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Tesis por compendio / Los microorganismos son esenciales para poder llevar a cabo la producción intensiva de langostinos marinos, ya que son los encargados de mantener el agua con una calidad óptima para los animales. Además, los microorganismos permiten minimizar o eliminar las necesidades de realizar cambios de agua. Entre todos los microorganismos presentes en los sistemas intensivos, sin renovación de agua, las bacterias y las microalgas son los más abundantes y los que tienen un papel más importante en el sistema de producción. En esta tesis se profundiza en el conocimiento de la dinámica de las microalgas presentes en un sistema de preengorde y en un sistema de bioflóculos, para el cultivo intensivo de langostinos marinos, utilizando la metodología HPLC/CHEMTAX. Además, se testa si la bacteria Bacillus amyloliquefaciens puede tener un papel probiótico en los sistemas de bioflóculos y si es capaz de mejorar el proceso de maduración del sistema. Los resultados obtenidos han determinado que la metodología HPLC/CHEMTAX es una forma rápida y eficiente para analizar el fitoplancton y el perifiton en los sistemas intensivos de langostinos, siempre que predominen los procesos autotróficos. Esta técnica, ha permitido detectar por primera vez prasinofíceas y primnesiofíceas en el perifiton que crece sobre los tanques de policloruro de vinilo (PVC). También se ha observado la influencia del estado trófico del sistema y la exposición a la luz sobre la dinámica de las microalgas en los sistemas de bioflóculos. Por lo que respecta a la aplicación de Bacillus amyloliquefacien a los sistemas de bioflóculos, esta implica un refuerzo del sistema inmunológico de los langostinos, sumando sus efectos a los producidos por los bioflóculos. Estos beneficios, ocurren incluso al aplicar dosis del orden de 103 ufc/mL, siendo esta menor a la recomendada para otras bacterias probióticas. A pesar de que los resultados de otras investigaciones apuntaban que Bacillus amyloliquefaciens podría participar en la formación de bioflóculos, la mejora de la calidad del agua y el crecimiento de los langostinos, la aplicación de esporas sobre el agua no mostró efectos estadísticamente significativos sobre la dinámica del sistema, la calidad del agua o los parámetros zootécnicos. / Microorganisms are essential for the intensive production of marine shrimps, since they are responsible for maintaining the water with an optimum quality for them. Also, these microorganisms minimize or eliminate water renewal. Among all the microorganisms present in intensive culture systems, without water renewal, microalgae and bacteria play the most important role and are the most abundant ones. In this thesis we analyze the microalgae dynamics using HPLC/CHEMTAX methodology in two intensive culture systems of marine shrimp, a shrimp nursery and a biofloc system. We also test the role of the bacteria Bacillus amyloliquefaciens as a probiotic and its capacity to improve the maturation process of the biofloc system. The results showed that the HPLC/CHEMTAX methodology is a fast and efficient way to analyze phytoplankton and periphyton in intensive culture systems of shrimp, provided that autotrophic processes are predominant. This technique allowed to detect prasinophytes and prymnesiophytes for the first time in the periphyton that grows on the polyvinyl chloride (PVC) tanks. Also, it has allowed to study the influence of trophic state and light exposition on microalgae dynamic in the biofloc systems. The application of Bacillus amyloliquefaciens in biofloc systems strengthens the immune system of shrimp and adds its probiotic effects to that produced by bioflocs. These benefits happen even with doses of 103 cfu/mL, lower than recommended doses for other probiotic bacteria. Other researches indicated that Bacillus amyloliquefaciens might have other effects such as participating in the biofloc system formation, improving water quality and shrimp growth; however, the application of spores in the culture water did not show any statistically significant effect on the system dynamic, water quality or zootechnic parameters. / Els microorganismes són essencials per a poder dur a terme la producció intensiva de llagostins marins, ja que són els encarregats de mantenir l'aigua amb una qualitat òptima per als animals. A més a més, els microorganismes permeten minimitzar o eliminar les necessitats de realitzar canvis d'aigua. Entre tots els microorganismes presents en els sistemes intensius, sense renovació d'aigua, les bactèries i les microalgues són les més abundants i les que tenen un paper mes important en el sistema de producció. En aquesta tesis es profunditza en el coneixement de la dinàmica de les microalgues presents als sistemes de preengreixament i en un sistema de bioflòculs, per al cultiu intensiu de llagostins marins, utilitzant la metodologia HPLC/CHEMTAX. A més a més, es prova si la bactèria Bacillus amyloliquefaciens pot tenir un paper probiòtic en els sistemes de bioflòculs i si es capaç de millorar el procés de maduració del sistema. Els resultats obtinguts han determinat que la metodologia HPLC/CHEMTAX és una forma ràpida i eficient per a analitzar el fitoplàncton i el perifíton als sistemes intensius de llagostins, sempre que predominen els processos autotròfics. Aquesta tècnica, ha permès detectar per primera vegada prasinofícies i primnesiofícies al perifíton que creix sobre els tancs de policlorur de vinil (PVC). També s'ha observat l'influencia del estat tròfic del sistema i l'exposició a la llum sobre la dinàmica de les microalgues als sistemes de bioflòculs. Pel que respecta a l'aplicació de Bacillus amyloliquefaciens en els sistemes de bioflòculs, aquesta implica un reforç del sistema immunològic dels llagostins, sumant els seus efectes als produïts pels bioflòculs. Aquestos beneficis, permaneixer al aplicar dosis de l'ordre de 103 ufc/mL, sent aquesta menor a la recomanada per a altres bactèries probiòtiques. Encara que els resultats d'altres investigacions apunten que Bacillus amyloliquefaciens podria participar en la formació de bioflòculs, la millora de la qualitat de l'aigua i el creixement dels llagostins, l'aplicació d'espores a la columna d'aigua no va mostrar ningún efecte estadísticament significant sobre la dinàmica del sistema, la qualitat de l'aigua o els paràmetres zootècnics. / Llario Sempere, F. (2018). IMPORTANCIA DE LOS MICROORGANISMOS EN LA PRODUCCIÓN DE LANGOSTINOS MARINOS EN SISTEMAS DE BIOFLÓCULOS Y SIN RENOVACIÓN DE AGUA [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/111825 / Compendio

Viele Bacillus Stämme sekretieren Phytasen, diese katalysieren die Dephosphorylierung von myo-Inositolhexakisphosphat (phytate). Das monocystronische phyC Gen aus dem im Boden lebenden Bacillus amyloliquefaciens FZB45 konnte als Teil des, durch Phosphatmangel induzierbaren, PhoPR-Regulons identifiziert werden. Der Transkriptionsstart des SigmaA-abhängigen Promotors wurde 27 bp upstream von Translationsstart bestimmt. Der Promotor weist jedoch eine ungewöhnliche Struktur auf, da die –35 und die –10 Region durch ein 21 bp Fenster voneinander getrennt sind. Die in vitro Transkriptionsanalyse zeigte, dass PhoP-P für die Initiation der phyC-Transkription notwendig ist. Die PhoP-Bindungsstellen der meisten B. subtilis Promotoren, die durch PhoP aktiviert werden, besitzen mindestens vier TTAACA-ähnliche Sequenzmotive, welche durch 5-6 bp- Intervalle getrennt sind. Die upstream Region von phyC aus B. amyloliquefaciens FZB45 weicht von dieser Struktur ab. Hier konnten nur zwei solcher Motive, die für die Bindung eines PhoP-Dimeres zuständig sind und die Positionen -47 und -35 überlagern, identifiziert werden. Ein weiteres Motiv befindet sich zwischen -13 und –8 und überlappt um eine Base mit der –10 Region. Die Funktionen der drei Bindungsstellen konnten durch DNaseI-Footprinting ermittelt werden. Ein PhoP-Dimer besetzt die –35 Region und dirigiert dabei die RNA-Polymerase wahrscheinlich in Richtung –10 Region. Durch die Bindung von PhoP an die PhoP-Erkennungsstelle bei –10 wird die Transkription gehemmt. Diese PhoP-vermittelte duale Kontrolle des phyC-Promotors scheint bis lang einzigartig für Pho-Regulongene zu sein. Der globale Regulator AbrB konnte als weiterer Repressor des phyC identifiziert werden. Die Bindungsstelle befinden sich upstream von –147 und downsteam von +29, müssen aber in weiteren Experimenten genauer spezifiziert werden. Diese Arbeit stellt als erste die Modelle der phyC-Regulation durch PhoP und AbrB vor. / Several Bacillus strains secrete phytase, an enzyme catalysing dephosphorylation of myo-inositol hexakisphosphate (phytate). The monocistronic phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 was identified as a member of the phosphate-starvation inducible PhoPR regulon. The transcriptional start was determined downstream of a sigmaA-like promoter region located 27 bp upstream of the translation start codon. Inspection of the phyC promoter sequence revealed an unusual structure, since the -35 and -10 region are separated by a window of 21 bp. In vitro transcription analysis established that PhoP-P is necessary to initiate the transcription from phyC promoter. PhoP binding boxes occurring in most B. subtilis promoters activated by PhoP consist of at least four TTAACA-like sequences repeated at intervals of 5-6 bps. The upstream region of the B. amyloliquefaciens FZB45 phyC gene deviates from this general architecture in that there is only one appropriate binding site for the dimeric PhoP protein, which consists of two motives centered at -47 and -35 and separated by five base pairs. A single PhoP binding site is located at -13 to -8, nearly matching the -10 consensus. Functionality of the three PhoP binding boxes was demonstrated by DNaseI footprinting suggesting that a pair of dimeric PhoP molecules cover the -35 region directing the RNA-polymerase to the –10 region. Binding of PhoP at a single PhoP binding site covering the -10 consensus repress the transcription. It seems to be a unique feature of the phyC promoter structure and has not been reported for any other member of the PhoPR regulon, previously. Furthermore an inhibitory effect via AbrB, a global regulator, could be verified. The binding regions could be determine upstream of –147 and downstream of +29, but have to be specify in further experiments. This work presents for the first time the models for the regulation of phyC in Bacillus via PhoP and AbrB.

Bioaugmentation promises benefits for agricultural production as well as for remediation and phytomining approaches. Thus, this study investigated the effect of soil inoculation with the commercially available product RhizoVital®42, which contains Bacillus amyloliquefaciens FZB42, on nutrient uptake and plant biomass production as well as on the phytoaccumulation of potentially toxic elements, germanium, and rare earth elements (REEs). Zea mays and Fagopyrum esculentum were selected as model plants, and after harvest, the element uptake was compared between plants grown on inoculated versus reference soil. The results indicate an enrichment of B. amyloliquefaciens in inoculated soils as well as no significant impact on the inherent bacterial community composition. For F. esculentum, inoculation increased the accumulation of most nutrients and As, Cu, Pb, Co, and REEs (significant for Ca, Cu, and Co with 40%, 2042%, and 383%, respectively), while it slightly decreased the uptake of Ge, Cr, and Fe. For Z. mays, soil inoculation decreased the accumulation of Cr, Pb, Co, Ge, and REEs (significant for Co with 57%) but showed an insignificant increased uptake of Cu, As, and nutrient elements. Summarily, the results suggest that bioaugmentation with B. amyloliquefaciens is safe and has the potential to enhance/reduce the phytoaccumulation of some elements and the effects of inoculation are plant specific.

Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde. / Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.

This study is an investigation of the effect of soil bioaugmentation (inoculation) on a field scale with the commercially available product RhizoVital®42, containing Bacillus amyloliquefaciens FZB4, on element bioavailability, plant biomass production, as well as accumulation of rare earth elements (REEs), germanium, and selected trace elements. Zea mays and Helianthus annuus were selected as test plants. Post-harvest, results showed inoculation increased biomass production of Z. mays and H. annuus by 24% and 26%, albeit insignificant at p ≤ 0.05. Bioaugmentation enhanced Z. mays shoot content of P, Cd, and Ge by percentages between 73% and 80% (significant only for Ge) and decreased shoot content of REET, Pb, and Cu by 28%, 35%, and 59%, respectively. For H. annuus grown on bioaugmented soil, shoot content of Ca, Cu, Ge, REET, and Pb increased by over 40%, with a negligible decrease observed for Cd. Summarily, results suggest that bioaugmentation with Bacillus amyloliquefaciens FZB42 could enhance biomass production, increase soil element bioavailability enhance, and increase or reduce plant accumulation of target elements. Additionally, differences in P use efficiency could influence bioaugmentation effects on P accumulation.

Made available in DSpace on 2014-06-11T19:25:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T19:52:56Z : No. of bitstreams: 1 alves_gcs_me_jabo.pdf: 1113816 bytes, checksum: 1eb10624020c5b9792319a590c0fc737 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil os nematóides-chave da cana-de-açúcar são Meloidogyne incognita, M. javanica e Pratylenchus zeae. Dentre as práticas de manejo o controle biológico é uma alternativa importante para as grandes culturas. Assim o objetivo deste trabalho foi, realizar um estudo da dinâmica de população de P. zeae em área de cultivo de cana-de-açúcar, no período de março a novembro de 2009. avaliar in vitro a ação de 21 isolados de rizobactérias sobre a eclosão e motilidade de juvenis de segundo estádio (J2) de M. incognita, M. javanica e sobre a motilidade de formas ativas de P. zeae e avaliar em campo, a influência do plantio na linha e na entrelinha com nematicidas sintéticos e um isolado bacteriano sobre a população de P. zeae. Os isolados bacterianos FCAV 6, FCAV 8 e FCAV 10 apresentaram ação ovicida para M. javanica. Os isolados FCAV 5, FCAV 7 e Bacillus amyloliquefaciens mostraram-se promissores no controle biológico de P.zeae. No campo o tratamento com rizobactéria FCAV 9 + adubo organomineral se mostrou superior aos demais em relação número de perfilhos. Estes resultados confirmam a potencialidade das rizobactérias para o controle de nematóides / In Brazil, Meloidogyne incognita, M. javanica and Pratylenchus zeae are key nematodes sugar cane. Among management practices for decreasing nematode infestation, biological control is an important alternative for the major crops. So, the objective was to carry a study of population dynamics of P. zeae area in sugar cane in growing, to evaluate the in vitro the action of 21 isolates of rhizobacteria on hatching and motility of second stage juveniles (J2) of M. incognita, M. javanica and on the motility of active forms of P. zeae and to assess in the field, the influence of planting in lines and rows with synthetic nematicides and a bacterial isolate on the population of P. zeae. The bacterial isolates FCAV 6, FCAV 8 and FCAV 10 showed ovicidal action for M. javanica. Isolate C116 showed promise for the control of J2 of M. javanica. Isolates FCAV 5, FCAV 7 and Bacillus amyloliquefaciens have shown promise for the biological control of P.zeae. In the treatments 5 (Isolate FCAV 9 in the rows) and 6 (Isolate FCAV 9 between rows), were superior to others for the biometric analysis. These results confirm the potential of rhizobacteria for control of nematodes

Resumo: No Brasil os nematóides-chave da cana-de-açúcar são Meloidogyne incognita, M. javanica e Pratylenchus zeae. Dentre as práticas de manejo o controle biológico é uma alternativa importante para as grandes culturas. Assim o objetivo deste trabalho foi, realizar um estudo da dinâmica de população de P. zeae em área de cultivo de cana-de-açúcar, no período de março a novembro de 2009. avaliar in vitro a ação de 21 isolados de rizobactérias sobre a eclosão e motilidade de juvenis de segundo estádio (J2) de M. incognita, M. javanica e sobre a motilidade de formas ativas de P. zeae e avaliar em campo, a influência do plantio na linha e na entrelinha com nematicidas sintéticos e um isolado bacteriano sobre a população de P. zeae. Os isolados bacterianos FCAV 6, FCAV 8 e FCAV 10 apresentaram ação ovicida para M. javanica. Os isolados FCAV 5, FCAV 7 e Bacillus amyloliquefaciens mostraram-se promissores no controle biológico de P.zeae. No campo o tratamento com rizobactéria FCAV 9 + adubo organomineral se mostrou superior aos demais em relação número de perfilhos. Estes resultados confirmam a potencialidade das rizobactérias para o controle de nematóides / Abstract: In Brazil, Meloidogyne incognita, M. javanica and Pratylenchus zeae are key nematodes sugar cane. Among management practices for decreasing nematode infestation, biological control is an important alternative for the major crops. So, the objective was to carry a study of population dynamics of P. zeae area in sugar cane in growing, to evaluate the in vitro the action of 21 isolates of rhizobacteria on hatching and motility of second stage juveniles (J2) of M. incognita, M. javanica and on the motility of active forms of P. zeae and to assess in the field, the influence of planting in lines and rows with synthetic nematicides and a bacterial isolate on the population of P. zeae. The bacterial isolates FCAV 6, FCAV 8 and FCAV 10 showed ovicidal action for M. javanica. Isolate C116 showed promise for the control of J2 of M. javanica. Isolates FCAV 5, FCAV 7 and Bacillus amyloliquefaciens have shown promise for the biological control of P.zeae. In the treatments 5 (Isolate FCAV 9 in the rows) and 6 (Isolate FCAV 9 between rows), were superior to others for the biometric analysis. These results confirm the potential of rhizobacteria for control of nematodes / Orientador: Jaime Maia dos Santos / Coorientador: Pedro Luiz Martins Soares / Banca: Marineide Mendonça Aguillera / Banca: Júlio Cesar Galli / Mestre

We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...