Um método baseado na extração de rRNA, seguido de RT-PCR rRNA 16S, clonagem e ARDRA foi otimizado e validado para a identificação das bactérias ativas em biofilmes. O método foi analisado primeiro com consórcios artificiais de três organismos. As etapas de clonagem e RT não causaram variações importantes na composição destes consórcios, do contrário da etapa de PCR, onde foi necessária a redução de 30 para 10 ciclos para limitar a distorção da proporção de templates. A análise de biofilmes reais indicou que clones dominantes podem ser identificados com o critério de ocorrência de >2% na biblioteca, mas que a reprodutibilidade de análises ainda é insatisfatória, possivelmente devido a fatores como a micro heterogeneidade espacial do biofilme, viés na reação de PCR e formação de mais de um clone de ARDRA por organismo. O armazenamento do biofilme a -20 °C por 2 meses não levou à alterações expressivas em sua composição. O perfil de clones detectado com o kit (Mo Bio) de extração de RNA foi muito diferente do perfil detectado com o método otimizado neste trabalho. / A method based on extraction of rRNA, followed by RT-PCR of 16S rRNA, cloning and ARDRA was optimized and validated for identification of bacteria active in biofilms. The method was first tested with artificial three-membered consortia. Cloning and RT did not lead to significant changes in the composition of the artificial consortia, but a reduction in cycle number in the PCR reaction from 30 to 10 was necessary for limiting the distortion in the proportion of amplicons relative to that of the templates. Analysis of real biofilms revealed that clones from active organisms occurred in frequencies >2% in the clone library, but reproducibility of analysis was unsatisfactory, probably due to factors such as the spatial heterogeneity of colonization of biofilms by microbes, PCR bias and more than one ARDRA clone per organism. Storage of biofilm samples at -20 °C for 2 months did not lead to important changes in composition. Very different clone profiles were obtained in the analysis of the same biofilm sample with the optimized method and with a kit (Mo Bio) for extraction of RNA.

No description available.

Myrmarker har en stor roll i regleringen av den globala kolbalansen och koncentrationerna av koldioxid och metan i atmosfären, vilket gör dem till speciellt viktiga ekosystem ur ett klimatförandringsperspektiv. Förändringar av myrmarker genom naturlig utveckling eller antropogen påverkan kan därför få långtgående störningar av myrars klimatreglerande funktion. Mikroorganismer har en avgörande roll i biogeokemiska processer genom att t ex bryta ned organisk material i mark och därmed styra kolets kretslopp. För att förstå hur myrsystemen reagerar på störningar är det därför väsentligt att veta hur mikroorganismsamhällena reagerar genom förändringar i sammansättning och biogeokemisk aktivitet. Målet för studierna, som ligger till grund för denna avhandling, var att undersöka hur mikroorganismsamhällen i myrar reagerar på uppvärmning genom klimatförändring och ökade kväve- (N) och svavel- (S) halter i nederbörd. High through-put sekvensering användes för att studera taxonomiska och funktionella egenskaper hos mikroorganismerna i myrar och quantative PCR användes för att mer specifikt studera de metanbildande arkeorna. Två fältkampanjer vardera omfattande tre ombrotrofa myrar med olika klimatförhållanden och olika mängder N och S inederbörden användes för att undersöka lokala och storskaliga effekter på myrars mikrobiella samhällen. Resultaten visade att latudinell variation i geoklimatologiska förhållanden (temperatur ochnederbördsmängd) och deposition av näringsämnen hade en påverkan på sammansättningen av de mikrobiella samhällena och aktiva metanbildare förr än variationen i den kemiska miljön inom varje specifik myr. Myrväxtsamhällenas sammansättning för en specifik myr visades sig i stor utsträckning styra sammansättningen av motsvarande mikrobiella samhälle i torvprofilen. Detta framgick klart av i en analys av samexisterande nätverk av mikroorganismsamhällen och motsvarande växtsamhällen i en studie av tre geografiskt skilda myrar med olika kvävedeposition. Effekterna av klimatförändring och nederbörd med olika mängder av N och S studerades mer specifikt genom att analysera de mikrobiellasamhällena i  ett långliggande (18 år) försök. Påverkan av var och en av dessa manipulationer antingen förstärktes eller minskades, när de förekom i kombinationer. Ökad kvävedeposition var den faktor som hade starkast effekt. De långvariga störningarna medförde stora förändringar i den mikrobiella taxonomin inom samhällena. Detta återspeglades dock inte i den fysiologiska kapaciteten, vilket visar att det finns en stark buffring i myrarnas mikrobiella funktion. Detta tyder på att framtida utveckling av myrar i relation till olika störningar sannolikt inte kommer att påverka myrarnas roll för kolbalans och växthusgasutbyte med atmosfären. / Peatlands play a substantial role in regulating the global carbon balance and concentrations of the greenhouse gases CO2 and CH4 in the atmosphere, and are thus of utmost importance from a climate change perspective. Any changes of peatland functions due to natural or anthropogenic perturbations may result in changes in these ecosystem services. Soil microbial communities are essential drivers of biogeochemical processes, including the carbon cycle. In order to fully understand the effect of environmental perturbations on peatland functions, it is essential to understand how microbial communities are affected. The aim of the research presented in this thesis was to investigate the responses of the peat microbial communities to climate change and increased precipitation of nitrogen(N) and sulfur (S) compounds. High-throughput sequencing approaches were used to investigate the taxonomic and functional composition of microbial communities, and quantitative PCR was used to specifically target the methanogen community. Two field studies including three ombrotrophic peatlands each that differed in climatological conditions and atmospheric N and S depositions, were used to investigate and compare the effect of large- and local-scale environmental conditions on microbial communities. The results show that the variation in geo-climatological (temperature and precipitation) and atmospheric deposition conditions along the latitudinal gradient modulate the peat microbial community composition and the abundance of active methanogens to a greater extent thansite-related microhabitats. Furthermore, a tight coupling between the plant community composition of a site and the composition of its microbial community was observed, and was found to be mainly driven by plants rather than microorganisms. These co-occurrence networks are strongly affected by seasonal climate variability and the interactions between species in colder areas are more sensitive to climate change. The long-term effects of warming and increased N and S depositions on the peat microbial communities were further investigated using an 18-year in-situ peatland experiment simulating these perturbations. The impacts of each of these perturbations on the microbial community were found to either multiply or counteract one another, with enhanced N deposition being the most important factor. While the long-term perturbations resulted in a substantial shift in the taxonomic composition of microbial communities, only minor changes occurred in genome-encoded functional traits, indicating a functional redundancy. This could act as a buffer maintaining ecosystem functioning when challenged by multiple stressors, and could limit future changes in greenhouse gases and carbonexchange.

Les communautés microbiennes du sol peuvent améliorer la croissance de la plante en augmentant la disponibilité en nutriments du sol, favorisant ainsi leur prélèvement par la plante. Dans le contexte d’une production agricole à bas niveau d’intrants, la nutrition de la plante est susceptible de reposer de plus en plus sur les interactions plante-communautés microbiennes de la rhizosphère, qui peuvent être modulées par le génotype de la plante. Pourtant, très peu d’études se sont intéressées aux modifications des communautés microbiennes de la rhizosphère dans leur globalité et ce en relation avec à la fois le génotype et le phénotype de la plante. Ces travaux de thèse ont été consacrés à étudier l’effet du génotype de la plante sur la structure génétique des communautés microbiennes de la rhizosphère en relation avec les stratégies nutritionnelles de la plante.L’interaction plante-communautés microbiennes de la rhizosphère a été évaluée par une approche multidisciplinaire alliant écophysiologie et écologie microbienne. L’effet du génotype de la plante sur la structure génétique des communautés microbiennes de la rhizosphère qui lui sont associées a été analysé par DNA fingerprint. Les différentes stratégies nutritionnelles de la plante ont été analysées par une approche de type structure/fonction prenant en compte la mise en place des structures (feuilles, racines) et leur fonctionnement (photosynthèse, rhizodéposition, prélèvement spécifique d’azote).Dans une première expérimentation réalisée sur sept génotypes de Medicago truncatula, nous avons montré qu’à un stade précoce du développement de la plante, le génotype de Medicago truncatula affectait la structure génétique des communautés bactériennes du sol. En revanche, à ce stade précoce, peu de différences de croissance ont été observées entre les différents génotypes étudiés. Ces derniers ont par contre présenté des stratégies nutritionnelles contrastées. Les descripteurs fonctionnels sont donc plus efficaces que les descripteurs structurels pour discriminer les génotypes de plantes à un stade précoce du développement de la plante. De plus, nous avons montré un lien entre les stratégies nutritionnelles de la plante et la sélection des communautés bactériennes associées. Cette étude nous a également permis de développer un cadre d’analyse écophysiologique appliqué à l’étude des interactions plante-communautés microbiennes de la rhizosphère.Outre l’effet majeur du génotype de la plante dans les interactions plante-communautés bactériennes de la rhizosphère, nous avons également montré qu’il y avait un effet important de l’environnement, comme la disponibilité en azote minéral du sol. En effet, la disponibilité en azote minéral du sol a affecté la structure génétique des communautés bactériennes rhizosphériques via un effet indirect de la plante dépendant du génotype considéré. Les effets des différents génotypes de Medicago truncatula et de leurs stratégies de réponses à des contraintes environnementales, comme la disponibilité de l’azote du sol, se sont révélées être des composantes majeures de la sélection des communautés microbiennes. [...] / The soil microbial communities can improve plant growth by increasing soil nutrient availability, thereby promoting their uptake by the plant. In an overall context of input reduction, the plant nutrition should be increasingly based on plant- rhizosphere microbial communities’ interactions. Yet, very few studies have examined the entire rhizosphere microbial communities in relationship with both plant genotype and phenotype. The aim of this thesis was to study the plant genotype effect on the rhizosphere microbial communities in relationship with the plant nutritional strategies.To do so, the plant-rhizosphere microbial communities’ interaction was assessed by a multidisciplinary approach combining ecophysiology and microbial ecology. The plant genotype effect on the genetic structure of the associated rhizosphere microbial communities was analyzed by DNA fingerprinting. The different plant nutritional strategies were analyzed by a structural/functional approach taking into account both structure establishment e.g. leaves and functions e.g. photosynthesis.In a first experiment carried out on seven genotypes of Medicago truncatula, we showed that the Medicago truncatula genotype affected the genetic structure of the rhizosphere bacterial communities very early relatively to the plant development stages. However, at this early stage, few growth differences could be observed among the different genotypes. Yet, those genotypes presented contrasted nutritional strategies. Therefore, the functional descriptors were more efficient than the structural ones to discriminate plant genotypes at an early developmental stage. In addition, we showed that a link existed between the plant nutritional strategies and the rhizosphere bacterial communities selection. Finally, this study enabled to develop a multidisciplinary framework applied to the study of the plant- rhizosphere microbial communities’ interactions.In addition to the plant genotype effect, we showed that there is an environmental effect e.g. soil mineral nitrogen availability on the rhizosphere bacterial communities. Indeed, the soil mineral nitrogen availability affected the genetic structure of the rhizosphere bacterial communities via an indirect effect of the plant depending on its genotype. The effects of the different Medicago truncatula genotypes and their response strategies to environmental constraints (soil mineral nitrogen availability), proved to be a major component of the selection of the rhizosphere microbial communities.In order to identify the genetic determinisms of the interaction between the plant and the rhizosphere microbial communities, a second experiment was conducted on a core collection of 184 genotypes of Medicago truncatula. Initial results enabled to identify and characterize four groups of genotypes with contrasted phenotypes for their growth and their specific nitrogen uptake. Thanks to high-throughput sequencing, we will analyze the rhizosphere microbial communities’ diversity associated with the different Medicago truncatula genotypes. These results should determine if the plant genotype influences the selection of beneficial rhizosphere microbial communities. Moreover, when the whole genome sequencing data would be available for the 184 genotypes of the Medicago truncatula core collection, a genome-wide association study will be proceed. The creation of plant ideotypes, which will promote beneficial interactions with rhizosphere microbial communities, will be possible. Plant growth and yield will be improved without the concomitant increase of agricultural inputs.

Untersuchungsgegenstand der vorliegenden Dissertation sind biomassebasierte Kohlen (Biokohlen, biochar), welche für eine langfristige Kohlenstoffspeicherung in Böden mit dem gleichzeitigen Ziel der zusätzlichen Bodenverbesserung hergestellt werden. Die Auswahl der Kohlen umfasste Kohlen aus Pyrolyse- und hydrothermale Carbonisierung (HTC). In dieser Arbeit werden einige zentrale Phänomene, die bei deren Ausbringung in einem bestehenden Bodenökosystem auftreten können, nähergehend untersucht. Einerseits beeinflusst das fremde Material den Stoffwechsel und die Abundanz und Vielfalt innerhalb der mirkobiellen Gemeinschaft im Boden; im Gegenzug spielen die Mikroorganismen eine aktive Rolle beim Abbau des neuen Substrats. Diese beiden Aspekte sind größer Bedeutung, um bewerten zu können, wie erfolgsversprechend der Einsatz einer bestimmten Kohle im Boden hinsichtlich der Langlebigkeit, der gewünschten Ertragseffekte sowie möglicher Nebenwirkungen ist. Daraus ergeben sich die beiden folgenden zwei Fragestellungen, auf die diese Arbeit fokusiert ist: • Welche Faktoren beeinflussen die Abbaubarkeit der Kohlen im Boden? • Welche Wirkungen haben die Kohlen auf die Bodenatmung, auf den Boden-C-Gehalt, auf die mikrobielle Abundanz und auf die Dynamik der mikrobiellen Gemeinschaft? Als mögliche Einflussgrößen für die Abbaubarkeit der Kohlen wurden die Art der Kohlenherstellung, eine mögliche Nachbehandlung, der Alterungsprozess sowie die Zugabe einer Nährstoff- und einer labilen Kohlenstoffquelle getestet. Für diese Studie wurden Pyrolyse- und HTC-Kohlen aus Mais-Silage in einen Sandboden ausgebracht. Grundlage aller Versuche war die Untersuchung der Respirationsdynamik in unterschiedlichen Boden-Kohle-Gemischen, die durch Infrarotspektrometrie ermittelt wurde. Sie diente als Indikator für die mikrobielle Aktivität und dem daraus resultierenden Abbau der Substrate. Ergänzend wurde am Anfang und am Ende jedes Versuchs der Boden-Kohlenstoffgehalt gemessen. Die Versuche erfolgten auf verschiedenen Skalen: • Kurzzeit-Laborinkubationen (10 Tage) unter konstanten klimatischen Bedingungen in einem automatisch gesteuerten Durchflusssystem, an das das Messgerät direkt angeschlossen wurde. • Parzellenversuch (2 Jahre) im Freiland im Nordwesten Brandenburgs, bei dem die Bestimmung der Bodenatmung mittels wiederholter Beprobung aus auf der Ackerfläche gestellten geschlossenen Hauben erfolgte. In einer Laborinkubation wurde zusätzlich eine qPCR (quantitative Echtzeit Polymerase Kettenreaktion) zur Bestimmung der Abundanz ausgewählter mikrobieller Gruppen eingesetzt. Im Feldversuch wurde außerdem die Abundanz der stabilen Kohlenstoff-Isotopen (12C und 13C) im Boden und im freigesetzten CO2 ermittelt, um den Abbau der Kohlen vom Abbau des bodenorganischen Kohlenstoffs, der durch die Kohlen beeinflusst sein kann (priming), zu unterscheiden. Die Ergebnisse bestätigen die erhöhte Stabilität beider Kohlen im Vergleich zum Ausgangsmaterial, vor allem für die Pyrolyse-Kohle, deren Abbau sowohl im Labor als auch im Freiland am langsamsten erfolgte. Bei beiden Kohlen sank die Abbaubarkeit mit ihrer Alterung. Anhand der Abbauraten im zweiten Jahr des Feldversuchs wurden für die Pyrolyse- und HTC-Kohle Halbwertszeiten von 81 bzw. 60 Jahren ermittelt. Im Gegensatz zur Pyrolyse-Kohle wies der Abbau der HTC-Kohle eine komplexere Dynamik auf, was im Lauf der 10-tägigen Inkubationsversuche mit einer Verschiebung der mikrobiellen Gemeinschaft einherging. Im ersten Jahr des Freilandversuchs kam es bei der HTC-Kohle zur Ausgasung flüchtiger und leicht abbaubarer Kohlenstoffverbindungen, wodurch die Stabilität im Folgejahr deutlich erhöht wurde. Eine Nachbehandlung der Kohlen durch anaerobe Fermentierung führte zu einer deutlichen Verminderung der kurzzeitigen Ausgasung bei HTC-Kohle, sowohl im Freiland als auch im Labor, jedoch zu einer langfristigen Reduktion der Stabilität beider Kohlen: die ermittelten Halbwertszeiten für die fermentierte Pyrolyse- und HTC-Kohle nach dem zweiten Jahr des Feldversuchs betrugen 14 bzw. 13 Jahren. Die Wirkung der unbehandelten Kohlen auf die Abundanz der untersuchten mikrobiellen Gruppen im C-armen Boden war stark reduziert im Vergleich zum Ausgangsmaterial, und unter C-reichen Bedingungen kam es zu einer Hemmung der Aktivitätssteigerung. Die Zugabe leicht verfügbaren Kohlenstoffs wie Glukose zum reinen Boden in einem Inkubationsversuch steigerte die Bodenatmung erheblich und erhöhte die Variationsbreite der mikrobiellen Gemeinschaft. In Gegenwart der Kohlen war dies allerdings weniger stark ausgeprägt. Bei Zugabe mineralischen Stickstoffs in Gegenwart von Kohlen wurde hingegen keine signifikante Veränderung der Bodenatmung nachgewiesen. Die Inkubationsversuche haben es ermöglicht, die Kurzzeitdynamik der Bodenatmung und die Anpassung der mikrobiellen Gemeinschaft nach Zugabe der Kohlen und zusätzlicher C- und N-Quellen nachzuweisen. Im Freilandversuch konnte die Abbaudynamik von Kohlenstoffverbindungen unter Praxisbedingungen untersucht werden und durch die Messung der stabilen Isotope differenzierte Aussagen über die langfristige Stabilität von zugesetzten Kohlen und der bodenorganischen Substanz getroffen werden. Eine langfristige Festlegung von Kohlenstoff ist im Boden in Form von Biokohlen ist möglich. Allerdings hängt die Dauer der Festlegung von einer Vielzahl von Faktoren wie der Art der Ausgangsstoffe, den Prozessbedingungen, den Interaktionen zwischen Kohlepartikeln und Bodenorganismen und nicht zuletzt der Versuchsdauer ab. Während Kurzzeitversuche eine gute Möglichkeit darstellen, um die Effekte veränderter Bedingungen im Boden aufzuzeigen, kann die Kohlestabilität im Boden und damit das C-Sequestrierungspotenzial am zuverlässigsten nur in Langzeitstudien im Freiland abgeschätzt werden. / The object of the present thesis is charred biomass (biochar) produced for double aim of carbon storage in soil and improvement of soil properties. The chosen chars included chars from pyrolysis and hydrothermal carbonisation (HTC). The present work investigates closely some basic phenomena which can occur upon application of chars into an existing soil ecosystem: on the one hand, the allochthonous material affects the metabolism and the relative abundance of different microbial groups; on the other hand the microorganisms play an active role in the degradation of the new substrate. These two aspects are crucial to evaluate the suitability of the application of a specific char in the soil, particularly as concerns its stability, the length of time the char remains in the soil, the expected effects on crop yields, as well as possible side effects on the soil ecosystem. Based on this, two research questions arise which have been investigated in this thesis: • What factors affect the degradability of chars in soil? • How do the chars influence soil respiration, soil carbon content, microbial abundance and the dynamics of the microbial community? The production process, a post-treatment, the ageing process as well as the addition of a source of nutrients and a source of labile carbon were assessed as possible factors in determining the degradability of chars. For the present study, pyrolysis char and HTC char from maize silage were applied to a sandy soil. The basis of all experiments was an investigation of the respiration dynamics in different soil/char mixtures, measured through an infrared spectrometer, which was used to track the microbial activity and the substrate degradation. As a complement, soil carbon was also measured at the beginning and at the end of each experiment. The investigations were performed at different scales: • Short-term laboratory incubations (10 days) under constant climatic conditions in an automatic multi-channel flowthrough system, with direct plug-in for the measurement instrument. • A plot-wise investigation (2 years) in an agricultural field in North-West Brandenburg, where the soil respiration was measured by a repeated sampling from static chambers placed hermetically on the field. For one incubation study, qPCR (qunatitative real time polymerase chain reaction) was additionally applied to determine the abundance of selected microbial groups. Moreover, for the field investigation the abundance of stable carbon isotopes (12C und 13C) in the soil and in the released CO2 was recorded, to differentiate between the degradation of the chars and the degradation of soil organic carbon, which might be affected by the presence of chars (priming). The results confirm the higher stability of both chars in comparison to the feedstock, in particular for pyrolysis char, whose decay was the slowest both in the laboratory and in the field. The degradability of both chars decreased with their ageing. Based on the decay rates in the second year of the field investigation, decay half-lives for pyrolysis char and HTC char amounted respectively to 81 years and 60 years. Other than pyrolysis char, the degradation of HTC char revealed a more complex dynamics, which was accompanied by a shift of the microbial community within the 10 days incubation. During the first year of the field experiment, an intensive release of volatile and labile compounds took place, which led to an increased stability during the following year. A post-treatment of the chars via anaerobic fermentation led to a reduction in the initial degasing of the HTC char, both in the laboratory and in the field, but also to a decrease in stability for both chars: the calculated half-lives for fermented pyrolysis char and fermented HTC char on the basis of the second year of the field investigation were respectively 14 years and 13 years. The effects of the untreated chars on the abundance of the selected microbial groups in the carbon-poor soil used was also strongly reduced in comparison to the feedstock, while in a situation of carbon abundance a inhibition of the activity increase took place. Addition of readily available carbon in the form of glucose increased soil respiration tremendously and magnified the variation amplitude of the microbial community, which was however much reduced in the presence of chars. Instead, after addition of mineral nitrogen in presence of chars, no significant variation in the soil respiration could be observed. The incubation experiments made it possible to report the short-term dynamics of the soil respiration and the adaptation of the microbial community after application of char and additional carbon and nitrogen sources. In the field experiment the decay dynamics of char compounds could be investigated in a situation of common agricultural practice and the measurement of stable isotopes has given differentiated outcomes about the long-term stability of the added chars and of the soil organic matter. Storage of carbon in the soil in the form of char for a long period is possible. How long carbon can actually be stored depends on a number of factors such as the feedstock, the carbonisation process parameters, the interactions between char particles and soil microorganisms and the duration of the investigation itself. Short-term experiments represent a good possibility to highlight the effects of modified soil conditions, while the stability of char in soil and thus the potential carbon sequestration can be estimate in the most reliable way only through long-term studies in field. / Oggetto della presente tesi sono i carboni prodotti da biomasse (biochar) e utilizzati per lo stoccaggio del carbonio nel suolo e allo stesso tempo come ammendanti per terreni agricoli. I carboni considerati sono derivati da pirolisi o carbonizzazione idrotermale (HTC). Nel presente lavoro vengono esaminati in dettaglio alcuni importanti fenomeni che si possono manifestare in seguito all’applicazione su un ecosistema preesistente nel terreno: da un lato il materiale estraneo influenza il metabolismo della materia organica, l’abbondanza e la varietà all’interno della comunità microbica nel suolo, dall’altro i microorganismi giocano un ruolo fondamentale nella degradazione del nuovo substrato. Questi due aspetti sono essenziali per valutare quanto sia opportuno l’utilizzo di un determinato carbone dal punto di vista della sua longevità, degli effetti attesi sulla resa agricola e di eventuali effetti collaterali sull’ecosistema. Da queste premesse sono emerse le seguenti domande, su cui è focalizzato il presente lavoro: • Quali fattori determinano la degradabilità dei carboni nel suolo? • Che effetti possono avere i carboni sulla respirazione del terreno, sul suo contenuto di carbonio, sull’abbondanza dei microorganismi e sulla dinamica della comunità microbica? Come possibili variabili indipendenti per la degradabilità dei carboni sono state considerate: il processo di produzione, un possibile posttrattamento, l’invecchiamento dei substrati, l’aggiunta di nutrienti e di carbonio biodisponibile. Per questo studio sono stati applicati carboni derivati da pirolisi e da HTC di insilato di mais in un terreno sabbioso. Il fondamento di tutti gli esperimenti riportati è lo studio della dinamica della respirazione microbica in diverse miscele terreno/carbone, misurata tramite spettroscopia a infrarossi, che vale come tracciante per l’attività microbica e per la degradazione del substrato. In aggiunta è stato periodicamente misurato il contenuto di carbonio nel terreno. Gli esperimenti sono stati condotti su due diverse scale: • Incubazioni in laboratorio (10 giorni) in condizioni climatiche controllate, all’interno di un apparato per la ventilazione a flusso continuo, con presa diretta per lo strumento di misura. • Esperimento in campo parcellizzato (2 anni) nel Brandeburgo nordoccidentale, dove la misura della respirazione è avvenuta per campionamento ripetuto da camere opache poggianti ermeticamente sul suolo. Per una delle incubazioni è stata anche eseguita una qPCR (reazione a catena della polimerasi quantitativa in tempo reale) per quantificare l’abbondanza di determinati gruppi tassonomici di microorganismi. Nel campo è stata inoltre misurata l’abbondanza degli isotopi stabili del carbonio (12C e 13C), sia nel terreno sia nella CO2 liberata, per differenziare la degradazione dei carboni da quella del carbonio organico nel suolo, che in principio può essere influenzata dalla presenza dei carboni (priming). I risultati confermano l’aumentata stabilità di entrambi i carboni in confronto al materiale di partenza, in particolare del carbone pirolitico che si è degradato piú lentamente, sia in laboratorio sia in campo. La degradabilità di entrambi i carboni si è in ogni caso ridotta con l’invecchiamento. Basandosi sulle emissioni del secondo anno della sperimentazione in campo, sono stati calcolati dei tempi di dimezzamento di 81 anni e 60 anni, rispettivamente per il carbone pirolitico e per il carbone da HTC. La degradazione del carbone da HTC ha rivelato una dinamica piú complessa, che testimonia un adattamento della comunità microbica nell’arco dei 10 giorni di incubazione. Nel primo anno in campo è stata rilevata un’elevata emissione di composti volatili e labili, che ha portato a un incremento della stabilità nell’anno seguente. Il posttrattamento dei carboni tramite fermentazione anaerobica ha comportato una notevole riduzione dell’iniziale mineralizzazione del carbone da HTC, ma una diminuzione della stabilità di entrambi i carboni sul lungo periodo: i tempi di dimezzamento calcolati per il carbone pirolitico fermentato e per il carbone da HTC fermentato nel secondo anno dell’esperimento sul campo valgono rispettivamente 14 anni e 13 anni. Nel terreno usato, povero di carbonio, gli effetti dei carboni sull’abbondanza dei gruppi microbici selezionati è stata nettamente ridotta rispetto al materiale non carbonizzato, mentre la reazione all’aggiunta di carbonio labile è stata tendenzialmente inibitoria. Infatti, se l’aggiunta di glucosio ha incrementato considerevolmente la respirazione e l’ampiezza delle variazioni nella comunità microbica, in presenza dei carboni le variazioni sono state fortemente ridotte. L’aggiunta di azoto inorganico non ha invece portato a variazioni apprezzabili nella respirazione. Gli esperimenti basati su incubazioni hanno consentito di determinare la dinamica a breve termine della respirazione e l’adattamento della comunità microbica in seguito ad aggiunta dei carboni e di altre sorgenti di carbonio e azoto. Nell’esperimento su campo si è potuta osservare la dinamica di degradazione dei composti carboniosi in condizioni di prassi agricola e grazie alla misura degli isotopi stabili si sono potuti ottenere risultati differenziati sulla stabilità a lungo termine dei carboni e della sostanza organica del suolo. È quindi possibile immagazzinare il carbonio in modo duraturo nel suolo sotto forma di carbone. La durata stimabile di questo immagazzinamento dipende però da molteplici fattori tra cui la materia prima, il processo di carbonizzazione, le interazioni tra particelle carboniose e microorganismi del suolo e non da ultimo la durata della sperimentazione. Gli esperimenti a breve termine sono un mezzo efficace per rilevare le conseguenze immediate di modifiche del terreno; la stabilità dei carboni e quindi il loro potenziale per il sequestro del carbonio può essere determinata nel modo piú affidabile solo in studi a lungo termine sul campo.

Les communautés microbiennes procèdent au recyclage des nutriments et à la degradation de la matière organique, et sont ainsi essentielles aux cycles biogéochimiques dans le sédiment et plus largement dans les océans. La contamination chronique aux hydrocarbures représente près de 80% des déversements totaux dans les océans. Toutefois, en comparaison des marées noires, son impact sur les communautés microbiennes est encore mal compris. Dans cette étude, nous avons d’abord utilisé une approche de type méta-analyse pour élucider l’effet global de la contamination aux hydrocarbures dans différents habitats. La réponse des communautés bactériennes à la contamination s’est révélée être dépendante du type d’habitat, les sols étant plus impactés que d’autres habitats, comme par exemple les sédiments marins. Nous nous sommes ensuite intéressés aux communautés microbiennes des trois domaines du vivant de sédiments côtiers provenant des côtes méditerranéennes et atlantiques. La contamination chronique n’influençait que marginallement les communautés benthiques, et la diversité alpha n’était pas réduite dans les sédiments contaminés. Cedendant, la comparaison des réseaux de co-occurrence des échantillons contaminés et non-contaminés a montré que le réseau des communautés contaminées présentait une topologie différente, indiquant une vulnérabilité plus importante à d’éventuelles perturbations environnementales. Des indicateurs potentiels de la contamination identifiés avec la méta-analyse ont été ciblés pour étudier l’impact de la contamination chronique aux hydrocarbures sur les services écologiques qu’ils assurent (i.e. la dégradation de la matière organique et des hydrocarbures) en utillisant la technique de Micro-FISH. / Within the sediment, microbial communities play a pivotal role by driving essential processes such as nutrient cycling and organic matter degradation. Chronic hydrocarbons contamination represents almost 80% of the total input in the oceans. However, as compared to oil spills, its impact on microbial communities remains poorly understood. In this study, we first used a meta-analysis approach to decipher the global effect of hydrocarbons contamination in different habitats. Bacterial community response to the contamination was found to be dependant of the habitat studied, with soils being more impacted than other habitats, like marine sediments. Because bacteria are in interactions with other important members of microbial communities such as Archaea and Eukaryotes, we focused on microbial communities from the three domains of life in coastal marine sediments from the Mediterrranean and the French Atlantic coasts. Independently of the domains of life, chronic hydrocarbons contamination appeared to be a poor driver of communities structuration, and alpha diversity was not reduced in contaminated sediments. However, the comparison of co-occurences networks of contaminated and non-contaminated samples showed that the network from the contaminated samples exhibited a different topology, which suggests a higher vulnerability to eventual environmental perturbations. Potential indicators species identified using the meta-analysis approach were targeted to study the impact of chronic contamination on the ecological services they provide (i.e. organic matter and hydrocarbons degradation) using the Micro-FISH method.

Au niveau des fonds océaniques, souvent considérés comme des déserts aux conditions de vie extrêmes, des oasis luxuriants de vie existent pourtant. Les sources hydrothermales et les suintements froids, principalement localisés au niveau des dorsales océaniques et des marges continentales, vont permettre le développement de communautés microbiennes et animales très particulières. Le Bassin de Guaymas, situé dans le Golfe de Californie (Mexique) présente la particularité de regrouper à la fois une zone de sources hydrothermales et une zone de suintements froids, situées à une soixantaine de kilomètres l’une de l’autre, et toutes deux recouvertes par une épaisse couche sédimentaire. Ces deux zones sont également colonisées par des étendues de bivalves, des buissons de vers tubicoles ainsi que de tapis microbiens blancs ou colorés comparables. Afin de mieux comprendre le fonctionnement global de ces deux types d’écosystèmes et notamment le rôle structurant des communautés microbiennes sédimentaires sur la répartition des différents assemblages de surface, les travaux entrepris dans cette thèse se proposaient d’étudier les communautés microbiennes sédimentaires associés à la zone de suintements froids (Marge de Sonora) et la zone de sources hydrothermales (Southern Trough) du Bassin de Guaymas. Pour cela, la diversité des communautés microbiennes (Bacteria et Archaea) de différents habitats caractérisés par une faune et des profils géochimiques particuliers, a été étudiée grâce à l’utilisation d’une technique de séquençage haut-débit, le pyroséquençage par la technique du 454, combinée à d’autres techniques comme le FISH ou la PCR quantitative. Cette étude a permis de déterminer que la structure et la diversité des communautés microbiennes dans ces sédiments étaient très spécifiques de ces environnements. Par ailleurs, les colonisateurs présents en surface des sédiments reflétaient des profils géochimiques et des communautés microbiennes très différentes au sein des sédiments. Dans les sédiments colonisés par les tapis microbiens, riches en méthane, les communautés microbiennes dominantes (ANME, Deltaproteobacteria…) utiliseraient notamment les émissions de méthane des couches sédimentaires profondes et produiraient d'importantes concentrations de sulfures, nécessaires à l’installation des communautés microbiennes thiotrophes de surface formant les tapis. Ces fortes concentrations en sulfure excluraient en revanche les communautés animales de ce périmètre. A contrario, dans les sédiments présentant de faibles concentrations en méthane et en sulfure, permettant l'installation d’assemblages faunistiques variés, les communautés microbiennes méthanotrophes anaérobies et productrices de sulfure étaient minoritaires. L'activité et le métabolisme de ces colonisateurs de surface pourraient par ailleurs permettre le développement des lignées microbiennes détectées dans ces habitats (MBG-D, Chloroflexi…). L'analyse des larges jeux de données obtenus au cours de cette étude nous a donc permis de mettre en évidence un système dynamique complexe fonctionnant en équilibre entre les communautés microbiennes sédimentaires, les organismes colonisant la surface du sédiment et la composition géochimique des eaux interstitielles. / Whereas the deep-sea environment is often considered to be a desert, hydrothermal vents andcold seeps provide “oases” of biological activity on the ocean floor. Vent and seep ecosystems support complex food webs based on microbial chemoautotrophic primary production. These hydrothermal vent and cold seeps ecosystems both release hydrocarbon- and sulfide-rich fluids,fueling various surface assemblages such as mat-forming giant bacteria or symbiont-bearinginvertebrates (e.g. bivalves, tubeworms). In the Guaymas Basin, the nearby presence at a few tens of kilometers of cold seeps and hydrothermal vents coupled with comparable sedimentary settings and depths offer a unique opportunity to assess and compare the microbial community composition of these ecosystems. Tobetter understand their overall functioning, we studied sedimentary microbial communities associated with cold seep and hydrothermal vent areas in the Guaymas Basin. The diversity of microbial communities inhabiting sediments was studied using high throughput sequencing (454pyrosequencing), combined with complementary approaches, such as FISH and quantitative PCR. This study reveals that sediments found in the Guaymas Basin were colonized by microbial communities typically found in these types of ecosystems. Our results revealed a high similarity between microbial communities composition associated with the cold seep and hydrothermal vent areas as a probable consequence of the sedimentary context. Nonetheless, thermophilic and hyperthermophilic lineages (e.g.: Thermodesulfobacteria, Desulfurococcales, etc) were exclusively identified in hydrothermally influenced sediments highlighting the strong influence of temperature gradients and other hydrothermally-related factors on microbial community composition. Furthermore, sediments populated by different surface assemblages show distinct porewater geochemistry features and are associated with distinct microbial communities. Indeed, in the sediments underlying microbial mats characterized by high methane porewater concentrations,microbial communities were dominated by anaerobic methane oxidizers (ANME), known to produce sulfide which provides high fluxes of sulfide to the seafloor. In contrast, sediment associated microbial communities underlying faunal assemblages were characterized by a lower biomass and lower methane porewater concentrations in sediments, limiting porewater sulfide concentrations. Without elevated and toxic sulfide concentrations, faunal assemblages can colonize the surface. Together, geochemical and microbial surveys indicate that porewater methane concentrations play an important role in the microbial community structure and subsequently in the establishment of the surface colonizers. Furthermore, presence and activity of the surface colonizers influence the underlying microbial communities probably because of modification of energy source availabilities. Finally, the existence of similar microbial populations between the two ecosystems also raises the question of their dispersal mechanisms. Our results support the hypothesis of a potential continuity among deep-sea ecosystems. In absence of physical borders, environmental conditions (temperature, specific compounds associated withhydrothermal fluids) might select specific and highly adapted microorganisms from the pool of microorganisms dispersed globally across the seafloor.

Le sol est une des composantes essentielles au soutien du fonctionnement de l’écosystème. Il est lesupport de la production primaire, un habitat pour la diversité biologique et remplit de nombreux processus écologiques. Les activités anthropiques, et parmi elles, les pratiques agricoles intensives, ont conduit à des déséquilibres au sein du sol causant un fort déclin de leur biodiversité. En réponse à ces effets, de nouvelles pratiques agricoles ont été développées pour préserver les sols et les services écosystémiques qu’ils fournissent. Ces pratiques dites innovantes s’appuient principalement sur la réduction du travail du sol, une gestion intégrée des résidus de culture, l’installation d’un couvert végétal permanent ou encore l’introduction de légumineuses dans les rotations de culture. C’est dans ce contexte de mutation des pratiques agricoles que s’inscrit ce sujet de thèse qui vise à mieux comprendre les effets de l’adoption de ces gestions innovantes sur (i) la diversité des communautés de la macro- et de la mésofaune du sol et (ii) sur le fonctionnement du sol en termes de stockage ducarbone organique (source de nourriture de base), de structure du sol et de distribution de la taille des pores (milieu de vie). Au cours des différents chapitres de ce travail, nous avons mis en évidence trois actions liées à des pratiques agricoles innovantes combinant des approches expérimentales in et ex situ. L’objectif de la première action visait à étudier l’effet combiné de l’introduction de légumineuse (féverole) et de la réduction du travail du sol sur le compartiment endogé. Pour y répondre, nous avons développé une conception expérimentale empirique faite avec la combinaison des situations rencontrées dans les fermes. Nos résultats ont montré que l’interaction entre les deux pratiques innovantes n’a pas entrainé d'augmentation significative de la diversité de la faune du sol. Les fonctions microbiennes ainsi que la dynamique du carbone (C) et de l’azote (N) ne semblent pas non plus être affectées par ces pratiques. Il s’avère que les effets bénéfiques de ces pratiques innovantes ne peuvent être envisagés sans prendre en compte la manière dont elles sont mises en oeuvre à l’échelle des rotations culturales et des itinéraires techniques. Cela nous a permis de valoriser une seconde action dont l’objectif était de suivre dans les mêmes conditions pédoclimatiques et mêmes itinéraires techniques, l'effet à long terme (quatre ans) de cinq régimes de gestion différents sur les communautés de faune du sol. Les traitements comprenaient des combinaisons de différentes cultures (annuelles, vivaces), de différents taux de fertilisation azotée (ajout d'azote, azote réduit),incorporation ou élimination de résidus de culture et différentes intensités de labour (labour vs. travail superficiel). Les résultats ont montré que la réduction de l’intensité du travail du sol a favorisé la diversité des collemboles et ses groupes fonctionnels grâce à l’amélioration des conditions de l’habitat à travers l’augmentation de la biomasse microbienne de carbone (source trophique). Par ailleurs, les autres composantes de la gestion, soit la « réduction des apports d’azote » et la « restitution / exportation des résidus de culture», n’ont pas eu d’effet sur les collemboles (abondance et richesse), notamment sur ceux vivants dans le sol, qu'ils soient hémi ou euédaphiques. Toutefois, cette étude démontre que la dissemblance dans la composition des assemblages de collemboles augmente avec la différenciation temporelle des pratiques agricoles et le travail du sol est le premier facteur responsable de cette trajectoire. Cette assertion a donné lieu à une troisième action dont l’objectif était de suivre sur une saison de végétation la conversion de la perturbation mécanique résultant du travail du sol sur la résistance et résilience fonctionnelle des assemblages biotiques édaphiques. / Soil is one of the essential components supporting the functioning of the ecosystem. It is the support of primary production; an habitat for biodiversity, and it fulfills many ecological processes. Human activities, including intensive agricultural practices, have led to impaired the soil causing a sharp decline in their biodiversity. In response to these effects, new agricultural practices have been developed to preserve the soils and the ecosystem services they provide. These so-called innovative practices rely mainly on the reduction of tillage, integrated management of crop residues, the installation of permanent plant cover or the introduction of legumes into crop rotations. It is in this context of changes in agricultural practices that this thesis topic lays. This thesis aims to better understand the effects of the adoption of these innovative management practices on (i) the diversity of the macro and mesofauna communities soil and (ii) soil functioning in terms of organic carbon storage (basic food source), soil structure and pore size distribution (living environment). During the different chapters of this work, we highlighted three actions related to innovative agricultural practices combining in situ and ex situ experimental approaches. The aim of the first action was to study the combined effect of the introduction of legume (faba bean) and the reduction of tillage on the endogenous compartment. To answer this question, we developed an empirical experimental design made with the combination of situations encountered on farms. Our results showed that theinteraction between the two innovative practices did not result in a significant increase in the diversity of soil fauna. Microbial functions and the dynamics of carbon (C) and nitrogen (N) also do not seem to be affected by these practices. It turns out that the beneficial effects of these innovative practices cannot be considered without taking into account how they are implemented at the scale of crop rotations and technical itineraries. This allowed us to promote a second action whose objective was to follow, under the same pedoclimatic conditions and the same technical itineraries, the long-term effect (four years) of five different management regimes on the communities of soil fauna. Treatments included combinations of different crops, different rates of nitrogen fertilization, incorporation or removal of crop residues and different plowing intensities. The results showed that the reduction of tillage intensity favored the diversity of Collembola and its functional groups by improving habitatconditions through the increase of microbial carbon biomass (trophic source). In addition, the other components of management, namely the "reduction of nitrogen inputs" and the "restitution / export of crop residues", had no effect on springtails (abundance and wealth), in particular on those living in the soil, whether hemi or euedaphic. However, this study demonstrates that the dissimilarity in the composition of Collembola assemblages increases with the temporal differentiation of agricultural practices, and tillage was the main factor responsible for this trajectory.

Les écosystèmes marins côtiers sont constament soumis à des pollutions, notamment aux hydrocarbures, du fait de leur localisation et de leurs caractéristiques environnementales. Le rôle clé des microorganismes dans la dégradation de ces polluants est aujourd’hui très bien décrit. Toutefois, les conditions d’oxygénation fluctuantes dans ces environnements côtiers, dues aux marées et aux activités de bioturbation de la macrofaune, influencent les communautés microbiennes.Ainsi, ce travail de thèse a eu pour objectif de caractériser, l’assemblage de communautés microbiennes hydrocarbonoclastes de sédiments marins côtiers soumises à des oscillations oxie/anoxie en présence de pétrole lors d’une expérience en bioréacteurs. L’adaptation des bactéries marines hydrocarbonoclastes notamment des genres Alcanivorax et Cycloclasticus vis-à-vis de ces variations d’oxygène a pu être investiguée par oligotypage. Des écotypes ont été identifiés en fonction des conditions d’oxygénation démontrant ainsi les capacités d’adaptation aux conditions oscillantes d’oxygène de ces deux genres. La structure des communautés archéennes (séquençage des transcrits du gène de l’ARNr 16S) n’a pas montré de modification évidente liée aux conditions d’oxygénation démontrant ainsi des capacités d’adaptation et/ou de résistance plus importantes chez ces microorganismes comparées aux communautés bactériennes. Enfin, les analyses métagénomiques ont mis en évidence une réponse fonctionnelle spécifique aux oscillations oxie/anoxie. Ainsi, ces travaux de thèse apportent de nouvelles connaissances sur l’influence des variations d’oxygénation sur les communautés microbiennes et par conséquent sur la dégradation des hydrocarbures au sein des écosystèmes marins côtiers. / Coastal marine ecosystems are constantly subject to pollution, particularly hydrocarbons, because of their location and their environmental characteristics. The key role of microorganisms in the degradation of these pollutants is now well described. However, fluctuating oxygenation conditions in these coastal environments, due to tides and macrofauna bioturbation activities influence microbial communities.Thus, this thesis work aimed to characterize the assembly of microbial hydrocarbonoclastic communities of coastal marine sediments subjected to oxic/anoxic oscillations in the presence of oil during a bioreactor experiment. The adaptation of MOHCB, particularly of Alcanivorax and Cycloclasticus genera, to these oxygen variations has been investigated by oligotyping. Ecotypes were identified according to the oxygenation conditions demonstrating adaptation capacities of these two genera to the oscillating oxygen conditions. The structure of archaeal communities (16S rRNA transcript sequencing) did not show any modification related to the oxygenation conditions thus demonstrating greater adaptation and/or resistance capacities in these microorganisms compared to the bacterial communities. Finally, metagenomics analyses revealed a specific functional response to oxic/anoxic oscillations. Thus, this thesis provides new insights into the influence of oxygenation variations on microbial communities and consequently on the degradation of hydrocarbons in coastal marine ecosystems.

Abstract Community assembly results from a combination of deterministic and stochastic mechanisms, whose relative effects can vary in response to environmental heterogeneity, connectivity, disturbance regimes and anthropogenic stressors. Understanding how community assembly mechanisms vary in response to environmental changes and connectivity is crucial for the management and conservation of river ecosystems. In this thesis, I tested the effects of connectivity and natural flow disturbances on riverine invertebrate communities by assessing assembly mechanism changes in response to (I) habitat connectivity, (II) seasonal flow intermittency and (III) inter–annual hydrological variability. I also conducted a field experiment to test for (IV) the effects of human–induced nutrient enrichment on community assembly of microorganisms (diatoms and aquatic fungi) and stream ecosystem functioning under different environmental settings. Invertebrate community assembly changed gradually with habitat connectivity. While limited dispersal resulted in higher community variability in the most isolated streams, mass effects caused community homogenization in the most connected ones. Natural and human induced disturbances lead to changes in the relative importance of deterministic and stochastic factors but often through different, or even opposite, mechanisms depending on the natural background of the ecosystem and organism type considered. For instance, seasonal drying and high–flow periods in intermittent Mediterranean rivers promoted deterministic and stochastic assembly processes respectively, whereas environmental sorting and stochastic processes respectively dominated during high and low flow years in boreal streams. Diatom and fungal communities responded differently to nutrient enrichment, with detrital processes and fungal communities responding more in naturally acidic than in circumneutral streams. The results of this thesis highlight the complexity of community assembly mechanisms: they tend to be highly context dependent and temporally variable. Therefore, stream bioassessment and conservation will benefit from explicitly incorporating connectivity and natural disturbance regimes. Assessing the interactive effects of connectivity and disturbances at the river network scale would provide a greater understanding of community assembly mechanisms and river ecosystem functioning. / Tiivistelmä Eliöyhteisöjen koostumus heijastelee determinististen ja stokastisten mekanismien vuorovaikutusta. Niiden suhteellinen merkitys vaihtelee suhteessa yhteisöjen kytkeytyneisyyteen sekä luontaisiin ja ihmisen aiheuttamiin häiriöihin. Yhteisöjen säätelymekanismit vaihtelevat jokiverkoston eri osissa ja tietoa tästä vaihtelusta tarvitaan jokiekosysteemien hoidon kehittämiseksi. Tässä tutkielmassa testasin elinympäristöjen kytkeytyneisyyden ja luontaisten häiriöiden (virtaamavaihtelut) vaikutuksia jokien selkärangatonyhteisöihin. Suoritin myös kenttäkokeen, jossa testattiin ihmisen aiheuttaman rehevöitymisen vaikutuksia mikro–organismeihin (piilevät, mikrobit) ja ekosysteemitoimintoihin erilaisissa ympäristöoloissa (luontaisesti happamat vs. neutraalit purot). Selkärangattomien yhteisökoostumus muuttui asteittain jokiverkostossa. Yhteisökoostumuksen vaihtelu oli suurinta eristäytyneimmissä latvapuroissa, kun taas isommissa, uomaston keskivaiheilla sijaitsevissa koskissa voimakas levittäytyminen eri suunnista (ns. massatekijät) aiheutti yhteisöjen rakenteen homogenisoitumista. Kuivuusjaksot ja niitä seuraavat korkean virtaaman jaksot edistivät determinististen prosessien merkitystä Välimeren alueen joissa, kun taas boreaalisissa puroissa Pohjois–Suomessa äärevät virtaamaolot, erityisesti poikkeuksellisen kuivat kesät, edistivät satunnaismekanismien vaikutusta. Perustuottajat (piilevät) ja hajottajat (akvaattiset sienet) vastasivat eri tavoin ravinnelisäykseen. Sienten hajotustoiminta nopeutui ravinnelisäyksen myötä, mutta vain luontaisesti happamissa puroissa. Tämän opinnäytetyön tulokset korostavat yhteisön kokoonpanomekanismien monimutkaisuutta: ne ovat usein erittäin tilanneriippuvaisia ja ajallisesti vaihtelevia. Siksi jokien ekologisen tilan arvioinnissa tulisi huomioida tutkimuspaikkojen kytkeytyneisyys jokimaisemassa.