RESEARCH PLAN Antibacterial Fractions From Marine Phytoplankton (Melosira sp., Navicula sp., Nitzschia sp.) Collected From Bali Strait
Antibacterial
Fractions From Marine Phytoplankton
(Melosira sp., Navicula sp., Nitzschia sp.)
Collected From Bali Strait
By:
Ni
Putu Intan Apsari [1] and Cok Laksmi Pradna Paramita [2]
[1,2]Denpasar 3rd State Senior
High School, Denpasar, Bali-Indonesia
A.
Question
or Problem being addressed
As a maritime country, 75% Indonesia’s surface
area is covered by ocean. The area is about 5.8 million km² and it has a coastline
length about 81,000 km[1]. That condition leads to the large
potential of marine biodiversity. Indonesia’s ocean, known as the coral
triangle area, is the highest marine biodiversity in the world. The highest
potential of marine biodiversity means highest chemicals diversity. Therefore,
the uniqueness of chemical structure in Indonesia’s water biodiversity can be marine
bio-prospecting efforts which potential as source of new drugs. The secrete of
Indonesian marine resources should be unfold to find a useful material
especially in health sector[2].
One
of useful material is antimicrobial resistance. It can threatens the effective
prevention and act as a treatment of an ever-increasing range of infections
caused by bacteria, parasites, viruses and fungi. Cuurently, those infections
has become serious threats to global public health that requires action across
all government sectors and society. Hence, the existance of antimicrobial
resistance has become valuable present in all parts of the world.
However,
new resistance mechanisms have emerged and have spreaded globally. There are
high proportions of antibiotic resistance in bacteria that cause common infections
(e.g. urinary tract infections, pneumonia, bloodstream infections) in all
regions of the world. A high percentage of hospital-acquired infections are
caused by highly resistant bacteria such as methicillin-resistant Staphylococcus
aureus (MRSA) or multidrug-resistant Gram-negative bacteria. As an example,
patients with infections caused by drug-resistant bacteria mostly have higher
risk of worse clinical conditions and they should consume more health-care
resources than other non drug-resistant patients who are infected with the same
bacteria
According to WHO (World Health
Organization) (2014), antibiotic resistance is a serious public health problem
in 114 countries. This cases are due to improper use of dose and function[3].
Therefore, investigating the new source of antibiotic should be conducted
especially from the new targeted marine resources such as marine phytoplankton.
In
Bali Island, Indonesia, Balineses that live near coastal areas have a local
knowledge to use the boiled water with green algae extract inside for treating
skin itchy after sailing. The preliminary study identified that in this green
algae extract contained Melosira sp, Navicula
sp and Nitzschia sp.
These green algae–associated as phytoplankton, are used as antiseptic, even
though they have not been investigated yet as an antibiotic source. There
wasn’t a scientific data that supported the potency of Melosira sp, Navicula sp
and Nitzschia sp. as source of
antibiotic agent. Therefore, this study aims to investigate a new sources of
antibiotic from phytoplankton collected in Bali Strait.
B.
Research
Question
The
research questions of this research are as follows:
1. Are Melosira
sp, Navicula sp and Nitzschia sp. collected from Bali strait extracts can
be used as antibiotic agent?
2. How does Melosira
sp, Navicula sp and Nitzschia sp. extracts
work effectively to against pathogenic bacteria S.aureus
and E.coli?
C.
Enviro-Engineering
Goals
The goals of this research are as follows:
1.
To obtain the scientific data that supported the
potency of Melosira sp, Navicula sp and Nitzschia sp. collected from
Bali strait as antibiotic producers.
2.
To determine the effectiveness of Melosira sp,
Navicula sp, Nitzschia sp. extracts against pathogenic bacteria S.aureus
and E.coli.
D. Expected
Outcomes:
The
expected result of this study is to obtain a new source of antibiotic from
Indonesia’s phytoplankton. These data will be used for further research to
develop a new antibiotic from marine phytoplankton.
E. Hypothesis
The hypothesis of this research are:
1. Melosira sp,
Navicula sp, Nitzschia sp. phytoplankton extract
containing antibacterial substance, as
an active compound to inhibit S.aureus
and E.coli. growth.
2. Melosira sp,
Navicula sp, Nitzschia sp. extract has the potency to be a new sources of
antibiotic.
F.
Description
in detail of method or procedures
1. Isolation & Identification of phytoplankton
o Reviewing literature to know the
characteristics of Melosira sp,
Navicula sp, Nitzschia sp. phytoplankton [4].
o
Collecting
and obtaining of the Melosira sp, Navicula sp, Nitzschia sp. phytoplankton in Bali strait.
·
Identifying the phytoplankton in Bali strait to get
species of Melosira sp, Navicula sp, Nitzschia sp. phytoplankton.
·
Isolating
Melosira sp, Navicula sp, Nitzschia sp. phytoplankton.
2. Cultivation of Phytoplankton
·
Cultivation Melosira
sp, Navicula sp, Nitzschia sp. phytoplankton using F/2 medium (Guillard and Rhyter, 1963). Ready
culture that has been harvested within two weeks.
3. Extraction and Separation
o The culture
solution were centrifuged at 4°C for 15 minutes (6,000 rpm). Then separated
between
o Extraction
of secondary metabolits Melosira sp, Navicula sp, Nitzschia sp. phytoplankton pellet and supernatant
4. GC-MS analysis
·
Analyzing the antibacterial activity by
antimicrobial activity test (in-vivo)
for S.aureus and E.coli, MIC (Minimum Inhibitory Concentration) Test using nano quant infinite M200 pro, BSLT
(Brine Shrimp Lethality Test) using Mayer method.
·
Analyzing the antibacterial compound
using column
chromatography, Fractination (n-heksana < EtOAc < MeOH), Thin Layer Chromatography (TLC), and
GC-MS Test (Gas Cromatography-Mass Spectometri).
H.
Hazardous
chemicals, Activities and Devices
a)
Detail
of chemical substances : MeOH (Methanol), EtOH (Ethanol), EtOAc (Ethyl acetate),
n-Hexane, H2SO4 (Sulfur acid), CH2CL2 (Diklorometan).
b)
Biological
agents: Staphylococcus aureus and
E. coli
c)
Device
and equipment: Centrifuge, rotary evaporator, nano quant infinite M200 pro.
d)
Method
Of Disposal : the heavy metals and other solution waste is placed in the
special bottle for disposal liquid. All of organic waste was collected and
recycled using distillation process. Acid and base wastes were neutralized
before dispose in sewer. Others chemical substance is also treated well. All of
microbial cultured waste was killed or autoclaved before disposed.
a)
Some
substances are toxic material. It is harmful by inhalation and if swallowed may
cause eye, skin, and respiratory tract irritation. The first aid procedures for
inhalation, move to the fresh air. If breathing is difficult used oxygen mask.
Do not use mouth-to-mouth resuscitation if victim ingested or inhaled the
substance; induce artificial respiration with a respiratory medical device.
Immediate medical attention is required. If there is skin contact, rinse
immediately with plenty of water, also under the eyelids, for at least 1,5
minutes.
b)
Using
rubber gloves, laboratory coat and mask when preparing and reacting for the
samples and chemicals in the fume hood.
c)
Eye
protection using UV-protected glass and Standard Operating Procedure (SOP) must
be taken when using UV-rays.
d)
All
of microbial supplies were sterilized after used, researcher should know the
standard microbiology practices/asepthic technic (lab coat, glove, and mask).
E. Research Analysis Method
Research analysis method will use
experiment at the laboratory and field observation. The results of
antibacterial assay will be analyzed using quantitative method.
POST
PROJECT SUMMARY
Antibacterial
Fractions From Marine Phytoplankton
(Melosira sp., Navicula sp., Nitzschia sp.)
Collected From Bali Strait
By:
Ni
Putu Intan Apsari [1] and Cok Laksmi Pradna Paramita [2]
[1,2]Denpasar 3rd State Senior
High School, Denpasar, Bali-Indonesia
RESULT
AND DISCUSSION
1 Isolation
& Identification
Cultivation was done to obtain the
single species of phytoplankton. Identification using morphology technic showed
that the isolated phytoplankton were Melosira sp., Navicula sp., and Nitzchia sp. [DSM1]
Identification of isolated
phytoplankton was base to the following chategory;
Observation Fitoplankton Profile
1.1 Melosira sp.
Table
4.3 Phytoplankton Melosira sp. Profile
Classification
|
|
General Classification
|
Close centric diatom
|
Description
|
|
Shapes
|
almost
spherical shaped pills
|
Size
|
11 - 30 μm Length, 17 - 70 μm diameter
|
Colour
|
Yellow-Brown
|
Lid
|
Bantalan
mukosa
|
Flagella
|
None
|
Chloroplast
|
A lot in cell wall.
|
Behavior
|
|
Lifestyle
|
Photosynthesis,
sexsual/asexsual, produce
auxospores.
|
Blooming
|
None
|
Hazardous Effect
|
None
|
Distribution
|
|
Habitate
|
Sea, river, or lake near by sea.
|
Geographical
|
Widely distributed; are present in
many parts of the world.
|
Season
|
Abundant in summer
|
Growth Condition
|
|
Salinity
|
23-36
|
Temperatur
|
-1 – 250C
|
|
1.2 Navicula sp.
Classification
|
|
General Classification
|
Pennate
diatome
|
Description
|
|
Shape
|
Shaped
like a boat
|
Size
|
32 - 130 μm length,
7 - 21 μm wide
|
Colour
|
Yellow-Brown
|
Silika frustules (silicified diatom
cell wall, which consists of two valves or sections overlap)
|
|
Flagella
|
None
|
Chloroplast
|
Two chloroplasts, the other one in
the middle of the raphe (In some diatom pennate, one or two longitudinal
slits along the [long] axis or around the margins of the valve)
|
Behavior
|
|
Lifestyle
|
Photosynthesis, sexual / asexual
|
Blooming
|
Do not cause blooming
|
Hazardous Effect
|
None
|
Distribution
|
|
Habitate
|
Benthic (ecological zone in the
bottom of the water)
|
Geographical
|
Widely distributed; are present in
many parts of the world.
|
Season
|
Present throughout the year, but more
abundant in spring and autumn.
|
Growth Condition
|
|
Salinity
|
16-39
|
Temperature
|
-2 –
290C
|
|
1.3 Nitzschia sp.
Classification
|
|
General Classification
|
Pennate
diatom
|
Description
|
|
Shape
|
Small
and boat-shaped
|
Size
|
25 - 160 μm length , 0.5 - 8 μm wide
|
Colour
|
Yellow-Brown
|
Lid
|
Silica
frustules (silicified diatom cell wall, which consists of two valves or
sections overlap)
|
Flagella
|
None
|
Chloroplast
|
Two chloroplasts, the other one in
the middle of the raphe (In some diatom pennate, one or two longitudinal
slits along the [long] axis or around the margins of the valve)
|
Behavior
|
|
Lifestyle
|
Photosynthesis, sexual/asexual
|
Blooming
|
None
|
Hazardous Effect
|
None
|
Distribution
|
|
Habitate
|
There is a fairly a superficial
marine waters close to the beach
|
Geographical
|
Widely distributed;
are present in many parts of the world.
|
Season
|
Plenty in the spring
|
Growth Condition
|
|
Salinity
|
18-38
|
 Temperature |
-2 – 290C
|
|
Each
cultivation solution from 3 strains phytoplankton could be obtained the dry
weight of biomass around 13-14 grams.
Table 4.1 showed the result of cultivation
Table 4.1 Biomass from Each Sample
No.
|
Sample
|
Sample Description
|
Biomass
|
1.
|
A
|
Navicula
sp.
|
13,6350 gram
|
2.
|
B
|
Nitzchia sp.
|
13,6211 gram
|
3.
|
C
|
Melosira
sp.
|
14,8924 gram
|
.3 Phytoplankton Extraction Result
Solvent extraction process has been
done to obtain the selected potential antibacterial agent. The yield of extract
was measured by using the following formula:
Yield =
Weight extract x 100%
Biomass
In this experiment, the pellet
contained microalaga biomass was extracted using methanol and the water
solution of supernatant was extracted using ethyl acetate. The yield in each
extraction were described in Table 4.6.
Table 4.6 Yield
calculation table from each extract
No
|
Sample
|
Extract Weight (mg)
|
Biomass
(mg)
|
Yield
|
1.
|
Pellet Navicula sp.
|
2.1
|
13635.0
|
0.015 %
|
2.
|
Pellet Nitzchia sp.
|
2.9
|
13621.1
|
0.021 %
|
3.
|
Pellet Melosisa sp.
|
3.6
|
14892.4
|
0.024 %
|
4.
|
Supernatant Navicula sp.
|
5.4
|
13635.0
|
0.039%
|
5.
|
Supernatant Nitzchia sp.
|
5.3
|
13621.1
|
0.039%
|
6.
|
Supernatant Melosira sp.
|
5.2
|
14892.4
|
0.034%
|
The highest yield
among these phytoplankton extract was reached by supernatant extrat of Navicula sp and Nitzchia sp. This result indicated that the most of the secondary
metabolite was expressed in the extracellular.
4. The Antibacteria Activity Result
The
antibacterial activity test of pellet
and supernatant extract of Melosira
sp., Navicula sp., and Nitzschia sp. against the pathogenic
bacteria Staphylococcus aureus and E. coli showed that almost all of the
pellet and supernatant extract can inhibit Staphylococcus
aureus and E. coli. Table 4.7 & 4.8. shows the results of diameter inhibition
that indicated antibacterial activity of pellet and supernatant extracts
against Staphylococcus aureus and
E. coli.
Tabel [DSM7] 4.7 Anti
bacterial activity of Melosira
sp., Navicula sp., and Nitzcschia s. extracts againts pathogenic bacteria Staphylococcus
aureus
No.
|
Sample
Code
|
Sample
Description
|
The diameter inhibition against S. aureus (mm) in 100 µg
extract
|
Description
|
1
|
a
|
Pellet extract of Navicula
sp.
|
9,775
|
+
|
2
|
b
|
Pellet extract of Nitzschia
sp.
|
11.05
|
+++
|
3
|
c
|
Pellet extract of Melosira
sp.
|
10
|
+
|
4
|
d
|
Supernatant extract of Navicula sp.
|
12.5
|
+++
|
5
|
e
|
Supernatant extract of Nitzschia sp.
|
12.4
|
+++
|
6
|
f
|
Supernatant extract of Melosira sp.
|
13.85
|
+++
|
7
|
amp
|
Ampicilin ( 10 μg)
|
50.7
|
++++
|
Descriptiom
: + = Weak
++ = Medium
+++ = Strong
++++ = Very Strong
Table
4.7 described that the
supernatant extracts of three phytoplankton
contained the stronger anti-Staphylococcus
aureus substances than pellet extracts. The highest antibacterial extract
was reached by supernatant of Melosira sp. extract against Staphylococcus aureus with the diameter
inhibition 13.85 mm.
Tabel[DSM8] 4.8 Anti
bacterial activity of Melosira sp.,
Navicula sp., and Nitzcschia s. extracts
againts pathogenic bacteria Staphylococcus aureus
No.
|
Sample
Code
|
Sample
Description
|
The Average Diameter Inhibitor zone (mm)
|
Description
|
1
|
a
|
Pellet extract of Navicula sp.
|
11.05
|
+++
|
2
|
b
|
Pellet extract of Nitzschia sp.
|
21.8
|
++++
|
3
|
c
|
Pellet extract of Melosira
sp.
|
11.85
|
+++
|
4
|
d
|
Supernatant extract of Navicula sp.
|
9.65
|
++
|
5
|
e
|
Supernatant extract of Nitzschia sp.
|
11.05
|
+++
|
6
|
f
|
Supernatant extract of Melosira sp.
|
12.75
|
+++
|
7
|
amp
|
Ampicilin (20 μg)
|
29.15
|
++++
|
Description
: + = Weak
++ = Medium
+++ = Strong
++++ = Very Strong
Table
4.8 indicated that all of pellet and supernatant extract of Melosira sp., Nitzschia sp., and Navicula
sp. could inhibit the growth of E.coli.
The highest activity showed by pellet
extract of Nitzschia sp. which has a
diameter inhibition approximately 21.8 mm. (Picture 4.4)
|
5. MIC
(Minimum Inhibitory Concentration) Result
The results of MIC (Minimum Inhibitory
Concentration) of extracts against Staphylococcus
aureus can be seen in Table 4.9
Tabel [DSM10] 4.9 MIC test result of Melosira
sp., Navicula sp., Nitzschia sp. extracts againts pathogenic bacteria Staphylococcus aureus
No.
|
Sample Code
|
Sample Description
|
MIC (mg/mL)
|
1.
|
A
|
Pellet
extract of Navicula sp.
|
2.5
|
2.
|
B
|
Pellet
extract of Nitzchia sp.
|
5.0
|
3.
|
C
|
Pellet
extract of Melosira sp.
|
>5.0
|
4
|
D
|
Supernatant
extract of Navicula sp.
|
5.0
|
5.
|
E
|
Supernatant
extract of Nitzchia sp.
|
5.0
|
6.
|
F
|
Supernatant
extract of Melosira sp.
|
5.0
|
MIC
(Minimum Inhibitory Concentration) in this experiment means the lowest
concentration of extract which inhibit the growth of Staphylococcus aureus. The lower value of MIC from the antibiotic, the higher sensitivity of extracts
against phatogenic bacteria. Each sample were adjusted to the following
concentration : 250μg, 125μg, 62,5μg, 31,25μg, 15,625μg, 7,8125μg, 3,90625μg,
and the smallest concentration were 1,953125μg in 50 µL. The absorbance
intensity of control without bacterial inoculation was around 0.8 to 0.9. This
absorbance number was used as the benchmarks to determine the value of MIC in
the 96 well. Table 4.9 showed that the lowest MIC number against Staphylococcus aureus was reached by
pellet extract of Navicula sp.
6. BSLT (Brine Shrimp Lethality Test) Result
The results
of BSLT test analysis showed that LC50 values of pellet extract of Navicula sp. was 19.69 pg / ml, pellet extract of Nitszchia sp. was.32.34 pg / ml, pellet
extract of Melosira sp. was 27.78 pg
/ ml. The supernatant extract of Navicula sp. was 46.43 pg / ml,
supernatant extract of Nitszchia sp.
was 102.51 pg/ml and supernatant extract of Melosira
sp. was 44.27 pg / ml.
|
 |
||||
|
||||
Extract
|
Sample
Description
|
y
|
a
|
b
|
x
|
LC 50 =
anti log dari x
|
A
|
Pellet extract of Navicula
|
5
|
2.375
|
1.926
|
1.294
|
19.69
|
B
|
Pellet extract of Nitzschia
|
5
|
2.195
|
1.686
|
1.510
|
32.34
|
C
|
Pellet extract of Melosira
|
5
|
2.24
|
1.766
|
1.444
|
27.78
|
D
|
Supernatant extract of Navicula
|
5
|
2.08
|
1.533
|
1.667
|
46.43
|
E
|
Supernatant extract of Nitzschia
|
5
|
1.86
|
1.26
|
2.011
|
102.51
|
F
|
Supernatant extract of Melosira
|
5
|
2.08
|
1.576
|
1.646
|
44.27
|
Table 4.10 described that
based on the cytotoxic test using the BSLT method, six extracts tested
categorized as toxic substances. The categorizing of toxicity properties was indicated by LC50 value less than 100 ppm. Pellets extract
of Navicula sp. has LC50
approximately 19.69 ppm, pellet extract of Nitzschia
sp. has 32.34 ppm, pellet extract of Melosira
sp. has 27.78 ppm,,supernatant extract of Navicula
sp. has 46.43 ppm, supernatant extract of Nitzchia
sp. has 102.51 ppm, and the supernatant
extract of Melosira sp. has 44.27
ppm. The most toxic extract was indicated by pellet of Navicula extract with
the LC50 19.69 ppm. This substance according to Meyer was very potential to be
anticanter as LC50 value <1000 pg / ml.
7.
Result of Open Column Chromatography separation
Table
4.11 Open column fractionation of pellet
extract of Nitzchia sp and antibacterial
activity against Staphylococcus aureus
Fraction
|
Ratio
of Solvent
|
Colour
|
Fraction
Weight
|
Diameter
Inhibitor
|
F1
|
N-Hexana
100%
|
Clear
|
10 mg
|
5.65 mm
|
F2
|
N-Hexana : Ethyl Acetate
20% : 75%
|
Cream
|
22 mg
|
10.9 mm
|
F3
|
N-Hexana : Ethyl Acetate
50% : 50%
|
Broken White
|
10 mg
|
10.6 mm
|
F4
|
N-Hexana : Ethyl Acetate
75% : 25%
|
Broken White
|
0 mg
|
7.75 mm
|
F5
|
Ethyl Acetate
100%
|
Clear
|
11 mg
|
7.65 mm
|
F6
|
Ethyl Acetate : Methanol
50% : 50%
|
Yellow
|
353 mg
|
11.8 mm
|
F7
|
Methanol
100%
|
Yellowish
|
700 mg
|
13.95 mm
|
Table 12. Open column chromatography of
Melosira sp extract.
And antibacterial activity against Staphylococcus
aureus
Fraction
|
Ratio
of Solvent
|
Colour
|
Fraction
Weight
|
The
Average Diameter Inhibitor zone (mm)
|
F1
|
N-Hexana
100%
|
Clear
|
3 mg
|
8.7 mm
|
F2
|
N-Hexana : Ethyl Acetate
20% : 75%
|
Dark Green
|
38 mg
|
7.8 mm
|
F3
|
N-Hexana : Ethyl Acetate
50% : 50%
|
Yellow
|
3 mg
|
12.55 mm
|
F4
|
N-Hexana : Ethyl Acetate
75% : 25%
|
Light Green
|
14 mg
|
12.15 mm
|
F5
|
Ethyl Acetate
100%
|
Green
|
2 mg
|
7.9
mm
|
F6
|
Ethyl Acetate : Methanol
50% : 50%
|
Dark Green
|
265 mg
|
7.35 mm
|
F7
|
Methanol
100%
|
Green
|
215 mg
|
11.5 mm
|
8. GC–MS analysis
Sampel
|
retention
time (min)
|
molecular
weight
|
compound
|
F3(S)
|
2.801
|
166
|
ankilostin
|
41.993
|
480.5
|
Hexadecanoic
acid, hexadecyl ester
|
|
F4(S)
|
33.175
|
282
|
eicosan
|
4.752
|
120
|
1,2,4-trimethylbenzene
|
|
F(6) S
|
4.269
|
281
|
cyclotetrasiloxane
|
F7(P)
|
37.553
|
396
|
beta
sitossterol
|
The important compounds contained in
active fractions were ankilostin,
eicosan, 1,2,4 trimethyl bezene , cyclotetrasiloxane and β-sitosterol. Some of
these compound also reported before contained in the active antibacterial
fractions from Sedum Pallidum ( Abbas
et. al. 2012)
Conclusion and
suggestion
Final
conclusion resulting from this study was seawater extract containing Melosira sp, Nitzchia sp and Navicula
sp. in Bali and Lombok sea has potential to develop as antibiotic source against gram-negative S.aureus
and gram-posittive E.coli.
Antibacterial
assay test showed that three extract active against Staphylococcus aureus and E.
colli . The highest antibacterial extract presented by Nitzchia sp with the MIC against S. aureus was 2.50 mg/mL. The diameter inhibition of pellet extract
of Nitzchia sp against E colli was 21.80 mm and 13.85 mm
against S aureus. The BSLT test
showed that Navicula sp extract very
potential for anticancer sources with the LC 50 value was 19.96 ppm. GC-MS analysis of active antibacterial
fraction of Nitzchiasp contained
cyclotetrasilloxaneoctamethyl, hexadecanoicacid, hexadecyl ester, β-
sitossterol, ankilostin.
Afterall,
Melosira sp,
Nitzchia sp and Navicula sp. proved that contain antibacterial compounds, so it can
be used as base to the further research for finding new candidate of new
antibiotics source.
Further separation was needed to know
the undefined compounds in active fractions
such as using HPLC for obtain a single compound derived from Melosira sp., Navicula sp., Nitzschia sp.
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