RESEARCH

Niyaz AhamedIsolation and identification of secondary metabolites producing organisms from marine sponge,Discovery, 2012, 1(1), 14-17, www.discovery.org.inhttp://www.discovery.org.in/d.htm 2012 discovery publication. All rights reserved

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Marine sponges:There are more than7000 species alivetoday living in bothfreshwater andmarine environments,and are the oldestknown multicellularanimals. Spongeslive in all types ofregions all over theworld. 99% of allsponges live in themarineenvironment. Thereare a higher numberof sponge individualsand sponge speciesin the tropics of allregions because thewater is warmer.They do best inclearer waters as siltor sediment ladenwater may clog thepores of the spongescausing the demiseof the sponge as itcannot get itsnutrition and oxygento survive.

Niyaz Ahamed MI*Department of Biotechnology, Hindustan College of Arts & Science, Kelambakkam-603103, Tamil Nadu, India, Email:*Corresponding Author: Niyaz Ahamed MI, Department of Biotechnology, Hindustan College of Arts & Science, Kelambakkam-603103,Tamilnadu. Email: niyazbt@gmail.comReceived 08 April; accepted 18 May; published online 25 June; printed 29 June 2012

ABSTRACTSponges are among the simplest of multicellular animals and often described as the most primitive of all. This study is focusedon the identification of the organisms which produce secondary metabolites and extraction of metabolites from marine spongeswhich exhibit antibacterial activities. This work also analyzes the confirmation of the secondary metabolites by thin layerchromatography. The cross streak method revealed that Halobacterium Spp1, Halobacterium Spp2, Halobacterium Spp3,Marinomonas spp has better antagonistic activity. TLC result shows that the secondary metabolites emit fluorescence whileabsorbing ultraviolet rays.Key words: Marine sponge; Antagonism; Secondary metabolites; Therapeutic agent.Abbreviations: TLC - Thin Layer Chromatography; ZMA - ZoBell Marine Agar; SMA - Skim Milk Agar; NA - Nutrient Agar; I -Indole; MR - Methyl Red; VP - Voges Proskauer; CU - Citrate Utilization; O - Oxidase; C - Catalase

1. INTRODUCTIONSponges are sessile marine animals which arecommonly found in seas where there are rocks,coral or other suitable substrata. There are morethan 5000 species and they are found in almostevery sea from mid-tide levels to the deepest partsof the oceans. Sponges act as hosts to a varietyof symbiotic/parasitic organisms including blue-green algae and bacteria. Marine invertebrateshave been recognized as an important source ofbioactive compounds having medicinal potential.Although no major therapeutic drugs have yetbeen developed from the sea, several compoundshave so far entered clinical trials as anticancerdrugs. Many of the compounds are present insmall quantities and have complicated structuresthat preclude economical industrial syntheses.The development of biotechnological production ofmarine natural product such as aquaculture(bryostatin, esteinacidin), through symbioticmicroorganism production (swinholide,manzamine) and also development of syntheticalproduction (Ara-A) might become the solution ofthose plagues. Thus prospects of marine naturalproducts as future medicine are still promising.Marine organisms have attracted special attentionin the last years for their ability to produceinteresting pharmacological lead compounds.

Belarbi et al. (2003) reported that Sponges aremost primitive of the multi-celled animals that haveexisted for 700-800 million years. Of theapproximately 15,000 sponge species, most occurin marine environments. Only about 1% of thespecies inhabits freshwater. Pawlik et al. (2002)revealed that sponges produce secondarymetabolites to repel and deter predators, competefor space with other sessile species and forcommunication and protection against infection.Hellio et al. (2005) reported that potentiallytherapeutic compounds identified in spongesinclude anticancer agents and immunomodulators.Some sponges seem to produce potentially usefulantifouling agents. The objective of the work is toisolate and screen the microorganisms from thesponges which produce maximum secondarymetabolites and showing antagonistic effect. Thiswork also analyzes the extraction of secondarymetabolites and confirm with thin layerchromatography.

2. MATERIALS AND METHODS2.1. Collection of spongesMarine sponge Dendrilla nigra was collected fromthe department of microbiology in BharathidasanUniversity, Trichy. The samples were stored at4oC until use.

RESEARCH Discovery, Volume 1, Number 1, July 2012

Isolation and identification of secondary metabolitesproducing organisms from marine sponge

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RESEARCH

Niyaz AhamedIsolation and identification of secondary metabolites producing organisms from marine sponge,Discovery, 2012, 1(1), 14-17, www.discovery.org.inhttp://www.discovery.org.in/d.htm 2012 discovery publication. All rights reserved

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Symbioticmicroorganisms:Symbioticmicroorganisms bacteria, fungi(yeasts), andprotozoans playan important role inthe lives ofmany insects. Theymay be harbored inthe gut or in specialcells (mycetocytes)which are oftengrouped together toform organs calledmycetomes. Mosttermites havesymbioticprotozoans in thehind part of the gut.These activelyingest the woodparticles that thetermite has eatenand break themdown, releasingsubstances that thetermites can absorb.Experiments showsthat the termitesdepend on theprotozoans dependon the protozoansfor much of theirfood, and when thelatter have beenremoved, so that thetermite has none inits gut, it losesweight rapidly anddies.

2.2. Isolation of sponge associatedbacteria1g of sponge sample was taken and ground wellwith distilled water in sterile mortar and pestle andthe extract was taken. Nutrient agar was preparedwith 2%Nacl and spread plate method wasperformed. Plates were kept for incubation at 37ocfor 24 hours. Colonies were counted and subcultured.

2.3. Screening of antibacterial activity bycross streak methodSecondary metabolites producing bacteria werescreened by cross streak method. Nutrient agarwith 2%Nacl was prepared and the marinebacteria isolated was streaked at the centre of thepetri plate and incubated for 48hrs at roomtemperature. After incubation, the test organismswere streaked at both the sides of the marinebacteria which was streaked already andincubated for 24hrs at 37oc. After 24hrs ofincubation the organisms showed maximumactivity were selected and sub cultured.

2.4. Identification of antagonisticorganismsThere are many practical applications foridentifying antagonistic organisms.Characterizations of the selected isolates areconfirmed by various physiological andbiochemical test. Cultural characteristics of theorganisms were studied by cultivating theorganism in ZoBell marine agar, nutrient agar with2%Nacl and skim milk agar with 2%Nacl.

2.5. Production of secondary metabolitesfrom the bacteriaTryptic digest broth was prepared with 2%Nacl indifferent conical flasks. The selected colonieswere inoculated in the broth. Then the conicalflasks were kept in the shaking incubator for 3days at 180rpm. After 3days secondarymetabolites was extracted.

2.6. Extraction of secondary metabolitesby solvent extraction methodAfter the production of secondary metabolites, thebroth was aseptically transferred to sterilecentrifuge tubes and centrifuged at 10,000 rpm for10mins. The supernatant was collected in sterileconical flask and the pellet was discarded. Thesupernatant was transferred to the separating

funnel and mixed with ethyl acetate in the ratio 1:1(supernatant: ethyl acetate). Then the mixture inthe separating funnel was shaked continuously for15mins. After 15mins, keep the separating funnelundisturbed for 10-15mins. After 15mins, threelayers were formed and the middle layer wasremoved in a sterile petri plate. The petri platecontaining secondary metabolites in thesuspension was allowed to dry in the air. Thisdried sample contains the secondary metabolites.

2.7. Antibiotic sensitivity assay bywell diffusion methodThe Muller Hinton agar medium was prepared andthe test organisms (Vibrio cholerae, klebsiellapneumoniae, staphylococcus aureus) were spreadon individual plates. Then wells were made withhelp of gel puncher. 100microlitre of crude extractfrom each sample was added in a separate well. Itwas incubated at 37oC for 24hrs. Zone of inhibitionwas noted and the diameter was measured. Ethylacetate served as control.

2.8. Extraction of alkaloids from thesponge by solvent extraction methodThe marine sponge was taken and cut into smallpieces. Then the sponges were ground with ethylacetate. The extract was collected in the sterilecentrifuge tube and was transferred in to theseparating funnel. Add an equal volume of ethylacetate and the mixture in the separation funnelwas shaked continuously for 15mins. After that theseparation funnel was kept in a burette stand for5-10mins. After 10mins three layers were formedand the middle layer was removed in a sterilecentrifuge tube, which was used for furtheranalysis.

2.9. SolubilityThe solubility of the secondary metabolites wastested using distilled water, petroleum ether, ethylacetate and chloroform.

2.10. UV absorbing compound in thinlayer chromatographySilica gel was prepared and poured in the glassslide and spreaded evenly using spreader andkept in hot air oven for 45 minutes. The crudesample of sponge was mixed with ethyl acetateand was added to the TLC slide. The slide wasplaced in the beaker at one end of the slideimmersed in the solvent prepared. The prepared

SECONDARY METABOLITESSecondary metabolites are organic compounds that are not directly involved in the normal growth, development,or reproduction of an organism. Unlike primary metabolites, absence of secondary metabolites does not result in immediatedeath, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in nosignificant change at all. Secondary metabolites are often restricted to a narrow set of species withina phylogenetic group. Secondary metabolites often play an important role in plant defense against herbivory and otherinterspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs. Most of thesecondary metabolites of interest to humankind fit into categories which classify secondary metabolites based on theirbiosynthetic origin. Since secondary metabolites are often created by modified primary metabolite synthases, or "borrow"substrates of primary metabolite origin, these categories should not be interpreted as saying that all molecules in thecategory are secondary metabolites (for example the steroid category), but rather that there are secondary metabolites inthese categories.

RESEARCH

Niyaz AhamedIsolation and identification of secondary metabolites producing organisms from marine sponge,Discovery, 2012, 1(1), 14-17, www.discovery.org.inhttp://www.discovery.org.in/d.htm 2012 discovery publication. All rights reserved

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solvent contains ethyl acetate, methanol, anddistilled water in the ratio of 10:0.25:0.1 byvolume. Run the TLC plate for more than 30minutes and remove the slides from the solvent.The fluorescence was observed and viewed underUV trans illuminator.

3. RESULTS AND DISCUSSIONThe secondary metabolites producing organismwas isolated from marine sponge and wasscreened by spread plate method followed bycross streak method. Efficient antagonisticbacteria were identified by staining method,cultural characteristic and biochemical test (Table1). Antibacterial activity among marine bacteria isa well-known phenomenon and has beendemonstrated in a number of studies (Isnansetyoet al., 2003: Uzair et al., 2006). The cross streakmethod revealed that Halobacterium Spp1,Halobacterium Spp2, Halobacterium Spp3,Marinomonas spp has better antagonistic activity.Solubility test shows that the extracted secondary

metabolites are insoluble in chloroform and ethylacetate, petroleum ether and water showsmoderate solubility (Table 2). TLC result showsthat the extracted secondary metabolites from thesponges have the capability to absorb theultraviolet rays and emit fluorescence (Fig.1).4. CONCLUSIONMarine bacteria have been recognized as animportant and untapped resource for novelbioactive compound .The chemical compound ofmarine organism are less well known than thoseof their terrestrial counterparts however, in the lastdecade several bioactive compound have beenisolated from marine bacteria and are newresources for the development of medically usefulcompound. The cross streak method revealed thatHalobacterium Spp1, Halobacterium Spp2,Halobacterium Spp3, Marinomonas spp has betterantagonistic activity. Sponge associated bacteriawere identified as Halomonas spp andMarinomonas spp. Secondary metabolites wereproduced from these bacteria using tryptic digestbroth. They showed broad spectrum activityagainst both Gram positive and Gram negativeorganisms, Compound nature was screened usingTLC method which showed UV absorbingcompounds

SUMMARY RESEARCH1. This work has done to analyze the confirmation of the secondary metabolites in marine sponges by thin layer

chromatography.2. The cross streak method revealed that Halobacterium Spp1, Halobacterium Spp2, Halobacterium Spp3, Marinomonas spp

has better antagonistic activity.3. TLC result shows that the secondary metabolites emit fluorescence while absorbing ultraviolet rays..

FUTURE ISSUES1. In the future, metabolite production from cultured sponge cells and primmorphs may become feasible.2. Is the use of microorganisms for biological purpose has become an effective alternative to control pathogens for all diseases?

DISCLOSURE STATEMENTThere is no financial support for the proposed research work.

Table 1 Identification of secondary metabolites producing organismsCultural characteristic Biochemical test OrganismZMA SMA NA I MR VP CU O C

Milky white orange Milky white - - - - - + Halobacterium spp 1Milky white orange Milky white - + - - - + MarinomonasMilky white orange Milky white - - - - - + Halobacterium Spp 2Milky white colorless Milky white - - - - - - Halobacterium Spp 3

Table 2 Solubility test for the extractedsecondary metabolites

SOLVENT SOLUBILITYPetroleum ether Moderately solubleChloroform InsolubleEthyl acetate InsolubleWater Moderately soluble

Figure 1Fluorescence emitting secondary metabolitesfrom sponges in TLC plate

RESEARCH

Niyaz AhamedIsolation and identification of secondary metabolites producing organisms from marine sponge,Discovery, 2012, 1(1), 14-17, www.discovery.org.inhttp://www.discovery.org.in/d.htm 2012 discovery publication. All rights reserved

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Belarbi et al., 2003: Thisreview focuses onmethods of producingsponge biomass toovercome supplylimitations. Productiontechniques discussedinclude aquaculture in thesea, the controlledenvironments ofaquariums, and culture ofsponge cells andprimmorphs. Cultivation inthe sea and aquariumsare currently the onlypracticable and relativelyinexpensive methods ofproducing significantquantities of spongebiomass.

ACKNOWLEDGMENTSI would like to thank the management, Project Coordinator, Faculties from Department of Biotechnology, Hindustan college ofArts and science, Chennai, for guiding and providing me an opportunity, to complete my research work successfully.

REFERENCES

1. Belarbi EH, Gomez AC, Chisti Y, Camacho FG, Grima EM. Producing drugs from marine sponges. Biotechnol.Adv 2003, 21, 585-598

2. Hellio C, Tsoukatou M, Marechal JP, Aldred N, Beaupoil C, Clare AS, Vagias C, Roussis V. Inhibitory effects ofmediterranean sponge extracts and metabolites on larval settlement of the barnacle Balanus amphitrite. MarBiotechnol 2005, 7, 297-305

3. Isnansetyo A, Kamei Y. Pseudoalteromonas phenolica sp. nov., a novel marine bacterium that produces phenolicanti-methicillin-resistant Staphylococcus aureus substances. Int. J. Syst. Evol. Microbiol 2003, 53(Pt 2), 583-8

4. Pawlik JR, McFall G, Zea S. Does the odor from sponges of the genus Ircinia protect them from fish predators?. JChem Ecol 2002, 28, 1103-1115

5. Uzair B, Ahmed N, Ahmed V, Kousar F. A new antibacterial compound produced by indigenous marine bacteria;fermentation, isolation and biological activity. Nat Pro Res 2006, 20(14), 1326-1331

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