Advance Sustainable Science, Engineering and Technology (ASSET)
Vol. 2, No.2, October 2020, pp. 0200205-1 ~ 0200205-5
ISSN: 2715-4211 DOI: https://doi.org/10.26877/asset.v2i2.7041

Characterization of Yellow Pigmented Bacteria Associated with
Gracilaria sp.
Arina Tri Lunggani, Susianna Purwantisari, Siti Nur Jannah
Biology Departement, Faculty of Science and Mathemathics Diponegoro University,
Jl. Prof H. Soedharto, SH, Tembalang, Semarang 50275, Indonesia
rynatri7@gmail.com
Abstract. Research on the kinship analysis of endophytic bacterial isolated from Gracillaria sp
has been carried out. The presence of bacteria associated with Gracilaria sp. has enabled the use
of these bacteria as a source of new bioactive compounds, such as biopigments. The research
aims to isolated bacteria from Gracilaria sp., screened their symbiont bacteria that could
potentially produce pigments. Sampling Gracilaria sp. conducted in the waters of the Island of
Karimunjawa, Jepara. Furthermore, bacterial isolation was carried out, screening for pigmentproducing bacteria and 16S rRNA sequence analysis. Research result showed that the symbiont
bacteria isolate TK 373 produced consistent pigments after several regenerations, in several types
of growth media incubated at room temperature. The results of 16S rDNA identification showed
that the TK 373 isolate had the closest relationship with Pseudoalteromonas sp. with 98.72 %
homology.

Keywords: symbiont bacteria, pigment, Gracilaria sp.

(Received 15 August 2020, Accepted 10 September 2020, Available Online by 30 September 2020)

1. Introduction
Indonesia gets a big advantage because it has a very large marine. These advantages include in the form
of biodiversity, both in the form of community, species and genetic diversity. Indonesia's abundant
natural wealth and biodiversity have prospects for this nation to develop bio-industry. One of these
potential genetic resources is algae.
Various natural products from macro algae metabolites plays a very important role in the natural
product discovery process [ 1,2,3 ]. Algae, has been widely used as a bioactive source metabolites such
as proteins, lipids, mineral salts, polyphenols and polysaccharides which are very useful in various
industries of food, pharmacy, cosmetics [ 4,5 ]. Red algae is a group of algae that has extraordinary
potential, including the pigment content of red algae which has pharmaceutical, nutraceutical and
cosmeceutical prospects [ 6, 7 ].
Marine microorganisms have a very large role in the life cycle at sea. One of them is as epiphytic
microbes or microbes associated with marine organisms. Various scientific publications show that there
are associations of microorganisms with marine organisms that also synthesize secondary metabolites
such as their host organisms, including red algae symbionts [ 8, 9 ]. This is a consideration to explore

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the red algae symbiont bacteria with the hope of providing a wider opportunity to get red algae bacterial
associan that have biological abilities like their host.
2. Methods
2.1. Sampling site
Algae samples of Gracilaria sp. were collected from Karimunjawa island Indonesia. After collection,
algae materials were placed onto sterile bottles containing autoclaved seawater and brought in chilled
condition, to the Integrated Biotechnology Laboratory, Diponegoro University.
2.2. Isolation and purification of bacteria from algae
Fresh algae samples were washed with marine steriled water. Bacteria associated with Gracilaria sp.
isolated according to the method of [ 10 ]. Using a sterile scalpel, seaweed tissue is cut in a size of about
0.1 cm3 and sprayed three times with sterilized sea water. These pieces are then rinsed for surface
sterilization with sterile sea water. Zobell media are used to place Gracilaria sp tissue in an incubator
at 28° C for 72 hours. Different colonies appear morphologically to be separated and purified.
2.3. Phylogenetic Analysis
Genomic DNA was extracted using the standard chelex protocol according to [ 11 ]. The 16S rRNA
gene of the isolate was amplified with the bacterial universal 16S rRNA primers 27F and 1492R. The
optimizations used were: 93° C for 30 seconds, 54.5° C for 30 seconds, and 72 ° C for 1.5 minutes. run
30 cycles. The presence of PCR products was confirmed by electrophoresis on 1 % agarose gels. Basic
Local Alignment Search Tool (BLAST) is used to determine bacterial species that are closely related to
potential bacterial isolates. Phylogenetic trees with bootstrap sampling were reconstructed by the
neighbor-joining method, with 1,000 bootstrap replications was run in MEGA 7 [12, 13 ].
3. Results and Discussion
Efforts to obtain bacterial isolates pigmented brown algae symbionts were carried out using culturedependent methods. This method is a physiological identification process of microbes by first isolating
and purifying the bacteria from the host. Based on the results of isolation and purification, it was found
that several isolates produced pigment. However, after several isolates regeneration processes were
carried out, it turned out that only one isolate was able to show fertile growth and a consistent color,
namely the TK 373 isolate.This isolate produced a bright yellow color when grown on Marine agar and
Nutrient Agar media, incubated at room temperature ( Figure 1 )

Figure 1. Colony characteristics of TK 373 isolates on ZMA media were incubated for 48 hours at room
temperature
The BLAST results showed that the TK 373 isolate had the highest similarity with Pseudoalteromonas
with 98.72% similarity (Table 1). 97-99% similarity for 16S rRNA sequences for bacteria showed
similarity at the genus level, whereas> 99% similarity in gene sequences was the criterion used to
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identify isolates at the species level [14]. The results of phylogenetic reconstruction of bacterial isolates
based on partial gene sequences of 16S rRNA showed the closeness of TK 373 to Pseudoalteromonas
sp. ( Figure 2).
Table 1. Homology of partial gene sequences of 16S rRNA isolate TK 373 with GenBank Database
Isolate
code
TK 373

100%
100%

Homology
Pseudoalteromonas sp.
strain S14

Query
cover (%)
91%

Score Evalue
2084 0,0

Per
Ident
98,72
%

Ascension
number
KX989351.1

MH283805.1 Pseudoalteromonas flavipulchra strain SCSIO 43721
KX529477.1 Pseudoalteromonas flavipulchra strain ES8
JF314511.1 Pseudoalteromonas sp. J021(2011)

100%

MG652490.1 Pseudoalteromonas sp. strain CDA22
100%

MG652484.1 Pseudoalteromonas sp. strain CDM8
KF746896.1 Pseudoalteromonas sp. W-137-6
isolat TK 373

100%
100%
100%

KX989351.1 Pseudoalteromonas sp. strain S14
KP301098.1 Pseudoalteromonas sp. ZJ2406

Figure 2. Reconstruction of the TK 373 phylogenetic tree with reference strains obtained
from NCBI. A neighbor joining tree was calculated using partial 16S rRNA gene sequences.
Many researchers have reviewed the Pseudoalteromonas. [ 15 ] stated that pseudoalteromonas are
true marine bacteria, this is because the Pseudoalteromonas require a sea water base for growth.
Pseudoalteromonas are gram-negative bacteria, motile and have polar flagella, are heterotrophic, and
have GC content between 38 - 50% [16 ]
The genus Pseudoalteromonas is an interesting topic of study. This is due to its potential and
uniqueness. This potential is the productive capacity of its metabolite production, while its uniqueness
is seen in terms of its association with other organisms [17 ]. Based on these two points of view, it can
be said that their ability to associate with other organisms is the key to the abundance of potential
metabolites from the Genus Pseudoalteromonas [15 ]. The results of this study also support this
argument. Isolate TK 373 was related to Genus Pseudoalteromonas, isolated from Gracilaria sp. on
sampling sites in marine waters.
The metabolite production capacity in Pseudoalteromonas is usually associated with pigmentation,
for example, P. tunicata CCUG 26757 and P. rubra DSM 6842 strains produce various pigments that are
involved in antibacterial and antifungal activity [18,19,20,21]
Isolate TK 373 produces a bright yellow color. This of course gives hope that these isolates are
potential. Screening for bioactive potential needs to be done as part of the bioprospection of potential
local isolates that can be applied in the industrial sector.

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4. Conclusion
Research on molecular characterization and morphotypes of local bacterial isolates with symbionts in
Gracilaria sp. has provided findings that TK 373 isolate has the closest similarity to Pseudoalteromonas,
one of the potential genera where most of its members are able to produce pigments that can be explored
further for its potential.

Acknowledgements
For all assistance during the research, Siu Semar Saratu Langden was greatly appreciated.
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