Дисертації з теми "Nitrogen fixation and transfer"

The studies presented in this thesis include studies of nucleotide-dependent conformations of the electron donor protein in nitrogenase and dark-operative protochlorophyllide reductase (DPOR) characterized using small-angle x-ray scattering and x-ray diffraction methods. Nitrogen fixation and chlorophyll synthesis are involved in the reduction of high energy bonds under physiological conditions. Both make use of elegant reaction mechanisms made possible by complex enzyme systems which are evolutionarily related. Nitrogenase reduces nitrogen to ammonia and is a two-component metalloenzyme composed of Fe protein and MoFe protein. For nitrogen reduction, the Fe protein and MoFe protein associate and dissociate in a manner concomitant with hydrolysis of at least two MgATP molecules and enables the concomitant transfer of at least one electron from Fe protein to MoFe protein. During chlorophyll biosysnthesis, the rate limiting step is catalyzed by a two-component metalloenzyme called DPOR. The two components of DPOR are BchL and BchNB proteins and these share high level of sequence similarity with the Fe protein and the MoFe protein, respectively. Based on this sequence similarity and biochemical data available, it is proposed that the reaction mechanism is similar to nitrogenase mechanism in which the components of DPOR associate and dissociate in a nucleotide dependent manner, to enable intercomponent electron transfer. Fe protein and BchL present as unique examples of proteins that couple nucleotide dependent conformational change to enable electron transfer for high energy bond reduction. The present studies have been directed at studying the low resolution studies of MgATP-bound wild-type Fe protein and its comparison to the structure of the proposed mimic, i.e, L127 Delta Fe protein. The studies presented show evidence of the MgATP-bound wild-type Fe protein having a conformation very different from the L127 Delta Fe protein. The chapters also include detailed characterization of the structure of BchL in both MgADP bound and nucleotide-free states which offer detailed insights in the structure based mechanism of BchL, with primary focus on identifying key residues involved in componenet docking and in electron transfer. Together, the studies on the Fe protein and BchL have furthered our understanding of mechanism of electron transfer in these complex enzyme systems.

Nitrogen is a critical nutrient for growth and reproduction in living organisms. Although the Earth’s atmosphere is composed of ~80% nitrogen gas (N2), it is inaccessible to most living organisms in that form. Biological nitrogen fixation, however, can be performed by microbes that harbor the enzyme nitrogenase. This enzyme converts N2 into bioavailable ammonia (NH3) and accounts for at least half of the “fixed”nitrogen on the planet. The other major contributor to ammonia production is the industrial Haber-Bosch process. While the Haber-Bosch process has made significant advances in sustaining the global food supply through the generation of fertilizer, it requires high temperature and pressure and fossil fuels. This makes nitrogenase an ideal system for study, as it is capable of performing this challenging chemistry under ambient conditions and without fossil fuels. Nitrogenase requires energy and electrons to convert N2into NH3. The work presented here examined how the enzyme receives electrons to perform the reaction. It was discovered that some microbes employ a novel mechanism that adjusts the energy state of the electrons so that nitrogenase can accept them. Further, the slowest step that takes place in nitrogenase once the electrons are taken up was identified. Finally, by capitalizing on fundamental knowledge, a biohybrid system was designed to grow nitrogen-fixing bacteria in association with electrodes for light-driven production of fixed nitrogen that has potential to be used as a fertilizer for plant growth. Gaining an in-depth understanding of nitrogenase provides insight into one of the most challenging biological reactions, and the newfound knowledge may be a catalyst in developing more efficient systems for sustainable ammonia production.

In this study, six annual legumes and the perennial, red clover (Trifolium pratense L.) were monocropped (MC) and intercropped (IC) with barley in a field study with three N levels, 0, 30 and 60 kg N ha$ sp{-1}$. At O kg N ha$ sp{-1}$, N$ sb2$-fixation and N transfer were estimated by the $ sp{15}$N isotope dilution (ID) method. At 60 kg N ha$ sp{-1}$, a direct $ sp{15}$N labelling method was employed to study N transfer. The hypotheses were that the annual species would be more productive within one growing season than red clover, that increased N levels would increase herbage dry matter (DM) and crude protein (CP), that the proportion of N derived from N$ sb2$-fixation in IC-legumes would be higher than that of MC-legumes and that within intercrops there would be evidence of N transfer. In neither year was the total DM yield of red clover, MC or IC, less than the rest of the legumes. In 1991, the total DM yield of intercrops responded to 30 kg N ha$ sp{-1}$; in neither year did the estimated total CP yield of MC-legumes or intercrops respond to N levels. Only in 1992 was there evidence of N$ sb2$-fixation and the proportion of N derived from fixation by IC-legumes was 145% higher than that of MC-legumes. Only the $ sp{15}$N direct labelling method gave evidence of N transfer, to associated legume and barley plants in 1991, and to associated legume plants in 1992.

β-N-methylamino-L-alanine (BMAA) is one of the more recently discovered bioactive compounds produced by cyanobacteria. BMAA is a non-protein amino acid reported present in human brain tissues of patients deceased from a neurodegenerative disease, such as Alzheimer´s disease or amyotrophic lateral sclerosis (ALS). This observation in combination with its neurotoxic effects in eukaryotes (in vivo and in vitro) and its potential to incorporate into (human) proteins, causing protein aggregation, suggests BMAA as a possible causative environmental agent for neurodegenerative diseases. Due to the ubiquitous nature of cyanobacteria with a wide occurrence in both aquatic and terrestrial environments, BMAA could be globally spread. Hence, investigations of a possible coupling between BMAA and neurodegeneration are urgently needed as well as to identify sources of BMAA in Nature. The aim of this thesis was to examine the potential occurrence of BMAA in bloom forming cyanobacteria of the Baltic Sea and its possible transfer to other organisms of this ecosystem. Of importance was also to reveal any likely routes for human BMAA exposure in the Baltic Sea region and to further investigate BMAA as a triggering agent for neurodegenerative diseases. Acknowledged difficulties of analysing BMAA in biological samples also inferred method development as part of the experimental studies. Investigating the role of BMAA in its producers was another purpose of the thesis, which may be crucial for future management of BMAA-producing cyanobacteria. By screening natural populations of the major filamentous bloom forming cyanobacteria of the Baltic Sea, we discovered the presence of BMAA throughout the entire summer season of two consecutive years, using a highly specific analytical method (liquid chromatography-tandem mass spectrometry; LC-MS/MS). BMAA was found to bioaccumulate in zooplankton and fish, as well as in mussels and oysters from the Swedish west coast. To improve the understanding of BMAA analyses in natural samples, the formation of carbamate adducts in the presence of bicarbonate was examined. Using two derivatization techniques in combination with LC-MS/MS, we could show that BMAA detection was not hindered by carbamate formation. Exogenously added BMAA inhibited nitrogen fixation in the model cyanobacterium Nostoc sp. PCC 7120, which was also hampered in growth and displayed signs of nitrogen starvation. Finally, BMAA was detected in cerebrospinal fluid in three of 25 Swedish test individuals, and represents the first confirmation of BMAA in the human central nervous system using LC-MS/MS as the primary analytical method. However, the association of BMAA to neurodegenerative diseases could not be verified as BMAA was present in both control individuals (two) and in one ALS-patient. Nevertheless, the finding of a known neurotoxic compound in the human central nervous system is alarming and potential consequences should be investigated. The discovery of the neurotoxic compound BMAA in Baltic Sea organisms, and in the central nervous system of humans potentially consuming fish from this ecosystem is concerning and warrants continued investigations of BMAA occurrence and human exposure. Further knowledge on the function and regulation of BMAA may help in developing strategies aiming to minimise human exposure.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Chaque année, plus de 3,7 millions de tonnes d'engrais azotés sont utilisées par l'agriculture française. Bien qu'étant un élément essentiel au bon développement et à la bonne croissance des plantes, la fertilisation azotée est de plus en plus décriée en raison des nombreuses nuisances aux écosystèmes et à la biodiversité qu'il induit. Le colza (Brassica napus L.) est l'une des plantes de grandes cultures les plus exigeantes en azote (N), en raison de sa faible efficience d'usage de cet élément. Dans le but de maintenir une croissance et des rendements optimaux de la culture de colza tout en limitant les apports en fertilisants azotés, il est essentiel de mettre au point de nouvelles pratiques culturales plus respectueuses de l'environnement. Dans ce contexte, l'utilisation combinée des cultures associées à base de Fabacées et de produits biostimulants constitue une alternative intéressante aux pratiques actuelles de la culture de colza. Du fait de ses nombreux effets bénéfiques lors de stress biotiques et abiotiques, le silicium (Si) semble être un biostimulant pertinent. Les objectifs de ces travaux de thèse visent à observer les effets d'un apport en Si sur (i) les performances agronomiques du colza et du trèfle en monoculture cultivés en conditions contrôlées et (ii) sur une possible potentialisation du fonctionnement de l'association colza-trèfle incarnat. Dans un premier temps, nos travaux ont permis de montrer qu'en condition de carence en soufre (S), un apport de Si s'accompagne d'une baisse du stress oxydatif et d'une modulation de l'expression des gènes codant les transporteurs racinaires de sulfate qui permet aux plantes traitées au Si d'être plus efficaces pour absorber le S lorsque celui-ci est à nouveau disponible. Dans un second temps, l'apport de Si à une culture de trèfle incarnat soumis à une carence en S ou cultivé avec une solution nutritive dépourvue d'N a un impact positif sur la nodulation et les capacités de fixation du diazote atmosphérique. Le Si induit une augmentation significative du nombre de nodules racinaires ainsi que de leur teneur en nitrogénase. En outre, l'étude du ionome et du protéome des nodules a permis de mieux comprendre les effets d'un apport en Si en montrant notamment l'accumulation d'éléments et de protéines impliqués directement ou indirectement dans la fixation de diazote atmosphérique. Enfin, nos travaux ont mis en lumière les effets bénéfiques d'un apport en Si sur une association colza-trèfle notamment en augmentant le transfert d'N de la Fabacée vers la Brassicacée et en améliorant le rendement grainier du colza<br>Over 3.7 million tonnes of N fertilizers are used in French agriculture every year. Although N fertilization is an essential element for the growth of crops, it is increasingly criticized for its harmful effects on ecosystems and biodiversity. rapeseed (Brassica napus L.) is one of the most N requiring field crops, due to its low N use efficiency. To maintain optimum the growth and the yield of rapeseed with lower N fertilizer inputs, it is crucial to develop new cultural practices more respectful of environment. In this context, the combination of rapeseed-clover intercrops and biostimulants is an interesting alternative to the current cultivation practices of rapeseed. Considering its numerous beneficial effects under biotic and abiotic stresses, Silicon (Si) appears to be a relevant biostimulant. The aim of this PhD work was to observe the effects of Si supply on (i) the agronomic performance of monoculture rapeseed and clover grown under controlled conditions, and (ii) the possible potentiation of the functioning of the rapeseed-clover intercrop. Firstly, our work has shown that under sulfur (S) deficiency conditions, the supply of Si leads to a reduction in oxidative stress and modulation of the expression of genes encoding root sulfate transporters, allowing +Si plants to take up S more efficiently when it becomes available again. Secondly, the Si supply to crimson clover subjected under S-deficiency or or grown without N in the nutrient solution, had a positive impact on nodulation and atmospheric dinitrogen fixation capacities. Si induces a significant increase in the number of root nodules and their nitrogenase content. In addition, the study of the nodule ionome and proteome has enhanced our knowledge of the benefit effects of Si supply, particularly by showing the accumulation of elements and proteins directly or indirectly involved in the fixation of atmospheric dinitrogen. Finally, our work has highlighted the beneficial effects of Si supply on a rapeseed-clover intercrop, notably by increasing the N transfer from the Fabaceae to the Brassicaceae and improving the yield of the rapeseed

A Transferência Horizontal de Genes (THG) é um dos principais mecanismos de evolução bacterianos, impactando a evolução de praticamente todas famílias gênicas. Neste trabalho identificamos e avaliamos padrões de possíveis transferências horizontais de genes pertencentes a duas classes funcionais de dois níveis taxonômicos distintos. Caracterizamos a ocorrência e evolução de 45 genes importantes para a fixação de N2 em 479 genomas de Proteobacteria. Identificamos cinco potenciais aquisições de genes ligados a fixação de N2 por linhagens de Proteobacteria, as quais foram identificadas consistentemente em 36 dos genes analisados. Realizamos predições de transferências horizontais dos 45 entre todos os 479 genomas de Proteobacteria e identificamos possíveis enriquecimentos de THG, provavelmente ligados à sinais filogenéticos e ecológicos. Desenvolvemos um pipeline para identificação semi-automática de efetores do Sistema Secretor do Tipo III em Aeromonas, o qual reportou 21 famílias de potenciais efetores presentes em 105 genomas. Entre os 21 efetores identificados 17 foram descritos pela 1º vez em Aeromonas, corroborando a sensibilidade de nosso pipeline. Com o auxílio de nossos colaboradores foram realizados testes de citotoxidade para efetores identificados in silico, e apenas quatro não inibiram o crescimento de Saccharomyces cerevisiae. Por fim, desenvolvemos um método para agrupamento de famílias gênicas com histórias evolutivas similares que não requer a reconstrução de árvores filogenéticas, aumentando a eficiência computacional. Aplicamos o método desenvolvido para reconstrução da filogenia de Aeromonas, o qual mostrou-se compatível com dados presentes na literatura.<br>Horizontal Gene Transfer (HGT) is one of main mechanisms of bacterial evolution, affecting virtually all gene families. In this document we identified and assessed putative horizontal transfers of genes from two functional classes from two distinct taxonomic levels. We characterized the distribution and evolution of 45 genes important to N2 fixation among 479 Proteobacteria genomes. We identified five potential distinct acquisitions of such genes by Proteobacteria lineages. The distinct origins are consistently identified in 36 out of the 45 assessed genes. We computed possible horizontal transfers of the 45 genes among the 479 Proteobacteria genomes, and we identified enrichments of HGT, likely related to phylogenetic and ecological signals. We developed a semi-automated pipeline to identify effectors of the Type III Secretion System within Aeromonas, which reported 21 putative effector families distributed among 105 genomes. Among the 21 likely effectors 17 have been described in Aeromonas for the first time, highlighting the sensibility of our pipeline. Our colaborators performed cytotoxicity tests for the 21 likely effector families identified by in silico analysis, and only four did not inhibited Saccharomyces cerevisiae growth. Lastly, we developed a method to cluster gene families according to shared evolutionary history, without the requirement of phylogenetic tree reconstruction, increasing computational efficiency. We applied this proposed method during Aeromonas phylogenetic reconstruction, and it showed up compatible with data available on the literature.

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere. Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

While bacterioplankton indisputably control vital biogeochemical paths in the cycling of carbon and nutrients in the world’s oceans, our knowledge about the functional and genetic diversity of bacterioplankton communities is negligible. In this thesis, molecular and more traditional microbiological methods were used to study the specific function of N2-fixation and in a general sense diversity of marine bacterioplankton species. Most oceans are nitrogen limited and, therefore, adaptive to bacterioplankton capable of N2-fixation. Recent studies have found nifH genes (coding for the nitrogenase enzyme) related to diverse heterotrophic bacteria in oceanic seawater samples indicating that, along with cyanobacteria, also heterotrophic bacteria benefit from N2-fixation. Here, molecular and cultivation methods were used to examine diazotrophic bacterioplankton in the Baltic Sea. We successfully isolated heterotrophic N2-fixing bacteria belonging to the γ-proteobacterial class by means of low-nitrogen plates and semi-solid diazotrophic medium tubes. The isolates required low-O2 conditions for N2-fixation. Using Real-time PCR it was found that heterotrophic bacterioplankton carrying the nifH gene was abundant (3 x 104 nifH gene copies L seawater-1) at locations in the Southwest Baltic proper. With the aim to identify the main N2-fixing organisms in Baltic Proper surface waters, a clone library of nifH gene transcripts (RNA) was generated. Clone inserts were exclusively related to Aphanizomenon sp. and Nodularia sp. Using quantitative real-time PCR it was found that the nifH gene expression from Nodularia sp. was highly variable between stations in the Baltic Proper but was 10-fold higher during mid summer relative to early summer and fall. A diel study showed a 4-fold increase in Nodularia transcript concentrations at early to mid day relative to rest of the day. Real-time PCR was found to be a powerful and highly sensitive method for measuring gene expression. Since nucleic acids are a prerequisite for molecular analyses of bacterioplankton dynamics a protocol to extract DNA from seawater samples was developed with the aim to maximize the yield of high-quality DNA. Each step in the protocol was important for the efficiency of extraction. The obtained extraction efficiencies were up to 92% for seawater samples and up to 96% for isolates. The protocol provides a guideline for DNA extraction from seawater samples for other studies. In a global sampling campaign (9 locations from polar, tropical and temperate regions) we sampled DNA from surface water and constructed 16S rRNA gene libraries to investigate diversity and biogeography of bacterioplankton. Approx. 80% of the sequences found were similar to sequences already deposited in GenBank, indicating that a large fraction of the marine bacterioplankton already has been sampled, which in turn suggests a limited global bacterioplankton diversity. This thesis have improved our knowledge about the composition and nifH gene expression of the diazotrophic bacterioplankton community in the Baltic Sea and contribute significantly to the discussion on global marine bacterioplankton diversity and biogeography.<br>Östersjön är ett av världens största brackvattensystem. Den ekologiska balansen i detta hav är hotad på grund av övergödning. Mycket arbete har därför fokuserats på att reducera utsläppen av näringsämnen, speciellt kväve. Dessa ansträngningar kan dock motverkas av bakterier som har förmåga att omvandla luftens kväve till metaboliskt användbart ammonium (kvävefixering). På sommaren är Östersjöns primärproduktion begränsad av kväve, med följden att det årligen uppstår massiva blomningar av kvävefixerande bakterier, framför allt cyanobakterier. Dessa är främst Aphanizomenon och Nodularia, men inte endast de fototrofa cyanobakterierna har förutsättningar att fixera N2. NifH gener (genen som kodar för nitrogenas) bärs också av heterotrofa bakterioplankton, vilket har visats i studier i främst Atlanten och Stilla havet. Med hjälp av två olika odlingsmetoder lyckades vi isolera heterotrofa kvävefixerande bakterier tillhörande klassen γ-proteobakteria från Östersjön. Svårigheten med att finna dessa bakterier ligger i att de kräver en miljö med mycket låg syrehalt för att kunna fixera kväve. Resultaten från denna studie ledde oss vidare till att undersöka vilka organismer som uttrycker nifH genen (och då troligen även fixerar kväve) i Östersjön. En av de bakterier som isolerats kunde påvisas med Realtids PCR i ett relativt stort antal (3 x 104 nifH genkopior per liter) vid en av de ursprungliga provtagningsstationerna. För att söka rätt på de olika organismtyper som uttrycker nifH skapades ett klonbibliotek baserat på mRNA extraherat från havsvatten. Det visade sig då att alla de närmare 100 kloner som sekvenserades tillhörde antingen Aphanizominon eller Nodularia. De heterotrofa bakteriernas nifH genuttryck var troligen i jämförelse med dessa cyanobakterier alltför lågt för att kunna detekteras. Realtids PCR mätningar av Nodularias nifH genuttryck visade på en stor variation mellan de olika provtagningsstationerna samt mellan de olika provtagningstillfällena. Vi fann dock en kraftig ökning under juli med en nedgång igen i augusti. En dygnscykelstudie visade att Nodularia nifH genuttrycket ökade under förmiddagen med en topp mitt på dagen för att sedan minska igen. Detta troligen med anledning av att den energikrävande kvävefixeringsprocessen sker under de ljusa timmarna då cellen får energi från fotosyntesen. I de molekylärbiologiska metoderna som används för att få information om identitet och aktivitet hos skilda organismer krävs att DNA och RNA kan extraheras från prover tagna i naturliga vattenmiljöer. Även om antalet bakterier tillsynes är högt, så är mängden DNA och RNA per liter havsvatten relativt låg, därför krävs ett väl fungerande protokoll för denna extraktion. I en inledande studie i denna avhandling optimerades en metod för att utvinna DNA. Ett antal sådana protokoll finns publicerade men dessa har ofta lågt utbyte. Det nya protokollet har hög effektivitet, vilket gör att små provvolymer kan användas (2 ml jämfört med tidigare flera liter) och därmed ökar hanterbarheten. Vi visar i denna studie att varje steg 7 i DNA-extraktionsprotokollet är viktigt för att ge en hög effektivitet. Detta protokoll kan med fördel användas som vägledning för många olika typer av studier. På grund av att många havsbakterier inte kan bilda kolonier och alltså inte växa på traditionella medier har det varit svårt att få en klar bild av artrikedomen. Molekylärbiologin har dock gjort det möjligt att identifiera bakterier med hjälp av 16S rRNA genen, en enorm mängd gensekvenser från världens alla hav har inkommit till den gemensamma databanken (GenBank). År 2002 gjordes en studie där man sammanställde informationen i denna databank, för att få en bild av artrikedomen i världshaven. Resultatet av denna studie var att det i världshaven fanns färre bakterietyper än vad många forskare har spekulerat i. I denna avhandlig har vi utfört en studie där vi gjorde en stor global provtagning för att se om denna undersökning överensstämde med den datainformativa. Provtagning från nio lokaliteter gjordes i de tempererade, tropiska och polarhaven. Ett genbibliotek från varje lokal gjordes och kloner sekvenserades. Resultatet visar i likhet med den datainformativa undersökningen på en begränsad artrikedom. 80% av gensekvenserna fanns redan i databanken, vilket tyder på att de flesta arter redan har blivit funna. Dessutom visade det sig att få av bakterierna återfanns på alla ställen och många återfanns endast på ett ställe. Utöver detta visade det sig att det fanns en ökad artrikedom ju närmare ekvatorn man kom, vilket tidigare har visats för större organismer. Studierna i denna avhandling har ökat förståelsen för hur sammansättningen av det kvävefixerande bakteriesamhället i Östersjön ser ut samt bidragit till diskussionen om den globala artrikedomen bland bakterioplakton och dess utbredning.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences, 2009.<br>Includes bibliographical references (p. 163-185).<br>Oceanic nitrogen fixation and biogeochemical interactions between the nitrogen, phosphorus and iron cycles have important implications for the control of primary production and carbon storage in the ocean. The biological process of nitrogen fixation is thought to be particularly important where the ocean is nitrogen limited and oligotrophic. This thesis examines some of the mechanisms responsible for the distribution, rates and temporal variability of nitrogen fixation and its geochemical signature in the modern ocean. I employ simple analytical theories and numerical models of ecosystems and biogeochemical cycles, and closely refer to direct observations of the phytoplanktonic community and geochemical tracers of the marine nitrogen cycle. Time-series observations of geochemical tracers and abundances of nitrogen fixers (or diazotrophs) in the northern subtropical gyres suggest variability in nitrogen fixation on interannual and longer timescales. I use a highly idealized, two-layer model of the nitrogen and phosphorus biogeochemistry and ecology of a subtropical gyre to explore the previously proposed hypothesis that such variability is regulated by an internal biogeochemical oscillator. I find, in certain parameter regimes, self-sustained oscillations in nitrogen fixation, community structure and biogeochemical cycles even with perfectly steady physical forcing. The period of the oscillations is strongly regulated by the exchange rate between the thermocline and mixed-layer waters, suggesting a period of several years to several decades for the North Pacific subtropical gyre regime, but would likely be shorter (only a year or so) for the North Atlantic Ocean.<br>(cont.) Geochemical tracers such as DINxs (=NO3--16PO3-) measure the oceanic departure from the Redfield ratio. DINx, is often used to estimate the rate of nitrogen fixation in the ocean, by quantifying the tracer accumulation along isopycnals. However this tracer reflects an interwoven set of processes including nitrogen fixation, but also denitrification, atmospheric and riverine sources, differential remineralization and complex transport pathways. I examine analytical solutions of the prognostic equation of DINx, and an idealized three-dimensional model of the basin-scale circulation, biogeochemical cycles and ecology of the North Atlantic Ocean. The two approaches demonstrate that the observations of a subsurface maximum in the North Atlantic Ocean and the temporal variability at the station BATS of DINxs can be explained simply by preferential remineralization of organic phosphorus relative to nitrogen. A further analysis reveals that the current geochemical estimates based on inorganic forms of phosphorus and nitrogen underestimate integrated nitrogen fixation rates by a factor of two to six, by neglecting the preferential remineralization effect. Most current understanding of oceanic nitrogen fixation is based on the Trichodesmium, though unicellular cyanobacteria, diatom-diazotroph associations (DDA) and heterotrophic bacteria might be as important in adding nitrogen into the ocean. I employ a self-assembling global ocean ecosystem model to simulate diverse phytoplanktonic diazotrophs in the global ocean and examine how temperature, oligotrophy, iron and phosphate limitations influence the global marine diazotroph distribution.<br>(cont.) Analogs of Trichodesmium, unicellular diazotrophs and DDA are successful in the model, showing very similar distributions with observations. The total diazotrophic population is distributed over most of the oligotrophic warm (sub)tropical waters in the model. The model demonstrates that temperature is not the primary control, but suggests instead that diazotroph biogeography is restricted to the low fixed nitrogen oceanic regions which have sufficient dissolved iron and phosphate. The theory of resource competition is used to map out regions of iron and phosphate regulation of diazotroph distribution. The theory suggests that diazotrophs are largely regulated by iron availability, in particular in the Pacific and Indian Oceans. The iron cycle is currently not well enough constrained to confidently predict the diazotroph distribution in global ocean models.<br>by Fanny Monteiro.<br>Ph.D.

The compatibility of potentially nitrogen fixing associations between ten Rhizobium strains and six Sesbania accessions (species) was studied under glasshouse conditions. The rates of N₂ (C₂ H₂) fixation (u moles C₂ H₄ /plant/h) were determined. The various Sesbania accessions responded differently to inoculation with the strains tested. The ANOVA test revealed that there are real accessions (P = 0.01) and strains (P = 0.05) differences. In general the results obtained indicated that the highest mean rate of N₂ (C₂ H₂) fixation and the highest degree of compatibility with strains under test was shown by Sesbania bispinosa (accession BA12). Sesbania grandiflora (accession GL 2.02) ranked next. The performance of Sesbania pachycarpa (accession PCI), Sesbania macrantha (accession MNI), and Sesbania sesban (accession SBIO) in the N₂ (C₂ H₂) assay is lower than that of accessions BAI2 and GL2.02, thus indicating the possibility of lack of compatibility between these three accessions and almost all of the Rhizobium strains studied. Plants of Sesbania rostrata (accession RSI) produced either extremely low or no ethylene (C₂ H₄) quantities in the N₂ (C₂ H₂) assay thus indicative of high specificity or that this legume is not promiscuous at all. However, inoculated and fertilized Sesbania rostrata performed quite satisfactorily and formed profuse N₂-fixing nodules on roots and stems when grown in potted soil under Central Saudi Arabia climatic conditions. The results obtained indicated high variability among treatments in nodule number.

Thesis (M.S.) -- University of Maryland, College Park, 2004.<br>Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

When grown under constant illumination, <I>Gloeothece</I> ATCC 27152, a unicellular cyanobacterium, can use nitrate, nitrite, dinitrogen or ammonium as the sole N-source for growth. The uptake systems for nitrate and nitrite were fully active and ammonium-repressible in N<SUB>2</SUB>-fixing cultures. Nitrite uptake was mediated via two pH-dependent systems: passive diffusion of HNO<SUB>2</SUB> and active transport of nitrite. Nitrate uptake was highly light-dependent. Ammonium was also transported by passive diffusion of ammonia and active uptake of ammonium, depending on the pH of the medium. Ammonium inhibited nitrate uptake almost completely, but inhibited nitrite uptake only partially. The true inhibitor was a product of ammonium assimilation, possibly glutamine, rather than ammonium itself, since L-methionine-DL-sulphoximine, an inhibitor of glutamine synthetase blocked GS activity very quickly and decreased the inhibitory effect of ammonium. On the other hand, the inhibitory effect of nitrite on ammonium uptake was stronger than that of nitrate. Nitrate and nitrite competitively inhibited each other's assimilation, occurring at the uptake stage. This suggested that nitrate and nitrite were transported by a common transporter in <I>Gloeothece</I>. The rates of nitrate and nitrite uptake were similar, but the uptake of ammonium was much faster than that of either nitrate of nitrite. In contrast to the uptake systems, systems of nitrate and nitrite reductions were substrate-inducible. Ammonium, either generated intracellularly or supplied exogenously, was assimilated via the GS-GOGAT pathway. Nitrite and ammonium inhibited N<SUB>2</SUB> fixation rapidly. Nitrate inhibited N<SUB>2</SUB> fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of nitrate and ammonium could be prevented by L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N<SUB>2</SUB> fixation was an assimilatory product of ammonium rather than ammonium or nitrate itself.

Two sets of field experiment were conducted at the Lincoln University's agroforestry trial. The first experiment was to study nitrogen (N) uptake by radiata pine and pasture, and soil total N changes with time. The second experiment was to assess the magnitude of input from biological nitrogen fixation (BNF) and factors affecting BNF. Lucerne was found to be the most severe competitor with trees. It reduced tree height, root collar diameter and diameter at breast height, and occasionally reduced fascicle dry weight and foliar N content. However, lucerne had a high dry matter yield (DMY), nitrogen concentrations, nitrogen yields, and amounts of nitrogen fixed. It had lower percentage of N derived from atmosphere (%Ndfa) than clover which resulted in a high N removal from the lucerne plot, when the herbage was removed as silage. Clover has high %Ndfa during spring and summer ranging from 83 to 97%. Radiata pine did not affect total N concentration of pastures and %Ndfa of the legumes. However, radiata pine reduced seasonal DMY of the pastures and seasonal and annual DMY of legumes, which led to the reduction of N yield and amount of N fixed. Clovers in ryegrass/clover, cocksfoot/clover and phalaris/clover were estimated to fix 134,71, and 75 kg N ha ⁻¹ year ⁻¹ which were lower than lucerne which was estimated to fix 230 kg N ha ⁻¹ year ⁻¹. The variations of amounts of N fixed by clover in different grass/clover mixtures were due to the persistence and productivity of the clover in pasture mixtures. Nitrogen balance in all pasture treatments was negative showing that N removal in herbage exceeded N input from BNF. Similarly, the total N in the soil decreased with time. Biological nitrogen fixation was important to stabilise N balance in pasture by minimising soil N removal and to ensure a high pasture productivity. Soil moisture and N were likely to be the important resources competed for by pasture plants and the trees. However, the effect of competition was more apparent on altering N status of the trees than that of the pastures. The N status of radiata pine grown with pastures was occasionally marginal. Soil moisture content close to the row of trees was lower than that at the midway between two rows of trees. Rain shadow effect from trees further lowered the moisture content of soil to the north side of trees. Overall the use of ¹⁵N isotope dilution technique for measuring %Ndfa and percentage of grass N derived from transfer (%Ndftrans) has given satisfactory results. Nitrogen transfer from clover to ryegrass/clover was considered as insignificant (<1.5 g m ⁻² annually). The atom % ¹⁵N enrichment in the soil decreased with time.

Dry Beans (Phaseolus vulgaris L.), is the second-most important grain legume, and North Dakota has ranked first in dry bean production. The overarching research question of my project is whether we can increase the nitrogen (N) fixing potential of dry bean cultivars with inoculation and compare the nitrogen (N) fixing potential of common dry bean cultivars. Field experiment was conducted to compare peat and liquid inoculants during 2016 and 2017 growing seasons. In the field study, N2 fixation was estimated by stable isotope (15N) dilution technique. Liquid inoculant (70.5?5.9 kg ha-1) showed similar potential to the peat based inoculant (60.5?4.7 kg ha-1) for N2 fixation, however, inoculation treatments did not increase the N2 fixation over uninoculated (control). Further, it was investigated the nifH gene (marker for N2 fixation) expression in the dry bean-Rhizobium phaseoli symbiotic system. It was found that the relative normalized nifH gene expression significantly correlates (r = 0.82) with the total amount of N2 fixed, indicating the genetic control of symbiotic efficiency. Study on N-assimilatory genes, NR for nitrate reductase and GS for glutamine synthetase, showed that N2 fixation alone could not support plant N need in the later stages of growth (i.e. late flowering) and supplementary application of mineral-N is necessary for better plant growth and economic-yield. Dry bean cultivars did not response to inoculation and genetically, they differed significantly in N2 fixation potentials.<br>Specialty Crop Block Grant

The growth and Nitrogen fixation by one low salt tolerant alfalfa (Medicago sativa L.) and two germination salt tolerant selections inoculated with were investigated at two salt levels (0, -0.6 Mpa) and two N rates (1, 5ppm) using a system which automatically recirculates a nutrient solution. The high level of salinity (-0.6 Mpa osmotic potential of culture solution) resulted in substantial reduction in the N fixation percentage and total fixed N. The effect of salinity was more pronounced for later cuttings than for the earlier cutting. The N fixation percentages were substantially decreased by increasing N level and the reduction was enhanced by time. The N treatment levels did not exhibit a significant effect on total fixed N. Cultivars did not differ in either growth or N fixation. However, the interaction of N and salinity significantly decreased the percentage and amount of N fixation.

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Normal"<br>mso-tstyle-rowband-size:0<br>mso-tstyle-colband-size:0<br>mso-style-noshow:yes<br>mso-style-priority:99<br>mso-style-qformat:yes<br>mso-style-parent:""<br>mso-padding-alt:0in 5.4pt 0in 5.4pt<br>mso-para-margin-top:0in<br>mso-para-margin-right:0in<br>mso-para-margin-bottom:10.0pt<br>mso-para-margin-left:0in<br>line-height:115%<br>mso-pagination:widow-orphan<br>font-size:11.0pt<br>font-family:"Calibri","sans-serif"<br>mso-ascii-font-family:Calibri<br>mso-ascii-theme-font:minor-latin<br>mso-fareast-font-family:"Times New Roman"<br>mso-fareast-theme-font:minor-fareast<br>mso-hansi-font-family:Calibri<br>mso-hansi-theme-font:minor-latin<br>mso-bidi-font-family:"Times New Roman"<br>mso-bidi-theme-font:minor-bidi<br>} </style> <![endif]--> <p class="MsoNormal"><span style="font-size: 12pt<br>line-height: 115%<br>font-family: &quot<br>Times New Roman&quot<br>,&quot<br>serif&quot<br>">The heterogeneous photocatalytic oxidation of dinitrogen to nitrate, NO3 and/or nitrite, no2 using peroxy species of titanium (IV) in aqueous suspensions has been investigated. The photocatalysts used were titanium peroxide and Degussa p25 TiO2 pretreated with H2O2. These photocatalysts were investigated by SEM and FTIR spectroscopy. <span style="">&nbsp<br></span><o:p></o:p></span></p> <span style="font-size: 12pt<br>font-family: &quot<br>Times New Roman&quot<br>,&quot<br>serif&quot<br>"><o:p></o:p></span></p> </p>

Population pressure on food production motivates the search for new ways to increase the productivity of arable land, especially land rendered marginal by salinity or aridity. The global thesis motivating this work is that nanotechnology can benefit agriculture. My specific thesis is that that part of nanotechnology concerned with nanoparticle production can benefit soybean yield. I have focused on symbiotic nitrogen fixation, and systematically investigated the effects thereon of magnetite nanoparticles introduced into the rhizosphere. My main finding is that the presence of these nanoparticles increases nodulation - both the number of nodules and the size of individual nodules. Since the experiments were carried out on plants provided with minimal nutrients, there was no corresponding increase in vegetative growth. Some evidence was obtained for the nanoparticles enhancing the "molecular dialogue" between soybean root and the Bradyrhizobia that become incorporated in the nodules. A secondary finding is that the nanoparticles enhance the growth rate of Bradyrhizobia in culture, which is advantageous for the preparation of inocula. Furthermore, coating soybean seeds with nanoparticles and Bradyrhizobia prior to planting enhances survival of the bacteria, and therefore increases the efficiency of subsequent nodulation.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2009.<br>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Submitted. Härtill 4 uppsatser.

Rhizobia are soil-dwelling bacteria capable of infecting legume roots and forming a symbiotic association. Inside root nodules, low O2 levels trigger expression of the nitrogenase enzyme complex in rhizobia, allowing them to fix atmospheric N2 into NH3, and secrete this reduced nitrogen source to the host plant. Rhizobial N2 fixation is activated via the transcriptional regulator NifA, which is itself induced by low O2 concentration in legume root nodules, most commonly via the two-component sensor-regulator FixLJ or a modified version of this regulon. For rhizobia in the genus Mesorhizobium, it is currently unclear how low O2 tension is sensed and transduced into a signal to activate N2 fixation, with the FixLJ system shown to be dispensable for N2 fixation. Mesorhizobium ciceri CC1192, which forms a symbiosis with Cicer arietinum (chickpea) and is the commercial inoculant for this crop legume in Australia, harbours fixV, a putative LacI/GalR transcriptional regulator which may directly control the expression of nifA in this strain. To investigate the role of fixV and nifA within CC1192, inactivation vectors were constructed in the suicide vector pJQ200SK via Gibson Assembly and fixV and nifA deletion mutants generated, along with a nitrogenase null mutant (nifH) as a non-N2-fixing control. nifH mutants were symbiotically defective on C. arietinum, with nitrogenase activity completely abolished. Deletion of fixV resulted in reduced, but not abolished, nitrogenase activity on a per nodule basis and reduced foliage dry weights, while the ΔnifA mutant phenotype remains to be tested. FixV, therefore, is not essential for N2 fixation in CC1192 but likely plays a vital role alongside another as yet unidentified regulator.

Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.

Bibliography: leaves 186-209.<br>xiii, 218 leaves : ill. (some col.), 1 col. map ; 30 cm.<br>Title page, contents and abstract only. The complete thesis in print form is available from the University Library.<br>Thesis (Ph.D.)--Adelaide University, Dept. of Agronomy and Farming Systems, 2000

Nitrogen (N) fixation by white clover (Trifolium repens L.) was measured in the ley phases of three experimental organic ley-arable rotations at two sites, between 1997 and 2000, using the 15N dilution technique. Soil nitrate-N, ammonium-N, and total soluble N were measured at one of the sites between 1999 and 2001. Variation of soil N was assessed in different ages of ley, year, month, rotation, site and microsite type (grass or clover). Data for soil pH, organic matter, P, K, and Mg, was also compared, as was weather data for the period. Variation of N fixation, grass and clover dry matter, proportion of nitrogen derived from the atmosphere, grass and clover N concentrations, grass and clover non symbiotic N-yield, and N transfer from clover to grass were compared in different ages of ley, years, months, rotations and sites. Flowering rates and presence of clover tap-roots were also examined. Above ground N fixation varied between 26 and 75 kg ha'1 over the course of the growing season. Estimated total N fixation ranged from 70-214 kg ha'1. N fixation was closely related to yield of clover. There were clear relationships between soil nitrate and the behaviour of the grass and clover components of the leys. Nitrate-N was slightly higher under clover patches than under grass patches in mid-late summer. White clover and perennial ryegrass were grown in circular chambers, divided radially, and their invasion into microsites with different vegetation, different management and different N treatments was observed. Ryegrass was not capable of rapidly colonising microsites by vegetative spread alone. Clover was more likely to successfully invade microsites containing grass with no added N than microsites with added N, or bare soil microsites.

When cultures of <i>Gloeothece</i> grown under constant light, were transferred to dark anerobic conditions, the rate of acetylene reduction dropped within 1h to 25-30% of that in the light, and reached a minimum after 10-12h. The only source at ATP for nitrogenase, under such conditions, is substrate level phosphorylation utilising endogenous material. All attempts to support N<SUB>2</SUB>-fixation by an exogenous source of energy proved unsuccessful. Although <i>Gloeothece</i>was able to fix nitrogen under dark anaerobic conditions, no nitrogenase synthesis was observed. <i>Gloeothece</i> survived under dark anaerobic conditions for at least 96h, but they appeared to lose the ability to protect nitrogenase against inactivation by O<SUB>2</SUB> over this period. Thus, when cultures of <i>Gloeothece</i> maintained under dark anaerobic conditions for more than 12h, were transferred to the light under anaerobic conditions, the nitrogenase activity which subsequently appeared was oxygen-sensitive. ATP is a necessary source of energy for N<SUB>2</SUB>-fixation. On the other hand, ADP inhibits nitrogenase activity. Therefore, the relative amounts of ADP and ATP may control nitrogenase activity. A routine method for measuring adenine nucleotides in <i>Gloeothece</i>, based on the luciferin-luciferase technique, was developed as existing methodology was found unsuitable. Under constant illumination, the observed fluctuations in nitrogenase activity in <i>Gloeothece</i> were not found to be related to changes in the ADP/ATP ratio. However, the ADP/ATP ratio had an indirect effect, possibly <i>via</i> synthesis, on the nitrogenase activity in <i>Gloeothece</i> grown under an alternating cycle of 12h light and 12h darkness. On the other hand, acetylene reduction was inversely related to the ADP/ATP ratio when cultures of <i>Gloeothece</i> were transferred from dark anaerobic conditions to dark aerobic and then to light aerobic conditions.

Morphological and growth observations made on landraces of African yam bean (AYB) used in this study confirm that this species is the most morphologically variable in the genus (Potter, 1992). Morphological characters such as seed colour, stem colour, internode length, leaf size and number of leaves per plant were found to vary between landraces. Growth and development was controlled by both genotype and environment. Flowering was observed only when plants were grown at 25°C, rather than at 30°C, with a 12 h photoperiod. Tuber formation occurred only in AYBS and not in other landraces. Growth rates differed between landraces and between environments with plants grown in the soil displaying faster growth than those grown in pots. The response to the environment (pot and soil experiment) differed between landraces, i.e. AYB1 performed better than AYB2 in the pot experiment, whereas it was surpassed by AYB2 in the soil experiment. Clonal propagation protocols were developed using nodal explants/propagules to reproduce material with a high level of genetic uniformity from existing shoot meristems. Clonal propagation was investigated using macro (leafy stem cuttings) and micro (in vitro propagation from nodal stem segments) approaches. Axenic shoot cultures have been achieved from stem nodal segments sterilised with 10% "Domestos" bleach and grown in MS-based medium fortified with cytokinins. Amongst the cytokinins used, BAP (6-benzylaminopurine) was found to be more suitable than TDZ (N-phenyl-N'-1,2,3thidiazol-5-ylurea) and 2iP (6-(y, y-dimethylallylamino)purine) at both culture establishment and shoot multiplication stages, although optimisation of the protocol for shoot multiplication requires further study. There was persistent callus proliferation at both the establishment/initiation of cultures and the multiplication stage and the use of other plant growth regulators, such as GA3 (Gibberellic acid) and TIBA (2,3,5-triiodobenzoic acid), known to counter callus growth in cultures, did not give positive results. Although in vitro adventitious root formation was erratic, some shoots were able to root when exposed to auxins (IBA [indole-3-butyric acid] and NAA [αnaphthaleneacetic acid]) and were established in compost. IBA was preferable to NAA, as it induced more root formation. Overall, AYB cuttings produced adventitious roots relatively easily with or without auxins. Auxins at low concentrations induced rapid formation of roots in high numbers. Unlike in vitro rooting, adventitious rooting of cuttings was as high as 100% without any auxin treatment, suggesting a possibility of other factors involved in the rooting process in vitro. A cheap source of nitrogen for AYB is in the form of biological nitrogen fixation. AYB nodulated profusely with strains of both a slow growing Bradyrhizobium sp. and a fast growing Rhizobium sp., plants forming nitrogen fixing nodules with strains ORS302, CP279 and NGR234. Nitrogen fixed from the atmosphere accounted for 79-98% of the plant nitrogen and supported plant growth by an increase of up-to 1547% of dry matter in shoots.

In order to study the influence on blue-green algal nitrogenase activity of environmental variables in deepwater rice-fields (DWR), a laboratory study was planned on a DWR isolate of Calothrix (D764). The variables chosen were light, oxygen, combined nitrogen, phosphorus and iron. As availability of P is likely to play an especially important role for growth and nitrogen fixation in DWR, studies on phosphatase activity of the isolate were also included. The method used for measuring nitrogenase activity was acetylene reduction assay (ARA). In order to convert nitrogenase activity to nitrogen fixation, the conversion ratio of N(_2) : C(_2)H(_2) reduced was determined by comparing the total amount of N fixed with total C(_2)H(_2) reduced. The ratio was 1 : 4.1 and 1 : 5.2 at 85 and 10 µmol photon m(^-2) s(^-1), respectively. Changes in nitrogenase activity in batch culture were studied in relation to growth characteristics. Maximum activity (10.5 nmol C(_2)H(_4) mg d. wt(^-1) min(^-1)) was observed after two days of growth. During this period, juvenile trichomes (hence maximum heterocyst frequency) were abundant and cyanophycin granules were absent; chl a, phycobiliprotein and algal N decreased. It is suggested that the juvenile filament is the most active nitrogen-fixer during the growth of the alga. The response of nitrogenase to changes in light flux (down- or upshift) was rapid. The alga showed a marked drop in nitrogenase activity in the dark, but subsequent changes were slow, with detectable activity after 24 h. Higher nitrogenase activity was observed when the dark grown alga was re-illuminated, than the maximum activity found under continuous illumination. Nitrogen fixation and heterocyst differentiation were suppressed when 10 mg 1(^-1) NH(_4)-N was added to a batch culture. Fe-deficient cultures had lower nitrogenase activity and N content than Fe-sufficient cultures. Fe- deficiency led to the development of a series of new heterocysts apical to the basal ones. Addition of Fe to Fe-deficient cultures led to a marked increase in nitrogenase activity and loss of the degenerated basal heterocysts. The alga was capable of using a number of organic P substrates as the sole source of phosphorus and showed both cell-bound phosphomono- and phosphodiesterase activities. In batch culture, phosphatase activity was detected when cellular P content dropped to 0.98%. A brief study on the influence of the environmental factors on cell-bound phosphatase activities of the alga has been included. A brief comparison in nitrogenase activity of a UK field Rivularia population and bacterised laboratory isolate Rivularia D403 was made and probable behaviour of algae in DWR is discussed.

The fixABCX genes are essential for nitrogen fixation in R. leguminosarum bv. viciae 3841. Comparison of FixABCX to homologous proteins across the Kingdoms of Life suggests a role in electron transport to nitrogenase, which requires eight electrons per molecule of dinitrogen fixed. Mutation of this operon leads to bacteroids unable to fix nitrogen in symbiosis with P. sativum (pea). Electron microscopy revealed a drastically altered bacteroid morphology in fixAB mutants, revealing insights into the developmental response of both plant and bacteria to a lack of nitrogen fixation. Observations from electron microscopy were coupled to data obtained using single-cell Raman microscopy in order to understand metabolite production in nitrogen fixing and non-fixing bacteroids. The promoter controlling fixA has been characterised to a minimal region consisting of binding sites for NifA, the general transcriptional activator of nitrogen fixation, and RpoN (σ54), its cognate sigma factor. Mutation analysis reveals that fixABCX is part of a larger operon including the nifA gene. Promoter analysis of the downstream genes has identified a set of basal promoters found within the fixCX region, which control expression of the nifA gene. Control of nitrogen fixation occurs at the post-transcriptional level, whereby NifA is able to activate nitrogen fixation genes, including the fixABCXnifA operon, autoregulating its own expression under nitrogen-fixing conditions. Pull-down assays have revealed protein-protein interactions between FixAB and nitrogenase, as well as an interaction with both pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. FixAB may interact with these dehydrogenases and via electron bifurcation couple the exergonic reduction of the quinone pool to the endergonic reduction of ferredoxin and subsequently nitrogenase. Furthermore, FixAB, nitrogenase and pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase may form a supra-molecular complex within nitrogen fixing bacteroids.

Pterocarpus indicus is a promising tropical woody legume for the establishment of forest plantations in Malaysia. Woody legumes that form symbiotic relationships with nitrogen-fixing bacteria also play an important role for forest restoration on degraded land. Although P. indicus has been widely planted as an amenity tree in SE Asia, its silvicultural requirements have not been determined. There are no recommendations for fertilizer or for inoculation with nitrogen-fixing bacteria. This thesis explores the phosphorus (P) and nitrogen (N) requirements of seedlings and identifies a range of nitrogen-fixing bacteria capable of forming root nodules under glasshouse conditions. Four glasshouse experiments were undertaken on two soil types: Yalanbee sandy gravel (YB) and yellow sand (YS) to determine the P and N concentration ranges in the foliage of deficient and healthy plants and to define critical nutrient concentrations for the diagnosis of deficiency. There was a narrow range in rates of P fertilizer, supplied as aerophos, Ca(H2PO4)2.H2O, between deficiency and toxicity in both soil types. The relationship between yield and P concentration in the youngest fully expanded leaf (YFEL) enabled critical P concentrations for the diagnosis of deficiency (0.17%) and toxicity (0.41%) to be determined at 90% maximum yield from linear regressions fitted to the data. The foliar P concentration ranges for deficiency and toxicity were similar to other nitrogen-fixing trees. Only plants in YS responded to inorganic N fertilizer, and soil analysis suggested that seedlings may take up ammonium-N in preference to nitrate-N at luxury supply. A critical concentration for the diagnosis of N deficiency was not able to be determined due to the lack of data points. Plants with adequate N fertilizer had YFEL N concentrations of 2-3.5% dry weight. To determine whether P. indicus is more sensitive to luxury soil P supply than other fast-growing legume trees, the P response of P. indicus in YB was compared with three other woody legumes, Pterocarpus macrocarpus, Acacia mangium and Sesbania formosa. The sensitivity of P. indicus to high P was confirmed and the response shown to be similar to P. macrocarpus. Both species showed severe symptoms of P toxicity, namely leaf necrosis and stunted growth. In contrast, shoot and root yields of A. mangium and S. formosa were not reduced at luxury P supply and yield x fertilizer relationships were able to be fitted to the Mitscherlich model. Critical P concentrations for the diagnosis of P deficiency in A. mangium and S. formosa, derived using the Mitscherlich model, were estimated to be 0.2-0.3% dry weight for the YFELs. Two glasshouse inoculation trials were carried out using diverse strains of root nodule bacteria in order to identify strains suitable for inoculation in the nursery. There were eight strains from Bradyrhizobium, five from Rhizobium, three from Sinorhizobium and two Mesorhizobium strains. P. indicus formed nodules with strains from Bradyrhizobium, Rhizobium, Sinorhizobium and Mesorhizobium, which suggests it is a promiscuous host. Nodules formed were globose, single and of aeschynomenoid type. In the first trial, Bradyrhizobium strain WSM 2096, promoted shoot growth while in the second trial, Bradyrhizobium strain WSM 3712 promoted shoot growth. Growth stimulation was similar to the uninoculated control supplied with inorganic N as KNO3 but was inferior to plants given (NH4)2SO4. The response of P. indicus to low soil P in inoculated and uninoculated plants was studied in a pot trial comprising two P treatments (nil, just adequate). Plants were grown for 3 and 6 weeks. At nil fertilizer P, uninoculated P. indicus seedlings had higher total root length and root dry weight than those with adequate P. Inoculation with WSM 3712 suppressed root growth relative to uninoculated plants. Information gathered in this thesis has application for the production of planting stock in forest nurseries. Firstly, care needs to be taken to ensure that rates of P supplied as hard or liquid fertilizer are not in the range likely to cause toxicity. Secondly, if any starter inorganic N fertilizer is to be used then it should be supplied either as ammonium-N or as urea. Thirdly, more research is required in order to identify effective strains of rhizobia for widespread commercial application. Fourthly, the critical foliar P concentrations identified for the diagnosis of P deficiency and P toxicity can be used to help interpret foliar analysis of seedlings in the future.

The responses of two cowpea (Vigna unguiculata (L.) Walp.) cultivars inoculated with granular inoculum (a mixture of rhizobia) at different levels of applied nitrogen were evaluated. California Blackeye 5 (Ca5) and Knuckle Purple Hull (KPH) cowpeas were examined under field conditions for percent nodulation, nodule number, and nodule mass per plant, and nitrogenase activity at various times during the 1983 and 1984 growing seasons. Data were also obtained for shoot and root dry weights, seed yield, and protein content. Significant differences between inoculated and uninoculated plots were found at all nitrogen treatments for both cultivars with respect to nodulation percentage, nodule number, nodule mass, and nitrogenase activity. An inverse trend linear and quadratic was noted between nitrogen increments and nodulation (nodule number and nodule mass) of Ca5 and KPH cowpeas. High N (168 kg N ha('-1)) was more inhibitory to nodulation than low (28 kg N ha('-1)) N applied. Nitrogenase activity of both cultivars was significantly stimulated with low and inhibited by high levels of ammonium nitrate. Effects of nitrogen treatments on nodulation and nitrogenase activity were influenced by the stage of growth and cowpea cultivar. At the pod-fill stage, higher nodule numbers and nitrogenase activity were recorded for Ca5 as compared to KPH cowpeas. Morphological differences, with Ca5 plants being erect with few vines, whereas KPH plants were semi-erect and bushy, were observed between inoculated and uninoculated plants of Ca5 and KPH cowpeas at all levels of applied nitrogen. Dry weights of vegetative components of both cultivars were significantly improved when low (28 kg N ha('-1)) rather than high (168 kg N ha('-1)) nitrogen was added. Inoculation significantly increased seed yield of Ca5 cowpeas. When conditions were favorable seed yield of inoculated Ca5 exceeded that of inoculated KPH in all comparisons within the same increments of applied N. Seed yield of both cultivars was adversely affected when high levels of nitrogen were applied. Seed, leaf, petiole, and stem protein content were significantly higher with respect to inoculated than uninoculated plots. However, nitrogen fertilizers tended to decrease protein content of the measured parameters for Ca5 and KPH cowpeas.