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.

Fouling Mechanism

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


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.

Metals Copper

Zinc

Oxidizing Materials

Chlorine ( gas and sodium hypochlorite).

Chloramines.

Bromine.

Chlorine Dioxide.

Hydrogen Peroxide.

Ozone.

Potassium Permenganate.

Aim of the work

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 Bacillus alvei 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

Lo

g c

ell n

um

ber

Distilled water

sea water

Fig. 2: Growth curve of Bacillus brevis using media in distilled and sea

water

0.0

2.04.0

6.08.0

10.0

12.014.0

16.0

0 20 40 60 80 100 120

Time / hours

Lo

g c

ell n

um

ber

Distilled water

Sea water

Fig. 3: Growth curve of Bacillus thuringiensis using media in distilled

and sea water

10.010.511.0

11.512.012.513.0

13.514.014.515.0

15.516.0

0 20 40 60 80 100 120

Time / hours

Lo

g c

ell n

um

ber

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

Lo

g c

ell n

um

be

r

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

Lo

g c

ell n

um

be

r

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 mussels Inoculum Size (ml) Time / hrs

8 6 4 2

t c t c t c t c

10 0 0 0 0 0 0 1.0 0 5.0

30 0 0 0 0 2.0 0 1.0 0 5.5

40 0 0 0 0 2.0 0 2.0 0 6.0

40 0 0 0 0 2.0 0 2.0 0 6.5

60 1.0 1.0 1.0 1.0 2.0 0 2.0 0 7.0

70 1.0 0 1.0 0 3.0 0 2.0 0 7.5

70 1.0 0 1.0 0 3.0 0 2.0 0 8.0

80 1.0 0 2.0 0 3.0 0 2.0 0 8.5

90 1.0 0 2.0 0 3.0 0 3.0 0 9.0

100 1.0 0 2.0 0 3.0 0 3.0 0 9.5

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

8 6 4 2

t c t c t c t c

10 1.0 0 0 0 0 0 0 0 6.50

10 1.0 0 0 0 0 0 0 0 6.75

10 1.0 0 0 0 0 0 0 0 7.00

10 1.0 0 0 0 0 0 0 0 7.25

30 3.0 0 0 0 0 0 0 0 7.50

40 1.0 0 3.0 0 0 0 0 0 7.75

40 0 0 2.0 0 2.0 1.0 0 0 8.0

80 2.0 0 2.0 0 2.0 0 2.0 0 8.25

100 2.0 1.0 2.0 0 3.0 0 3.0 0 8.50

c: control samples (10)

t: treated samples (10)

Table 3: Effect of temperature on Bacillus thuringiensis affecting mortality of Brachidontes

variabilis

Mortality %


Temp. oC

Time / hrs

8 6 4 2

t c t c t c t c

0 0 0 0 0 0 0 0 0 20

40 4.0 0 0 0 0 0 0 0 25

80 2.0 0 2.0 0 2.0 0 2.0 0 30

100 0 1.0 0 0 10.0 0 0 0 35

100 0 0 0 0 0 0 10.0 0 40

100 0 0 0 0 0 0 10.0 0 45


Table 4: Effect of carbon source on Bacillus thuringiensis affecting mortality

of Brachidontes variabilis

Mortality %


Carbon Source (5g/lit.)

Time / hrs

8 6 4 2

t c t c t c t c

100 0 0 2.0 0 4.0 0 4.0 0 Glycerol

100 0 0 2.0 0 0 0 8.0 0 Glucose

100 0 0 6.0 0 2.0 0 2.0 0

Water sol. starch

100 0 0 4.0 0 4.0 0 2.0 0

Malt extract

100 0 0 6.0 0 0 4.0 0

Sun flower oil

100 0 0 0 0 6.0 0 4.0 0 Molasses

c: control samples (10)

t: treated samples (10)

Table 5: Effect of concentration of the nutrient molasses on Bacillus thuringiensis affecting

mortality of Brachidontes variabilis

Mortality %


Concentration g/lit.

Time / hrs

8 6 4 2

t c t c t c t c

100 6.0 0 4.0 0 0 0 0 0 2.0

100 0 0 6.0 0 4.0 0 0 0 4.0

100 0 0 0 0 6.0 0 4.0 0 6.0

100 0 0 0 0 5.0 0 5.0 0 8.0

100 0 0 0 0 4.0 0 6.0 0

10.0

100 0 0 0 0 0 0 10.0 0 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 %, 2hrs Nitrogen nutrient Mortality %, 2hrs Nitrogen nutrient

30 Ammonium chloride 30 Ammonium chloride

100 Amm.dihydrogen orthophosphate 100 Amm.dihydrogen orthophosphate

20 Amm. Oxalate 20 Amm. Oxalate

40 Amm. Carbonate 40 Amm. Carbonate

40 Corn steep liquor 40 Corn steep liquor

60 Glutamic acid 60 Glutamic acid

Table 7: Effect of salinity on Bacillus thuringiensis affecting mortality of Brachidontes

variabilis

Mortality %

Number of killed mussels, 2 hrs Salinity

‰ t c

100 10.0 0 20

100 10.0 0 25

40 4.0 0 30

20 2.0 0 35

0 0 0 40

100 10.0 0 45

100 10.0 0 50

70 7.0 0 55

50 5.0 0 60


Table 8: Effect of γ-irradiation on Bacillus thuringiensis affecting mortality of


Mortality % , 2hrs

γ-irradiation (Gy)

t

c

10.0 0 0.5

0 0 0.2

0 0 10.0

0 0 50.0

0 0 70.0


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

24 18 12 8 6 4 2

t c t c t c t c t c t c t c

100 0 0 0 0 2.0 0 2.0 0 2.0 0 2.0 0 2.0 0 2

100 0 0 0 0 1.0 0 1.0 0 3.0 0 3.0 0 2.0 0 5

100 0 0 0 0 0 0 2.0 0 2,0 0 3.0 0 3.0 0 10

100 0 0 0 0 0 0 0 0 2.0 0 4.0 0 4.0 0 15

100 0 0 0 0 0 0 0 0 0 0 4.0 0 6.0 0 20

100 0 0 0 0 0 0 0 0 0 0 4.0 0 6.0 0 25


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

26.8%

0.45 %

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

Treated

Control

Biochemcial Composition

25.5 12.75 Total protein (µg /µl)

2.48 5.12 Total carbohydrates (mg/dl)

114.7 93.2 Total lipid (mg/dl)

0.0557 0.1125 Phospholpid 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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