Selective Isolation of Actinomycetes from Mangrove Forest of

International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia)
Selective Isolation of Actinomycetes from
Mangrove Forest of Pahang, Malaysia
Nurfathiah Abdul Malek, Ahmed Jalal Khan Chowdhury, Zarina Zainuddin, and
Zaima Azira Zainal Abidin

uneconomical rediscovery of known compounds. Thus, a new
resource is essential to circumvent this issue. Thereby,
researchers are now looking forward on the underexplored
habitats such as marine ecosystem, dunes, volcanic zones,
deserts and mangrove forests as new reservoirs of putative
actinomycetes.
Mangrove forests are highly productive ecosystems which
comprise of unique woody plant communities and located in
tropical and subtropical coastal area [1],[8]. According to Ara
[9], mangroves form unique saline environments under the
influence of tidal flow, hence the muddy alluvial soil due to
the intermittent flooding. Mangrove ecosystems are
nutritionally versatile as they are highly rich in organic matter,
nitrogen and sulfur content which can be used by living
microorganisms. Thus, it is believed that mangrove
ecosystems have the potential of becoming new reservoir for
highly diverse actinomycetes as demonstrated by the isolation
of Micromonospora rifamycinica [10] and Verrucosispora
wenchangensis [11]. Located in the world tropical belt,
Malaysia is blessed with abundant mangrove forest,
particularly in the west coast of Peninsular Malaysia [12].
Pahang mangrove forest which is situated on the east coast
of Penisular Malaysia, is a poorly studied ecosystem.
Assessment of actinomycetes diversity in Pahang mangrove
forest is not attempted so far, hence this location is foreseen to
be able to provide rich source of actinomycetes. The primary
aim of this study was to determine the diversity of culturable
actinomycetes from mangrove forest of Pahang. The
efficiency of various selective isolation procedures employed
were also discussed.
Abstract—Actinomycetes which abundantly found in nature are
renowned for their unique capabilities to produce biologically active
metabolites. Poorly studied mangrove ecosystem offers a promising
natural resource of new actinomycetes due to their high species
diversity. This study aims to assess the diversity of actinomycetes
from mangrove forest of Pahang, Malaysia. Various selective
isolation media and pre-treatments were employed for maximum
isolation of mangrove actinomycetes. A total of 4850 actinomycete
isolates had been successfully recovered. It was observed that
inorganic-salt starch agar (ISP4) displayed the highest percentage of
actinomycetes recovery (31.7 %). Starch-yeast extract (SYE) and
starch casein agar (SCA) were found to be more efficient in isolating
wide variety of actinomycetes based on their morphological
appearance. It was also observed that wet heat pre-treatment was
more effective in isolating mangrove actinomycetes as 81.4 % of
total isolates were yielded using this technique. On the other hand,
dry heat treatment was better in the enumeration of spore-forming
actinomycetes despite lower number of isolates recovered. Of both
pre-treatments, station 1 which is located within the deep mangrove
forest area recorded the highest recovery (23.7 %), with
Streptomyces and Micromonospora-like isolates being the most
predominant observed from all sampling sites. The presence of
relatively
large
number
of
isolates
suggests
that
Pahang mangrove forest is a potentially rich source of actinomycetes.
Keywords—Mangrove actinomycetes, Pahang, pre-treatments,
selective isolation.
I. INTRODUCTION
Filamentous bacteria within the order of Actinomycetales or
else known as actinomycetes are aerobic and Gram-positive
bacteria that belong to the class Actinobacteria [1], [2]. These
slow growing prokaryotes are widely distributed in nature.
They can be found virtually in every natural substrate; the
water, air, soil, foodstuffs, manure and composts [3].
Actinomyetes have been continuously reported as the main
prolific producers of microbial bioactive secondary
metabolites for potential agricultural, pharmaceutical and
industrial applications [4]-[6]. More than 70% of the
commercially available active compounds originated from
actinomycetes [7]. Extensive isolation and screening of
actinomycetes from soil however, has lowered the rate of
discovery novel biological active compounds owing to the
II. MATERIAL AND METHODS
A. Study Area and Location of Sampling Sites
Pahang mangrove forest which covers an area of 343 ha is
located in Kuantan, the capital state of Pahang, Malaysia. It is
situated within Kuantan estuary which mainly influenced by
Kuantan river that flows out to South China Sea. This study
area experience semidiurnal tides that lies in wet tropical area
[12]. According to Jalal et al. [13], the brackish water
ecosystem surrounding the Pahang mangrove forest serves as
a natural resource of diverse species of microorganism
communities. Seven locations (S1, S2, S3, S4, S5, S6 and S7)
were identified as sampling sites as shown in Fig. 1. The
locations of the sampling sites were noted using Global
Positioning System (GPS).
Nurfathiah Abdul Malek, Ahmed Jalal Khan Chowdhury, Zarina
Zainuddin, Zaima Azira Zainal Abidin, are with Kulliyyah of Science,
International Islamic University Malaysia, Malaysia. (Email Id:
nurfathiah90@gmail.com, jkchowdhury@iium.edu.my,
zzarina@iium.edu.my, zzaima@iium.edu.my)
http://dx.doi.org/10.17758/IAAST.A1214007
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International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia)
Fig. 1 Location of the Sampling Sites III. RESULTS AND DISCUSSION
B. Collection of Samples
At each location, three sediment core samples were
collected at a depth of 0 – 30 cm. The sediment samples were
then immediately transported back to the laboratory using
sterile polythene bag.
A total of 4850 actinomycete isolates had been successfully
recovered from all seven sites of Pahang mangrove forests.
These dull, tough, chalky, leathery in appearance and earthly
smell colonies were enumerated using 2 pretreatment
procedures and 8 selective isolation media supplemented with
nystatin (50 µg ml-1) to combat the growth of fungus. An
average of 19 mycelium-forming colonies per plate was
observed when only considering those plates that yielded
actinomycetes. These relatively large numbers of isolates
suggested that Pahang mangrove forest is a prominent
resource of actinomycetes.
The percentage of actinomycetes recovered from each sites
was presented in Fig. 2. It was clearly observed that highest
number of isolates were obtained from S1 (23.7%), which is
located within the deep untapped Kuantan Mangrove Reserve
Forest having rich alluvial soil, while the lowest number are
recovered from both S6 and S7 (6.9%). Similar observation
was reported by Mitra et al. [14], whereby isolation from the
intertidal region furthest from the coastal line yielded
maximum number of isolates. According to Kathiresan and
Bingham [15], such forests were influenced with the
movement of higher amount of freshwater with fluvial
nutrients and consequently making it an eminently productive
ecosystem. Low recovery of actinomycete colonies were
observed from S6 and S7; the sites that are near the fishing
village occupied with fishing activity. Contaminants from
domestic wastewater and urban runoff from nearby fishing
village might be deposited to these two mangrove sites [12],
subsequently lowering the number of bacterial counts
recovered.
C. Pre-treatments of the Sediment Samples
A total of 21 sediment samples were air dried at room
temperature for 1 week to constant weight. The sediments
were then ground and sieved to exclude large mineral and
organic matter particles before pre-treatment. For wet heat
pre-treatment; soil suspension containing 1 gm of dried soil
diluted in 9mL of sterilized sea water was heated in a 60 oC
water bath for 20 minutes. Meanwhile, for dry heat pretreatment, 1gm of dried sediment was heated at 120 oC for 60
minutes in a hot air oven.
D. Selective Isolation of Actinomycetes from the Sediment
Samples
The pre-treated sediment samples were diluted 1:10 v/v
with sterile saline, serially diluted down to 10-6 and plated
onto a set of selective isolation plates in triplicate. The
selective media that were used in this study include starchyeast extract agar (SYE), yeast extract-malt extract agar
(ISP2), oatmeal agar (ISP3), inorganic salt-starch agar (ISP4),
starch-casein agar (SCA), glucose asparagine agar (GAA),
actinomycete isolation agar (AIA) and marine agar (MA). All
media were supplemented with nystatin (50 µg ml-1) as
antifungal agent. The inoculated plates were incubated at 28 –
30 oC for 2 – 3 weeks. Typical actinomycete colonies on the
isolation plates were picked out based on morphological
bases, streaked and purified on the original media.
http://dx.doi.org/10.17758/IAAST.A1214007
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International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia)
isolation recovery (31.7%), followed by AIA (24.1%) as seen
in Fig. 3. A total of 1538 colonies were picked from ISP4 agar
based on actinomycete-like morphology, of which around 735
isolates formed powdery colonies with well-developed aerial
hyphae fragmented into spore-chains. As described by
Bredholt et al. [20], these isolates are tentatively called as
Streptomyces-like colonies. ISP4 contains starch, calcium
carbonate and ammonium sulphate that support good growth
of actinomycetes, particularly the Streptomyces-like group
[23]. Enhancement of actinomycetes growth by addition of
calcium carbonate to the culture medium has also been
suggested previously by Arifuzzaman et al. [24] and Kitouni
et al. [25].
Fig. 2 Recovery of Actinomycetes at Different Sampling Sites of
Pahang Mangrove Forest
Previous studies had reported that pre-heating regimes
generally stimulate the isolation of actinomycetes by
eliminating the dominance of unwanted microorganisms
which could inhibit the colonization of slow growing
actinomycetes [16]-[19]. Treatment of the samples might also
promote the germination of rare genera as previously reported
by Bredholt et al. [20]. Hence, pre-heating treatment had
become a useful standard practise for routine isolation. In this
study, two pre-heat procedures; wet heat and dry heat pretreatment were employed for maximum isolation of
actinomycetes. It was stipulated that wet heat pre-treatment at
60 oC for 20 minutes was more effective in enumerating
actinomycetes from Pahang mangrove forest. More than twothird (84.11%) of the total isolates have been successfully
yielded using this moist heating technique. This was in line
with the findings of Naikpatil and Rathod [21] whereby
recovery of actinomycetes increased up to 50% of the total
microorganisms upon mild heating treatment of the sediment
suspension. On the other hand, dry heat treatment was better
in the isolation of spore-forming actinomycetes and greater in
suppressing the growth of bacterial colonies, despite of low
actinomycetes recovery. Elevated temperature may promote
the germination of highly resistant endospores; subsequently
support their survival through this heating procedure at 120 oC
for 60 minutes.
Nutrient availability is one of the main factors that
determine the growth of actinomycetes. Most actinomycetes
can use wide variety of compounds such as glucose, starch,
proteins and amino acids as their energy source; unlike other
bacterial groups that only favour simple carbon and nitrogen
compound [22]. Various types of selective isolation media
were employed in order to increase the number of nonSteprtomyces actinomycetes relative to the non-actinomycetes
microbial flora [20]. In this sudy, the efficiency of culture
media was evaluated using eight different selective isolation
media to assess the optimal nutrient conditions for isolation of
actinomycetes of Pahang mangrove forest.
ISP4 was the most effective medium in the isolation of
Pahang mangrove actinomycetes as it showed the best
http://dx.doi.org/10.17758/IAAST.A1214007
Fig. 3 Percentage of Actinomycetes Recovery Using Various
Selective Isolation Media
However, the use of SCA and SYE agar result in better
recovery of wide variety of actinomycetes, based on their
morphological characteristics. This was also supported by the
finding of Naikpatil and Rathod [21], whereby SCA improved
the enumeration of actinomycetes from mangrove sediment of
Karwar, India. According to Kim [26], SCA possesses high
carbon-to-nitrogen ratio and contains resistant complex carbon
such as starch and casein which was very suitable for
actinomycete isolation as these organisms are able to utilize
such high molecular weight polymers. Despite containing
simple carbon and nitrogen source; the presence of seawater
in SYE medium might promote the growth of actinomycetes
by mimicking the high salinity mangrove environment.
Actinomycetes isolated from SYE agar should be further
tested for their sea water requirement that could be a unique
and crucial component for their growth condition. GAA was
the least effective medium for the isolation of Pahang
mangrove actinomycetes with 3.0% of recovery (Fig. 3). This
clearly showed that L-asparagine, the main component of
GAA might not be a good nitrogen source of mangrove
actinomycetes.
Of all isolation procedures, 140 representative isolates have
been selected based on their distinct morphology appearances,
from seven sediment samples of Pahang mangrove forest.
These isolates are further categorized into seven series
according to the colour of mature aerial mycelium; namely
white, grey, red, yellow, green, black and orange series.
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International Conference on Agriculture, Biology and Environmental Sciences (ICABES'14) Dec. 8-9, 2014 Bali (Indonesia)
IV.
ACKNOWLEDGMENTS
This research was funded by Fundamental Research Grant
Scheme (FRGS13-039-0280). The authors would like to
acknowledge Ministry of Higher Education for the scholarship
given.
Fig. 4 Some Actinomycetes Colonies Successfully Enumerated from Pahang Mangrove Forest
Members of white and grey series tend to have
unfragmented substrate mycelium and abundant aerial
mycelium with long spore chains, suggesting that they are
members of Streptomyces-like group as seen in Fig. 4(a) –
Fig. 4(f). They represented the highest number of isolates
being 67 out of 140 representative isolates (47.7%). The
orange series or else known as Micromonospora-like group
(tiny, solid colonies with unusual aerial hyphae) (Fig. 4(g),
Fig. 4(h)) were also found in all sampling sites representing
25.0% of the total isolates. The relative abundance of these
two groups compared to the other series, indicated that
Streptomyces-like and Micromonospora-like actinomycetes
were dominant in Pahang mangrove forest. Red (2.1%),
yellow (2.1%) and green (0.7%) series were the least abundant
actinomycete isolates where they were only found in some
sampling sites.
As proposed by Hong et al. [1], mangrove forest might
become an important source for discovering novel
actinomycetes owing to their adaptation towards salinity and
tidal gradients. Presence of huge numbers of actinomycete
with various distinct morphological appearances (Fig. 4)
suggests that Pahang mangrove forest is a promising resource
of actinomycetes. This study was not intended to construct a
detailed inventory of actinomycetes kinds found in the
mangrove ecosystem, but it serves as a foreground to study
the biocapability of mangrove actinomycetes. Nevertheless,
this study provide the most comprehensive survey of
culturable actinomycetes present in Pahang mangrove forest,
resulting from the use of broad selective isolation procedures.
http://dx.doi.org/10.17758/IAAST.A1214007
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