Inhibition of marine biofouling by aquatic Actinobacteria and coral-associated marine bacteria

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Malaysian Journal of Microbiology, Vol xx(x) xxxx, pp. xxx-xxx
Malaysian Journal of Microbiology
Published by Malaysian Society for Microbiology
(In since 2011)
XXX ISSN (print): 1823-8262, ISSN (online): 2231-7538
*Corresponding author
Inhibition of marine biofouling by aquatic actinobacteria and coral-associated
marine bacteria
Diana Elizabeth Waturangi1*, Jessen Purwa Hariyanto1, Windy Lois1, Rory Anthony Hutagalung1, Jae Kwan
Hwang2
1Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jenderal Sudirman no. 51, 12930 Jakarta,
Indonesia.
2Biomaterial Research Laboratory, Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu,
Seoul 120-749, Korea
Email: diana.waturangi@atmajaya.ac.id
Received XXX; Received in revised form XXX; Accepted XXX
ABSTRACT
Aims: Biofouling is a common biology phenomenon occuring on ship surface. This phenomenon has become serious
threat in marine industries because of great economic loss. Tributyltin has been used to prevent biofouling, but it turned
to cause the environmental problem. Therefore, the discovery of alternative environment-friendly compound is
necessarily needed.
Methodology and Results: Five Actinobacteria isolates and fourteen marine bacteria isolates were tested against the
biofilm formation of eight biofouling bacteria isolates that isolated from boat surface and the attachment of three
biofouling diatoms (Amphora, Navicula, Nitzschia). Actinobacteria CW17 supernatant showed the broad spectrum
activity against all fouling bacteria, whereas BC 11-5 supernatant was the only marine bacteria that capable to inhibit
biofilm formation of V. neocaledonicus. Moreover, three representative diatoms attachment could be inhibited by the
bioactive compounds produced by Actinobacteria and marine bacteria. CW01 supernatant showed broad spectrum and
high activity against all three representative diatoms which is very promising. Molecular identification based on 16S
rDNA gene sequence showed eight fouling bacteria isolates were biofilm-forming bacteria.
Conclusions, Significance and Impact of study: This research showed aquatic Actinobacteria and coral-associated
marine bacteria have the potential to prevent biofouling formation. Further studies are needed to purify and characterize
these antibiofouling compounds for environmental application.
Keywords: Biofouling, Antibiofouling, Actinobacteria, Marine Bacteria, Diatom
INTRODUCTION
Marine transportations and structures are easily colonized
by fouling organism in a process called biofouling. This is
a serious problem for marine industries all over the world
that creates great disadvantages and economic loss.
Fouling organism form a complex layer on submerged
substrates, like ship hulls, which increases the surface
roughness, resulting in increased frictional resistance and
fuel consumption because the top speed and the range of
the ship is decreased (Müller et al., 2013). Biofouling also
causes the distribution of non-indigenous species (NIS)
by ship transport (Hong and Cho, 2013). To prevent this
biological phenomenon, antifouling coatings had been
developed for water-exposed surfaces. Copper oxide and
tributyltin oxide (TBT) were found to be the most effective
agents against biofouling. Unfortunately, these compound
not environmentally friendly due to the fact that they are
not quickly degraded naturally and attack both target and
non-target species (Müller et al., 2013). This led the
International Maritime Organization (IMO) to ban their
application on ships since 2008 (Qian et al., 2010). Since
then, the demand for new antibiofouling compounds that
environmentally friendly is increased.
Biofouling is formed by the adhesion and interaction of
fouling organism, which consist of microfoulers (i.e.
bacteria and diatoms) and macrofoulers (i.e. barnacles,
mussels, polychaete worms, bryozoans, and seaweed).
The process of biofouling formation is divided into four
main steps; (i) formation of conditioning film composed of
organic materials (such as protein, polysaccharide, and
proteoglycan) on water-exposed surface, (ii) the
settlement of microfoulers, (iii) formation of biofilm, and
(iv) attachment of marcofoulers larvae. Many organisms
involved in biofouling makes it hard to removed (Cao et
al., 2011; Müller et al., 2013).

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XXX ISSN (print): 1823-8262, ISSN (online): 2231-7538
Bacterial and diatom biofilm formation was the initial
step biofouling formation. Biofilm consists of extracellular
polymeric substance (EPS) secreted by bacteria and
diatom. Biofilm leads to irreversible bacteria adhesion and
stronger diatom attachment (Cao et al., 2011). Moreover,
biofilm formation will stimulate the attachment of
invertebrates and algae to submerged marine surfaces.
Microbial biofilms in particular provide biochemical signals
that larvae employ in selecting a settlement site, attaching
to it, and undergoing metamorphosis (Zardus et al.,
2008). Therefore, inhibiting formation of the bacteria
biofilm was the one of the important thing to prevent the
biofouling formation.
Diatom was the dominant eukaryotic marine fouling
organisms. Diatom does passive movement to approach
on a surface because their lack of flagella. Electrostatic
interactions such as coulomb attraction and van der
Waals force was involved in diatom attachement. After
the diatom land on the substrate, it will secret EPS and
reorient themselves along the surface into better
positions, this movement called diatom gliding. EPS of
diatom is composed of carboxylated or sulfated acidic
polysaccharides. Diatom would secret mucilage strand at
their central pore to tightly bind on the substrate (Cao et
al., 2011). Diatom attachment would be prevent because
when they are abundant, it can promote bio-corrosion of
the surface (Silva-Aciares and Riquelme 2008).
Actinobacteria are the group of filamentous bacteria
which are recognized as source of bioactive metabolites.
According to Bérdy (2012), about 13,700 microbial
metabolites are reported derived from this group of
bacteria. Many metabolites have been reported to have
antibiotics, antivirals, and anticancer activity. However,
aquatic and marine Actinobacteria are not explored
widely. Therefore, many novel bioactive compounds can
be harvested (Kumaran et al., 2011). Marine bacteria
group also have been reported produce many type of
bioactive compounds. Satheesh et al. (2012) isolated
coral-associated bacteria with antifouling activity from
Sigmadocia sp.
In the present study, antibiofouling compounds from
Actinobacteria and marine bacteria are still scarce. There
are more novel Actinobacteria and marine bacteria which
have bioactive metabolites that have not studied yet
which may have potential activity against biofouling.
MATERIALS AND METHODS
Biofouling bacteria isolation
Biofouling sample was collected by scrapping everything
that covered the fisherman boat surfaces in Segara Ayu
Beach, Sanur, Bali, Indonesia and transported to
laboratory. One gram of biofouling sample was serially
diluted using sterile seawater. 10-3 to 10-5 dilutions were
spread to Marine Agar (Difco™) and incubated at 28°C
for 1-3 days. Morphologically-different bacterial colonies
were selected, purified, and sub-cultured (Gopikrishnan et
al., 2013). Each biofouling bacteria isolates were tested
for biofilm formation activity using static biofilm assay
(detailed explanation in section 2.5). Biofilm-forming
isolates were used for further assay and identification.
Molecular identification of biofouling bacteria
Biofouling bacteria isolates were identified using
polymerase chain reaction (PCR) amplification of 16S
rDNA gene. The method is optimized from Marchesi et al.
protocol (Marchesi et al., 1998). The PCR master mix and
conditions are described in Table 1 and Table 2,
respectively. Samples were sent to 1st Base Sequencing,
Malaysia for DNA sequencing analysis. DNA sequences
then were processed with SeqTrace 0.9.0 software for
basic local alignment search tool (BLAST) purpose. The
16S rDNA gene sequences were submitted into the
GenBank.
Table 1: Master mix for 16S rDNA amplification.
Solution
Volume (µL)
GoTaq Green Master Mix
12.5
Primer 63f’
1
Primer 1387r’
1
Nuclease free water
9.5
DNA template
1
Total volume
25
Table 2: Polymerase chain reaction condition for 16S
rDNA.
Steps
Time (minutes)
Temperature (oC)
Pre-Denaturation
7
95
Denaturation
0.5
95
Annealing
0.5
55
Elongation
1
72
Post-Elongation
20
72
Hold
∞
4
Cycles
30 cycles
Diatom culture
Three representative biofouling species of diatom
(Nitzschia sp., Navicula sp., and Amphora sp.) were
obtained from the culture collection of Faculty of
Biotechnology, Atma Jaya Catholic University of
Indonesia. The diatoms were isolated from soft coral
Dendronephthya sp. and identified morphologically that
based on size, form, and color characteristics
(Hutagalung et al., 2014). The cultures were grown in f/2
media (Guillard 1975) at room temperature with 24 h light,
maintained in 5 mL glass tubes and sub-cultured every
two weeks.
Crude extract production
Five Actinobacteria isolates (CW01, CW17, SW03,
SW12, and TB12) were obtained from previous study,
which isolated from various aquatic environments (Table
3). Each isolates were sub-cultured in Glucose Yeast Malt
Extract Starch Agar (GYMS) (glucose 4 g/L, yeast extract

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XXX ISSN (print): 1823-8262, ISSN (online): 2231-7538
4 g/L, malt extract 10 g/L, starch 20 g/L, CaCO3 2 g/L,
and bacteriological agar 12 g/L) and incubated at 28°C for
7 days. Fermentation was done using Tryptone Soy Broth
(Oxoid) at 28°C 120 rpm for 7 days. After that, the broths
were centrifuged at 7,798 × g, 4°C for 15 minutes. Then,
fourteen coral-associated marine bacteria (BB 08-1, BF
04-2, BF 06-2, BF 08-2, BF 09-2, BF 13-4, BF 14-2, BF
15-2, BB 07-6, BC 10-1, BC 11-5, BC 12-4, BC 13-2, and
BF 05-4) from previous studies were used. These
specimens were isolated from hard coral and soft coral in
Indonesia (Table 4). Each marine bacteria isolates were
inoculated in Marine Broth (Oxoid) and incubated at
28°C125 rpm for 3 days. The cultures were centrifuged at
8,000 rpm, 4°C for 20 minutes. Collected supernatants
were kept under 4°C until further assay were performed.
Table 3: Actinobacteriaisolates origin and genus.
Isolates
Origin
Genus/Species
Accession
Number
CW01
CuncaWulang
River at West
Flores
Arthrobacter
sp.
JX434848
CW17
CuncaWulang
River at West
Flores
Streptomyces
sp.
JX434845
SW03
Paddy Field at
Gancahan 8
Village, Sleman
Streptomyces
sp.
JX434841
SW12
Paddy Field at
Gancahan 8
Village, Sleman
S. carpaticus
JX434849
TB12
TelagaBiru Lake at
Green Botanical
Garden, Cibodas
A. mysorens
JX434842
Table 4: Marine bacteria isolates origin
Isolates
Coral Species
Origin
Soft Coral
BB 08-1
Lobophytum sp.
Bali
BF 04-2
Scleronephthya sp.
Lampung
BF 06-2
Sinulariamollis
Lampung
BF 08-2
Scleronephthya sp.
Kapuran
BF 09-2
Heteroxenia
Bali
BF13-4
Studeriotes sp.
Kapuran
BF 14-2
Nephthyigorgia sp.
Kapuran
BF15-2
Sinularia lobata
Seribu Island
Hard Coral
BB 07-2
Tubastrea micrantha
Cilegon
BC 10-1
Acropora simplex
Lombok
BC 11-5
A. desalwi
Bali
BC 12-4
A. echinata
Kendari
BC 13-2
Haliclona sp.
Karawang
BF 05-4
Brotyllus sp.
Kapuan
Biofilm inhibition assay
Biofilm-forming bacteria were grown in Brain Heart
Infusion Broth (Oxoid) with 1% glucose supplementation
and incubated overnight at 28°C, 125 rpm. Bacterial
densities were measured until reach absorbance value
OD600 = 0.132 (McFarland 0.5) using spectrophotometer
and dilution were done if needed. Biofilm inhibition assay
were done using static biofilm assay using 96-well
microplate (IWAKI). Each well contains 200 μL
suspensions with 10% (v/v) supernatants. After two days
of incubation at 28°C, spent medium was discarded, and
rinsed twice using sterile distilled water. Adherent biofilm
was then stained with crystal violet solution for 30
minutes, and subsequently rinsed five times using sterile
distilled water and air dried. Crystal violet solutions were
then solubilized with 200 µL absolute ethanol. Then, 200
µL solubilized crystal violet were transferred to new
microplate, and the optical density were determined at
595 nm using microplate reader Biorad 680 Microplate
Reader (Stepanović et al., 2007). Biofilm inhibitory
activities of each supernatant were determined with
Equation (1).
(1)
Note:
1. Positive control is biofouling bacteria growth in
medium without supernatants added.
2. Negative control is 200 μL medium.
Diatom attachment inhibition assay
Assay of diatom attachment inhibition were followed Hong
and Cho (Hong and Cho, 2013) with some modifications.
The initial cell density of diatom cell suspension was
counted and necessary dilutions using f/2 medium were
made to obtain 1 × 105 cells/mL. Thirty milliliter of diatom
suspension was transferred to 50 mL centrifugal tube and
10% (v/v) supernatants were added. Then, sterile object
glass (2.5 × 7.5 cm) was placed in the tube for facilitating
the attachment. The tubes were lied down statically and
incubated at room temperature with 24 h light conditions.
The object glass was completely drowned and not moved.
After three days of incubation, the object glasses were
removed and the cells attached on the object glasses
were scraped, then it was diluted in 1 mL of aquades. The
cells were counted using hemocytometer. Diatom
attachment inhibition activities of the supernatants were
determined with Equation (2).
(2)
RESULTS
Isolation and identification of biofouling bacteria
Eight of eleven biofouling isolates showed the ability to
form biofilm. Molecular identification using BLASTN
revealed that those bacteria are closely related (99%
similarity) with members of genus Vibrio, Pseudomonas
and Shewanella (Table 5). This result showed gram-
negative bacteria, especially Vibrio, were dominant
bacteria in biofuling community.

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Table 5: Biofouling biofilm-forming bacteria molecular
identification using PCR 16S rDNA gene.
Isolate
Closest relatives
Similarity
(%)
Accession
Number
FB1
Vibrio neocaledonicus
99
KP744374
FB2
Vibrio neocaledonicus
99
KP744375
FB3
Pseudomonas stutzeri
99
KP744376
FB5
Vibrio neocaledonicus
99
KP744377
FB6
Vibrio alginolyticus
99
KP744378
FB7
Vibrio natriegens
99
KP744379
FB8
Shewanella algae
99
KP744380
FB9
Shewanella algae
99
KP744381
Biofilm inhibition activity
Supernatants extracted from Actinobacteria showed
inhibition activity against attachment of fouling bacteria
(Figure 1). CW17 supernatant was the only one who had
the inhibition activity against all fouling bacteria biofilms,
followed by CW01 (no inhibition against FB7) and TB12
(no inhibition against FB6). However, the overall activity
of CW17 was lower than TB12 supernatant, except
against FB6 and FB9.
Supernatants from fourteen marine bacteria showed
different inhibitory activity against eight fouling bacteria
(Figure 2). The highest inhibition (95%) of supernatant
was shown by BC 13-2 against biofilm formation of FB3
and (94%) BC 11-5 against biofilm formation of FB 2. On
the other hand, BC 13-4 showed the lowest inhibition
activitiy (3.15%) against FB3 biofilm formation.
Meanwhile, inhibition against FB7 biofilm formation was
shown by the most marine of bacteria; i.e., BB 08-1, BF
04-2, BF 06-2, BF 08-2, BF 09-2, BF 13-4, BF 14-2, and
BF 15-2.This result showed that bioactive compounds
from these marine bacteria have antibiofilm activity
against specific fouling bacteria.
Diatom attachment inhibition activity
Five Actinobacteria and fourteen marine bacteria
supernatants showed inhibition activity against Amphora,
Navicula, and Nitzchia. These species are common
fouling diatoms found on biofouling surface (Yang et al.,
2014). From the five isolates, CW01 and CW17
supernatants showed broad spectrum activity, whose
activity was above 60%, especially for CW01 having the
highest inhibition against Amphora (83.7%). However, the
highest inhibition activity against Navicula (98.35%) and
Nitzschia (99.28%) were generated by TB12 supernatant
(Figure3). Following to the result, almost all of the marine
bacteria supernatants could inhibit the three
representative diatoms attachment on the substrate.
Highest attachment inhibition activity (91.8%) was shown
by BB 08-1 against Nitzchia, while BF 13-4 showed the
lowest attachment inhibitory activity (19.96 %) against
Amphora (Figure 4).
Figure1: Biofilm inhibition activity of 10% (v/v) Actinomycetes extracts against fouling bacteria isolates.

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Figure 2: Biofilm inhibition activity of 10% (v/v) marine bacteria extracts against fouling bacteria isolates.
Figure 3: Attachment inhibition activity of 10% (v/v) Actinobacteria extract against fouling diatoms.
Figure4: Attachment inhibition activity of 10% (v/v) marine bacteria extract against fouling diatoms.

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DISCUSSION
The eight isolated and identified biofouling bacteria (FB1,
FB2, FB3, FB5, FB6, FB7, FB8, and FB9) were known to
have biofilm-forming activity (Casey et al., 2000; Snoussi
et al., 2008; Chalkiadakis et al., 2013; Martín-Rodríguez
et al., 2014), except Pseudomonas stutzeri. However, P.
stutzeri has flagella and pili structure that help in bacteria
attachment (Lalucat et al., 2006). This leads to false
perception of biofilm formation by FB3. Some members of
genus Bacillus, Pseudomonas, Staphylococcus, Vibrio,
Aeromonas, Micrococcus, Alcaligenes, Proteus, and
Shewanella have been successfully isolated from marine
biofouling samples (Dhanasekaran et al., 2009; Kumaran
et al., 2011; Gopikrishnan et al.,2013). All of our bacterial
isolates are belongs to those genera. Moreover, S. algae
was reported as contributors in biofouling formation
(Martín-Rodríguez et al., 2014). Therefore, these isolates
can represent biofouling bacteria as model organisms.
There were three supernatants could inhibit FB3
completely. This might related to the inability of
Pseudomonas stutzeri to form biofilm (Lalucat et al.,
2006). The absence of biofilms on FB3 might allow the
inhibition of the adhesion directly (Lalucat et al., 2006).
FB8 isolate could be inhibited by every Actinobacteria
supernatants, while FB9 could not. FB1, FB2, and FB5,
which share same similarity, also showed different
inhibition results between them. This can be deduced that
they have different properties in mechanism or structure
involved in biofilm formation. Therefore, further
researches are needed to reveal the distinct properties
between bacteria, which will lead into strain classification.
CW17, SW03, and SW12 are belongs to
Streptomyces genus, while CW01 and TB12 are
Arthrobacter genus. Chen et al. (2013), reported that
Streptomyces sp. and Arthrobacter sp. had the ability to
interfere quorum sensing, which is known as quorum
quenching. They could produce acyl homoserine lactone
(AHL)-degrading enzyme, like acylase and lactonase.
Some strains producing those enzymes have been
identified in Streptomyces, while in Arthrobacter only
lactonase-producer was found (Chen et al., 2013). In
other study, Streptomyces libani and several marine
Actinobacteria were screened to have inhibition activity
against biofouling bacteria (Kumaran et al., 2011).
Streptomyces filamentous also has been reported to have
good antifouling activity (Bavya et al., 2011). Molecular
analysis should be done to reveal their shared traits and
genetic diversity, which leads to find a specific
mechanism in antifouling activity.
Several marine Actinobacteria were found to produce
furanone compounds as biofilm inhibition mechanisms. 2-
furanone structure in the compounds responsible in
interrupting quorum sensing, which is correlated with
biofilm formation (Xu et al., 2010). Other mechanism was
reported from two Actinobacteria members, Streptomyces
akiyoshinensis and Actinobacterium sp. They produced
potent biofilm inhibitor of Streptococcus pyogenes by
reducing cells surface hydrophobicity, which plays
important role in cell adhesion and colonization
(Nithyanand et al., 2010). These characteristics may be
used as screening method for novel Actinobacteria with
antibiofouling activity.
As for marine bacteria, only BC 11-5 supernatant
could inhibit the biofilm formation of V. neocaledonicus
while the other marine bacteria crude extract did not show
that activity. V. neocaledonicus is a new member of the
Vibrio genus bacteria, recent study showed V.
neocaledonicus could produce a different type of extra
polymeric substance (EPS). This EPS exhibits a high N-
acetyl-hexosamines and uronic acid content with a low
amount of neutral sugar. The different EPS structure of V.
neocaledonicus may happen due to the evolution. It is
lead to the better defense mechanism that refer to the
result showed limited bacteria can inhibit this bacteria
(Chalkiadakis et al., 2013).
Many types of coral-assosciated bacteria was isolated
from Acroporadigitifera which Bacillus genus was
represent the most abundant (Thenmozhi et al., 2009).
Their Bacillus genus crude extract show promising result
that can be used for antibiofilm and quorum quenching
agent. In fact, marine bacteria with antifouling activity
were often found associated with coral (Thenmozhi et al.,
2009). These coral-associated bacteria mostly play an
important role for the coral and sponge. The sponges, as
sessile filter-feeder animals, could not produce
antibiofouling agent by itself. Positive symbiotic with coral-
associated bacteria that produce antibiofouling agent is
evolved antifouling strategies to protect themselves
against micro- and subsequent macrobiofouling
processes (Müller et al., 2013). Many bioactive
compounds of marine bacteria with antibiofouling activity
have been characterized and identified. Biosurfactant was
the common bioactive compound that marine bacteria
produce to inhibit the formation biofilm. Biosurfactant can
reduce the surface or interfacial tension for resist
attachment of biofilm in the surface (Dusane et al., 2011).
Amphora was the least inhibited diatoms by
Actinobacteria and marine bacteria crude extracts. This
result might be appropriate with the properties of
Amphora, which has two raphes on one side (ventral
surface), while Navicula and Nitzschia have single raphes
on ventral and dorsal side of the cell (Arce et al., 2004;
Wigglesworth-cooksey and Cooksey, 2005; Jin et al.,
2013). This might lead to stronger attachment for
Amphora to the substrate. The varieties of diatom
morphological structure could generate different
mechanism of attachment in every diatom (Hilaluddin et
al., 2011).
Our study did not analyse the mechanism or
determine the spesific compound that responsible for
inhibition of diatom attachment. Recent study reported
that two furanone derivatives had been successfully
isolated from Streptomyces violaceoruber SCH-09 and
showed antifouling activities against Navicula annexa and
other fouling organism. This marine Actinobacteria was
isolated from seaweed Undaria pinnatifida surface. The
result also showed these compounds had no effect
against non-target organism at the same concentration
(Hong and Cho, 2013). Coral-associated bacteria was the

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XXX ISSN (print): 1823-8262, ISSN (online): 2231-7538
one natural potential for prevent the diatom attachment on
substrate. Polybrominated diphenyl ethers had been
identified in a marine sponge (genus Dysidea) causing an
inhibition of diatom growth (Ortlepp et al., 2008).
CONCLUSION
Extracts with broad spectrum activity against biofouling
bacteria was extracted from Streptomyces sp. (CW 17),
whereas Arthrobacter sp. (CW 01) against diatoms, and
extracts with overall highest activity was extracted from
Arthrobacter mysorens (TB 12). Extracts of marine
bacteria BC 11-5 had the most promising ability to inhibit
biofilm among others. Even though our study has not
discovered either the compound or mechanism of the
inhibition activity, the promising results showed they have
great potential, especially in inhibiting marine biofouling
pioneer organisms, which will become the alternative
solution against biofouling. Hence, further study is needed
to characterize these compounds, in order to find out the
inhibition mechanism, and screen inhibition activity with
other organism contributed to the formation of biofouling.
ACKNOWLEDGEMENTS
Authors appreciated the help of Cindy Ciputra for
collecting the samples. Authors also thanked Atma Jaya
Catholic Univerisity of Indonesia for providing the
research facility. This work was supported in part by the
Ministry of Land, Transport and Maritime Affairs, South
Korea and Hibah Kompetensi research grant 2014 from
Directorate General of Higher Education, Indonesia.
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