Crude Cacterial Consortium Biosurfactants Potent For Bioemulsification of Oils and Petroleum Hydrocarbons

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International Journal of Research (IJR) Vol-1, Issue-7, August 2014 ISSN 2348-6848
CRUDE CACTERIAL CONSORTIUM BIOSURFACTANTS POTENT FOR BIOEMULSIFICATION OF OILS AND
PETROLEUM HYDROCARBONS Jyotsna Kiran Peter, Abhishek Kumar Rao and Ritu Kumari

Crude Cacterial Consortium Biosurfactants Potent for
Bioemulsification of Oils and Petroleum Hydrocarbons

Jyotsna Kiran Peter
1
*, Abhishek Kumar Rao
1
and Ritu Kumari
1

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

Abstract
Crude biosurfactants from Pseudomonas
aeruginosa, Serratia marscecens and Bacillus
subtilis along with the consortium of the three
mentioned bacteria were harvested after batch
mode submerged fermentation. The
emulsification was found highest by consortium
crude biosurfactants as compared to individual
bacterial crude biosurfactants. The stability was
also found persistant from 0h to 96 h in case of
crude consortium biosurfactants.

Key words: Pseudomonas aeruginosa, Serratia
marscecens, Bacillus subtilis, crude consortium
biosurfactants and emulsification

Introduction
Hydrocarbons are commonly used as the
substrate for the production of biosurfactants. It
has been postulated that the biological function of
surface-active compounds is related to
hydrocarbon uptake, and therefore a
spontaneous release occurs with these
substrates (Mu et al., 2009). Biosurfactants are
produced by several types of microorganisms,
such as bacteria, fungi and yeasts Namir et al.,
2009]. Bacillomycin F, D, L [Marion and Maget-
Dana, 1985], surfactin [Schneider et al., 1988],
lichenysin [Grangemard et al., 1999], iturin
[Peypoux et al., 1978], halobacillin [Grangemard
et al., 1999], and plipastatin [Nishikiori et al.,
1986] are lipopeptide biosurfactants produced by
Bacillus strain. Surfactin, one of the most
effective biosurfactants, was isolated from the
cell-free culture medium after growth of Bacillus
subtilis on glucose [Cooper et al., 1981]. This
biomolecule is usually a cyclic compound
consisting of seven amino acids bonded to a lipid
moiety. Surfactin is effective in lowering the
surface tension of water to30 mN/m [Fox and
Bala, 2000], which is comparable with the values
obtained by conventional synthetic surfactants.
Additionally, surfactin preparations have other
interesting characteristics, including antibiotic
and antiviral properties [Davis et al., 1999].
Surfactin isoforms produced by Bacillus subtilis
HSO121 have been studied extensively in our
laboratory [Haddad et al., 2008; Liu et al., 2007].
Pseudomonas species are known to produce
different types of BS viz., rhamnolipids, cyclic
lipopeptides- putisolvins, lipopolysaccharide.
Two types of cyclic lipopeptides (putisolvins I and
II) are produced by P. putida PCL1445, which
possess surfactant activity and also plays
significant role in biofilm formation and
degradation. Serratia is one of the well-studied
bacterium in terms of molecular genetic studies of
BS production. Serratia, a Gram-negative or-
ganism is known to produce extracellular surface
active(Matsuyama et al., 1987) and surface
translocating agents (Horng et al., 2002) S.
marcescens produces a cyclic lipopeptide BS
Serrawettin which contains 3-hydroxy-C10 FA
side chain. BS production is correlated with
populational surface migration (Wei et al., 2004)

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Materials and Methods
Place of work done
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.
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.
Name of bacteria
Pseudomonas aeruginosa
Bacillus subtilis
Serratia marscecens
Maintenance of bacterial culture
The bacterial cultures were sub cultured routinely
at 15 days interval on Nutrient agar incubated at
35C for 24 hours.
Drop Collapse assay
Petrol/Diesel oil (2l) was added to Petriplate lid.
The lid was pre-equilibrated for 1 hour at room
temperature, and then 5l of the culture
supernatant was added to the surface of oil. The
shape of the drop on the oil surface was
inspected after 1 minute. Biosurfactant containing
cultures will give flat drops, thus indicating a
positive result.
Production, extraction and Partial purification
of biosurfactants
Biosurfactant production was conducted in
submerged batch culture operation using starter
culture of 10% of the bacteria in grown in Nutrient
broth medium. The seed culture was grown till
stationary phase and inoculated to 100 ml of
Bushnell Haas broth supplemented with 2%
Mustard oil as Soul Carbon Source in 250 ml
capacity Erlenmeyer flasks duly cotton plugged.
The flasks were kept in shaker incubator at 35C
and 160 rpm for 10 days. An uninoculated flask
was used to check any contamination.
Crude extraction of biosurfactants
After 10 days of incubation the contents of the
flask were centrifuged at 8,000 rpm for 30 mins.
The cell pellet was discarded that contained the
bacterial or consortium cell 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.
Results
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 Serratia
marscecens. Consortia was prepared in Nutrient
broth and inoculated as 10% inoculum into the
Bushnell Haas broth. The biosurfactants from
each flask is obtained during late stationary
phase of the bacterial growth curve as the
biosurfatant is a secondary metabolite secreted
out by the bacterial cell into the medium. The
transparency of the medium is reduced to opaque
with increasing time of incubation and increase in
biosurfatant production. The oil supplemented to
the medium was 2% is completely degraded after
10-15 days of incubation.

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Plate 1: Degradation of oil and
simultaneous production of biosurfactant
from Consortia

from Bacillus subtilis

from Pseudomonas aeruginosa

from Serratia marscecens

Drop collapse assay of crude biosurfactants
Crude biosurfactants from the individual strains of
Pseudomonas aeruginosa, Serratia marscecens
and Bacillus subtilis along with the consortium of
the three mentioned bacteria showed positive
drop collapse assay (Table1). The drop collapse
assay signifies the biosurfactant activity of crude
biosurfactants. An oil drop acquires a convex
shaped drop while resting on a calibrated glass
slide. The convex shape of the drop is distorted
or the drop collapse due to biosurfactants activity
as the nature of biosurfactant is to align and orient
them into various configurations at oil aqueous
interphase and reduces the surface tension of oil
drop so that the drop collapses. The assay has
been used by several authors to screen the
biosurfactant activity of either microorganism or
partially or crude or purified biosurfactants.
Table1: Drop collapse assay of crude biosurfactants
Biosurfactants source
P
e
t
r
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Pseudomonas aeruginosa
+
+ + + + + + +
Serratia marscecens
+ + + + + + + +
Bacillus subtilis
+ + + + + + + +
Consortium
+ + + + + + + +

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Emulsification index of crude biosurfactant
of Pseudomonas aeruginosa
Crude biosurfactants obtained from
Pseudomonas aeruginosa was used for detection
of hydrocarbon (Kerosene, Petrol, Diesel and
Mobil oil) emulsification and vegetable
emulsification (Mustard oil, Coconut oil, Soy bean
oil and Jasmine oil). The emulsification stability
was observed from 0h to 96h as E0, E24, E48, E72
and E96. The highest emulsified hydrocarbon was
mobil oil followed by diesel and petrol while
kerosene was not emulsified by the crude
biosurfactant. The stability of the three emulsified
hydrocarbon gradually declined from 0-96h. All
the four vegetable oils were emulsified and found
stable till 96h of incubation. The highest
emulsisfid was mustard oil followed by soy bean
oil, jasmine oil and then coconut oil.
Table 2: Emulsification index of crude biosurfactant of Pseudomonas aeruginosa
Emulsification index of crude biosurfactant of Pseudomonas aeruginosa
Substrate E0 E24 E48 E72 E96
Hydrocarbons Kerosene 0 0 0 0 0
Petrol 25 22.22 17.64 12.5 5.88
Diesel 51.72 50 48.14 48 41.66
Mobil oil 65.71 44.11 40.62 34.37 33.33
Vegetable oils Mustard oil 56 36 30.43 27.27 22.22
Coconut oil 50 45.83 45.45 40.9 38.88
Soy bean oil 53.57 50 48.14 46.15 40.9
Jasmine oil 52 48 45.83 43.47 36.36

Fig:1 Emulsification index of crude biosurfactant of Bacillus subtilis for hydrocarbons

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P a g e | 786


Plate:5 Emulsification activity of crude biosurfactant of Pseudomonas aeruginosa showing
emulsification of vegetable oils

Plate:6 Emulsification activity of crude biosurfactant of Pseudomonas aeruginosa showing
emulsification of hydrocarbons
Emulsification index of crude biosurfactant of
Serratia marscecens
Crude biosurfactants obtained from Serratia
marscecens was used for detection of
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P a g e | 787
hydrocarbon (Kerosene, Petrol, Diesel and Mobil
oil) emulsification and vegetable emulsification
(Mustard oil, Coconut oil, Soy bean oil and
Jasmine oil). The emulsification stability was
observed from 0h to 96h as E0, E24, E48, E72 and
E96. Among hydrocarbons highest emulsified was
mobil oil followed by diesel, petrol and kerosene.
Among vegetables most emulsified oil was
mustard while slightly less emulsified were
coconut and soybean oil. Jasmine was not
emulsified by crude Serratia marscecens
biosurfactant.
Table 3: Emulsification index of crude biosurfactant of Serratia marscecens
Emulsification index of crude biosurfactant of Serratia marscecens
Hydrocarbons Kerosene 19.04 13.63 8.69 4.76 0
Petrol 10.52 9.09 0 0 0
Diesel 46.42 37.03 26.92 24 17.39
Mobil oil 70.83 56.52 52.17 45.45 44.44
Vegetable oils Mustard oil 60 46.15 40.74 40 29.62
Coconut oil 51.72 48.27 44.82 40.74 36.36
Soy bean oil 50 45.45 42.85 40 33.33
Jasmine oil 0 0 0 0 0

Fig:3 Emulsification index of crude biosurfactant of Serratia marscecens for hydrocarbons

Fig:4 Emulsification index of crude biosurfactant of Serratia marscecens for hydrocarbons
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Plate:7 Emulsification activity of crude biosurfactant of Serratia marscecens showing emulsification
of vegetable oils

Plate:8 Emulsification activity of crude biosurfactant of Serratia marscecens showing emulsification
of hydrocarbons

Bacillus subtilis
Crude biosurfactants obtained from Bacillus
subtilis was used for detection of hydrocarbon
(Kerosene, Petrol, Diesel and Mobil oil)
emulsification and vegetable emulsification
E96. The most stable and highly emulsified oil was
mobil among the hydrocarbons the stability was
recorded till 96h. Lower emulsification activity
was recorded for kerosene petrol and diesel
whose stability declined to 0 at 96h of incubation.
Among vegetable oil jasmine was not emulsified
and soy bean was the highest emulsified oil with
stability showing till 96h as 42.85%.

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Table 4: Emulsification index of crude biosurfactant of Bacillus subtilis
Emulsification index of crude biosurfactant of Bacillus subtilis
Hydrocarbons Kerosene 14.81 11.11 7.69 3.84 0
Petrol 13.63 11.76 9.09 0 0
Diesel 9.09 6.25 3.57 3.33 0
Mobil oil 86.95 61.53 48 45.83 37.03
Vegetable oils Mustard oil 44 41.66 39.13 36.36 33.33
Coconut oil 16 12 8.33 4.54 4.54
Soy bean oil 51.85 48.14 46.15 44 42.85
Jasmine oil 0 0 0 0 0


Fig:6 Emulsification index of crude biosurfactant of Bacillus subtilis for vegetable oils
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P a g e | 79

Plate:9 Emulsification activity of crude biosurfactant of Bacillus subtilis showing emulsification of
hydrocarbons
consortium
Crude biosurfactants obtained from consortium
was used for detection of hydrocarbon
(Kerosene, Petrol, Diesel and Mobil oil)
emulsification and vegetable emulsification
E96. The crude consortium biosurfactant was able
to emulsify all hydrocarbons and vegetable oils
that were found stable till 96h of incubation. The
stability of the emulsion formed from 0h remained
high till 96h in all cases. The consortium
biosurfactant showed high emulsification index as
well as stability in all hydrocarbons and vegetable
oils tested.
Table 5: Emulsification index of crude biosurfactant of consortium
Emulsification index of crude biosurfactant of consortium
Hydrocarbons Kerosene 71.42 69.23 68 65.21 59.09
Petrol 59.09 60 52.38 47.61 42.1
Diesel 75 73.07 72 70.83 65.21
Mobil oil 70 69.56 65.21 63.63 60
Vegetable oils Mustard oil 45.83 41.66 40.9 40 38.88
Coconut oil 57.14 43.33 39.28 34.61 25.92
Soy bean oil 66.66 66.33 60 58.62 55.55
Jasmine oil 68.96 67.85 64 60.86 57.14

P a g e | 79!

Fig:8 Emulsification activity of crude biosurfactant of consortia showing emulsification of hydrocarbons

Fig:9 Emulsification activity of crude biosurfactant of consortia showing emulsification of vegetable oils

Plate:10 Emulsification activity of crude biosurfactant of consortia showing emulsification of vegetable oils
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P a g e | 792

Plate:11 Emulsification activity of crude biosurfactant of consortia showing emulsification of
vegetable oils

Discussion
Biosurfactants can be synthesized using different
microorganisms and carbon sources and
production is influenced by the composition of the
medium and by culture conditions (Desai and
Banat, 1997; Franzetti et al., 2009). 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 C), even showing
emulsification activity at temperature of 10 -100
C. 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

P a g e | 793

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. 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 b-
oxidation 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. Micelles are capable of
dissolving hydrophobic contaminants in their
hydrophobic core, which results in an increased
apparent aqueous solubility of the pollutants
(Edwards et al., 1991; Prak and Pritchard, 2002).

Conclusion
Conclusively, the research revealed the
emulsifying potential of crude biosurfactants
from Pseudomonas aeruginosa, Serratia
marscecens and Bacillus subtilis along with the
consortium of the three mentioned bacteria.
The consortium was more potent of producing
stable emulsion in the aforementioned
hydrocarbons and vegetable oils.
Therefore it could be recommended from the
study that the crude consortium or individual
crode biosurfactants could be used as strong
emulsifying agents.
Acknowledgements
The authors offer gratuitous thanks to Honble
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.
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Crude Cacterial Consortium Biosurfactants Potent For Bioemulsification of Oils and Petroleum Hydrocarbons

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