Journal of Environmental Protection, 2011, 2, 1245-1249
doi:10.4236/jep.2011.29143 Published Online October 2011 (http://www.scirp.org/journal/jep)
Antifouling Activity of Bacterial Symbionts of
Seagrasses against Marine Biofilm-Forming
Bacteria
Bintang Marhaeni1,2, Ocky Karna Radjasa3,4*, Miftahuddin Majid Khoeri3, Agus Sabdono4,
Dietriech G. Bengen1, Herawati Sudoyo3
1Graduate School of Marine Sciences, Bogor Agricultural University, Bogor, West Java, Indonesia; 2Department of Fisheries and
Marine Science, Soedirman University, Purwokerto, Central Java, Indonesia; 3Marine Microbiology Unit, Eijkman Institute for Mo-
lecular Biology, Jakarta, Indonesia; 4Department of Marine Science, Diponegoro University, Semarang, Central Java, Indonesia.
Received September 6th, 2011; revised October 5th, 2011; accepted November 3rd, 2011.
ABSTRACT
Marine biofouling has been regarded as a serious problem in the marine environment. The application of TBT and
other heavy metal-based antifoulants has created another environmental problem. The present study explored the possi-
ble role of baterial symbionts of seagrasses Thalassia hemprichii, and Enhalus acoroides, which were successfully
screened for antifouling activity against marine biofilm-forming bacteria isolated from the surrounding colonies of
seagrasses. Bacterial symbionts were isolated and tested against biofilm-forming bacteria resulted in 4 bacterial sym-
bionts capable of inhibiting the growth biofilm-forming isolates. Molecular identification based on 16S rRNA gene se-
quences revealed that the a ctive bacterial symbionts belonged to the members of the genera Bacillu s and Virgibacillus.
Further tests of the crude extracts of the active bacterial symbionts supported the potential of these symbionts as the
alternative source of environmentally friendly marine antifoulants.
Keywords: Biofouling, Antifoulant, Bacterial Symbionts, Seagrasses
1. Introduction
A regularly observed phenomenon, marine biofouling, is
not only a natural process as a result of organism growth
on underwater surfaces [1], but is also undesirable due to
massive economic losses to marine industries. Primary
biofilm, another phenomenon and is part of marine mi-
crofouling creates further threat by facilitating the atta-
chment and metamorphosis of fouling organisms [2].
Bacteria as part of the marine microbial population, have
been considered as the primary colon izers within biofilm
and play a role as dominant components [3]. It is then
reasonable to manipulate the role of these bacterial film
by disrupting their growth by applying marine antifou-
lants.
Along with long-term application of tributyl-tin (TBT)-
based antifouling paints, there has been an increased
concerns due to the increased concentrations resulted in
environmental hazards to marine marine ecosystems. It
was then the reason for EU countries in 1989, followed
by International Maritime Organization (IMO) and the
Maritime Environment Protection Committee (MPEC) to
restrict the use of TBT [4]. Since then, the development
of alternative environmentally friendly antifoulants has
been carried out to find alternative replacements.
Sea grasses are one of the most productive coastal eco-
systems and a rich source of secondary metabolites with
ecologically important roles such as preventing surface
fouling [5]. As an effort to explore the potential of
marine antifoulants produced by seagrasses [6], it was
reported the potential of seagrasses Cymodocea serrulata
and Syringodium isoetifolium in inhibiting the growth of
marine biofilm-forming bacteria. This finding has
confirmed the possible manipulation of biofilm-forming
bacteria in reducing the effects of marine biofouling.
The fact that the problem of supply has hampered the
development of most secondary metabolites from marine
organisms and plants, thus, it is important to highlight
the possible role of marine bacteria associated with sea-
grasses in providing an alternative to the commercial
metal-based antifouling coatings. Bacteria-seagrass asso-
ciation that occurs on the seagrass surface then could be

Antifouling Activity of Bacterial Symbionts of Seagrasses against Marine Biofilm-Forming Bacteria 1246
of great interest to search for potential use as commercial
antifoulants.
In this work, we report the potential o f marine bacteria
associated with seagrasses Thalassia hemprichii, and En-
halus acoroides for controlling the growth marine bio-
film-forming bacteria. Antibacterial assays of the bac-
terial symbionts and their crude extracts confirmed the
potential role of these symbionts as the source of envi-
ronmentally friendly marine antifoulants.
2. Materials and Methods
2.1. Sampling and Isolation of Bacterial
Symbionts of Seagrasses
Colonies of seagrasses Thalassia hemprichii, and Enha-
lus acoroides were collected from seagrass beds in the
Teluk Awur waters, Jepara, Central Java, Indonesia (Fi-
gure 1) by hands. Upon collection seagrass colonies
were put into sterile plastic bags (Whirl-Pak, Nasco,
USA. The colonies were then rinsed with sterile seawater
and scraped off with a sterile knife for the isolation of
epiphytes. As for isolation of endophytes, the surfaces of
seagrass leafs were sterilized with 70% alcohol, and the
leafs were cut to open the inner parts. The inner surfaces
were scraped off with a sterile cutter. The resultant tis-
sues were serially diluted, spread on 1/2 strength ZoBell
2216E marine agar medium and incubated at room tem-
perature for 48 hours. Purification and isolation of bac-
terial colonies were performed by using streak plates on
the basis of morphological features [7].
2.2. Isolation of Marine Biofilm-Forming
Bacteria
Isolation was carried out according to a modified method
[8]. Four pre-sterilized wooden slides had been de-
ployed in 4 different directions around each seagrass co-
lonies for a week. The biofilm developed in these woo-
den slides were then aseptically rinsed with sterile sea-
water, scrapped off with a sterile knife and diluted. One
hundred µl of each dilution was spreaded onto 1/2
strength ZoBell 2216E and incubated at room tempe-
rature for 48 hours. Colonies with distinguished feature
were selected and purified.
2.3. Extraction of Active Bacterial Symbionts
Each of active bacterial symbiont was grown in a 500 ml
flask containing ZoBell2216E broth medium and in-
cubated for 4 d in a shaker. The cultures were then cen-
trifuged at a speed of 12,000 rpm for 5 minutes and the
supernatants were collected. Supernatants were then
extracted based on liquid-liquid extraction on separatory
funnel (4 supernatan: 1 n-hexane). The extracts were
concentrated by using a rotary evaporator and kept in
freezer until antifouling activity was performed.
2.4. Antifouling Activity Test
Antifouling test of bacterial symbionts of seagrasses against
marine biofilm-forming bacteria was performed by using
an overlay method. Culture of each marine biofilm-forming
bacterium in the logarithmic phase (ca. 10 9 cells·ml–1) was
Figure 1. Sampling site at Teluk Awur, Jepara, Central Java, Indonesia.
C opyright © 2011 SciRes. JEP

Antifouling Activity of Bacterial Symbionts of Seagrasses against Marine Biofilm-Forming Bacteria1247
mixed with ZoBell 2216E soft agar medium (1% v/v),
which were then poured on to the respective agar surface
previously inoculated with bacterial symbionts and in-
cubated for 4 d. The plates were then incubated at room
temperature for 48 hours. As for bacterial extracts, an
agar diffusion method [9] was performed. Tweenty mic-
rolitters of each extract was poured onto a paper disk (8
mm, Advantec, Toyo Roshi, Tokyo, Japan) previously
put on agar surfaces containing each biofilm-forming
bacterium. Antibacterial activity was defined by the for-
mation of inhibition zones around the bacterial colonies.
2.5. PCR Amplification and Sequencing of 16S
rRNA Gene Fragments
PCR amplification of partial 16S rRNA gene of active
strains and marine biofilm-forming bacteria, purification
of PCR products and subsequent sequencing analysis
were performed [9]. The determined DNA sequences of
strains were then compared for homology to the BLAST
database. A phylogen etic tree was constructed using ma-
ximum-likelihood analysis and Phylogenetic analysis
was performed with the PAUP software package [10].
2.6. Nucleotide Sequence Accession Number
The 16S rRNA gene sequences of the active bacterial
symbionts of seagrasses have been deposited into the
DNA Database Bank of Japan with the following acces-
sion numbers: AB665240-43.
3. Results and Discussion
This work represents an effort towards finding alternative
solution to the problem of marine biofouling, a serious
problem faced by worldwide maritimes industries [11]
and leads to huge economic losses [12].
Four bacterial symbionts of seagrasses were found to
inhibit the growth of marine biofilm-forming bacteria
ranging from 1 - 6 bacteria (Table 1). Out of 4 active
isolates, 3 isolates were obtained from seagrass E. co-
roides and 1 isolate was from seagrass T. hemprichii.
Interestingly, the crude extracts of these active bacterial
symbionts showed more pronounced growth inhibition
with a range of 6 - 14 b ac te r ia (Table 1).
The potential of seagrass species of Cymodocea serru-
lata and Syringodium isoetifolium collected from the
coastal area of Tuticorin has confirmed the biological
activity of these seagrasses against biofilm-forming bac-
teria [6]. It is very reasonable that biofilm-forming bac-
teria have been the primary target for controlling bio-
fouling due to their important cues for larval settlement
and development of biofouling organisms [13]. However,
considering the issue of bottleneck of most marine na-
tural products and the need of sustainable use of coastal
ecosystems as well as the concerns regarding the colle-
ction marine resources for discovery and development of
marine natural products [14], has turned marine microor-
ganisms such as marine microbial symbionts as a potent
source of marine antifoulings. Unfortunately, less data
are available of the antifouling compounds produced by
marine bacteria [15-17].
The present work show the biological activity against
marine biofilm-forming bacteria from the surrounding
collonies of these seagrass species that were exposed into
the same ecological conditions. Both endophite and epi-
phytes were found to inhibit the growth of marine bio-
film-forming bacteria. Interestingly, all extracts from the
active isolates showed broad spectrum of antifouling
activity against marine biofilm-forming bacteria. This
finding is very important in regards to the fact that bac-
teria are the primary colonizers and play significant roles
in the mediatory effects on the settlement and deve-
lopment of biofouling [13,18]. The higher number of
biofilm-forming bacteria inhibited by the extracts of
active symbionts may indicate the chemical diversity of
secondary metabolites produced by the bacterial sym-
bionts. In addition, the fact that non-polar bacterial ex-
tracts were found to be active, is a higly desirable in term
of possible application of in the marine environment so
that it won’t be washed out easily. Furthermore, th e work
also confirmed the bacterial symbionts of seagrass spe-
cies T. hemprichii and E. acoroides provide sustainable
use of seagrass ecosystems in term of developing a large-
scale cultures of bacterial symbionts for possible com-
mercial development.
The active bacterial isolates were closely related to th e
members of genus Bacillus and Virgibacillus with high-
est homology of 98% - 99% (Table 2). Molecular phylo-
genetic (Figure 2) shows that all active isolates belonged
to the family Bacillaceae. It is revealed that isolates
EA2.1 and EA.6 belong to Bacillus aquamaris and Baci-
llus sp., respectively. On the other hand, isolates ESJ.5
and TT.9 belong to Virgibacillus olivae and Virgibacillus
marismortui.
A report on the biological activity of bacterial sym-
bionts of sponge Pseudoceratina purpurea shows that
they actively inhibited th e growth of fouling bacteria iso-
lated from natural marine biofilm. The 3 active isolates
belonged to genus Bacillus and 1 isolate belonged to Vir-
gibacillus and were also found to show a broad spectrum
biological activity against marine biofilm-forming bac-
teria [19].
In another study, various marine bacteria associated
with a range of marine surfaces were screened and
showed potential application as natural antifoulants and
found that the isolates were related to Bacillus mojaven-
sis and Bacillus firmus [18]. A screening on bacterial
symbionts of softcoral Sinularia sp.and it was found

Antifouling Activity of Bacterial Symbionts of Seagrasses against Marine Biofilm-Forming Bacteria 1248
Table 1. Growth inhibition of biofilm-forming bacteria by bacterial symbionts and their crude extracts.
No Isolate Type of symbiont No of biofim b a c t e ria inhibited by
bacterial symbiont No of biofilm bacteria inhibited by extract of symbiont
1 EA2.1 Ephyphite 3 8
2 EA.6 Ephyphite 1 14
3 ESJ.5 Endophite 6 6
4 TT.9 Ephyphite 2 9
Table 2. Molecular identification of active bacterial symbionts of seagrasses.
No Isolate Source Closest relative Similarity (%) Accession No.
1 EA2.1 E. acoroides Bacillus aquamaris 99 EU443752
2 EA.6 E. acoroides Bacillus sp. 98 HQ704707
3 ESJ.5 E. acoroides Virgibacillu s olivae 99 HM179216
4 TT.9 T. hemprichii Virgibacillus maris mortui 99 GU172145
Figure 2. Phylogenetic affiliation of bacterial symbionts of seagrasses.
C opyright © 2011 SciRes. JEP

Antifouling Activity of Bacterial Symbionts of Seagrasses against Marine Biofilm-Forming Bacteria1249
that the member of genus Virgibacill us inhibited the
growth of MDR strain Staphylococcus aureus [19].
In conclusion, the present study represents the poten-
tial of bacterial symbiont of seagrass species T. hem-
prichii and E.acoroides as the alternative source of en-
vironmentally friendly marine antifoulant. The so far un-
characterized secondary metabolites from these active
bacterial symbionts deserve futher work on the bioassay-
guided isolation and purification of the active antifouling
compounds.
4. Acknowledgements
We appreciated the help of the technicians of Marine Sta-
tion of Diponegoro University during the sampling. This
work was partly supported by a research grant from Bio-
security Engagement Program (BEP), grant #IDB1-
21003-JA-08 through the US Civilian Research & De-
velopment Foundation (CRDF). Dr. Radjasa was awarded
grants from International Foundation for Science (IFS),
Sweden (Contract. No. F/3965-2) and Directorate Ge-
neral of Higher Education, Ministry of National Edu-
cation within “Hibah Strategis Nasional” scheme.
REFERENCES
[1] R. C. Pereira, A. G. V. Carvalho, B. A. P. Gama and R.
Coutinho, “Field Experimental Evaluation of Secondary
Metabolites from Marine Invertebrates as Antifoulants,”
Brazilian Journal of Biology, Vol. 62, No. 2, 2002, pp.
311-320. doi:10.1590/S1519-69842002000200015
[2] M. E. Callow and J. A. Callow, “Marine Biofouling: A
Sticky Problem,” Biologist, Vol. 49, 2002, pp. 1-4.
[3] H. Dang and C. R. Lovell, “Bacterial Colonization and
Early Succession on Surfaces in Marine Waters as Deter-
mined by Amplified rRNA Gene Restriction Analysis and
Sequence Analysis of 16S rRNA Genes,” Applied and
Environmental Microbiology, Vol. 66, No. 2, 2000, pp.
467-475.
[4] I. K. Konstantinou and T. A. Albanis, “Worldwide Oc-
currence and Effects of Antifouling Paint Booster Bio-
cides in the Aquatic Environment: A Review,” Environ-
ment International, Vol. 30, No. 2, 2004, pp. 235-248.
doi:10.1016/S0160-4120(03)00176-4
[5] P. R. Jensen, K. M. Jenkins, D. Porter and W. Fenical,
“Evidence that a New Antibiotic Flavone Glycoside
Chemically Defends the Sea Grass Thalassia testudinum
against Zoosporic Fungi,” Applied and Environmental
Microbiology, Vol. 64, No. 4, 1998, pp. 1490-1496.
[6] P. Mayavu, S. Sugesh and V. J. Ravindran, “Antibacterial
Activity of Seagrass Species against Biofilm Forming
Bacteria,” Research Journal of Microbiology, Vol. 4, No.
8, 2009, pp. 314-319.
[7] M. T. Madigan, J. M. Martinko, J. Parker and T. D. Brock,
“Biology of Microorganisms,” Prentice-Hall, Inc., New
Jersey, USA, 2000.
[8] O. K. Radjasa, T. Martens, H.-P. Grossart, T. Brinkoff, A.
Sabdono and M. Simon, “Antagonistic Activity of a Ma-
rine Bacterium Pseudoalteromonas luteoviolacea TAB4.2
Associated with Coral Acropora sp,” Journal of Biologi-
cal Sciences, Vol. 7, No. 2, 2007, pp. 239-246.
[9] O. K. Radjasa and A. Sabdono, “Ecological Role of a
Softcoral-Associated Bacterium Arthrobacter sp. on Ma-
rine Biofilm-Forming Bacteria,” Microbiology Indonesia,
Vol. 2, No. 2, 2008, pp. 7-12.
[10] S. Abarzua, S. Jakubowski, S. Eckert and P. Fuchs, “Bio-
technological Investigation for the Prevention of Marine
Biofouling II. Blue-Green Algae as a Potential Producers
of Biogenic Agents for the Growth Inhibition of Microo-
fouling Organisms,” Botanica Marina, Vol. 42, 1999, pp.
459-465.
[11] D. M. Yebra, S. Kiil and K. Dam-Johansen, “Antifouling
Technology-Past, Present and Future Steps towards Effi-
cient and Environmentally Friendly Antifouling Coat-
ings,” Progress in Organic Coatings, Vol. 50, No. 2, 2004,
pp. 75-104. doi:10.1016/j.porgcoat.2003.06.001
[12] P.-Y. Qian, S. C. K. lau, H. -U. Dahms, S. Dobrestsov and
T. Harder, “Marine Biofilms as Mediators of Colonization
by Marine Macrooganisms: Implications for Antifouling
and Aquaculture,” Marine Biotechnology, Vol. 9, No. 4,
2007, pp. 399-410.
[13] Sukarmi and O. K. Radjasa, “Bioethical Consideration in
the Search for Bioactive Compounds from Reef’s Inver-
tebrates,” Journal of Applied Sciences, Vol. 7, No. 8,
2007, pp. 1235-1238. doi:10.3923/jas.2007.1235.1238
[14] N. Fusetani, “Biofouling and Antifouling,” Natural
Product Reports, Vol. 21, 2004, pp. 94-104.
[15] S. Dobretsov, H.-U Dahms and P.-Y. Qian, “Inhibition of
Biofouling by Marine Microorganisms and Their Metabo-
lites,” Biofouling, Vol. 22, No. 1, 2006, pp. 43-54.
[16] V. J. Paul, M. P. Puglisi and R. Ritson-Wlliams, “Marine
Chemical Ecology,” Natural Product Reports, Vol. 23,
2006, pp. 153-180.
[17] M. Kanagasabhapathy, H. Sasaki, K. Nakajima, K. N agata
and S. Nagata, “Inhibitory Activities of Surface Associ-
ated Bacteria Isolated from the Marine Sponge Pseudo-
ceratina purpurea,” Environmental Microbiology, Vol. 20,
2005, pp. 178-185.
[18] B. O. Ortega-Morales, M. J. Chan-Bacab, E. Miranda-
Tello, M.-L. Fardeau, J. C. Carrero and T. Stein, “Anti-
fouling Activity of Sessile Bacilli Derived from Marine
Surfaces,” Journal of Industrial Microbiology and Bio-
technology, Vol. 35, 2008, pp. 9-15.
doi:10.1007/s10295-007-0260-2
[19] S. Sulistiyani, S. A. Nugraheni., M. H. Khoeri, A. Sab-
dono, and O. K. Radjasa, “Antibacterial Activity of Bac-
terial Symbionts Softcoral Sinularia sp. against Patho-
genic Resistant Bacteria,” Journal of Coastal Develop-
ment, Vol. 13, No. 2, 2010, pp. 113-118.