solid.
Their
unique
property
like
nontoxic,
easily
Biosurfactants
are
produced
from
variety
of
the
biosurfactant
producing
bacteria
from
oil
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
soil
samples
was
from.
collected
solid.
Their
unique
property
like
nontoxic,
easily
Biosurfactants
microorganisms.
are
produced
from
variety
of
encoding
such
functions.
The
effectiveness
of
population
in
or
consortium
environment.
When
can
few
be
or
enriched
no
and
indigenous
with
this
problem
associated
with
biosurfactant
ether or ester group. Among the glycolipids, the best known are
rhamnolipids
sophorolipids
and
trehalolipids.
Lipopeptide
that
biosurfactant
activities
are
not
influenced
by
suited
for
the
environmental
applications
such
as
lethal
to
50%
of
test
species)
against
the
surfactant.
In
larger
a
toxicity
particular
of
study
the
chemically
where
toxicity
derived
of
six
applicability,
make
it
different
from
chemical
and
Organic
Chemicals,
Pharmaceuticals,
Foods
Mining
and
Beverages,
and
Metallurgy,
Agrochemicals
and
Fertilizers,
Environmental
Control
and
The
ecology
of
hydrocarbon
degradation
by
burying,
evaporation,
dispersion,
and
washing.
such
as
and
soil
binding
fertility,
water-holding
capacity.
The
capacity,
contamination
of
overcome
these
environmental
problems,
microbial
development.
However,
the
oil
exploration
and
These
synthetic
surfactants
are
usually
toxic
for
development
and
practical
application
of
Review of literature
Life in our planet is sustained in a fragile biological balance;
microorganisms play an important role on nutritional chains that
contaminant
decomposition
or
immobilization
by
functions
derived
through
selection,
or
by
the
population
or
consortium
can
be
enriched
and
maintained
in
environment.
When
few
or
no
indigenous
mechanical,
burying,
evaporation,
dispersion,
and
such
and
as
soil
binding
fertility,
water-holding
capacity.
The
capacity,
contamination
of
sequence
analysis
combined
with
morphological
high
degradation
ability
for
bioremediation
of
the
Both
mono-
bioremediation.
and
mixed-cultures
However,
higher
rates
can
of
be
used
for
degradation
of
isolated
from
the
environment
that
needs
mechanisms
in
the
breakdown
of
diesel
oil
consortium
diversity
of
the
and
monocultures,
microbial
community
knowledge
present
of
in
the
soils
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
with
this
problem
associated
with
biosurfactant
by
microbes
like
bacteria,
fungi
and
yeast.
of
performance
their
under
production
intense
on
large
conditions
scale,
and
selectivity,
their
future
wide
variety
of
biosurfactant
can
be
produced
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,
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.
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
lethal
to
50%
of
test
species)
against
the
surfactant.
In
larger
a
toxicity
particular
of
study
the
chemically
where
toxicity
derived
of
six
applicability,
make
it
different
from
chemical
and
Agrochemicals
Organic
Chemicals,
Pharmaceuticals,
and
Fertilizers,
Foods
Mining
and
Beverages,
and
Metallurgy,
Environmental
Control
and
isolated
biosurfactant
producing
bacteria
from
they
isolated
three
unknown
biosurfactant
the hydrocarbon
such
as hexadecane,
heptadecane,
from
lactobacilli,
they
were
found
that
the
because
GRAS
and
these
are
microorganisms
already
used
in
are
usually
many
food
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.
(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.
COLONY MORPHOLOGY
After incubation check the morphology of growing bacteria.
(a)
SHAPE
(b) MARGIN
(c)
(d)
ELEVATION
(e)
(f)
1. GRAM STAINING
Reagents
Crystal Violet,
Iodine,
Ethyl alcohol (95%),
Safranin
tool
microorganisms.
for
classification
and
differentiation
of
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.
2. 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.
MR TEST
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
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
100 ml
Solution B
Potassium Hydroxide
16 g
Distilled water
100 ml
tubes
were
observed
for
change
in
colour,
the
4. INDOLE TEST
Materials
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.
75 mL
Concentrated HCl
25 mL
P-dimethylaminebenzaldehyde
5 g.
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.
Procedure
For
assaying
contaminated
oil
degradation
activity
of
microorganisms
which
could
be
employed
for
the
Biosurfactants
are
produced
from
variety
of
discrete colony each from first and second plate and record form,
elevation, pigmentation and size of the colony.
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.
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,
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
(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.
After the addition of kovacs reagent to the culture was observed no any
colour change. Both the spp. was unable to produce indole.
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,
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.
solid.
Their
unique
property
like
nontoxic,
easily
Biosurfactants
are
produced
from
variety
of
A
B
Fig 8: (A)
Micro-organism
Diesel
11
Bacillus sp.
24
Pseudomonas sp.
18
32
Biosurfactants
or
microbial
surfactants
are
surface-active
activity
than
Bacillus
sp.
environment
contain
large
amount
of
Oil
contaminated
hydrocarbons
and
the
oil
contaminated
soil.
Biosurfactants
have
gained
Collapsing
done for
determination
of
After
that
added
identified
bacterial
culture
of
Biosurfactants
are
amphiphilic
compound
(possessing
both
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
from
oil
contaminated
soil
and
their
degradation
biostimulation
is
an
effective
method
of
reducing
References
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Microorganismos. 1998. 8. P. 726.
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318
4. Alvarez P., Vogel M. Biodegradation. 1991. 2. P. 43.
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Stanley
GA.
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Environ.
&
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of
micro-organism
from
oil
M.
Biodegradation
and
Bioremediation,
24.
M.,
Paquot,
scale
M.,
Jacques,
organization
P.,
Thonart,
of
P.,
mixed
33.
peptidolipid
surfactant
produce
by
Bacillus
Production
by
Bacillus
sp.Isolated
from
Zhenyu
Wang.
41.
study
of
biosurfactant
produced
by