ANTIFOULING ACTIVITY OF BACTERIA ASSOCIATED WITH THE SURFACE OF MARINE INVERTEBRATES AND SEAWEEDS

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ANTIFOULING ACTIVITY OF BACTERIA ASSOCIATED WITH
THE SURFACE OF MARINE INVERTEBRATES AND SEAWEEDS
By Gayathri.R Reg.No09MBT08
Under the guidance of Dr. S. SATHEESH
Lecturer International Centre for
Nanobiotechnology Manonmaniam Sundaranar
University Rajakkamangalam 629 502 Kanyakumari
District Tamil Nadu, India
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• Introduction,
• Objective,
• Materials and methods,
• Results,
• Discussion,
• Summary
• References.

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Biofilm are architecturally complex communities
of microorganisms in which the cells are held
together by an extracellular matrix
called slime, typically containing
exopolysaccharides ( EPS ), proteins, and even
nucleic acids. This process of undesirable
accumulation of microorganisms, plants, animals
and algae, most often in aquatic environment is
called biofouling.
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Impacts on biodiversity, habitats or ecological
processes, Economic impacts and Impacts on
health ( humans, plants and animals ).
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Anti fouling is the process of removing or
preventing the accumulation of microorganisms.
Preventing the initial settlement of fouling
species by repelling or killing them,
Preventing the development of settled
organisms by killing them, inhibiting their
growth or reducing their adhesion ability and
Removing the fouling through cleaning.
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Physical methods, Chemical methods,
Mechanical cleaning, Heat treatment, Water
velocity, Enzymes as antifoulants, Coatings
methods, Non coating methods and Natural
Product Antifoulants ( NPA ).
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To screen the crude extract of the bacterial
strain isolated from the marine living sources
against biofilm bacteria. To observe the
inhibitory activity of the crude extract
by adhesion assay, To prepare a bacterial
extract mixd antifouling coatings for biofilm
development assay, To study the anti
settlement activity of the crude extract against
gastropods and To characterize the crude
extract by TLC, HPLC and NMR.
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Isolation of bacteria from marine samples,
Biochemical characterization of the isolated
bacterial strains, Grams staining,
Motility Test, Indole production Test,
Methyl-Red Test , Voges-Proskauer Test,
Citrate Utilization Test, Starch
hydrolysis, Urea hydrolysis,
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Catalase Test and Gelatin Hydrolysis
Test. Preparation of crude bacterial
extract, Antibacterial assay against biofilm
bacteria, Influence of crude bacterial extract
on the EPS production in biofilm bacteria,
Influence of crude bacterial extract on the
adhesion of biofilm bacteria onto the
surface, Biofilm development assay,
Gastropods settlement assay and
Characterization of crude extract by TLC, HPLC
and NMR.
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Strain A3 - Gastropods II Strain B3 - Sea
cucumber II ( 2 ) Strain C2 - Seaweeds Strain D -
Polychaetes Strain E - Sea lilly
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Table 1 - Biochemical and physiological
characteristics of the five marine surface
associated bacterial isolates used in the present
study
Sl.No.
Biochemical and Physiological tests
Organisms
Strain A3
Strain B3
Strain C2
Strain D
Strain E
1.
Gram staining
-




2.
Morphology
rod
cocci
cluster
cocci
cocci
3.
Motility
non motile
non motile
non motile
non motile
non motile
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Gelatin Hydrolysis

-
-

-
5.
Starch Hydrolysis
-

-

-
6.
Catalase Activity





7.
Citrate Utilization


-
-

8.
Indole production
-
-
-
-
-
9.
Methyl Red





10.
Voges Proskauer


-
-
-
11.
Glucose
-
-
-


12.
Fructose
-
-
-


13.
Lactose
-
-
-
-
-
14.
Sorbitol

-
-

-
15.
Urease
Acid





Alkaline
-
-
-
-
-
16.
Triple Sugar Iron (TSI)
Acid





Alkaline


-
-

Gas
-
-
-


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Table 2 - Antimicrobial activity of crude
bacterial extract produced by different
bacterial strains against target bacteria
S.No.
Organisms
Target Bacteria
Bacillus sp
E.coli
Klebsiella sp
V.harveyi
V.parahaemolyticus
1.
Strain A3
-

-
-

2.
Strain B3

-
-

-
3.
Strain C2

-

-
-
4.
Strain D

-


-
5.
Strain E
-
-
-

-
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Table 3 - Carbohydrate concentration of crude
bacterial extract produced by five bacterial
strains
S.No.
Bacterial Strains
Concentrations
50 µg
100 µg
1.
A3
2.7944
2.5988
2.
B3
3.128
2.794
3.
C2
3.236
2.667
4.
D
3.571
2.392
5.
E
3.571
2.706
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Table 4 - Protein concentration of crude
bacterial extract produced by five bacterial
strains
S.No.
Bacterial Strains
Concentrations
50 µg
100 µg
1.
A3
0.611
0.5775
2.
B3
0.479
0.573
3.
C2
0.405
0.42
4.
D
0.61
0.575
5.
E
0.490
0.460
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Table 5 Gastropod Settlement Assay
S.No.
Strains
Patella sp
Babylonia sp
1.
A3


2.
B3


3.
C2


4.
D
-

5.
E
-

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Table 6 - Rf values of compounds observed in TLC
S.No.
Isolated Strains
Rf Values (cm)
1.
Strain A3
0.8421
2.
Strain B3
0.6111
3.
Strain C2
0.7282
4.
Strain D
0.8
5.
Strain E
0.8877
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Figure 1 Antimicrobial activity of crude
extract isolates from the strain A3 against
target bacteria
Vibrio parahaemolyticus
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Figure 2 Antimicrobial activity of crude
extract isolates from the strain B3 against
target bacteria
Bacillus sp
Vibrio harveyi
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Figure 3 Antimicrobial activity of crude
extract isolates from the strain C2 against
target bacteria
Bacillus sp
Klebsiella sp
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Figure 4 Antimicrobial activity of crude
extract isolates from the strain D against
target bacteria
Bacillus sp
Klebsiella sp
V.harveyi
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Figure 5 Adhesion assay using crude extract of
the strain E ( number of bacterial cells adhered
on glass coupons )
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Figure 6 Biofilm development assay using
crude extract mixed coated coupons.
Strain D
Strain A3
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Figure 7 Gastropod settlement assay using
crude extract
Patella sp
Babylonia sp
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Figure 8 Thin layer chromatogram of the crude
extract isolated from the strains A3, B3, C2, D
and E.
Strains A3
Strain B3
Strain C2
Strain D
Strain E
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Figure 9 HPLC spectrum of crude extract
isolated from the strain A3
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Figure 10 HPLC spectrum of crude extract
isolated from the strain B3
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Figure 11 HPLC spectrum of crude extract
isolated from the strain D
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Figure 12 HPLC spectrum of crude extract
isolated from the strain E
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Fig 13 1H NMR spectrum of the crude extract
isolated from the strain D
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Fig 14 13C NMR spectrum of the crude extract
isolated from the strain D
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Natural products and their synthetic analogs
exhibiting anaesthetic, repellant and settlement
inhibition properties, but non toxic to the
non target organisms, are preferred as
potential antifouling agents. Sessile, soft
bodied marine organisms maintaining a
clean surface were identified as possible sources
of natural product antifoulants ( NPA ).
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The present study was carried out to assess the
antifouling activity of surface associated
bacteria. Five bacterial strains ( Bacillus
sp, Klebsiella sp, E.coli, V.harveyi and
V.parahemolyticus ) were used as target organisms
for the screening. Of 19 surface associated
bacteria isolated, five bacterial extract were
found to possess inhibitory activity against
target bacteria. These five bacterial extract
were also found to possess inhibitory activity
against the settlement of gastropods and one
bacterial extract from sealilly was found to
possess inhibitory activity against an adhesion
of bacteria onto the surfaces. The bacterial
extract was characterized by TLC, HPLC and NMR.
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