Int.J.Curr.Microbiol.App.

Sci (2013) 2(6): 1-18

ISSN: 2319-7706 Volume 2 Number 6 (2013) pp. 1-18

http://www.ijcmas.com

Original Research Article

Environmental studies on the microbial degradation of oil
hydrocarbons and its application in Lebanese oil polluted coastal and
marine ecosystem
Darin Maliji, Zakia Olama* and Hanafy Holail

Biological and Environmental Science Department, Beirut Arab University, Beirut, Lebanon
*Corresponding author e-mail: zakia.olama@bau.edu.lb

ABSTRACT

One of the major environmental problems today is hydrocarbon contamination

resulting from the activities related to the petrochemical industry, therefore,
accidental releases of petroleum products are of particular concern in the
environment, which could lead to consequences for the biotic and abiotic
Keywords components of the ecosystem if not restored. Remediation of hydrocarbon-
contaminated system could be achieved either by physiochemical or biological
Biostimulation method. Present study aims to use biological methods for the remediation of the
stratagem; contaminated sites. Screening study for the isolation of the promising hydrocarbon
promising degraders and optimization experiments to evaluate the best environmental and
hydrocarbon physiological factors that lead to maximum degradation of hydrocarbons revealed
degraders; that, Bacillus cereus A , Bacillus cereus B and Bacillus sp. ZD the most promising
degradation of hydrocarbon degraders were isolated from Lebanese marine ecosystem and
hydrocarbon; identified using 16S rRNA. Bacillus cereus A showed maximum diesel oil
Bacillus degradation (82.41% and 81.56% of aliphatic and aromatic hydrocarbons) after 2
cereus; days incubation under shaken condition, at pH 7, 100 ml culture volume and 2%
Plackett- inoculum size. Seven nutritional factors were examined for their significance on
Burman; hydrocarbon degradation using a statistical design known as Plackette-Burman.
Maximum diesel oil degradation produced by Bacillus cereus A was revealed by
the statistical design. Immobilization technique showing that Bacillus cereus A can
be used for diesel oil degradation on large environmental scale, solving by this one
of the problems of oil spill management that results from the petrochemical
industry and saving the environment from additional water and soil pollution.
.

Introduction
Petroleum which is the major source of recognized, and named as : aromatic,
energy for industry and daily life is an saturate or aliphatic, asphaltic and resins
extremely complex mixture of (Gopinathan et al., 2012). However, after
hydrocarbons. From the hundreds of the sinking of the super tanker Torney
individual components, four classes, based Canyon in 1967 the attention of the
on related chemical structures can be scientific community was drawn towards

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 Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 1-18

the problems of oil pollution. These Biodegradation, is a mineralization of

pollution problems often result in huge organic chemicals, which ultimately
disturbances of both the biotic and abiotic leading to the formation of CO2, H2O and
components of the ecosystems (Al- biomass (Hamdi et al.,2007).
Jumaily and Al- wahab, 2012), Biodegradation being an economical and
furthermore some hydrocarbon eco-friendly approach, has emerged as the
components have been known to belong to most advantageous soil and water clean-up
a family of carcinogenic and neurotoxic technique for contaminated sites
organopollutants (Tevvors and Saier, containing oil spills, is applied with
2010). Thus, release of hydrocarbons into different strategies, but well accomplished
the environment whether accidentally or with a process called biostimulation, that
due to human activities is a main cause of is, the addition of several nutrients and
water and soil pollution (Kumar et al., fertilizers to a contaminated matrix (Tyagi
2008). et al., 2011).

Several physical and physiochemical However, a number of limiting factors

techniques have been used to clean up the have been recognized to affect the
oil residues but, compared to biodegradation of petroleum
physiochemical methods; biological hydrocarbons. The success of
method such as biodegradation offers to bioremediation is dependent upon physical
be non-invasive, simple to maintain, and chemical conditions such as
applicable over large areas and relatively nutritional requirements (carbon, nitrogen
cost effective method for the treatment of and phosphorous), oxygen, pH and
oil contamination because : first, the biosurfactant.
majority of molecules in the fuel oil and
refined products are biodegradable The present study aimed for isolation and
(Prince, 2002), and oil-degrading selection of hydrocarbon degrading
microorganism are ubiquitous (Chaîneau bacteria from oil polluted seawater and
et al., 2000 and Joo et al., 2008). studies the environmental and
physiological factors of the most
Although many microorganisms have the promising and selected isolates lead to
ability to utilize hydrocarbons as sole maximum hydrocarbon degradation and its
sources of carbon and energy for application for the biodegradation as a
metabolic activities, but the microbial biostimulation strategy for oil
utilization of hydrocarbons depends on the bioremediation in polluted seawater.
chemical nature of the compounds within
the petroleum mixture and on Materials and Methods
environmental determinants and
biodegradability of the these compounds Water sample
generally decreases in the following order:
n-alkanes, branched-chain alkanes, Oil contaminated seawater sample were
branched alkenes, low molecular- weight collected under aseptic conditions from
n-alkyl aromatics monoaromatics, cyclic Saida port in south Lebanon at 50 cm
alkanes polycyclic aromatic hydrocarbons depth from seawater surface in sterile
asphaltenes (Van Hamme et al., 2003). containers.

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Oil samples seawater nutrient agar media plates

containing either diesel or fuel oil as sole
Diesel oil and fuel oil Samples were carbon and as energy for metabolic
obtained from the Jieh power plant in activities and incubated at 30°C for 48 hrs
south Lebanon. (Sathishkumar et al., 2008; Jyothi et al.,
2012).
Microorganisms
Maintenance of the Microorganisms
The microorganisms used throughout the
present investigation were isolated from All the bacterial strains used throughout
seawater at Saida port in south Lebanon the present investigation were maintained
and identified genotypically using 16S on nutrient agar slants and stored at 4°C
rRNA as three different Bacillus sp. with regular transfer at monthly intervals.

Media Preparation of seed culture

Unless otherwise indicated, all media were Transfer from single slant cultures (48
prepared with distilled water, adjusted to hour old) were taken into 50 ml aliquots of
initial pH 7 and sterilized by autoclaving the seed medium containing (g/l): Beef
for 20 min at pressure of 15 lb/inch2 to extract, 1; Yeast extract, 2; Pepton, 5 and
raise the temperature to 121°C. 1L of distilled water. Dispensed in 250 ml
of Erlenmeyer flask to initiate the growth
Nutrient agar medium (NA): was used for (OD 1). Standard inocula 2% (v/v) were
the bacterial cultures and had the taken from the latter liquid culture after
following composition (g/l); peptone,5; growth for 18 hours at 30 °C on a
yeast extract,2; sodium chloride, 5 and reciprocal shaker to start growth in the
bacteriological agar, 15 (Goldman and fermentation flask which is equivalent to
Green, 2009). 1.5 x 108 colony forming unit/ml
(CFU/ml) according to McFarland scale
Fermentation medium: used for the growth 0.5.
of seawater autochthonous oil degraders
and contained (g/l): NH4Cl, 2.5; KH2PO4, Screening for hydrocarbon degrading
0.3; Na2HPO4, 0.7; MgSO4.7H2O, 0.2; activities of the isolates under
Yeast extract, 0.1; Thiamine HCl, investigation
0.001(Dutta and Harayama 2000).
The cultivation of the isolated and purified
Isolation and purification of oil bacterial strains was achieved in replicate
degrading bacteria in 250 ml Erlenmeyer flasks each
containing 50 ml of fermentation medium
Serial dilutions-agar plating technique of .The media were sterilized by autoclaving
the seawater sample for 15 min at 121°C then 2ml of the diesel
2 3 4 5
(1/10,1/10 ,1/10 ,1/10 and 1/10 ) were and fuel oil was added one at a time with a
prepared in sterile distilled water and chemical intermediate tween 20, which is
plated on nutrient agar media plates and act as emulsifying agent and incubated
incubated at 30°C for 48 hrs. The bacterial with 2 % (v/v) inoculum level unless
colonies obtained were further purified on otherwise stated and then incubated at

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 Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 1-18

30°C for different time interval 12, 24, determine the quantity of residual diesel
36, 48 and 72 hours respectively under oil/fuel oil by difference. The second time,
shaken conditions using rotary shaker(160 extraction done in same manner but with
rpm.) and static conditions. dichloromethane (40 ml) for the extraction
of aromatic hydrocarbon fractions.
Residual hydrocarbon analysis
The percentage of aliphatic or aromatic
Analytical determination of oil fraction in diese/fuel oils degraded at
hydrocarbons different time course was determined from
the equation: % degraded of aliphatic or
Cell free extract preparation aromatic fraction = (weight of aliphatic or
aromatic fractions degraded / original
At the end of incubation period the culture weight of diesel oil or fuel oil introduced)
was sampled and the cells were removed × 100. Where the weight of aliphatic or
by centrifugation at 6000 r.p.m for 15 min. aromatic fraction degraded was
The supernatant (cell free extract) was determined as original weight of diesel
used for analysis of residual oil fractions oil/fuel oil minus weight of residual
immediately or stored at 4°C for further aliphatic or aromatic fraction obtained
analysis. after evaporating the extractant (Nwaogu
et al., 2008; Kebria et al., 2009).
Quantification and Extraction of
aliphatic and aromatic fractions in both Morphological characterization
diesel and fuel oil
Growth characterization of the cultures
The residual aliphatic and aromatic was determined on the basis of color
fractions in both diesel and fuel oil from appearance, size and shape of colonies
culture flasks (supernatent) were extracted developed, and the reaction towards the
twice in separatory funnel, one time with stain.
one volume of n- hexane (40 ml) for the
extraction of aliphatic hydrocarbon Genotypic characterization
fractions and shaken vigorously for 3 min
and allowed to settle for 5 min. The The genomic DNA of isolate (A,B,C)
solvent layer (liquid phase) was separated were isolated according to Sambrook et
by allowing the (diesel oil or fuel oil al., (1989). The 16S rRNA was amplified
"aliphatic fractions" n-hexane) to pass by polymerase chain reaction (PCR) using
gradually through a funnel fitted with filter universal eubacteria specific primers,
paper. Anhydrous sodium sulphate, spread designated to amplify 1500bp fragment of
on the filter paper was employed to the 16S rRNA regions. The forward
remove any moisture in the mixture. The primer was: 27F
liquid phase was collected in a 50-ml flask (5 AGAGTTTGATCMTGGCTCAG 3 )
.The flask containing the extract (aliphatic and the reverse primer was: 1492R
fractions"- n-hexane) was placed in an hot- (5 TACGGYTACCTTGTTACGACTT3 )
plate and the extractant solvent (n- , which yielded a product of approximately
hexane), allowed to evaporate at 90 °C. 1500 bp.Successful amplification was
The residual diesel/fuel oils were poured confirmed by agarose gel electrophoresis
in pre-weighted vials and weighed to and the remnant mixture was purified

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 Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 1-18

using QIA quick PCR purification Exi=( Mi+ - Mi-)/N

reagents (Qiagen). DNA sequence were
obtained using an ABI PRIMSE 377 DNA Where Exi is the variable main effect, Mi+
Sequencer and ABI PRIMSE BigDye and Mi- are percentage of consumed oil in
Terminator Cycle Sequencing (Perkin trials where the independent variable (xi)
Elmer). The PCR product was sequenced was present in high and low levels,
using the same PCR primers. Blast respectively, and N is the number of trials
program was used to assess the DNA divided by 2.
similarities and multiple sequence
alignment and molecular Phylogeny were The standard error (S.E) of the variables
performed using BioEdit software (Hall, was the square root of variance and the
1999). The Phylogenetic tree was significance level (Pvalue) of each
displayed using TREEVIEW program variables calculated by using the t- Test:
(Page. 1996). t = Exi/ S.E

Environmental and Physiological where Exi is the effect of the tested

factors affecting diesel oil degradation variable. The variable with higher
confidance levels were considered to
Different environmental factors such as influence the response or output variable.
culture volume, inoculum size, pH and
carbon source were tested and screened for Immobilization of Bacillus cereus A by
maximum diesel oil degradation. adsorption on sponge and luffa

Optimization of the nutritional factors Sponge and luffa of a known mass were
affecting biodegradation using cut into small cubes were added
multifactorial statistical design individually into a 250 ml Erlenmeyer
(Plackett-Burman design) flask containing 100 ml fermentation
media inoculated with the selected
Plackett-Burman design (Placket and bacterium and allowed to grow for 48 hrs
Burman,1946), well established statistical at 30°C under shaken condition (160 rpm).
technique for medium component
optimization (Xiong et al.,2005) was Application in a bioreactor
applied to screen the medium components
critically affecting degradation of diesel A glass column was sterilized with its
oil by the selected bacterium. Seven caps. A known mass of sponge and luffa
independent variables were screened in cubes loaded with the selected bacterium
eight combinations organized according to cells were transferred under aseptic
the Plackett-Burman design matrix conditions to the columns. Fifty ml of
described in the result section. For each distilled water fortified with 1 ml diesel oil
variable, a high (+) and low(-) was tested. were added to the bioreactors. After oil
All trials were performed in duplicates and degradation was observed, the medium
the percentage of hydrocarbon was drained. The process was repeated for
consumptions was treated as the response several times until degradation of oil was
for each trial. The main effect of each decreased. In every run medium taken was
variable was determined with the centrifuged, and the supernatant was used
following equation: to measure the degradation of oil.

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Results and Discussion

Screening for hydrocarbon degrading

activities of the isolates under
investigation

Data revealed that the highest oil

degradation (82.41% and 81.56% of
aliphatic and aromatic diesel oil
hydrocarbons respectively), was achieved
with isolates A when grown on
fermentation medium fortified with diesel Phenotypic characterization
oil for 2 days under shaken conditions,
followed by isolate B( 79.51% and The cultural and colonial characteristics
78.64% of aliphatic and aromatic diesel oil showed that the colonies were circular,
hydrocarbons respectively) than by isolate whitish, medium in size with serrated
C(76.44% and 76.25% of aliphatic and edge, raised elevation and smooth
aromatic diesel oil hydrocarbons surfaces. Morphological characterization
respectively when grown under the same showed gram positive, long rod shape,
conditions.(Figure 1). spore-forming isolates were identified as
Bacillus sp.
Figure.1 Diesel oil degrading capacity of
the bacterial isolates under test using Genotypic characterization and
shaken conditions phylogeny

The most promising bacterial isolates

A,B,C were identified by sequencing PCR
amplified 16S rRNA. The obtained
sequences submitted to FASTA3 data base
in order to find homologies with other 16S
rRNA. Tables (1,2,3)shows the similarities
percentages and accession numbers
obtained after comparing the sequences of
the tested strains(isolate A, isolate B and
isolate C) to the submitted sequences in
gene bank respectively. The tested strains
were affiliated to the genus Bacillus with
99%, 99% and 93% similarity to Bacillus
cereus respectively. The phylogeny of the
tested strains and closely related species
were analyzed using multi sequence
alignment program (TREEVIEW
program) and the results are presented in
phylogenetic cladogram (Figure 2,3, 4
respectively). Isolate A showed 99%
identity to different strains of Bacillus
cereus so it is named Bacillus cereus A.
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Table.1 Bacillus strains showing 99 % identity to Bacillus cereus A

Accession number Description Identity (%)
FJ686822.1 Bacillus cereus strain LB5 99
EU272862.1 Bacillus cereus strain MK30 99
JN206612.1 Bacillus sp. SR2 99
HQ844479.1 Bacillus cereus strain JSYM28 99
JN182694.1 Bacillus sp. ANIOT10 99

Table.2 Bacillus strains showing 99 % identity to Bacillus cereus B

Accession number Description Identity (%)

GU471204.1 Bacillus sp. Q1BY4 99
EU162024.1 Bacillus cereus isolate PGBw4 99
EF503534.1 Bacillus cereus strain HPC1414 99
N206612.1 Bacillus sp. SR2 99
HQ844479.1 Bacillus cereus strain JSYM28 99
JF728871.1 Bacillus cereus strain Tcb1 99
JF736840.1 Bacillus cereus strain Tcb2 99
JF895490.1 Bacillus cereus strain Cr-50 99
JF939041.1 Bacillus cereus strain AIMST Nse4 99

Figure.2 Phylogenetic relationships among representative experimental isolate A and the

most related bacteria based on 16S rRNA sequences.

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Figure.3 Phylogenetic relationships among representative experimental isolate B and the

most related bacteria based on 16S rRNA sequences

Figure.4 Phylogenetic relationships among representative experimental isolate C and the

most related bacteria based on 16S rRNA sequences

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However Isolate B also showed 99% inoculum level was selected to carry out
identity to different strains of Bacillus the next part of the research.
cereus so it was named Bacillus cereus B,
while Isolate C showed 93% identity to Low inoculum size required longer time
Bacillus cereus so it was named Bacillus for cells to multiply and produce the
sp ZD. desired product (Jiff et al., 1998). A small
amount of inoculum can lead to
Environmental and Physiological insufficient number of microbial cells and
factors affecting hydrocarbons a reduced amount of the secreted enzymes
degradation by Bacillus cereus A: while a much higher inoculum could lead
to or cause a lack of oxygen and depletion
The aim of the present study is to evaluate of nutrients in the culture media (Abusham
the optimum physiological and et al., 2009).
environmental factors for maximum
hydrocarbon degradation by Bacillus Effect of pH
cereus A.
Maximum diesel oil degradation by
Effect of culture level Bacillus cereus A (85.99% and 83.84% of
aliphatic and aromatic hydrocarbon) was
Bacillus cereus A has attained maximum at pH 7 (Figure 7). Therefore pH 7 was
diesel oil degradation (85.89% and selected for further experimentation.
83.33% of aliphatic and aromatic Higher or lower pH values showed inferior
hydrocarbon respectively with 100 ml results; metabolic processes are highly
culture volume (Figure 5) that was utilized susceptible to even slight changes in pH
as the optimum culture volume in the next (Wang et al., 2012).
experiments. The finite volume of the Meredith et al., (2000) and Rahman et al.,
culture medium means the limitation of (2002) were reported that any extremes in
the nutrients for the microorganism. The pH were shown to have a negative
consumption of the nutrients is largely influence on the ability of microbial
dependent on the bacterial population. To populations to degrade hydrocarbons. A
ensure high diesel oil degradation in change in pH has an effect into the
limited culture volume, the inoculum size biodegradative activity of microbial
should therefore be controlled (Abusham populations, as well as on the
et al., 2009). solubilization and absorption/desorption of
ions and pollutants (San Martín, 2011).
Effect of Inoculum Size
Effect of carbon source
Results revealed that maximum diesel oil
degradation by Bacillus cereus A (85.89 % The maximum hydrocarbon degradation
and 83.33 % of aliphatic and aromatic by Bacillus cereus A was achieved in the
hydrocarbon) was achieved with 2ml presence of arabinose as a carbon source
inoculum level/flask; however, minimal (88.03 % aliphatic and 84.28 % aromatic
diesel oil degradation(50.1 % aliphatic and hydrocarbons), while lactose and mannose
48.51 % aromatic hydrocarbon) was were not favorable for bacterial growth as
achieved with 4ml inoculum level/flask compared to the other tested carbon
(Figure 6). Accordingly, 2ml/100ml sources(Figure 8).

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Table.3 Bacillus strains showing 93 % identity to Bacillus sp ZD

Accession number Description Identity (%)

HQ844639.1 Bacillus cereus strain AIMST 1.Imp.5 93

HQ694287.1 Bacillus cereus strain AIMST 7.M18.2 93

HM480311.1 Bacillus cereus strain WIF15 93

FJ686822.1 Bacillus cereus strain LB5 93

AF385082.1 Bacillus sp. F26 93

HM461192.1 Bacillus sp. enrichment culture clone HSL67 93

GQ344805.1 Bacillus cereus strain DC3 93

GQ344803.1 Bacillus cereus strain DC1 93

EU557028.1 Bacillus cereus strain KU206-3 93

EU368183.1 Bacillus cereus strain TpP-2 93

EU368181.1 Bacillus cereus strain MpP-1 93

Table.4 The experimental design using Plackett-Burmans method for screening of medium
components affecting diesel oil degradation by Bacillus cereus A

Variables MgSO4.7 Yeast Thiamine % of oil degredation

NH4CL KH2PO4 Na2HPO4 Oil
Run H2O Extract HCL aromatic aliphatic
1 - + + - - + + 82.26 83.3
2 + - + + - - + 96.54 96.90
3 + + - + + - - 74.45 75.67
4 - + + - + + - 98.30 98.78
5 - - + + - + + 70.44 71.86
6 + - - + + - + 62.50 63.64
7 + + - - + + - 82.37 83.47
8 - - - - - - - 57.02 58.82

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Figure.5 Effect of the culture volume (ml) Figure.8 The effect of different carbon
on diesel oil degradation by Bacillus cereus sources on diesel oil degradation by Bacillus
A. cereus A

Figure.9 Main effect of the medium

components factors on diesel oil degradation
Figure.6 Effect of the inoculum size on the by Bacillus cereus A based on Plackett
diesel oil degradation by Bacillus cereus Burman design's result
A.

Figure.10 Effect of interaction between %

of aliphatic diesel oil degradation,Na2HPO4
and KH2PO4 by Bacillus cereus A.
Figure.7 The effect of pH on diesel oil
degradation by Bacillus cereus A (Aliphatic)

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Bayoumi et al., 2011, reported that shown that potassium dihydrogen phosphate
maximum yield of biosurfactant and and disodium hydrogen phosphate had a
degradation of crude oil is occurred in the significant effect on diesel oil degradation
presence of sucrose. Batista et al., (2006) (Figure 10, 11), whereas the other factors
reported that, glucose is a better carbon affected slightly the hydrocarbon
source than fructose and sucrose for degradation process.
biosurfactant production and biodegradation
of crude oil by Gram-positive and Gram- Figure.11 Effect of interaction between %
negative bacteria. of aromatic diesel hydrocarbon
degradation,Na2HPO4 and KH2PO4 by
Optimization of the Best Nutritional Bacillus cereus A.
Factors Affecting Hydrocarbon
Biodegradation Using Multifactorial
Statistical Design

Sequential optimization approaches were

applied in the present part of the study. The
approach was to optimize the nutritional
factors that control hydrocarbon degradation
process. The best culture conditions such as,
incubation time for 2 days; initial pH at 7; 2
ml inoculum level; 100ml culture volume
and 30ºC incubation temperature were used
for the optimization of the nutritional factors
using the Plackett-Burman statistical design.
The main effect that was estimated as a
Evaluation of the Factors Affecting diesel difference between both average of
oil degradation measurements made at the high level (+1)
and at the low level (-1) of the factor of the
In screening and optimizing the factors examined factors affecting diesel oil
affecting diesel oil degradation, it is very degradation was calculated and presented
important to test as much factors as possible graphically (Figure 9).
and to identify the significance of each of
them. Plackett-Burman design offers good On the analysis of the regression coefficients
and fast screening procedure and of the seven variables, ammonium chloride,
mathematically computes the significance of potassium dihydrogen phosphate, disodium
large number of factors in one experiment, hydrogen phosphate, yeast extract and
which is time saving and maintain thiamine HCl had shown a positive effect,
convincing information on each component whereas magnesium sulfate and oil had
(Srinivas et al., 1994). The design is shown a negative effect.
recommended when more than five factors
are under investigation (Abdel-Fattah et al., In the present study, nitrogen and
2005). phosphorus supply promoted the
biodegradation of oil hydrocarbons with
The influences of seven factors on diesel oil lower level for nitrogen and higher level for
degradation were tested (Table 4). It was phosphorous. These results are in

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accordance with that obtained by Dongfeng activity and the net result of the effect
et al., (2011), Onuoha et al., (2011) and created by the metals (Mg+2) used was
Al-Jumaily and Al- wahab (2012) which favoring petroleum bioremediation by the
explained that excess amount of nitrogen Penicillium strain. On the other hand
fertilizer could suppress the microbial Dongfeng et al., (2011) which showed that
growth, leading to a reduced ability to an excessively high concentration of metal
degrade petroleum hydrocarbons by ions (Mg+2) could have an intoxicating
decreasing its ability for production of effect on growth of the strain KL2-13 to
metabolic enzymes. However, the data of reduce the strain s ability to utilize
the present study indicated that KH2PO4 and petroleum hydrocarbons.
Na2HPO4 showed a significant effect on
diesel oil biodegradation which is in The influence of some additives on
agreement with that obtained by Haines et hydrocarbon biodegradation was tested;
al., (2003) and Farag and Soliman (2011) present investigation revealed that the
who stated that the presence of phosphate addition of yeast extract in its higher level
plays a critical role and its inadequate promoted the biodegradation of oil
supply may result in slowing the rate of hydrocarbons and this is in agreement with
biodegradation and phosphorus can be used Abdel-Fattah and Hussein (2002) who
as source of storage energy in the form of reported that the addition of yeast extract to
ATP. On contrary Wenxiang et al., (2012) the medium components was also induced
reported that the presence of an excess the bioremediation process since the
amount of phosphorus decreased diesel oil microorganisms require a primary growth
removal efficiency through inhibition of substrate to cooxidize hydrocarbon
bacterial growth. Furthermore, it would compounds. Nicolson & Fathepure (2004)
increase the cost of bioremediation and may reported that the addition of yeast extract to
cause eutrophication in sea ecosystems. The halophilic and halotolerant bacteria
phosphorus addition can stimulate the enhanced the biodegradation of benzene,
biodegradation of petroleum hydrocarbons, toluene, ethylbenzene and xylene (BTEX)
however some sources (phosphate and compounds. In addition to that Arulazhagan
ortho- phosphate) can have diverse effect on et al., (2010) studied the influence of yeast
the biodegradation, depending on its toxicity extract as an additional substrate by the
and solubility (Changyi et al., 2009). bacterial consortium and they reported that
yeast extract is the water soluble portion of
Magnesium (principal inorganic cation in autolyzed yeast containing vitamins,
cells and constitutes approximately 1 % of nitrogen, amino acids and carbon for
the dry weight of the microbial cell) plays a bacterial growth which promote the
role as a cofactor for many enzymatic degradation of PAHs. On the other hand ,
reactions or cell wall components; it Okeke and Frankenberger (2003)
stimulate enzyme reactions associated with a demonstrated that good growth and
synthesis of cell materials (Cameotra et al ., degradation rate of crude oil in presence of
2008). In the present investigation , 1% yeast extract as nitrogen source. On the
magnesium in its low level negatively contrary Khleifat (2006) reported that the
affected the biodegradation which is in nitrogen sources supply, except yeast extract
agreement with Abdel-Fattah and Hussein and casein, led to the enhancement of the
(2002) who reported that metal ion (Mg+2) phenol biodegradation of Ewingellaamerica.
may favor fungal and bacterial enzymatic
The present study also revealed that the
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addition of Thiamine HCl in its higher level conditions achieved nearer to optimum one
favor the biodegradation of oil hydrocarbons for diesel oil biodegradation by Bacillus
.On other hand Todar (2008) reported that cereus A were (g/l): NH4CL, 2.5; KH2PO4,
some bacteria do not require any growth 0.6; Na2HPO4, 1.4 ; MgSO4.7H2O, 0.2 ;
factors; they can synthesis all essential Yeast Extract, 0.2; Thiamine HCL, 0.002
vitamins, starting with carbon source, as a and diesel oil, 2ml (with few drops of
part of their own intermediary metabolism. tween 20). Flasks with 100 ml culture
volume were inoculated with 2 % (v/v) of
Diesel oil used as a carbon and energy the bacterial suspension equivalent to 0.5
sources was negatively significant factor Macfarland and incubated at 30°C for 48 hrs
once it present in its lower level (2ml/ under shaken condition(160r.p.m.).
100ml). Atlas and Hazen (2011)
microorganisms are able to consume Biodegradation of diesel oil using
petroleum hydrocarbons as the sole source immobilized Bacillus cereus A
carbon and energy for metabolism, although
inhibition to microorganisms by toxins is It was aimed in the present part to test the
also possible( Wenxiang, 2012). When potentiality of immobilized cells in diesel oil
crude oil in the environment reached a degradation. Different immobilizing agents;
definite concentration it would have toxic sponge and luffa were used as natural
effect on microorganisms, because the strain support to immobilize the living cells of
could not tolerate the high concentration of Bacillus cereus A as previously described.
crude oil, which would inhibit the normal To study the adsorption of diesel oil on
microbial growth to dramatically reduce the immobilization matrix, an experiment was
hydrocarbon degradation rate (Changyi et carried out with cells free sponge and luffa.
al., 2009). Ferreira et al., (2012) reported The sponge was transferred to glucose
that some compounds in petroleum can be containing medium and kept for 48 hrs. The
toxic to Y. lipolytica IMUFRJ 50682 above a same process was done for other
certain concentration, inhibiting the immobilization matrix (luffa).
metabolism of the microorganism. An
increase in diesel degradation corresponded Use of immobilized Bacillus cereus A by
to an increase in cell number during the adsorption on luffa and sponge
degradation processes demonstrating the
microbial ability of utilizing diesel as the Immobilized Bacillus cereus A on luffa and
energy source (Zhu et al., 2001). Thus sponge were added individually to
reinforcing the micobial ability to utilize bioreactor containing distilled water
both aliphatics and aromatics as sole source amended with diesel oil (1ml/50ml) and oil
of carbon and energy. Biodegradation of degradation was determined after 48 hours.
crude oil by microorganisms appears to be Maximum percentage of oil degradation
the natural process by which the bulk of the (80.67% and 78.83% of aliphatic and
polluting oil is used as an organic carbon aromatic hydrocarbons respectively) was
source, causing the breakdown of petroleum recorded with Bacillus cereus A
components to lower molecularcompounds immobilized on luffa. Eight successive runs
or transformed into the other organic were overloaded with the oil and the results
compounds such as biosurfactants (Zhang et showing decrease in oil degradation until it
al., 2005). reach (39.58 % and 32.76% of aliphatic and
aromatic hydrocarbons respectively).
By the end of the present study, the

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 Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 1-18

Whereas maximum percentage of oil Studies showed that natural support such as
degradation was recorded with Bacillus pumice, modified rice straw and luffa are a
cereus A immobilized on sponge (71.97% good biosorbent for some pollutants in
and 68.63% of aliphatic and aromatic water, such as oil-spills (Sun et al., 2002),
hydrocarbons respectively). Six successive dyes (Gong et al., 2008) and heavy metal
runs were overloaded with the oil and the ions (Rocha et al., 2009). In a trial to apply
results showing decrease in oil degradation fed-batch cultures, a bioreactor was used.
until it reach 36.95 % and 30.43 % of Immobilized (75.67% and 73.83% of
aliphatic and aromatic hydrocarbon) (Figure aliphatic and aromatic hydrocarbons
12). The major advantages of an adsorption respectively) cells on luffa were repeatedly
system for water pollution control are less used to degrade diesel oil.
investment in terms of initial cost, and easy
operation (Namasivayam and Yamouna, At the end of this study, it was concluded
1992). Tao et al., (2010) found that that Saida port seawater autochthonous
immobilization of strain GY2B with rice bacteria had high potential to degrade
straw possesses a good application potential petroleum hydrocarbons and the present
in the treatment of wastewater and study provided an evidence to recommend
bioremediation of estuary and offshore that biostimulation of autochthonous
environment contaminated by phenanthrene, microorganisms of the oil contaminated
since the adsorption of phenanthrene onto water is a good strategy to bioremediation
the carrier promoting the degradation oil polluted seawater using the free and
efficiency of the strain immobilized on it. immobilized bacterial cells on natural
support as sponge and luffa.

Figure.12 Oil degradation by immobilized Bacillus cereus A as affected by using different

immobilizing agents through several cycles.

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 Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 1-18

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