A indústria sucroenérgetica gera grande quantidade de resíduos, sendo a torta de filtro e as cinzas os principais resíduos sólidos. Uma das tecnologias desenvolvidas para o manejo destes resíduos é a compostagem. A aplicação do composto tem mostrado efeitos positivos na cultura da cana-de-açúcar no que diz respeito à fertilização fosfatada. Os objetivos do presente trabalho foram: I. Observar o comportamento das comunidades bacterianas durante a compostagem e o efeito da inoculação de estirpes bacterianas mobilizadoras de fósforo sobre a disponibilidade de fósforo no composto final; II. Avaliar o efeito da aplicação de diferentes fontes de P e de bactérias mobilizadoras de fósforo no desenvolvimento de plantas de cana-de-açúcar e o efeito sobre as comunidades bacterianas do solo. Nesse sentido, pilhas de compostagem foram instaladas na Usina São José da Estiva em Novo Horizonte, SP. Os tratamentos consistiram de pilhas com e sem rocha fosfática; e pilhas com e sem a inoculação das estirpes Pseudomonas aeruginosa PSBR12 e Bacillus sp. BACBR1. Amostragens foram realizadas quinzenalmente durante 60 dias. Adicionalmente determinou-se a atividade enzimática, e parâmetros químicos do composto. A comunidade bacteriana foi acessada por meio da técnica independente de cultivo T-RFLP (restriction fragment length polymorphism) e sequenciado por meio da plataforma de nova geração MiSeqTM System (Illumina). Com o objetivo de avaliar o efeito do composto e da inoculação de bactérias mobilizadoras de P no desenvolvimento da cana-de-açúcar foi instalado um experimento em casa de vegetação com plantas de cana de açúcar, utilizando-se composto, rocha fosfática e super fosfato triplo como fontes de P, além da inoculação dos seguintes: Inoculante 1: Pseudomonas sp. PSBR10, Azotobacter sp. AZTBR19, Rhizobium sp. RIZBR01; e o Inoculante 2: Bacillus simplex BACBR04, Bacillus sp. BACBR06, Rhizobium sp. RIZBR01. O experimento foi conduzido durante 75 dias. Ao fim do experimento foram analisados os seguintes parâmetros: biomassa seca, acúmulo de P, N, e K na parte aérea, e fosfatases no solo. A estrutura das comunidades bacterianas no solo foram avaliadas por meio do sequenciamento na plataforma Illumina. A aplicação de bactérias mobilizadoras de P durante a compostagem diminuiu o P ligado ao Ca. A mudança nas comunidades bacterianas durante a compostagem foi temporal, no início dominaram membros da ordem Lactobacillales, após 15 dias as ordens Bacillales e Clostridiales passaram a dominar o processo. As comunidades bacterianas são influenciadas principalmente pelos parâmetros pH, temperatura e umidade. Quanto ao experimento em casa de vegetação, o uso dos inoculantes (principalmente o inoculante 2) aumentou o acúmulo de P, N e K na parte aérea nos tratamentos que receberam composto e super fosfato triplo como fonte de P. A aplicação dos inoculantes e a adição do composto modificou a estrutura das comunidades bacterianas do solo; essa alteração quando os inoculantes são aplicados pode estar relacionada com o incremento no acúmulo de nutrientes. O uso de bactérias mobilizadoras de P é uma tecnologia potencial para o uso na agricultura, tanto na compostagem de resíduos da indústria sucroenergética com rocha fosfática, como no aumento da eficiência da fertilização fosfatada na cana-de-açúcar. / Sugarcane industry generates large amounts of waste, filter cake and ashes being the main solid wastes. One of the technologies developed for the management of this waste is composting. The application of compost has shown positive effects on the sugarcane culture with regard to phosphorus fertilization. The objectives of this study are: I. To study the diversity of bacterial communities during composting and the effect of inoculation of phosphorus mobilizing bacterial strains on the phosphorus availability in the final compost; II. To evaluate and compare the effects of the application of different sources of phosphorus and P mobilizing bacteria on the development of sugarcane and the effect on soil bacterial communities. Therefore, compost piles were installed at Usina São José da Estiva in Novo Horizonte, Brazil. Treatments consisted of piles with and without rock phosphate and with and without inoculation of Pseudomonas aeruginosa PSBR12 and Bacillus sp. BACBR1 strains. Samples were taken every two weeks for 60 days. In addition, the enzymatic and chemical composition of the compost was determined. The bacterial community was assessed through T-RFLP (restriction fragment length polymorphism) and was sequenced through the new generation platform MiSeqTM System (Illumina). The abundance of bacteria was evaluated by qPCR. In order to evaluate the effect of compost and of inoculating P mobilizing bacteria on sugarcane development, a greenhouse experiment was installed with sugarcane using compost, rock phosphate and triple superphosphate as P sources, besides inoculations, as follows: Inoculant 1: Pseudomonas sp. (PSBR10) Azotobacter sp. (AZTBR19), Rhizobium sp. (RIZBR01); and Inoculant 2: Bacillus simplex (BACBR04), Bacillus sp. (BACBR06), Rhizobium sp. (RIZBR01). The experiment was conducted during 75 days. At the end of the experiment the following parameters were analyzed: dry plant biomass, accumulation of P, N, and K in the shoot, and phosphatases in soil. Bacterial soil communities were sequenced through the new generation Illumina. The application of P mobilizing bacteria during composting decreased the Ca-linked P. Changes of the bacterial communities during composting were temporal. In the beginning, members of the order Lactobacillales were dominant and after 15 days a succession of bacterial communities occurred, when Bacillales and Clostridiales began to dominate. Bacterial communities are mostly influenced by the parameters pH, temperature and humidity. Moreover, in the greenhouse experiment, the use of inoculants (mainly inoculant 2) increased the accumulation of P, N and K in shoots in the treatments that received compost and triple superphosphate as P source. Inoculant application and compost addition modified soil bacterial community structures. Changes in the soil microbiome when inoculant was applied may be related to the increase in nutrients accumulation. The use of P mobilizing bacteria is a potential technology for use in agriculture, when composting waste from the sugarcane industry with rock phosphate or when increasing P fertilization efficiency in sugarcane in the field.

La microbiologia de l’aigua és un camp científic que ha ajudat enormement a millorar la seguretat sanitària de les aigües destinades als usos humans. De fet, augmentar el coneixement sobre les poblacions microbianes presents a l’aigua tant pel que fa a les poblacions autòctones com a les al•lòctones, pot ser de gran ajuda de cara a garantir els estàndards de qualitat, que cada vegada són més exigents. Tanmateix, la gran heterogeneïtat dels sistemes aquàtics i de les problemàtiques lligades als seus usos, fan necessari un estudi individualitzat de les seves poblacions microbianes. Així doncs, el propòsit d’aquesta tesi doctoral va ser estudiar diferents poblacions microbianes associades a diferents sistemes aquàtics: aigües minerals naturals, aigües recreatives i aigües regenerades. Pel que fa a les aigües minerals naturals, es van estudiar les poblacions bacterianes aeròbies heterotròfiques autòctones de tres fonts a 22ºC i 37ºC durant l’hivern i l’estiu. Totes les soques aïllades es van fenotipar mitjançant les microplaques PhP-48 del Phene-Plate System®. Es van poder aïllar un elevat nombre de gèneres poc descrits en aquests ambients. Això afegit als canvis de composició observats entre estacions, confirma que es tracta d’ambients complexos, i que encara hi ha un gran desconeixement sobre la seva composició i dinàmica. D’altra banda, es va confirmar que cada aigua mineral natural conté una microbiota autòctona pròpia, l’estructura i composició de la qual és prou específica per poder diferenciar-la amb mètodes de caracterització fenotípica de la d’altres aigües minerals naturals. D’altra banda es va avaluar la capacitat de la ISO 16266:2006 per identificar Pseudomonas aeruginosa en aigües minerals naturals. En general, el procediment de la ISO 16266:2006 identifica adequadament la major part dels aïllats de P. aeruginosa, llevat dels casos de soques no pigmentades. Els resultats recomanen la incorporació dels assaigs de creixement en agar King A i a 4°C i 42°C en les anàlisis rutinàries d’aigües envasades, i s’aconsella l’ús de les galeries API®20NE en la confirmació d’aquells aïllats que generin dubtes en base als criteris de la ISO 16266:2006. En l’àmbit de les aigües recreatives, es van caracteritzar les poblacions microbianes associades a quatre piscines naturalitzades d’ús privat. Tres d’elles presentaven a l’estiu concentracions d’E. coli o enterococs superiors als límits establerts a les recomanacions. La contaminació fecal relacionada amb la fauna salvatge s’apunta com un contribuïdor significatiu en les piscines naturalitzades privades. A més, el sistema de depuració natural de les piscines naturalitzades sembla ser força limitat alhora de disminuir la contaminació fecal a les piscines, cosa que posa en dubte la seva aplicabilitat en piscines de major volum i obertes al públic. Pel que fa a les aigües regenerades, es van avaluar la capacitat del fenotipatge bioquímic de coliforms fecals i enterococs per determinar l’origen de la contaminació fecal en dues basses d’aigua regenerada utilitzades en la irrigació d’un camp de golf. La caracterització bioquímica i l’estudi d’inactivació van proporcionar evidències que la contaminació fecal detectada a les basses a l’estiu no tenia relació amb les poblacions que entraven a través de l’aigua regenerada, sinó que semblava estar relacionat amb una aportació de material fecal animal, probablement ocells, generat al propi sistema. El fenotipatge bioquímic d’enterococs i coliforms fecals és una eina útil per a l’estudi de l’origen de la contaminació fecal en aquells casos on la càrrega fecal és baixa, fet que és rellevant, tenint en compte que molts altres mètodes presenten problemes en ser aplicats per sota d’aquestes concentracions. En general, els estudis realitzats donen peu a continuar aprofundint en l’estudi de la diversitat microbiana en diferents ambients aquàtics, tant pel que fa a les poblacions autòctones com a les al•lòctones. / "Characterization and diversity of microbial populations associated to with natural mineral waters, recreational waters and reclaimed waters" SUMMARY: Microbial populations from natural mineral waters, recreational waters and reclaimed waters were characterized to increase the knowledge about their indigenous and introduced microorganisms. Regarding natural mineral waters, we characterized the aerobic heterotrophic bacteria from three sources at 22ºC and 37ºC during summer and winter. The study of the similarities showed that growing temperatures and seasons caused significant differences in structures and composition. In addition, several bacterial species were isolated and identified, some of them rarely isolated in natural mineral waters, revealing the complexity and lack of knowledge of these ecosystems. Furthermore, we evaluated the procedure included in ISO 16266:2006 to identify presumptive P. aeruginosa isolates in natural mineral waters. The results showed that ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained. Additional test assayed such as King A medium, growth tests at 4 °C and 42 °C and API® 20NE correctly discriminated all the studied strains. As far as recreational waters are concerned, we analyzed the microbial populations in four private natural swimming pools. Three of them exceeded the E. coli or enterococci limits stated in the recommendations for natural swimming pools. The concentrations of P. aeruginosa and aerobic heterotrophic bacteria were acceptable. The results suggest that wildlife was an important source of fecal pollution in the pools. Since there is a lack of regulations on these systems, and the health risks are higher than in conventional swimming pools, further research is needed to establish the parameters for ensuring safe bathing in private and public natural swimming pools. Finally, we aimed to identify the origin of the fecal contamination in two reclaimed-water open-air ponds used to irrigate a golf course. Although the concentration of fecal bacterial indicators was low, the biochemical fingerprinting used provided evidence that the origin of the fecal contamination in the ponds was not related to the reclaimed water. Furthermore, a mesocosm assays indicated that none of the microbial fecal indicators was able to regrow in the ponds. Finally, the study highlighted the fact that reclaimed water may be recontaminated in open-air reservoirs.

No description available.

No description available.

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.

O histórico agronômico da cultura, em geral, explica a comunidade microbiana presente na massa ensilada, entretanto, a diversidade e o grau de contaminação da população microbiana epifítica pode auxiliar na compreensão do padrão de fermentação da silagem e da estabilidade desse produto quando em exposição ao ambiente aeróbio. No presente trabalho, foram avaliados a influência do genótipo, maturidade e período de estocagem na composição da comunidade bacteriana em silagens de grãos de milho. Para isso, dois cultivares de milho AG 1051 (\"dent\") e IAC 8390 (\"flint\") foram colhidos em três estágios de maturidade (ponto de silagem de planta inteira, ponto de silagem de grão úmido e ponto de grão seco), moídos e ensilados por 0, 7 e 120 dias. Atualmente, a aplicação de técnicas de microbiologia molecular permite acessar alterações causadas nestas comunidades de maneira independente do cultivo bacteriano, por esse motivo, a comunidade bacteriana foi avaliada por meio da técnica de Eletroforese em Gel de Gradiente Desnaturante (DGGE) e sequenciamento dos produtos de PCR via sistema MiSeqTM Illumina. Foi demonstrado que em silagens de grãos de milho contendo alta umidade, os diferentes estágios de desenvolvimento da cultura, e por conseguinte, do grão, são os principais determinantes da composição da comunidade bacteriana deste, sendo menos importantes o genótipo das plantas e os tempos de estocagem das silagens. Aos 120 dias de estocagem nas amostras de grão seco reconstituído, as sequências afiliadas ao gênero Clostridium representaram total de aproximadamente 40% das sequências afiliadas aos gêneros encontrados, enquanto o gênero Lactobacillus representou menos de 7% das sequências afiliadas a este gênero. Provavelmente, grãos secos sofrem mais estresse a campo, o que consequentemente, pode interferir na qualidade higiênico sanitária das silagens desses grãos. Com base nestes resultados fica evidenciada a possibilidade de realização de recomendações potenciais de aditivos específicos para ensilagem de grãos de milho, direcionados para cada ponto de maturidade da cultura. / The agronomic background of crops in general, explains the microbial community present in silage, however, diversity and contamination status may help on understanding the silage fermentation profile and aerobic stability. In the present work, the influence of factors such as different genotypes, different stages of plants development and storage time in the composition of bacterial communities were evaluated. On this way, maize cultivars AG 1051 (\"dent\") and IAC 8390 (\"flint\") were harvested in three physiological stages (whole plant silage, wet grain silage and dry grain), the grain were grounded and ensiled for 0, 7 and 120 days. Nowadays, the applications of techniques of molecular microbiology allow assessing the shifts caused on these communities by a culture independent approach, therefore, bacterial community were evaluated by Denaturing Gradient Gel Electrophoresis technique (DGGE), and PCR products were sequenced by Illumina MiSeqTM System. It was demonstrated that in high moisture corn silage, different stages of plants development are main determinants of bacterial community composition rather than the plants genotypes and storage time. In addition in the samples of reconstituted dry grain, it was demonstrated that after 120 days of storage, sequences affiliated to the gender Clostridium accounted for a total of approximately 40% of total sequences affiliated to genera found, while the genus Lactobacillus represented less than 7% of sequences affiliated to this gender. Probably dried grains suffer more stress at field conditions, which in turn can interfere with the sanitary hygienic quality of silages obtained from these grains. At least, based on these results it is clear the possibility of performing potential specific additives recommendations, unique at each stage of maize plants development.

Climate change is expected to increase the precipitation ~30% in higher latitudes during the next century, increasing the land runoff via rivers to aquatic ecosystems. The Baltic Sea will receive higher river discharges, accompanied by larger input of allochthonous dissolved organic matter (DOM) from terrestrial ecosystems. The salinity will decrease due to freshwater dilution. The allochthonous DOM constitute a potential growth substrate for microscopic bacterioplankton and phytoplankton, which together make up the basal trophic level in the sea. The aim of my thesis is to elucidate the bacterial processing of allochthonous DOM and to evaluate possible consequences of increased runoff on the basal level of the food web in the Baltic Sea. I performed field studies, microcosm experiments and a theoretical modeling study. Results from the field studies showed that allochthonous DOM input via river load promotes the heterotrophic bacterial production and influences the bacterial community composition in the northern Baltic Sea. In a northerly estuary ~60% of bacterial production was estimated to be sustained by terrestrial sources, and allochthonous DOM was a strong structuring factor for the bacterial community composition. Network analysis showed that during spring the diversity and the interactions between the bacteria were relatively low, while later during summer other environmental factors regulate the community, allowing a higher diversity and more interactions between different bacterial groups. The influence of the river inflow on the bacterial community allowed “generalists” bacteria to be more abundant than “specialists” bacteria.    Results from a transplantation experiment, where bacteria were transplanted from the northern Baltic Sea to the seawater from the southern Baltic Sea and vice versa, showed that salinity, as well as the DOM composition affect the bacterial community composition and their enzymatic activity. The results showed that α-proteobacteria in general were favoured by high salinity, β-proteobacteria by low salinity and terrestrial DOM compounds and γ-proteobacteria by the enclosure itself. However, effects on the community composition and enzymatic activity were not consistent when the bacterial community was retransplanted, indicating a functional redundancy of the bacterial communities.  Results of ecosystem modeling showed that climate change is likely to have quite different effect on the north and the south of the Baltic Sea. In the south, higher temperature and internal nutrient load will increase the cyanobacterial blooms and expand the anoxic or suboxic areas. In the north, climate induced increase in riverine inputs of allochthonous DOM is likely to promote bacterioplankton production, while phytoplankton primary production will be hampered due to increased light attenuation in the water. This, in turn, can decrease the production at higher trophic levels, since bacteria-based food webs in general are less efficient than food webs based on phytoplankton. However, complex environmental influences on the bacterial community structure and the large redundancy of metabolic functions limit the possibility of predicting how the bacterial community composition will change under climate change disturbances.

A l’échelle de l’agro-écosystème, la productivité primaire est sous la dépendance du recyclage des matières organiques du sol (MOS) par l’action des organismes indigènes décomposeurs, qui minéralisent les composés organiques libérant ainsi les nutriments nécessaires à la croissance végétale. A une échelle plus globale, le recyclage des MOS détermine les flux de carbone entre le sol et l’atmosphère, avec des conséquences majeures sur la qualité de l’environnement et les changements globaux. Malgré le rôle central des microorganismes indigènes dans ces processus, la composante microbienne est encore mal connue et souvent considérée comme une boîte noire en termes de diversité et de fonctionnalité. Par conséquent, pour mieux comprendre et prédire l’évolution des MOS et donc les flux de carbone (C) dans les agro-écosystèmes, il est nécessaire de mieux connaitre les populations et les mécanismes microbiens impliqués dans leur dégradation et transformation. Dans ce contexte, l’objectif de cette thèse était de progresser dans la connaissance de la réponse des communautés microbiennes telluriques à l’apport de résidus de culture. Cette réponse des communautés microbiennes a été abordée en termes de (i) succession des populations impliquées dans les processus de dégradation de ces MOF (matières organiques fraîches), (ii) lien avec leur fonction de dégradation et répercussion sur la dynamique des matières organiques, et (iii) rôle dans les processus de stockage/déstockage du carbone via les processus de « priming effect ». Différents paramètres pouvant moduler la dégradation des résidus et la dynamique des communautés ont été pris en compte : modalité d’apport des résidus (pratiques culturales), qualité biochimique des résidus (différentes espèces végétales), et température. La stratégie globale de recherche développée repose sur des expérimentations de terrain et au laboratoire impliquant différentes échelles spatiales (du microcosme de sol à la parcelle agronomique) et temporelles (du temps de génération microbien aux cycles culturaux). La réponse des communautés microbiennes à l’apport de résidus a été évaluée par l’utilisation de méthodes moléculaires permettant de caractériser sans a priori la diversité des microorganismes du sol (empreintes moléculaires, clonage/séquençage, séquençage haut débit). En parallèle, un suivi quantitatif et qualitatif de la matière organique du sol, par des méthodes de biochimie ou de spectroscopie, a été réalisé afin d’établir le lien entre la dynamique des communautés microbiennes et le devenir de la matière organique dans le sol. Les deux premiers chapitres du manuscrit portent sur des expérimentations réalisées au terrain (conditions naturelles) afin d’évaluer l’influence de la localisation des résidus (résidus de blé incorporés vs. laissés en surface ; site expérimentale INRA Mons) d’une part et d’autre part de la qualité biochimique des résidus (résidus de blé, colza et luzerne incorporés, site expérimentale INRA Epoisses) sur la dynamique des communautés microbiennes du sol. Les résultats obtenus mettent en évidence une forte influence de la localisation comme de la qualité biochimique des résidus sur les successions de populations microbiennes induites suite à l’apport. Des populations/groupes microbiens stimulés spécifiquement dans chaque modalité ont été identifiés. Les résultats de diversité ont été mis en regard des dynamiques de décomposition des résidus, afin de faire le lien entre les successions de populations et l’évolution des ressources trophiques. La troisième partie du travail correspond à une expérimentation en conditions contrôlées (microcosmes de sol) nous permettant de coupler des outils moléculaires et isotopiques (ADN-SIP) pour cibler spécifiquement les populations microbiennes activement impliquées dans la dégradation des résidus de culture - etc / The effect of the location of wheat residues (soil surface vs. incorporated in soil) on their decomposition and on soil bacterial communities was investigated by the means of a field experiment. Bacterial-Automated Ribosomal Intergenic Spacer Analysis (B-ARISA) of DNA extracts from residues, detritusphere (soil adjacent to residues), and bulk soil evidenced that residues constitute the zone of maximal changes in bacterial composition. However, the location of the residues influenced greatly their decomposition and the dynamics of the colonizing bacterial communities. Sequencing of 16S rRNA gene in DNA extracts from the residues at the early, middle, and late stages of degradation confirmed the difference of composition of the bacterial community according to the location. Bacteria belonging to the -subgroup of proteobacteria were stimulated when residues were incorporated whereas the -subgroup was stimulated when residues were left at the soil surface. Moreover, Actinobacteria were more represented when residues were left at the soil surface. According to the ecological attributes of the populations identified, our results suggested that climatic fluctuations at the soil surface select populations harboring enhanced catabolic and/or survival capacities whereas residues characteristics likely constitute the main determinant of the composition of the bacterial community colonizing incorporated residues. Microbial communities are of major importance in the decomposition of soil organic matter. However, the identities and dynamics of the populations involved are still poorly documented. We investigated, in a eleven-month field experiment, how the initial biochemical quality of crop residues could lead to specific decomposition patterns, linking biochemical changes undergone by the crop residues to the respiration, biomass and genetic structure of the soil microbial communities. Wheat, alfalfa and rape residues were incorporated into the 0-15 cm layer of the soil of field plots by tilling. Biochemical changes in the residues occurring during degradation were assessed by near infrared spectroscopy (NIRS). Qualitative modifications in the genetic structure of the bacterial communities were determined by Bacterial-Automated Ribosomal Intergenic Spacer Analysis (B-ARISA). Bacterial diversity in the three crop residues at early and late stages of decomposition process was further analyzed from a molecular inventory of the 16S rDNA. The decomposition of plant residues in croplands was shown to involve specific biochemical characteristics and microbial communities dynamics which were clearly related to the quality of the organic inputs. Decay stage and seasonal shifts occurred by replacement of copiotrophic populations/bacterial groups such as proteobacteria successful on younger residues with those successful on more extensively decayed material such as Actinobacteria. However, relative abundance of proteobacteria depended greatly on the composition of the residues, with a gradient observed from alfalfa to wheat, suggesting that this bacterial group may represent a good indicator of crop residues degradability and modifications during the decomposition process...

Les hydrocarbures aromatiques polycycliques (HAP) sont des composés ubiquitaires issus de la combustion incomplète de matières organiques. Ils sont à l'origine de pollutions de l'environnement, surtout liées à l'exploitation des produits pétroliers, car ce sont des composés toxiques pour les êtres vivants et pour l'homme en particulier. De nombreuses bactéries capables de dégrader les HAP ont été isolées et étudiées, mais celles qui les dégradent in situ sont mal connues, car moins de 5% des bactéries du sol sont cultivables en laboratoire. Le premier objectif de cette étude était d'identifier les bactéries qui dégradent les HAP dans le sol par des méthodes moléculaires indépendantes de la culture. A cette fin, une stratégie de marquage isotopique in situ a été mise en œuvre qui repose sur l'utilisation du phénanthrène, un HAP à trois cycles, dans lequel l'isotope naturel du carbone a été remplacé par le 13C. Cette molécule a été introduite comme traceur dans des microcosmes contenant du sol provenant d'un bassin de rétention des eaux de ruissellement d'autoroute. Les bactéries ayant incorporé le 13C ont ensuite été identifiées par séquençage des gènes d'ARNr 16S amplifiés à partir de l'ADN marqué extrait du sol. Les résultats montrent que des Betaprotéobactéries peu étudiées à ce jour, appartenant aux genres Acidovorax, Rhodoferax, Hydrogenophaga et Thiobacillus, ainsi que des Rhodocyclaceae, étaient les principaux acteurs de la dégradation du phénanthrène. La prépondérance des Betaprotéobactéries a été établie par des mesures de PCR quantitative. Une analyse dynamique de la diversité bactérienne a montré que celle-ci changeait en fonction de la biodisponibilité du phénanthrène. En outre, la diversité d'arène-dioxygénases impliquées dans la dégradation des HAP a été explorée sur le plan phylogénétique et fonctionnel. Nous avons ainsi détecté des séquences nouvelles, pour la plupart apparentées à des dioxygénases de Sphingomonadales et de Burkholderiales. Grâce à la construction et l'expression d'enzymes hybrides, il a été possible, pour la première fois, d'associer une activité catalytique d'oxydation des HAP à des séquences partielles de gènes, amplifiées à partir de l'ADN du sol. Les résultats obtenus et les outils mis au point dans cette étude pourront servir à développer des méthodes de diagnostic et de suivi de biodégradation de polluants, par exemple dans le cadre d'opérations de bioremédiation de sites pollués par les HAP. / Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by 13C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the 13C were then identified by 16S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera Acidovorax, Rhodoferax, Hydrogenophaga and Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaprotéobactéries was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating dioxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to dioxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences amplified from soil DNA, by constructing hybrid enzymes and assaying their activity The results obtained and the tools implemented in this study may be used to develop methods for the diagnostic and monitoring of pollutant biodegradation in processes such as bioremediation of PAHs contaminated sites.

La fertilité de terres agricoles dépend en large partie du recyclage des nutriments dans le sol. Généralement, ce recyclage est effectué en grande partie par les communautés bactériennes du sol. On assume donc souvent que la diversité bactérienne du sol peut constituer un indicateur de sa santé/fertilité. Cependant, certaines pratiques agricoles conventionnelles nuisent à la diversité bactérienne du sol. Parmi ces pratiques, le labourage et les applications d’intrants chimiques tels que les pesticides, les antibiotiques et les engrais influencent négativement la diversité microbienne. Par conséquent, des recherches actives sont menées pour développer des façons de rétablir la diversité microbienne dans les sols en agriculture conventionnelle. Plusieurs alternatives biologiques ont été développées au fil des ans, aboutissant à des produits commerciaux en tant que des biostimulants incluant des substances d’origines biologiques, des microorganismes ou la combinaison des deux. Entre autres, le thé de compost a été développé et suggéré comme étant un produit riche en microorganismes bénéfiques, ayant les capacités d’améliorer les cultures et la durabilité des systèmes agricoles biologiques. Cependant, sa performance et son application à grande échelle dans les systèmes de production conventionnelle demeurent peu étudiées. L’objectif de ce mémoire et d’évaluer l'effet du thé de compost sur l'abondance, la diversité et la structure des communautés bactériennes du sol et les rendements, dans un essai en champs de la production du soja dans un système conventionnel de monoculture. Dans un champ d’environ trois hectares est subdivisé en six blocs, chacun contenait deux parcelles: l'une a été traitée par le thé de compost frais et l'autre a été utilisé comme témoin avec thé de compost stérilisé à la chaleur pour tuer les microorganismes. Notre hypothèse est que le thé de compost frais améliore la croissance du soja et son rendement avec l’apport de microorganismes bénéfiques et l’enrichissement des communautés bactériennes des sols. Le séquençage à haut débit de l’ADN ribosomique 16S bactérien extrait de différents échantillons (thé de compost, sol traité et sol témoin), associé aux analyses bio-informatiques et statistiques, a démontré que le traitement du thé de compost frais n'a pas influencé de manière significative les communautés bactériennes, ni par des changements dans la diversité alpha, ni dans la structure de la communauté de celles-ci. De plus, les résultats des analyses de croissance des plantes et de rendement ont eu aucun effet significatif du thé de compost frais sur la biomasse végétative des plantes ou le poids des graines de soja. Nos résultats de recherche indiquent que le thé de compost frais utilisé dans notre expérience n’a pas modifié les communautés bactériennes des sols traités et n’a pas influencé la croissance des plantes ni le rendement en grain. Notre hypothèse n’est pas supportée par ces résultats qui suggèrent que les bénéfices relatifs à l’application du thé de compost frais ne sont pas dus aux microorganismes vivants mais plutôt à un apport potentiel des nutriments. L’absence d'effets positifs dans notre étude pourrait être attribué spécifiquement à notre conception expérimentale, au thé de compost utilisé, ou à la dose ou la fréquence d'application de celui-ci. D’autres expériences sont nécessaires afin de tirer des conclusions robustes quant à l’effet et la performance du thé de compost sur des cultures conventionnelles. / The fertility of agricultural lands largely depends on the recycling of nutrients in the soil. Usually, this recycling is carried out largely by bacterial communities in the soil, that their diversity is an important indicator of the health and fertility of agricultural soils. However, some agricultural practices, especially in conventional production systems, harm the essential functions of these soil bacterial communities. Among these practices, tillage and the applications of chemical inputs such as pesticides, antibiotics and fertilizers negatively influence the diversity and structures of microbial communities. As a result, the abundance and diversity of these beneficial microorganisms and the potential services they provide decrease in these soils. Several biological alternatives have been developed over the years, resulting in commercial products as biostimulants including substances of biological origin, microorganisms or a combination of the two. Among others, compost tea has been developed and suggested as a product rich in beneficial microorganisms, with the capacity to improve crops and the sustainability of organic farming systems. However, its performance and large-scale application in conventional production systems remains little studied. The objective of this master’s thesis is to assess the effect of fresh compost tea on the abundance, diversity and structure of soil bacterial communities and yields, in a field trial of soybean production in a conventional system of monoculture. In a field of about three hectares is subdivided into six blocks, each one contained two plots: one was treated with fresh compost tea and the other was used as a control with heat sterilized compost tea to kill microorganisms. Our hypothesis is that fresh compost tea improves soybean growth and yield with the addition of beneficial microorganisms and the enrichment of bacterial communities in soils. High throughput sequencing of bacterial 16S ribosomal DNA extracted from different samples (compost tea, treated soil and control soil), combined with bioinformatics and statistical analyzes, demonstrated that processing of compost tea did not significantly influenced bacterial communities, neither by changes in alpha diversity nor in their community structure. In addition, the results of plant growth and yield analyzes had no significant effect of fresh compost tea on plant vegetative biomass or soybean weight. Our research results indicate that the fresh compost tea used in our experiment did not change the bacterial population in the treated soils and it did not show a significant effect on either plant growth or yield. Our hypothesis is not supported by these results which suggest that the relative benefits of the application of compost tea are not due to living microorganisms but rather to a potential supply of nutrients. The lack of positive effects in our study could be attributed specifically to our experimental design, the compost tea used, or the dose or frequency of its application. More experiments are needed in order to draw robust conclusions about the effect and performance of compost tea on conventional crops.