Investigation of an Antifouling Compound from Sponge Siphonodictyon

It is a complex phenomenon

diversity of marine species which constitute communities whose growth dynamic is driven by physical and biological processes [3].Marine is an environment rich in biological resources, inspiring humans to explore and exploit to get a variety of biological materials as natural products from the results of primary and secondary metabolites in the form of bioactive substances from marine biota.Various marine organisms in Indonesian waters were investigated for their bioactivity, including algae, soft corals, mollusks, crustaceans, echinoderms, sponges, and ascidians.The use of marine resources, especially in natural products, has only developed from the past until recent years [4][5][6].These natural products are commonly used in the pharmaceutical industry and the environment.The sponge is one of the marine organisms that have various bioactive substances.More than 6,000 new compounds were isolated from marine organisms, and 33% were isolated from sponges [4,7].The sponge-derived bioactive compounds are reported with pharmacological activities or effects, including antiviral, antibacterial, cytotoxic, and anti-inflammatory [8].
Sponges are also reported to have activities that play a role in ecology; one is antifouling.Several research groups reported antifouling activity obtained from sponges [8][9][10][11][12][13].Previously, the Siphonodictyon coralliphagum, A marine sponge species growing in Salibabu Beach, showed potent antibacterial activity against a series of Gram-positive and negative bacteria [14].

Extraction
The sponge sample of S. coralliphagum, collected from the Salibabu Waters, was extracted using ethanol as a solvent using the maceration technique.Previously, the sample was washed using fresh water to reduce the salt content from the sponge body.The sponge was sliced, put into a PTE plastic bottle, and soaked in ethanol for 7x24 hours.The extract was then decanted and evaporated using Rotary Vacuum Evaporator.The extract was weighed before being dissolved with 98% ethanol for further analysis.

Antifouling activity testing
Antifouling activity testing was carried out using the method described in Moningka (2000) with some modification [2,16,17] which was done by painting on the top surface of a 10.5 x 21 cm concrete paving block with the Avianpaint tm brand as a base layer.After the base layer is dried, ethanolic sponge extract S. coralliphagum is mixed with the paint above with increased concentrations of 5 (further documented as treatment 1), 10 (further documented as treatment 2), and 25% w/v (further documented as treatment 3), as treatments.
Extract suspension was then re-applied to the surface of the paving block with three replications in each treatment.The negative control is applied using the same paint without adding extract.Positive control was using Nipponpaint tm copper antifouling paint.The paint on the paving block was allowed to dry for three days.Paving blocks were placed in a subtidal area where the objects were completely submerged in seawater during the experiment.The antifouling activity test was conducted at coordinates 1 ° 26'36.7 "LU and 125 ° 12'8.4"East (Figure 3).The study was conducted for three months, with intensive data collection every seven days in the first month of testing.Data was collected on the 7 th , 14 th , 21 st, and 28 th days after laying on a subtidal area.The following paving-block tests were left for 60 days to observe biofouling growth.The observation was stopped on the 90 th day to see the attachment process and development of macro-foulers.Data is collected by following the treated paving surfaces.

Fractionation
Fractionation of sponge extract S. coralliphagum performed by using the Vacuum Liquid Chromatography (VLC) method using the solvent hexane, dichloromethane: ethyl acetate; ethyl acetate: methanol to obtain 11 fractions with 500 ml each eluent.Furthermore, each obtained fraction was dried.The obtained fractions were tested for their activity against the Gram-positive marine isolates Bacillus megaterium DSM32T and Gram-negative Escherichia coli DSM498.

Antimicrobial Assays
The fractions obtained in the previous procedure were individually tested against Bacillus megaterium DSM 32 T and Escherichia coli DSM 498 using Agar Diffusion Assay for antimicrobial assays.The strains were grown in 10 ml slope nutrient agar and incubated at 37˚ C overnight.The cultures were harvested, and their density was measured.The initial OD600 was set to 1 to reach a concentration of 8x10 8 cells/ml.The cultures were diluted to 8x10 6 cells/ml, and 1 ml of the bacterial culture was subjected and seeded into the Luria Betani Agar (LB Agar).After solidification, 20 μl of the sponge fractions were spotted on the plates.Chloramphenicol (1mg/ml) and 96% ethanol for 20 µL each served as positive and negative controls, respectively.All samples were performed in triple.Plates were incubated at 37°C for 18 h and checked for inhibition zones.

NMR experiments
NMR experiments were performed at 25°C in a Bruker Avance DRX 300 MHz spectrometer with an indirect 5 mm triple TBI 1H/[9]/13C probe head using standard pulse sequences available in the Bruker software.The samples were dissolved in 700 μL of 99.8% MeOD.1D 1H spectra were recorded at 300 Mhz with a 30° pulse, a delay D1 of 2s and 64 scans.As an external standard, chemical shifts were expressed in ppm relative to TMS (Tetrametylsilane).Double-quantum filtered 1H-1H correlated spectroscopy (DQF COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) with a 60-ms mixing time were performed according to standard pulse sequences to assign 1 H and 13 C resonances.

Antifouling observations
Observations from in-situ testing after subtidal submersion were presented in Figure 4. Seven days after submersion, the paving surfaces looked relatively clean, and no fouling organisms were observed in the paving blocks of each treatment or both controls.Microscopic observations of the 14 th day of submerged paving blocks of the scraped paving surface showed the fiberlike structure produced by bacteria observed in treatments 1 (5%) and 2 (10%) of sponge extracts and negative control.It is likely the initial formation of a biofilm from bacteria.The distribution of fiber-like structure formation was observed to decrease with an increasing percentage of the extract on paving blocks.The scraped paving surface in treatment 3 (25%) and positive control did not show the presence of hyphae threads produced by bacteria-producing hyphae such as Actinomycetes (Figure 6).Biofilm formation is a complex process with several stages [19,20].The procedure typically starts with the settlement of bacteria on the submerged surface.From here, a conditioning film is formed, which involves the adsorption of organic compounds (i.e., proteins, carbohydrates, and nucleic acids) to the surface [21,22].The following stage in biofilm formation is the attachment of cells to the surface.Afterward, cell proliferation leads to the formation of a micro-colony, and cell-cell chemical signaling activates biofilm genes which enhance micro-colonies ability to form and adhere to the solid interfaces.Established biofilms can then detach from the tangible interfaces to facilitate the multiplication and distribution of cells and biofilm microcolonies [23].The result of the 28 th -day observation is presented in Figure 8.At 28 days after subtidal immersion, no macroscopic organisms were attached to all treatments and in both controls.The observed tunicate Corella eumyota which had previously begun to grow in the negative control was no longer kept (Figure 7); it was very likely to be predated by litoral fish or Ophiolepsis sp., which was also found on the paving.Microscopic observations showed that some of the thalli of green algae were seen on the paving surface in treatment 1 (5%) and in the negative control.The presence of the thalli indicates the settlement process of microalga; the environmental changes at the microscopic level of the paving surface are marked by the initial formation of a biofilm layer by bacteria that occurred on the 14 th day to be most likely to provide nutrients that allow the process to take place [20].After the 90th day, it was observed that macro-foulers could only develop in the negative and positive control (Figure 10).The development of fouling organisms was not observed in all treatments 5, 10, or 25% extract of S. coralliphagum.Fouling organisms observed were juvenile gastropods Pinctada margaritifera, P. maxima, and Cerithidea rizophorarum.From the sponge species, Niphates erecta, and the tunicate species, Corella eumyota.In contrast, the alga Ulva lactuca was observed only in negative control (Figure 10).

Figure 11. Microscopic appearance of the surface of negative control paving block after 90 days of subtidal immersion
Microscopic observations show the formation of a mycelium structure from the fungus Fusarium cf.tricintum with fiberlike pink color; this structure forms a biofilm layer on top of organic material underneath it, which is very likely formed from bacterial biofilm that grew previously from day 14 (Figure 11).We have also observed settled micro alga that have developed and are attached to the surface of the substrate (indicated by red arrows).
Tables 1 and 2 summarize the organisms found on the paving blocks with different treatments.

NMR analysis
The active fraction of S. coralliphagum was then sent to 1H NMR to analyze its chemical structure (Figure 12).In general, the active fraction still contains long fatty acid compounds.Furthermore, the active fraction might consist of a cyclic aliphatic compound with keto-enol functional groups attached to the system, which was shown in 4.0-4.5 ppm, followed by several methyl aliphatic groups at the end of it their chain (appears at 1.5 ppm) and has been observed based on 1 H NMR spectrum.Due to the less amount of the fraction, we are not able to elucidate the exact chemical structure of the active compound

CONCLUSIONS
The extract sponge S. coralliphagum has vigorous antifouling activity against marine fouling organisms.The extract was fractionated using VLC and continued by testing their activity against Gram-negative bacteria where DCM:EtoAC=3:7 fraction is active.Investigation of NMR spectra of Fr.DCM:EtoAC=3:7 shows a cyclic aliphatic compound with keto-enol functional groups attached to the system followed by several methyl aliphatic groups at the end of its chain.

Figure 3 .
Figure 3.In situ testing location, Harbor of the Indonesian Navy Patrol, Bitung, Indonesia.Red dot = in situ location.

Figure 4 .
Figure 4. Display of paving blocks with different treatments after seven days of subtidal immersion.

Figure 5 .
Figure 5. Display of paving blocks with different treatments after 14 days of subtidal immersion.

Figure 9 .Figure 10 .
Figure 9. Microscopic appearance of the surfaces of paving block after 28 days of subtidal immersion, A) Negative control; B). 5% extract.Red arrows = thalli formation of green microalga.

Table 3 .
Based on the result, only DCM:EtoAc=3:7 fraction show activity against tested microorganisms.The polarity of the active compound of S. coralliphagum belongs to semi-polar.No other fractions detected no activity[24].