Many infectious diseases caused by Escherichia coli and Staphylococcus aureus

which turned into a resistant pathogen. A symbiotic fungi of marine sponge Axinella sp.,

Aspergillus sydowii from the waters of Riung, East Nusa Tenggara, Indonesia showed

antibacterial activity, cultured on the four media, MEB (ST), Noni Juice Media (MG), avocado

leaves media (AL), and Soursop leaves media (SR). The symbiotic fungi was cultured for 14

days on each media. The largest weight of symbiotic fungi biomass on ST media 138,95gr and

at least 99,12gr of AL media. Purification of bioactive compound is carried out using

separatory funnel, and column chromatography. The highest rendemen of extracts on SR media

was 3,67%, while the lowest in ST media was 1,22%. The bioactive test used diffusion agar

method. Fungi extracts from four mediums have bioactivity against, E. coli and S. aureus. The

biggest inhibition zone obtained from the extract of MG KN-15-3-1-3, with inhibition zone

10.71mm and 10.98mm against E. coli and S. aureus

Radjasa, Ocky Karna

Trianto, Agus

Widyaningsih, Sekar

Wittriansyah, Khoeruddin

English

Diponegoro University Institutional Repository

IOP Conference Series: Earth and Environmental SciencePAPER • OPEN ACCESSAntibacterial Activity Symbiotic Fungi of MarineSponge Axinella sp., Aspergillus Sydowii on FourGrowth MediumTo cite this article: S Widyaningsih et al 2018 IOP Conf. Ser.: Earth Environ. Sci. 116 012084 View the article online for updates and enhancements.Related contentSymbiotic Fungus of Marine SpongeAxinella sp. Producing Antibacterial AgentA Trianto, S Widyaningsih, OK Radjasa etal.-Antimicrobial activity of tempeh gembushydrolyzateA Noviana, F F Dieny, N Rustanti et al.-Development of extract library fromindonesian biodiversity: exploration ofantibacterial activity of mangrovebruguiera cylindrica leaf extractsK A Audah, J Amsyir, F Almasyhur et al.-This content was downloaded from IP address 103.213.129.245 on 13/05/2019 at 04:361Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.Published under licence by IOP Publishing Ltd12345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/012084Antibacterial Activity Symbiotic Fungi of Marine Sponge Axinella sp., Aspergillus Sydowii on Four Growth Medium  S Widyaningsih1, A Trianto2.4, OK Radjasa2, K Wittriansyah3  1Oceanography Department, Faculty of Engineering and Marine Science, Hang Tuah University, Surabaya, Indonesia 2Marine Science Department, Faculty of Fisheries and Marine Science, Diponegoro University, Semarang, Indonesia 3Fishery Mechanical Engineering, Cilacap, Indonesia 4Natural Product Laboratory, Centre of Research and Services (Laboratorium Terpadu) Diponegoro University, Indonesia  Email: sekar.widyaningsih@hangtuah.ac.id  Abstract. Many infectious diseases caused by Escherichia coli and Staphylococcus aureus which turned into a resistant pathogen. A symbiotic fungi of marine sponge Axinella sp., Aspergillus sydowii from the waters of Riung, East Nusa Tenggara, Indonesia showed antibacterial activity, cultured on the four media, MEB (ST), Noni Juice Media (MG), avocado leaves media (AL), and Soursop leaves media (SR). The symbiotic fungi was cultured for 14 days on each media. The largest weight of symbiotic fungi biomass on ST media 138,95gr and at least 99,12gr of AL media. Purification of bioactive compound is carried out using separatory funnel, and column chromatography. The highest rendemen of extracts on SR media was 3,67%, while the lowest in ST media was 1,22%. The bioactive test used diffusion agar method. Fungi extracts from four mediums have bioactivity against, E. coli and S. aureus. The biggest inhibition zone obtained from the extract of MG KN-15-3-1-3, with inhibition zone 10.71mm and 10.98mm against E. coli and S. aureus. Keywords: antibacterial,  sponge, symbiotic fungi, Aspergillus sydowii  1. Introduction Axinella sp. is one of marine sponge that have many biological activities [1], such as antifungi, anti-microbial, aniviral, insecticidal activity, cytotoxicity, and antioxidant [2-7]. Axinella is also source of bioactive compounds [1]. Some compounds from marine sponge Axinella sp. are Axinastatin, Cyclonellin, and Daminin [8-10].  Only few of them passed to the preclinical and clinical stages [11]. Supply is a major problem for drug development. Therefore, another alternative is required, by utilizing microorganisms such as fungal symbionts [12-13]. Kelecom (2001) [14] mentioned that symbiotic microorganisms like fungi symbiont produce secondary metabolites that are similar to those produced by its host. Fungi symbiont can be isolated and cultured [15].  Growth media is one of the major factors in isolating and growing microorganisms. This is because it can affect the content of compounds in these microorganisms [16-17]. Atalla et al. (2008) [18], stated that the formation of a secondary metabolite in micro-organisms will be affected by conditions and growth media. 212345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/012084This paper describes the effect of growth media on the biomass and antibacterial activity of symbiotic fungi of marine sponge Axinella sp., Aspergillus sydowii  from the waters of Riung, East Nusa Tenggara, Indonesia.  2. Materials and methods  2.1. Sample collection Symbiotic Fungi of Marine Sponge Axinella sp., Aspergillus sydowii was sample collection of Natural Product Laboratory, Faculty od Fisheris and Marine Science, Diponegoro University, Semarang.  2.2. Antibacterial screening The MDR bacteria, Escherichia coli and Staphylococcus aureus, was obtained from Microbiology Laboratory, Kariadi Hospital, Semarang. Each MDR bacteria was cultured in the logarithmic phase, be mixed in Zobell soft agar medium (1% v/v), and poured on the agar surface that previously inoculated with fungi symbiont. The inhition zone shown after 24 h incubation and the isolates active as antibacterial [19]. (the data were not shown in this paper).  2.3. Isolation and Cultured of Fungi Isolation and cultured of fungi symbiont Aspergillus sydowii conducted with modification methods of Trianto et.al. [20]. There are four mediums that used for mass cultured, Malt Extract Broth/MEB (ST), Noni Juice Media (MG), Avocado leaves media (AL), and Soursop leaves media (SR). Symbiotic fungi Aspergillus sydowii was cultured on growth media for 7 days or until the maximum growth at ambient temperature.  2.4. Extraction The media was filtered to obtain the micellium that was extracted with methanol at room temperature. The solvent was filtered using filter paper and then dried with rotary evaporator [21].   2.5. Purification 2.5.1. Separatory funnel The solvents used in the separatory funnel method are water and ethyl acetate with a ratio of 1: 3, and dried with rotary evaporator [22].  2.5.2. Thin layer chromatography (TLC) Thin layer chromatography were performed on silica-gel 60 TLC plate and developed using different solvent systems to obtain best separation. There were two methods to visualize the compound. Using  UV-light and vanilin sulfuric acid that sprayed at plat TLC and heated on hot plate until the color changes on the TLC plate [23].  2.5.3. Open Column chromatography (OCC) Purification by column chromatography conducted with modification methods of Jafarzade et al. [24]. The solvents used were n-hexane (1), chloroform (2), chloroform: ethyl acetate (1: 1) (3), ethyl acetate (4), and ethyl acetate: methanol (2:1) (5).    312345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/0120842.6. Bioassay Bioassay based on the method proposed by Safaeian et al. [25] with slight modification. The extracts were tested against S.aureus and E.coli using difussiun agar method at concentration 100 and 50 µg/disk with two times repeated. The pathogenic bacteria were inoculated by spread method on MEA media, after 30 minutes incubation, the paper disks contained the extract were placed on the agar surfaced.  3. Result and discussion Fungi of marine sponge, Aspergillus sydowii, code KN-15-3 is isolate collection of Natural Product Laboratory, Faculty of Fisheris and Marine Science, Diponegoro University, Semarang. The isolate was be morphology and molecular identify. Further studies were done after antagonistic test (The process was not performed in this paper).  The isolate fungi was isolated dan cultured on four liquid mediums, Malt Extract Broth/MEB (ST), Noni Juice Media (MG), Avocado Leaves Media (AL), and Soursop Leaves Media (SR) for 14 days (on optimum growth). Then be extracted with methanol. The result of isolation and mass cultured, known that the largest wet weight of fungi symbionts were 138.95gr on ST medium, and the least were 99.12gr on AL medium with filtrate volume 750mL. The largest rendement extract from SR medium (Table 1). Table 1. Crude extracts of symbiontic fungi on four growth media Isolate Media Weight wet of fungi (gr) Filtrate volume (mL) Crude extract (gr) Rendement (%) KN 15-3 ST 138.95 750 1.707 1.23 AL 99.12 3.122 3.15 SR 103.07 3.783 3.67 MG 126.59 4.244 3.35  The crude extract were separated again with separatory funnel. From the separation, fraction of ethyl acetate AL medium had the highest amount, and the lowest was ST medium (Table 2).  The result of separation was checked with TLC plate (Figure 1), and bioassay test result on Figure 2. Table 2. Separatory funnel (fraction extract) Isolate Media Crude extract (gr) Fractions Weight of fraction extract (gr) KN-15-3 ST 1.7068 Ethyl acetate 0.5384 Water 1.1684 AL 3.1221 Ethyl acetate 0.9569 Water 2.1652 SR 3.7834 Ethyl acetate 1.9192 Water 1.8642 MG 4.2442 Ethyl acetate 0.8461 Water 3.3981 412345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/012084 Figure 1. Visualization of TLC, vanilin sulfuric and UV rays (left: ST, SR, AL,and MG) (a, b) Kloroform : Etil Asetat (4 : 1); (c, d) N-Heksan : Etil Asetat (1 : 1).  The visualization of TLC plate showed the spot change color and and the spots formed on each media were almost identical. The Spot colors on the TLC plate are dominated by blue, green, and red (Figure 1). The blue green spot on the TLC plate indicated fraction containing steroid and red spot were indicating terpenoid compounds [26].   Figure 2. Diagram Inhibition zone of separatory funnel ethyl acetat fractions (KN-15-3-1).  The biggest E.coli inhibition zone was ST-KN-15-3-1 of 3.88mm, and the biggest S.aureus inhibition zone was MG-KN-15-3-1 of 2.83mm (Figure 2). All of the ethyl acetate fractions were separated again using open column chromatography (Table 3). The largest weigth was MG-KN-15-3-1-3 of 383.8mg and the smallest was ST-KN-15-3-1-4 of 34mg.  The different between wet weight and rendement extract on four growth were influenced by the nutrient contents in each growth medium (Table 1-3). The nutrient contents on each growth media affected the growth quantity of microorganisms [27].  024681012Control MEB (ST) AvocadoLeaves Media(AL)SoursopLeaves Media(SR)Noni JuiceMedia (MG)Inhibition zone (mm)E.coliS.aureusd c b a 512345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/012084Table 3. Open Column Chromatography (fraction extract) Isolate Media Ethyl acetate fraction (separatory funnel) (gr) Fraction (OCC) Weight of the OCC fraction extract (mg) KN-15-3-1 ST 0.5384 1 120.9 2 75.7 3 183.8 4 34 5 116.1 AL 0.9569 1 157.4 2 103.9 3 329.8 4 35.6 5 64.3 SR 1.9192 1 210 2 245.9 3 168.9 4 262.2 5 148.4 MG 0.8461 1 120.1 2 178.4 3 383.8 4 47.1 5 105.2    Figure 3. Diagram inhibition zone of Open Column Chromatography against E.coli.  0246810121-1 1-2 1-3 1-4 1-5Inhibition zone(mm)FractionsST-KN-15-3AL-KN-15-3SR-KN-15-4MG-KN-15-3612345678903rd International Conference on Tropical and Coastal Region Eco Development 2017 IOP PublishingIOP Conf. Series: Earth and Environmental Science 116 (2018) 012084  doi :10.1088/1755-1315/116/1/012084 Figure 4. Diagram inhibition zone of Open Column Chromatography against S.aureus.  The biggest E.coli and S.aureus inhibition zone were MG-KN-15-3-1-1 of 10,71mm and 10,98mm (Figure 3-4). Growth media also affected the quality and ability to inhibit pathogenic bacteria, E.coli and S.aureus, extracts of symbiotic fungi of marine sponge Axinella sp., Aspergillus sydowii (Figure 2-4). The content of nutrients in growth media affected the content of bioactive compounds from microorganisms [28-29].  4. Conclusion Symbiotic fungi of marine sponge Axinella sp., Aspergillus sydowii could be grown on four media, Malt Extract Broth/MEB (ST), Noni Juice Media (MG), Avocado leaves media (AL), and Soursop leaves media (SR). Growth media affected the quantity and quality of the symbiotic fungi extract, Aspergillus sydowii and its ability to inhibit pathogenic bacteria,  E.coli  and S.aureus.  References [1] Yalçın F N 2007 Biological activities of the marine sponge Axinella Hacettepe University, J Faculty of Pharmacy, 27(1), 47-60. [2]  Tsukamoto S, Tane K, Ohta T, Matsunaga S, Fusetani N, and Van Soest R W M 2001 Four new bioactive pyrrole-derived alkaloids from the marine sponge Axinella brevistyla. J. Nat. Prod., 64, 1576-1578. [3] Wratten S J, and Meinwald J 1981 Antimicrobial metabolites of the marine sponge Axinella polycopella. Cell. Mol. 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Antibacterial Activity Symbiotic Fungi of Marine Sponge

Axinella sp., Aspergillus Sydowii on Four Growth Medium

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Antibacterial Activity Symbiotic Fungi of Marine Sponge Axinella sp., Aspergillus Sydowii on Four Growth Medium

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