Research Journal of Biotechnology Vol.

8 (3) March (2012)

Res. J. Biotech

Isolation, Characterization and Extraction of

antimicrobial compound from marine actinomycete
Streptomyces hygroscopicus BDUS 49
Parthasarathi S.,1* Sathya S.,2 Bupesh G.,3 Manikandan M.,4 Kim C.J.,5 Manikandan T.1 and Balakrishnan K.1
1. Department of Biotechnology, Bharathidasan University, Tiruchirrappall, Tamil Nadu, 620 024, INDIA
2. Department of Bionanotechnology, Kyungwon University, Gyeonggi-Do 461 701, SOUTH KOREA
3. Department of Animal Science, Bharathidasan University, Tiruchirrappall, Tamil Nadu, 620 024, INDIA
4. SriKrishna Arts and Science college, Kuniamuthur, Coimbatore 641 008, INDIA
5. Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305 806, SOUTH KOREA
*sarathimeister@gmail.com

Abstract pharmaceutically relevant microorganis ms, actinomycetes

A total of eight actinomycetes colonies were isolated
from near sea shore marine environment locations of
Bigeum Island, South West coast of South Korea.
Among them, 4 isolates were morphologically distinct
on the basis of spore mass colour, reverse side colour,
aerial and substrate mycelia formation and
production of diffusible pigment. The majority of these
isolates were assigned to the genus Streptomyces of
which one isolate showing broad spectrum of
antimicrobial was on the basis of their morphological,
physiological and biochemical properties. Phylo
genetic analysis of a 16S rRNA gene sequence showed
that strain Streptomyces hygroscopicus BDUS 49
forms a distinct clade within the Streptomyces 16S
rRNA gene tree and is closely related to Streptomyces
hygroscpicus subsp sub species.
This strain possessed a broad spectrum of
antimicrobial activity against Gram-positive, Gram-
negative bacteria and fungi. The UV spectra of the
active compounds in ethyl acetate showed peaks
between 200 to 295 nm. The bioactive region was
detected on the TLC plate (Rf 0.40). The structure of
the bioactive components was further determined
using FTIR, MS, 13C NMR and 1H NMR. The
molecular formula of the given compound was
identified as 7, demethoxy rapamycin C50H75NO12+NA
(MW 905.12).
Keywords: Actinomycetes, Antimicrobial activity, UV/
VIS, FTIR, MS and NMR spectrum.
Introduction
Marine organisms represent a promising source for natural
products of the future due to the incredible diversity of
chemical compounds that were isolated. The oceans cover
almost 70% of the earths surface and over 90% of volume
of its crust1. Marine actinomycetes have been traditionally
a rich source for biologically active metabolites. Although
heavily studied over the past three decades, actinomycetes
continue to prove themselves as reliable sources of novel
bioactive compounds. Among the well characterized
remain major sources of novel, therapeutically relevant
natural products2. The majority of these compounds
demonstrate one or more bioactivities, many of them
developed into drugs for treatment of wide range of
diseases in human, veterinary and agriculture sectors3.
Searching for novel actinomycetes constitutes an essential
component in natural product-based drug discovery.
Analytical methods continue to improve to allow the rapid
elucidation of structures and make natural products
valuable components of modern drug discovery. The
isolated compounds from marine actinomycetes have a
broad spectrum of biological activities such as antibiotic,
antifungal, toxic, cytotoxic, neurotoxic, antimitotic,
antiviral and antineoplastic activities.4 Recently, new
targets have been added to the general screening like AIDS,
immunosuppression, anti-inflammation, Alzheimer disease,
aging processes and some tropical diseases and resulted in
discovery of several drugs.5 Studies on marine
actinomycetes are facing some unexpected problems.
For many of the marine actinomycetes, the taxonomy of the
strain is very poorly defined so that binomial identifications
are frequently uneasy to be carried out. Streptomycetes,
Gram (+) filamentous bacteria, are widely distributed in a
variety of natural and man-made environments and
constitute a significant component of the microbial
population in most soil.6 The results of extensive screening
have been the discovery of about 4000 antibiotic substances
from bacteria and fungi, many of which have found
applications in human medicine, veterinary medicine and
agriculture. Most of them are produced by streptomycetes.7
Most streptomycetes and also other actinobacteria produce
a diverse array of antibiotics, including aminoglycosides,
macrolides, peptides, polyenes, polyether, tetracyclines etc.
In searching for new antibiotics, over 1000 different
bacteria (including actinobacteria), fungi and algae have
been investigated. To prevent exponential emergence of
microorganisms becoming resistant to the clinically
available antibiotics already marketed, the periodic
replacement of existing antibiotics is necessary.
There are very few reports on antimicrobial activity from
marine microorganisms from unpolluted Bigeum Island,

(40)
Research Journal of Biotechnology
South West Coast of South Korea. In bio prospecting point
of view, Bigeum Island marine soil samples possess neutral
to alkaline with pH ranging from 6.33 to 7.94 and CaCO3
concentration in soil samples ranged from 1,200 to 9,600
ppm. Interestingly, Syed et al8 isolated few novel
microorganisms namely, Frigoribacterium mesophilum sp.
nov., Nocardioides halotolerans sp. nov., Leifsonia
bigeumensis sp. nov. Phycicoccus bigeumensis sp. nov.,
Phycicoccus bigeumensis sp. nov., Leifsonia bigeumensis
sp. nov., Nocardioides islandiensis sp. nov., Nocardioides
tritolerans sp. nov., Nocardioides islandiensis sp. nov.,
Leifsonia bigeumensis sp. nov. and Nocardioides dilutes sp.
isolated from the Bigeum Island sample. Recently, a
yellow-pigmented actinobacterium, designated strain
Leifsonia kribbensis sp. nov., was isolated from a soil
sample collected from Bigeum Island.9
Thus, it is crucial that new groups of marine
microorganisms from hitherto unexplored habitats be
considered as sources of novel bioactive secondary
metabolites. Hence the present investigation deals with the
screening and characterization of producers of
antimicrobial substances by marine microorganisms from
the sea waters of Bigeum Island, South West Coast of
South Korea. Further, the identified antagonistic
actinomycetes were characterized based on morphological,
biochemical, physiological, molecular and elemental
analysis.
Material and Methods
Collection of sample: The marine soil sediment samples
were collected at the depth of 20 cm, 20 meter near the sea
side in Bigeum island, South west in Korea / grid 3445'
02.23'' N 125 53' 42.32''. Samples were kindly provided by
Korea Research Institute of Bioscience and Biotechnology
(KRIBB), Daejeon, South Korea. The collected soil
sediment samples were placed in sterile polyethylene bags,
closed tightly and stored in the refrigerator at 4C until use.
For the isolation of actinomycetes, soil sample were first
mixed, suspended in sterile distilled water (1 g in 100 ml)
homogenized by vortexing and finally treated 510 min by
sonication according to Ouhdouch methodology10. The
treated samples were serially diluted up to 10-6 and spread
(0.1 ml) over the surface of starch casein agar (SCA)
medium (pH 7.2) supplemented with 25 g ml1
cycloheximide to inhibit fungal growth and 10 g /ml
nalidixic acid to inhibit the bacteria capable of swarming,
without affecting the growth of actinomycetes. All of the
inoculated plates were incubated at 30C for 710 days.
Morphological and physiological characteristics:
Cultural characteristics of strain Streptomyces
hygroscopicus were examined by eyes of 14-day-old
culture grown on various International Streptomyces
Project (ISP) media11 and Bennetts agar medium. Micro
morphology and sporulation were observed under light
microscope by inclined cover slip technique12 on starch
casein agar after incubating at 30C for 7 days. Colours of
(41)
Vol. 8 (3) March (2012)
Res. J. Biotech
aerial and substrate mycelia were determined and recorded
using National Bureau of Standards (NBS) Colour Name
Charts. The spore chain morphology and spore surface
ornamentation were examined by scanning electron
microscopy (JEOL) of 14-day-old cultures grown on
oatmeal agar (Figure is not shown).
All physiological tests were carried out at 30C and at pH
7.0 unless otherwise specified. The effect of different
temperatures and pH conditions on growth and the
tolerance of strain S. hygroscopicus to salt were determined
by using plates made with modified Bennetts agar
medium. Production of melanoid pigments was tested on an
ISP-7 plate.11 Utilization of carbon source (L-arabinose, D-
xylose, D-glucose, D-fructose, L-maltose, sorbital, sucrose,
glycerol and mannitol) was examined using ISP-9 as a
basal medium. Decomposition of substrates such as nitrate,
starch, gelatine, Milk coagulation, melanin production,
pectin and xylan was detected in modified Bennetts
medium. Resistance to some antibiotics was detected by
disc diffusion method.
Chemotaxonomic analysis: Amino acid and sugar
analyses of whole cell hydrolysates were performed.13,14
Fatty acid methyl esters were prepared from 50 mg of wet
cells. Mixtures of fatty acid methyl esters were prepared
and analyzed with the MIDI system. Menaquinones were
extracted, purified and identified by HPLC.15
DNA extraction: The actinomycete isolate was grown in
25 ml Bennetts medium at 28C for 7 days. An early
grown actinomycete colony was picked up and suspended
in 5% chelax resin. After that, the sample was heated in
boiling water for 10 min, cooled in an ice bath for 5 min
and centrifuged at 7,500 g for 3 min. The supernatant (300
l) was recovered, transferred to a new tube and stored at
4C until use.16
PCR amplification: PCR amplification of 16S rRNA gene
was performed by Maxime PCR PreMix kit (i-Star Taq)
protocol. The specific actinomycete primers, F27 (5-
AGAGTTTGATCCTGGCTCAG-3) and R1492 (5-
GGTTACCTTGTTACGACTT-3) were used to amplify
16S rDNA. The amplification reaction was performed in a
final volume of 20 l containing 10pmol/ l of each primer,
2.5mM each dNTP, 2.5 units Taq DNA polymerase, 16 l
of sterile distilled water, 1.5 mM MgCl2 and 2 l DNA
sample in 1x Taq polymerase buffer. The amplification was
performed on a thermal cycler (PTC-100TM programmable
Thermal Controller, MJ Research Inc, USA). The mixture
was first denatured at 94C for 5 min. Then, 40 cycles of
PCR were performed by denaturation at 94C for 15 sec,
annealing at 55C for 30 s and extension at 72C for 90 s.
At the end of the last cycle, the mixture was incubated at
72C for 10 min and then cooled to 4C.
For each reaction, a negative control lacking DNA template
was included. PCR products of the expected size were
Research Journal of Biotechnology
revealed on 1% agarose gels containing Ethidium bromide
(10g/ml). The resultant 16S rRNA gene sequences were
aligned manually with corresponding almost complete
sequences from Streptomyces strains retrieved from the
GenBank database as described previously. Phylogenetic
tree analysis was performed using the MEGA software
version 4.017.
Screening for antimicrobial activity: Antimicrobial
activities of isolate were tested preliminarily by cross streak
method18. Actinomycetes isolates were streaked across
diameter on starch casein agar plates. After incubation at
28C for 7 days, 24 hrs cultures of Bacillus subtilis,
Staphylococcus aureus, Escherichia coli, Salmonella typhi,
Candida albicans, Aspergillus niger and Saccharomyces
cerevisiae were streaked perpendicular to the central strip
of actinomycetes culture. All plates were again incubated at
30C for 24 hrs and zone of inhibition was measured.
Extraction of antimicrobial compounds: The selected
antagonistic actinomycete isolates were inoculated into
starch casein broth and incubated at 28C in a shaker (200-
250 rpm) for seven days. After incubation the broths were
filtered through Whatmann No.1 filter paper and then
through Millipore filter (Millipore Millex-HV Hydrophilic
PVDF 0.45 m). The filtrate was transferred aseptically
into a conical flask and stored at 4C for further assay. To
the culture filtrate, equal volume of various solvents (viz.,
chloroform, ethyl acetate and methanol) was added
separately and centrifuged at 8000 rpm for 10 min at 4C to
extract the antimicrobial compound19. The compound
obtained from each solvent was tested for their activity
against the test pathogens (Bacillus subtilis, Staphylococcus
aureus, Escherichia coli, Salmonella typhi, Candida
albicans, Aspergillus niger and Saccharomyces cerevisiae)
by well diffusion method. After incubation for 24 and 48
hours the zone of inhibition was measured.
Isolation of antimicrobial metabolites: Antibacterial
compound was recovered from the filtrate by specific
solvent extraction like ethyl acetate. Ethyl acetate was
added to the filtrate in the ratio 1:1 (v/v) and shaken
vigorously for 1 hour for complete extraction. The ethyl
acetate phase that contains an antibiotic was separated from
the aqueous phase. It was evaporated to dryness in a water
bath at 80-90C and the residue obtained was weighed.
Thus obtained compound was used to determine the
antimicrobial activity. Filter paper disks (6mm in diameter)
were impregnated with extracted broth, dried and placed
onto BSM plates previously seeded with Bacillus subtilis.
The plates were incubated at 37C for 48h and examined
for zones of inhibition.
The absorption spectrum of each active extract was
determined in the UV region (200-400nm) by using a
Perkin-Elmer Lambda 15 UV/VIS spectrophotometer. The
structure of the bioactive components was further
determined using FTIR, MS, 13C NMR and 1H NMR. The
(42)
Vol. 8 (3) March (2012)
Res. J. Biotech
molecular formula of the given compound was identified as
7, demethoxy rapamycin C50H75NO12+NA (MW 905.12).
Results and Discussion
South Pacific coast of Philippines, especially from
Mindanao has wide range of salinities and was selected as
an ecosystem for studying the diversity of actinomycetes
and their antimicrobial properties. All the 68 actinomycetes
were identified at a generic level based on the colony
morphology and microscopic morphology. Identification of
strains by both morphological and cultural characteristics
revealed that most (54%) of the isolates belonged to white
and grey colour series. Out of 68 isolates, 66% of isolates
were assigned to the genus Streptomyces sps and the
remaining were identified as Nocardiopsis sps (18%),
Micromonospora sps (11%) and Actinopolyspora sps (5%)
(Fig. 1).
Fig. 1: Percentage frequency of isolated actinomycetes
genera
The present study revealed that among the isolates
Streptomyces was the dominant genera. Frequency and
dominance of Streptomyces among actinomycetes in
various soil types were reported by several workers.20-22
Similarly the present study also determined that out of 68
strains, 23 isolates had antimicrobial activity, of which 12
isolates showed antibacterial activity, 9 isolates showed
antifungal activity against C. albicans. However, only one
isolates namely Streptomyces hygroscopicus sps BDUS 49
had broad spectral antimicrobial activity and was selected
for further studies and they represented marine seashore
soil sample (Fig. 2).
25
20
% of activity
15
10
5
0
Activity against
Antimicrobial activity Antibacterial activity
Antifungal activity Gram positive
Gram negative Both antibacterial and antifungal
Fig. 2: Antimicrobial activity of actinomycetes isolate
Research Journal of Biotechnology
Strain Streptomyces hygroscopicus BDUS 49 showed ext-
ensively branched substrate mycelia and aerial hyphae und-
er light microscopy. The scanning electron micrograph of
strain Streptomyces hygroscopicus BDUS 49 revealed that
aerial mycelia were highly branched with tight spirales and
rectiflexibile spore chain. Each spore chain consisted of
5060 short rod (0.50.60.70.8 m) hairy surface spores.
The cultural characteristics are shown in table 1. Strain S.
hygroscopicus BDUS 49 grew well on most media except
on inorganic salt starch agar. Particularly, the colonies
were convex and some part of the aerial mycelia and spore
chains could be observed around the colony margin. Aerial
mycelia were initially white, becoming pink to purple
colour depending on the medium used and gradually dark-
ened in old culture. Likewise, vegetative mycelium colour
was yellowish-pink to brown depending on the medium.
Melanin and other soluble pigments were not produced.
The physiological properties are shown in table 2. Strain S.
hygroscopicus BDUS 49 showed growth in the pH range
6.0-8.2, with optimal growth occurring at pH 7.0. The
temperature range for growth was 2040C with optimal
growth occurring at 30C. Good growth was shown at
NaCl concentration up to 8% which could be designed as
moderate halophilic actinomycete. No melanoid pigments
were produced. It showed positive results for utilization of
many carbon sources such as D-glucose, L-arabinose, D-
xylose, fructose, maltose and glycerol. However, carbon
sources sucrose and sorbital could not be utilized. It
showed sensitive to most antibiotics except Bacitracin and
Chloramphenical. All the tested carbon sources could be
decomposed by strain S. hygroscopicus BDUS 49 except
pectin and xylan.
Chemotaxonomic tests showed that whole-cell hydrolysates
of strain S. hygroscopicus BDUS 49 were rich in LL-
diaminopimelic acid (LL-DAP) with no characteristic sugar
and indicated a wall chemotype I. The diagnostic polar
lipids were phosphatidylethanolamine, diphosphati
dylglycerol, phosphatidylinositol and phosphatidylinositol
mannoside. Predominant menaquinone was MK9 (H8).
Chemotaxo-nomic and morphological properties of strain
S. hygroscopicus BDUS 49 are in line with its classification
in the genus Streptomyces12. The assignment of this strain
to the genus Streptomyces was also supported by 16S
rRNA gene sequence data (Fig. 3). An almost full sequence
of 16S rRNA gene of strain S. hygroscopicus BDUS 49
(1,404 nucleotides) was obtained and submitted to the
GenBank database under an accession number GU195049.
Comparison of the sequence of strain S. hygroscopicus
BDUS 49 with the corresponding sequences of
representative strains of the genus Streptomyces showed
that this organism formed a distinct phyletic line with a
clade encompassed by Streptomyces hygroscopicus and
Streptomyces lydicus (Fig. 4).
The antimicrobial efficacy of the isolates was tested by
(43)
Vol. 8 (3) March (2012)
Res. J. Biotech
using 5 different solvent extracts and ethyl acetate extract
produced maximum inhibitory zone against all the
pathogens tested followed by methanol, ethy acetate and
chloroform extracts. Ethyl acetate extract of the strain
Streptomyces hygroscopicus BDUS 49 showed maximum
activity against followed by S. aureus (35 mm), B. subtilis
(25 mm), Aspergillus niger (23 mm), Saccharomyces
cerevisiae (28 mm), Escherichia coli (10 mm), S. typhi (9
mm) and C. albicans (8 mm) (Table 4). Similarly, various
solvents were used for the extraction of antibiotics from
actinomycetes by many workers using ethyl acetate and
methanol and chloroform.23-25
The UV spectral data for the ethyl acetate extract of
selected active fermented broth are shown in figure 5.
Maximum absorbance peaks range between 200-295 nm
and the characteristics of absorption peaks indicate a highly
polyene nature. The bioactive regions were detected on the
TLC plate (Rf 0.70). The bioactive compound exhibited a
maximum UV absorption at 210 and 225 nm in ethyl
acetate. The strain Streptomyces hygroscopicus BDUS 49
produced either a broad-spectrum anti-microbial compound
or several compounds with different activities.
The antimicrobial compounds in the ethyl acetate were
purified and separated by column and thin layer
chromatography. Single separated band was observed in the
sample. The Rf value of S. hygroscopicus BDUS 49 was
0.40 in thin layer chromatographic-separation. The
separated S. hygroscopicus compound was brownish colour
and the melting point was 180C-185 C. The compound
was stable at pH from 7.0 to 8.0 and at temperatures
ranging from 20-40C, S. hygroscopicus BDUS 49
compound revealed that the absorption maximum was 200
to 295 nm in ethyl acetate. The UV spectrum of S.
hygroscopicus BDUS 49 is shown in fig.5. FTIR spectrum
of S. hygroscopicus BDUS 49 compound showed two
absorption peaks in the region of 3500 and 1730 cm.-1
The spectrum indicates that the compound had OH and
possibly lactone carbonyl group. Another two absorption
peaks were shown in the region of 1700 (carbonyl) and
between 1610 and 1630 cm-1. This peaks indicates that the
compound had carbonyl and alkenes (C=C) group. The
absence of carboxylic acid (COOH) and ester (COOR),
alkynes was confirmed by the lack of bands in the region of
1670-1674 and 1700-750 cm-1 respectively (Fig. 6). The
mass spectrum showed that the molecular ion peak at 912
m/z (Fig. 7). The large number of peaks through out the 1-
5 was observed in 13C and 1H NMR spectrum of purified
compound from S. hygroscopicus BDUS 49 (Fig. 8 a and b
respectively). Based on the spectral datas, the given
compound was identified as 7-demethoxy rapamycin with
molecular weight and formula as 905.12
(C50H75NO12+NA).
Conclusion
In conclusion, the use of new screening approaches to
Research Journal of Biotechnology
identify microorganisms from original ecosystems is
essential for the discovery of novel natural substances with
valuable biological activities. In our work, the use of
marine sea shore soil sample from particular Bigeum Island
biotopes enabled us to isolate a moderately halophilic
Streptomyces hygroscopicus BDUS 49 strain secreting an
active molecule that exhibits broad spectrum antimicrobial
activity.
Vol. 8 (3) March (2012)
Res. J. Biotech
Acknowledgement
The authors wish to thank Dr. J. C. Lee and D. J. Park for
their help. The selected isolates were identified by Korea
Research Institute of Bioscience and Biotechnology,
Daejeon and Seoul National University, Seoul, Republic of
Korea.

Fig. 3: Amplified fragment of 16S rRNA gene of strain S. hygroscopicus BDUS 49 (1,404 nucleotides);
M: Marker DNA.

Fig. 4: Neighbor-joining tree based on almost complete 16S rRNA gene sequence showing relationships between
streptomycete isolate Streptomyces hygroscopicus BDUS 49 and related members of the genus Streptomyces. The
numbers at the nodes indicate the level of bootstrap support based on a neighbor-joining analysis of 1,000 resampled
datasets; only values above 50% are given. Scale bar indicates 0.1 substitutions per nucleotide position

(44)
Research Journal of Biotechnology Vol. 8 (3) March (2012)
Res. J. Biotech

Fig. 5: UV - spectrum of S. hygroscopicus BDUS 49

Fig. 6: FTIR - spectrum of S. hygroscopicus BDUS 49

Fig. 7: Mass spectrum of S. hygroscopicus BDUS 49

(45)
Research Journal of Biotechnology Vol. 8 (3) March (2012)
Res. J. Biotech

13
Fig. 8(a): C NMR - spectrum of S. hygroscopicus BDUS 49

Fig. 8(b): 1H NMR spectrum of S. hygroscopicus BDUS 49

Table 1
Cultural characteristic of Streptomyces hygroscopicus BDUS 49
Medium Growth Aerial mycelium Substrate mycelium
Yeast extractmalt extract agar Good Whitish, Pale grayish Pale yellow, brown
ISP-3 Good White to chalk white Pale yellow
ISP-4 Good No aerial mycelium Yellow to brown
ISP-5 Good Whitish to pale candy Brownish yellow
Bennetts agar Good Whitish Pale to dark grey
Potato dextrose agar Good Sandal white Yellowish brown
Starch casein agar Good Whitish Yellowish brown

(46)
Research Journal of Biotechnology Vol. 8 (3) March (2012)
Res. J. Biotech
Table 2
Biochemical and Physiological characteristics of Streptomyces hygroscopicus BDUS 4
Characteristics Results
Sporophore morphology Spirally twisted
Spore surface Smooth
Colour of aerial mycelium Dull white
Colour of substrate mycelium Dull yellowish brown
Spore mass White
Gram staining +
Acid fast Non acid fast
Motility Non motile
Decomposition of
Nitrate +
Starch +
Pectin -
Gelatin liquefactions +
Xylan -
Milk coagulation +
Melanin production +
Carbon source utilization (1% w/v)*
D-glucose +
L-arabinose +
Sucrose _
D-xylose +
Fructose +
Maltose +
Glycerol +
Mannitol +
sorbital _
Glycerol
Resistance to antibiotics (g ml1)
Bacitracin (10 i.u) Resistance
chloramphenical(5 i.u.) Resistance
Amikacin(50) Sensitive
Amphicilin (10) Sensitive
Streptomycin (10) Sensitive
Tetracyclin(30) Sensitive
Temperature for Growth (C)
Range 20-40C
Optimum 28C
pH for growth*
Range 6.0-8.2
Optimum 7.0
Effect of NaCl concentration (w/v)
2% +
4% +
6% +
8% +
10% _
*: Growth of the strain was measured as dry weight of the mycelium, +: positive; -: Negative.

(47)
Research Journal of Biotechnology
Table 3
Antimicrobial efficacy of Streptomyces hygroscopicus BDUS 49
Zone of inhibition (mm)
Name of the test organism
Ethyl acetate
Bacillus subtilis 25
Staphylococcus aureus 35
Escherichia coli 20
Salmonella typh 9 5
Candida albicans 8 4
Aspergillus niger 23
Saccharomyces cerevisiae 28
References
1. Fenical W., Chemical studies of marine bacteria developing a
new resource, Chem Rev., 93, 16731683 (1993)
2. Jensen P.R. and Fenical Marine W., Microorganisms and Drug
Discovery Current Status and Future Potential, In Drugs from the
Sea, Fusetani N., ed., Karger, Basel, 6-29 (2000)
3. Bernan V.S. Marine microorganisms as a source of new natural
products, Adv Applied Microbiol., 43, 57-90 (1997)
4. Newman D.J. and Cragg M.G., Natural products as sources of
new drugs over the last 25 years, J Nat Prod., 70, 461-477 (2007)
5. Kelecom A., Chemistry of marine natural products Yesterday,
today and tomorrow, AnAcad Bras Cienc., 71, 249-263 (1999)
6. Watve M.G. R. et al, How many antibiotics are produced by
the genus Streptomycetes, Arch Microbiol., 176, 386-390 (2001)
7. Demain A.L., Pharmaceutically active secondary metabolites
of microorganisms, Appl Microbiol Biotechnol., 52, 455 (1999)
8. Syed G., Dastager Jae-Chan Lee, Yoon-Jung Ju, Dong-Jin
Park and Chang-Jin Kim, Frigoribacterium mesophilum sp. nov.,
a mesophilic actinobacterium isolated from Bigeum Island,
Korea, International Journal of Systematic and Evolutionary
Microbiology, 58, 18691872 (2008)
9. Syed G. et al, Leifsonia kribbensis sp. nov., isolated from soil,
International Journal of Systematic and Evolutionary
Microbiology, 59, 1821 (2009)
10. Ouhdouch Y., Barakate M. and Finance C., Actinomycetes
of Moroccan habitats isolation and screening for antifungal
activities, Eur J. Soil Biol., 37, 69-74 (2001)
11. Shirling E.B. and Gottlieb D., Methods for characterization of
Streptomyces species, Int J Syst Bacteriol., 16, 313-340 (1966)
12. Williams S.T. et al, Bergeys manual of systematic
bacteriology, Williams and Wilkins, Baltimore (1989)
13. Hasegawa T., Takizawa M. and Tanida S.A., rapid analysis
for chemical grouping of aerobic actinomycetes, J. Gen. Appl.
Microbiol., 29, 319-322 (1983)
14. Staneck J.L. and Roberts G.D., Simplified approach to
identification of aerobic actinomycetes by thin-layer
chromatography, Appl Microbiol., 28, 226-231(1974)
(48)
Vol. 8 (3) March (2012)
Res. J. Biotech
Methanol Chloroform
18 19
24 27
14 16
7
5
16 18
21 22
15. Collins M.D., Isoprenoid quinone analysis in bacterial
classification and identification, In Goodfellow M., Minnikin
D.E., eds., Chemical methods in bacterial systematic, London,
267-287 (1985)
16. Cook A.E. and Meyer P.R., Rapid identification of
filamentous actinomycetes to the genus level using genus -
specific 16S rRNA gene restriction fragment patterns, Int J Syst
Evol Microbiol., 53, 1907-1915 (2003)
17. Tamura K., Dudley J., Nei M. and Kumar S.S., MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA) software
version 4.0, Mol. Biol. Evol., 24, 1596-1599 (2007)
18. Lemos M.L., Toranzo A.E. and Barja J.L., Antibiotic activity
of epiphytic bacteria isolated from intertidal seaweeds, Microbiat
Ecol., 11, 149-163 (1985)
19. Sambamurthy K. and Ellaiah P., A new Streptomycin
producing Neomycin (B and C) complex, S. marinensis (part-1),
Hind Antibiot Bull, 17, 24-28 (1974)
20. Balagurunathan R., Antagonistic actinomycetes from Indian
shallow sea sediments with reference to , unsaturated -
lactone, type of antibiotic from Streptomyces griseobrunncus, Ph.
D. thesis, Annamalai University, Tamil Nadu (1992)
21. Saadoun I. F., Al-Momani Frequency and dominance of grey
series Streptomyces in Jordan soils, Actinomycetes, 61 (1998)
22. Peela S., Bapiraju Kurada V.V.S.N. and Terli R., Studies on
antagonistic marine actinomycetes from the Bay of Bengal, World
J. Microbiol Biotechnol, 21, 583-585 (2005)
23. Taechowisan T., Lu C., Shen Y. and Lumyong S., Secondary
metabolites from endophytic Streptomyces aureofaciens
eMUAc130 and their antifungal activity, Microbiology, 151,
1651-1695 (2005)
24. Ilic S.B., Kontantinovic S.S. and Todorovic Z.B., UV/VIS
analysis andantimicrobial activity of Streptomyces isolates, Facta
Universitatis Med Biol., 12, 44-46 (2005)
25. Thangadurai D. et al, Antimicrobial screening of Decalepis
hamiltonii Wight and Arn, (Asclepiadaceae) root extracts against
food-related microorganisms, J. Food Safety, 24, 239-245 (2004).
(Received 26th June 2011, revised 15th April 2012,
accepted 27th October 2012).
Research Journal of Biotechnology Vol. 8 (3) March (2012)
Res. J. Biotech

(49)