Optimization of Lipopeptide Biosurfactant Production by Bacillus clausii J1G0B and Its Application in Ex-situ Microbial Enhanced Oil Recovery (MEOR)
Mukhammad Asy’ari(1*), Nabila Rona Nur Kamila(2), Adyuna Riana Putri(3), Agustina Lulustyaningati Nurul Aminin(4), Marcelinus Christwardana(5)
(1) Department of Chemistry, Faculty of Science and Mathematics, Universitas Diponegoro, Jl. Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia
(2) Department of Chemistry, Faculty of Science and Mathematics, Universitas Diponegoro, Jl. Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia
(3) Department of Chemistry, Faculty of Science and Mathematics, Universitas Diponegoro, Jl. Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia
(4) Department of Chemistry, Faculty of Science and Mathematics, Universitas Diponegoro, Jl. Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia
(5) Department of Chemistry, Faculty of Science and Mathematics, Universitas Diponegoro, Jl. Prof. Soedharto SH, Tembalang, Semarang 50275, Indonesia
(*) Corresponding Author
Abstract
The decline in global oil production has increased interest in microbial enhanced oil recovery (MEOR) using biosurfactant-producing microorganisms to improve oil mobilization. This study investigates Bacillus clausii J1G0B, a halophilic bacterium from Indonesian salt ponds, for optimized lipopeptide biosurfactant production and ex-situ MEOR application. Production conditions were optimized by varying carbon and nitrogen sources, identifying 2% palm oil and 15 mM urea as optimal for biosurfactant synthesis. The produced biosurfactant exhibited strong emulsification activity, achieving EI24 values of 94.70 ± 0.81% for palm oil and 80.00 ± 1.15% for crude oil. Biosurfactant produced in tryptone-supplemented medium (BS1) showed higher emulsification performance than that produced in urea medium (BS2), reaching up to 95.00 ± 1.15% toward palm oil. FTIR analysis confirmed the lipopeptide structure of the biosurfactant, while stability tests showed an initial decrease in the soluble fraction within 24 h, followed by relatively stable behavior up to 72 h. In ex-situ oil-washing assays, the biosurfactant achieved 67.70 ± 1.64% oil removal at 20,000 ppm. These results highlight the complementary roles of emulsification and oil washing and support the feasibility of crude production process from inexpensive substrates for cost-effective, sustainable ex-situ MEOR.
Keywords
References
[1] Nagy, R., Hartyányi, M., Bejczi, R., Bartha, L., and Puskás, S., 2025, Recent aspects of chemical enhanced oil recovery, Chem. Pap., 79 (5), 2695–2716.
[2] Ragab, A., and Mansour, E.M., 2021, “Enhanced Oil Recovery: Chemical Flooding” in Geophysics and Ocean Waves Studies, Eds., Essa, K.S., Di Risio, M., Celli, D., and Pasquali, D., IntechOpen, London, UK.
[3] Zhou, Y., Yin, D., Chen, W., Liu, B., and Zhang, X., 2019, A comprehensive review of emulsion and its field application for enhanced oil recovery, Energy Sci. Eng., 7 (4), 1046–1058.
[4] She, H., Kong, D., Li, Y., Hu, Z., and Guo, H., 2019, Recent advance of microbial enhanced oil recovery (MEOR) in China, Geofluids, 2019 (1), 1871392.
[5] Xiao, M., Sun, S.S., Zhang, Z.Z., Wang, J.M., Qiu, L.W., Sun, H.Y., Song, Z.Z., Zhang, B.Y., Gao, D.L., Zhang, G.Q., and Wu, W.M., 2016, Analysis of bacterial diversity in two oil blocks from two low-permeability reservoirs with high salinities, Sci. Rep., 6 (1), 19600.
[6] Al-Sayegh, A., Al-Wahaibi, Y., Joshi, S., Al-Bahry, S., Elshafie, A., and Al-Bemani, A., 2016, Bioremediation of heavy crude oil contamination, Open Biotechnol. J., 10 (1), 301–311.
[7] Putra, W., and Hakiki, F., 2019, Microbial enhanced oil recovery: Interfacial tension and biosurfactant-bacteria growth, J. Pet. Explor. Prod. Technol., 9 (3), 2353–2374.
[8] Joshi, S.J., and Desai, A.J., 2013, Bench-scale production of biosurfactants and their potential in ex-situ MEOR Application, Soil Sediment Contam.: Int. J., 22 (6), 701–715.
[9] Purwasena, I.A., Amaniyah, M., Astuti, D.I., Firmansyah, Y., and Sugai, Y., 2024, Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: A green chemical for microbial enhanced oil recovery (MEOR), Sci. Rep., 14 (1), 10270.
[10] Wu, B., Xiu, J., Yu, L., Huang, L., Yi, L., and Ma, Y., 2022, Biosurfactant production by Bacillus subtilis SL and its potential for enhanced oil recovery in low permeability reservoirs, Sci. Rep., 12 (1), 7785.
[11] Pathak, K.V., and Keharia, H., 2014, Application of extracellular lipopeptide biosurfactant produced by endophytic Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) in microbially enhanced oil recovery (MEOR), 3 Biotech, 4 (1), 41–48.
[12] Al-Sulaimani, H., Al-Wahaibi, Y., Al-Bahry, S., Elshafie, A., Al-Bemani, A., Joshi, S., and Zargari, S., 2011, Optimization and partial characterization of biosurfactants produced by Bacillus species and their potential for ex-situ enhanced oil recovery, SPE J., 16 (3), 672–682.
[13] Daryasafar, A., Jamialahmadi, M., Moghaddam, M.B., and Moslemi, B., 2016, Using biosurfactant producing bacteria isolated from an Iranian oil field for application in microbial enhanced oil recovery, Pet. Sci. Technol., 34 (8), 739–746.
[14] Xi, W., Ping, Y., and Alikhani, M.A., 2021, A review on biosurfactant applications in the petroleum industry, Int. J. Chem. Eng., 2021 (1), 5477185.
[15] Englerová, K., Bedlovičová, Z., Nemcová, R., Király, J., Maďar, M., Hajdučková, V., Styková, E., Mucha, R., and Reiffová, K., 2021, Bacillus amyloliquefaciens—Derived lipopeptide biosurfactants inhibit biofilm formation and expression of biofilm-related genes of Staphylococcus aureus, Antibiotics, 10 (10), 1252.
[16] Sarwar, A., Brader, G., Corretto, E., Aleti, G., Abaidullah, M., Sessitsch, A., and Yusuf Hafeez, F., 2018, Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity, PLoS One, 13 (7), e0201624.
[17] Saravanakumar, D., Thomas, A., and Banwarie, N., 2019, Antagonistic potential of lipopeptide producing Bacillus amyloliquefaciens against major vegetable pathogens, Eur. J. Plant Pathol., 154 (2), 319–335.
[18] Bartal, A., Vigneshwari, A., Bóka, B., Vörös, M., Takács, I., Kredics, L., Manczinger, L., Varga, M., Vágvölgyi, C., and Szekeres, A., 2018, Effects of different cultivation parameters on the production of surfactin variants by a Bacillus subtilis strain, Molecules, 23 (10), 2675.
[19] Alvarez, V.M., Guimarães, C.R., Jurelevicius, D., de Castilho, L.V.A., de Sousa, J.S., da Mota, F.F., Freire, D.M.G., and Seldin, L., 2020, Microbial enhanced oil recovery potential of surfactin-producing Bacillus subtilis AB2.0, Fuel, 272, 117730.
[20] Sari, C.N., Hertadi, R., Gozan, M., and Roslan, A.M., 2019, Factors affecting the production of biosurfactants and their applications in enhanced oil recovery (EOR). A review, IOP Conf. Ser.: Earth Environ. Sci., 353 (1), 012048.
[21] Zhang, J., Xue, Q., Gao, H., Lai, H., and Wang, P., 2016, Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery, Microb. Cell Fact., 15 (1), 168.
[22] Zhu, Z., Zhang, B., Chen, B., Cai, Q., and Lin, W., 2016, Biosurfactant production by marine-originated bacteria Bacillus subtilis and its application for crude oil removal, Water, Air, Soil Pollut., 227 (9), 328.
[23] Deng, Z., Jiang, Y., Chen, K., Li, J., Zheng, C., Gao, F., and Liu, X., 2020, One biosurfactant-producing bacteria Achromobacter sp. A-8 and its potential use in microbial enhanced oil recovery and bioremediation, Front. Microbiol., 11, 247.
[24] Sari, I.P., Basyiruddin, M.I., and Hertadi, R., 2018, Bioconversion of palm oil into biosurfactant by Halomonas meridiana BK-AB4 for the application of corrosion inhibitor, Indones. J. Chem., 18 (4), 718–723.
[25] Hsu, C.Y., Mahmoud, Z.H., Hussein, U.A.R., Abduvalieva, D., Alsultany, F.H., and Kianfar, E., 2025, Biosurfactants: Properties, applications and emerging trends, S. Afr. J. Chem. Eng., 53, 21–39.
[26] Janek, T., Gudiña, E.J., Połomska, X., Biniarz, P., Rodrigues, L.R., Rymowicz, W., and Lazar, Z., 2021, Sustainable surfactin production by Bacillus subtilis using crude glycerol from different wastes, Molecules, 26 (12), 3488.
[27] Karmainski, T., Lipa, M.K., Kubicki, S., Bouchenafa, A., Thies, S., Jaeger, K.E., Blank, L.M., and Tiso, T., 2024, Optimized feeding strategies for biosurfactant production from acetate by Alcanivorax borkumensis SK2, Fermentation, 10 (5), 257.
[28] Nikolova, C., and Gutierrez, T., 2020, Use of microorganisms in the recovery of oil from recalcitrant oil reservoirs: Current state of knowledge, technological advances and future perspectives, Front. Microbiol., 10, 2996.
[29] Wood, D.A., 2019, Microbial improved and enhanced oil recovery (MIEOR): Review of a set of technologies diversifying their applications, Adv. Geo-Energy Res., 3 (2), 122–140.
[30] Théatre, A., Cano-Prieto, C., Bartolini, M., Laurin, Y., Deleu, M., Niehren, J., Fida, T., Gerbinet, S., Alanjary, M., Medema, M.H., Léonard, A., Lins, L., Arabolaza, A., Gramajo, H., Gross, H., and Jacques, P., 2021, The surfactin-like lipopeptides from Bacillus spp.: Natural biodiversity and synthetic biology for a broader application range, Front. Bioeng. Biotechnol., 9, 623701.
[31] Berberov, K., Boyadzhieva, I., Yakimova, B., Petkova, H., Stoineva, I., Nacheva, L., and Kabaivanova, L., 2026, Exploring biosurfactant production from halophilic bacteria, isolated from Burgas salterns in Bulgaria, Mar. Drugs, 24 (1), 53.
[32] Christwardana, M., Asy’ari, M., Saputra, M.R., and Hadiyanto, H., 2025, Exploring halophilic bacteria Bacillus clausii isolated from Madura salt pond: Challenges of utilization in hypersaline microbial fuel cells for fish processing wastewater treatment, Appl. Sci. Eng. Prog., 18 (3), 7663.
[33] Suryanti, V., Masykur, A., Setyono, H.A., and Ramadani, S., 2021, Production and characterization of biosurfactant produced by LactoBacillus lactis grown in media containing crude palm oil (CPO), Biodiversitas, 22 (12), 5501–5506.
[34] Alyousif, N.A., Al-Tamimi, W.H., and Abd Al-Sahib, M.A., 2022, Evaluation of the effect of various nutritional and environmental factors on biosurfactant production by Staphylococcus epidermidis, Biodiversitas, 23 (7), 3533–3538.
[35] Schalchli, H., Lamilla, C., Rubilar, O., Briceño, G., Gallardo, F., Durán, N., Huenchupan, A., Diez, M.C., 2023, Production and characterization of a biosurfactant produced by Bacillus amyloliquefaciens C11 for enhancing the solubility of pesticides, J. Environ. Chem. Eng., 11 (6), 111572.
[36] Li, H., Fang, C., Liu, X., Bao, K., Li, Y., and Bao, M., 2023, Quantitative analysis of biosurfactants in water samples by a modified oil spreading technique, RSC Adv., 13 (15), 9933–9944.
[37] Sari, C.N., Hertadi, R., Harahap, A.F.P., Ramadhan, M.Y.A., and Gozan, M., 2020, Process optimization of palm oil mill effluent-based biosurfactant of Halomonas meridiana BK-AB4 originated from Bledug Kuwu mud volcano in Central Java for microbial enhanced oil recovery, Processes, 8 (6), 716.
[38] Novák, P., and Havlíček, V., 2016, “Protein Extraction and Precipitation” in Proteomic Profiling and Analytical Chemistry: The Crossroads: Second Edition, Eds. Ciborowski, P., and Silberring, J., Elsevier, Boston, US, 51–62.
[39] Ramcharan, T., and Bissessur, A., 2016, Analysis of linear alkylbenzene sulfonate in laundry wastewater by HPLC–UV and UV–vis spectrophotometry, J. Surfactants Deterg., 19 (1), 209–218.
[40] Bhattarai, K., Majer, T., Haussmann, M., Schollmeyer, D., Kramer, M., Oni, F.E., Höfte, M., Voget, R., Gütschow, M., Ruetalo, N., Schindler, M., Straetener, J., Wannenwetsch, T., Brötz-Oesterhelt, H., Karongo, R., Masberg, B., Lämmerhofer, M., Hennessy, R.C., Muletz-Wolz, C.R., and Gross, H., 2025, Salamandamide lipodipeptides are biosynthetic intermediate shunt products of the nonamodular nonribosomal peptide assembly lines of the viscosin family, J. Nat. Prod., 88 (4), 1012–1022.
[41] Purwasena, I.A., Astuti, D.I., Syukron, M., Amaniyah, M., and Sugai, Y., 2019, Stability test of biosurfactant produced by Bacillus licheniformis DS1 using experimental design and its application for MEOR, J. Pet. Sci. Eng., 183, 106383.
[42] Cooper, D.G., and Goldenberg, B.G., 1987, surface-active agents from two Bacilllus species, Appl. Environ. Microbiol., 53 (2), 224–229.
[43] Li, Z., Zhang, Y., Lin, J., Wang, W., and Li, S., 2019, High-yield di-rhamnolipid production by Pseudomonas aeruginosa YM4 and its potential application in MEOR, Molecules, 24 (7), 1433.
[44] Montgomery, D.C., 2019, Design and Analysis of Experiments, 10th Edition, John Wiley & Sons, Inc., Hoboken, NJ, US.
[45] Cohen, J., 2013, Statistical Power Analysis for the Behavioral Sciences, Academic Press, Cambridge, UK.
[46] Uyar, E., and Sağlam, Ö., 2021, Isolation, screening and molecular characterization of biosurfactant producing bacteria from soil samples of auto repair shops, Arch. Microbiol., 203 (8), 4929–4939.
[47] Hiller, E., Off, M., Hermann, A., Vahidinasab, M., Benatto Perino, E.H., Lilge, L., and Hausmann, R., 2024, The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes, Microb. Cell Fact., 23 (1), 260.
[48] Ng, Y.J., Khoo, K.S., Chew, K.W., Tang, D.Y.Y., Alharthi, S., Mayol, A.P., and Show, P.L., 2025, Solution towards low yield biosurfactant production from bacteria, a novel approach using microalgae–bacteria consortium, Microb. Cell Fact., 24 (1), 220.
[49] Nurfarahin, A.H., Mohamed, M.S., and Phang, L.Y., 2018, Culture medium development for microbial-derived surfactants production—An overview, Molecules, 23 (5), 1049.
[50] Ali, N., Pang, Z., Wang, F., Xu, B., and El-Seedi, H.R., 2022, Lipopeptide biosurfactants from Bacillus spp.: Types, production, biological activities, and applications in food, J. Food Qual., 2022 (1), 3930112.
[51] Puhm, M., Ainelo, H., Kivisaar, M., and Teras, R., 2022, Tryptone in growth media enhances Pseudomonas putida biofilm, Microorganisms, 10 (3), 618.
[52] Wang, Q., Ren, Y., Cui, Y., Gao, B., Zhang, H., Jiang, Q., Loor, J.J., Deng, Z., and Xu, C., 2022, Bacillus subtilis produces amino acids to stimulate protein synthesis in ruminal tissue explants via the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta–serine/threonine kinase–mammalian target of rapamycin complex 1 pathway, Front. Vet. Sci., 9, 852321.
[53] He, H., Li, Y., Zhang, L., Ding, Z., and Shi, G., 2023, Understanding and application of Bacillus nitrogen regulation: A synthetic biology perspective, J. Adv. Res., 49, 1–14.
[54] Furukawa, K., Oba, M., Toyama, K., Opiyo, G.O., Demizu, Y., Kurihara, M., Doi, M., and Tanaka, M., 2017, Low pH-triggering changes in peptide secondary structures, Org. Biomol. Chem., 15 (30), 6302–6305.
[55] Kumar, A.P., Janardhan, A., Viswanath, B., Monika, K., Jung, J.Y., and Narasimha, G., 2016, Evaluation of orange peel for biosurfactant production by Bacillus licheniformis and their ability to degrade naphthalene and crude oil, 3 Biotech, 6 (1), 43.
[56] Xu, H., Wang, H., Jia, W., Ren, S., and Wang, J., 2019, Application of Bacillus subtilis strain for microbial-enhanced oil recovery, Int. J. Green Energy, 16 (7), 530–539.
[57] Pereira, J.F.B., Gudiña, E.J., Costa, R., Vitorino, R., Teixeira, J.A., Coutinho, J.A.P., and Rodrigues, L.R., 2013, Optimization and characterization of biosurfactant production by Bacillus subtilis isolates towards microbial enhanced oil recovery applications, Fuel, 111, 259–268,
[58] Mondal, M.H., Sarkar, A., Maiti, T.K., and Saha, B., 2017, Microbial assisted (Pseudomonas sp.) production of novel bio-surfactant rhamnolipids and its characterisation by different spectral studies, J. Mol. Liq., 242, 873–878.
[59] Piotr, D., Natalia, R., Kinga, P., Tomasz, B., and Olga, M.M., 2025, Genomic and functional characterization of a lipopeptide-producing endophytic Bacillus velezensis EL isolated from Euphorbia cyparissias L, Microb. Cell Fact., 24 (1), 250.
[60] Joshi, S., Bharucha, C., Jha, S., Yadav, S., Nerurkar, A., and Desai, A.J., 2008, Biosurfactant production using molasses and whey under thermophilic conditions, Bioresour. Technol., 99 (1), 195–199.
[61] Leal, E., Teixeira, J.A., and Gudiña, E.J., 2024, Development of foam-free biosurfactant production processes using Bacillus licheniformis, Fermentation, 10 (7), 340.
[62] Fenibo, E.O., Douglas, S.I., and Stanley, H.O., 2019, A review on microbial surfactants: Production, classifications, properties and characterization, J. Adv. Microbiol., 18 (3), 1–22.
[63] Sass, G., Groleau, M.C., Déziel, E., and Stevens, D.A., 2023, Simple method for quantification of anionic biosurfactants in aqueous solutions, Front. Bioeng. Biotechnol., 11, 1253652.
[64] Bochynek, M., Lewińska, A., Witwicki, M., Dębczak, A., and Łukaszewicz, M., 2023, Formation and structural features of micelles formed by surfactin homologues, Front. Bioeng. Biotechnol., 11, 1211319.
[65] Duan, C., Wang, M., Ghobadi, A., Eike, D.M., and Wang, R., 2025, Quantifying the critical micelle concentration of nonionic and ionic surfactants by self-consistent field theory, J. Colloid Interface Sci., 700, 138592.
[66] Sultana, S., Sultana, R., Al-Mansur, M.A., Akbor, M.A., Bhuiyan, N.A., Ahmed, S., Yasmin, S., and Shofiul Islam Molla Jamal, A.H.M., 2024, An industrially potent rhamnolipid-like biosurfactant produced from a novel oil-degrading bacterium, Bacillus velezensis S2, RSC Adv., 14 (34), 24516–24533.
[67] Tian, Y., Zhou, J., He, C., He, L., Li, X., and Sui, H., 2022, The formation, Stabilization and separation of oil–water emulsions: A review, Processes, 10 (4), 738.
[68] Nayarisseri, A., Singh, P., and Singh, S.K., 2018, Screening, isolation and characterization of biosurfactant producing Bacillus subtilis strain ANSKLAB03, Bioinformation, 14 (6), 304–314.
[69] Meena, K.R., Dhiman, R., Singh, K., Kumar, S., Sharma, A., Kanwar, S.S., Mondal, R., Das, S., Franco, O.L., and Mandal, A.K., 2021, Purification and identification of a surfactin biosurfactant and engine oil degradation by Bacillus velezensis KLP2016, Microb. Cell Fact., 20 (1), 26.
[70] Wang, Q., Zhang, H., Han, Y., Cui, Y., and Han, X., 2023, Study on the relationships between the oil HLB value and emulsion stabilization, RSC Adv., 13 (35), 24692–24698.
[71] Qi, G.N., Qin, W.Q., Li, G.J., Ma, T.T., Liu, Y.F., Zhou, L., Liu, J.F., Gang, H.Z., Yang, S.Z., and Mu, B.Z., 2024, A new bacterial strain producing both of the surfactin and fengycin lipopeptide biosurfactant with strong emulsifications on crude oil, Appl. Biochem. Biotechnol., 197 (2), 1192–1208.
[72] Melikoglu, M., 2026, Biosurfactants for enhanced oil recovery and bioremediation in the modern petroleum industry: A global review, JCIS Open, 21, 100168.
[73] Pei, H., Shan, J., Zhang, G., Zheng, J., and Zhao, J., 2021, Selection of optimum surfactant formulations with ultralow interfacial tension for improving the oil washing efficiency, ACS Omega, 6 (37), 23952–23959.
[74] Ali, F., Das, S., Hossain, T.J., Chowdhury, S.I., Zedny, S.A., Das, T., Ahmed Chowdhury, M.N., Uddin, M.S., 2021, Production optimization, stability and oil emulsifying potential of biosurfactants from selected bacteria isolated from oil-contaminated sites, R. Soc. Open Sci., 8 (10), 211003.
[75] Amani, H., 2015, Study of enhanced oil recovery by rhamnolipids in a homogeneous 2D micromodel, J. Pet. Sci. Eng., 128, 212–219.
[76] Datta, P., Tiwari, P., and Pandey, L.M., 2020, Oil washing proficiency of biosurfactant produced by isolated Bacillus tequilensis MK 729017 from Assam reservoir soil, J. Pet. Sci. Eng., 195, 107612.
[77] Dong, H., Xia, W., Dong, H., She, Y., Zhu, P., Liang, K., Zhang, Z., Liang, C., Song, Z., Sun, S., Zhang, G., 2016, Rhamnolipids produced by indigenous Acinetobacter junii from petroleum reservoir and its potential in enhanced oil recovery, Front. Microbiol., 7, 1710.
[78] Dong, H., Zheng, A., He, Y., Wang, X., Li, Y., Yu, G., Gu, Y., Banat, I.M., Sun, S., She, Y., and Zhang, F., 2022, Optimization and characterization of biosurfactant produced by indigenous BreviBacillus borstelensis isolated from a low permeability reservoir for application in MEOR, RSC Adv., 12 (4), 2036–2047.
[79] Pacwa-Płociniczak, M., Płaza, G.A., Piotrowska-Seget, Z., and Cameotra, S.S., 2011, Environmental applications of biosurfactants: Recent advances, Int. J. Mol. Sci., 12 (1), 633–654.
[80] Liu, X., Kang, Y., Yan, L., Tian, J., Li, J., and You, L., 2022, Implication of interfacial tension reduction and wettability alteration by surfactant on enhanced oil recovery in tight oil reservoirs, Energy Rep., 8, 13672–13681.
Article Metrics
Copyright (c) 2026 Indonesian Journal of Chemistry

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Indonesian Journal of Chemistry (ISSN 1411-9420 /e-ISSN 2460-1578) - Chemistry Department, Universitas Gadjah Mada, Indonesia.












