MICROBIAL BIODEGRADATION OF

CRUDE PETROLEUM OIL AS

AFFECTED BY SALINITY
Lobna A. Moussaa; Ahmed Z. Abdel Azeizb*and
Journal Sameh E. Hassanienc
a
Soils, Water and Environment Research Institute,
Agriculture Research Center (ARC), Giza, Egypt.
J. Biol. Chem. b
College of Biotechnology, Misr University for Science and
Environ. Sci., 2017,
Technology (MUST), 6th October City, Egypt.
Vol. 12(1): 545-558 c
Bioinformatics & Computer Networks Dept., Agricultural
www.acepsag.org
Genetic Engineering Research Institute (AGERI), Agriculture
Research Center (ARC), Giza, Egypt

ABSTRACT
The petroleum oil spills during oil extraction processes causes
considerable environmental pollution. The aim of the present study
was isolation and characterization of halo-tolerant petroleum oil-
biodegrading bacteria to be used for bioremediation of petroleum oil
contaminated non-saline and saline soil and seawater. Nineteen
bacterial isolates were isolated from petrol contaminated soil on
Mineral Salt Medium (MSM) supplemented with crude petroleum oil
as a sole source of carbon. The total hydrocarbons biodegradation
(THB) activity was determined by 2,6-dichlorophenol indophenol
(2,6-DCPIP) method. The most active three isolates were identified by
16S-rRNA gene sequencing as Bacillus axarquiensis, Bacillus
thuringiensis and Bacillus cereus. The aromatic petroleum compounds
were completely degraded by the consortium of B. thuringiensis and
B. cereus after only 14 days of incubation under non-saline MSM
condition as appeared from the GC/MS analysis. Presence of
dioxygenases genes was detected in-silico in the three bacterial strains
as well as activity determination. B. thuringiensis and B. cereus can be
effectively used individually or co-culture for petroleum oil
biodegradation in the contaminated environment including sea-water
and salty soil.
Key words: B.axarquiensis,B.cereus,B. thuringiensis, bioremediation,
dioxygenases, petroleum oil.
546 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL

INTRODUCTION
During oil production and exploration operations the crude
petroleum oil spills are inevitable. The biodegradation of
hydrocarbons by microorganisms may represent one of the
remediation mechanisms, by which, these pollutants can be eliminated
from the contaminated field sites. The major advantages of
biodegradation methods compared with the other alternative
technologies, such as incineration or chemical extraction, are lower
cost, greater safety, and less environmental disturbance (Farahat and
El-Gendy, 2008). The biodegradation of petroleum hydrocarbons in
salty soil was a major aim of several studies (Prasanna, et al., 2006;
Dariush, et al., 2009 and Qin, et al., 2012).
The general degradation pathway for an alkane involves
sequential formation of an alcohol, an aldehyde and a fatty acid,
which in turn, metabolised by the β-oxidation pathway. The general
pathway for aromatic hydrocarbons biodegradation involves cis-
hydroxylation of the ring structure forming a diol (e.g. catechol) using
dioxygenase. The ring is oxidatively cleaved by dioxygenases,
forming a dicarboxylic acid (e.g. muconic acid) (Hamme, et al.,
2003).
The aim of this study was to isolate halo-tolerant petroleum oil
biodegrading bacteria from petroleum oil polluted soil to be used for
bioremediation of petroleum oil contaminated salty soil and seawater.

MATERIALS AND METHODS

Isolation of petrol biodegrading bacteria:
Soil samples were collected from Mustorod district beside
petroleum companies group, EL-Kalyobia Governorate, Cairo, Egypt.
The petroleum oil-biodegrading bacteria were isolated by pouring
technique in Minimal Salt Medium (MSM) supplemented with 10% of
crude petroleum oil. The MSM composed of (g /l-1): NH4NO3, 3.0;
K2HPO4, 1.5; KH2PO4, 0.5; MgSO4.7H2O, 0.2g; NaCl, 0.5; FeSO4,
0.02g; CaCl2, 0.05; pH 7 (Chao, et al., 2006).
Crude petroleum oil source:
The petroleum oil was obtained from Alexandria Petrol
Company, Egypt. It has the following properties and composition:
Density: 0.82g l-1, Viscosity at 100ºF: 34 Sec., Pour point: -3 ºC,
 J. Biol. Chem. Environ. Sci., 2017, 12(1), 545-558 547

Vapor Pressure Reid: 0.85 K g cm-2. Composition (% w/w): Sulfur:

0.84, salt: 0.0016, water: zero, sediments: 0.02, asphaltenes: 1.42 and
ash: 0.04.
Preparation of seed inoculum:
Fifty ml of nutrient broth medium was sterilized and inoculated
with a loop of each bacterial strain, separately. The flasks were
incubated for 1-day at 28ºC on rotary shaker at 150 rpm. The cells
were then harvested by centrifugation at 4000 rpm for 10 min at 4 ºC.
The bacterial pellet was re-suspended in 10 ml of sterilized saline
(0.85%) to give absorbance of 0.2 at wavelength 600 nm. One ml of
this bacterial suspension is used for inoculation of all flasks in this
investigation.
Biodegradation efficiency test:
The hydrocarbon degradation efficiency by the isolated bacteria
was tested by the reduction of 2, 6-dichlorophenol indophenol
(DCPIP) (Hanson, et al., 1993). One ml of the seed inoculum was
used separately to inoculate 50-ml of sterile Bushnell-Hass (BH)
medium (Difco, 1984). This medium composed of (g L-1): MgSO4:
0.2, CaCl2: 0.02, KH2PO4: 1.0, KHPO4: 1.0, NH4 NO3: 1.0, FeCl3:
0.05, DCPIP: 50µg and 5 mL of crude petroleum oil. A non-
inoculated medium was served as control. The flasks were kept under
agitation (150 rpm) at 28.0±1.0 °C. (Mariano, et al., 2007). The color
of the medium was observed, and evaluated as positive for microbial
hydrocarbon degrading ability if colorless (degraded) and negative for
microbial hydrocarbon-degrading ability if blue (not degraded). The
optical density (O.D.) of all tubes was measured at 600 nm against
control tube without the dye. Three replicates were made from each
test.
Bacteria identification:
The best three bacterial isolates were identified by the 16S
rRNA sequencing method (Baker, et al., 2003). Bacterial DNA was
isolated and recovered from a single colony using the Lego DNA
Isolation kit (Top-Bio, Czech Republic) according to the
manufacturer’s instruction (Sauer, et al., 2005). The used primers are
(5'- AGA GTT TGA TCC TGG CTC AG -3') as a forward primer, and
(5'- GGT TAC CTT GTT ACG ACT T -3') as a reverse one for PCR
reactions. The PCR products were purified using Gene Jet Gel
548 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL

Extraction Kit (Fermentas, k0691). Sequencing of the PCR products

using the chain termination method, was performed with the ABI
PRISM Big Dye terminator kit (PE Applied Biosystems, USA) in
conjunction with ABI PRISM (310 Genetic Analyzer). The nucleotide
sequences were compared with sequence database using the BLASTN
algorithms (Altschul, et al., 1998).
Biodegradation by individual and consortium bacteria:
In 250-ml conical flask, 100-ml portions of MSM supplemented
with 10% petroleum oil were added. Another set of MSM were
prepared by the same manner with addition of 6% NaCl. The pH was
adjusted to 6. After sterilization, the flasks were inoculated with one
ml of each bacterial suspension (from the seed inoculum) individually,
0.5 ml of two bacterial suspensions or 330 ul of each of the three
bacterial suspensions. The cultures were then incubated for 14 days at
28ºC on rotary shaker at 150 rpm. Three replicates were made from
each test.
Petroleum oil fractionation:
The saturate and aromatic fractions were separated from the
petroleum oil samples before and after biodegradation in all
treatments by using silica gel columns (Anupama and Padma, 2009).
The residues weight of either saturate and aromatics fractions were
determined after solvent evaporation.
GC/MS analysis:
The GC/MS analysis was performed using Agilent 5890 GC,
equipped with 5971 Mass detector. The separation column was HP-
5MS, 0.25 mm X 30m X 0.25µm. Helium was used as a mobile phase
at 1 ml min-1 flow rate. Oven temperature program for saturate
analysis: initial temperature 70ºC, for 2 min, 6ºC/min. rising up to
280ºC for 7 min. The same oven temperature program was used for
separation of aromatic compounds, except the initial temperature was
100ºC. Percentages of each compound were calculated from the peak
areas.
Determination of dioxygenases activity:
The activity of catechol 1,2-dioxygenase was determined
spectrophotometrically at 260 nm by quantifying the formation of
cis,cis-muconic acid (CCMA) (εCCMA =16 800 M–1 cm–1) at 40°C.
Catechol 2,3-dioxygenase activity was determined by quantitating the
 J. Biol. Chem. Environ. Sci., 2017, 12(1), 545-558 549

formation of 2-hydroxymuconic semialdehyde (2-HMS) at 375 nm

(ε2-HMS = 36 000 M–1 cm–1) (Hegeman, 1966). A control flask was
prepared by adding 10% of crude petroleum oil in 100ml MSM
without inoculation. Three replicates were made from each test. One
unit of each enzyme was expressed as the enzyme amount that
produces 1µM of the product/min./ml. The results were statistically
analyzed by one-way Analysis of Variance (ANOVA) and
comparisons based on the Least Significant Differences (LSD) at 5%,
were performed using the SAS system.

RESULTS AND DISCUSSION

1. Isolation of petrol biodegrading bacteria:
Nineteen bacterial isolates able to degrade the petroleum oil
were isolated. The most active three isolates (named P1, P3 and P5)
that showed the highest DCPIP color reduction were selected for
identification and studying their individual and consortium biodegradation
activities under saline and non-saline conditions and determination of
dioxygeanse enzymes.
2. Bacteria identification:
The three bacterial isolates were identified after BLAST analysis
(http://blast.ncbi.nlm.nih.gov/Blast.cgi)asB.axarquiensis,
B. thuringiensis and B. cereus.
3. Biodegradation of saturate and aromatic compounds by using
individual and bacterial consortium under not-saline and salinity
conditions:
The GC/MS analysis results for the saturate fractions showed presence of
22 aliphatic compounds in both control and all treatments with different
percentages. The total saturate residue in the non-saline MS ranged from 1.21 to
2.30 g/100 ml, as compared with 2.17 to 5.21 g/100ml in the saline MSM, in all
of the tested bacteria (Table 1). This reflects the higher biodegradation
efficiency in the non-saline conditions. The increase in total saturate compounds
in the some bacterial combination treatments such as combined culture of B.
axarquiensis and B. cereus (5.21 g/100 ml culture) than the control (1.88
g/100ml) (Table 1) may be due to that the aliphatic compounds can be obtained
from the degradation of the higher molecular weight one, as well as the
degradation of the alkyl-aromatic and poly-aromatic compounds. The low
degradation efficiency of saturate compounds under salinity conditions may be
due to inhibition of alkane mono-oxygenase enzyme that catalyzes conversion of
alkane to alcohol, which is the initial reaction in the biodegradation of alkanes.
550 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL
 J. Biol. Chem. Environ. Sci., 2017, 12(1), 545-558 551

The GC/MS analysis for the aromatic fractions showed presence

of 18 alkyl-benzene derivatives (Fig. 1). Table (2) illustrates the
remaining amount (g/100 ml medium) of each aromatic compound. In
the non-saline MSM the maximum degradation was obtained by the
consortium of B. thuringiensis and B. cereus (T6), which was able to
degrade all aromatic compounds (degradation percentage 100%),
although each bacterium alone was not able to degrade any of them
completely under the same conditions. This result strongly
recommended that there is integration between the enzymatic systems
for both of these bacteria that are working in serial enabling the
complete degradation of all aromatic compounds by this consortium.
Furthermore, the degradation by the consortium of the three bacteria
in the non-saline MSM showed effective degradation for all aromatic
compounds, where the degradation percentage was 74.67% (Table 2).
Under the high osmotic pressure environment, the microbial
metabolism is oriented to provide osmoprotection. The intracellular
concentrations of these solutes rise as the environmental salinity rises
to confer protection from osmotic stress (Lai and Gunsalus, 1992).
Some of these compounds may be working as bio-surfactants and
enhance the petrol solubility, which will be reflected on the
degradation activity. For this reason, the salinity may enhanced the
activity of all individual bacteria especially, B. thuringiensis and B.
cereus; where the maximum degradation of total aromatic compounds
in saline MSM was obtained from B. thuringiensis (87.9%) followed
by B. cereus (82%), compared with only 41 % and 67.6% in the non-
saline MSM for each bacterium, respectively (Fig. 2). However,
salinity appeared to break-down the integration between B. thuringiensis
and B. cereus consortium; where it showed a very low degradation
activity (only 2%) in the saline MSM.
552 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL
 J. Biol. Chem. Environ. Sci., 2017, 12(1), 545-558 553

4. in-silico detection of dioxygenase:

Since the biodegradation of aromatic compounds got much
attention than aliphatic compounds due to its degradation resistibility.
Most of the aromatic compounds, including polyaromatics, are known
to be metabolized into a common intermediate, catechol, which is then
oxidized through the two ring-cleavage pathways (ortho and meta
cleavage pathways) catalyzed by catechol 1,2-dioxygenase and
catechol 2,3-dioxygenase, respectively. Therefore, it has been
proposed that catechol dioxygenase genes are good markers for the
detection of a whole range of aromatic compound degrading bacteria
(Sei, et al., 1999). Therefore, it was very important to confirm
presence of dioxygenases in the isolated three bacteria in-silico;
especially, they showed valuable degradation activities of the aromatic
compounds. Therefore, we screened the published genome sequences
of the identified isolates B. axarquiensis, B. thuringiensis and B.
cereus for presence of a dioxygenase enzyme.
B. axarquiensis is an organism has no data on Genomes database
(http://www.ncbi.nlm.nih.gov/genome) yet. So, we used its lineage to
screen exist published genome data for the presence of dioxygenases
activity. One of Bacillus subtilis group (B. subtilis taxonomy ID:
653685) genome accession no. AP012495.1. For B. thuringiensis and
B. cereus (taxonomy ID: 1428, 1396 and genome acc. No.
AE017355.1 and AE016877.1, respectively) published genome data
were used for screening their ability for dioxygenases enzymatic
554 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL

activities. Dioxygenases (EC: 1.13.11.-) have two CATH

(http://www.cathdb.info/) Superfamilies: (1) CATH Superfamily
2.60.130.10 (Aromatic compound dioxygenase), and (2) CATH
Superfamily 3.10.180.10 (2,3-Dihydroxybiphenyl 1,2-Dioxygenase,
domain 1).
For CATH Superfamily 2.60.130.10 (Aromatic compound
dioxygenase) there are two representative Functional Families
(Domains) involved in it, which are 3pcnO00 and 1dmhB00. For
CATH Superfamily 3.10.180.10 (2,3-Dihydroxybiphenyl 1,2-
Dioxygenase, domain 1), nine representative Functional Families
(Domains) involved in it, which are, 1sqdA02, 1mpyB02, 1sp8C01,
3hq0A01, 1kw9B02, 1kw8B01, 1cjxC01, 3hq0A01 and 3ey8A01.
Screening the genome sequences of the identified accession numbers
for represented functional domains indicated presence of each single
domain in every identified genome. Therefore, the dioxygenases were
determined in all of these strains to confirm this result.
5. Production of dioxygenases as affected by salinity:
As shown in Table (3), there was a significant difference in
catechol 1,2-dioxygenase (C1,2D) produced by all bacterial strains in
all treatments as compared with the control. There was no significant
difference in the C 1,2D production by each of three individual
bacteria in the non-saline MSM, where the production was ranged
from 0.060 to 0.068 U/ml. The same result was also noticed in the
saline MSM, where the production was ranged from 0.040 to 0.049
U/ml by each of the three individual bacteria. The C1,2D production
by the consortium of the three bacteria was also significantly higher in
the non-saline MSM (0.081U/ml) than the saline MSM (0.045U/ml).
From this experiment, it was clear that the salinity inhibited the
catechol 1,2-dioxygenase production by each of B. axarquiensis, B.
thuringiensis and the consortium of B. thuringiensis and B. cereus as
well as the consortium of the three bacteria. The catechol 2,3-
dioxygenase production was very small in all treatments, where it was
ranged from 0 to 0.0032 U/ml. The high degradation activity of
aromatic compounds in the saline-MSM by the thuringiensis and B.
cereus consortium, although the low dioxygenase activity, may be due
to production of biosurfactants under saline condition, that enhance
the aromatic compounds biodegradability.
 J. Biol. Chem. Environ. Sci., 2017, 12(1), 545-558 555

In similar previous investigations, two strains of Bacillus showed

a degradation efficiency of 80-90% of crude oil (5g/l) within 5 days
(Ijah and Antai, 2003). They reported that Bacillus
sp. being the predominant isolates of all the crude oil utilizing
bacteria. It was postulated that Bacillus species are more tolerant to
high level of hydrocarbons in soil due to their resistant endospore. B.
cereus has been studied for its biodegradation activity of alkanes. B.
cereus ACE4 degraded almost all the n-alkanes (C10–C20) and many
of the branched alkanes found in diesel oil (Rajasekar, et al., 2007).
Salinity has been regarded as a very important factor that determines
the survival of bacterial inoculum seeded to soils (Kastner, et al.,
1998 and Obayori, et al., 2008). Therefore, several research studies
had been conducted to isolate halo-tolerant bacteria for degradation of
aromatic compounds. Effect of different NaCl concentrations (0%–
5%) on poly aromatic hydrocarbons reduction from the heavy crude
oil-contaminated soil was studied (Dariush, et al., 2009); the highest
total crude oil degradation was obtained at 0% NaCl concentration and
decreased as the NaCl concentration increases. (Fig. 2).
556 MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL

Table (3): Production of dioxygenases (U/ ml medium) by B.

axarquiensis, B. thuringiensis and B. cereus in MSM supplemented
with 10% of crude petroleum oil as affected by salinity.

Conclusion: B. axarquiensis, B. thuringiensis and B. cereus

isolates able to degrade the aromatic fr action of the crude petroleum
oil under both non-saline and saline conditions. The consortium of B.
thuringiensis and B. cereus was able to completely degrade the
volatile aromatic compounds after two weeks of incubation in non-
salty MSM. Therefore, it is recommend use of B. thuringiensis and B.
cereus in bioremediation of petroleum oil contaminated environments
including the salty soil and sea water.

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‫‪558‬‬ ‫‪MICROBIAL BIODEGRADATION OF CRUDE PETROLEUM OIL‬‬

‫التكسير الميكروبي لسيت البترول الخام تحت تأثير األمالح‬

‫]‪[3‬‬
‫لبنى عبد العسيس أحمد موسى ]‪ [1‬و أحمد زين عبد العسيس]‪ [2‬و سامح حسنين‬

‫]‪ [1‬يعٓذ بذٕد األساضي ٔ انًيبِ ٔ انبيئت يشكض انبذٕد انضساعيت ببنجيضة‪ -‬يصش‬

‫]‪ [2‬كهيت انخكُٕنٕجيب انذيٕيت جبيعت يصش نهعهٕو ٔ انخكُٕنٕجيب ببنسبدط يٍ اكخٕبش‪ -‬يصش‬

‫]‪ [3‬يعٓذ بذٕد انُٓذست انٕساريت يشكض انبذٕد انضساعيت ببنجيضة‪ -‬يصش‬

‫حعخبش انبقع انًُسكبت يٍ انبخشٔل ارُبء عًهيت اسخخشاجّ يٍ يهٕربث انبيئت‪ .‬انٓذف يٍ ْذزِ‬
‫انذساست عضل ٔ حعشيف انبكخشيب انخي نٓب انقذسة عهى حكسيش انًٕاد انبخشٔنيت في انخشبت انًبنذذت‬
‫ٔ غيش انًبنذت ٔ يبء انبذذش‪ .‬حذى عذضل حسذعت عزذش عضنذت يذٍ انخشبذت انًهٕرذت بضيذج انبخذشٔل ٔ‬
‫حًُيخٓب عهى بيئت االيالح انًعذَيت ٔ انًضٔدة بضيج انبخشٔل انخذبو كًصذذس ٔديذذ نهكشبذٌٕ‪ .‬حذى‬
‫‪2,6-dichlorophenol‬‬ ‫حقذذذذيش كةذذذبءة حكسذذذيش انًذذذٕاد انٓيذسٔكشبَٕيذذذت ببسذذذخخذاو شيقذذذت‬
‫)‪ ٔ indophenol (2,6-DCPIP‬حذذى حعشيذذف اَز ذ رالرذذت عذذضالث فذذي انخكسذذيش ببسذذخخذاو‬
‫شيقذت حخذببع جذيٍ ‪ٔ 16S-rRNA‬كذبَٕا كذبيحي‪Bacillus axarquiensis, Bacillus :‬‬
‫‪ . Bacillus cereusٔthuringiensis‬ديذذذذ ٔجذذذذ بٌ يشكبذذذبث انبخذذذشٔل انع شيذذذت‬
‫(ايسٔيبحيت) حى حكسيشْب ببنكبيم بإسخخذاو انخهقذيخ انًزذخشب بذيٍ ‪B. ٔ B. thuringiensis‬‬
‫‪ cereus‬بعذ اسبعت عزش يٕيب يٍ انخذضيٍ حذج ظشٔف انبيئت انغيش يبنذذت ٔ حذى ايذبط رنذ‬
‫ببسذذخخذاو جٓذذبص ‪ ٔ .GC/MS‬اظٓذذشث انُخذذبجو ٔجذذٕد َزذذب اَذذضيى ‪ dioxygenases‬فذذي ‪3‬‬
‫عضالث ٔ حى حأكيذ ٔجذٕدد انَذضيى ‪ ٔ .in-silico‬بُذبءا عهذى رنذ فإَذّ يًكذٍ اسذخخذاو كذال يذٍ‬
‫انسذذالنخيٍ ‪ B. cereus ٔ B. thuringiensis‬يُةذذشديٍ أ يهي ذذب بيًُٓذذب فذذي حكسذذيش انًذذٕاد‬
‫انبخشٔنيت بةبعهيت في انبيئت انًهٕرت ٔ انخي حخضًٍ انخشبت انًخأرشة ببأليالح ٔ يبء انبذش‪.‬‬