Nesreen Ph.D. Presentation

A STUDY ON BACTERIAL CONTROL
OF BRACHIODONTES VARIABILIS THE
BIOFOULING CAUSATIVE IN SOME
PETROLEUM REFINERIES

Nesreen Abd-Elhameed Fatth-Allah
Supervisors:
 Late Prof. Dr. Erian George Kamel,
Prof. of Zoology , Zoology Dept., Women College for Arts, Science and
Education, Ain Shams Uinversity.
 Prof. Dr. Mohamed Fouad Abd-Elaziz Salama,
Prof. of Applied Organic Chemistry, Dept. of Processes Development ,
Petroleum Biotechnology Lab, Egyptian Petroleum Research Institute
(EPRI).
 Prof. Dr.Faika Ibrahim Kossa,
Prof. of Zoology , Zoology Dept., Women College for Arts, Science and
Education, Ain Shams Uinversity.
 Dr. Mohamed Ahmed Zaki
Researcher of Marine Toxins, National Institute of Oceanography and
Fisheries, Suez.

Fouling
 Fouling is a leading cause of diminished
efficiency and productivity in refineries.
 Fouling of a heat transfer equipment is
defined as the formation of deposits on heat
exchanger surfaces which impede the
transfer of heat and increase the resistance
to fluid flow. The accumulation of these
deposits causes thermal and hydrodynamic
performance of heat transfer equipment to
decline with time.

 The total fouling cost results in
 1. Need for additional costs for anti fouling equipment,
such as the installation of on-line cleaning devices,
 2.Extra fuel costs due to increase in fuel burning,
 3. Maintenance costs for removing fouling deposits,
and coasts for chemicals or other operating costs of
antifouling .
 4.Production losses during planned and unplanned shut-
down due to fouling .

Fouling Classification
1. Precipitation Fouling.
2. Particulate Fouling.
3. Chemical reaction Fouling.
4. Corrosion Fouling.
5. Biological Fouling.
6. Freezing Fouling.

A: Initially clean surface exposed to a turbulent flow of fluid containing
microorganisms and associated material.
B: Adsorption of organic material from the bulk fluid.

C: Flux and attachment of microbial cells to the surface from the bulk fluid.
D: Continued flux of microbial cells to the surface with simultaneous growth
process occurring.

E: Continued flux of microbial cells to the surface and simultaneous growth
opposed by attachment of biomass due to fluid shear.
F: Summary of biofouling process: organic adsorption ; particle transport ;
attachment ; growth .

COMMON BIOFOULING
BIVALVES
 Brachidontes variabilis
 Brachidontes striatulus
 Corbicula fluminea
 Modiolus auriculatus
 Modiolus barbatus
 Mytilus edulis
 Mytilus galloprovincialis
 Perna viridis

PREDOMINANT BIOFOULING
MUSSEL IN PETROLEUM
REFINERIES AT SUEZ
Brachidontes variabilis

Brachidontes variabilis
 It is considered to be the principal macro-
biofoulant in petroleum refineries at Suez.
 These mussels are pest organisms because they
not only attach to one another, but also to man-
made objects, including water intakes, cooling
systems, heat exchangers and power stations in
different companies that deal with water.

3◦ Paints and
Coatings
2◦ Physical
4◦ Chemical
1◦ Mechanical
Fouling Control Methods

1. Mechanical Control
 Screens.
 Strainers.
 Filters.

2. Physical Control
 Thermal treatment.
 Salinity.
 pH.

3. Paints and Coatings
 TBTO Based Coatings.
 TBTF Based Coatings.
 Zinc Based Coatings.

4. Chemical Control
 Metals.
 Oxidizing materials.
 Non- Oxidizing materials.

Oxidizing Materials
 Chlorine ( gas and sodium hypochlorite).
 Chloramines.
 Bromine.
 Chlorine Dioxide.
 Hydrogen Peroxide.
 Ozone.
 Potassium Permenganate.

 It was aimed to control the mussel Brachidontes variabilis, the
causative agent of biofouling using different bacterial strains.
 Optimization of the different cultural conditions for growth of the
strain having the higher molluscicidal capacity (including pH,
temperature, nutrients, salinity, mutation ….etc.) was an essential
target.
 Extraction of the crude toxins out of that bacterium was also another
target to evaluate the molluscicidal potency against the mussel
Brachidontes variabilis.
 However, it was essential to investigate the different histological and
histochemical alterations which might affect the organs of the mussel
Brachidontes variabilis, a sub-lethal dose of the highly potent
bacterium is applied.
 It was substantial when regarding the ecological considerations, to
assay the bacterial toxicity of the tested species having the higher
mortality against the non-target sea organisms.

Screening of Efficacy of some
Bacteria against Brachidontes
variabilis

Fig. 1: Growth curve of Bacillusalvei using media in distilled and sea
water
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0 20 40 60 80 100 120
Time / hours
Logcellnumber
Distilled water
sea water

Fig. 2: Growth curve of Bacillusbrevisusing media in distilled and sea
water
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0 20 40 60 80 100 120
Time / hours
Logcellnumber
Distilled water
Sea water

Fig. 3: Growth curve of Bacillus thuringiensis using media in distilled
and sea water
10.0
10.5
11.0
11.5
12.0
12.5
13.0
13.5
14.0
14.5
15.0
15.5
16.0
0 20 40 60 80 100 120
Time / hours
Logcellnumber
Distilled water
Sea water

Fig. 4: Growth curve of Bacillus subtilis using media in distilled and
sea water
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0 20 40 60 80 100 120
Time / hours
Logcellnumber
Distilled water
Sea water

Fig. 5: Growth curve of Bacillus megatarium using media in distilled
and sea water
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0 20 40 60 80 100 120
Time / hours
Logcellnumber
Distilled water
Sea water

Optimization of Different Parameters Controlling
Cultural Conditions of Bacillus thuringiensis on
Mortality of Brachidontes variabilis
1. Inoculum size
2. pH value
3. Temperature
4. Different carbon sources
5. Different nitrogen sources
6. Salinity
7. -Irradiation

Table 1: Effect of inoculum size of Bacillus thuringiensis on mortality of Brachidontes
variabilis
Mortality
%
Number of killed musselsInoculum
Size
(ml) Time / hrs
8642
tctctctc
100000001.005.0
3000002.001.005.5
4000002.002.006.0
4000002.002.006.5
601.01.01.01.02.002.007.0
701.001.003.002.007.5
701.001.003.002.008.0
801.002.003.002.008.5
901.002.003.003.009.0
1001.002.003.003.009.5
c: control samples (10)
t: treated samples (10)

Table 2: Effect of pH values on Bacillus thuringiensis affecting mortality of
Mortality
%
Number of killed mussels
pH
value
Time / hrs
8642
tctctctc
101.000000006.50
101.000000006.75
101.000000007.00
101.000000007.25
303.000000007.50
401.003.0000007.75
40002.002.01.0008.0
802.002.002.002.008.25
1002.01.02.003.003.008.50

Table 3: Effect of temperature on Bacillus thuringiensis affecting mortality of Brachidontes
variabilis
Mortality
%
Temp.
oC
Time / hrs
8642
tctctctc
00000000020
404.0000000025
802.002.002.002.0030
10001.00010.000035
10000000010.0040
10000000010.0045

Table 4: Effect of carbon source on Bacillus thuringiensis affecting mortality
of Brachidontes variabilis
Mortality
%
Carbon
Source
(5g/lit.)
Time / hrs
8642
tctctctc
100002.004.004.00Glycerol
100002.00008.00Glucose
100006.002.002.00
Water sol.
starch
100004.004.002.00
Malt extract
100006.0004.00
Sun flower
oil
10000006.004.00Molasses

Table 5: Effect of concentration of the nutrient molasses on Bacillus thuringiensis affecting
mortality of Brachidontes variabilis
Mortality
%
Concentration
g/lit.
Time / hrs
8642
tctctctc
1006.004.000000
2.0
100006.004.0000
4.0
10000006.004.00
6.0
10000005.005.00
8.0
10000004.006.0010.0
10000000010.00
12.0

Table 6: Effect of different nitrogen sources on Bacillus thuringiensis affecting mortality of
Nitrogen Source (peptone base)Nitrogen Source (beef extract base)
Mortality %, 2hrsNitrogen nutrientMortality %, 2hrsNitrogen nutrient
30Ammonium chloride30Ammonium chloride
100Amm.dihydrogen orthophosphate100Amm.dihydrogen orthophosphate
20Amm. Oxalate20Amm. Oxalate
40Amm. Carbonate40Amm. Carbonate
40Corn steep liquor40Corn steep liquor
60Glutamic acid60Glutamic acid

Table 7: Effect of salinity on Bacillus thuringiensis affecting mortality of Brachidontes
variabilis
Mortality
%
Number of killed mussels, 2 hrs
Salinity
‰
tc
10010.0020
10010.0025
404.0030
202.0035
00040
10010.0045
10010.0050
707.0055
505.0060

Table 8: Effect of γ-irradiation on Bacillus thuringiensis affecting mortality of
Mortality % , 2hrs
γ-irradiation
(Gy)
tc
10.000.5
000.2
0010.0
0050.0
0070.0

Effect of Bacillus
thuringiensis toxins against
Brachidontes variablilis

Table 9: Effectiveness of Bacillus thuringiensis crude toxins on mortality of Brachidontes
variabilis
Mortality
%
Dose
(ppm)
Time / hrs
2418128642
tctctctctctctc
10000002.002.002.002.002.002
10000001.001.003.003.002.005
1000000002.002,003.003.0010
100000000002.004.004.0015
10000000000004.006.0020
10000000000004.006.0025

Estimation of total proteins and
nitrogen percentage in crude toxins
of Bacillus thuringiensis

Histological Alterations in
Infected Brachidontes
variabilis

Histological alterations in gills

Histological alterations in digestive
gland

Histological alterations in ovary

Histochemical Analysis of
Infected Mussels

Table 10: Histochemical analysis of infected mussels
TreatedControlBiochemcial Composition
25.512.75Total protein (µg /µl)
2.485.12Total carbohydrates (mg/dl)
114.793.2Total lipid (mg/dl)
0.05570.1125Phospholpid phosphorus (mg/dl)

Bioassay of Bacillus thuringiensis
Against Non-Target Sea
Organisms
 Fish larvae
 Amphipode larvae
 Isopode larvae
≥16.7 %
15% mortality
≥16.7 %
20% mortality
2hrs
72hrs

Nesreen Ph.D. Presentation

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