Jurnal Ilmu Kelautan P-ISSN: 2460-0156
SPERMONDE (2021) 7(2): 38-43 E-ISSN: 2614-5049

ANTIBACTERIAL ACTIVITY OF CAULERPA RACEMOSA ENDOPHYTIC
FUNGI FROM LEMUKUTAN ISLAND WATERS
Syarif Irwan Nurdiansyah
1
, Mega Sj Sofiana
1
, Melia Trianasta
1
, Warsidah
1
*, Maulana Hidayat
1

Submitted: September 4, 2021 Accapted: December 23, 2021 Online Published: February 14, 2022
1
Department of Marine Science, FMIPA, University Tanjungpura
Corresponding Author:
*Warsidah
E-mail:[email protected]
ABSTRACT
Green macroalgae Caulerpa racemosa from the waters of Lemukutan Island was isolated to obtain endophytic fungi.
Endophytic fungi were explored to determine the antibacterial activity. Isolation of endophyte fungi was carried out by
dilution method using PDA (Potato Dextrose Agar) seawater media. Endophyte fungi with different morphology were
obtained 11 isolates, namely CRF01, CRF02, CRF03, CRF04, CRF05, CRF06, CRF07, CRF08, CRF09, CRF10 and
CRF11. Antibacterial activity testing was carried out by agar diffusion method. Endophyte fungi isolate CRF09 showed
the highest activity against both Escherichia coli and Staphylococcus aureus test bacteria with inhibition zone diameters
of 15.96 mm and 16.47 mm respectively. Endophyte fungi isolates identified from the green macroalgae Caulerpa
recemosa were of the genus Trichocladium sp., Aspergilus sp., Chaetomium sp., Coprinus sp., Hymenochaete sp.,
Rhizopus sp., Trenella sp. and Zygorhynchus sp.
Keywords: antibacterial. endophyte fungi, Caulerpa racemosa, Lemukutan Island, activity
INTRODUCTION
Macroalgae (seaweed) is a plant-like organism that
lives in coastal waters and grows attached to
substrates (Meriam et al., 2016; Kepel and Mantiri,
2019). Based on macroalgae morphology including
Thallophyta plants where roots, stems and leaves
have not been clearly identified (not yet true)
(Meriam et al., 2016). According to Rahmat et al.
(2020), macroalgae generally consists of three
classes namely Chlorophyta class (green
macroalgae), Phaeophyta (brown macroalgae) and
Rhodophyta (red macroalgae). Green macroalgae
have branched filament-shaped thallus. It has
chlorophyll pigments a, b, carotene, lutein and
zeaxanthin (Meriam et al., 2016).
One of the green macroalgae species in coastal
waters is Caulerpa racemosa. It has green thallus
composed of ramuli, stolon and holdfast.
Macroalgae produces secondary metabolites as a
form of self-defense (Minarti et al., 2019).
According to Ridhowati and Asnani (2016), there
are about 500 chemical compounds derived from
macroalgae have been identified and most of them
are bioactive compounds derived from secondary
metabolites. C. racemosa has secondary metabolite
compounds such as alkaloids, phenols, flavonoids,
tannins, saponins, terpenoids and steroids. They
showed antibacterial activities (Wulandari, 2017;
Marfuah et al., 2018; Indayani et al., 2019).
Secondary metabolites produced from marine
organisms also produced by associated
microorganisms such as fungal endophyte.
Endophytic fungi are fungi that live intracellularly
in plant tissues by forming colonies in tissues
without causing harmful effects on their hosts
(Murdiyah, 2017). They protect their hosts and
produce secondary metabolite compounds that can
kill pathogens (Andriani, 2015). Endophytic fungi
can produce bioactive compounds that have
biological activities such as antioxidants,
anticancer, antibacterial, antifungi, antimalaria and
antivirus (Rollando et al., 2017).
Antibacterial is a compound that can be used to
control the growth of pathogenic bacteria (Marfuah
et al., 2018). Based on research conducted by
Andriani (2015) that C. racemosa from Takalar
Regency, South Sulawesi obtained 1 isolate of
endophytic fungi that have antibacterial activity
against Escherichia coli bacteria and
Staphylococcus aureus with diameter of inhibition
zone of 18.5 mm and 18.35 mm, respectively. The
ability of endophytic fungi to produce secondary
metabolite compounds similar to their host plants is
a solution to find antibacterial sources without
damaging existing ecosystems. The aim of this
research is exploring of antibacterial fungi
endophytes from the macroalgae C. racemosa in the
waters of Lemukutan Island, Bengkayang Regency,
against E. coli and S. aureus.
MATERIALS AND METHODS
This research was conducted in December 2020-
March 2021. Macroalgae sampling of C. racemosa
in the waters of Lemukutan Island, Bengkayang
Regency, West Kalimantan (Figure 1). Isolation
and testing of antibacterial activity was conducted
in the Laboratory of Microbiology, Technical

P-ISSN: 2460-0156
E-ISSN: 2614-5049
Syarif Irwan Nurdiansyah et al. 39

Implementation Unit of The Implementation of
Quality of Fishery Products, West Kalimantan
Province.

Figure 1. The research site
Sampling of Macroalgae C. Racemosa
Sampling of C. racemosa was taken using basic
diving equipment. Samples are taken at a depth of
1-2 m, then washed with sea water. The sample is
put in a sterile plastic sample containing sea water,
then stored in a cool box containing ice cubes with
the aim of maintaining the freshness of the sample
and subsequently in the analysis in the laboratory.
Isolation of Endophyte Fungi
Macroalgae samples of C. racemosa as the source
of isolates to be isolated weighed as much as 10 g
(Handayani et al., 2019). Furthermore, macroalgae
samples are washed with sterilized water and
soaking the sample into a solution of sodium
hypochlorite 1% for 5 minutes, ethanol 70% 1
minute then cleaned with sterile aquades
(Nurzakiyah, 2016). The sample was smoothed
using mortal after fine samples were inserted into
the erlenmeyer and added sterile sea water until 100
mL. Samples suspended in stock solution taken 1
mL then put in 9 mL sterile sea water to produce
dilution of 10
-1
, 10
-2
, 10
-3
, 10
-4
and 10
-5
. Then, 1 mL
samples suspended by dilution 10
-3
, 10
-4
and 10
-5

were inoculated on Potato Dextrose Agar (PDA)
media dissolved in seawater by pouring method.
Chloramphenicol is added as much as 50 mg/L as
an antibiotic (Rizky et al., 2019) to the PDA media
with the aim of inhibiting bacterial growth. Then,
samples incubated for 5-7 days at 25-27 °C.
Antibacterial activity test
Antibacterial activity test conducted using diffusion
method agar. The isolate colony of endophytic
fungi C. racemosa was grown for 7 days then the
colony was cut round with a diameter of 6 mm and
placed in the media NA (Nutrient Agar) which had
previously been scratched with test bacteria and
incubated at a temperature of 37 °C for 2 days.
Isolates grown in petri dish are analyzed with
diameter inhibition zone measurement (Rizky et al.,
2019).
Identification of endophyte fungi
The identification of endophyte fungal isolates were
carried out based on macroscopic and microscopic
observations. Macroscopic observations based on
colony color and colony shape (Yunaedi et al.,
2016). Microscopic observations of endophytic
fungi isolates are seen directly under binocular
microscopes with magnification of 100x. The
identification of isolation-based endophytic fungi
based on the pictoral atlas of soil and seed fungi
morphologies of Cultured Fungi and Key to
Species, Watanabe (2010), referring to the journal
Nie et al. (2017) and Zhao et al. (2019).
RESULTS AND DISCUSSION
Sampling of green macroalgae C. racemosa
Sampling of green macroalgae C. racemosa was
taken in the waters of Lemukutan island,
Bengkayang Regency of West Kalimantan (Figure
2). The sampling location was carried out at N
00'46'48.46'' E 108°42'23.981''. The C. racemosa
sample was taken at a depth of 0.9-1.3 m with
murky water conditions. The conditions coincide
with high tides and large ocean waves.

Figure 2. C. racemosa in the waters of Lemukutan Island
Isolation of endophytic fungi in C. racemosa
The endophyte fungal of C. racemosa were isolated
using a multilevel dilution method. A series dilution
aims to minimize or decrease the number of
microbes suspended in the sample. Isolation of
endophytic fungi using PDA media (Potato
Dextrose Agar) dissolved using seawater. PDA
media is a common medium used for the isolation
and cultivation of fungi (Rohmi et al., 2019). The
isolation of endophytic fungi from C. racemosa was
obtained 11 isolates, namely CRF01, CRF02,
CRF03, CRF04, CRF05, CRF06, CRF07, CRF08,
CRF09, CRF10, and CRF11 (Figure 3).

Jurnal Ilmu Kelautan
SPERMONDE (2021) 7(2): 38-43
40 Syarif Irwan Nurdiansyah et al.

Figure 3. Endophytic fungi of C. racemosa a) CRF01; b) CRF02; c) CRF03; d) CRF04; e) CRF05; f) CRF06; g) CRF07;
h) CRF08; i)CRF09; j)CRF10 and k) CRF11
Antibacterial Activity Test
Antibacterial activity tests of endophytic fungi
isolate C. racemosa showed that 11 endophytic
fungi isolates had antibacterial activity against S.
aureus bacteria. Six of 11 isolates were active
against E. coli bacteria. Antibacterial activity was
characterized by the presence of clear zones and fog
zones. The clear zone indicates the presence of
antibacterial activity, while the fog zone indicates
weak antibacterial activity.
CRF03, CRF06 and CRF09 isolates were active
against E. coli with clear zones diameter of 9.62
mm, 7.45 mm, 15.96 mm, respectively. CRF01,
CRF10 and CRF11 isolates forms fog zone. While
CRF02, CRF04, CRF05, CRF07 and CRF08
isolates do not form clear zones indicating that there
is no antibacterial activity. CRF09 isolates were
inhibited S. aureus growth with clear zone diameter
of 16.47 mm (Table 1).
Test antibacterial activity using diffusion agar
method. The antibacterial compounds diffuse into
the medium and inhibit the growth of test bacteria.
The category of antibacterial activity determined by
measuring the clear zone. According to Djakatara et
al. (2019), the clear zone >21 mm (antibacterial
activity is very strong), 11-20 mm (strong
antibacterial activity), 6-10 mm (moderate
antibacterial activity) and the <5 mm (weak
antibacterial activity). Based on the results of
endophytic fungi isolate C. racemosa have
antibacterial activity against E. coli and S. aureus
test bacteria with categories of antibacterial activity
ranging from moderate to strong.
Table 1. Antibacterial activity of endophytic fungi C.
racemosa against E.coli and S. aureus
Isolate
Diameter zone of inhibition
(mm)
E. coli S. aureus
CRF01 ± ±
CRF02 - ±
CRF03 9,62 ±
CRF04 - ±
CRF05 - ±
CRF06 7,46 ±
CRF07 - ±
CRF08 - ±
CRF09 15,96 16,47
CRF10 ± ±
CRF11 ± ±
The highest antibacterial activity possessed by
CRF09 isolates against posisive gram bacteria,
namely S. aureus (Figure 4). Gram-positive bacteria
have a cell wall with a peptidoglycan layer located
on the outer membrane. Testing the antibacterial
activity of endophytic fungi isolates begins with
preparing a 7 day old fungi isolate, then it tested for
antibacterial with an incubation time of 24 hours.
The purpose of its were get secondary metabolite
compounds in the stationary phase (Roosheroe et
al., 2016).

P-ISSN: 2460-0156
E-ISSN: 2614-5049
Syarif Irwan Nurdiansyah et al. 41


Figure 4. Antibacterial activity test of CRF09 isolate
against S. aureus bacteria
Identify Endophytic Fungi Isolate C. racemosa
The CRF01 isolated morphological micrographs
have oval-shaped spores and directly in laterally
arranged hyphae. Its belongs to the genus
Trichocladium. The morphological micrographs of
CRF02 and CRF05 isolates have spores shaped like
flowers and form round vesicles with 4 apical
fragments, while CRF11 isolates have flower-
shaped spores with circles located on hyphae.
Morphology in all three isolates belongs to the
genus Aspergillus. The CRF03 isolates have round-
shaped spores with spore surfaces covered by
hyaline hair, these features are owned by the genus
Chaetomium.
The CRF04 isolated morphological micrographs
have radially aligned hyaline and subhyaline
hyphae. Hyphae does not clump, narrow and does
not debauch, these characteristics belong to the
genus Coprinus. The morphological micrographs of
CRF06 and CRF08 isolates have dense hyphae and
arranged in a branching frame with cylindrical
form. Spores are short and curved cylindrical. The
characteristics exhibited by both isolates belong to
the genus Hymenochaete. CRF09 isolated
morphological micrographs have a single, branched
and grouped hyaline hyphae. Spores are round in
shape, have cells 2-4 and a smooth spore surface.
These features belong to the genus Tremella.
The morphological micrograph of CRF07 isolates
has hiphae that is upright branched, rooted and has
spores. Spores are located at the top of hyphae with
a triangular shape with sharp ends such as thorns.
These features belong to the genus Rhizopus. The
morphological micrograph of CRF10 isolate has
upright, branched hyaline hyphae. Spores are
located in hyphae with a rough surface and have
spiral hair. These traits belong to the genus
Zygorhynchus. Isolation and identification of fungi
in ocean waters has been widely reported. The
genus Aspergillus is a fungi found in many marine
waters. The fungi genus Aspergillus is found in the
macroalgae C. racemosa in the Indian Sea
(Suryanarayanan et al., 2010; Venkatachalam et al.,
2015). Morphology of endophytic fungal colonies
can be seen in Figure 5.

Figure 16. Morphological micrographs isolate endophytic fungi with light microscop binocular of 100x CRF01 (a),
CRF02 (b), CRF03 (c), CRF04 (d), CRF05 (e), CRF06 (f), CRF07 (g), CRF08 (h), CRF09 (i), CRF10 (j)
and CRF11 (k)
Inhibition zone

Jurnal Ilmu Kelautan
SPERMONDE (2021) 7(2): 38-43
42 Syarif Irwan Nurdiansyah et al.
CONCLUSION
Isolation of fungal endophyte C. racemosa from the
waters of Lemukutan Island, Bengkayang Regency
West Kalimantan obtained as many as 11 isolates.
The best antibacterial activity of the green
macroalgae endophytic fungi C. racemosa against
E. coli and S. aureus bacteria is the CRF09 Isolate

with a clear zone diameter of 15.96 mm and 16.47
mm, respectively. Identification of the green
macroalgae endophyte fungi isolate C. racemosa
from the waters of Lemukutan Island is known to be
the genus Trichocladium, Aspergillus,
Chaetomium, Coprinus, Hymenochaete, Rhizopus,
Tremella and Zygorhynchus.
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