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A Study on the Phylogeny and the Ecology of Ammonia Oxidizing Bacteria Using a New Molecular Marker Based on the Gene Amob

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A Study on the Phylogeny and the Ecology of Ammonia Oxidizing Bacteria Using a New Molecular Marker Based on the Gene Amob A STUDY ON THE PHYLOGENY AND THE ECOLOGY OF AMMONIA-OXIDIZING BACTERIA USING A NEW MOLECULAR MARKER BASED ON THE GENE AMOB Laia CALVÓ PERXAS ISBN: 84-689-3087-3 Dipòsit legal: GI-872-2005 A study on the phylogeny and the ecology of ammonia oxidizing bacteria using a new molecular marker based on the gene amoB Laia Calvó Perxas 2005 A study on the phylogeny and the ecology of ammonia oxidizing bacteria using a new molecular marker based on the gene amoB Memòria redactada per Laia Calvó Perxas, inscrita al programa de doctorat de Biotecnologia de l’Institut de Tecnologia Agroalimentària, Campus Agroalimentari de Girona, Institut d’Ecologia Aquàtica, Departament de Biologia i Departament EQATA, per optar al grau de Doctor en Biologia per la Universitat de Girona. El present treball s’ha realitzat al laboratori de Microbiologia Molecular de l’Institut d’Ecologia Aquàtica de la Universitat de Girona sota la direcció del Dr. L. Jesús Garcia-Gil. Vist-i-plau El Director de la Tesi Autora Dr. L. J. Garcia-Gil Laia Calvó Perxas Catedràtic de Microbiologia Girona, 2005 Aquesta tesi ha estat realitzada gràcies a una beca de Formació de Personal Investigador, i finançada pel projecte CICYT REN2000 del Ministerio de Ciencia y Tecnología. Primera edició: abril 2005 Edita: Publicacions de la UdG © Laia Calvó Perxas Dipòsit legal: B-17.482-2005 Universitat de Girona. Publicacions Ed. Les Àligues - Pl. Sant Domènec, 3. 17071 Girona Tel. 972 41 80 99 - Fax. 972 41 80 31 http://www.udg.edu/publicacions [email protected] Els antics costums i les faules de les velles són coses vulgars, però no s’han de sepultar en l’oblit. Encara es pot trobar en elles certa veritat i alguna cosa útil. A més, és un autèntic plaer comprovar que les generacions anteriors cometien tants errors…com la d’ara. John Aubrey Para llegar a ser sabio, es preciso querer experimentar ciertas vivencias, es decir, meterse en sus fauces. Eso es, ciertamente, muy peligroso; más de un sabio ha sido devorado al hacerlo. Friedrich Nietzsche Els problemes són una guia per aconseguir objectius Anònim AGRAÏMENTS Voldria agrair especialment a en Jesús Garcia-Gil el donar-me l’oportunitat de conèixer el món de la microbiologia i de la recerca. Agrair-li la confiança que ha dipositat en mi i en tots els projectes en què m’ha ofert de participar. Per les idees, les discussions i per deixar-me portar la contrària en més d’una ocasió. A en Carles Abellà gràcies per dirigir-me la tesina, amb la que em vaig posar de ple en el món dels nitrificants quasi sense ni adonar-me’n. A en Bo, per les xerrades de llibres i música i per ensenyar-me a ensenyar durant les pràctiques, però sobretot per l’ajuda que mai m’ha negat i pels bons consells. A en Catxo, per les bones estones al laboratori i per ser el millor company de despatx durant l’època de fer gràfiques per la tesina (encara no entenc com saps tan bé què ha d’anar a les files i què ha d’anar a les columnes!). A en Xavi Vila, per haver estat al meu costat en començar amb els nitris, i per les converses que ens van portar a tenir els primers resultats. A tots els “micros de dalt” amb qui porto ja uns anys compartint despatx. La Marga, per fer de McGiver amb mi a Tarragona i per suportar amb bona cara el dia a dia en un despatx ple d’històries. La Tiky, compi de l’època d’escriptura de tesi amb qui he compartit preocupacions i alegries i molts moments de “no acabem!”. A en Tri, que sempre té a punt bones paraules, per l’empenta i per 7 A study on the phylogeny and the ecology of AOB seguir-se emocionant amb tot. A la Noemí, per ser un sol i el somriure amb potes del despatx, pel suport moral i els detalls que només ella sap tenir. A la Geno i en Xavi Fuentes, per les xerrades constructives sobre docència i per intentar arreglar el món. A la Susi, que tot i anar sempre atabalada va ser una bona companya de despatx i sempre tenia un moment per somriure. A la Gemma Deulofeu, no només per congelar-se amb mi mostrejant, sinó per ser una bona amiga. A tots els micros que ja no hi són, especialment als “cabezones” Xevi Cristina i Javi Rodríguez, no només per haver estat els meus mestres a peu de poiata, sinó per tants mostratges, tantes “batallites”, tantes acollonades, tants cafès i tants farts de riure... que bé m’ho havia passat amb vosaltres! També vull recordar a en Jordi Figueras i en Frederic Gich que sempre tenien paraules d’ànim en els moments difícils. Als micros d’espais comuns, l’Olaya, en Gela, en Marc, l’Àlex, en Joaquín, la Nuri i l’Anna, per aportar noves il·lusions al grup i per haver-me acompanyat en aquesta darrera etapa del trajecte. A la Laia Mauricio, per l’eficiència amb les comandes i per tenir-ho tot sempre a punt. A en Vicenç Oliveras per la seqüenciació i la real-time a última hora. A la Teresa Roura, gràcies per inscriure’ns als congressos i saber moure el papeleo que et passem sempre massa tard. Als LEQUIA, per les mostres i les mil reunions sobre els biodiscs. A tota la gent del passadís de baix i de dalt, especialment en José-Luís García pel seu coneixement i ajuda amb l’anàlisi poligènica. A la Noe per ser de bona pasta i aguantar calors i freds cada matí. En un altre ordre també vull donar les gràcies a en Martí Cortey, no només per haver estat allà des que acabàvem la carrera, sinó per l’inestimable ajuda amb l’estadística de les anàlisis de seqüències i per les filosofades. Sembla que al final ens n’hem sortit tots dos, eh? 8 Agraïments A tota la Komuna Kemal pels sopars i les festes, especialment a l’Steffi per viure i veure l’estiu kaòtic, a la Sònix per les nits de Mariscal i la contundència, i a l’Alba per compartir preocupacions i per l’idealisme. No voldria deixar-me els “locus” de Begur. A l’Àlex per totes les grans converses de ciència i per preocupar-se de totes les epidèmies que ens han de matar a tots un dia d’aquests. A en Soro per ser com és i saber treure ferro als assumptes. A la gent del Rostei amb qui he compartit cansament durant tres anys: hem currat, hem xuscat, però ho hem passat bé! A la Natàlia per ser la roca amiga que tot ho aguanta, i a en JM per les trobades glamouroses i les que no ho han estat tant. Em resta donar les gràcies a la família Alsina-Bardagí, especialment a en Carlos i la Magda per acollir-me els estius com una més i per preocupar-se de què tingués un espai i un temps per escriure la tesi. Finalment, amb tot el cor, gràcies a la meva família, especialment als meus pares i germà, que m’han encoratjat en tot moment i sense els qui no m’hauria atrevit mai a embrancar-me ni en la tesi ni en molts altres projectes. Només en Lluís ha sabut recolzar-me com ells i ha confiat sempre en mi més que jo mateixa. GRÀCIES A TOTS. 9 ABBREVIATIONS AMO Ammonia monooxygenase amoA Gene encoding the subunit A of AMO amoB Gene encoding the subunit B of AMO amoC Gene encoding the subunit C of AMO AmoA Subunit A of AMO AmoB Subunit B of AMO AmoC Subunit C of AMO AOB Ammonia Oxidizing Bacteria CI Chlorophorm Isoamyl alcohol CY3 Fluorescent Indocarbocyanine DAPI 4’,6-diamiine-2-phenylindole DGGE Denaturing Gradient Gel Electrophoresis EDTA Etilenediamine-sodium tetraacetate FISH Fluorescent in situ hybridization MOB Methane Oxidizing Bacteria Nc Nitrosococcus Nm Nitrosomonas NOB Nitrite Oxidizing Bacteria Ns Nitrosospira 11 A study on the phylogeny and the ecology of AOB pb base pair PBS Posphate buffered saline PCR Polymerase Chain Reaction pmoA Gene encoding the subunit A of AMO pmoB Gene encoding the subunit B of AMO pmoC Gene encoding the subunit C of AMO r.p.m. revolutions per minute RDP Ribosomal Database Project SBR Sequencing Batch Reactor TBE Tris-Borate-EDTA buffer TEMED N,N,N’,N’-tetramethylendiamide Tris Tris-hydroxymethyl-aminomethane WWTP Wastewater Treatment Plant 12 SUMMARY Human activities such as farming and industrialization have produced a significant increase in the number of ammonium-rich environments. The presence of nitrogenated compounds reduces water quality causing toxicity problems, deteriorating the environment and even affecting human health. Consequently, nitrification has recently become a widespread process involving the cycling of nitrogen in the biosphere, which is mainly due to microbial activities. Ammonia oxidizing bacteria (AOB) are an essential component of the global cycling of nitrogen, being responsible for the aerobic oxidation of ammonium to nitrite. Although the first ammonia oxidizing bacteria were isolated by the end of the XIX century, the slowness of their growth and the difficulties in culturing impeded achieving a full knowledge of this bacterial group until the 80s, when the first studies based on the gene 16S rDNA where performed. Nowadays, the databases contain huge numbers of entries of 16S rDNA sequences belonging to ammonia oxidizing bacteria. The aim of this work was to find, develop, and evaluate useful and reliable tools for the study of ammonia oxidizers in environmental samples. In this work we describe the use of Fluorescence In Situ Hybridization (FISH), based on the use of DNA probes specifically targeting the ammonia- oxidizers 16S rRNA molecule.
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