TITLE: Biodegradation potential of Bacillus subtilis strains isolated from a petroleum oil-

contaminated soil from Pandacan Oil Depot

chemical industries in recent years has

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