Stable isotope probing (SIP) is an established technique that can be applied to identify the metabolically active micro-organisms within a microbial population. The SIP method utilises an isotopically-labelled substrate and PCR techniques to discern the members of a microbial community that incorporate the isotope into their DNA or RNA. The current literature gap around using 15N isotopes with RNA-SIP offers real potential and advantages for targeting and identifying active members from mixed communities involved in global biogeochemical nitrogen cycling. This study specifically investigated whether nitrogen based compounds can be used as substrates in RNA-SIP methodologies and whether they can in turn be used to probe mixed community environments known to be actively fixing nitrogen. The nitrogen-limited systems targeted represented an ideal opportunity to assess the suitability of 15N-RNA-SIP approaches due to their known high nitrogen fixation rates. Identifying these nitrogen-fixing bacteria could provide a better representation analysis of the community, leading to an improved prediction on how to manage and optimise the treatment performance of target waste systems and to exploit the unique bioconversion properties of these types of organisms. Initially, the project undertook methodological proof of concept by using a soluble nitrogen source, 15NH4Cl, to label the RNA of Novosphingobium nitrogenifigens and a mixed microbial community. Successful separation of the 14N- (control) and 15N-RNA was achieved for both pure and mixed communities using isopycnic caesium trifluoroacetate (CsTFA) gradients in an ultracentrifuge. The usefulness of this technique to identify active diazotrophs in real environmental samples was tested using a nitrogen-fixing community from a pulp and paper wastewater treatment system. After growing the mixed culture with 15N2 as the sole nitrogen source, the labelled RNA was extracted and fractionated using isopycnic centrifugation in CsTFA gradients. The community composition of the active nitrogen-fixing community in the 15N2 enriched fraction was analysed by establishing a 16S rRNA gene clone library containing over 200 members. These were analysed by comparison with published sequences and by phylogenetic analysis. It was found that the more isotopic label substrate incorporated, the further the buoyant density (BD) separation between 15N- and 14N-RNA. Novosphingobium nitrogenifigens gave an average BD shift of 0.03 + 0.004 g ml-1 (95.0 atom % 15N) with 15NH4Cl. For mixed communities the average BD shift was 0.02 + 0.004 g ml-1 (80.0 atom % 15N) with 15NH4Cl and 0.013 + 0.002 g ml-1 (32.6 atom % 15N) when using 15N2. Clone library analysis of 16S rRNA genes present in the enriched 15N-RNA fraction of the mixed community was shown to consist of a diverse population of bacteria as indicated by a Shannon Weaver index value of gt;2.8. Three dominant genera (Aeromonas, Pseudomonas and Bacillus) were identified by comparison with published sequences and phylogenetic analysis. Many other groups not known as archetypal nitrogen-fixing bacteria were also identified, demonstrating that 15N2-RNA-SIP provides a useful tool for the identification of important and previously unknown contributors to nitrogen fixation in a range of environments. Overall, this project has established that nitrogen based RNA-SIP is a powerful tool that can be used successfully and reproducibly with both pure and complex mixed microbial communities to study active diazotrophs in environmental samples.

<p>Discoveries in recent years have disclosed the importance of marine cyano-bacteria in the context of primary production and global nitrogen cycling. It is hypothesized here that microbial mats in tropical coastal habitats harbour a rich diversity of previously uncharacterized cyanobacteria and that benthic marine nitrogen fixation in coastal zones is substantial.</p><p>A polyphasic approach was used to investigate cyanobacterial diversity in three tropical benthic marine habitats of different characters; an intertidal sand flat and a mangrove forest floor in the Indian Ocean, and a beach rock in the Pacific Ocean. In addition, nitrogenase activity was measured over diel cycles at all sites. The results revealed high cyanobacterial diversity, both morphologically and genetically. Substantial nitrogenase activity was observed, with highest rates at daytime where heterocystous species were present. However, the three habitats were dominated by non-heterocystous and unicellular genera such as <i>Microcoleus</i>, <i>Lyngbya</i>, <i>Cyanothece</i> and a large group of thin filamentous species, identified as members of the Pseudanabaenaceae family. In these consortia nocturnal nitrogenase activities were highest and <i>nifH</i> sequencing also revealed presence of non-cyanobacterial potential diazotrophs. A conclusive phylogenetic analysis of partial nifH sequences from the three sites and sequences from geographi-cally distant microbial mats revealed new clusters of benthic potentially ni-trogen-fixing cyanobacteria. Further, the non-heterocystous cyanobacterium <i>Lyngbya majuscula</i> was subjected to a physiological characterization to gain insights into regulatory aspects of its nitrogen fixation. The data demon-strated that nitrogenase activity is restricted to darkness, which called upon a re-evaluation of its diazotrophic behaviour.</p>

<p>Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by <i>Aphanizomenon </i>sp<i>.</i> and the toxic <i>Nodularia spumigena.</i> Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N₂-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed.</p><p>The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of <i>Aphanizomenon</i> and <i>Nodularia</i> was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in <i>Aphanizomenon</i> was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.</p>

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

Switchgrass, Panicum virgatum, is being developed as an economically and ecologically sustainable biomass crop. Nitrogen is considered one of the most limiting inputs of switchgrass. Alternatives to synthetic nitrogen fertilization may be nitrogen-fixing legumes interseeded into switchgrass. The objectives of this research were: (1) develop efficient legume management strategies for switchgrass production systems, (2) evaluate and identify cool and warm-season legumes that can be grown compatibly with switchgrass, (3) determine whether switchgrass yields are increased by legume N-fixation, and (4) determine N-fixation of common (Vicia sativa) and hairy vetch (Vicia villosa). This study examined the establishment and persistence of ten different legume species in ‘Alamo’, a lowland variety of switchgrass in two switchgrass production systems: a one-cut biomass harvest and a two-cut forage/biomass harvest. Cool-season legumes were alfalfa (Medicago sativa), arrowleaf clover (Trifolium vesiculosum), common vetch, crown vetch (Securigera varia), red clover (Trifolium pretense), hairy vetch, and crimson clover (Trifolium incarnatum). Warm-season legumes were Illinois bundle flower (Desmanthus illinoensis), trailing wild bean (Strophostyles helvula), and partridge pea (Chamaechrista fasciculata). Red clover showed the highest plant densities and increase in switchgrass yields when interseeded into existing switchgrass stands in both harvest systems. Crude protein levels were highest in the 135 kg N ha-1 treatment in the forage cut of the two-cut harvest system. Arrowleaf clover, crimson clover, and red clover had high stand densities with annual reseeding. A combination of cool-season legumes, crimson clover and common vetch, in combination with warm-season partridge pea, were established in existing switchgrass stands after one year. Common vetch was evaluated for its nitrogen fixing capacity, seed germination, establishment, and effects on yield of switchgrass. Scarification by sulfuric acid had higher seed germination than other scarification treatments, except 100 grit sandpaper treatment for one minute at 0.7 kg of pressure. Common and hairy vetch nitrogen contributions were 59.3 and 43.3 kg N ha-1 respectively at seeding rates of 6.7 kg PLS ha-1. Switchgrass yields can increase with common and hairy vetch seeding rates of 7.6 and 10.4 kg PLS ha-1 to achieve 67 kg N ha-1, the recommended rate of N-fertilization for switchgrass stands.

Adaptability is one of the reasons for the success of bacteria, allowing them to survive in conditions where no other organisms would be able to thrive. Nitrogen deficiency, for example, can be a limiting factor for the growth of micro-organisms, as this element is an essential part of almost all types of biomolecules. As such, some bacteria have evolved specific mechanisms to overcome nitrogen limitation. Nitrogen fixing bacteria, or diazotrophs, use a specific enzyme complex, nitrogenase, in order to harness this element from the enormous reservoir that is the Earth’s atmosphere. However, nitrogen fixation is a demanding process for the cells, requiring vast amounts of energy and tight regulation. In this thesis we explore the mechanisms regulating nitrogen fixation in Rhodospirillum rubrum, a purple non-sulphur photosynthetic bacterium. Using proteomics tools, we show how the regulation of both the nitrogen and carbon fixation processes is interconnected, possibly in order to maintain the intracellular redox balance. Using a new detergent molecule, we also demonstrate how nitrogen availability affects the chromatophore membrane proteome. Our studies have revealed the crucial role of the cellular pool of 2-oxoglutarate (2OG) for adequate signaling through the PII proteins and the effects resulting from artificially manipulating this metabolite’s concentration. In R. rubrum nitrogenase is also subjected to post-translational control (the “switch-off” effect) and this work shows for the first time that the enzyme modifying nitrogenase (Dinitrogenase Reductase ADP-ribsosyl Transferase or DRAT) forms a complex with the PII protein GlnB. This complex allows DRAT activation and its formation – and, therefore, DRAT activity – is regulated by binding of ADP:ATP and 2OG to GlnB. Upon light withdrawal, nitrogenase activity anaerobically in the dark is also here demonstrated to be dependent on the activity of the pathway starting in pyruvate formate-lyase and we show how different nitrogen sources influence the switch-off response. This response can, in some conditions, be modified by addition of pyruvate and we have studied how this metabolite influences nitrogenase activity and switch-off regulation. This study allows a better understanding of the underlying processes controlling the metabolic routes in R. rubrum and also provides new insights into regulation of enzyme activity, paving the road for the complete establishment of the mechanisms regulating nitrogenase switch-off. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Submitted. Paper 4: Manuscript. Paper 5: Submitted.</p>

Discoveries in recent years have disclosed the importance of marine cyano-bacteria in the context of primary production and global nitrogen cycling. It is hypothesized here that microbial mats in tropical coastal habitats harbour a rich diversity of previously uncharacterized cyanobacteria and that benthic marine nitrogen fixation in coastal zones is substantial. A polyphasic approach was used to investigate cyanobacterial diversity in three tropical benthic marine habitats of different characters; an intertidal sand flat and a mangrove forest floor in the Indian Ocean, and a beach rock in the Pacific Ocean. In addition, nitrogenase activity was measured over diel cycles at all sites. The results revealed high cyanobacterial diversity, both morphologically and genetically. Substantial nitrogenase activity was observed, with highest rates at daytime where heterocystous species were present. However, the three habitats were dominated by non-heterocystous and unicellular genera such as Microcoleus, Lyngbya, Cyanothece and a large group of thin filamentous species, identified as members of the Pseudanabaenaceae family. In these consortia nocturnal nitrogenase activities were highest and nifH sequencing also revealed presence of non-cyanobacterial potential diazotrophs. A conclusive phylogenetic analysis of partial nifH sequences from the three sites and sequences from geographi-cally distant microbial mats revealed new clusters of benthic potentially ni-trogen-fixing cyanobacteria. Further, the non-heterocystous cyanobacterium Lyngbya majuscula was subjected to a physiological characterization to gain insights into regulatory aspects of its nitrogen fixation. The data demon-strated that nitrogenase activity is restricted to darkness, which called upon a re-evaluation of its diazotrophic behaviour.

Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by Aphanizomenon sp. and the toxic Nodularia spumigena. Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N2-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed. The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of Aphanizomenon and Nodularia was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in Aphanizomenon was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.

On the Canadian prairies, organic production generally includes the use of annual green manure (GrM) crops, which are terminated using tillage to add nutrients and organic matter to the soil. However, in a GrM plough-down year, farmers face loss of income. As an alternative to growing traditional GrM crops, legumes can be grown alone or intercropped with cereals and harvested as green feed forage (GF) for use on-farm or for sale to other producers without depleting soil nitrogen (N) for the subsequent crop. We hypothesized that the GF system would have similar biomass, and N yield, and ultimately would return N into the soil. Furthermore, by intercropping a legume with a cereal, biological N2-fixation will be enhanced in the legume.<p> Field experiments, conducted over two years, were established at Vonda and Delisle, Saskatchewan, Canada. The experiment was conducted using a randomized complete block design (RCBD) with 16 treatments and four replicates in which field pea (<i>Pisum sativum</i> cv 40-10 silage pea), oat (<i>Avena sativa</i> L.cv AC Morgan), and triticale (X <i>Triticosecale</i> Wittmack cv Pika) were grown alone or in combination and managed as GrM or GF. Wheat and tillage fallow served as cropped and uncropped controls, respectively. The tillage fallow-control system was tilled twice in the growing season using a small tractor disc. The intercropped oat was seeded at three densities (50, 100, and 150 plants m-2) to determine whether increasing cereal density stimulated N2-fixation in the field pea.<p> The GrM system was sampled and incorporated (when the field pea was at full bloom) two weeks earlier than the GF system. Consequently, at both sites, all treatments in the GF system consistently yielded more dry matter and accumulated more N than treatments in the GrM system. At the Delisle site, where percent nitrogen derived from the atmosphere (%Ndfa) was compared, increasing cereal density did not increase N2-fixation in both management systems. However, pea in the GF system accumulated more than twice the amount of N (kg ha-1) from fixation as compared to pea in the GrM system, presumably because of the longer growth period.<p> Wheat grown following the GrM treatments produced more biomass and accumulated more N than wheat following the GF treatments. Wheat grown after the monoculture field pea as a GrM had greater yield than all treatments. As well, the GrM system returned more N to the soil than did the GF system. The extra two weeks of growth in the GF system resulted in the extraction of significant amounts of nutrients and probably moisture from the soil, which adversely affected yield and nutrient composition of the following wheat crop.<p> Although organic farmers may lose income in the plough-down year, on a longterm soil sustainability basis, the GrM system is a better option than the GF system as it returns nutrients to the soil, thus providing improved plant biomass, and N accumulation of subsequent crops. However, organic farmers growing GF for hay may benefit from the increased productivity of this system on a short-term basis. Thus, farmers pursuing GF options may need to adopt other means of sustaining soil productivity on a longer term. The tilled fallow-control system resulted in high amounts of biomass and N accumulation by the subsequent wheat crop, probably due to the fact that there were no nutrients taken up in the previous year and moisture was conserved in these treatments. However, this system may have less long-term benefits compared to the GrM regime, as no nutrients are returned through ploughing down a crop.

Nitrogen (N) is an essential plant nutrient, widely applied as N-fertilizer to improve yields of agriculturally important crops. An alternative to fertilizer use could be the exploitation of plant growth-promoting bacteria, capable of enhancing growth and yield of many plant species. Azospirillum and Gluconacetobacter are root colonizing, free-living, N2-fixing bacteria (diazotrophs) with the potential to transfer fixed N to associated plants. The purpose of this study was to evaluate the agronomic efficiency of two diazotrophs, Azospirillum lipoferum and Gluconacetobacter azotocaptans, inoculated onto wheat. Physiological parameters and yield components were evaluated. The objectives of this study were to: 1) determine the survival of each diazotroph species on wheat seeds over time; 2) determine the survival of A. lipoferum and G. azotocaptans inoculated on wheat seed treated with a fungicide seed treatment, Dividend® XL RTA®; 3) determine if inoculation of wheat with the diazotrophs under controlled conditions causes an increase in dry matter, N2-fixation and N uptake; 4) determine if fertilizer N applied at three levels influences atmospheric N2-fixation by A. lipoferum or G. azotocaptans; 5) determine if inoculation of wheat with A. lipoferum or G. azotocaptans under field conditions causes any increase in dry matter, N2-fixation and N uptake; 6) determine if N-fertilization levels under field conditions influenced N2-fixation by A. lipoferum or G. azotocaptans. In order to meet these objectives lab, growth chamber, and field studies were completed. Laboratory investigations revealed that the decline in recovery of colony forming units (CFU) of G. azotocaptans was not significantly different (P<0.05) for any seed treatment. There was a general decrease in CFU over time regardless of seed treatment. Analysis of the recovered CFU of A. lipoferum over time showed that there was a significant difference (P<0.05) between both the non-sterilized seed and the Dividend® XL RTA® treated seed when compared sterilized seed. Recovery of CFU on sterilized seed declined at a more rapid rate compared to the other two seed treatments. Gluconacetobacter azotocaptans and A. lipoferum were not negatively influenced by the Dividend® XL RTA® seed treatment. Also, both diazotrophs were able to compete with other microorganisms that may have been on the seed coat of unsterilized seeds. Azospirillum lipoferum and G. azotocaptans were able to fix atmospheric N, but, there were no significant (P<0.05) differences between the diazotroph species. Additions of fertilizer N enhanced N2-fixation, in both the growth chamber and field studies. As the amount of fertilizer N increased, so did the %Ndfa and N uptake. In the growth chamber study, inoculated wheat, and fertilized with 12.2 and 24.5 µg N g-1 had the highest %Ndfa of 25.5%, and wheat fertilized with 24 µg N g-1 had the highest N uptake (1.3 g pot-1) at maturity. In the field study, inoculated wheat fertilized with of 80 kg N ha-1 had significantly higher (P<0.05) %Ndfa (10.5%) compared to wheat grown with the other fertilizer levels, which also corresponded to the highest N uptake in wheat plants (47 kg ha-1). The diazotrophs also affected the partitioning of N in the wheat plants differently. Wheat inoculated with A. lipoferum had significantly higher (P<0.05) amounts of N accumulated in heads of plants, and wheat inoculated with G. azotocaptans had significantly higher (P<0.05) amounts of N accumulated in stems of plants. However, this trend was not evident in the field study.