www.ijecs.in
International Journal Of Engineering And Computer Science ISSN:2319-7242
Volume 3 Issue 6 June, 2014 Page No. 6476-6490
Consortium based Biosurfactant development for
degradation and emulsification of oils and
Petroleum Hydrocarbons
Jyotsna Kiran Peter1*, Abhishek Kumar Rao1 and Ritu Kumari1
Department of Microbiology and Fermentation Technology (MBFT), Jacob School of Biotechnology and Bioengineering (JSBB), Sam
Higginbottom Institute of Agriculture Technology and Sciences (SHIATS), Naini, Allahabad, Uttar Pradesh, India, 211007
Corresponding author: Jyotsna Kiran Peter1*, jyots.kp@gmail.com, 9793475020
Abstract: To develop the consortium three bacterial strains were used namely, Pseudomonas aeruginosa, Bacillus subtilis and
Serratia marscecens. Among the three Serratia marscecens yielded high quantity of biosurfactant obtained after partial
purification. Emulsification activity was determined from 0h to 96h for Poly aromatic hydrocarbon (against Petrol, Diesel, Mobil
oil, Kerosene) and vegetable oils (Mustard oil, Soybean oil, Coconut and Jasmine oil). Among the hydrocarbons most efficient
emulsification by three bacterial strains and consortium was seen for mobil oil then kerosene and petrol while diesel was least
emulsified but consortium biosurfactant emulsified Diesel and was stable till 96h. Among vegetable oils highly emulsified oil
was mustard and soy bean followed by Coconut and Jasmine. The study revealed the emulsification capacity of biosurfactant
from consortium for poly aromatic hydrocarbons and vegetable oils.
Key words: Consortium, Emulsification activity, Poly aromatic hydrocarbon, vegetable oil
1.
Introduction
the hydrophobic effect, the process of micelle selfassembly
Surfactants are surface active agents that have a tendency to
is also mediated by van der Waals, hydrogen-bonding, and
adsorb at the interfaces in the form of a monolayer and to
screened electrostatic interactions (in the case of charged
lower the free energy of the phase boundary (Yan et al.;
surfactants) [Stephenson et al. ]. Micelles were extensively
Cserháti et al., 2002; Colomer et al., 2011; Paluch and
and successfully used as models for biological membranes
Gzyl, 1996) The lowering of the surface tension of solvent
[Fendler, 1982]. Microorganisms produce a wide variety of
stops when surfactant molecules begin to form micelles in
high- and low-molecular weight biosurfactants (reviewed
the bulk solution [Mulligan, 2005]. Surfactant molecules
by Banat, 1995; Lin, 1996; Desai and Banat, 1997;
self assemble into different micellar aggregates, with their
Cameotra and Makkar, 1998; Rosenberg and Ron,
hydrophobic tails shielded from water in the micellar
1999). The low molecular weight biosurfactants are
interior and their hydrophilic heads exposed to water at the
generally glycolipids or lipopeptides and are more effective
micellar surface. The hydrophobic effect, which is of
in lowering the interfacial and surface tension (Lin, 1996).
entropic nature, is the primary driving force responsible for
The high molecular weight biosurfactants, which are
surfactant self-assembly in aqueous solution. In addition to
amphipathic polysaccharides, proteins, lipopolysaccharides
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6476
and lipoproteins, are effective stabilizers of oil-in-water
plants (Compant et al., 2005; Haas and Défago 2005;
emulsions (Gutnik and Shabtai, 1987; Ward et al., 2006).
Pieterse et al., 2002; Ryu et al., 2003). The diverse life
Soybean oil is a mixture of triglycerides consisting of
styles of Pseudomonas spp. and the complexity of their
saturated and unsaturated fatty acids. The presence of
interactions with multiple hosts have been the subject of
double bonds allows for the modification of the structure of
numerous molecular, biochemical, and ecological studies
soybean oil. Epoxidation is a method commonly used to
(Ramos, 2004). Rhamnolipids produced by P. aeruginosa
modify soybean oil. The double bonds are converted into
strains have four main structural types: monorhamnolipid
more reactive epoxide or oxirane ring groups by reacting
acid, monorhamnolipid methyl ester, dirhamnolipid acid
with peracids or peroxides. The benefits of epoxidized
(dR-A), and dirhamnolipid methyl ester (dR-Me). These
soybean oil (ESO) include its low toxicity, low irritancy,
rhamnolipids can be produced in mixtures that vary in
and lack of carcinogenic or non-genotoxic effects (BIBRA,
composition (Zhang and Miller, 1995). Serrawettin W1
1988; EFSA, 2004).
produced by Serratia marcescens is a surface-active
The spore-forming bacterium, Bacillus subtilis, is an
exolipid resulting in a lot foam formation during the 2, 3-
important organism for the molecular genetic study of
butanediol (2, 3-BD) fermentation Process (Zhang et al.,
peptide synthesis. Lipopeptides represent a unique class of
2010).
bioactive microbial secondary metabolites, and many of
them show attractive therapeutic and biotechnological
properties [Maget Danna and Peypox, 1994; Katz and
2.
Materials and Methods
2.1 Place of work done
Demain, 1977]. The surfactin family encompasses structural
variants but all members are heptapeptides interlinked with
a bhydroxy fatty acid to form a cyclic lactone ring structure
[Peepox et al., 1999]. They are powerful biosurfactants with
exceptional emulsifying and foaming properties. Because of
their amphiphilic nature, surfactins can also readily
associate and tightly anchor into lipid layers and can thus
interfere with biological membrane integrity in a dosedependent manner. Studies on lipid vesicles suggest that at
low concentration [surfactin-to-lipid mole ratio (Rb) <0.04
in the membrane] surfactins insert exclusively in the outer
leaflet of the membrane inducing only limited perturbation.
At intermediate concentration (Rb 0.05–0.1) they provoke a
transient
permeabilization,
but
membranes
The study was conducted at laboratory of Department of
Microbiology and Fermentation Technology (MBFT), Jacob
School of Biotechnology and Bioengineering (JSBB), Sam
Higginbottom Institute of Agriculture Technology and
Sciences (SHIATS), Naini, Allahabd, India.
2.2 Study materials
Bacterial strains were procured from Microbial Culture
Collection Bank (MCCB), Department of Microbiology and
Fermentation Technology (MBFT), Jacob School of
Biotechnology
and
Bioengineering
(JSBB),
Sam
Higginbottom Institute of Agriculture Technology and
Sciences (SHIATS), Naini, India in Nutrient agar slants.
reanneal.
Irreversible pore formation then occurs at higher ratios (Rb
Name of bacteria
0.1–0.2) because of the insertion of surfactin-rich clusters in
Pseudomonas aeruginosa
the membrane. Further addition of surfactins to reach the
Bacillus subtilis
critical micellar concentration (CMC) leads to complete
disruption and solubilization of the lipid bilayer with
Serratia marscecens
formation of mixed micelles (at Rb 0.22) [Heerklotz and
2.3 Maintenance of bacterial culture
Seeling, 2007; Carrillo, 2003].
The genus Pseudomonas not only harbors plant- and
humanpathogenic species, but also accommodates species
The bacterial cultures were sub cultured routinely at 15 days
interval on Nutrient agar slants (COMPOSITION :)
incubated at 35˚C for 24 hours.
that promote plant growth, degrade xenobiotic compounds,
antagonize plant pathogenic fungi, or induce resistance in
2.4 Study Design
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6477
The study was conducted with following objectives
I.
To Produce and partially purify biosurfactant from
bacterial consortium and individual bacteria in
To characterize the biochemical
nature of
biosurfactants produced
III.
Then the contents were centrifuged at 8000 rpm for 30
minutes. The pellet was obtained at the bottom of centrifuge
tube. The pellet was collected as a partially purified
submerged batch mode operation
II.
allowed to stand for 10 hours at 4°C (Ilori et al., 2005).
biosurfactant and stored in a screw capped bottle at
refrigerating condition for further use.
To analyze emulsification activity of individual
biosurfactant of Bacillus subtilis and Serratia marscecens
and consortium bio surfactants
was
partially
purified
using
–methanol
Chloroform
extraction system in ratio of 2:3 added to the supernatant.
2.5 Drop Collapse assay
Then the pH of the contents was dropped to 2 using conc.
Petrol/Diesel oil (2µl) was added to Petriplate lid. The lid
HCl. The flask was kept in refrigerator at 4˚C overnight.
was pre-equilibrated for 1 hour at room temperature, and
Then the contents were centrifuged at 8000rpm for 30
then 5µl of the culture supernatant was added to the surface
minutes. The pellet was obtained at the junction of organic
of oil. The shape of the drop on the oil surface was inspected
and aqueous phase. The pellet was collected as a partially
after 1 minute. Biosurfactant containing cultures will give
purified biosurfactant and stored in a screw capped bottle at
flat drops, thus indicating a positive result.
refrigerating condition for further use.
2.6 Production, extraction and Partial purification of
For extraction of biosurfactant from consortium a solvent
biosurfactants
extraction method was used. Solvent were selected on the
Biosurfactant production was conducted in submerged batch
basis of the prior extraction methods. A solvent system was
culture operation using starter culture of 10% of the bacteria
developed as 3parts acetone, two parts chloroform and one
in grown in Nutrient broth medium. The seed culture was
part of methanol to extract the biosurfactants from
grown till stationary phase and inoculated to 100 ml of
consortium. The pellets were obtained at bottom of
Bushnell Haas broth supplemented with 2% Mustard oil as
centrifuge tube as well as at junction of the aqueous and
Soul Carbon Source in 250 ml capacity Erlenmeyer flasks
organic phase. The pellets were collected in a screw capped
duly cotton plugged. The flasks were kept in shaker
bottle to be used further.
incubator at 35˚C and 160 rpm for 10 days. An uninoculated
2.7 Characterization based on biochemical composition
flask was used to check any contamination.
of
2.6.1 Crude extraction of biosurfactants
biosurfactant
After 10 days of incubation the contents of the flask were
The supernatant was analyzed for protein and carbohydrate
centrifuged at 8,000 rpm for 30 mins. The cell pellet was
content.
discarded that contained the bacterial or consortium cell
2.7.1 Determination of dry weight of surfactant
biosurfactant
Biochemical
composition
of
debris. The supernatant was filtered using Whatmann no 1
filter paper that contained the biosurfactant which was used
as crude biosurfactant and was further purified using solvent
extraction method.
Sterilized petriplate was taken and the weight of the plate
was measured in grams. Now the sediment was poured on
the plate and placed in the hot air oven for drying at 100 °C
for 30 minutes. After drying the plates were weighed
2.6.2 Solvent extraction and precipitation:
Biosurfactant purification from Pseudomonas aeruginosa
(Anandraj and Thivakaran, 2010). The dry weight of the
biosurfactants was calculated using the following formula:
was done through chilled acetone precipitation method for
which the clear sterilized supernatant of the crude
Dry weight of biosurfactants = Weight of the plate
after drying - weight of the empty plate
biosurfactant was precipitated using three 2ml of chilled
Acetone added to one part of supernatant i.e. 1:3 ratio and
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6478
2.7.2 Estimation of total protein content of the
Concentration
biosurfactant
of BSA (mg/ml)
Protein content in the sample was estimated by the Lowry‟s
Sample size: 5
method (Lowry, 1951). The total protein concentration is
exhibited by a colour change of the sample solution in
proportion to protein concentration, which can then be
measured using colorimetric techniques. Standard stock
solution (1mg/ml) of Bovine Serum Albumin (BSA) was
prepared and transferred to different test tubes in different
Intercept (a): -0.038999999999999
Slope (b): 1.175
Regression line equation: y=1.175x0.038999999999999
aliquots of 0, 0.2, 0.4, 0.6. 0.8 and 1ml and final volume in
2.7.3 Determination of carbohydrate content of the
each test tube was made up with distilled water to 1ml to
biosurfactant
produce concentration range of 0, 0.2mg/ml, 0.4mg/ml,
0.6mg/ml, 0.8mg/ml, 1mg/ml. In another test tube 0.5ml of
Determination of WSC using phenol-sulphuric acid (Dubois
sample was taken and again its volume was made up to 1ml
et. al., 1956) is based on the absorbance at 490 nm of a
by distilled water. After this, fresh reagents were prepared
coloured aromatic complex formed between phenol and the
i.e. Reagent A, Reagent B, Reagent C and Reagent D, then
carbohydrate. The amount of WSC present was determined
5ml of Reagent C was added in each test tube and all test
by comparison with a calibration curve using a calorimeter.
tubes was kept at room temperature for 10min under dark.
1 mg/ml of standard stock solution of glucose was prepared
Then 0.5ml of Reagent D (F-C Reagent) was added in each
in distilled water. The aliquots was transferred to 10
test tube and again kept at room temperature for 30-40min
different 10ml tubes in 5- increments ranging from 5 to 50
under dark after proper vortexing. Optical Density
µl and volume in each tube was made up with distilled water
(absorbance was measured) was taken at 750nm (A750) using
to a final volume of 50µl with an accurate pipette. Similarly,
a calorimeter. Then absorbance against BSA concentration
5µl of the Petrol/Diesel sample was taken in a 10 ml tube
was plotted to get a standard calibration curve. At last,
and its final volume was also made up to 50µl by addition of
absorbance of unknown sample was measured and the
distilled water. To all the tubes, 500 µl of 4% Phenol
concentration of the unknown sample was determined using
followed by 2.5 ml 96% Sulfuric acid was added and the
the standard curve plotted. The intercept of the A750 of the
tubes was kept at room temperature for 10min. A tube
Petrol/diesel sample with the calibration line will represent
having 4% Phenol and 2.5 ml 96% sulfuric acid was taken
the amount (µg) of protein present in the
as blank. After this, the solutions from the test tubes was
biosurfactant of
Petrol/Diesel sample
transferred to the cuvette on by one after setting colorimeter
to 0 by blank tube and the absorbance was measured at
X  dilution factor
Total Protein content of the sample
(mg/ml) =
__________________________
490nm (A490) of the sugar standards and Petrol/Diesel
sample. To calculate the concentration of WSC present in
the food sample, a graph plotting A490 versus sugar weight
Amount of sample
(µg)/ concentration of the sugar standard was prepared. The
intercept of the A490 of the Petrol/diesel sample with the
calibration line will represent the amount (µg) of WSC
O.
D.
at
75
0
nm
present in the Petrol/Diesel sample.
Total carbohydrate content of the sample (mg/ml)
X  dilution factor
= ______________________________
Amount of sample
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6479
reagent was added to each tube and mixed well. All the
tubes were incubated at 37°C for 15 minutes in water bath.
The optical density was measured at 540 nm.
Calculation:
O. D. unknown_________= O.D. standard____________
Concentration unknown
concentrationstandard
Sample size: 5
ean x x 0.6
ean y
0.672
Intercept (a): -0.041999999999999
Slope (b): 1.19
Regression line equation: y=1.19x0.041999999999999
3.
Results
3.1.1 Production, extraction and partial purification of
biosurfactants
Plate 1, 2, 3, 4 shows the laboratory scale submerged
production of biosurfactants in Bushnell Haas broth
supplemented with 2% Mustard oil by individual bacteria
namely, Bacillus subtilis, Pseudomonas aeruginosa and
2.7.4 Estimation of lipid contents
Serratia marscecens. Consortia was prepared in Nutrient
Lipids upon reaction with sulfuric acid forms carbonium
broth and inoculated as 10% inoculum into the Bushnell
ion which after reacting with vanillin phosphate ester forms
Haas broth. The biosurfactants from each flask is obtained
a purple complex. A setup of three test tubes was made
during late stationary phase of the bacterial growth curve
among which the one with 20 µl of distilled water will
as the biosurfatant is a secondary metabolite secreted out
serve as blank. In another tube 20 µl of standard (olive oil/
by the bacterial cell into the medium. The transparency of
cholesterol) was taken and to the third tube 20 µl of
the medium is reduced to opaque with increasing time of
unknown sample was taken. To each tube 20 µl of
incubation and increase in biosurfatant production. The oil
sulphuric acid was added and mixed using a vortex mixer.
supplemented to the medium was 2% is completely
The set was allowed to be incubated for 10 minutes and
degraded after 10-15 days of incubation.
then cooled to room temperature. 10 ml of phosphovanillin
Plate 1: Degradation of oil and simultaneous production of biosurfactant from Consortia
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6480
Plate 2: Degradation of oil and simultaneous production of biosurfactant from Bacillus subtilis
Plate 3: Degradation of oil and simultaneous production of biosurfactant from Pseudomonas aeruginosa
Plate 4: Degradation of oil and simultaneous production of biosurfactant from Serratia marscecens
Extraction and partial purification by solvent extraction method
The biosurfactant production was obtained after 15 days of
Partially purified fractions of the biosurfactants were
obtained from the bacterial cultures after solvent
incubation time. Among the four Serratia marscecens gave
extraction method.
highest yield.
3.1.2
Yield of biosurfactants in g/l of production
medium
Table: 1 Yield of biosurfactants in g/l of production medium
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6481
Biosurfactants source
Yield (g/l)
Pseudomonas aeruginosa
5.445
Serratia marscecens
36.515
Bacillus subtilis
43.76
Consortia
24.41
Yield (g/l)
Pseudomonas aeruginosa
24.41
Serratia marscecens
5.445
Bacillus subtilis
Consortia
36.515
43.76
Biochemical characterization of biosurfactants
Table: 2 Biochemical characterization of biosurfactants
Partially purified biosurfactant
Protein
source
(mg/ml)
Carbohydrate
(mg/ml)
0.296
0.187
0.489
0.607
Pseudomonas aeruginosa
Serratia marscecens
Bacillus subtilis
Consortium
Lipid
(µl/20µl
sample)
0.967
4.032
0.113
1.45
0.518
0.314
0.254
0.692
Drop collapse assay was performed to evaluate the surface
activity of biosurfactants obtained from Pseudomonas
aeruginosa, Serratia marscecens, Bacillus subtilis and the
All bacterial isolates were screened for biosurfactants
consortium developed from the three strains. The assay was
production by drop collapse assay.
performed against Petrol, Diesel, Mobil oil, Kerosene,
Mustard oil, Soybean oil, Jasmine oil and coconut oil. For
3.1.3. a Drop collapse assay of partially purified
all the oils and hydrocarbons a positive drop collapse assay
biosurfactants
was found.
Table: 3 Drop collapse assay of partially purified biosurfactants
Biosurfactants source
Petrol
Diesel
Mobil oil
Kerosene
Mustard
Soybean
Jasmine
Almond
3.1.3 Screening of partially purified biosurfactants for
surface activity
Pseudomonas aeruginosa
Serratia marscecens
Bacillus subtilis
Consortia
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3.2
Emulsification
biosurfactants
index
of
partially
purified
Partially purified biosurfactants obtained from Pseudomonas
aeruginosa, Serratia marscecens,
Bacillus subtilis and the consortium was used to determine
the emulsification activity against oils and poly aromatic
hydrocarbons.
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6482
and that declined to 85% after 24h while after 96 h the
emulsification was stable to 73.68%. Among vegetable oils,
Mustard oil had highest emulsification i.e 92.30% at 0h that
declined to 47.61% after 96h.
3.2.1 Emulsification index of partially purified
biosurfactant of Serratia marscecens
Highest emulsification was found after 24h of incubation
then gradually declined till 96h of incubation. Among
hydrocarbons Mobil oil was emulsified with highest
intensity showing 100% emulsification at 0h of incubation
Table: 4 Emulsification index of partially purified biosurfactant of Serratia marscecens
Substrate
category
Hydrocarbons
Vegetable oils
Emulsification index of partially purified biosurfactant of Serratia
marscecens
Substrate
E0
E24
E48
E72
Kerosene
21.7
18.18
14.28
5
Petrol
17.39
15.78
11.76
6.66
Diesel
12
8.33
4.34
0
Mobil oil
100
85
80
75
Mustard oil
92.30
52
50
47.82
Coconut oil
16.66
9
4.76
0
Soy bean oil
42.30
37.5
34.78
27.27
Jasmine oil
13.6
9.52
5
0
Emulsification index (%)
Kerosene
120
100
80
60
40
20
0
100
Petrol
Diesel
E96
0
0
0
73.68
47.61
0
23.8
0
Mobil oil
85
80
75
73.68
21.7
12
17.39
18.18
15.78
8.33
11.76
14.28
4.34
6.66
5
0
0
E0
E24
E48
Time of incubation (h)
E72
E96
Emulsification Index (%)
Fig: 4 Emulsification index of partially purified biosurfactant of Serratia marscecens
100
50
Mustard oil
92.3
Coconut oil
Soy bean oil
42.3
52
37.5
50
34.78
16.66
13.6
9.52
9
4.76
5
E0
E24
Jasmine oil
47.82
27.27
47.61
0
0
E72
E96
23.8
0
E48
Incubation time (h)
Fig: 4 Emulsification index of partially purified biosurfactant of Serratia marscecens
3.2.2 Emulsification index of partially purified
biosurfactant of Pseudomonas aeruginosa
Emulsification index was highest at 0h and 24h of
incubation which after 24h till 96h. the stability of
Partially purified biosurfactant from Pseudomonas
among hydrocarbons while the highest emulsification index
aeruginosa was capable to emulsify hydrocarbons
and stability was recorded for coconut oil among the
(Kerosene, Petrol, Diesel and Mobil oil) as well as vegetable
vegetable oils.
oil (Mustard, coconut, soy bean and Jasmine oil).
Table: 5 Emulsification index of partially purified biosurfactant of Pseudomonas aeruginosa
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6483
Substrate
category
Hydrocarbons
Vegetable oils
Emulsification index of partially purified biosurfactant of Pseudomonas aeruginosa
Substrate
E0
E24
E48
E72
Kerosene
45.83
39.13
36.36
33.33
Petrol
54.54
50
47.36
35.29
Diesel
21.42
15.38
12
8.33
Mobil oil
54.54
52.38
45
40
Mustard oil
30.43
27.27
27.27
25
Coconut oil
36
33.33
30.43
27.27
Soy bean oil
12
8.69
4.54
0
Jasmine oil
23.07
16.66
13.04
8.69
Emulsification Index (%)
Kerosene
60
50
54.54
45.83
Petrol
52.38
50
47.36
45
36.36
39.13
40
30
21.42
20
Diesel
15.38
Mobil oil
40
35.29
33.33
12
E96
26.31
13.33
8.33
31.57
21.05
23.80
0
4.54
31.57
26.31
8.33
13.33
8.33
E72
E96
10
0
E0
E24
E48
Time of Incubation (h)
Fig: 4 Emulsification index of partially purified biosurfactant of Pseudomonas aeruginosa
Emulsification Index(%)
Mustard oil
30
36
30.43
23.07
20
12
40
Coconut oil
33.33
27.27
16.66
8.69
Soy bean oil
30.43
27.27
27.27
25
13.04
8.69
4.54
10
Jasmine oil
23.8
21.05
0
4.54
0
E72
E96
0
E0
E24
E48
Time of Incubation in hours
Fig: 4 Emulsification index of partially purified biosurfactant of Pseudomonas aeruginosa
3.2.3 Emulsification index of partially purified
biosurfactant of Bacillus subtilis
Partially purified biosurfactant from Bacillus subtilis
emulsified both categories i.e. hydrocarbons and vegetable
oils and the emulsification tend to decline from 0h to 96h of
incubation gradually. Among hydrocarbons kerosene was
highly emulsified and the emulsification stability was high
as 54.54% till 96h. Among vegetable oils, highest
emulsification was recorded for Mustard oil in range of 100
to 71.42% from 0h to 96h of incubation. It was observed
that diesel emulsification was unstable after 24h and no
emulsification was observed from 48h to 96h.
Table: 6 Emulsification index of partially purified biosurfactant of Bacillus subtilis
Emulsification index of partially purified biosurfactant of Bacillus subtilis
Substrate
category
Substrate
E0
E24
E48
E72
Kerosene
68
66.66
65.21
63.63
Petrol
58.38
50
45
36.84
Hydrocarbons
Diesel
13.04
9.09
0
0
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
E96
54.54
29.41
0
Page 6484
Mobil oil
Mustard oil
Coconut oil
Soy bean oil
Jasmine oil
Vegetable oils
45
100
28
15.38
48
Kerosene
Emulsification Index (%)
80
Petrol
66.66
65.21
45
50
40
45
36.84
26.31
73.91
13.63
4.54
38.09
16.66
71.42
9.52
0
35
Mobil oil
63.63
54.54
40
13.04
36.84
75
18.18
8.69
43.47
Diesel
68
58.38
60
20
40
80
21.7
12.5
45.83
36.84
26.31
29.41
16.66
9.09
0
0
0
E48
E72
E96
0
E0
E24
Time of Incubation (h)
Fig: 4 Emulsification index of partially purified biosurfactant of Bacillus subtilis
Emulsification Index (%)
Mustard oil
120
100
80
60
40
20
0
Coconut oil
Soy bean oil
Jasmine oil
100
80
75
73.91
71.42
48
28
15.38
45.83
43.47
38.09
21.7
12.5
35
18.18
8.69
13.63
4.54
9.52
0
E0
E24
E48
E72
E96
Time of Incubation (h)
Fig: 4 Emulsification index of partially purified biosurfactant of Bacillus subtilis
3.2.4 Emulsification index of partially purified
biosurfactant of consortium
Partially purified biosurfactant was obtained from the
bacterial consortium developed using Pseudomonas
aeruginosa, Serratia marscecens and Bacillus subtilis. The
bacterial consortium was potent for emulsification of the
selected hydrocarbons (Kerosene, Petrol, Diesel and Mobil
oil) and vegetable oils(Mustard, coconut, soy bean and
Jasmine oil).
Table: 7 Emulsification index of partially purified biosurfactant of consortium
Substrate
category
Hydrocarbons
Vegetable oils
Emulsification index of partially purified biosurfactant of consortium
Substrate
E0
E24
E48
E72
Kerosene
40.90
31.81
28.57
25
Petrol
34.78
31.57
25
13.35
Diesel
28.57
25.92
23.07
20
Mobil oil
84
45.83
43.47
36.36
Mustard oil
28
27.27
22
20
Coconut oil
19.23
16.66
13.04
4.54
Soy bean oil
38.46
37.5
34.78
27.27
Jasmine oil
33.33
30.43
22.72
19.04
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
E96
21.05
7.69
16.66
25
20
0
23.80
14.28
Page 6485
Kerosene
Petrol
Diesel
Mobil oil
84
100
Emulsification Index (%)
80
60
40
40.9
34.78
28.57
45.83
31.81
31.57
25.92
43.47
28.57
25
23.07
E0
E24
E48
20
36.36
25
20
13.35
25
21.05
16.66
7.69
E72
E96
0
Time of Incubation (h)
Fig: 4 Emulsification index of partially purified biosurfactant of consortium
Emulsification Index (%)
Mustard oil
50
40
30
38.46
33.33
28
19.23
20
Coconut oil
37.5
30.43
27.27
16.66
Soy bean oil
Jasmine oil
34.78
22.72
22
13.04
27.27
20
19.04
4.54
10
23.8
20
14.28
0
0
E0
E24
E48
E72
E96
Time of Incubation (h)
Fig: 4 Emulsification index of partially purified biosurfactant of consortium
Plate: 5 Emulsification activity partially purified biosurfactant from consortia
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6486
Plate: 6 Emulsification activity crude biosurfactant from consortia
Plate: 7 Emulsification activity crude biosurfactant from consortia
Plate:8 Emulsification activity partially purified biosurfactant from consortia
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6487
Plate: 9 Emulsification activity crude biosurfactant from consortia
Plate: 10 Emulsification activity crude biosurfactant from consortia
4. Discussion
Techaoei et al. (2007) did preliminary screening of
preliminary screening of biosurfactant producing
microorganisms isolated from hot spring and garages in
northern Thailand and reported the emulsification at 24 36
and 48 h of incubation. The deposition of material at the
edge of evaporating droplets, known as the „coffee ring
effect‟, is caused by a radially outward capillary flow. This
phenomenon is common to a wide array of systems
including colloidal and bacterial systems. The role of
surfactants in counteracting these coffee ring depositions is
related to the occurrence of local vortices known as
Marangoni eddies. Here we show that these swirling flows
are universal, and not only lead to a uniform deposition of
colloids but also occur in living bacterial systems.
Experiments on Pseudomonas aeruginosa suggest that the
auto-production of biosurfactants has an essential role in
creating a homogeneous deposition of the bacteria upon
drying (Sempels et al., 2013). Bodour et al. reported that
the drop collapse method may be used to detect
biosurfactant producing microorganisms in natural
environments. The results of the present study also
suggested that the drop collapse method suits well as a
primary screening method for biosurfactant production and
the oil spreading method is good to quantify the
biosurfactant. The Biosurfactant production may be also
detected by using emulsification index. Surface activity and
emulsification activity have direct correlation [Surachai et
al., 2007]. Biosurfactants have the ability to reduce surface
tension and emulsify hydrocarbons, thereby, boosting the
bioavailability quotient of the contaminating hydrocarbons.
According to the study of Chandran and Das, 2011, the
stability of formed emulsions was found to be more than one
month in room temperature without changing emulsification
activity. Interestingly, the crude biosurfactants gave the
highest emulsification activity (E24) on diesel, probably
because it was produced by diesel oil as a carbon source.
The emulsification index values of biosurfactants were also
measured at different temperatures, pH and NaCl
concentrations. The optimum temperature for emulsification
activity of biosurfactants was at room temperature (28 ºC),
even showing emulsification activity at temperature of 10 100 ºC. This mechanism is of importance when two
immiscible phases (oil and water) are present and direct
substrate uptake is plausible (Neu, 1996; Franzetti et al.
2009). The presence of biosurfactants may also lead to a
potential enhancement of biodegradation efficiency. In this
concept, the biosurfactant molecules act as mediators, which
increase the mass transfer rate by making hydrophobic
pollutants more bioavailable for microorganisms (Inakollu
et al. 2004; Whang et al. 2009). Alternatively,
biosurfactants may also induce changes in the properties of
cellular membranes, resulting in increased microbial
adherence. Hydrocarbons are organic compounds made of
carbon atoms bound to each other forming a backbone with
hydrogen atoms attached to the remaining sites on carbon.
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6488
The carbon backbone can be straight or normal, branched, or
cyclic (Olah and Molnar, 1995). Most hydrocarbons used
as an energy source are degraded under aerobic conditions
to carbon dioxide and water. This degradation process is a
catabolic process and the degradation to inorganic
compounds is called mineralization. However, some
hydrocarbons are not mineralized but transformed into
simpler compounds (Ferrari et al. 1996). The hydrocarbons
used as a carbon source are degraded to smaller compounds
and incorporated into the cell materials. This degradation
process is a combination of catabolic and anabolic processes
(Brock et al., 1994). Cometabolism is another mode of
degradation, which is observed in the degradation of
hydrocarbons. In cometabolism the hydrocarbon is
transformed, but the organisms does not gain any energy or
nutrients (Field et al.,1991, Juhasz et al., 1996). The
specific degradation mechanisms are determined by the
compound structure. Linear alkanes degrade through boxidation in which the backbone is broken up two carbons at
a time and the resulting acetyl-CoA is mineralized in the
TCA cycle. Some cyclic alkanes degrade through
cometabolism (Juhasz et al. 1996). Aromatic compounds are
generally degraded via a dioxygenase enzyme, which
converts the compound to a catechol followed by ring
fission in the ortho or meta positions (Prince, 1993).
Emulsification is a process that forms a liquid, known as an
emulsion, containing very smalldroplets of fat or oil
suspended in a fluid, usually water. The high molecular
weight biosurfactants are efficient emulsifying agents. They
are often applied as an additive to stimulate bioremediation
and removal of oil substances from environments.
5. Conclusion
Conclusively the study revealed the idea of degradation of
poly aromatic hydrocarbon and vegetable oils using a potent
consortium capable of biosurfactant production. The three
strains viz. could be used to degrade or emulsify a range of
oils and hydrocarbons.
6. Acknowledgments
The authors offer gratuitous thanks to Hon‟ble Vice
Chancellor, Most Rev. Prof. R.B. Lal, SHIATS, Naini,
Uttar Pradesh, India for provision of research
conductance. Heartfelt thanks to Head, Department of
Microbiology and Fermentation Technology, JSBB,
SHIATS, Allahabad, Uttar Pradesh, India for the kind
cooperation towards the research.
7.
References
[1] Colomer, A. Pinazo, M.A. Manresa, M.P.
Vinardell, M. Mitjans, M.R. Infante, L. Pérez,
Cationic surfactants derived from lysine: effects of
their structure and charge type on antimicrobial and
hemolytic activities, J. Med. Chem. 54: pp. 989–
1002, 2011.
[2] B.C. Stephenson, A. Goldsipe, K.J. Beers, D.
Blankschtein, Quantifying the hydrophobic effect.
1.
A
computer
simulation−molecularthermodynamic model for the self-assembly of
hydrophobic and amphiphilic solutes in aqueous
solution, J. Phys. Chem. B. 111:1025–1044.
[3] Banat IM. Biosurfactant production and possible
uses in microbial enhanced oil recovery and oil
pollution remediation. Biores Technol .pp. 51:1-12,
1995.
[4] Brock, T. D., Madigan, M. T., Martinko, J. M.,
and Parker, J. Biology of Microorganisms.
Prentice Hall Inc. Englewood Cliffs, NJ. 1994.
[5] C.N. Mulligan, Environmental applications for
biosurfactants, Environ. Pollut. 133, pp. 183–198,
2005.
[6] Cameotra SS, Makkar RS. Synthesis of
biosurfactants in extreme conditions. Appl
Microbiol Biotechnol . 50, pp. 520–9, 1998;.
[7] Carrillo, C. et al. Molecular mechanism of
membrane permeabilization by the peptide
antibiotic surfactin. Biochim. Biophys. Acta 1611,
pp. 91–97,2003.
[8] Chandran P. and Das, P. Characterization of
sophorolipid biosurfactant produced by yeast
species grown on diesel oil. International Journal
of Science and Nature. 2(1), pp. 63-71, 2011.
[9] Compant, S., Duffy, B., Nowak, J., Clement, C.,
and Barka, E. A. Use of plant growth-promoting
bacteria for biocontrol of plant diseases: Principles,
mechanisms of action, and future prospects. Appl.
Environ. Microbiol. 71, pp. 4951-4959, 2005.
[10] Desai JD, Banat IM. Microbial production of
surfactants and their commercial potential.
Microbiol Mol Biol Rev.;61, pp. 47–64, 1997.
[11] EFSA, Opinion of the Scientific Panel on Food
Additives, Flavourings, Processing Aids and
Materials in Contact with Food (AFC) on a request
from the Commission related to the use of
epoxidised soybean oil in food contact materials.
EFSA J. 64, pp. 1–17, 2004.
[12] Ferrari, M. D., Neirotti, E., Albornoz, C.,
Mostazo, M. R., and Cozzo, M. “Biotreatment of
Hydrocarbon from Petroleum Tank Bottom
Sludges in Soil Slurries.” Biotechnology Letters,
18(11), pp. 1241-1446.
[13] Field, S. D., Marks, R. E., and Wojtanowicz, A.
K. “Advanced Biological Treatment and
Separation of Hazardous Constituents from
Petrochemical Sludges.” Journal of Hazardous
Materials, 28, pp. 101-113.
[14] Franzetti A, Caredda P, Ruggeri C, La Colla P,
Tamburini E, Papacchini M, Bestetti G.
Potential applications of surface active compounds
by Gordonia sp. strain BS29 in soil remediation
technologies. Chemosphere 75, pp. 801–807, 2009.
[15] Gutnik DL, Shabtai Y. Exopolysaccharide
bioemulsifiers. In: Kosaric N, Cairns WL, Gray
NCC, editors. Biosurfactants and biotechnology.
New York: Dekker. pp. 211–46, 1987.
[16] H. Yan, S.-L. Yuan, G.-Y. Xu, Ch.-B. Liu,
Effect of Ca2+ and Mg2+ ions on surfactant
solutions investigated by molecular dynamics
simulation, Langmuir 26, pp. 10448–10459.
[17] Haas, D., and Défago, G. Biological control of
soil-borne pathogens by fluorescent pseudomonads.
Nat. Rev. Microbiol. 3, pp. 307-319, 2005.
[18] Heerklotz, H. and Seelig, J. Leakage and lysis of
lipid membranes induced by the lipopeptide
surfactin. Eur. Biophys. J. 36, pp. 305–314, 2007.
[19] Inakollu S, Hung H, Shreve GS. Biosurfactant
enhancement of microbial degradation of various
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6489
strructural classes of hydrocarbon in mixed waste
systems. Environ Eng Sci 21, pp. 463–469, 2004.
[20] J.H. Fendler, Membrane Mimetic Chemistry,
Wiley-Interscience, New York, 1982.
[21] Juhasz, A. L., Britz, M. L., and Stanley, G. A.
“Degradation of High olecular Weight Polycyclic
Aromatic
Hydrocarbons
by
Pseudomanas
Cepacia.” Biotechnology Letters, 18(5), pp. 577582, 1996.
[22] Katz E, Demain AL. The peptide antibiotics of
Bacillus: Chemistry, biogenesis, and possible
functions. Bacteriol Rev .41, pp. 449–474, 1977.
[23] Lin S-C. Biosurfactants: recent advances. J Chem
Technol Biotechnol . 66, pp. 109–20, 1996.
[24] M. Paluch, B. Gzyl, Properties of mixed
adsorption films of 2,2,2-trichloroethanol– ethanol
at the water/air interface, J. Colloid Interface Sci.
179, pp. 51–56, 1996.
[25] Maget-Dana R, Peypoux F. Iturins, a special class
of poreforming lipopeptides: Biological and
physicochemical properties. Toxicology. 87, pp.
151–174, 1994.
[26] Neu TR, Significance of bacterial surface-active
compounds in interaction of bacteria with
interfaces. Microbiol Rev 60, pp. 151–166, 1996.
[27] Olah, G. A. and Molnar, A. (1995). Hydrocarbon
Chemistry. John Wiley & Sons, Inc., New York,
NY, 1-53.
[28] Peypoux, F. et al. Recent trends in the
biochemistry of surfactin. Appl. Microbiol.
Biotechnol. 51, pp. 553–563, 1999.
[29] Pieterse, C. M. J., Van Wees, S. C. M., Ton, J.,
Van Pelt, J. A., and Van Loon, L. C. Signaling in
rhizobacteria-induced systemic resistance in
Arabidopsis thaliana. Plant Biol. 4, pp. 535-544,
2002.
[30] Prince, R. C. “Petroleum Spill Bioremediation in
arine Environments.” Critical Reviews in
Microbiology, 19, pp. 217-242, 1993.
[31] Ramos, J.-L. 2004. Pseudomonas, Vol. 1–3.
Kluwer Academic/Plenum Publishers, New York,
U.S.A.
[32] Rosenberg E, Ron EZ. High- and low-molecularmass microbial surfactants. Appl Microbiol
Biotechnol. 52, pp. 154–62, 1999.
[33] Ryu, C.-M., Hu, C.-H., Reddy, M. S., and
Kloepper, J. W. Different signaling pathways of
induced resistance by rhizobacteria in Arabidopsis
thaliana against two pathovars of Pseudomonas
syringae. New Phytol. 160, pp. 413-420, 2003.
[34] Sempels, W., De Dier, R., Mizuno, H., Hofkens,
J. and Vermant, J.
Auto-production of
biosurfactants reverses the coffee ring effect in a
bacterial system. Nature Communications. 4(1757),
pp. 1-8, 2013.
[37] Techaoei,
S.,
Leelapornpisid,
P.
and
Santiarwarn, D. Preliminary screening of
biosurfactantproducing microorganisms isolated
from hot spring and garages in northern Thailand.
KMITL Sci. Tech. J. 7(S1), pp. 38-43, 2007.
[38] The British Industrial Biological Research
Association (BIBRA), Toxicity profile:
epoxidised soy bean oil. Carlshalton, Surrey, UK,
1988.
[39] Ward, O.P., Singh, A. and Hamme, J.D.V.
Physiological aspects Part 1 in a series of papers
devoted to surfactants in microbiology and
biotechnology. Biotechnology Advances- Elsevier.
24, pp. 604–620, 2006..
[40] Whang L-M, Liu P-WG, Ma C-C, Cheng S-S.
Application of rhamnolipid and surfactin for
enhanced diesel biodegradation- Effects of pH and
ammonium addition. J Hazard Mater.164, pp.
1045–1050, 2009.
[41] Zhang, L., Sun, J., Hao, Y., Zhu, J., Chu, J.,
Wei, D.and Shen, Y. Microbial production of 2,3butanediol by a surfactant (serrawettin)-deficient
mutant of Serratia marcescens H30. J Ind
Microbiol Biotechnol. 37, pp. 857–862, 2010.
[42] Zhang, Y and Miller. R M. Effect of
Rhamnolipid
(Biosurfactant)
Structure
on
Solubilization and Biodegradation of n-Alkanes.
Applied and Environmental Microbiology. 61(6),
pp. 2247–2251, 1995.
Author Profile
Jyotsna Kiran Peter, received university project
from Sam Higginbottom Institute of Agriculture
Technology and Sciences, Naini, Uttar Pradesh,
India during the year 2013-2014.the work was
conducted in the research Laboratory of
Department of Microbiology and Fermentation
Technology, Jacob School of Biotechnology and
Bioengineering, SHIATS, Naini, India.
[35] Surachai T, Pimporn L, Dammrong S, et al.
Preliminary screening of biosurfactant producing
microorganisms isolated from hot Spring and
garages in Northern Thailand. Kmitl sci Tech J 7,
pp. 38–43, 2007.
[36] T. Cserháti, E. Forgács, G. Oros, Biological
activity and environmental impact of anionic
surfactants, Environ. Int. 28,337–348, 2002.
Jyotsna Kiran Peter1 IJECS Volume 3 Issue 6 June, 2014 Page No.6476-6490
Page 6490