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BIOEDUSCIENCE
ISSN: 2614-1558
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Isolation and Activity Testing of Proteolytic Bacteria Associated with

The Sponge Stylotella sp. and Its Potential as an Antibacterial
Maria Septiana Seuk 1*, Lukas Pardosi 1, Gergonius Fallo 1, Adelya Irawan Manalu 1
1 Biology Study Program, Faculty of Agriculture, Science and Health, University of Timor
Jalan Km. 09, Sasi Village, Kefamenanu City District, North Central Timor Regency, East
Nusa Tenggara Province
* Correspondence: lukaspardosi51@[Link]

Abstract

Background: Background: The province of East Nusa Tenggara (NTT), Indonesia, boasts a significant
expanse of marine territory, making its waters a potential treasure trove of aquatic resources. One such
marine resource is the Stylotella sp. sponge, which is found in this region. Sponges like Stylotella sp.
often have intricate relationships with microorganisms, including bacteria. Method: This research was
to characterize and identify proteolytic bacteria and test bioactive compounds from isolates of
proteolytic bacteria from the sponge Stylotella sp. Proteolytic bacterial strains were rejuvenated first in
2% TSA + Nacl medium and then characterized microscopically and biochemically using catalase and
oxidase tests. The diffusion method used the antibacterial activity test against Escherichia coli and
Staphylococcus aureus. Result: The results showed that proteolytic bacterial isolates had irregular and
rounded colony shapes, wavy and intact colony edges, hilly and convex colony elevation, and white
color, while biochemically, it was declared positive for the catalase and oxidase tests. Antibacterial
activity tests on proteolytic bacterial strains showed the ability to inhibit the growth of S. aureus with
an inhibitory capacity of 9.5 mm and [Link] with an inhibitory capacity of 7 mm. Conclusion: Hasil
Article history
Received: 22 Nov 2023 isolasi bakteri proteolitik menunjukkan bahwa keenam isolat bakteri yaitu SP0, SP1, SP3, SP4, dan SP5
Accepted: 02 Apr 2024 umumnya mempunyai bentuk koloni bulat dan tidak beraturan, berwarna putih, tepinya berwarna
Published: 30 Apr 2024 putih.

Publisher’s Note: Keywords: Antimicrobial; Proteolytic Bacteria Isolate; Stylotella sp. Sponge
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Introduction
Indonesia is the largest archipelagic country in the world, with an area of around 3.1
Citation:
Seuk et al., (2024). Isolation million square kilometers. This means that Indonesia also has a vast sea. The sea contains
and Activity Testing of various kinds of natural resources in it. Natural resources in the sea are natural resources
Proteolytic Bacteria Associated that have great potential and need to be developed and managed optimally (Jo et al., 2016).
with The Sponge Stylotella sp.
Marine resources are biological resources with great potential that must be developed and
and Its Potential as an
Antibacterial. BIOEDUSCIENCE, managed optimally. Indonesia's vast sea areas contain a wealth of marine diversity that can
8(1), 75-83. doi: be explored, including mangroves, coral reefs, and seagrass, along with other types of
10.22263/jbes/12980 marine biota such as fish, mollusks, crustaceans, sponges, algae, and turtles (Suparno, 2005).
One of Indonesia's provinces with a giant sea area is the province of East Nusa Tenggara
(NTT). This makes NTT waters rich in potential marine resources such as fisheries, seaweed,
©2024 by authors. Licence coral reefs, and sponges (Pardosi et al., 2022). Sponges are a type of porous invertebrate and
Bioeduscience, UHAMKA, belong to the genus Porifera. Sponges act as filter feeders, looking for food, among other
Jakarta. This article is open-
access distributed under the things, by filtering or absorbing sea water containing food through pores (ostia). Sponges or
terms and conditions of the porifera are animals from the phylum Porifera, one of the components in coastal and marine
Creative Commons Attribution
(CC-BY) license. ecosystems, especially on coral reefs. Sponges have bioactive potential, including
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antibacterial, anticancer, and antifungal properties (Rumampuk, 2017). This is by (Utami

et al., 2016), which states that bioactive compounds from sponges can be used as
medicine, for example, antibacterial, anticancer, antifungal, anti-inflammatory, cytotoxic,
antibacterial, antiviral, antimalarial, anti-inflammatory, cancer and immunosuppressive.
Exploration of microbes in symbiosis with sponges from various regions in
Indonesia is one thing that needs to be done. Isolation of bacteria in symbiosis with
sponges, molecular characterization, and characterization of bioactive compounds
produced by bacteria from sponges are strategies that can be used to explore the role of
sponges. According to Mahdiyah (2021), Sponges produce compounds that have the
potential to act as antibacterials on a large scale. Sponges harbor various types of
bacteria, up to 40% of their biomass (Sable et al.,2017). Bacteria in symbiosis with
sponges serve as a source of extracellular enzymes (Maharsiwi et al., 2020). Extracellular
enzymes are active outside bacterial cells and have various functions, such as breaking
down large molecules into smaller ones, an essential process in degrading nutrients
sponges need (Wicaksana & Rachman, 2018). This shows that there is a complex
symbiotic relationship between sponges and bacteria that can provide benefits to sponge
organisms. With the presence of bacteria, sponges become more resistant to bacterial
infections than other land and marine organisms (Mahdiyah, 2021).
In addition, the extracellular enzymes these bacteria produce are used to break
down nutrients and act as chemical defenses against predators (Wicaksana & Rachman,
2018). Proteolytic bacteria are a type of bacteria that can produce extracellular protease
enzymes (Agrawal et al., 2016). Protease enzymes are a group of enzymes that have an
essential role in the breakdown or digestion of proteins, but they also have wide
applications in the industrial field (Rizaldi, 2016). Regarding this, it is necessary to
identify and characterize proteolytic bacteria and test bioactive compounds from isolates
of proteolytic bacteria from the sponge Stylotella sp.

Methods
Tools and materials
The tools used in this research are scissors, incubator, laminar airflow, micropipette,
knife, dropper pipette, test tube, beaker, petri dish, autoclave, microscope, object glass,
oven, analytical balance, Erlenmeyer, Bunsen, tube needle. horn spoon, mortar, match,
mask, tweezers, tube rack. The materials used in this research were Stylotella sp. sponge
samples and distilled sodium chloride (NaCl) salt solution with a concentration of 0.9%,
cotton, label paper, iodine solution, tissue, 96% alcohol, disc paper, crystal violet dye,
solution dye Lugol, Methylene Blue (MB) dye, and safranin dye.
Sponge Sampling
Sponge samples were taken from Oenggae Waters, Pantai Baru District, Rote Ndao
Regency. They were taken randomly by diving into the seabed. The samples were then
put into sterile plastic. Next, the sponge was taken to the laboratory to be characterized
and isolated (Rizaldi, 2016).
Media Creation
The media used is Tryptic soy agar media, made by dissolving 6 grams of TSA media in
150 ml of distilled water and 3 ml of Nacl and heating it on a hot plate. Then, the press is
sterilized in an autoclave for 15 minutes at a temperature of 121oC (Retnowati et al.,
2011).
Bacterial Isolation
Isolation of bacteria associated with the sponge was carried out by washing it with
running water and rinsing it with sterile distilled water. Then, the sample was cut into
small pieces, weighed 10 grams, and crushed using a mortar. Next, a 10-0 to 10-5 dilution
is made. The results of the dilutions 10-0, 10-1, 10-2, 10-3, 10-4, and 10-5 were then
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taken 0.1 ml and inoculated in TSA media + 3 ml Nacl, incubated for 24 hours at 30ºC
(Pastra et al., 2012; Marzuki et al., 2014). The growing bacterial isolates were observed
for colony morphology, including shape, edges, elevation, and color.
Purification of Bacterial Isolates
Take one cycle of bacterial culture that grows on TSA media in a petri dish, streak it on
Tryptic soy agar medium + 3ml NaCl, and incubate at 30ºC for 24 hours (Massinai, 2013).
Microscopic Identification of Potential Isolates
Microscopic observations include gram-staining biochemical tests, catalase tests, and
oxidase tests.
Gram stain
Identification of proteolytic bacterial isolates includes gram staining using the smear
method. Bacterial cultures are taken in one dose and spotted on a glass object to be made
into smears and added with dye. The smear that has been made is then observed under a
microscope to be observed under a microscope at a magnification of 10 x 0.25.
Catalase Test
Take a bacterial sample from the culture medium and place it on an object glass
cleaned with alcohol. Drip 1-3 drops of 3% H2O2 solution onto the bacterial sample in the
object glass. When H2O2 is added, oxygen gas bubbles will form. These gas bubbles will
appear as foam or air bubbles rising upward from the bacterial sample. If gas bubbles
form, this indicates that the bacteria have the catalase enzyme and the test result is
positive (catalase positive). If no gas bubbles form, then the test is negative.
Oxidase Test
The bacterial culture to be tested is planted on Trypticase Soy Agar and then
incubated at 37ºC for 1x24 hours. After 24 hours, bacterial colonies growing on the TSA
medium were flooded with oxidase reagent. If these bacteria produce the oxidase
enzyme, the colony will experience a color change after the application of the oxidation
reagent, which will become light red, dark red, and black.
Proteolytic bacterial activity assay
The proteolytic bacterial activity test was carried out using the disk diffusion method.
The bacterial isolate to be screened is first cultured for 1x24 hours in TSA+ 2% NaCl
media. Next, isolate cultures were made as dots on the surface of Skim Milk Agar media
and incubated for 1x24 hours. The clear zone around the bacterial isolate is then
observed and measured (Hudzicki, 2013; Rizaldi, 2016).
Antibacterial activity test
Antibacterial testing of proteolytic bacterial isolates was done by culturing
proteolytic bacterial isolates in TSA+2% NaCl media, testing bacteria in NA median, and
incubating for 1x24 hours. Proteolytic bacterial isolate cultures were then prepared in
suspension form following McFarland standards. Then, 10 µl of the suspension was
pipetted and dropped onto a paper disc with a diameter of 0.6 cm, which was placed on
Mueller Hinton Agar, which had been etched with S. aureus and E. coli. The diameter of
the inhibition zone is measured using the formula:

Information:
DV: Vertical diameter
DH: Horizontal diameter
DC: Disc diameter
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Data Analysis
Data from research on proteolytic bacterial activity tests and antibacterial activity
tests against E. coli and S. aureus was obtained from the sponge Stylotella sp. The data
obtained and analyzed is descriptive. The analysis results are presented in various
formats, explained narratively in text form, and presented in tables and figures.

Result and Discussion

Isolation and Identification
The isolation of bacteria associated with the Stylotella sp. sponge resulted in 6
bacterial isolates. The results of observations of proteolytic bacterial colony isolates from
the sponge Stylotella sp. have morphological characteristics, including shape, edges,
elevation, and color. SP0 bacterial isolates have colony characteristics that include
irregular shapes, wavy edges, and hilly elevations and have a white color, whereas SP1,
SP2, SP3, SP4, and SP5 bacterial isolates have round-shaped colonies, flat edges, and
convex elevations and colors. White. This agrees with Pahriyani et al. (2020), who stated
that bacterial isolates associated with sponges, which include colony color and colon
shape, orange, yellow, and white colony colors, and circular colony shapes with a nucleus
in the middle, the edges of the isolate are smooth. , irregular, has curved elevations and
convex elevations, and elevations like drops, raised, and hilly (Pardosi et al., 2022). The
results of colony morphology identification of 6 bacterial isolates associated with the
Stylotella sponge can be seen in (Table 1).

Table 1. Bacterial isolates associated with the sponge Stylotella sp. macroscopically.
Bacterial Colony morphology
isolate Shape Margin Elevation Pigmentation
SPO Irregular Undulate Hilly White
SP1 Circular Entire Convex White
SP2 Circular Entire Convex White
SP3 Circular Entire Convex White
SP4 Circular Entire Convex White
Sp5 Circular Entire Convex White
I
Dentification of proteolytic bacteria microscopically
The results of the gram test on bacterial isolates SP0 and SP1 were found to have a
cocci form and were gram-positive, while isolates SP2, SP3, SP4, and SP5 were in the form
of bacilli with gram-negative characteristics (Table 2). Pelczar and Chan (1986) stated
that 95% of marine bacteria are gram-negative, some are motile, motile nature refers to
the ability of bacteria to move actively, which can be used to find sources of nutrition or
respond to environmental changes, 70% contain pigments. The pigment content can give
bacteria a specific color and role in bacterial photosynthesis or adaptation to the marine
environment (Kuddus, 2019).
The results of observations showed that bacillus-shaped bacteria were the dominant
ones found in this study. This is probably because these bacteria have flagella, so they can
stick to sponges, whereas bacteria not equipped with flagella, non-flagellum, can move by
gliding. Aryulina (2005) suggested that bacteria that have flagella can help these bacteria
find environmental conditions that are more suitable for their growth. According to
Fardiaz (1989), marine bacteria associate with various solid surfaces, including algae,
sponges, seagrass, coral, and mangrove plants, to obtain the necessary attachment places.
Bacteria in cocci form do not have locomotion, such as flagella. Therefore, bacteria tend to
stick to objects in the water as a survival strategy (Rizaldi, 2016). Bacteria in the form of
coccus, which do not have locomotion, tend to live attached to specific substrates,
including sponges. The coccus shape of this bacteria is caused by the presence of a slimy
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material, which causes the bacterial cells to bond or combine to form a firm or solid
surface (Megawati et al., 2019).

Table 2. Results of Gram Staining and Biochemical Tests

D Gram staining Biochemical Test
Isolate
e Catalase Oxidase
Code Picture Form Gram
s Test Test
c
r
i SPO
p Cocci + + +
t
i
o
n

:
SP1 Cocci + + -
(
+
)

P
o SP2 Basil - + _
s
i
t
i
v
e SP3 Basil - + _
;

(
-
)
SP4 Basil - + _
N
e
g
a
t
Sp5 Basil - + _
i
v
e

Biochemical testing is carried out using 2 test methods: the catalase and oxidase
tests. In the catalase test results, six isolates of proteolytic bacteria were found, producing
air bubbles on the colony's surface. Air bubbles indicate positive bacteria produce the
catalase enzyme (Figure 2. a). Proteolytic bacterial isolates that do not produce air
bubbles on the surface of their colonies can be declared catalase-negative, meaning that
the bacteria do not degrade the H2O2 provided and, therefore, dorres not produce
oxygen. According to Cappuccino & Sherman (2017), positive catalase test results for all
isolates tested indicated the presence of air bubbles on the colony's surface when dipped
with 3% H2O2 solution. This shows that these bacteria live in aerobic conditions, such as
S. aureus, Bacillus sp., and Pseudomonas sp. (Kuddus, 2019).
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The results of the oxidase test on proteolytic bacterial isolates carried out on six
bacterial isolates showed that the isolate that produced the cytochrome oxidase enzyme
was isolate code SP0, which was marked by colonies that changed color to red.
Meanwhile, isolates SP1, SP2, SP3 and SP4, SP5 showed no color change. According to Lay
(1994), positive bacteria in the oxidase test contain the enzyme (Kuddus, 2019).

(a) (b)
Figure 2. (a)Catalase test results; (b) Oxidase results.
Proteolytic Bacterial Activity Test
Table 3. Proteolytic activity test results of associated bacterial isolates with the sponge
Stylotella sp.
Isolate code Proteolytic activity test Proteolytic index
SPo + 37,5 mm
SP1 + 40 mm
SP2 _ 0
SP3 _ 0
SP4 _ 0
SP5 _ 0
Description: (+) exists, (-) does not exist

Based on the results of the proteolytic activity test, it showed that the isolates
producing the protease enzyme were isolates SP0 and SP1, which were characterized by
explicit zone activity around the paper disc with a proteolytic index for isolate SP0 of 37.5
mm and bacterial isolate SP1 with a proteolytic index of 40 mm (Figure 3). This can be
calculated using the following formula:

The results of precise zone measurements on proteolytic bacterial isolates showed

significant variations between these isolates. The high proteolytic index of the SP0 isolate
may be due to the isolate's rapid ability to synthesize and degrade amino acids. According
to (Rizaldi, 2016), bacteria from the genus Bacillus, such as Bacillus sp., produce protease
enzymes. The formation of clear zones in media containing proteins, such as skim milk, is
a typical sign of this proteolytic activity. Therefore, clear zones in proteolytic bacterial
isolates associated with the sponge Stylotella sp. indicate these bacteria' ability to
produce protease enzymes.
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SPO SP1
Proteolytic

Clear
Zone

Figure 3. Proteolytic activity assay

Antibacterial Activity Test
Testing the antibacterial activity of proteolytic bacterial isolates is an essential step in
evaluating the potential of these isolates to inhibit the growth of pathogenic bacteria
(Nioede et al., 2021). The results of this test show that proteolytic bacterial isolates can
produce effective antibacterial active compounds. This clear zone indicates that the
antibacterial compounds produced by these isolates can inhibit the growth of the
pathogenic bacteria tested (Figure 4).

[Link] [Link]
Clear Zone

Clear Zone

Figure 4. Antibacterial Activity Test Results of Proteolytic Bacterial Isolates

Proteolytic bacterial isolates tested against pathogenic bacteria had antibacterial

activity, with a barrier diameter of 7 mm against E. coli and 9.5 mm against S. aureus
(Table 4).

Table 4. Antibacterial activity test results of proteolytic bacterial isolates

Inhibition Zone
Isolate Code Tested bacteria Repetition
Diameter (mm)
1 8
E. coli 2 6
Bacteria Average 7
Proteolytic 1 10
S. aureus 2 9
Average 9.5

According to Abubakar et al. (2011), isolated from the mesohyl and superficial parts
of Jaspis sp. show antibacterial properties because it can inhibit the growth of S. aureus,
Vibrio harveyii, E. coli, and P. aeruginosa (Pardosi et al., 2022). Gratia et al. (2019) stated
that bacterial isolates associated with sponges had antibacterial activity against the test
bacteria E. coli at 13.5 mm and S. aureus at 14.25 mm.
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Conclusions
Based on the research results, it was concluded that the results of proteolytic bacterial
isolation showed that the six bacterial isolates, namely SP0, SP1, SP3, SP4, and SP5,
generally had round and irregular colony shapes, were white, the edges of the isolates
were wavy and flat, the colony elevations were hilly and convex and were colored. White.
The proteolytic bacterial activity test results showed two bacterial isolates, SP0 and SP1.
The SP0 bacterial isolate had a proteolytic index of 37.5 mm, while the SP1 bacterial
isolate had a proteolytic index of 40 mm. The results of testing the antibacterial activity of
bacterial isolates SP0 and SP1 showed an average resistance of 9.5 mm against S. aureus
and 7 mm against E. coli.

Acknowledgment
The research team would like to thank the Ministry of Education and Culture and the
Central Institute for Research and Community Service, University of Timor, for financial
support for this fundamental research.

Declaration statement
The authors reported no potential conflict of interest.

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