Bidang Unggulan : Ketahanan Pangan
LAPORAN AKHIR HASIL PENELITIAN UNGGULAN PERGURUAN TINGGIHIBAH BERSAIING (TAHUN KE-1) TAHUN ANGGARAN 2012
Judul
:
Mobilisasi 3-Isolat Bakteri Untuk Memacu Proses Nitrifikasi Pada Budidaya Tambah Udang
Ketua
:
Dr. Ir. Anik. M. Ariati, MSc
Anggota
:
1. Ir. Dewa Gede Raka Wiadnya, M.Sc 2. Ating Yuniarti, S.Pi.m M.Aqua
Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Kementerian Pendidikan dan Kebudayaan, Melalui DIPA Universitas Brawijaya nomor : 0636/023-04.2.16/15/2012, tanggal 9 Desember 2011, dan berdasarkan SK Rektor Universitas Brawijaya Nomor : 058/SK/2012 tanggal 8 Pebruari 2012
LembagaPenelitiandanPengabdiankepadaMasyarakat UniversitasBrawijaya 2012
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ABSTRAK
Nitrifikasi telah dipelajari secara ekstensif untuk signifikan di dalam siklus nitrogen.Seringkali penurunan nitrogen dari air limbah yang bermasalah karena laju pertumbuhan yang rendah dari bakteri yang terlibat. Amobilisasi merupakan metode yang efisien untuk mempertahankan pertumbuhannya bakteri yang lambat pada reaktor. Sel-sel amobilreaktor terperangkap dalam gel manik-manik membentuk alternatif tersebut terpasang-sel sistem. Beberapa teknik untuk evaluasi diberikan, termasuk tingkat konsumsi substrat dan bahan pendukung (matriks). Bakter ini trifikasi diamobilisasi dengan alginat, karagenan, polivinil alkohol (PVA) dan serbuk gergaji yang digunakan sebagai matriks. Reaktor matriks dapat mengoksidasi TAN berkisar 63-78%. Matrik salginat memiliki efisiensi nitrifikasi sebesar 10-20 persen lebih tinggi dari matriks lainnya. Penggunaan matriks amobil secara efektif dapat mengoksidasi TAN sampai tiga.
ABSTRACT
Nitrification has been studied extensively for its significance in the nitrogen cycle. Often nitrogen removal from wastewater is problematic because of the low growth rate of thebacteria involved. Immobilization is an efficient method to retain slow-growing organisms in reactors.Immobilized-cell reactor cells entrapped in gel beadsform an alternative to these attached-cell systems.Several techniques for evaluation are given, includingsubstrate consumption rates and support material (matrix).Nitrifying microorganisms, immobilized by alginate, carrageenan, polyvinyl-alcohol (PVA) and sawdust were used as a matrix. The matrix reactor removed ranging from 63-78%of TAN.Alginate matrix have the nitrification efficiency of 10-20 percent higher than other matrix. The matrix can effectively remove of TAN until three times used.
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RINGKASAN Amobilisasi bakteri nitrifikasi dapat menjadi metode yang efektif untuk menurunkan total amonia-nitrogen (TAN) dari budidaya udang. Hal ini dapat membantu meningkatkan kualitas air untuk budidaya udang dan engurangi biaya modal dan operasional. Tujuan dari penelitian ini adalah untuk mendapatkan matrik penjerap bakteri nitrifikasi (AOB dan NOB) dalam proses amobilisasi yang mampu menurunkan TAN yang paling efektif. Bakteri yang digunakan dalam penelitian ini adalah Nitrosomonas, Nitrobacter dan Nitrospira. Bakteri ini hasil dari isolasi sedimen tambak udang. Untuk keperluan amobilisasi, masing-masing bakteri ditumbuhkan pada medianya. Bakteri yang digunakan untuk proses amobilisasi diambil pada fase ekspon ensial yaitu pada inkubasi jam ke 12 – 18. Pada penelitian ini menggunakan beberapa matrik seperti alginate, karagenan, polivnilalkohol (PVA) dan serbuk gergaji sebagai penjerap bakteri. Pengujian laju nitrifikasi dilakukan dengan sebuah bioreaktor nitrifikasi dengan media yang telah ditentukan konsentrasi TAN sebesar 1,6mM. Untuk mengetahui efisiensi penggunaan bakteria mobil, dilakukan pengujian berulang dari manik-manika mobil yang sama. Hasil penelitian menunjukkan bahwa kemampuan matrik untuk menjerap bakteri berkisar antara 70-80%. Sedangkan kekuatan gel yang terbentuk paling tinggi pada matrik alginate dan PVA. Laju penurunan TAN berkisar antara 0,032 sampai 0,042 mM/menit. Laju penurunan TAN sangat dipengaruhi oleh matrik penjerap. Secara ratarata penggunaan penjerap alginate, karagenan, PVA dan serbuk gergaji dapat menurunkan TAN secara berturut sebesar 0,042; 0,036; 0,033 dan 0,032 mM/menit. Sedangkan untuk periode penggunaan manik-manika mobil, kemampuan laju nitrifikasinya menurun secara drastis setelah penggunaan lebih dari tiga kali. Efisiensi penurunan TAN juga dipengaruhi oleh matrik penjerapnya. Nilai efisiensi penurunan TAN berkisar antara 62 sampai 78%, dengan efisiensi paling tinggi pada matrik alginat. Sedangkan efisiensinya menurun sampai 50% setelah penggunaan lebih dari tiga kali. Dari hasil ini dapat disimpulkan bahwa: Alginat, karagenan, PVA dan serbuk gergaji merupakan bahan yang dapat digunakan sebagai matrik penjerap untuk bakter initrifikasi. Kemampuan nitrifikasi yang tertinggi dicapai pada penggunaan matrik penjerap alginate. Matrik penjerap alginate mempunyai efisiensi nitrifikasi 10-20 persen lebih tinggi dibanding dengan penjerap karagenan, PVA dan serbuk gergaji. Sehingga dalam penelitian lanjutan dapat disarankan bahwa Alginat relative efektif dalam nitrifikasi, tetapi harganya cukup mahal. Sebaliknya serbuk gergaji relative murah dengan kemampuan 20% lebih rendah. Kedua jenis penjerap ini perlu diuji kemampuan nitrifikasinya pada budidaya udang.
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SUMMARY Immobilization of nitrifying bacteria can be an effective method to reduce the total ammonia-nitrogen (TAN) from shrimp farming. This can help improve the quality of water for shrimp farming and reduce capital and operational costs. The purpose of this study was to obtain a matrix entrapment nitrifying bacteria (AOB and NOB) in animmobilization process that can effectively reduce of the TAN concentration. The bacteria used in this study wereNitrosomonas, Nitrobacter and Nitrospira. This bacteriaare the result of the isolation from the shrimp pond sediment. For the purposes of immobilization, each bacterium was grown in the specific medium. The bacteria that are used toimmobile processtaken from the exponential growth phase at 12 to 18 hours incubation time.Several metrics such as alginate, carrageenan, polyvinyl alcohol (PVA) and sawdust were used as bacteria entrapped. Nitrification rate was conducted by using of nitrification bioreactor with media specified TAN concentration of 1.6 mM. To determine the efficiency of the use of immobilized bacteria, repeated testing of same immobilized beads was applied. The results showed that the ability of the matrix to adsorb bacteria ranged from 70-80%. While the strength of the gel formed highest in alginate and PVA matrix. The removal rate of TAN ranged from 0.032 to 0.042 mM/min. The TAN removal rate is influenced by the matrix entrapped. The matrix of alginate, carrageenan, PVA and sawdust can removed TAN at 0.042; 0.036; 0.033 and 0.032 mM/min, respectively. While for the use of immobilized beads, the ability of nitrification rate decreases dramatically after the use of more than three times. TAN removal efficiency is also affected by the type of matrix. TAN removal efficiency values ranged from 62 to 78%, with the highest efficiency in the alginate matrix. While the removal efficiency decreased to 50% after the use of more than three times. From these results it can be concluded that: Alginate, carrageenan, PVA and sawdust are a material that can be used as a matrix for bacterial immobilization. The ability of nitrification of the highest achieved in the use of alginate matrix. Alginate matrix have the nitrification efficiency of 10-20 percent higher than matrix of carrageenan, PVA and sawdust. Thus, in further research can be suggested that Alginate relatively effective in nitrification, but the price is quite expensive. In contrast with the relatively low cost sawdust 20% lower capacity. Both types of matrix need to be tested for their nitrification on shrimp culture
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DAFTAR PUSTAKA
Avnimelech, Y. and G. Ritvo. 2001. Aeration, mixing and sludge control in shrimp ponds. Glob. Aquac. Alliance Advocate 4: 51– 53. Avnimelech, Y. and G. Ritvo. 2003. Shrimp and fish pond soils: processes and management. Aquaculture 220: 549–567 Bartosch, S., I. Wolgast, E. Spieck & E. Bock, (1999) Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase. Appl Environ Microbiol 65: 4126-4133 Bartosch, S., C. Harttwig, E. Spieck and E. Bock. 2002. Immunological detection of Nitrospira-like bacteria in various soils. Microbiol. Ecol. 43: 26-33. Baticados, M. C. L., Curz-Lacierda, E. R., de la Cruz, M. C., Duremdez-Fernandez, R. C., Gacutan, R. Q., Lavilla-Pitogo, C. R. and Lio-Po, G. D., Diseases of Penaeid Shrimp in the Philippines, Aquaculture Extension Manual No. 16. Aquaculture Department, Southeast Asian Fisheries Development Centre, Philippines, 1990, p. 46. Bédard, C., and R. Knowles. 1989. Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers. Microbiol. Rev. 53:68-84 Bendtsen, J. D., H. Nielsen, D. Widdick, T. Palmer & S. Brunak, (2005) Prediction of twin-arginine signal peptides. BMC Bioinformatics 6: 167. Berks, B. C., M. D. Page, D. J. Richardson, A. Reilly, A. Cavill, F. Outen & S. J. Ferguson, (1995) Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop. Mol Microbiol 15:319-331 Blackburn, T.H. and N.D. Blackburn, 1992. Model of nitrification and denitrification in marine sediments. FEMS Microbiology Letters, 100:517-522. Bollmann, A., I. Schmidt, A. M. Saunders & M. H. Nicolaisen, (2005) Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis. Appl Environ Microbiol 71: 1276-1282. Boyd, C.E., M.E. Tanner, M. Madkour and K. Masuda. 1994. Chemical characteristics of bottom soils from freshwater and brackishwater aquaculture ponds. J. World Aquac. Soc. 25: 517–534. Boyd, C. 1995. Shrimp pond bottom soil and sediment management. Soil and Sediment Mangt. Reviews: 166-181 Boyd, C.E., C.W. Wood, and T. Thunjai, 2002. Pond soil characteristics and dynamics of soil organic matter and nutrients. In: K. McElwee, K. Lewis, M. Nidiffer, and P. Buitrago (Editors), Nineteenth Annual Technical Report. Pond dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, pp. 1–10. Briggs, M.R.P., and Funge-Smith, S.J., 1994. A nutrient budget of some intensive marine shrimp ponds in Thailand. Aquaculture and Fisheries Management 25, 789– 6
811. Burford, M.A., Longmore, A.R., 2001. High ammonium production from sediments in hypereutrophic shrimp ponds. Marine Ecology Progress Series 224, 187–195. Burford M.A. and, K. Lorenzen. 2004. Modeling nitrogen dynamics in intensive shrimp ponds: the role of sediment remineralization. Aquaculture 229 (2004) 129–145 Burford, M.A., and Williams, K.C., 2001. The fate of nitrogenous waste from shrimp feeding. Aquaculture 198, 79– 93. Burrell, P.C., J. Keller and L.L. Blackall. 1998. Microbiology of a nitrite-oxidizing bioreactor. Appl. Environ. Microbiol. 64: 1878-1883. Chen J.C. & Chen S.F. (1992) Effects of nitrite on growth and molting of Penaeus monodon juveniles. Comparative Biochemistry and Piujsiology C— Comparative Pharmacology andToxicoIogy 101.453-458. Daims, H., S. Lucker & M. Wagner, (2006) daime, a novel image analysis program for microbial ecology and biofilm research. Environ Microbiol 8: 200-213. Daims, H., J. Nielsen, P.H. Nielsen, K.H. Schleifer and M. Wagner. 2001. In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67: 5273-5284. Deizman, M.M. and S. Mostaghimi, 1991. A model for evaluating the impacts of land application of organic waste on runoff water quality. Research Journal WPFC, 63:17-27. Denac, M., Uzman, S., Tanaka, H. and Dunn, I. J. Modeling of experiments on biofilm penetration effects in a fluidized bed nitrification reactor. Biofechnol. Bioeng. 1983, 25, 1841-1861 Ehrich, S., D. Behrens, E. Lebedeva, W. Ludwig and E. Bock. 1995. A new obligately chemolithoautotrophic, nitrite-oxidizing bacterium, Nitrospira moscoviensis sp. nov. and its phylogenetic relationship. Arch. Microbiol. 164: 16-23. El Sheikh, A. F. & M. G. Klotz, (2008) Ammonia-dependent differential regulation of the gene cluster that encodes ammonia monooxygenase in Nitrosococcus oceani ATCC 19707. Environ Microbiol 10: 3026-3035. Foesel, B. U., A. Gieseke, C. Schwermer, P. Stief, L. Koch, E. Cytryn, J. R. de la Torre, J. van Rijn, D. Minz, H. L. Drake & A. Schramm, (2008) Nitrosomonas Nm143like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm. FEMS Microbiol Ecol 63: 192-204 Friedrich, T., (1998) The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. Biochim Biophys Acta 1364: 134-146. Hariati, A.M., D.G.R. Wiadnya, A. Prajitno, M. Sukkel, J.H. Boon, and M.C.J. Verdegem 1995. Recent development of shrimp, Penaeus monodon (Fabricus) and Penaeus merguensis ( de Man ), culture in East Java. Aquaculture research 26; 819-829 pp., Elsevier Scien. B.V . Amsterdam Hariati, A.M., D.G.R. Wiadnya, M.W.Tanck, J.H. Boon and M.C.J. Verdegem. 1996a. Penaeus monodon ( Fabricus) production related to water quality in East Java , Indonesia. Aquaculture Res. 27;255-260
7
Hariati, A.M., D.G.R. Wiadnya, M.W. Tanck, J.H. Boon and M.C.J. Verdegem. 1996b. Pond production of Penaeus monodon (Fabricus) in relation stocking density, survival rate and mean weight at haverst in East Java. Aquaculture 27:277-282. Hariati, A.M., D.G.R. Wiadnya, R.K. Rini, J.H. Boon and M.C.J. Verdegem. 1998. Penaeus monodon (Fabricus) and Penaeus merguensis (de Man) biculture in East Java, Indonesia. Aquaculture Research 29: 1-8 Hariati, A.M. 2008. Aplikasi probiotik terhadap karakteristik sedimen dan produksi tambak udang intensif. Seminar Nasional Perikanan dan Kelautan UB. Hariati, A.M. dan A. Yuniarti. 2008. Isolasi dan identifikasi bakteri indigeneus dari udang Vanname, Litopenaeus Vannamei sebagai kandidat probiotik. Seminar Nasional Hasil Penelitian Perikanan dan Kelautan UGM. Heylen, K., B. Vanparys, D. Gevers, L. Wittebolle, N. Boon & P. De Vos, (2007) Nitric oxide reductase (norB) gene sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated denitrifiers. Environ Microbiol 9: 1072-1077. Hynes, R. K., and R. Knowles. 1982. Effect of acetylene on autotrophic and heterotrophic nitrification. Can. J. Microbiol. 28:334-340 Jackson, C., Preston, N., Thompson, P., Burford, M., 2003. Nitrogen budget and effluent nitrogen components at an intensive shrimp farm. Aquaculture 218, 397– 411 Jamu, D.M. and R.H. Piedrahita, 1995. Aquaculture pond modeling for the analysis of integrated Aquaculture/agriculture systems. In: H. Egna, B. Goetze, D. Burke, M. McNamara, and D. Clair (Editors), Thirteenth Annual Technical Report, Pond Dynamics/Aquaculture CRSP, Office of International Research and Development, Oregon State University, Corvallis, Oregon, USA, pp. 142-147 Jaya, I.B.M.S. 1994a. Organic matter and aquaculture: a review. ASEAN-EEC Aquaculture development and coordination programme (AADCP), component 1: metdodology of coastal zone assessment for brackishwater aquaculture. Pp 66. Jaya, I.B.M.S., C.K. Sutrisno dan N. Hamid. 1994. Analisis mutu sediment di kawasan tambak desa Turunrejo, Kendal, Jawa Tengah. Majalah Ilmiah Perikanan, BBAP, Jepara. 1: 1-9 Jime´nez-Montealegre, R., Verdegem, M.C.J., van Dam, A., and Verreth, J.A.J., 2002. Conceptualization and validation of a dynamic model for the simulation of nitrogen transformations and fluxes in fish ponds. Ecological Modelling 147, 123– 152. Juretschko, S., G. Timmermann, M. Schmid, K-H. Schleifer, A. Pommering-Röser, H-P. Koops and M. Wagner. 1998. Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 64: 3042-3051 Kleiner, D. (1981). The transport of NH3 and NHz 4 across biological membranes. Biochim Biophys Acta 639, 41–52. Kochba, M., S. Diab, and Y. Avnimelech, 1994. Modeling nitrogen transformations in intensively aerated fish ponds. Aquaculture, 120:95-104 Lebedeva, E. V., M. Alawi, F. Maixner, P. G. Jozsa, H. Daims & E. Spieck, (2008) 8
Physiological and phylogenetic characterization of a novel lithoautotrophic nitrite oxidizing bacterium, ‘Candidatus Nitrospira bockiana’. Int J Syst Evol Microbiol 58:242-250. Lefebvre, S., Bacher, C., Meuret, A., and Hussenot, J., 2001. Modeling approach of nitrogen and phosphorus exchanges at the sediment– water interface of an intensive fishpond system. Aquaculture 195, 279– 297. Lorenzen, K., 1999. Nitrogen recovery from shrimp pond effluent: dissolved N removal has greater overall potential than particulate nitrogen removal, but requires higher rates of water exchange than presently used. Aquaculture Research 30, 923– 927. Manju, N.J. , V. Deepest, Cini Achuthan, P. Rosamma and I.S. Bright Singh. 2009. Immobilization of nitrifying bacterial consortia on wood particle for bioaugmentating nitrification in shrimp culture systems. Aquaculture 294 (1009) 65-75 Maixner, F., D. R. Noguera, B. Anneser, K. Stoecker, G. Wegl, M. Wagner & H. Daims, (2006) Nitrite concentration influences the population structure of Nitrospira-like bacteria. Environ Microbiol 8: 1487-1495 Meincke, M., E. Bock, D. Kastrau & P. M. H. Kroneck, (1992) Nitrite oxidoreductase from Nitrobacter hamburgensis: redox centers and their catalytic role. Arch Microbiol 158: 127-131 Meincke1 M, Bock E, Kastrau D, Kroneck PMH (2004). “Nitrite oxidoreductase from Nitrobacter hamburgensis: redox centers and their catalytic role”. Arch Microbiol. 158 (2): 127–31. Milde K. and E. Bock. 1984. Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis.FEMS Microbiol Lett 21 : 137141 Millamena CM. (1990) Organic pollution resulting from excess feed and metabolite buildup: effect on P. monodon postlarvae. Aquacultural Engineering 9, 143-150. Munsiri, P., C.E. Boyd, and B.J. Hajek, 1995. Physical and chemical characteristics of bottom soil profiles in ponds at Auburn, Alabama, USA, and a proposed method for describing pond soil horizons. J. World Aquacult. Soc., 26:346–377. Nakamura, Y., H. Satoh, T. Kindaichi & S. Okabe, (2006) Community structure, abundance, and in situ activity of nitrifying bacteria in river sediments as determined by the combined use of molecular techniques and microelectrodes. Environ Sci Technol 40: 1532-1539 Seo, J.-K., Jung, I.-H., Kim, M.-R., Kim, B.J., Nam, S.-W., Kim, S.-K., 2001. Nitrification performance of nitrifiers immobilized in PVA(polyvinyl alcohol) for a marine recirculating aquarium system. Aquacultural Engineering 24, 181–194. Schramm, A., D. De Beer, M. Wagner and R. Amann. 1998. Identification and activities in situ of Nitrosospira and Nitrospira spp. As dominant populations in a nitrifying fluidized bed reactor. Appl. Environ. Microbiol. 64: 3480-3485. Schmidt, I., O. Sliekers, M. Schmid, E. Bock, J. Fuerst, J.G. Kuenen, M.S.M. Jetten, and M. Strous. 2003. New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS Microbiol. Rev. 27: 481-492
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Shin, M., Nguyen, T., Ramsay, J., 2002. Evaluation of support materials for the surface immobilization and decoloration of amaranth by Trametes versicolor. Applied Microbiology Biotechnology 60, 218–223 Sonnenholzner, S. and C.E. Boyd, 2000. Vertical gradients of organic matter concentration and respiration rate in pond bottom soils. J. World Aquacult. Soc., 31:376–380 Spector, T. 1978 Refinement of the coomassie blue method of protein quantification. Anal Biochem 86:142–146 Spieck E., J. Aamand, S. Bartosch and E. Bock. 1996a. Immunocytochemical detection and location of the membrane-bound nitrite oxidoreductase in cells of Nitrobacter and Nitrospira. FEMS Microbiol Lett 139:71–76 Spieck, E., C. Hartwig, I. McCormack, F. Maixner, M. Wagner, A. Lipski & H. Daims, (2006) Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge. Environ Microbiol 8: 405-415. Spieck E., S. Müller, A. Engel, E. Mandelkow, H. Patel. And E. Bock. 1996b. Twodimensional structure of membrane-bound nitrite oxidoreductase from Nitrobacter hamburgensis. J Struct Biol. 117:117–123 Spieck E, Ehrich S, Aamand J, Bock E (1998). “Isolation and immunocytochemical location of the nitrite-oxidizing system in nitrospira moscoviensis”. Arch Microbiol. 169 (3): 225–30 Sudirdjo, Marsoedi dan A.M. Hariati. 2001. Efektifitas bakteri super-Nb dalam mengendalikan laju akumulasi bahan 10lastic dan kualitas air media budidaya udang windu (Penaeus Monodon Fab.) BIOSAIN 1 (3): Sundermeyer-Klinger H., W. Meyer, B. Warninghoff and E. Bock. 1984. Membranebound nitrite oxidoreductase of Nitrobacter : evidence for a nitrate reductase system. Arch Microbiol. 140:153–158 Tanaka Y., Y. Fukumori and T. Yamanaka. 1983. Purification of cytochrome a1c1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium. Arch Microbiol. 135:265–271 Vanparys, B., E. Spieck, K. Heylen, L. Wittebolle, J. Geets, N. Boon & P. De Vos, (2007) The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis. Syst Appl Microbiol 30: 297-308. Watson, S.W., E. Bock, F.W. Valois, J.B. Waterbury and U. Schlosser. 1986. Nitrospira marina gen. nov. sp. nov.: a hemolithotrophic nitrite-oxidizing bacterium. Arch Microbiol. 144:1–7 Watson, S.W., E. Bock, H. Harms, H-P. Koops and A.B. Hooper. 1989. Nitrifying bacteria. Staley JT, Bryant MP, Pfennig N, Holt JG [Eds] Bergey’s Manual of Systematic Bacteriology. Wiadnya, D.G.R., P.J. Mous, R. Djohani, M.V. Erdmann, A. Halim, M. Knight, L. PetSoede, & J.S. Pet, 2005a. Marine Capture Fisheries Policy Formulation and the Role of Marine Protected Areas as Tool for Fisheries Management in Indonesia. Mar. Res. Indonesia (2005) 30:33-45
10
Wiadnya, D.G.R., R. Djohani, M.V. Erdmann, A. Halim, M. Knight, P.J. Mous, J.S. Pet, & L. Pet-Soede, 2005b. Kajian kebijakan pengelolaan perikanan tangkap di Indonesia: menuju pembentukan kawasan perlindungan laut. Jurnal Penelitian Perikanan Indonesia 11(3):65-77
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