Antibacterial Activity as Inhibitors Pathogen Bacterial on Pond Shrimp of Extract Marine Biota Collected From Maspari Island, South Sumatera, Indonesia

Marine biota has variety of bioactive compound that is potential to be an antibacterial for shrimp ponds diseases. Some of marine biota has potential as antibacterial i.e. soft coral Sarcophyton sp., Sponge Aaptos sp., seaweeds Sargassum sp. and Halimeda sp. and mangroves Avicennia sp. and Rhizophora sp. species. This study is purposed to find the most potential marine biota as antibacterial. The research method was as follows; sampling, sample identification, extraction and antibacterial activity test. The results is soft coral and sponge extract Sarcophyton sp. has bioactivity against of shrimp bacteria pathogen such as Vibrio spp2. bacteria for around 6.3±0.1 mm, while Aaptos sp. has bioactivity against Vibrio spp1. bacteria for around 7.9+0.1 mm, Vibrio spp2. bacteria for around 7.2± 0.1 to 7.9±0.1 mm, Vibrio spp6. bacteria for around 7.5±0.2 mm, Escherichia coli for around 7.2±0.1 mm and Staphylococcus aureus for around 15.9±0.2 mm. Seaweed extract activity found only of Sargassum sp. which has antibacterial activity against for around 7.1±0.0 mm for Vibrio spp6. and mangrove species activity has Rhizophora sp. extract which has bioactivity against Vibrio spp4. were 7.3±0.1 mm and E. coli bacteria were 6.7±0.1 mm. The most potential marine biota as antibacterial is showed on sponge Aaptos sp.. with an inhibitory zone for around 15.9±0.2 mm (for S. aureus bacteria).

Mangrove is a coastal plant that has various types and has the potential for antibacterial. Several types of mangroves that have been known to produce bioactive compounds for antibacterial are Rhizophora sp. and Avicennia sp. Species Rhizophora sp. is known to produce antibacterial compounds for fish and human pathogenic bacteria such as Proteus sp., P. florescence, Flavobacterium sp., K. oxytoca, V. parahaemolyticus, P. aeruginosa, B. subtilis, K. pneumonia, S. aureus and E. coli (Umashankari et al., 2012;Arunprabu et al., 2016;Powar and Gaikwad, 2013;Joel and Bhimba, 2010). While Avicennia sp are known to have resistance to bacteria such as Enterococcus faecium, S. pneumoniae, K. pneumoniae, S. aureus, Vibrio harveyi, Shigella flexneri and S. aureus (Behbahani et al., 2018;Thatoi et al., 2016).
In addition, antibacterials from the marine biota above are reported to be also used to treat some diseases in pond shrimp caused by pathogenic bacteria. Some studies on marine biota that makes antibacterial to shrimp bacteria such as seaweed Ulva sp. and marine sponge Dendrilla sp. as antibacterial to bacteria from shrimp (V. fischeri, E. coli, P. aeruginosa, A. hydrophila and M. luteus) showed that Ulva sp. can be used as prophylactic drugs and Dendrilla sp. can be used as a therapeutic agent (Yatip et al., 2018). Acanthus ilicifolius is also used as an antibacterial for shrimp and fish pathogenic bacteria (A. hydrophila, V. harveyi and E. coli) showing that the extract is effective for antibacterial (Rao et al., 2015). Therefore, this study was conducted to obtain and determine the most potential marine biota as antibacterial.

Sampling Site
The study was conducted on September 2017 to January 2018. Marine biota samples were taken from Maspari island, South Sumatera, Indonesia ( Figure 1). Maspari is a very small island with a size of approximately 3 km 2 that is not inhabited by humans. This island is located in the southern part of Bangka Strait which belongs to the Ogan Komering Ilir district, South Sumatera. Marine biota on this island are very diverse, where coral is found in many fringing reefs at the depth <5 m with a maximum depth waters about 15 m. For seaweeds is found in shallow waters <2 m by the coast with sandy and partially rocky substrate, while mangrove plants found in the north and south of the island.

Samples Collection
The marine biota samples collected from Maspari island were one species of soft coral Sarcophyton sp. and one sponge Aaptos sp. two species of seaweed Sargassum sp. and Halimeda sp., and two species of mangroves Avicennia sp. and Rhizophora sp., 250 (g) respectively (Figure 2A-F). Samples are inserted into plastic bags, labeled and stored in a cold box. then carried in the laboratory for macerations.

Sample Extraction
Marine biota samples were cleansed by dH2O waters, weighed 250 g and smoothly cut. The sample was macerated using an Ethyl Acetate (EtOAc) solvent at a ratio of 1:4 (w/v) for 48 hours at room temperature, and each was repeated three times. After that, maceration continued with Methanol (MeOH) solvent. The extraction was carried out using rotary evaporation drying at 60 o C until obtaining a crude extract.

Antibacterial Activity Test
Bacteria isolates used for the test were six isolates of Vibrio sp. (with code V. spp1 to spp6), isolates were isolated from Vannamei sp. shrimp ponds at shrimp farming location in Sungsang, South Sumatera. In addition, the performance is also tested on the bacteria S. aureus and E. coli using Nutrient Broth (NB) and Nutrient Agar (NA) growing media. Inhibition test is done with agar diffusion (10.000 ppm for extract concentration). The extracts were tested using paper disks and solvents as controls with repeated three times (Rozirwan et al., 2015).

Extracts Biomass
Six samples of marine biota extracted by biomass were varied in each solvent. The biomass extract from the methanol solvent was obtained vacant on the mangrove samples (Table 1).  Table 1 showed that the extract had high weight in ethyl acetate solvent, excepted species of soft coral Sarcophyton sp. and two species of seaweed (Halimeda sp. and Sargassum sp.). This showed that bioactive compounds had dominated semi polar, particularly of mangrove species (Avicennia sp. and Rhizophora sp.), and no found extracts of methanol solvent.
Marine and coastal biota were marine natural products because they have a wide variety of bioactive compounds, which one of its many potentials has been reported as antibacterial (McClintock et al. 2010). This study is purposed to see the potential of bioactive compounds in some marine biota collected from Maspari island such as soft coral (Sarcophyton sp.), sponge (Aaptos sp.), seaweed (Sargassum sp. and Halimeda sp.) and mangroves (Rhizophora sp. and Avicennia sp.) as antibacterial (Vibrio spp1 to 6, E. coli and S. aureus).   Soft coral extracts in Sarcophyton sp. and sponge Aaptos sp. species have antibacterial activity. In Sarcophyton sp. extract had antibacterial activity against Vibrio spp2 bacteria, while Aaptos sp. extract against Vibrio spp1,2,6, E. coli and S. aureus. The antibacterial activity of the Aaptos sp. extract was found to be better than Sarcophyton sp. This is probably caused by Aaptos sp. that produce Aaptamine alkaloids bioactive compounds (Shubina et al., 2009), while Sarcophyton sp. produce terpenoid bioactive compounds (Al-Footy et al., 2015;Cheng et al., 2015). Antibacterial activity found in also Aaptos sp. (Beesoo et al., 2017;Murniasih and Bayu, 2016;Rosmiati et al., 2014). Sarcophyton sp. is similar, although its antibacterial activity is shown only with one isolate (Vibrio spp2) but indicates its antibacterial potential (Gomaa et al., 2016;Afifi et al., 2016;Rozirwan et al., 2015). The extract Aaptos sp. in Ethyl Acetate solvent was shown that it has more diverse antibacterial activity, where the extract in Ethyl Acetate solvent was more effective in inhibiting compared to methanol solvents.
Antibacterial activity in seaweed extract is found only in Sargassum sp. species, which has formed an inhibitory zone against the Vibrio spp6 isolate. Antibacterial activity on Sargassum sp. showed better than other species of seaweed (Kim et al., 2016). In mangrove extract Rhizophora sp. has antibacterial activity against isolate Vibrio spp4 and E. coli. The Rhizophora sp. species was also more active as a bacterial inhibitor (Arunprabu et al., 2016;Ernawati, 2016;Powar and Gaikwad, 2013;Umashankari et al., 2012).
The potential of bioactive compounds can also be done with the exploration of microbial symbionts. Symbian microbes can be expected to produce bioactive compounds that are almost identical to their inhibitors. Antibacterial activity of microbial symbionts from marine biota has been done in many researches such as marine biota Sarcophyton sp. (ElAhwany et al., 2015), Aaptos sp. (He et al., 2017;Santos-Gandelman et al., 2014), Sargassum sp. (Susilowati et al., 2015) and Rhizophora sp. (Deivanai et al., 2014). For all samples of marine biota extract collected from Maspari Island, it was found that sponge extract of Aaptos sp. species showed the best antibacterial activity as inhibitor of shrimp pond bacteria and also pathogen bacteria.
The seaweed Halimeda sp. and mangrove Avicennia sp. showed no antibacterial activity, whereas the antibacterial activity of these species has been widely reported in Halimeda sp. (Basir et al., 2017;Sivaramakrishnan et al., 2017;Indira et al., 2013), and Avicennia sp. (Dhayanithi et al., 2012;Joel and Bhimba, 2010;Raju and Sreeramulu, 2017). The loss of antibacterial activity is suspected because the concentration is too small that the inhibitory zone is not formed and may also be due to resistance to other bacteria.

IV. CONCLUSIONS
There were four from six marine biotas collected from Maspari island found for having antibacterial activity for pond shrimp disease i.e. Sarcophyton sp. and Aaptos sp. species (soft coral and sponge), Sargasum sp. (seaweed) and Rhizophora sp. (mangrove). The best antibacterial activity was found in sponge extract of Aaptos sp. for around 7.9 ± 0.1 mm and 15.9 ± 0.2 mm for Vibrio spp2. and S. aureus bacteria, where both acted as inhibition of bacteria isolate of pond shrimp and bacterial pathogen.