Selection, Identification and Optimization of The Growth, Setyati et al. JPHPI 2014, Volume 17 Nomor 3 Selection, Identification and Optimization of the Growth Water Probiotic Consortium of Mangrove Ecosystems as Bioremediation and Biocontrol in Shrimp Ponds Wilis Ari Setyati*1, Erni Martani², Triyanto², Subagiyo³, Muhammad Zainuddin⁴ 1) Departement of Marine Science, Faculty of Fisheries and Marine Science Diponegoro University, Post-graduate Student, Gadjah Mada University 2)Departement of Biotechnology, Post-graduate, Gadjah Mada University. ³)Marine Science Laboratory, Marine Science Departement, Diponegoro University. ⁴)Natural Product Laboratory, UPT (Integrated Laboratory), Diponegoro University. Faculty of Fisheries and Marine – Tembalang – Semarang Telp 024 7474698 *Coresponding author: [email protected] Accepted April 15th, 2014/Approved Agustus 10th, 2014 Abstract Shrimp aquaculture is an activity that potentially generates organic waste. The accumulation of organic matter is becoming one of the main factors causing the emergence of disease. Problem-solving approach that is most effective is through bioremediation. The aims of this study were to select, identify and cultivate bacteria from mangrove sediments from Cilacap, Rembang and Banyuwangi which potentially as probiotic consortium of bioremediation activity and biocontrol. The results showed that total of 45 isolates (proteolytic), 35 isolates (amylolytic), 35 isolates (lipolytic), and 18 isolates (cellulolytic). There were 59 bacterial isolates had antibacterial activity of vibrio (V. harveyi, V. alginolyticus, V. vulnificus and V. anguilarum). Based on the identification of 16 S-rRNA genes, 4 isolates showed that the C2 isolate was identified as Bacillus subtilis, C11 isolate was identified as Bacillus firmus, C13 and C14 isolates were identified as B. Flexus. This study concluded that cultivation of Bacillus subtilis C2 optimum at 2% molase and yeast extract 0.5% at pH 8 and 30 0C. Bacillus firmus C11 optimum at 2% molase and yeast extract 0.5% at pH 8 and 30 0C. Bacillus flexus C13 optimum at 2% glucose and yeast extract 0.5% at pH 8 and 30 0C. Bacillus flexus C14 optimum at 4% molase and yeast extract 0.25% at pH 8 and 30 0C. The result of culture applications of 4 isolates showed an effect of increasing shrimp weight by 141, 9% compared by the control. Keywords: sediment, mangrove, bioremediation, biocontrol Introduction the sediment pond. The most effective approach Shrimp farm produces organic waste from is through bioremediation (microorganism’s the residue of the shrimp feed and feces. Jackson’s agent). Organic waste of feed are complex, so that, studies (2003) showed that the nitrogen efficiency bioremediation requires a consortium of different usage of feed was approximately 22%, whereas 78% microorganisms with the kind of variation of of nitrogen was discharged into the environment activity to clean up organic matter. and accumulated in the bottom of sediment In addition to environmental stress factors, pond. High organic matters content will spur on the problems are encountered in the development the growth of microorganisms, biodecomposition of shrimp farming is a factor of disease (Smith process and oxygen consumption (Avnimelech and Briggs 1993). So that the developing water and Ritvo 2003). Insufficient oxygen conditions probiotic consortium should have the function triggers the growth of anaerobic microorganisms to clean the organic material (biodecomposition) 2- which actively reducing SO4 into H2S that while controling the pathogen population inhibited the growth of domestic animals (biocontrol). Therefor, to produce probiotic (Mugnier et al. 2008). It is necessary to attempt to bacteria it is necessary to screen, optimization of overcome the accumulation of organic matter in growth mediums and probiotic application test. Masyarakat Pengolahan Hasil Perikanan Indonesia 243 JPHPI 2014, Volume 17 Nomor 3 Selection, Identification and Optimization of The Growth, Setyati et al. Materials and Methods the procedure according to Jacob & Gerstein The study was conducted by the laboratory (1960) in Bairagi et al. (2002) which used experimental method and design of complete 2216 E Zobell agar medium enriched with randomized study. Overall, the study will be carried starch (1%). Lugol’s iodine solution is poured in 5 stages: isolations, selections, identifications, above 1% of bacteria for agar identification of cultivations and applications. Insulating phase amylolytic activity (clear zone was formed). consists of the preparation, dilution and Cellulolytic activity test was performed using purification. Selection phase consists of selection agar medium enriched with 1% CMC. Congo of proteolytic activity, amylolytic, cellulolytic, red solution was poured into the bacteria for lipolytic, antibacterial and antaginis. Phase of identification of cellulose hydrolysis activity. identification of the molecular basis of bacterial Lipolytic activity test was done according to consortium-based on 16S rRNA gene. Cultivation the procedure Sangiliyi and Gunasekeran phase of probiotic consortium consists of 4 phases: (1996) in Bairagi et al. (2002) which used 2216 screening and selecting of probiotic growth E Zobel agar medium enriched with 80 tween. nutrients, optimization the nutrient concentrations Lipase activity was shown by the formation of of growth, optimization the pH growth, and fatty acid deposits around the bacteria. optimization the growth temperature. Consortium Application phase was conducted on a bench scale Bacterial Selection Based on Antibacterial (bench scale). Capability Antibacterial test for all isolates against Isolation of Bacteria pathogens (V. alginoliticus, V. harveyi, Sediment sampling was performed on V. anguilarum and V. vulnivicus ) were mangrove area in Cilacap, Rembang and performed by puncturing technique and Banyuwangi. Sediment samples were taken using overlaying (Isnansetyo 2004). the pure isolate a soil sampler at a depth of +10 cm. The Sediment cultures were inserted on the 2216 E Zobell samples were diluted up to 10-5, 10-6 and 10-7, agar medium by using a needle preparation respectively inoculation performed by pour plate and were incubated for 24 hours. After 24 method. Furthermore petri dishes were incubated hours of incubation, the overlay was done by for 2 x 24 hours. The colonies of bacteri which pouring the pathogen into Zobell 2216 E soft were formed on each petri dish from each dilution agar medium (70%). Double layered then were were isolated that showed different morphology. incubated for 24 hours. Antagonist activity Isolation and purification of bacterial isolates were was identified by the production of clear zone performed by the method of scratches (streak without growth (inhibition zone) around the method). puncture of colony. Bacteria Based Selection Capability Doing Antagonist Test Between Isolates Biodegradation of Organic Materials Antagonist tests were conducted using Screening will be done for bacteria streak on Zobell agar medium. Antagonist that are capable of altering or degrading activity was shown by the clear zone/barrier polysaccharides, proteins and fats by the zone around the colony. approaching the activity of protease enzymes, amylase, cellulase and lipase. Tests were Identification of Bacteria Consortium by carried out by the procedure of proteolytic 16s-rrna Gene Molecular activity by Jacob & Gerstein (1960) within The amplification of 16S-rRNA gene was Bairagi et al. (2002) by using 2216 E Zobell performed by using 1 mL of DNA template were medium enriched with skim milk (1%). amplified by PCR Beads kit RTG using a universal Amylolytic activity test was carried out by primer (Marchesi et al. 1998), there were 63F 244 Masyarakat Pengolahan Hasil Perikanan Indonesia Selection, Identification and Optimization of The Growth, Setyati et al. JPHPI 2014, Volume 17 Nomor 3 (5’-CAGGCCTAACACATGCAAGTC) and nitrogen sources with the optimum 1387r (5’-GGGCGGWGTGTACAAGGC). It concentration was adjusted to pH variation was made a master mix containing 1.5 units of of 6, 7 and 8 then were inoculated with a Tag DNA polymerase, 10 mM Tris HCl (pH starter isolates that gave OD 0.01 on A600 9.0 at room temperature), 50 mM KCl, 1.5 mM and incubation at room temperature. Each MgCl2, 200 mM on each dNTP and a stabilizer 12-hour intervals for 60 hours samples were and including BSA and 1 mL of DNA template. taken by 10 mL, then measuring the OD at Then, put into a PCR machine, GeneAmp PCR A600. Systems 2400 (Perkin Elmer Biosystems, USA), with the Pre-PCR conditions (94°C, 2 minutes), Optimizing of Temperature denaturation (92°C, 30 seconds), primer Medium was added with a carbon sources annealing (55°C, 30 seconds ), elongation and a nitrogen sources at the optimum (75°C, 1 min) and a Post PCR (75°C, 5 min), the concentration. It were inoculated with a cycle 30 times. The results of 16S-rRNA gene starter isolates that gave OD 0.01 on the amplification which produced the positif results A600. Incubation at varied temperature 30, were followed on positive sequencing analysis. 34 and 38 0C. every 12-hour intervals for 60 Homology analysis was done by online at http:// hours samples were taken by 7 mL then were www.ddbj.nig.ac.jp/. performed measuring OD at A600. Screening and Selecting of Nutrient Growth Tests of Consortium Application The methods used in this study followed The application consortium tests at the the procedure according to Polak-Bereka laboratory scale using a plastic tub, sea