation (150 rpm) at
30 °C for 4 days. The seed culture (50 ml) was inoculated in the
1L of fermentation medium containing 2% glycerol, 0.5% peptone,
and 3% yeast extract prepared in artificial sea water, and fermenta-
tion was carried out 12 days under agitation at 150 rpm at 28 °C
[18,19].
2.4. Medium optimization
The medium optimization was conducted in a series of experi-
ments changing one variable at a time, keeping the other factors
fixed at a specific set of conditions. Two factors were chosen aim-
ing to obtain higher productivity of the biosurfactant: carbon
source (C) and nitrogen source (N). The carbon sources were used
as glycerol, xylose, starch, mannitol, hexadecane (2% w/v), sucrose,
trehalose, maltose, dextrose, and glucose (20 g/l), with ammonium
chloride (NH4Cl) as nitrogen source. For evaluation of the most
appropriate nitrogen sources for the production of biosurfactants,
yeast extract, phenyl alanine, alanine, urea, peptone, tryptone,
ammonium sulfate, ammonium chloride, and beef extract were
employed at a concentration of 10 g/l with the optimum carbon
source. All the media and reagents used for study were procured
form Himedia, India [20,28–30].
2.5. Time course of biosurfactant production
The kinetics of biosurfactant production was followed in batch
cultures at optimum conditions. The experiment was designed
for 12 days starting from the log phase to stationary phase under
submerged culture conditions. The resultant cell free supernatant
was removed by filtration followed by cold centrifugation at
10,000 rpm at 4 °C for 20 min. The supernatant was analyzed for
biosurfactant production [11,28].
2.6. Effect of pH, temperature, and sodium chloride on biosurfactant
production
Effect of pH and temperature on production of biosurfactant
was studied by adjusting the pH and temperature of the basal
medium to different levels. Effect of NaCl on biosurfactant produc-
tion was studied by varying the concentrations of NaCl% (w/v)
added to the basal medium. Biosurfactant activity was expressed
as percentage relative activity [30].
2.7. Effect of oils, surfactants, and hydrocarbon on biosurfactant
production
The effect of crude oil and surfactant was evaluated for biosur-
factant production. The different oils such as castor oil, cod-liver
oil, clove oil, coconut oil, eucalyptus oil, senamom oil, (commercial
grade), and surfactants such as EDTA, CTAB, SDS, tween 20, 40, 80
were added separately in 1% (v/v) in optimized medium, and emul-
sification activity of medium was measured. The effect of different
hydrocarbons (1% v/v) were observed on production of biosurfac-
tant by using diesel, petrol, toluene, xylene, hexadecane, octade-
cane, cyclohexane and kerosene [16,30].
2.8. Production and purification of biosurfactant
Study on growth of the organism and biosurfactant production
in glycerol yeast extract medium with crude oil as sole carbon
source was carried out as described by Ilori et al. [30]. The growth
medium contained grams/100 ml: sucrose, 2.0 g; yeast extract, 1 g;
KH2PO4, 0.53 g; NaCl, 3.0 g; 7H2O and crude oil (2%, v/v) [31]. The
pH of the medium was adjusted to 7.0 before sterilization. Trace
elements solution (1 ml) of Bauchop and Elsden [32] was sterilized
separately and added aseptically to the medium. The medium
(100 ml) contained in an Erlenmeyer flask (250 ml) was inoculated
with the organism and incubated at 28 °C with shaking at 150 rpm
for 12 days. The culture broth was centrifuged (10,000 rpm,
20 min, 4 °C) to remove the cells. The biosurfactant was recovered
from the cell free culture supernatant acid precipitation as de-
scribed by Ilori et al. [30]. To purify the surface active compound,
the concentrated extract was subjected to column chromatography
on reverse phase silica gel (60–120 mesh) with step wise elution
using methanol 100% at a flow rate of 1 ml/min at room tempera-
ture. The active fraction was confirmed by the emulsification activ-
ity, and the purity was checked by thin layer chromatography.
Fractions of 10 ml each were collected and used for reading the
optical density at 280 nm using a UV-Spectrophotometer (JASCO,
Japan).
2.9. Determination of the critical micelle concentration (CMC)
The surface tension of the biosurfactant was measured by the
Du-Nouy ring method [20] at room temperature. The concentra-
tion at which micelles began to form was represented as the
CMC. At the CMC, a sudden change in surface tension was ob-
served. The CMC was determined by plotting the surface tension
as a function of the biosurfactant concentration [33,34].
 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318
2.10. Compositional analysis of purified biosurfactant
The total carbohydrate content of purified biosurfactant was as-
sayed by phenol–sulfuric acid method using glucose as standard
[35]. Protein content was determined by Lowry method [36]. Bo-
vine serum albumin (BSA) was used as calibration standard. The li-
pid content of biosurfactant was determined by gravimetric
estimation [37,38].
2.11. Determination of the effect of temperature, pH, and sodium
chloride on the activity of the biosurfactant
The thermal stability of the biosurfactant was determined by
maintaining the supernatant at constant different range of temper-
ature from 30–100 °C for 15 min and cooled at room temperature.
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 biosurfac-
tant was re-dissolved after purification with distilled water con-
taining the specific concentration of NaCl (0–9%, w/v) [18].
2.12. Antimicrobial activity
The crude biosurfactant was tested for antimicrobial activity
using well diffusion method [18], and area of the zone was calcu-
lated. Extracted active compounds were tested against human
pathogens such as Escherichia coli, B. subtilis, Pseudomonas aerugin-
osa, Staphylococcus aureus, and Candida albicans [29].
2.13. Fourier transforms infrared spectroscopy
Fourier transform infrared (FTIR) spectroscopy of the biosurfac-
tants obtained from Streptomyces sp. B3 was done on a Jasco FT-IR
4100 spectrometer by KBr pellet method [34]. The molecular char-
acterization was performed using lyophilized sample of biosurfac-
tant with 4 cm 1 resolution yielding IR traces over the range of
400–4000 cm 1.
3. Results and discussions
3.1. Characterization of strain B3
The strain B3 shows good growth in temperature range 25–
45 °C in 7 days on glycerol yeast extract agar medium [19,22].
The aerial mycelium at maturity formed chains of three to several
spores. Spores were non-motile. Initially, colonies were relatively
smooth surfaced but later they developed a weft of aerial myce-
lium that appears to be granular, powdery, or velvety and produce
a wide variety of pigments responsible for the color of the vegeta-
Fig. 1. Scanning electron microscopy of strain B3 (A) 4000 magnification, (B) 8000 magnification.
313
tive and aerial mycelia. Spores were oval and warty, seen like hairy
(Fig. 1A and B). By morphological, SEM, and 16S rDNA sequencing,
the isolated strain was found to be a member of Streptomyces
genus (Fig. 2) [23,24].
3.2. Screening of biosurfactant production
Hemolytic activity of strain B3 showed zone with diameter
23 mm around the colony. In the present study, a significant corre-
lation was established between the hemolytic activity and biosur-
factant production. According to Carrillo et al. [39] and Banat [5],
biosurfactant production of the new isolates was preliminary
screened by hemolytic activity. Blood–agar lysis has been used to
quantify surfactant and rhamnolipids [19]. Carrillo et al. [39] found
an association between hemolytic activity and surfactant produc-
tion, and they recommended the use of blood agar lysis as a pri-
mary method to screen biosurfactant production. In drop
collapsing test a flat drop and in oil displacement method, a clear
zone of 176.62 mm2 was observed (data not shown). From the
above observation, it was confirmed that the Streptomyces sp. B3
was a potent biosurfactant producer. Both the techniques have sev-
eral advantages such as small volume of samples was required, ra-
pid and easy to carry out and also do not require specialized
equipment [29].
3.3. Cultivation conditions and biosurfactant production
The isolated Streptomyces sp. B3 produces biosurfactant when
grown in various nutrients. The amount of biosurfactant produc-
tion was varied with respect to media composition. However, the
maximum biosurfactant production was observed in glycerol yeast
extract. The production of biosurfactant was not observed in min-
imal salt medium and potato dextrose medium. Similar result was
observed by Desai and Banat [2]. Therefore, glycerol yeast extract
was selected as the biosurfactant production medium for the strain
at 28 °C for 9 days.
3.4. Optimization of cultivation medium
3.4.1. Effect of carbon source
The production of biosurfactant was found to be dependent on
the composition of the medium. In shake-flask experiments, the
change of the carbon source in the medium was affected on both,
the amount of biomass produced and biosurfactant secretion. The
various carbon sources screened for biosurfactant production, out
of this sucrose, trehalose, dextrose, and fructose were found favor-
able. The highest biosurfactant production was achieved using su-
crose (2% w/v) being the sole source of the carbon (Fig. 3A) [38].
Screening of nutrient substrates showed that Streptomyces sup-
ports growth on all substrates although the yield was limited with
glycerol. The Streptomyces sp. B3 showed growth on starch as a
314 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318
Fig. 2. Neighbor-joining phylogenetic tree of strain B3 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.
substrate but did not produce the surfactant under similar condi-
tions. The synthesized biosurfactant decreased the surface tension
to 29 mN/m and showed 80% emulsifying activity. Similar results
were found with P. aeruginosa 44T1 [29].
3.4.2. Effect of nitrogen source
The choice of nitrogen source affects the biosurfactant produc-
tion as shown in Fig. 3B. In order to obtain high concentrations of
biosurfactant, it is necessary to have restrained conditions of the
macro-nutrients. Yeast extract was found to be the best source of
nitrogen for growth and biosurfactant synthesis. Ammonium salts
in the form of ammonium chloride was used for growth but not for
biosurfactant production and caused a significant decrease in pH
that results in decrease biosurfactant production. The maximum
emulsifying activity (285 EU/ml) and minimal surface tension
(30 mN/m) were reached in media with yeast extract [29].
3.5. Kinetics of biosurfactant production
The biosurfactant production and surface tension was depen-
dent 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 6 day of growth
(Fig. 4). On 4th day of growth, the surfactant concentration starts
to increase, reaching its maximum after about 6th day. The in-
crease in surface tension and the decrease in E24 after 12th day
of incubation showed that biosurfactant biosynthesis has been
stopped and is probably due to the production of secondary metab-
olites that could interfere with emulsion formation and the adsorp-
tion of surfactant molecules at the oil–water interface. These
results indicate that the biosurfactant biosynthesis occurred pre-
dominantly during the exponential growth phase, suggesting that
the biosurfactant is produced as primary metabolite accompanying
cellular biomass formation (growth-associated kinetics) [22].
3.6. Effect of pH, temperature, and salinity on biosurfactant production
The pH of the medium was important characteristic for cell
growth of organism and production of secondary metabolites.
The biosurfactant production was affected by initial pH of culture
medium. At pH 5, the biosurfactant production was severely de-
creased and the cell growth was significantly retarded. This low
pH created unfavorable growth conditions for the bacterial popula-
tion. When the initial pH was set to 7, the emulsification activity
increases (E24 = 85), if pH of medium increase more than 7 the bio-
surfactant production decreases [11,16]. The optimum pH for
growth and biosurfactant production was determined to be 7
(Fig. 5A). Similar result was observed by biosurfactant production
from bacteria P. aeruginosa MR01 by Lotfabada et al. [38]. The
strain B3 showed good growth between the temperature range of
25–45 °C. A change in temperature caused alterations in the com-
position of the biosurfactant in the case of Arthrobacter paraffineus
[24] and Pseudomonas sp. [25]. Temperature was one of the critical
parameters that have been controlled in bioprocess. The results in
the present study revealed that the biosurfactant activity reached
the highest when the strain was grown at 30 °C (E24 = 80%)
(Fig. 5B), and this clearly indicates moderately thermostable nature
of biosurfactant. The research was focused on the isolation of alka-
line biosurfactant from microbes because there is tremendous
potentiality of biosurfactant in detergent industry. The strain B3
was found to be moderately halophilic in nature; maximum bio-
surfactant production (E24 = 78%) was obtained in presence of 4%
(w/v) of NaCl and showed emulsification activity (E24 = 60%) in
presence of 9% (w/v) of NaCl (Fig. 5C).
3.7. Effect of oils, surfactants, and hydrocarbons on production of
biosurfactant
Fermentation was carried out with addition of different concen-
trations of oils, surfactant, and hydrocarbons in the fermentation
medium. It was observed that olive oil, tween 20, and xylene as a
substrate showed maximum activity against all test oils, surfac-
 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318 315

200 respectively (Fig. 6A and B). The organism was able to utilize all
Emulsification activity (EU/ml)
the hydrocarbons tested as sources of energy; growth was accom-
panied with biosurfactant production. The results of the hydrocar-
150
bon substrate specificity test revealed that the biosurfactant
production had very good emulsification activity with hexadecane
100 (Fig. 6C). The liquid aromatic hydrocarbons were particularly not
good substrates for emulsification. On other hand, alkenes were
found to be good substrates for emulsification by the biosurfactant.
50 Crude oils and diesel oils are mixtures of hydrocarbons and are
known to serve as an excellent sources of carbon and energy for
most hydrocarbonoclastic microorganisms. Potential toxicity has
0
been cited as possible reasons for the inability of most microorgan-
lu se
G se

al l
Xy e
Su se

eh se

St e
an h
H uc l

ec e
e
M ro

Fr nito
isms to grow on toluene [9]. Crude oil and hexadecane were also
s

os

ad os
an
M rc
o
co

to

lo
Tr cro
ce

a
tr

al

ex t
good substrates for emulsification by the biosurfactant. Most
ly
ex
G
D

microbial surfactants are substrate specific, solubilizing, or emulsi-

Carbon sources fying different hydrocarbons at different rates. An emulsion is
formed when one liquid phase is dispersed as microscopic droplets
[A] in another liquid continuous phase [2]. Poor emulsification of some
of the hydrocarbons might be due to the inability of the biosurfac-
400 tant to stabilize the microscopic droplets. Similar result was ob-
Emulsification activity (EU/ml)

served in biosurfactant production by oil degrading Aeromonas

300 sp. by Ilori et al. [30].

3.8. Critical micelle concentration (CMC) of biosurfactant

200
The CMC value of the biosurfactant was determined by sepa-
100
rately measuring the surface tension of different concentrations
(log of mg/l) of biosurfactant (Fig. 7). The Mili Q distilled water
was found to have the surface tension of 72 mN/m and the addition
0 of biosurfactant reduced its surface tension up to 30 mN/m. It is
worth mentioning that the CMC develops at the end of the expo-
C ate

A ide
Pe ine

e
a
t E ne

B par ct
en Ex e
A ct
ne
Tr re

in
on

yl tra
A xtra

nential phase. Although the production of the surfactant continues

as pto

ni
n

ee g
or
m lph

U
pt
la

la
hl

Ye y

after the exponential phase, its properties, such as the surface ten-
A Su

s
Ph f
m

sions, remain constant. This phenomenon is observed because of

m
m
A

the conditions in which CMC is approached. The CMC of biosurfac-

Nitrogen source tant isolated from the Streptomyces strain was found to be 110 mg/
[B] l. Rashedi et al. [40], was observed close result to that obtained in
the present work.
Fig. 3. (A) Effect of carbon source on biosurfactant production, (B) Effect of nitrogen
source on biosurfactant Production. 3.9. Preliminary characterization of biosurfactant

Compositional analyses revealed that the biosurfactant pro-

tants, and hydrocarbons respectively. The olive oil and tween 20 duced by Streptomyces might be a glycolipid primarily consisting
showed emulsification activity 201 EU/ml and 304 EU/ml of lipid with relative percent of 58% (w/w) and 33% (w/w) carbohy-

Fig. 4. Growth kinetics of Streptomyces cell growth (OD600) and biosurfactant production by Streptomyces sp. B3.
316 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318

Emulsification activity (EU/ml)

100 250

80 200
E 24 (%)

60 150

40 100

20
50

0
0
4

10

11

12

il

il

l
pH

oi

oi

oi

oi

oi
ro

eo

us

ve

ut

er

om
liv
te

on

liv
[A]

lo
pt
as

m
C
ly

oc

od
C

na
ca

Se
Eu
100
Oils

80 [A]

Emulsification activity (EU/ml)

E 24 (%)

60 400

40 300

20
200

0
100
4

20

25

30

35

40

45

0
Temperature ( C)
0
[B] TA

00

20

40

80

B
SD

TA
X1
ED

n
ee

ee

ee

C
n

Tw

Tw

Tw
ee

100
Tw

Surfactants
80
[B]
E 24 (%)

60
250
Emulsification activity (EU/ml)

40
200
20
150
0
1

100
NaCl (% w/v)

[C] 50

Fig. 5. (A) Effect of pH on biosurfactant production, (B) Effect of temperature on 0

biosurfactant production, (C) Effect of NaCl on biosurfactant production.
ne

ad ne

ad ne

Pe l
To rol

Xy e
ne
se
an

K en

en
xa

le
t
ie
H Oct

O ec

ec

lu

os
D
he

er
lo

ex

ct

drate. A minor fraction of protein (8% w/w) was found in some of

yc
C

extracted samples possibly arising from the existence of the resid-

ual cell debris in broth co-precipitated with biosurfactant during
the extraction process. Similar result was observed on the isolation
and surfactant production of different species of the genus Pseudo-
monas [35–38].
Hydrocarbons
[C]
Fig. 6. (A) Effect of oils on biosurfactant production, (B) Effect of surfactants on
biosurfactant production (C) Effect of hydrocarbons on biosurfactant production.

3.10. Stability studies
3.10.1. Temperature stability
The applicability of biosurfactants in several fields depends on
their stability at different temperatures and pH values. The stabil-
ity of biosurfactant was tested over a wide range of temperatures.
The biosurfactant produced by Streptomyces sp. was shown to be
thermostable (Fig. 8A). Heating of the biosurfactant to 100 °C
caused no significant effect on the biosurfactant performance.
The surface tension was quite stable at the temperatures 80 °C,
but the synthetic surfactants such as sodium dodecyl sulfate exhib-
its a significant loss of emulsification activity beginning at 70 °C.
Therefore, it can be concluded that this biosurfactant maintains
its surface properties unaffected in the range of temperatures be-
tween 30 and 100 °C. This activity was discovered indicating the
usefulness of the biosurfactant in food, pharmaceutical, and cos-
metics industries where heating to achieve sterility is of para-
mount importance [28,41].
 A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318 317

80 50

Surface tension (mN/m)

40
Surface tension (mN/m)

60
30

40
20

20 10

0
0

30

40

50

60

70

80

90

0
10
0 1 2 3 4 5
Temperature ( 0 C)
Conc. of biosurfactant (log of mg/l)
[A]
Fig. 7. Critical micelle concentration of biosurfactant.

50
3.10.2. pH stability
The surface activity of the biosurfactant remained relatively sta-

Surface tension (mN/m)

ble to pH changes between pH 8–12, showing higher stability at 40
alkaline pH 8 than acidic conditions. At pH 12, the surface tension
decreased up to 37 mN/m, whereas at pH 8 activities decreased to 30
29 mN/m. Fig. 8B shows the effect of pH on the biosurfactant prop-
erties. These results indicate that increase pH has a positive effect 20
on surface tension and emulsion stability. This could be caused by
a better stability of fatty acids surfactant micelles in the presence 10
of sodium hydroxide and the precipitation of secondary metabo-
lites at higher pH values. The effect of pH on surface activity has 0
been reported for biosurfactants for different microorganisms [41].
5

10

11

12
pH
3.10.3. Effect of salinity
The effect of sodium chloride addition on biosurfactant pro- [B]
duced from Streptomyces was studied. Maximum stability of bio-
surfactant observes at 6% NaCl concentration. Little changes were 50
observed in increase concentration of NaCl up to 9% (w/v)
Surface tension (mN/m)

(Fig. 8C). At higher concentration of NaCl, the biosurfactant retains 40

80% activity. The biosurfactant has stability at alkaline pH and high
salinity; such a biosurfactant may be useful for bioremediation of 30
spills in marine environment because of its stability in alkaline
condition and in the presence of salt [41]. 20

3.11. Antimicrobial activity of biosurfactant 10

Biosurfactant isolated from Streptomyces species showed a wide 0

activity against the pathogenic strains. The partial purified biosur-
1

factant showed activity against B. subtilis, P. aeruginosa, S. aureus, NaCl (% w/v)

and E. coli and strong activity toward the yeast C. albicans
(Fig. 9). According to Tsuge et al. [42], lipopeptide surfactants are
potent antibiotics mainly the surfactin, streptofactin, and gramici-
din produced by the microorganism and had the wide antimicro-
bial activity [43,44] compared to the glycolipid producing strain.
A glycolipid surfactant from the C. antartica has demonstrated anti-
microbial activity against Gram-positive bacteria [29].
3.12. Fourier transforms infrared spectroscopy
The presence of hydroxyl and amine groups of protein was con-
firmed by Infrared spectrum (data not showed) of the purified bio-
surfactant, which showed broad stretching peaks from 3458 to
3200 cm 1. Absorption around 3159 cm 1 was assigned to the
symmetric stretch ACH of CH2 and CH3 groups of aromatic chains.
Absorption around 2924 cm 1 was assigned to the symmetric
stretch ACH of CH2 and CH3 groups of aliphatic chains. Also, an in-
tense absorption band at 1738 cm 1 and a weak symmetric
stretching peak around 1645 cm 1 indicate the presence of ester
carbonyl groups (C@O in COOH) in the biosurfactant. The ester car-
[C]
Fig. 8. Effect of (A) Temperature, (B) pH and (C) salinity on biosurfactant stability.
bonyl group was also proved from the band at 1250 cm 1 that cor-
responds to C@O deformation vibrations, although other groups
also absorb in this region. It might be possible that the additional
bands at 1645 cm 1 and 1618 resulted from polypeptides origi-
nated from cell debris co-precipitated with the biosurfactant dur-
ing extraction process. Similarly, another strong IR absorption
found at 817 cm 1 was due to out of plane CAH bending, charac-
teristic of aromatic compounds [18,45]. The FTIR spectra of the bio-
surfactant obtained indicated that the isolated biosurfactant was
glycolipid in nature.
4. Conclusions
The biosurfactant produced by a marine Streptomyces B3 was
complex structure of proteins, carbohydrates, and lipids. The bio-
318
800
Area of the zone (mm 2 )
600
400
200
0 [10] P. Singh, S. Cameotra, Trends Biotechnol. 22 (2004) 142.
is
i
ol
eu
til
c
ub
ur
E.
a
.s
S.
B
a
P.
Pathogenic strains
Fig. 9. Antimicrobial activity of biosurfactant.
surfactant obtained from Streptomyces B3 employing olive oil as
substrate and having high lipid content may provide a promising
focus for further investigations on its application as a compound
with efficient biological activity for enhanced oil recovery [33].
The results highlight the usefulness of regulation of physiological
parameters and their effects on the biosurfactant production and
further emphasize on the unique biochemical properties of these
kinds of microbial natural products [27].
The importance of this biosurfactant for industrial uses was
shown by studying different physical properties, such as critical
micelle concentration, surface and interfacial tension, emulsifica-
tion activity, and it’s showed high stability to environmental fac-
tors such as temperature, pH, and salinity [33]. The isolated
biosurfactant reduced the surface tension of water from 72 mN/
m to 29 mN/m. The purified biosurfactant showed critical micelle
concentrations of 110 mg/l. The functional characterization of iso-
lated biosurfactant indicated that the biosurfactant produced was
glycolipid in nature [27,29]. The production of the biologically ac-
tive fraction may be preferred for the economical production of
these valuable molecules for therapeutic purposes such as biosur-
factant as an alternative antimicrobial or anticancer agent in the
medical field for applications against microorganisms responsible
for diseases [18]. From this study, it can be concluded that the bio-
surfactant obtained from marine Streptomyces sp. B3 can be used as
an alternative to chemical surfactants for bioremediation of spills
in marine environments [29].
Acknowledgments
The authors would like to acknowledge All India Council for
Technical Education (AICTE), HRD Ministry, New Delhi, Govt. of In-
dia for financial support to this research project under National
Doctoral Fellowship (NDF), 2010–2013.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jcis.2011.11.009.
A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318
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