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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.
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 2.5. Time course of biosurfactant production
the present study was production and characterizes the main
functional properties of the biosurfactant from marine Streptomy- The kinetics of biosurfactant production was followed in batch
ces. Characterization included the determination of minimum sur- cultures at optimum conditions. The experiment was designed
face tension, critical micelle concentration, effect of different for 12 days starting from the log phase to stationary phase under
hydrocarbon and oil on production, compositional analysis, func- submerged culture conditions. The resultant cell free supernatant
tional group detection, and stability to different factors such as was removed by filtration followed by cold centrifugation at
pH and temperature. Further, the antimicrobial activity of this bio- 10,000 rpm at 4 °C for 20 min. The supernatant was analyzed for
surfactant was assayed against different microorganisms. biosurfactant production [11,28].
2.1. Isolation and identification of marine actinomycetes strain Effect of pH and temperature on production of biosurfactant
was studied by adjusting the pH and temperature of the basal
Marine sediment samples were collected from the West coast of medium to different levels. Effect of NaCl on biosurfactant produc-
India. Different marine actinomycetes species were isolated by tion was studied by varying the concentrations of NaCl% (w/v)
using selective media such as glycerol yeast extract agar, starch added to the basal medium. Biosurfactant activity was expressed
casein agar, and maltose yeast extract agar [19–22]. The isolated as percentage relative activity [30].
strains were screened for biosurfactant production by using differ-
ent techniques. Identification of biosurfactant producing strain B3 2.7. Effect of oils, surfactants, and hydrocarbon on biosurfactant
was done by Scanning Electron Microscopy (SEM), 16S rDNA tech- production
nology, biochemical and cultural characterization. The slide culture
preparation for SEM was done as described by Williams and Davies The effect of crude oil and surfactant was evaluated for biosur-
[23,24]. 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
2.2. Screening methods for potential biosurfactant producers were added separately in 1% (v/v) in optimized medium, and emul-
sification activity of medium was measured. The effect of different
The potential biosurfactant producer was screened by different hydrocarbons (1% v/v) were observed on production of biosurfac-
method such as hemolytic assay, drop collapsing test, oil displace- tant by using diesel, petrol, toluene, xylene, hexadecane, octade-
ment test, and lipase activity [16,25–27]. Maximum biosurfactant cane, cyclohexane and kerosene [16,30].
producing marine actinomycetes sp. B3 was maintained on glyc-
erol yeast extract agar medium for further study. 2.8. Production and purification of biosurfactant
2.10. Compositional analysis of purified biosurfactant tive and aerial mycelia. Spores were oval and warty, seen like hairy
(Fig. 1A and B). By morphological, SEM, and 16S rDNA sequencing,
The total carbohydrate content of purified biosurfactant was as- the isolated strain was found to be a member of Streptomyces
sayed by phenol–sulfuric acid method using glucose as standard genus (Fig. 2) [23,24].
[35]. Protein content was determined by Lowry method [36]. Bo-
vine serum albumin (BSA) was used as calibration standard. The li- 3.2. Screening of biosurfactant production
pid content of biosurfactant was determined by gravimetric
estimation [37,38]. Hemolytic activity of strain B3 showed zone with diameter
23 mm around the colony. In the present study, a significant corre-
2.11. Determination of the effect of temperature, pH, and sodium lation was established between the hemolytic activity and biosur-
chloride on the activity of the biosurfactant factant production. According to Carrillo et al. [39] and Banat [5],
biosurfactant production of the new isolates was preliminary
The thermal stability of the biosurfactant was determined by screened by hemolytic activity. Blood–agar lysis has been used to
maintaining the supernatant at constant different range of temper- quantify surfactant and rhamnolipids [19]. Carrillo et al. [39] found
ature from 30–100 °C for 15 min and cooled at room temperature. an association between hemolytic activity and surfactant produc-
To determine the effect of pH on activity, the pH of the cell free tion, and they recommended the use of blood agar lysis as a pri-
broth was adjusted to different values using 1 N NaOH or 1 N mary method to screen biosurfactant production. In drop
HCl. The effect of addition of different concentration of NaCl on collapsing test a flat drop and in oil displacement method, a clear
the activity of the biosurfactant was investigated. The biosurfac- zone of 176.62 mm2 was observed (data not shown). From the
tant was re-dissolved after purification with distilled water con- above observation, it was confirmed that the Streptomyces sp. B3
taining the specific concentration of NaCl (0–9%, w/v) [18]. 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
2.12. Antimicrobial activity
equipment [29].
The crude biosurfactant was tested for antimicrobial activity
3.3. Cultivation conditions and biosurfactant production
using well diffusion method [18], and area of the zone was calcu-
lated. Extracted active compounds were tested against human
The isolated Streptomyces sp. B3 produces biosurfactant when
pathogens such as Escherichia coli, B. subtilis, Pseudomonas aerugin-
grown in various nutrients. The amount of biosurfactant produc-
osa, Staphylococcus aureus, and Candida albicans [29].
tion was varied with respect to media composition. However, the
maximum biosurfactant production was observed in glycerol yeast
2.13. Fourier transforms infrared spectroscopy extract. The production of biosurfactant was not observed in min-
imal salt medium and potato dextrose medium. Similar result was
Fourier transform infrared (FTIR) spectroscopy of the biosurfac- observed by Desai and Banat [2]. Therefore, glycerol yeast extract
tants obtained from Streptomyces sp. B3 was done on a Jasco FT-IR was selected as the biosurfactant production medium for the strain
4100 spectrometer by KBr pellet method [34]. The molecular char- at 28 °C for 9 days.
acterization was performed using lyophilized sample of biosurfac-
tant with 4 cm 1 resolution yielding IR traces over the range of 3.4. Optimization of cultivation medium
400–4000 cm 1.
3.4.1. Effect of carbon source
3. Results and discussions The production of biosurfactant was found to be dependent on
the composition of the medium. In shake-flask experiments, the
3.1. Characterization of strain B3 change of the carbon source in the medium was affected on both,
the amount of biomass produced and biosurfactant secretion. The
The strain B3 shows good growth in temperature range 25– various carbon sources screened for biosurfactant production, out
45 °C in 7 days on glycerol yeast extract agar medium [19,22]. of this sucrose, trehalose, dextrose, and fructose were found favor-
The aerial mycelium at maturity formed chains of three to several able. The highest biosurfactant production was achieved using su-
spores. Spores were non-motile. Initially, colonies were relatively crose (2% w/v) being the sole source of the carbon (Fig. 3A) [38].
smooth surfaced but later they developed a weft of aerial myce- Screening of nutrient substrates showed that Streptomyces sup-
lium that appears to be granular, powdery, or velvety and produce ports growth on all substrates although the yield was limited with
a wide variety of pigments responsible for the color of the vegeta- glycerol. The Streptomyces sp. B3 showed growth on starch as a
Fig. 1. Scanning electron microscopy of strain B3 (A) 4000 magnification, (B) 8000 magnification.
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- 3.6. Effect of pH, temperature, and salinity on biosurfactant production
tions. The synthesized biosurfactant decreased the surface tension
to 29 mN/m and showed 80% emulsifying activity. Similar results The pH of the medium was important characteristic for cell
were found with P. aeruginosa 44T1 [29]. 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
3.4.2. Effect of nitrogen source pH created unfavorable growth conditions for the bacterial popula-
The choice of nitrogen source affects the biosurfactant produc- tion. When the initial pH was set to 7, the emulsification activity
tion as shown in Fig. 3B. In order to obtain high concentrations of increases (E24 = 85), if pH of medium increase more than 7 the bio-
biosurfactant, it is necessary to have restrained conditions of the surfactant production decreases [11,16]. The optimum pH for
macro-nutrients. Yeast extract was found to be the best source of growth and biosurfactant production was determined to be 7
nitrogen for growth and biosurfactant synthesis. Ammonium salts (Fig. 5A). Similar result was observed by biosurfactant production
in the form of ammonium chloride was used for growth but not for from bacteria P. aeruginosa MR01 by Lotfabada et al. [38]. The
biosurfactant production and caused a significant decrease in pH strain B3 showed good growth between the temperature range of
that results in decrease biosurfactant production. The maximum 25–45 °C. A change in temperature caused alterations in the com-
emulsifying activity (285 EU/ml) and minimal surface tension position of the biosurfactant in the case of Arthrobacter paraffineus
(30 mN/m) were reached in media with yeast extract [29]. [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%)
3.5. Kinetics of biosurfactant production (Fig. 5B), and this clearly indicates moderately thermostable nature
of biosurfactant. The research was focused on the isolation of alka-
The biosurfactant production and surface tension was depen- line biosurfactant from microbes because there is tremendous
dent on growth of culture in the fermentation medium. The surface potentiality of biosurfactant in detergent industry. The strain B3
tension dropped rapidly after inoculation, reaching its lowest value was found to be moderately halophilic in nature; maximum bio-
(29 mN/m) during exponential phase after about 6 day of growth surfactant production (E24 = 78%) was obtained in presence of 4%
(Fig. 4). On 4th day of growth, the surfactant concentration starts (w/v) of NaCl and showed emulsification activity (E24 = 60%) in
to increase, reaching its maximum after about 6th day. The in- presence of 9% (w/v) of NaCl (Fig. 5C).
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- 3.7. Effect of oils, surfactants, and hydrocarbons on production of
olites that could interfere with emulsion formation and the adsorp- biosurfactant
tion of surfactant molecules at the oil–water interface. These
results indicate that the biosurfactant biosynthesis occurred pre- Fermentation was carried out with addition of different concen-
dominantly during the exponential growth phase, suggesting that trations of oils, surfactant, and hydrocarbons in the fermentation
the biosurfactant is produced as primary metabolite accompanying medium. It was observed that olive oil, tween 20, and xylene as a
cellular biomass formation (growth-associated kinetics) [22]. 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
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
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
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
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]
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
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
3.10. Stability studies The surface tension was quite stable at the temperatures 80 °C,
but the synthetic surfactants such as sodium dodecyl sulfate exhib-
3.10.1. Temperature stability its a significant loss of emulsification activity beginning at 70 °C.
The applicability of biosurfactants in several fields depends on Therefore, it can be concluded that this biosurfactant maintains
their stability at different temperatures and pH values. The stabil- its surface properties unaffected in the range of temperatures be-
ity of biosurfactant was tested over a wide range of temperatures. tween 30 and 100 °C. This activity was discovered indicating the
The biosurfactant produced by Streptomyces sp. was shown to be usefulness of the biosurfactant in food, pharmaceutical, and cos-
thermostable (Fig. 8A). Heating of the biosurfactant to 100 °C metics industries where heating to achieve sterility is of para-
caused no significant effect on the biosurfactant performance. mount importance [28,41].
A. Khopade et al. / Journal of Colloid and Interface Science 367 (2012) 311–318 317
80 50
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-
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)
800 References
Area of the zone (mm 2 )
ns
sa
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i
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Appendix A. Supplementary material