TITLE: Biodegradation potential of Bacillus subtilis strains isolated from a petroleum oil-
contaminated soil from Pandacan Oil Depot
Introduction
The contamination of the oil has been
considered as one of the most concerned
pollutions in the Philippines and even in the
world. Oil pollution accidents are nowadays
become a common phenomenon and have
caused ecological and social catastrophes
(Burger, 1993; Burns et al., 1993; Shaw,
1992). The production of the soil
contamination is widely produced through
various sources such as crude oil
production, improper waste disposal of oil
in some industrial establishment namely
the petrochemical metal and oil industry.
Oil contaminated soils are considered to be
harmful to the environment and most
especially in the human health. It contains
both aromatic and polycyclic hydrocarbons
that are used to be toxic and carcinogenic
Many studies have been conducted to be a
possible solution for the pollution of
petroleum. One of these is the
Bioremediation this technique is widely
used in the research, it is the process used
to treat contaminated media such as water,
soil and subsurface materials by degrading
the target hazardous materials that are
present in the media with the used of
microorganisms. The use of this technique
is helpful because it avoids the
environmental and technical disadvantages.
The demand of the petroleum as a source
of energy and as a primary raw material in
chemical industries in recent years has
resulted in an increase in world production
(K.V. Darsa, 2014). The possible reason
behind this uncontrollable increase of
environmental problems is the large
number of production, usage, and
distribution of the petroleum products
across the world. The prolonged existence
of the petroleum pollution is dependent on
the hydrogen mixture and to properties
that are contaminated in the ecosystem.
The ability to isolate high numbers of
certain oil-degrading microorganisms from
oil polluted environment is commonly taken
as evidence that these microorganisms are
active degraders in that environment
(Okerentugba et al; 2003)
Many species of bacteria have been
identified having the ability to degrade oil.
Because they are capable of converting
hydrocarbons substrates as their sole
source of carbon and energy.Bacillus sp
used to be considered as a petroleum
hydrocarbon degrader and known to be
naphthalene and pyrene degrader. Bacillus
subtilis grew on a large number of
hydrocarbon compounds as a source of
carbon and energy demonstrating this
strain might be efficient hydrocarbon
degraders because of its ability to produce
spores and emulsify hydrocarbons. There
are reports describing the effect of
exogenously added microbial biosurfactants
in enhancing the bioremediation of crude
oil-polluted soils by indigenous microbes.
This purpose of this study is to evaluate and
to know the potential of Bacillus stabilis
that was isolated from the contaminated
petroleum oil soil in Pandacan oil depot.
Materials and Methodology
Collection of Samples
The soil samples were collected from the
Pandacan oil depot and placed in a sterile
container and transported to the laboratory
for analysis.
Isolation of petroleum degrader bacteria
The collected soil samples were serially
diluted up to 10G7 and 0.1 mL from the
dilutions 10G5 and 10G6 were spread plated
on Bushnell hass mineral salt medium
(magnesium sulfate
0.2 g, calcium chloride 0.02 g, monopotassium
phosphate1 g, dipotassium phosphate 1 g,
ammonium nitrate 1 g
and ferric chloride 0.05 g) containing 2.5% of
petrol13.The plates were incubated at 37°C
for 24 h and from the developed colonies, one
was selected for further experiments.
Identification of petrol degrading strain
Gramstaining and biochemical tests like
catalase, oxidase, Voges proskaeur, indole
production and sporulation tests were carried
out for strain identification
Biodegradation studies
One hundred mL of minimal medium
(dextrose 1 g, ammonium sulphate 1 g,
dipotassium phosphate 7g, monopotassium
phosphate 2 g, sodium citrate 0.5 g and
magnesium sulphate 0.1 g) with petrol and
one mL inoculum from the overnight culture
maintained in nutrient broth during
the logarithmic phase were added to 250 mL
Erlenmeyer flasks which were subjected to a
period of sixteen days.Flasks were incubated
in a shaker at 30°C at 100 rpm.
After the specified time, flasks were taken
out. The ability of the isolated strain to
degrade petrol was studied by determining
the parameters pH, OD and CO in the 2
culture medium every four days.
pH estimation
Sample from the culture medium was
checked for pH after 0, 4, 8, 12 and 16 days of
treatment with the pH meter.
Optical density (growth rate) determination:
The optical density of the sample from culture
medium was determined at 610 nm after 0, 4,
8, 12 and 16 days of treatment using a
spectrophotometer.
Carbon dioxide estimation
From each petrol, the concentration was
taken after 4, 8, 12 and 16 days of treatment
and titrated against 0.05N NaOH solution.
Phenolphthalein was used as the indicator
and appearance of pink color was considered
as the endpoint. The amount of CO2 was
calculated using the following equation.
Statistical analysis
Two way ANOVA was performed for the
parameters pH, optical density and carbon
dioxide released using MS Excel. Variability
was considered only when the calculated F
value was greater than the

tabulated F-value at P is less than or equal to

0.0516.

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