Co-Existence and niche differentiation of sulfur oxidizing bacteria in marine environments

Reduced sulfur compounds and sulfur-oxidizing prokaryotes (SOP) are widely distributed in the marine environment. Diverse microbial lineages thrive on the oxidation of reduced sulfur. They co-exist successfully by the adaptive radiation into different physiological and ecological niches. However, the factors determining this differentiation and SOP distribution are largely unknown. Environmental factors, like pH, temperature and salinity, as well as the physiological capabilities of different SOPs for sulfur-oxidation and carbon assimilation likely govern the niche-differentiation. Therefore, as part of multiple collaborative studies, I studied the influence of substrate quality and availability on structuring sulfur-oxidizing microbial communities in different marine habitats. First, the role of elemental sulfur (S0), in particular cyclooctasulfur (S8), as substrate for SOPs in marine benthic habitats was examined (Chapter II). We observed a specific association between Sulfurimonas/Sulfurovum-related Epsilonproteobacteria and S0/S8 regardless of the habitat. We propose that substrate quality effects SOP diversity and niche differentiation, and the capability to oxidize S8 probably provides a competitive advantage to the Sulfurimonas/Sulfurovum-group. Moreover, we investigated the diversity and distribution SOPs along gradients of a sulfide, oxygen and light in a highly sulfidic marine karst lake (Lake Rogoznica, Chapter III). The comprehensive analysis of microbial diversity revealed a community shift from phototrophic to chemotrophic sulfur oxidation during holomixis and tight coupling between sulfide and oxygen concentration and the sulfur-oxidizing microbial community in Lake Rogoznica. In two further studies, we explored different aspects of carbon assimilation in hydrothermally influenced habitats dominated by thiotrophic Sulfurimonas/Sulfurovum-related Epsilonproteobacteria. We demonstrated the effects of temperature and/or substrate flux on carbon-isotope fractionation during CO2 assimilation in environmental samples (Chapter IV). Furthermore, we showed that these and other hydrothermal vent associated thiotrophs do not incorporate acetate (Chapter V), despite their heterotrophic potential. Other microorganisms, not involved in oxidative sulfur cycling at hydrothermal vents, showed high activity and growth after the input of organic substrate. In summary, this thesis contributes to the general understanding of microbial ecology in sulfur-rich environments by provides novel insights into diversity and niche in sulfur-oxidizing microbial communities

Agricultural impacts on plant beneficial pseudomonads

The soil microbiome is a dynamic and complex environment that offers numerous ecosystem services. Beneficial Pseudomonas spp. are agriculturally relevant bacteria with a plethora of plant growth promoting (PGP) traits, making them desirable targets for microbial inoculant development. Microbial inoculants have typically failed to produce reliable results, which can be attributed to the introduction of microbes into ecologically unsuitable environments. Its therefore important to better understand factors that can alter Pseudomonas spp. community structure and functioning. Crop domestication and land management have both played important roles in the development of agriculture over the last 10,000 years, however they have been associated with negative impacts on the soil microbiome. Here, the impacts of these agricultural components on soil pseudomonads was investigated. The study of 17 domesticated and ancestral wheat genotypes cultivated in a grassland soil revealed no clear difference in pseudomonad community structure within rhizosphere or bulk soil. The Highfield experiment at Rothamsted Research tests the impact of land management and revealed various impacts to soil properties, wheat physiology and total microbial abundance across grassland, arable and bare fallow managed soils. However, pseudomonad abundance was not found to significantly differ in bulk soil and rhizosphere communities. Additional studies looking at the more closely associated root compartment of wheat grown in soils from distinct land uses, revealed differences in abundance and phylogeny of cultivated pseudomonads. A range of PGP genetic and functional potentials including siderophore production, anti-fungal activity and phosphate solubilisation differed in isolates according to land use. The presence of the 1-Aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS) was of particular interest, due to its potential to reduce levels of stress ethylene in plants by degrading its precursor ACC. Intriguingly, acdS gene abundance, phylogeny and functional activity appeared to differ in pseudomonads associated with the different land uses. The rhizosphere and root compartments of wheat had a higher acdS gene abundance, particularly in the bare fallow soil which is known to have degraded soil properties. This suggests factors associated with wheat grown in different land managements were driving the selection of ACC deaminase producing pseudomonads. In vitro attempts to promote wheat growth under salt stress by applying ACC deaminase-containing isolates was not successful. Overall this thesis evidences the functional potential of pseudomonads for use in microbial inoculants, whilst providing an insight into the complexity of soil-plant-microbe interactions

Microbial community functioning at hypoxic sediments revealed by targeted metagenomics and RNA stable isotope probing

Microorganisms are instrumental to the structure and functioning of marine ecosystems and to the chemistry of the ocean due to their essential part in the cycling of the elements and in the recycling of the organic matter. Two of the most critical ocean biogeochemical cycles are those of nitrogen and sulfur, since they can influence the synthesis of nucleic acids and proteins, primary productivity and microbial community structure. Oxygen concentration in marine environments is one of the environmental variables that have been largely affected by anthropogenic activities; its decline induces hypoxic events which affect benthic organisms and fisheries. Hypoxia has been traditionally defined based on the level of oxygen below which most animal life cannot be sustained. Hypoxic conditions impact microbial composition and activity since anaerobic reactions and pathways are favoured, at the expense of the aerobic ones. Naturally occurring hypoxia can be found in areas where water circulation is restricted, such as coastal lagoons, and in areas where oxygen-depleted water is driven into the continental shelf, i.e. coastal upwelling regions. Coastal lagoons are highly dynamic aquatic systems, particularly vulnerable to human activities and susceptible to changes induced by natural events. For the purpose of this PhD project, the lagoonal complex of Amvrakikos Gulf, one of the largest semi-enclosed gulfs in the Mediterranean Sea, was chosen as a study site. Coastal upwelling regions are another type of environment limited in oxygen, where also formation of oxygen minimum zones (OMZs) has been reported. Sediment in upwelling regions is rich in organic matter and bottom water is often depleted of oxygen because of intense heterotrophic respiration. For the purpose of this PhD project, the chosen coastal upwelling system was the Benguela system off Namibia, situated along the coast of south western Africa. The aim of this PhD project was to study the microbial community assemblages of hypoxic ecosystems and to identify a potential link between their identity and function, with a particular emphasis on the microorganisms involved in the nitrogen and sulfur cycles. The methodology that was applied included targeted metagenomics and RNA stable isotope probing (SIP). It has been shown that the microbial community diversity pattern can be differentiated based on habitat type, i.e. between riverine, lagoonal and marine environments. Moreover, the studied habitats were functionally distinctive. Apart from salinity, which was the abiotic variable best correlated with the microbial community pattern, oxygen concentration was highly correlated with the predicted metabolic pattern of the microbial communities. In addition, when the total number of Operational Taxonomic Units (OTUs) was taken into consideration, a negative linear relationship with salinity was identified (see Chapter 2). Microbial community diversity patterns can also be differentiated based on the lagoon under study since each lagoon hosts a different sulfate-reducing microbial (SRM) community, again highly correlated with salinity. Moreover, the majority of environmental terms that characterized the SRM communities were classified to the marine biome, but terms belonging to the freshwater or brackish biomes were also found in stations were a freshwater effect was more evident (see Chapter 3). Taxonomic groups that were expected to be thriving in the sediments of the Benguela coastal upwelling system were absent or present but in very low abundances. Epsilonproteobacteria dominated the anaerobic assimilation of acetate as confirmed by their isotopic enrichment in the SIP experiments. Enhancement of known sulfate-reducers was not achieved under sulfate addition, possibly due to competition for electron donors among nitrate-reducers and sulfate-reducers, to the inability of certain sulfate-reducing bacteria to use acetate as electron donor or to the short duration of the incubations (see Chapter 4). Future research should focus more on the community functioning of such habitats; an increased understanding of the biogeochemical cycles that characterize these hypoxic ecosystems will perhaps allow for predictions regarding the intensity and direction of the cycling of elements, especially of nitrogen and sulfur given their biological importance. Regulation of hypoxic episodes will aid the end-users of these ecosystems to possibly achieve higher productivity, in terms of fish catches, which otherwise is largely compromised by the elevated hydrogen sulfide concentrations

Microbial community functioning at hypoxic sediments revealed by targeted metagenomics and RNA stable isotope probing

Microorganisms are instrumental to the structure and functioning of marine ecosystems and to the chemistry of the ocean due to their essential part in the cycling of the elements and in the recycling of the organic matter. Two of the most critical ocean biogeochemical cycles are those of nitrogen and sulfur, since they can influence the synthesis of nucleic acids and proteins, primary productivity and microbial community structure. Oxygen concentration in marine environments is one of the environmental variables that have been largely affected by anthropogenic activities; its decline induces hypoxic events which affect benthic organisms and fisheries. Hypoxia has been traditionally defined based on the level of oxygen below which most animal life cannot be sustained. Hypoxic conditions impact microbial composition and activity since anaerobic reactions and pathways are favoured, at the expense of the aerobic ones. Naturally occurring hypoxia can be found in areas where water circulation is restricted, such as coastal lagoons, and in areas where oxygen-depleted water is driven into the continental shelf, i.e. coastal upwelling regions. Coastal lagoons are highly dynamic aquatic systems, particularly vulnerable to human activities and susceptible to changes induced by natural events. For the purpose of this PhD project, the lagoonal complex of Amvrakikos Gulf, one of the largest semi-enclosed gulfs in the Mediterranean Sea, was chosen as a study site. Coastal upwelling regions are another type of environment limited in oxygen, where also formation of oxygen minimum zones (OMZs) has been reported. Sediment in upwelling regions is rich in organic matter and bottom water is often depleted of oxygen because of intense heterotrophic respiration. For the purpose of this PhD project, the chosen coastal upwelling system was the Benguela system off Namibia, situated along the coast of south western Africa. The aim of this PhD project was to study the microbial community assemblages of hypoxic ecosystems and to identify a potential link between their identity and function, with a particular emphasis on the microorganisms involved in the nitrogen and sulfur cycles. The methodology that was applied included targeted metagenomics and RNA stable isotope probing (SIP). It has been shown that the microbial community diversity pattern can be differentiated based on habitat type, i.e. between riverine, lagoonal and marine environments. Moreover, the studied habitats were functionally distinctive. Apart from salinity, which was the abiotic variable best correlated with the microbial community pattern, oxygen concentration was highly correlated with the predicted metabolic pattern of the microbial communities. In addition, when the total number of Operational Taxonomic Units (OTUs) was taken into consideration, a negative linear relationship with salinity was identified (see Chapter 2). Microbial community diversity patterns can also be differentiated based on the lagoon under study since each lagoon hosts a different sulfate-reducing microbial (SRM) community, again highly correlated with salinity. Moreover, the majority of environmental terms that characterized the SRM communities were classified to the marine biome, but terms belonging to the freshwater or brackish biomes were also found in stations were a freshwater effect was more evident (see Chapter 3). Taxonomic groups that were expected to be thriving in the sediments of the Benguela coastal upwelling system were absent or present but in very low abundances. Epsilonproteobacteria dominated the anaerobic assimilation of acetate as confirmed by their isotopic enrichment in the SIP experiments. Enhancement of known sulfate-reducers was not achieved under sulfate addition, possibly due to competition for electron donors among nitrate-reducers and sulfate-reducers, to the inability of certain sulfate-reducing bacteria to use acetate as electron donor or to the short duration of the incubations (see Chapter 4). Future research should focus more on the community functioning of such habitats; an increased understanding of the biogeochemical cycles that characterize these hypoxic ecosystems will perhaps allow for predictions regarding the intensity and direction of the cycling of elements, especially of nitrogen and sulfur given their biological importance. Regulation of hypoxic episodes will aid the end-users of these ecosystems to possibly achieve higher productivity, in terms of fish catches, which otherwise is largely compromised by the elevated hydrogen sulfide concentrations

Diversity, ecology and evolution of marine diazotrophic microorganisms

Premi extraordinari doctorat UPC curs 2017-2018. Àmbit d’Enginyeria Civil i AmbientalBiological N2 fixation, the reduction of dinitrogen (N2) gas to biologically available nitrogen, is a fundamental process since it represents a source of new nitrogen for marine life in areas where this important element can be limiting, supporting primary productivity and thus biological carbon export to the deep ocean. This process is performed by the nitrogen-fixing prokaryotic microorganisms (the so-called diazotrophs). However, very little is still known about the identity and ecology of diazotrophs, which largely limits our capacity to understand the global significance of this process, and to predict potential variations in nitrogen fixation upon changes in environmental conditions. In this thesis, we aimed at improving the knowledge on the diversity, ecology and evolution of the marine nitrogen-fixing microorganisms in the open ocean. Most current knowledge on diazotrophic diversity has been obtained using the nifH marker gene, which encodes for a structural protein of the enzymatic complex that performs the N2 fixation reaction. Thus, in Chapter 1 we first conducted a global exploration of the nifH gene extracted from metagenomic data derived from 68 globally distributed stations collected during the Tara Oceans expedition. This approach differs from previous studies in that it does not rely on primers to detect the nifH genes, and thus allows a more quantitative estimation of the contribution of these microorganisms and a more realistic view of their diversity. This study provides a first `primer-free¿ global map of the distribution of open ocean diazotrophic communities across ocean basins and throughout the water column, showing that diazotrophs often occurred at very low abundances, and that in general they were significantly more abundant in the mesopelagic than in photic waters. Likewise, we uncovered novel diversity that had remained unnoticed in all previous primer-based studies, since we demonstrate that more than half of the detected nifH variants cannot be captured by the primers used. This suggests that most diazotroph diversity studies may be disregarding an important fraction of the nitrogen-fixing community members. Among the diazotrophs detected in Chapter 1, the most abundant was the unicellular cyanobacterium C. Atelocyanobacterium thalassa (UCYN-A), which lives in symbiosis with a prymnesiophyte alga and has been shown to be a relevant player in nitrogen fixation. Thus, in Chapter 2 and Chapter 3, we explored aspects related to the ecology, diversity and evolution of this remarkable microorganism. We detected UCYN-A in the South Atlantic Ocean using not only metagenomic approaches but also microscopic visualization techniques (CARD-FISH). This allowed us to unveil that different UCYN-A lineages, UCYN-A1 and UCYN-A2, live in symbiosis with two distinct prymnesiophyte partners of different sizes. Both UCYN-A lineages showed a streamlined genome expression towards nitrogen fixation. We estimated that these two lineages diverged almost 100 Mya under a strong purifying selection process. Finally, in Chapter 3 we focused on the study of UCYN-A3, another lineage of which very little was known, to gain insight into its ecology. Using an array of methods (PCR, qPCR, CARD-FISH and metagenomes) we could visualize and identify for the first time UCYN-A3 and its association with an alga of different size, which suggests that different UCYN-A lineages occupy different planktonic compartments that are not always considered when nitrogen fixation of nifH diversity are studied. Finally, we manage to reconstruct a significant fraction of its genome, establishing that this lineage constitutes a new UCYN-A genomic species. Overall, this thesis has significantly contributed to expand the knowledge on marine diazotrophic organisms, unveiling new diversity and new planktonic compartments that could potentially lead to a better understanding of the marine nitrogen cycle.La fijación biológica de nitrógeno, es decir, la reducción del nitrógeno (N2) a amonio, es un proceso fundamental ya que representa una fuente de nitrógeno para la vida marina en áreas donde este elemento es limitante, posibilitando la producción primaria y por tanto la exportación de carbono al océano profundo. Este proceso se lleva a cabo por microorganismos procariotas, los llamados diazotrofos. Sin embargo, aún sabemos muy poco sobre la identidad y la ecología de estos microorganismos, lo que limita enormemente nuestra comprensión de la importancia global de este proceso, y nuestra capacidad de predecir cambios en la fijación de N2 ligados a cambios en el ambiente. El objetivo de esta tesis, por tanto, fue ahondar en el conocimiento de la diversidad, ecología y evolución de los microorganismos diazotrofos en el océano. La mayoría del conocimiento actual sobre la diversidad de diazotrofos se deriva del gen marcador nifH, que codifica una proteína estructural del complejo enzimático responsable de la fijación de nitrógeno. Por tanto, en el Capítulo 1 realizamos una exploración global del gen nifH usando datos metagenómicos de 68 estaciones muestreadas durante la campaña oceanográfica Tara Oceans. Nuestra aproximación se diferencia de los estudios anteriores ya que no se basa en el uso de cebadores para detectar el nifH y posibilita por tanto una cuantificación más precisa de la diversidad real. Este estudio representa el primer mapa global (no basado en cebadores) de la distribución de diazotrofos en el océano desde superficie hasta el mesopelágico. Aunque la abundancia de diazotrofos fue muy baja en general, era significativamente mayor en el océano profundo. Asimismo, descubrimos nuevos diazotrofos que habían pasado desapercibidos en los estudios basados en cebadores: más de la mitad de los diazotrofos detectados no se capturan por los cebadores para el nifH. Esto sugiere que la mayoría de estudios previos pueden haber obviado una fracción importante de las comunidades de fijadores de nitrógeno. Entre los diazotrofos detectados en el Capítulo 1, el más abundante fue la cianobacteria unicelular C. Atelocyanobacterium thalassa (UCYN-A), que vive en simbiosis con un alga primnesiofita y que juega un papel importante en la fijación de nitrógeno. En los capítulos 2 y 3 nos dedicamos a estudiar en detalle los aspectos relacionados con la ecología, diversidad y evolución de este diazotrofo. Mediante el análisis de metagenomas y de técnicas de visualización microscópicas como el CARD-FISH pudimos detectar UCYN-A en el atlántico sur, revelando que UCYN-A1 y UCYN-A2, dos linajes diferentes de UCYN-A, viven en simbiosis con dos hospedadores diferentes, dos primnesiofitas de tamaños distintos. Además, el análisis del perfil de expresión del genoma de ambos linajes mostró una dedicación optimizada a la fijación de nitrógeno. La edad de divergencia de UCYN-A se estimó en unos 100 millones de años, y presumiblemente ocurrió bajo presiones evolutivas de tipo estabilizadora. Por último, en el Capítulo 3, nos centramos en el estudio de UCYN-A3, otro linaje del que se sabe muy poco. Mediante el uso de varios métodos (PCR, qPCR, CARD-FISH y metagenomas) se logró visualizar e identificar por primera vez el linaje UCYN-A3 asociado con una alga de tamaño diferente, lo que sugiere que los distintos linajes de UCYN-A ocupan diferentes compartimentos planctónicos que no siempre se consideran en estudios de diversidad de nifH o de fijación de nitrógeno. Finalmente, pudimos reconstruir una fracción importante del genoma de UCYN-A3, estableciendo que representa una especie genómica diferente a las anteriores. En definitiva, esta tesis ha contribuido significativamente al conocimiento de los diazotrofos en el océano mediante el descubrimiento de nueva diversidad como de nuevos compartimentos del plancton donde puede darse la fijación de nitrógeno y que podrían ayudar a entender mejor el ciclo marino del nitrógeno.Award-winningPostprint (published version