Isolation and molecular identification of polyhydroxybutyrate (PHB) producing bacteria using waste cooking oil as carbon source

1. By
Nouf abdulaziz Almansour
Supervisor By
DR. Mona Othman Albureikan
ISOLATION AND MOLECULAR IDENTIFICATION OF
POLYHYDROXYBUTYRATE (PHB) PRODUCING BACTERIA
USING WASTE COOKING OIL AS A CARBON SOURCE

2. Outlines
Introduction
Aim of study
Materials and Methods
Results
Conclusion

3. Introduction

4. ■ Petroleum based plastics have become an
important part of our life (Sandström et
al., 2015; Leong et al., 2016).
■ Many activities in the modern life are
completely depending on petroleum
based plastic products like, in mobiles,
transport, clothes, and food packaging
(Jain and Tiwari, 2015; Le Guern, 2018).

5. • It become more economical to use than
other materials due to their properties:
• Lightweight.
• Strong.
• Durable.
• Resistance to degradation.
• Low cost.
Properties of petroleum
based plastics

6. ■ since the petroleum based plastic is
durable and non degradable
(Barnes et al., 2009).
■ Disposal of a large amount of
petroleum based plastic waste lead
to accumulated in the terrestrial and
marine environments and damaging
life’s (Thompson et al., 2009).
Environmental and Health impacts
to petroleum based plastics

7. ■ In the 1960s, the scientific reports showed
that a small piece of petroleum based
plastic (smaller than 5 mm) called
Microplastics which is a result of ultraviolet
sun light effect over time that brittle it into
smaller pieces and cause problems (Webb
et al., 2012; Rillig et al., 2017).

8. ■ A lot of animals ingest Microplastic with
their food and causes several problems
such as starving with full stomach,
choking, or suffering internal injuries (Cai
et al., 2017; Koelmans et al., 2017).

9. • Also, Burning of petroleum based
plastics are harmful to the
environment and health.
• It cause emissions of a high toxic
greenhouse gases like CO2, PCB that
leads to global warming and increase
human diseases (Verma et al., 2016).

10. v In Food Packaging, a toxic chemicals
such as BPA and PET can reach into
the food then to human causing
several health problems (Castle,
2007; Abdullahi, 2014).

11. Bio plastics
For this reasons, it is very important to use a biodegradable
plastics that are friendly to the environment (Zargoun et al., 2015).
v Is a family of a biodegradable
polymer.
v their properties are similar to
petroleum based plastics.
v Found as inclusion body in the
cytoplasme of the bacteria and
used as carbon and energy
sources.
Polyhydroxylalkanoate
(PHA)

12. Chemical structure of PHB
§ There are 155 of PHA monomers had been identified
(Nielsen et al., 2017).
§ Polyhydroxybutyrate (PHB) is the best characterized and
the most studied of PHA.

13. Carbon source of PHB production
§ The high cost of carbon source for PHB
production make the petroleum based
plastic more economical to use (Budde et
al., 2011).
§ Using materials waste can decrease the
environment problems and reduce the cost
of PHB production (Panchal, 2016).

14. § Plants oils are inexpensive carbon sources and higher productivity of PHB
compared with sugar due to the high number of carbon atoms in plants
oils (Jiang et al., 2016; Panchal, 2016).
§ Cooking oil waste found to be cheaper and produced a higher amount of
PHB than purified oils due to the high amount of free fatty acids (Jiang et
al., 2016).

15. ■ Some bacteria can secret extracellular enzyme called lipases to
degrade plants oils.
■ Catalyze the cleavage of carboxyl ester bonds of triacylglycerol
(TAG) into diacylglycerol, monoglycerol, glycerol, and free fatty
acids.
■ Then, the free fatty acids are transfer through the cell membrane
inside the bacterial cell and biodegraded by ß-oxidation process for
PHB production (Riedel, 2016; Jiang et al., 2016).

16. The aim of this study was to isolate the best PHB
producers that used cooking oil waste as a carbon source
Aim of Study

17. Materials and Methods

18. § Different soil samples were collected
from different gardens in Jeddah,
Kingdom of Saudi Arabia.
§ The soil samples were collected by a
hand shovel at 10 cm deep and kept it
in sterile plastic containers at 4ᵒC until
use.
1- Samples Collection

19. 2- Bacterial isolation from the
collected soil samples
A) Serial dilution method was used to isolate
the bacteria from soil samples.
B) Spreading and streaking plate’s method
were used to isolate the bacteria from the
diluted soil suspension by using Nutrient
Agar and modified 𝐂 𝟐M Agar until a pure
colony appears.

20. 3- Screening for PHB producers
BY Sudan Black B stain
A) Screening for PHB producers on solid
agar method:
• Nutrient Agar plates and modified 𝐂 𝟐M
Agar plates were used to culture the
bacterial colonies and incubated at
37℃ for 24 h.
• The Sudan Black B stain solution
spread over the plates and left it for 30
min.
• Then, the plates washed with 96 % of
ethanol to remove the excess stain.
• The PHB producer appears dark blue
colored.

21. B) Screening for PHB producers under
light microscope method:
• The bacterial colonies were spread on
a slide and heat fixed.
• Then, a few drops of Sudan Black B
solution added over the slide for 10
min.
• After that, the slide immersed in xylene
to completely decolorized.
• Then, the slide was stained with
safranine solution for 10 seconds,
washed with distilled water and
examined under the light microscope.
• The PHB producer showed a black
color which refers to the PHB granules
inside red cell.

22. 4- Growth of PHB
producers in liquid media
Pre culture:
The pre culture were prepared in 250 ml conical
flasks containing 50 ml of nutrient broth. Each
flask inoculated with the PHB producer bacteria
and incubated in a rotary shaker at 150 rpm
rate for 24 h at 37℃.
Growth of PHB producers in liquid media:
The 250 ml conical flasks contain 48 ml of modified
𝐂 𝟐M broth. Each conical flask was inoculated
with 2 ml of the pre cultures of the PHB
producer bacteria and incubated in a rotary
shaker at 150 rpm rate for 48 h at 37℃.

23. modified C2M broth media were used to
measure:
■ The bacterial growth was measured by
spectrophotometers at the optical density
520 nm.
■ The dry cell weight was measured by
centrifuge the C"M broth media and
discarded the supernatant. Then, the pellet
washed with distilled water and dried at
60℃.
5- Measurement of bacterial
growth and dry cell weight

24. 6- Extraction and assay of
PHB
The PHB was extracted by using the
hypochlorite method

25. ■ The PHB quantity were measured at the
absorbance 235 nm by UV_VIS
spectrophotometer.
■ The PHB percentage was calculated by
the following formula:
■ PHB % =
"#$ %&'()*)+ (-./01)
345 (./6)
×100

26. 7- Identification of the best PHB producer
■ Identification by Morphological, Physiological, biochemical
and Molecular characterization of the best PHB producer.

27. 8- Optimization of culture parameters
for PHB production
Effect of different pH values
on PHB production
Effect of different incubation
temperature on PHB production
Effect of different incubation
period on PHB production

28. Results

29. ■ Twenty two bacterial isolates were obtained from soil gardens in
different region in Jeddah, Kingdom of Saudi Arabia.
■ All bacterial isolates were screened by using Sudan Black B
stain.
■ The positive PHB producers founded to be (NM7, NM16, and
NM17).
Samples collection and Bacterial isolation
from the collected soil samples

30. positive PHB producers (NM7) stained by Sudan
Black B stain under light microscope.
Positive PHB producers
(NM7)
NA
C2M A

31. NA
Positive PHB producers
(NM16)
positive PHB producers (NM16) stained by Sudan Black B
stain under light microscope.
C2M A

32. Positive PHB producers
(NM17)
positive PHB producers (NM17) stained by Sudan
Black B stain under light microscope.
NA
C2M A

33. ■ Among the three bacterial isolates (NM7, NM16, NM17), NM17
produce the highest PHB %.
Bacterial
Isolates
Bacterial
growth
Dry cell
weight
(g/l)
PHB
quantity
(µg/ml)
PHB %
NM7 1.17 0.30 1.0249 34.16 %
NM16 1.18 0.31 1.0489 33.83 %
NM17 1.08 1.25 5.5075 44.06 %
Best PHB producer

34. Characters Results
Gram stain +
Acid fast -
Shape-Motion Bacilli,
motile
Color White
Colonies Circular
Colony edge Entire
Elevation Flat
Endospore +
Cyst -
The Morphological characterization of
the bacterial isolate NM17
The bacterial isolate NM17 stained with
Gram under light microscope

35. Characters Results Characters Results
Temperature
range
15-45 Hydrolysis of
esculin
-
Optimum pH 8-9 Hydrolysis of
Tween 80
-
NaCl tolerance 3-5 Hydrolysis of
starch
+
Catalase + Egg yolk
lecithinase
-
Urease + Hemolysis -
Methyl red + Cellulase +
Indole + Utilization of
casein
-
Voges-proskauer + Gelatin
liquefaction
-
Nitrate + H2S +
The physiological and biochemical
characterization of the bacterial isolates NM17

36. The sensitivity of some antibiotics of the bacterial
isolates NM17
Antibiotic Sensitivity
Chlorophenicol +
Tetracycline +
Ampicllin +
Penicillin G +

37. Molecular identification and
characterization of the bacterial isolate
NM17
■ Thermo Scientific Gene JET Genomic DNA
Purification Kit was used to extract the
DNA of the bacterial strain NM17.
■ The DNA fragment size was determined
on 1% TBE agarose gel electrophoresis.
■ Lane 1 is the DNA ladder range 500-
5000 bp, lane 2 is the DNA sample
bands of the bacterial strain NM17 which
was 1000 bp sequences.

38. ■ The 16S rRNA gene was amplified and
sequenced using two universal primers. The
forward was 27F (5′-AGAGTTTGATCMTGGCTCAG-
3′) and the reverse was 1492R (5′-
TACGGYTACCTTGTTACGACTT-3′).
■ PCR amplified of 16S rRNA gene were
determined by agarose gel electrophoresis.
■ Lane 1 is the PCR ladder at 500bp, lane 2 is
the PCR amplified 16S rRNA gene of the
bacterial strain NM17 founded to be 100 bp.

39. ■ The amplified 16S rRNA gene was send to Humanizing Genomics Macrogen
company at Korea and submitted to NCBI GEN BANK to identify the bacterial
isolate NM17 and find the most closely related strains.
■ The selected bacterial isolate (NM17) identified as Bacillus funiculus NM17.
Phylogenetic tree of the bacterial strain Bacillus funiculus NM17

40. Optimization of culture parameters for
PHB production

41. ■ It is clear that maximum growth
and PHB% were recorded at 37ᵒC .
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
10
20
30
40
50
60
70
80
90
100
20℃ 25℃ 30℃ 37℃ 40℃
GROWTH
%PHB
INCUBATION TEMPERATURE
% PHB Growth
incubation
temperature
Bacterial
growth
Dry cell weight
(g/L)
PHB quantity
(µg/ml)
PHB %
20℃ 0.65 0.32 0.4083 12.76 %
25℃ 0.73 0.82 1.4977 18.26 %
30℃ 0.87 0.36 0.7379 20.49 %
37℃ 1.07 1.25 5.5075 44.06 %
40℃ 0.49 0.36 0.5583 15.50 %
Effect of different incubation
temperature on PHB production

42. Effect of different incubation
period on PHB production
■ Maximum growth was after 2 days
while maximum PHB were after 3 days.
The lowest PHB % was recorded after 5
days of growth.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
10
20
30
40
50
60
70
80
90
100
day 1 day 2 day 3 day 4 day 5
GROWTH
%PHB
INCUBATION PERIOD
% PHB Growth
Incubation
period
Bacterial
growth
Dry cell
weight (g/L)
PHB quantity
(µg/ml)
PHB %
Day 1 1.01 0.97 1.5709 16.19 %
Day 2 1.07 1.25 5.5075 44.06 %
Day 3 0.44 0.32 1.4933 46.66 %
Day 4 0.39 0.17 0.4251 25.01 %
Day 5 0.34 0.13 0.2335 17.92 %

43. ■ It was found that maximum growth
and PHB % were recorded at pH 6.5.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
10
20
30
40
50
60
70
80
90
100
5.5 6 6.5 7 7.5 8 8.5
GROWTH
%PHB
PH VALUSE
% PHB Growth
pH values
Bacterial
growth
Dry cell
weight (g/L)
PHB quantity
(µg/ml)
PHB %
5.5 0.22 0.47 0.1939 4.12 %
6 0.49 0.45 0.8839 19.64 %
6.5 1.39 1.04 5.2113 50.10 %
7 1.07 1.25 5.5075 44.06 %
7.5 0.67 0.42 0.6621 15.76 %
8 0.56 0.32 0.3758 11.74 %
8.5 0.51 0.47 0.3082 6.55 %
Effect of different pH values
on PHB production

44. Bacterial Isolates pH Incubation
temperature
Incubation
period
Bacillus funiculus
NM17
6.5 37℃ 2 days
The optimal conditions of
PHB production

45. PHB was extracted and dried.
PHB EXTRACTION

46. Conclusion
■ Petroleum based plastics are non degradable polymers and causes many
problems to our environment and health since it is a wildly spread and
highly produced. For this reason, a biodegradable polymer similar to the
petroleum based plastic called PHB can be used and reduce these
problems.
■ Polyhydroxybutyrate (PHB) is macromolecules synthesized by bacteria.
They are inclusion bodies accumulated as reserve materials when the
bacteria grow under different stress conditions. Because of their fast
degradability under natural environmental conditions, PHB is selected as
alternatives for production of biodegradable plastics.

47. ■ Results of these studies confirmed that cheaply available oil can be
used for the production of PHB and reducing the cost of
biodegradable plastics, reducing environmental pollution problems
caused by the petroleum based plastics and solving disposal
problem of the oil wastes.
Conclusion

48. ‫ﺷ‬‫ﻛ‬‫ر‬‫و‬‫ﻋ‬‫ر‬‫ﻓ‬‫ﺎ‬‫ن‬
•‫ا‬‫ﻟ‬‫ﺣ‬‫ﻣ‬‫د‬‫ﷲ‬‫ر‬‫ب‬‫ا‬‫ﻟ‬‫ﻌ‬‫ﺎ‬‫ﻟ‬‫ﻣ‬‫ﯾ‬‫ن‬,‫ا‬‫ﻟ‬‫ﺣ‬‫ﻣ‬‫د‬0‫ﻋ‬‫ﻠ‬‫ﻰ‬‫ﻣ‬‫ﺎ‬‫أ‬‫ﻧ‬‫ﻌ‬‫م‬‫ﺑ‬‫ﮫ‬‫ﻋ‬‫ﻠ‬‫ﻲ‬‫ﻣ‬‫ن‬‫ﻓ‬‫ﺿ‬‫ﻠ‬‫ﮫ‬‫ا‬‫ﻟ‬‫ﺧ‬‫ﯾ‬‫ر‬‫ا‬‫ﻟ‬‫ﻛ‬‫ﺛ‬‫ﯾ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﻌ‬‫ﻠ‬‫م‬‫ا‬‫ﻟ‬‫و‬‫ﻓ‬‫ﯾ‬‫ر‬‫و‬‫أ‬‫ﻋ‬‫ﺎ‬‫ﻧ‬‫ﻧ‬‫ﻲ‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫إ‬‫ﻧ‬‫ﺟ‬‫ﺎ‬‫ز‬‫ھ‬‫ذ‬‫ا‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﺔ‬.‫ا‬‫ﺗ‬‫ﻘ‬‫د‬‫م‬‫ﺑ‬‫ﺧ‬‫ﺎ‬‫ﻟ‬‫ص‬
‫ا‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫ر‬‫ﻟ‬‫ﻛ‬‫ل‬‫ﻣ‬‫ن‬‫ﺳ‬‫ﺎ‬‫ھ‬‫م‬‫ﻓ‬‫ﻲ‬‫ا‬‫ﻧ‬‫ﺟ‬‫ﺎ‬‫ز‬‫ھ‬‫ذ‬‫ه‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﮫ‬‫ﺑ‬‫ر‬‫أ‬‫ي‬‫ا‬‫و‬‫ﺗ‬‫و‬‫ﺟ‬‫ﯾ‬‫ﮫ‬‫ا‬‫و‬‫د‬‫ﻋ‬‫م‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﻗ‬‫د‬‫م‬‫ﺧ‬‫ﺎ‬‫ﻟ‬‫ص‬‫ا‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫ر‬‫إ‬‫ﻟ‬‫ﻰ‬‫ا‬‫ﺑ‬‫ﻲ‬‫و‬‫ا‬‫ﻣ‬‫ﻲ‬‫ﻋ‬‫ل‬‫ﻣ‬‫ﺎ‬‫ﻗ‬‫د‬‫ﻣ‬‫و‬‫ه‬‫ﻣ‬‫ن‬‫د‬‫ﻋ‬‫م‬‫و‬‫ا‬‫ھ‬‫ﺗ‬‫ﻣ‬‫ﺎ‬‫م‬‫و‬‫ﺗ‬‫ﺣ‬‫ﻔ‬‫ﯾ‬‫ز‬‫ﻟ‬‫ﻠ‬‫ﻣ‬‫ﺛ‬‫ﺎ‬‫ﺑ‬‫ر‬‫ة‬‫و‬‫ا‬‫ﻻ‬‫ﺳ‬‫ﺗ‬‫ﻣ‬‫ر‬‫ا‬‫ر‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺧ‬‫ص‬‫ﺑ‬‫ﺎ‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫ﻣ‬‫ﺷ‬‫ر‬‫ﻓ‬‫ﺗ‬‫ﻲ‬‫ا‬‫ﻟ‬‫ﻐ‬‫ﺎ‬‫ﻟ‬‫ﯾ‬‫ﺔ‬‫د‬.‫ﻣ‬‫ﻧ‬‫ﻰ‬‫ﻋ‬‫ﺛ‬‫ﻣ‬‫ﺎ‬‫ن‬‫ا‬‫ﻟ‬‫ﺑ‬‫ر‬‫ﯾ‬‫ﻛ‬‫ﺎ‬‫ن‬‫ﻟ‬‫ﻛ‬‫ل‬‫ﻣ‬‫ﺎ‬‫ﺑ‬‫ذ‬‫ﻟ‬‫ﺗ‬‫ﯾ‬‫ﮫ‬‫ﻣ‬‫ن‬‫و‬‫ﻗ‬‫ت‬‫و‬‫ﺟ‬‫ﮭ‬‫د‬‫ﻓ‬‫ﻲ‬‫ﺳ‬‫ﺑ‬‫ﯾ‬‫ل‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫م‬‫ا‬‫ﻟ‬‫ﺗ‬‫و‬‫ﺟ‬‫ﯾ‬‫ﮭ‬‫ﺎ‬‫ت‬‫و‬‫ا‬‫ﻻ‬‫ر‬‫ﺷ‬‫ﺎ‬‫د‬‫ا‬‫ت‬‫ا‬‫ﻟ‬‫ﺗ‬‫ﻲ‬‫ﺳ‬‫ﺎ‬‫ھ‬‫ﻣ‬‫ت‬‫ﻓ‬‫ﻲ‬
‫ا‬‫ﻧ‬‫ﺟ‬‫ﺎ‬‫ز‬‫ھ‬‫ذ‬‫ه‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﺔ‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺗ‬‫ﻘ‬‫د‬‫م‬‫ﺑ‬‫ﺟ‬‫ز‬‫ﯾ‬‫ل‬‫ا‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫إ‬‫ﻟ‬‫ﻰ‬‫ﺻ‬‫ﺎ‬‫ﺣ‬‫ﺑ‬‫ﺔ‬‫ا‬‫ﻟ‬‫ﻘ‬‫ﻠ‬‫ب‬‫ا‬‫ﻟ‬‫ﺣ‬‫ﻧ‬‫و‬‫ن‬‫أ‬.‫د‬.‫ﻣ‬‫ﺎ‬‫ﺟ‬‫د‬‫ة‬‫ﻣ‬‫ﺣ‬‫ﻣ‬‫د‬‫ﻋ‬‫ﻠ‬‫ﻲ‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫ﻣ‬‫ﺎ‬‫ﻗ‬‫د‬‫ﻣ‬‫ﺗ‬‫ﯾ‬‫ﮫ‬‫ﻣ‬‫ن‬‫ﻋ‬‫ﻠ‬‫م‬‫ﻧ‬‫ﺎ‬‫ﻓ‬‫ﻊ‬,‫و‬‫ﻋ‬‫ط‬‫ﺎ‬‫ء‬‫ﻣ‬‫ﺗ‬‫ﻣ‬‫ﯾ‬‫ز‬,‫و‬‫إ‬‫ر‬‫ﺷ‬‫ﺎ‬‫د‬‫ﻓ‬‫ﻲ‬‫ا‬‫ﺗ‬‫ﻣ‬‫ﺎ‬‫م‬.‫ھ‬‫ذ‬‫ه‬
‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﺔ‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺗ‬‫ﻘ‬‫د‬‫م‬‫ﺑ‬‫ﺧ‬‫ﺎ‬‫ﻟ‬‫ص‬‫ا‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﻌ‬‫ر‬‫ﻓ‬‫ﺎ‬‫ن‬‫ﻟ‬‫ر‬‫ﺋ‬‫ﯾ‬‫ﺳ‬‫ﺔ‬‫ا‬‫ﻟ‬‫ﻘ‬‫ﺳ‬‫م‬‫د‬.‫ﻣ‬‫ﻧ‬‫ﻰ‬‫ا‬‫ﻟ‬‫ﺣ‬‫ر‬‫ﺑ‬‫ﻲ‬‫ﻟ‬‫ﻣ‬‫ﺳ‬‫ﺎ‬‫ﻋ‬‫د‬‫ﺗ‬‫ﻲ‬‫و‬‫ﺗ‬‫ﺳ‬‫ﮭ‬‫ﯾ‬‫ل‬‫أ‬‫ﻣ‬‫و‬‫ر‬‫ي‬‫ﻓ‬‫ﻲ‬‫ﻓ‬‫ﺗ‬‫ر‬‫ﺗ‬‫ﻲ‬‫ا‬‫ﻟ‬‫د‬‫ر‬‫ا‬‫ﺳ‬‫ﯾ‬‫ﮫ‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﻗ‬‫د‬‫م‬‫ﺷ‬‫ﻛ‬‫ر‬‫ي‬‫و‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫ر‬‫ي‬‫ا‬‫ﻟ‬‫ﻰ‬‫د‬.‫ﻧ‬‫ﺎ‬‫د‬‫ﯾ‬‫ﺔ‬‫ﻧ‬‫و‬‫ر‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫ﺣ‬‫ﺿ‬‫و‬‫ر‬‫ك‬‫و‬‫ﻣ‬‫ﺟ‬‫ﮭ‬‫و‬‫د‬‫ك‬‫و‬‫ﺗ‬‫ﺧ‬‫ﺻ‬‫ﯾ‬‫ص‬‫ﺟ‬‫ز‬‫ء‬‫ﻣ‬‫ن‬‫و‬‫ﻗ‬‫ﺗ‬‫ك‬‫ﻓ‬‫ﻲ‬‫ﺳ‬‫ﺑ‬‫ﯾ‬‫ل‬‫إ‬‫ﺗ‬‫ﻣ‬‫ﺎ‬‫م‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﺔ‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺷ‬‫ﻛ‬‫ر‬‫د‬.‫ﻧ‬‫ﺿ‬‫ﺎ‬‫ل‬‫ﻣ‬‫ﺣ‬‫ﻣ‬‫د‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫ﻣ‬‫ﺑ‬‫ﺎ‬‫د‬‫ر‬‫ﺗ‬‫ك‬‫ا‬‫ﻟ‬‫ﻛ‬‫ر‬‫ﯾ‬‫ﻣ‬‫ﺔ‬‫و‬‫ﻣ‬‫ﺛ‬‫ﺎ‬‫ﺑ‬‫ر‬‫ﺗ‬‫ك‬‫و‬‫ﻋ‬‫ط‬‫ﺎ‬‫ﺋ‬‫ك‬‫ﻓ‬‫ﻛ‬‫ﻠ‬‫ﻣ‬‫ﺎ‬‫ت‬‫ا‬‫ﻟ‬‫ﺛ‬‫ﻧ‬‫ﺎ‬‫ء‬‫ﻻ‬‫ﺗ‬‫و‬‫ﻓ‬‫ﯾ‬‫ك‬‫ﺣ‬‫ﻘ‬‫ك‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺷ‬‫ﻛ‬‫ر‬‫د‬.‫ﻣ‬‫ﺎ‬‫ﺟ‬‫د‬‫ة‬‫ﻗ‬‫ﻧ‬‫ش‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫و‬‫ﻗ‬‫ﺗ‬‫ك‬‫و‬‫ﻣ‬‫ﺟ‬‫ﮭ‬‫و‬‫د‬‫ك‬‫و‬‫ﺗ‬‫ﻌ‬‫ﺎ‬‫و‬‫ﻧ‬‫ك‬‫ﻓ‬‫ﻲ‬‫ﺗ‬‫ﯾ‬‫ﺳ‬‫ﯾ‬‫ر‬‫ﺗ‬‫ﺳ‬‫ﻠ‬‫ﯾ‬‫م‬‫ھ‬‫ذ‬‫ه‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﮫ‬.
•‫ﻛ‬‫ﻣ‬‫ﺎ‬‫ا‬‫ﺷ‬‫ﻛ‬‫ر‬‫ا‬‫ﺧ‬‫و‬‫ا‬‫ﻧ‬‫ﻲ‬‫و‬‫ا‬‫ﺧ‬‫و‬‫ا‬‫ﺗ‬‫ﻲ‬‫ﺳ‬‫ﻠ‬‫ط‬‫ﺎ‬‫ن‬‫و‬‫ﺳ‬‫ﺎ‬‫ر‬‫ا‬‫و‬‫ﻧ‬‫و‬‫ر‬‫ا‬‫و‬‫ﺧ‬‫ﺎ‬‫ﻟ‬‫ﺗ‬‫ﻲ‬‫ﻣ‬‫ﺷ‬‫ﺎ‬‫ﻋ‬‫ل‬‫و‬‫ﺻ‬‫د‬‫ﯾ‬‫ﻘ‬‫ﺗ‬‫ﻲ‬‫ا‬‫ﻓ‬‫ر‬‫ا‬‫ح‬‫ﺷ‬‫ﻛ‬‫ر‬‫ا‬‫ﻟ‬‫ﻛ‬‫م‬‫ﻋ‬‫ﻠ‬‫ﻰ‬‫ﻛ‬‫ل‬‫ﻣ‬‫ﺎ‬‫ﻗ‬‫د‬‫ﻣ‬‫ﺗ‬‫و‬‫ه‬‫ﻣ‬‫ن‬‫ﻣ‬‫ﺳ‬‫ﺎ‬‫ﻧ‬‫د‬‫ة‬‫و‬‫ﺗ‬‫ﺷ‬‫ﺟ‬‫ﯾ‬‫ﻊ‬‫ا‬‫ﺷ‬‫ﻛ‬‫ر‬‫ﻛ‬‫م‬
‫ﻣ‬‫ن‬‫ﻛ‬‫ل‬‫ﻗ‬‫ﻠ‬‫ﺑ‬‫ﻲ‬.
•‫و‬‫ﻟ‬‫ﻛ‬‫ل‬‫ﻣ‬‫ن‬‫ﻣ‬‫د‬‫ﻟ‬‫ﻲ‬‫ﯾ‬‫د‬‫ا‬‫ﻟ‬‫ﻌ‬‫و‬‫ن‬,‫أ‬‫و‬‫أ‬‫ﺳ‬‫د‬‫ى‬‫ﻟ‬‫ﻲ‬‫ﻣ‬‫ﻌ‬‫ر‬‫و‬‫ﻓ‬‫ﺎ‬‫أ‬‫و‬‫ﻗ‬‫د‬‫م‬‫ﻟ‬‫ﻲ‬‫ﻧ‬‫ﺻ‬‫ﯾ‬‫ﺣ‬‫ﺔ‬,‫أ‬‫و‬‫ﻛ‬‫ﺎ‬‫ﻧ‬‫ت‬‫ﻟ‬‫ﮫ‬‫ﻣ‬‫ﺳ‬‫ﺎ‬‫ھ‬‫ﻣ‬‫ﮫ‬‫ﺻ‬‫ﻐ‬‫ﯾ‬‫ر‬‫ة‬‫أ‬‫و‬‫ﻛ‬‫ﺑ‬‫ﯾ‬‫ر‬‫ة‬‫ﻓ‬‫ﻲ‬‫إ‬‫ﻧ‬‫ﺟ‬‫ﺎ‬‫ز‬‫ھ‬‫ذ‬‫ا‬‫ا‬‫ﻟ‬‫ر‬‫ﺳ‬‫ﺎ‬‫ﻟ‬‫ﺔ‬‫ﻓ‬‫ﻠ‬‫ﮫ‬‫ﻣ‬‫ﻧ‬‫ﻲ‬‫ﺧ‬‫ﺎ‬‫ﻟ‬‫ص‬
‫ا‬‫ﻟ‬‫ﺷ‬‫ﻛ‬‫ر‬‫و‬‫ا‬‫ﻟ‬‫ﺗ‬‫ﻘ‬‫د‬‫ﯾ‬‫ر‬.

49. !"#$%ً()*

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