Isolation and Identification of biosurfactant

producing bacteria from Oil Contaminated

Soil
Abstract
Biosurfactants are surface active compound that reduce the
interfacial tension between two liquids, or that between a liquid
and

solid.

Their

unique

property

like

nontoxic,

easily

biodegradable, eco-friendly and high stability, and wide variety of

industrial application makes them highly useful group of chemical
compound.

Biosurfactants

are

produced

from

variety

of

microorganisms. The objective of this study was to isolate and

characterize

the

biosurfactant

producing

bacteria

from

oil

contaminated soil. Oil contaminated soil samples was collected

from.

The sample was

screened for bacterial isolation using 1 % diesel in Nutrient agar

medium. Sample was incubated separately in shaking orbital
incubator at 37 C at 125 rpm up to 48 hours. For the
identification of growing bacteria go through different biochemical
test. Identification based on the Grams staining and various
biochemical tests from the key cited by Aneja, K.R from the
Bergeys manual of determinative Biology and on the basis of this
manual the species was predicted as pseudomonas and bacillus
species of bacteria. The two different bacterial species isolated

from oil contaminated soil were screened for their biosurfactant

activity by Drop Collapsing Test and Agar well diffusion method in
two different oils namely vegetable oil and diesel. In Agar well
diffusion method the organisms Pseudomonas and Bacillus
produced clear zone (Table 2) and in the drop collapse test the
samples were collapsed. This clearly indicated that the two
organisms produced biosurfactant. The results obtained that
Pseudomonas sp. recorded higher biosurfactant activity than
Bacillus sp. The oil degradation by Pseudomonas sp. was not
surprising not only because it was isolated from oil spilled soil but
also because it is known to possess a more competent and active
hydrocarbon degrading enzyme system than Bacillus sp. It is
known to be fast growing and is capable of degrading a wide
variety of organic compounds (Ijah & Okang, 1993).

INTRODUCTION
The objective of this study was to isolate and characterize the
biosurfactant producing bacteria from oil contaminated soil. The
ecology of hydrocarbon degradation by microbial populations in
the natural environment is reviewed, emphasizing the physical,
chemical,

and

biological

factors

that

contribute

to

the

biodegradation of petroleum and individual hydrocarbons. Oil

contaminated

soil

samples

was

from.

collected

The sample was

screened for bacterial oil degradation using 1 % diesel in Nutrient

agar medium. Sample was incubated separately in shaking orbital
incubator at 37 C at 125 rpm up to 48 hours.
Biosurfactants are surface active compound that reduce the
interfacial tension between two liquids, or that between a liquid
and

solid.

Their

unique

property

like

nontoxic,

easily

biodegradable, eco-friendly and high stability, and wide variety of

industrial application makes them highly useful group of chemical
compound.

Biosurfactants

microorganisms.

are

produced

from

variety

of

Life in our planet is sustained in a fragile biological balance;

microorganisms play an important role on nutritional chains that
are an important part of this biological balance. Adapting several
abilities, microorganisms have become an important influence on
the ecological systems, making them necessary for superior
organisms life in this planet. Ability of microorganisms to
transform and degrade many types of pollutants in different
matrixes (soil, water, sediments and air) has been widely
recognized during the last decades. Soil contamination with
hydrocarbons causes extensive damage of local ecosystems
since accumulation of pollutants in animals and plants tissues
may cause progenys death or mutation. In Mexico, an endless
number of contaminated sites exist as a result of more than 60
years of oil petroleum activity; in recent years this problem has
motivated researches to recover these contaminated sites.
Microorganisms survive in contaminated habitat because they are
metabolically capable of utilizing its resources and can occupy a
suitable niche. Contaminants are often potential energy sources
for microorganisms. Bioremediation, a process that exploits the
catalytic abilities of living organisms to enhance the rate or extent
of pollutant destruction, is an important tool in attempts to
mitigate environmental contamination. Bioremediation achieves
contaminant decomposition or immobilization by exploiting the
existing metabolic potential in microorganisms with catabolic
functions derived through selection, or by the introduction of
genes

encoding

such

functions.

The

effectiveness

of

bioremediation is often a function of the extent to which a

microbial
maintained

population
in

or

consortium

environment.

When

can
few

be
or

enriched
no

and

indigenous

degradative microorganisms exist in a contaminated area and

practically does not allow time for the natural enrichment of
suitable population, inoculation may be a convenient option.
Biosurfactants have unique property like biodegradability, low
toxicity, and more eco-friendly, large flexibility in operation etc.
But production on huge industry level is still challenge reason is
low economical than chemical surfactant, because of using
synthetic nutrient media is expensive than natural media. To
overcome

with

this

problem

associated

with

biosurfactant

production, researchers mainly focus on uses of industrial waste

for fermentation process like using of agro waste; molasses etc.
and using of optimize bioprocess like optimum temperature, pH,
and other parameters. Every year tons of hazards and nonhazards wastes are generated that needs to proper utilization to
prevent the world from pollution and other hazards impact.
Residues obtain from agriculture such as peels, hull, sugar beet,
sweet potato, residue from coffee processing unit, residue from oil
industries such as oil cake; can be used as substrate for
biosurfactant production.
Surfactants are surface active compound that reduce the
interfacial tension between two liquids, or that between a liquid
and a solid. Surfactants are organic compound that contain both

hydrophobic (head part of the surfactant) and hydrophilic (tail

part of the surfactant) moieties. Thus surfactant contains both
water insoluble i.e. water repellent groups as well as water
soluble i.e. water loving group. Biosurfactants are also surface
active compound

like chemical surfactants but unlike the

chemical surfactant, biosurfactant are synthesized by microbes

like bacteria, fungi and yeast. Biosurfactants comprise the
properties of dropping surface tension, stabilizing emulsions,
promoting foaming and are usually non-toxic and biodegradable.
Recently interest in biosurfactant has increased because of its
diversity, flexibility in operation, and more eco-friendly then
chemical surfactant. Furthermore possibility of their production on
large scale, selectivity, performance under intense conditions and
their future applications in environmental fortification also these
have been increasingly attracting the attention of the scientific
and industrial community. These molecules have a potential to be
used in a variety of industries like cosmetics, pharmaceuticals,
humectants, food preservatives and detergents.
Biosurfactants are classified in to two major groups one is low
molecular weight surface active agent call biosurfactant and high
molecular weight substance called bio-emulsifier that is especially
used as enhancement of emulsification of hydrocarbon. Further
these two major groups is divided in to six major group known as
glycolipids, lipopolysaccharides,
Mostly biosurfactants are glycolipids. They are lipids with a
carbohydrate attached. The connection is by means of either an

ether or ester group. Among the glycolipids, the best known are
rhamnolipids

sophorolipids

and

trehalolipids.

Lipopeptide

biosurfactants are cyclic compounds and they are mostly isolated

from Bacillus and Pseudomonas type bacteria Lipopeptides mainly
consist of hydrophilic peptides, generally they consist 7 and 10
amino acids long, linked to a hydrophobic fatty acid structure.
Bacillus cyclic lipopeptides consist of three major groups known
as the surfactin, iturin and fengycin families. Surfactin is the most
commonly studied and it contains 7 amino acid cyclic sequences
connected to a C13C16 fatty acid.
Biosurfactants are surface active compound that accumulate at
the boundary between two immiscible fluids or between a fluid
and a solid. By reducing surface (liquid-air) and interfacial (liquidliquid) tension they reduce the repulsive forces between two
different phases and allow them to mix and thus enhance the
solubility properties like chemical surfactant. In many cases it was
found

that

biosurfactant

activities

are

not

influenced

by

environmental condition such as temperature and pH. In 1990

McInerney suggested that lichenysin produced by B. licheniformis
was not affected by temperature (up to 50C), pH (4.59.0) and
by NaCl and Ca concentrations up to 50 and 25 g/l respectively.
Apart from theses above properties biosurfactant can be easily
degraded unlike the chemical surfactant and thus and they are
chiefly

suited

for

the

environmental

applications

such

as

bioremediation, and dispersion of oil spills. The toxicity of

biosurfactants is much lower and some of the researcher consider

as these are non toxic compounds. Very few literature are

available that describes the toxicity of biosurfactant and their
direct bad impact on environment. Therefore they are appropriate
for pharmaceutical, food and cosmetic uses. A study suggested
that a synthetic anionic surfactant (Corexit) showed an LC50
(concentration

lethal

to

50%

of

test

species)

against

Photobacterium phosphoreum ten times lesser than rhamnolipids,

suggesting

the

surfactant.

In

larger
a

toxicity

particular

of

study

the

chemically

where

toxicity

derived
of

six

biosurfactants was comparing with the toxicity of, four of the

synthetic surfactants and two commercial dispersants, it was
observed that mostly biosurfactants degraded quicker, except for
a synthetic sucrose-stearate that showed structure homology to
glycolipids and was degraded more rapidly than the biogenic
glycolipids.
The unique properties of biosurfactant (Microbial Surface Active
Agents) such as low toxicity, relative ease of preparation and
widespread

applicability,

make

it

different

from

chemical

synthetic surfactant and now it has become recently an important

product of biotechnology for industrial and medical applications
and they allow to replacement of chemical synthetic surfactant.
They can be used as emulsifiers, de-emulsifiers, wetting agents,
spreading agents, foaming agents, functional food ingredients and
detergents in various industrial sectors such as, Petroleum and
Petrochemicals,
Cosmetics

and

Organic

Chemicals,

Pharmaceuticals,

Foods
Mining

and

Beverages,

and

Metallurgy,

Agrochemicals

and

Fertilizers,

Environmental

Control

and

Management, and many others.

Petroleum refineries around the world have generated the solid
wastes during the refining process and stocking of crude oil
environment.

The

ecology

of

hydrocarbon

degradation

by

microbial populations in the natural environment is reviewed,

emphasizing the physical, chemical, and biological factors that
contribute to the biodegradation of petroleum and individual
hydrocarbons. Oil contaminated soil samples was collected
from.

The sample was

screened for bacterial oil degradation using 1 % diesel in Nutrient

agar medium. Sample was incubated separately in shaking orbital
incubator at 37 C at 125 rpm up to 48 hours.
Oil contamination is one of the most dangerous pollution factors
known today. It can cause a threat to the environment. It is very
feared by environmentalists and its very hard to control if it gets
out of hand. Oil spill have become a global problem in
industrialized and developing countries. Attention has been
focused on the marine environment, because of the largest and
most dramatic spills. Oil spills have been a major issue across
decades. Recent oil spill was in Mumbai (India) and caused due to
the leakage in Mumbai-Uran pipeline dated January 21, 2011 and
about 55 tons of oil was leaked in Arabian Sea. Various such
accidents occur throughout the years and it causes damage to
our surrounding. Diesel engine oil, which is one of the major
products of crude oil, constitutes a major source of pollution in our

environment. Diesel oil spills contaminated soils on agricultural

land generally reduce soil fertility and plant growth. Baker and
reduced germination to unsatisfactory soil condition due to
insufficient aeration of the soil because of the displacement of air
from the space between the soil particles by diesel engine oil.
Many indigenous microorganisms in water and soil are capable of
degrading hydrocarbon contaminants. The amount of natural
crude oil seepage was estimated to be 600,000 metric tons per
year with a range of uncertainty of 200,000 metric tons per year
of hydrocarbons into the environment whether accidentally or due
to human activities is a main cause of water and soil pollution.
The technology commonly used for the soil remediation includes
mechanical,

burying,

evaporation,

dispersion,

and

washing.

However, these technologies are expensive and can lead to

incomplete decomposition of contaminants. The process of
bioremediation, defined as the use of microorganisms to detoxify
or remove pollutants owing to their diverse metabolic capabilities
is an evolving method for the removal and degradation of many
environmental pollutants including the products of petroleum
industry.
Contamination of the soil by oil causes it to lose its useful
properties
permeability

such

as

and

soil

binding

fertility,

water-holding

capacity.

The

capacity,

contamination

of

groundwater is also a potential problem, which receives a lot of

untreated effluent from service stations containing oil and grease.
To

overcome

these

environmental

problems,

microbial

bioremediation is only way to preserve our nature. The purpose of

the present study was to investigate a possible exsitu method to
enhance the rate of biodegradation of diesel contaminated soil
sites. The main objectives of the study were to isolate a potential
strain which could be used in bioremediation of oil contaminated
sites and to find out the efficiency of the isolate in preliminary
screening of bioremediation.
This study also aimed to investigate the use of biosurfactant in
soil cleanup methods and to prove that biostimulation can be
effectively employed in the remediation of crude oil polluted soil
ecosystem. Crude oil is mainly composed of alkanes, cycloalkanes
and aromatic alkanes, which constitute about 50 % to 80 % of the
oil content. These constituents are called petroleum hydrocarbons
for short. More than 230 hydrocarbons have been identified in oil.
As an important energy source, oil has played an indispensable
role in industrial production. Therefore, the 20 th century was
named the oil century. Daqing is the largest oilfield in China,
whose crude oil is paraffin-based; with high wax content (20 % to
30 %), high freezing point (25 C ~30 C), high viscosity (the
ground viscosity in 35 cP) and low sulfur content (0.1 %). Over the
years, the Daqing oil field has provided large amounts of crude oil
for the country, which has made great contributions to Chinese
industrial

development.

However,

the

oil

exploration

and

transportation has contaminated the soil of the Daqing area in

various degrees. A numbers of alkali spots have formed, which
limit the vegetation cover and crop growth.

Moreover, petroleum hydrocarbons are absorbed by plant roots

and accumulated. From there, they could potentially get into
humans body through the food chain and pose a threat to human
health.
It is well known that a lot of soil bacteria and fungi can utilize
petroleum hydrocarbons as a carbon source. At the same time,
some aboriginal microbes have gradually adapted to the longterm oil contaminated soil and developed a superior community
which can make use of oil contaminants through special substrate
enrichment. Therefore, bioremediation of oil contaminated soil
has broad prospects because of its low cost, no secondary
pollution, processing in situ and so on.
Oil pollution and remediation technology has become a global
phenomenon of increasing importance. Most of the hydrocarbons
are insoluble in water and their degradation using microorganisms
have an important role in combating environmental pollution.
Hydrocarbon degrading microorganisms produce biosurfactants of
different chemical nature and molecular size which are surface
active compounds which increases the surface tension of the
hydrophobic water-insoluble substrates and thereby enhancing
their bioavailability and the rate of bioremediation. Almost all
surfactants currently produced are chemically derived from
petroleum.

These

synthetic

surfactants

are

usually

toxic

themselves and are hardly degraded by microorganisms. They

are, therefore, a potential source of pollution and damage to the
environment. These hazards associated with synthetic emulsifiers

have, in recent years, drawn much attention to the microbial

production of surfactants or biosurfactants. At present, microbial
remediation of oil contaminated soil has been carried out and
widely reported in the world, which provides a reference and
guidance

for

development

and

practical

application

of

bioremediation technology. In this research, 10 natural bacterial

strains were isolated from oil contaminated soil from the Daqing
oilfield in northern China, and identified by 16S rDNA sequence
analysis combined with morphological observation, physiological
and biochemical tests. The optimum growth condition, crude oil
biodegradation and degradation-related enzymes of these strains
were also investigated. The purpose was to select and provide
new strains with fast growth and high degradation ability for
bioremediation of the contaminated soil of the Daqing oilfield.

Review of literature
Life in our planet is sustained in a fragile biological balance;
microorganisms play an important role on nutritional chains that

are an important part of this biological balance [1]. Adapting

several abilities, microorganisms have become an important
influence on the ecological systems, making them necessary for
superior organisms life in this planet. Ability of microorganisms to
transform and degrade many types of pollutants in different
matrixes (soil, water, sediments and air) has been widely
recognized during the last decades [2, 3]. Soil contamination with
hydrocarbons causes extensive damage of local ecosystems since
accumulation of pollutants in animals and plants tissues may
cause progenys death or mutation [4]. In Mexico, an endless
number of contaminated sites exist as a result of more than 60
years of oil petroleum activity; in recent years this problem has
motivated researches to recover these contaminated sites [2].
Microorganisms survive in contaminated habitat because they are
metabolically capable of utilizing its resources and can occupy a
suitable niche. Contaminants are often potential energy sources
for microorganisms [1]. Bioremediation, a process that exploits
the catalytic abilities of living organisms to enhance the rate or
extent of pollutant destruction, is an important tool in attempts to
mitigate environmental contamination [3, 5]. Bioremediation
achieves

contaminant

decomposition

or

immobilization

by

exploiting the existing metabolic potential in microorganisms with

catabolic

functions

derived

through

selection,

or

by

the

introduction of genes encoding such functions. The effectiveness

of bioremediation is often a function of the extent to which a
microbial

population

or

consortium

can

be

enriched

and

maintained

in

environment.

When

few

or

no

indigenous

degradative microorganisms exist in a contaminated area and

practically does not allow time for the natural enrichment of
suitable population, inoculation may be a convenient option [5].
Oil contamination is one of the most dangerous pollution factors
known today. It can cause a threat to the environment. It is very
feared by environmentalists and its very hard to control if it gets
out of hand. Oil spill have become a global problem in
industrialized and developing countries. Attention has been
focused on the marine environment, because of the largest and
most dramatic spills [6].
Oil spills have been a major issue across decades. Recent oil spill
was in Mumbai (India) and caused due to the leakage in MumbaiUran pipeline dated January 21, 2011 and about 55 tons of oil was
leaked in Arabian Sea. Various such accidents occur throughout
the years and it causes damage to our surrounding [7]. Diesel
engine oil, which is one of the major products of crude oil,
constitutes a major source of pollution in our environment. Diesel
oil spills contaminated soils on agricultural land generally reduce
soil fertility and plant growth. Baker (1982) and reduced
germination to unsatisfactory soil condition due to insufficient
aeration of the soil because of the displacement of air from the
space between the soil particles by diesel engine oil [8]. Many
indigenous microorganisms in water and soil are capable of
degrading hydrocarbon contaminants. The amount of natural
crude oil seepage was estimated to be 600,000 metric tons per

year with a range of uncertainty of 200,000 metric tons per year

[9] of hydrocarbons into the environment whether accidentally or
due to human activities is a main cause of water and soil pollution
[10]. The technology commonly used for the soil remediation
includes

mechanical,

burying,

evaporation,

dispersion,

and

washing [11]. However, these technologies are expensive and can

lead to incomplete decomposition of contaminants. The process of
bioremediation, defined as the use of microorganisms to detoxify
or remove pollutants owing to their diverse metabolic capabilities
is an evolving method for the removal and degradation of many
environmental pollutants including the products of petroleum
industry [12].
Contamination of the soil by oil causes it to lose its useful
properties
permeability

such
and

as

soil

binding

fertility,

water-holding

capacity.

The

capacity,

contamination

of

groundwater is also a potential problem, which receives a lot of

untreated effluent from service stations containing oil and grease
[13]. To overcome these environmental problems, microbial
bioremediation is only way to preserve our nature. The purpose of
the present study was to investigate a possible exsitu method to
enhance the rate of biodegradation of diesel contaminated soil
sites. The main objectives of the study were to isolate a potential
strain which could be used in bioremediation of oil contaminated
sites and to find out the efficiency of the isolate in preliminary
screening of bioremediation. This study also aimed to investigate

the use of biosurfactant in soil cleanup methods and to prove that

biostimulation can be effectively employed in the remediation of
crude oil polluted soil ecosystem.
Crude oil is mainly composed of alkanes, cycloalkanes and
aromatic alkanes, which constitute about 50 % to 80 % of the oil
content. These constituents are called petroleum hydrocarbons
for short [14]. More than 230 hydrocarbons have been identified
in oil [15]. As an important energy source, oil has played an
indispensable role in industrial production. Therefore, the 20 th
century was named the oil century [16]. Daqing is the largest
oilfield in China, whose crude oil is paraffin-based; with high wax
content (20 % to 30 %), high freezing point (25 C ~30 C), high
viscosity (the ground viscosity in 35 cP) and low sulfur content
(0.1 %). Over the years, the Daqing oil field has provided large
amounts of crude oil for the country, which has made great
contributions to Chinese industrial development. However, the oil
exploration and transportation has contaminated the soil of the
Daqing area in various degrees. A numbers of alkali spots have
formed, which limit the vegetation cover and crop growth.
Moreover, petroleum hydrocarbons are absorbed by plant roots
and accumulated. From there, they could potentially get into
humans body through the food chain and pose a threat to human
health [17, 18]. It is well known that a lot of soil bacteria and
fungi can utilize petroleum hydrocarbons as a carbon source. At

the same time, some aboriginal microbes have gradually adapted

to the long-term oil contaminated soil and developed a superior
community which can make use of oil contaminants through
special substrate enrichment [19, 20]. Therefore, bioremediation
of oil contaminated soil has broad prospects because of its low
cost, no secondary pollution, processing in situ and so on [21-25].
At present, microbial remediation of oil contaminated soil has
been carried out and widely reported in the world [26], which
provides a reference and guidance for development and practical
application of bioremediation technology. In this research, 10
natural bacterial strains were isolated from oil contaminated soil
from the Daqing oilfield in northern China, and identified by 16S
rDNA

sequence

analysis

combined

with

morphological

observation, physiological and biochemical tests. The optimum

growth condition, crude oil biodegradation and degradationrelated enzymes of these strains were also investigated. The
purpose was to select and provide new strains with fast growth
and

high

degradation

ability

for

bioremediation

of

the

contaminated soil of the Daqing oilfield.

Amongst hydrocarbon pollutants, diesel oil (a complex mixture of
alkanes and aromatic compounds) is a frequently reported soil
contaminant leaking from storage tanks and pipelines or released
in accidental spills [27]. Bioremediation is considered the best
approach for restoring diesel oil contaminated soils in that the
technology is cost-effective and environmentally desirable. The
success of bioremediation is dependent upon the microbial ability

to degrade these complex mixtures and their rate limiting kinetics

[28].

Both

mono-

bioremediation.

and

mixed-cultures

However,

higher

rates

can
of

be

used

for

degradation

of

hydrocarbons are often achieved with a bacterial enrichment

consortium

isolated

from

the

environment

that

needs

biorestoration [29]. Bacterial consortia display a wide array of

metabolic

mechanisms

in

the

breakdown

of

diesel

oil

components, including production of surface-active agents and

emulsifiers [30]. To obtain an efficient diesel oil-degrading
bacterial

consortium

diversity

of

the

and

monocultures,

microbial

community

knowledge
present

of
in

the
soils

contaminated with diesel oil, their metabolic features and

capacity to degrade diesel oil are of paramount importance. One
of the important properties of hydrocarbon-degrading bacteria is
the production of surface-active agents (biosurfactants). These
agents are small surfactant molecules that affect the reduction of
the interfacial tension and amphiphilic macromolecules that
stabilize the emulsion [31]. In this work we report the diversity of
biosurfactant producing and hydrocarbon-degrading microbial
communities of two soils polluted with diesel oil. Furthermore, we
evaluated the emulsification capacity of the isolates.

BIOSURFACTANT
Biosurfactants have unique property like biodegradability, low
toxicity, and more eco-friendly, large flexibility in operation etc.
But production on huge industry level is still challenge reason is

low economical than chemical surfactant, because of using

synthetic nutrient media is expensive than natural media. To
overcome

with

this

problem

associated

with

biosurfactant

production, researchers mainly focus on uses of industrial waste

for fermentation process like using of agro waste; molasses etc.
and using of optimize bioprocess like optimum temperature, pH,
and other parameters. Every year tons of hazards and nonhazards wastes are generated that needs to proper utilization to
prevent the world from pollution and other hazards impact.
Residues obtain from agriculture such as peels, hull, sugar beet,
sweet potato, residue from coffee processing unit, residue from oil
industries such as oil cake; can be used as substrate for
biosurfactant production [32]. Surfactants are surface active
compound that reduce the interfacial tension between two liquids,
or that between a liquid and a solid. Surfactants are organic
compound that contain both hydrophobic (head part of the
surfactant) and hydrophilic (tail part of the surfactant) moieties.
Thus surfactant contains both water insoluble i.e. water repellent
groups as well as water soluble i.e. water loving group.
Biosurfactants are also surface active compound like chemical
surfactants but unlike the chemical surfactant, biosurfactant are
synthesized

by

microbes

like

bacteria,

fungi

and

yeast.

Biosurfactants comprise the properties of dropping surface

tension, stabilizing emulsions, promoting foaming and are usually
non-toxic and biodegradable. Recently interest in biosurfactant
has increased because of its diversity, flexibility in operation, and

more eco-friendly then chemical surfactant [33]. Furthermore

possibility

of

performance

their
under

production
intense

on

large

conditions

scale,
and

selectivity,

their

future

applications in environmental fortification also these have been

increasingly attracting the attention of the scientific and industrial
community. These molecules have a potential to be used in a
variety of industries like cosmetics, pharmaceuticals, humectants,
food preservatives and detergents [32]. But the production of
biosurfactant on industry level is still challenge because of using
high costly synthetic media for microbial growth. Biosurfactants
are classified on the basis of diversity in their structure and their
microbial origin. They contain a hydrophilic group, that contain an
acid, peptide cations, or anions, mono-, di- or polysaccharides and
a hydrophobic group of unsaturated or saturated hydrocarbon
chains or fatty acids. Biosurfactants produced by a variety of
microorganisms mainly bacteria, fungi and yeasts are diverse in
chemical composition and their nature and the amount depend on
the type of microbes producing a particular biosurfactant. And in
resulting

wide

variety

of

biosurfactant

can

be

produced

accordingly to the demand and uses.

Biosurfactant

Microorganism

Glycolipids

Pseudomonas aeruginosa

Lipopeptides

Bacillus subtilis

Surfactin/iturin/fengycin

P. fluorescens

Classification of Biosurfactant:
Biosurfactants are classified in to two major groups one is low
molecular weight surface active agent call biosurfactant and high
molecular weight substance called bio-emulsifier that is especially
used as enhancement of emulsification of hydrocarbon. Further
these two major group is divided in to six major group known as
glycolipids,

lipopolysaccharides,

lipoproteins-lipopeptides,

phospholipids, hydroxylated and cross linked fatty acids.

Glycolipids:
Mostly biosurfactants are glycolipds. They are lipids with a
carbohydrate attached. The connection is by means of either an
ether or ester group. Among the glycolipids, the best known are
rhamnolipids sophorolipids and trehalolipids.

Lipopeptides and lipoproteins:

Lipopeptide biosurfactants are cyclic compounds and they are
mostly isolated from Bacillus and Pseudomonas type bacteria
Lipopeptides mainly consist of hydrophilic peptides, generally
they consist 7 and 10 amino acids long, linked to a hydrophobic
fatty acid structure. Bacillus cyclic lipopeptides consist of three
major groups known as the surfactin, iturin and fengycin families.
Surfactin is the most commonly studied and it contains 7 amino
acid cyclic sequences connected to a C13C16 fatty acid [34].

Properties of Biosurfactant:
Biosurfactants are surface active compound that accumulate at
the boundary between two immiscible fluids or between a fluid
and a solid. By reducing surface (liquid-air) and interfacial (liquidliquid) tension they reduce the repulsive forces between two
different phases and allow them to mix and thus enhance the
solubility properties like chemical surfactant. The most effective
biosurfactants can reduce the surface tension of water from 72 to
30 mNm1 and the interfacial tension between water and nhexadecane from 40 to 1 mNm1 [35] Biosurfactant produces
from B. subtilis is able to lower the surface tension of water to 25
mN/m and interfacial tension of water/hexadecane to <1 mN/m
[36]. Further more, biosurfactants are more effective and efficient
and CMC of biosurfactant is about 1040 times lower than that of
chemical surfactants, so less amount surfactant is required to get

a maximum decrease in surface tension as compare to the

chemical surfactant [37].
In many cases it was found that biosurfactant activities are not
influenced by environmental condition such as temperature and
pH. In 1990 McInerney suggested that lichenysin produced by B.
licheniformis was not affected by temperature (up to 50C), pH
(4.59.0) and by NaCl and Ca concentrations up to 50 and 25 g/l
respectively. Apart from theses above properties biosurfactant
can be easily degraded unlike the chemical surfactant and thus
and they are chiefly suited for the environmental applications
such as bioremediation, and dispersion of oil spills. The toxicity of
biosurfactants is much lower and some of the researcher consider
as these are non toxic compounds. Very few literature are
available that describes the toxicity of biosurfactant and their
direct bad impact on environment. Therefore they are appropriate
for pharmaceutical, food and cosmetic uses. A study suggested
that a synthetic anionic surfactant (Corexit) showed an LC50
(concentration

lethal

to

50%

of

test

species)

against

Photobacterium phosphoreum ten times lesser than rhamnolipids,

suggesting

the

surfactant.

In

larger
a

toxicity

particular

of

study

the

chemically

where

toxicity

derived
of

six

biosurfactants was comparing with the toxicity of, four of the

synthetic surfactants and two commercial dispersants, it was
observed that mostly biosurfactants degraded quicker [35],
except for a synthetic sucrose-stearate that showed structure
homology to glycolipids and was degraded more rapidly than the

biogenic glycolipids. A biosurfactant from P. aeruginosa was

compared to a synthetic surfactant that is widely used in the
industry, regarding toxicity and mutagenic properties. Both
assays indicated a higher level of toxicity and mutagenic effect of
the chemically derived surfactant, whereas the biosurfactant was
considered to be slightly non-toxic and no mutagenic.

Advantages and Uses of Biosurfactant:

The unique properties of biosurfactant (Microbial Surface Active
Agents) such as low toxicity, relative ease of preparation and
widespread

applicability,

make

it

different

from

chemical

synthetic surfactant and now it has become recently an important

product of biotechnology for industrial and medical applications
and they allow to replacement of chemical synthetic surfactant.
They can be used as emulsifiers, de-emulsifiers, wetting agents,
spreading agents, foaming agents, functional food ingredients and
detergents in various industrial sectors such as, Petroleum and
Petrochemicals,
Cosmetics

and

Agrochemicals

Organic

Chemicals,

Pharmaceuticals,
and

Fertilizers,

Foods
Mining

and

Beverages,

and

Metallurgy,

Environmental

Control

and

Management, and many others.

There are many advantages of biosurfactant as compare to
chemically synthesized surfactants. Some of those are:
1. Biodegradability: Easy to biodegradable as compare to the
chemical surfactant [35].
2. Low toxicity

3. Biocompatibility and digestibility, that allows their application

in cosmetics, pharmaceuticals and food seasonings.
4. Easily availability of raw material: The raw material need for
production of biosurfactant are easily available, biosurfactant
producing microorganism can be isolated from the industrial
waste like oil contaminated soil, petrol pump spilled, and also can
be isolated from municipal waste.
5. Use in environmental control. Biosurfactants can be efficiently
used in handling industrial emulsions, control of oil spills,
biodegradation and lowering the toxicity of industrial discharges
and in bioremediation of polluted soil.
6. Specificity in their action, since biosurfactant has specific
organic functional group and often specific in their action. This is
particularly used in lowering the toxicity of the pollutant, used in
enhancing the emulsification property, used as raw material in
cosmetic, medicinal and foodstuff applications.
Ghayyomi

isolated

biosurfactant

producing

bacteria

from

petroleum contaminated soil and they observed that 160 strains

were able to producing biosurfactant, in which 59 strains showed
positive blood hemolysis test, 45 strains showed positive oil
spreading technique [38]. They found that emulsion and foaming
activity was maximum at 7 pH and 37C temperature. For the
isolation culture media was synthesized in lab by Banat method
[39]. Kevin has studied the emulsion properties of bacterial
biosurfactant,

they

isolated

three

unknown

biosurfactant

producing bacteria and their emulsification activity was compared

with the two artificial surfactant SDS and Triton X-100 [40].
In another research in which P. aeruginosa, was isolated from oil
contaminated sea water and it was seen that it was able to break
down

the hydrocarbon

such

as hexadecane,

heptadecane,

octadecane and nonadecane in sea water up to 47, 53, 73, and 60

% respectively [41]. In particular research [42], i.e production of
biosurfactant

from

lactobacilli,

they

were

found

that

the

production of biosurfactant not only depends on the type of

microorganism but also depend on the composition of mineral salt
media. It was noted that lactobacilli produce lower amounts of
biosurfactants as compare with other microorganisms, such as
Bacillus subtilis or Pseudomonas aeruginosa, and also they
consume many nutritional, they constitute a promising source of
biosurfactant,
considered

because

GRAS

and

these
are

microorganisms
already

used

in

are

usually

many

food

manufacturing and industrial process. Furthermore, it was noted

that the yield of biosurfactant production can be increased
through the optimization of culture condition.
In this research it was reported that yeast extract is an essential
component for the bacterial growth, whereas the peptone is for
the biosurfactant production. And the combination of yeast and
meat extract resulting in higher yield of biosurfactant [42].
In this research isolates three biosurfactant producing bacteria
from reservoir formation water [43], B. subtilis, P. aeruginosa, and
R. erythropolis, by using these three bacteria three biosurfactant

was extracted and studied using crude oil as a carbon source. P.

aeruginosa was noted that the overall biosurfactant production
rate, resistance and stability are extremely well than rest two
bacteria. This also attained emulsion index 80% for crude oil and
also reduce the surface tension of medium from 71.2 to 22.6
mN/m. P. aeruginosa showed14.3% oil recovery after water
flooding, in results of biosurfactant flooding experiment.

MATERIAL AND METHOD

SAMPLE COLLECTION

Procedure
Soil

samples

were

collected

from

contaminated

sites

of.
The soil at the sites had a characteristic black colour due to
continuous oil spillage and the soil surfaces were hard.
Collected samples were packed in sterile poly bags and
brought to the laboratory.
The entire sample was stored at refrigeration temperature
before the experimental work.

ISOLATION OF OIL DEGRADING BACTERIA

Procedure
The oil degrading bacteria was isolated by enrichment
technique.
Nutrient agar plates enriched with 1.0 % diesel were
prepared

(Jayashree,

Evany

Nithya,

Rajesh

Prasanna&

Krishnaraju, 2012).
1g of oil spill contaminated soil sample was weighed
aseptically and added to the 99ml of sterile distilled water.

 The flask was placed in a rotary shaker for about 30 minutes

at 30C.
Serial dilutions of the sample were performed separately
from 10-1 to 10-5.
Serially diluted sample of 10-3 plated on nutrient agar using
spread plate method.
A sterile micropipette tip was used to dispense 0.1 ml from
dilution onto duplicate nutrient agar plates.
A glass spreader dipped in alcohol, flamed and cooled was
used for spreading the plates.
The petriplate was then incubated at 37C for 24 to 48
hours.

COLONY MORPHOLOGY
After incubation check the morphology of growing bacteria.

(a)

SHAPE

(b) MARGIN

(c)

(d)

ELEVATION

TEXTURE: Hard, Brittle, Gumming

(e)

SURFACE: Smooth, Rough

(f)

OPACITY: Opaque, Transparent

IDENTIFICATION OF ISOLATED BACTERIA

After morphology characterization isolated colonies were streaked
in to the nutrient agar plates for purification and identification.
The isolated colonies were transformed to nutrient agar slants
and stored for further studies. For the identification of growing

bacteria go through different biochemical test. Identification

based on the Grams staining and various biochemical tests from
the key cited by Aneja, K.R from the Bergeys manual of
determinative Biology.

1. GRAM STAINING
Reagents
Crystal Violet,
Iodine,
Ethyl alcohol (95%),
Safranin

Principle: The Grams staining (developed by Dr. Hans Christian

Gram) is used to differentiate the bacterial cell into two major
groups Gram positive and Gram negative which makes it an
essential

tool

microorganisms.

for

classification

and

differentiation

of

Crystal violet is used as a primary stain and

iodine acts as a mordant which increases the affinity of the cells

for the stain. Ethyl alcohol 95% is used as de-colorizing agent,

which acts as lipid solvent and also as protein dehydrating agent,

Safranin is used as the secondary stain.

Procedure
Thin smears of the isolated different colonies were prepared,
air dried, and heat fixed.
Smear was covered with crystal violet for 60 seconds.
The stain was washed off using distilled water. The excess
water was drained off.
The smear was covered with Grams iodine solution and kept
for 60 seconds.
The Grams iodine was poured off and the smear was flooded
with 95% alcohol for 30 seconds. The slide was washed with
distilled water.
The counter stain Safranin was added to smear and was kept
for 60 seconds. The stain was washed gently for few seconds.

 The slide was air dried and, examined with a light microscope
under oil immersion.

Fig.5:- Gram Staining

2. CATALASE TEST

Reagents: Hydrogen Peroxide (3%).

Principle: During aerobic respiration in the presence of oxygen,
microorganisms produce hydrogen peroxide (H 2O2) which is lethal
to the cell. The enzyme catalase present in some organisms
breaks down hydrogen peroxide to water and oxygen and helps
them in survival. Catalase test is performed by adding H 2O2 to the

culture taken on a slide directly. Release of free oxygen gas

bubbles is a positive catalase test.

Procedure
Two-three drops of 3% hydrogen peroxide were taken on a
clean glass slide.
One loop full of the culture was just kept over the hydrogen
peroxide.
Slide was than observed for the appearance or absence of
gas bubbles.

3. METHYL RED TEST AND VOGES

PROSKAUER TEST
(MRVP BROTH):

Media: MRVP broth (Appendix).

MR TEST

Principal- Used to determine the ability of an organism to

produce mixed acid end products from glucose fermentations.
Some organisms produce large amounts of various acids (lactic,
acetic, succinic, formic) plus H2 and CO2. The large amounts of
acids lower the pH to lower than 5.0. These organisms also
produce great amounts of gas due to the presence of the enzyme
formic hydrogen lyase.

Procedure
The culture was inoculated in the tubes containing MRVP
broth, a control was also maintained.
The inoculated and uninoculated tubes were incubated at
370C, for 48 hours.
After incubation add 3-4 drop by drop methyl red indicator.
The

tubes

were

observed

for

change

in

colour,

the

development of red colour is indicative of MR positive test

while no change in colour is a negative test.

VP TEST
Reagents- Barritts reagent solution (A+B)
Principle: In the Voges-Proskauer test, the red color produced by
the addition of potassium hydroxide to cultures of certain
microbial species is due to the ability of the organisms to produce
a neutral end product, acetoin (acetylmethylcarbinol), from the
fermentation of dextrose. The acetone is oxidized in the presence
of oxygen and alkali to produce a red color. This is a positive
Voges-Proskauer reaction.

Procedure
Barritts reagent preparation (A+B)
Solution A

Alpha-naphtholin

6 gm

Ethyl alcohol (95%)

100 ml

Solution B
Potassium Hydroxide

16 g

Distilled water

100 ml

The culture was inoculated in the tubes containing MRVP

broth, a control was also maintained.
The inoculated and uninoculated tubes were incubated at
370C, for 48 hours.
After the incubation was over, 12 drops of VP A reagent and
2-3 drops of VP B reagent was added to the inoculated and
un-inoculated tubes.
Tubes were shaken gently for 30 seconds with the plugs off
to expose the media to oxygen.
The

tubes

were

observed

for

change

in

colour,

the

development of crimson to yellow colour is indicative of VP

positive test while no change in colour is a negative test.

4. INDOLE TEST

Materials

Sterilized test tubes

Conical flasks
Pipettes
Glass rod
Test culture
Kovacs reagent
Tryptone broth

Principle
Indole is a nitrogen-containing compound formed from the
degradation of the amino acid tryptophan. The indole test is
important because only certain bacteria form indole. Indole can
be easily detected with Kovacs reagent. After the addition of the
reagent and mixing the contents, the tube is allowed to stand.
The alcohol layer gets separated from this aqueous layer and,
upon standing, the reddening of the alcohol layer shows that
Indole is present in the culture. Thus, the formation of the red
layer at the top of the culture indicates the positive test.

Procedure
1. Preparation of tryptone broth:
Ingredients:
Tryptone

10 gm.

NaCl
Distilled water

-- 1 gm
1000 mL

Distribute 5 mL of the broth into the test tubes and plug with
cotton plugs. Sterilize it at 121C for 15 minutes.

2. Preparation of Kovacs reagent:

Ingredients:
N-amyl alcohol

75 mL

Concentrated HCl

25 mL

P-dimethylaminebenzaldehyde

5 g.

3. Inoculate the tubes with the test bacterial culture.

4. Incubate all the tubes for 48 hours at 37C.
5. Test for indole Add 0.3 mL of Kovacs reagent to each test
tube. Mix well by rotating the tubes between your hands. The
formation of a red layer at the top of the culture indicates a
positive test.

GROWTH AND MAINTENANCE OF

BACTERIA ISOLATES

Procedure
For

growth

and

maintenance

of

identified

bacteria

transferred in to broth.
A fresh single pure colony of each bacterial isolates was
transferred aseptically from agar plate into Nutrient Agar
broth medium using a sterile loop.
Prepare 1.5% of LB broth medium in a conical flask.
Autoclave and allow the medium to cool up to 45-50C. Flame
the neck of flask containing the medium.
Pick a single colony with the help of sterile loop from the
agar plate and culture the flask containing the medium.
The inoculated medium was then incubated at 37C at 100
rpm in orbital shaker.
All pure isolates were maintained in liquid and solid media.
They were regularly sub cultured into fresh medium for
short-term storage.

SCREENING FOR BIOSURFACTANT

ACTIVITY

Biosurfactant activity of isolated bacteria was detected by using

Drop Collapsing Test and oil spreading method in two different oils
namely vegetable oil and diesel.

1. DROP COLLAPSING TEST

Procedure
Biosurfactant production was screened using the qualitative
drop-collapse test described by (Youssef,Duncan, Nagle,
Savage, Knapp and McInerney, 2004).
For drop collapsing test used as 6-well Corning Costar plate.
The surface area of well was 9 cm2.
1 ml Diesel oil and vegetable oil was added to the well.
The plate was equilibrated for 1 h at 37C.
4 ml of the culture supernatant was added to the surface of
the oil in the well.
The shape of drop on the oil surface was observed after
1min.
The culture supernatant makes the drop collapsed was
indicated as positive result for biosurfactant presence and if
the drops remains intact indicates negative result.
Distilled water was used as negative control.

2. Agar well diffusion method

Reagent: Nutrient agar media

Procedure
For

assaying

contaminated

oil

degradation

activity

of

isolated bacteria, the agar well diffusion method of Perez et

al. (1990) and Rojas et al. (2006) will be used with minor
modifications.
1.5 gm of Nutrient agar media will be weighted and
dissolved in 60 ml distill water. Maintained the pH and
autoclaved it.
About 20 ml. of Nutrient agar media will be poured in to each
sterilized Petri plates and add 1% vegetale oil and diesel oil
in to the plate.
Once the agar solidified it will then punch with a six
millimeter diameter cork border to prepare wells .
These wells will be then filled with about 50 l of broth each
bacterial sp.
Sealed the plate with paraffin wax and incubated at 35C for
24 hours. Remove the plate with incubator and saw the Zone
of inhibition.

 The occurrence of a clear zone was indicates of positive

result (Rodrigues, Teixeira& Mei, 2006).

RESULTS AND DISCUSSION

The

microorganisms

which

could

be

employed

for

the

degradation of petroleum and its derivatives in minimizing

contamination due to oil leak and spill, has promoted a number
of investigators to study the process in the laboratory (Zo Bell ,
1946). Biosurfactants are surface active compound that reduce
the interfacial tension between two liquids, or that between a

liquid and a solid. Their unique property like nontoxic, easily

biodegradable, eco-friendly and high stability, and wide variety of
industrial application makes them highly useful group of chemical
compound.

Biosurfactants

are

produced

from

variety

of

microorganisms. Oil contaminated soil samples were collected

from...

. Two different bacterial species

were isolated from the oil contaminated soil samples by spread

plate method. There were several biochemical tests as catalase,
MRVP and Indole (Table 1). Standard biochemical tests were done
to identify the organisms. The two bacterial species were
identified as Pseudomonas sp. and Bacillus sp. The two different
bacterial species isolated from oil contaminated soil were
screened for their biosurfactant activity by Drop Collapsing Test
and Agar well diffusion method in two different oils namely
vegetable oil and diesel. In Agar well diffusion method the
organisms Pseudomonas and Bacillus produced clear zone and in
the drop collapse test the samples were collapsed. This clearly
indicated that the three organisms produced biosurfactant.

SPREAD PLATE METHOD

Observe all the inoculated plates as to the distribution of colonies
on each of the nutrient agar plates enriched with 1.0 % diesel,
some of the colonies will be free from each other. Select a

discrete colony each from first and second plate and record form,
elevation, pigmentation and size of the colony.

Fig 1: Sample 10-3 dilution

After the incubation of culture plate, two type of colony observed. On the nutrient agar
plate, the first was observed as a medium sized, light yellow almost clear colored
colony, identified as pseudomonas spp. The second was observed as white or cream in
colour and dry, flat and irregular with lobate margins identified as bacillus spp.

STREAK PLATE METHOD

After incubation, examine the plate for the growth of the colonies.
Isolated colonies were streaked in to the nutrient agar plates for

purification. The isolated colonies were transformed to nutrient

agar slants and stored for further studies. There was two type of
colonies were observed which streaked on nutrient agar plate. On
the basis of morphology light yellow colour colony identified as
pseudomonas spp and white colour as bacillus spp.

A
B
Fig 2: (A) Pseudomonas spp., (B)

Bacillus spp.

BIOCHEMICAL TEST

1.GRAMS STAINING
For the identification of the bacteria gram staining is the first in
the series of biochemical test. In gram staining test examine the
slides microscopically using oil-immersion objective. Identify the
gram reaction of both the cultures and classify them. Make
sketches for morphology of the cultures and describe the
morphology and arrangement of the cells. The result obtained
that pseudomonas spp showing as pink colour (grams +ve) and
bacillus spp as purple colour (grams -ve) and both the spp was
rod shaped.

A
B
Fig 3: Pink colour showing as Grams +ve and purple colour showing
as Grams-ve

2.CATALASE TEST
During

aerobic

respiration

in

the

presence

of

oxygen,

microorganisms produce hydrogen peroxide (H2O2). Catalase test

is the observation of free O 2 production. The result obtained that
pseudomonas spp was catalase positive and bacillus spp was
catalase negative. Production of gas bubbles is indicative of
catalase positive and negative. In figure right side the test of
pseudomonas spp. and left side bacillus spp.

Fig 4: Catalase test

3.METHYL RED TEST

MR test used to determine the ability of an organism to produce
mixed acid end products from glucose fermentations. Bacteria
able to ferment glucose give positive result. The result obtained
that both spp was unable to glucose fermentation and gives
negative result. There was no colour change showing that both
spp was MR negative.

Fig 5: MR test

4.VOGESPROSKAUER TEST
Certain microbial species is the ability to produce a neutral end
product, acetoin, from the fermentation of dextrose. By the
addition of potassium hydroxide to cultures the red color
produced. Changes in colour showing the bacteria are able to
ferment dextrose. The result obtained that pseudomonas spp was
VP negative and bacillus spp was VP positive. Pseudomonas spp
was not able to ferment dextrose and there was no any colour
change. But when the studied about bacillus spp. there was light
yellow colour found.

A
B

Fig 6: (A) VP ve,

(B)

VP +ve

5. INDOLE TEST
The indole test is important because only certain bacteria form
indole. Indole can be easily detected with Kovacs reagent. 5-10
drops of kovacs reagent are added to the tube. The reagent
reacts with indole to produce a ring that is cherry red in colour.
Thus, the formation of the red layer at the top of the culture
indicates the positive test. No colour changes showing the
negative test.

Fig 7: Indole negative

After the addition of kovacs reagent to the culture was observed no any
colour change. Both the spp. was unable to produce indole.

Table 1: Gram stain and Biochemical test results

Microorganism
Biochemical
test
Gram staining

Pseudomonas sp
Bacillus sp
Gram Negative, Rod
Positive, Rod

Catalase

Gram

Positive
Positive

Methyl Red
Voges

Negative
Negative
Negative

proskauer
Indole

Positive

Negative
Negative

From the Grams staining result, it was found that one strain is
negative and other is positive so for further characterization,

different bio-chemical tests were performed to identify the

species of strains. For the Identification and characterization of
isolate

bacterial

strains

Bergeys

manual

of

determinative

bacteriology was used and on the basis of this manual the species
was predicted as pseudomonas and bacillus species of bacteria.

SCREENING FOR BIOSURFACTANT

ACTIVITY
Biosurfactants are surface active compound that reduce the
interfacial tension between two liquids, or that between a liquid
and

solid.

Their

unique

property

like

nontoxic,

easily

biodegradable, eco-friendly and high stability, and wide variety of

industrial application makes them highly useful group of chemical
compound.

Biosurfactants

are

produced

from

variety

of

microorganisms. The two different bacterial species isolated from

oil contaminated soil were screened for their biosurfactant activity
by Drop Collapsing Test and Agar well diffusion method in two
different oils namely vegetable oil and diesel. In Agar well
diffusion method the organisms Pseudomonas and Bacillus
produced clear zone (Table 2) and in the drop collapse test the
samples were collapsed. This clearly indicated that the two
organisms produced biosurfactant.

A
B
Fig 8: (A)

Vegetable oil, (B) Diesel spread plate

Table 2: Oil Spreading Test for Pseudomonas sp. and

Bacillus sp.

Micro-organism

Zone formation in various oils tested

(diameter in mm)
Vegetable oil

Diesel

11

Bacillus sp.
24

Pseudomonas sp.

18

32

Biosurfactants

or

microbial

surfactants

are

surface-active

biomolecules that are produced by a variety of microorganisms.

The results obtained that Pseudomonas sp. recorded higher
biosurfactant

activity

than

Bacillus

sp.

environment

contain

large

amount

of

Oil

contaminated

hydrocarbons

and

biosurfactant producing\ microorganisms were naturally present

in

the

oil

contaminated

soil.

Biosurfactants

have

gained

importance in the fields of enhanced oil recovery, environmental

bioremediation, food processing and pharmaceuticals owing to
their unique properties such as high biodegradability and lower
toxicity.
Drop

Collapsing

Test was also

done for

determination

of

biosurfactant producing bacteria. In Drop Collapsing Test 6-well

Corning Costar plate was used. The surface area of wells is 9 cm 2.
One well contained diesel and other vegetable oil in required
amount.

After

that

added

identified

bacterial

culture

of

pseudomonas spp. The shape of drop on the oil surface was

observed after 1min. The culture supernatant makes the drop
collapsed was indicated as positive result for biosurfactant
presence and if the drops remains intact indicates negative result.

Fig 9: Drop Collapsing Test of pseudomonas sp.

Biosurfactants

are

amphiphilic

compound

(possessing

both

properties- hydrophilic and lipophilic), produced on living surfaces

mostly microbial cell surfaces and contain hydrophilic and
hydrophobic moieties that reduce surface tension and interfacial
surfaces between individual molecules at the surfaces.
The oil degradation by Pseudomonas sp. was not surprising not
only because it was isolated from oil spilled soil but also because
it is known to possess a more competent and active hydrocarbon
degrading enzyme system than Bacillus sp. It is known to be fast
growing and is capable of degrading a wide variety of organic
compounds (Ijah & Okang, 1993). In the case of Bacillus sp. which
is also known to possess the considerable efficiency to use it as

an oil degrader, but it requires more time compared to that of

Pseudomonas sp. The results were in accordance with the findings
of Van hamme, Singh, & Ward (2003) in which Pesudomonas
degraded 90.2 % of oil in 30 days followed by 82.3 % of oil
degraded by Bacillus, 78.8% of oil degraded by Serratia and
25.5% of oil degraded by Staphylococcus.

CONCLUSION
Cleaning up of petroleum hydrocarbons in the subsurface
environment is a real world problem. A better understanding of
the

mechanism

of

biodegradation

has

high

ecological

significance that depends on the indigenous microorganisms to

transform or mineralize the organic contaminants. Microbial
degradation process aids the elimination of spilled oil from the
environment after critical removal of large amounts of the oil by
various physical and chemical methods. This is possible because
microorganisms have enzyme systems to degrade and utilize
different hydrocarbons as a source of carbon and energy.
These results conclude that two different bacterial species were
isolated

from

oil

contaminated

soil

and

their

degradation

capability was checked individually by various screening methods

among which Pseudomonas sp. showed highest degradation
efficiency followed by Bacillus sp. Thus the above experiment
shows that bioremediation can be used effectively to treat oil
contaminated soil. By using biological processes, as in the case of
bioremediation, usually lowers the costs as compared to chemical
treatment processes for various contaminated sites. It is also less
disturbing to the environment. The toxicity and fertility of the soil
before and after treatment was also assessed, thereby proving
that

biostimulation

is

an

effective

method

of

reducing

environmental pollution. Therefore, based on the present study, it

may be concluded that microbial degradation can be considered

as a key component in the cleanup strategy for petroleum
hydrocarbon remediation.

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