In oligotrophic systems, where primary production is low and nitrogen is in short supply, nitrogen fixation process is intense. Although a few diazotrophs (eg. Trichodesmium) have been widely-studied, the rest of the diazotrophic community is still poorly understood. Furthermore, the global distribution of diazotrophs are yet to be clearly resolved. This dissertation assessed the distribution of diazotrophs in oligotrophic systems, particularly in the tropical and subtropical oceans, using genomics tools including next-generation sequencing. We first tested out a pair of nifH-specific primer that previously performed well in silico, but found that its application on seawater samples was biased towards paralogous, non-functional nitrogenase nifH genes. Instead, we found that the use of a nested PCR method using different primers sets to be more effective in amplifying functional nifH genes. Trichodesmium sp., UCYN-A and Pseudomonas sp. forms the core of the diazotrophic communities in oligotrophic oceans. Temperature is the primary driver of the abundances and distributions of these organisms in the Pacific, Atlantic and Indian Oceans, as well as in the oligotrophic Red Sea. Trichodesmium tends to dominate warm, surface waters, while UCYN-A prefers cooler environments and dwell in sub-surface waters in the Red Sea. Due to the dominance of Pseudomonas in the large-sized fraction samples, they are believed to be part of the Trichodesmium-associated consortia, although this requires further investigations. We also found non-cyanobacterial species of diazotrophs to be dominant previously-described hotspots of nitrogen fixation, and found evidence for the widespread of alternative nitrogenases (Cluster II). Using the Red Sea as an exemplar for future warming ocean, we found patterns of niche partitioning in the Red Sea diazotrophs, based on their distribution along seasons, latitude and depth. Our one-year observation of Red Sea Trichodesmium population witnessed the collapse of the population at temperatures above 32°C. This dissertation not only improve our understanding of the effects of future rising temperature on the natural populations of diazotrophs, but it also helps to establish a baseline understanding of the structure, spatial and temporal dynamics of Red Sea diazotrophs, which has not been discussed elsewhere.

The remarkable synchrony between changes in temperature recorded in Greenland ice cores and variations in N isotope records from sedimentary cores recovered from the Arabian Sea and the Eastern Tropical North Pacific (ETNP) has provided evidence for teleconnections between changes in marine denitrification in the tropics and climate variations in the northern high latitudes. Changes in tropical denitrification have been attributed to changes in productivity, changes in the source of intermediate waters and the flux of dissolved oxygen to suboxic zones. Variations in marine denitrification and anammox occurring at intermediate depths in proximity to productive continental margins have had profound effects on the N:P ratio of upwelled waters between stadials and interstadials, and may have indirectly affected carbon sequestration in the ocean by changing the balance of nutrients available to primary productivity. Competitive equilibrium, the changing stoichiometric balance of elements available as nutrients and the shorter residence time of N compared to P are factors that are believed to favour diazotrophs (N2-fixing organisms) during interstadials and shift the competitive advantage to non-N2-fixing ecosystems during stadials. This study presents a very high-resolution analysis of sedimentary nitrogen isotope records, phosphorus concentrations and bulk detrital element concentrations from two cores collected along the Pacific Mexican Margin. The results show that the oxygen minimum zone (OMZ) bathing intermediate waters in ETNP is modulated by the interaction of a Northern Hemisphere climate component with the “leakage” of heavy nitrate believed to derive from the Eastern South Pacific (ESP). This southerly component has a more “Antarctic” timing and is similar to records from the Peru-Chile margin. The sedimentary core recovered from the Mazatlan margin shows a “Greenland” timing of millennial-scale events, with reduced upwelling and reduced primary productivity, a less intense OMZ leading to reduced denitrification and a more southerly position of the mid-tropospheric subtropical ridge during stadials. This would have increased the onshore flow of moist air, ultimately leading to increased precipitation along the western Mexican Margin. Interstadials show a reversal of these conditions. In contrast to the Mazatlan core, the N isotope record from the core recovered from the Gulf of Tehuantepec records an element of “Antarctic” timing superimposed on local, millennial-scale variations in denitrification that are more similar in timing to Greenland temperature changes. In addition, the interpretation of observed variations in detrital elements from the Gulf of Tehuantepec highlights latitudinal displacements of the ITCZ that are consistent with those observed in the Cariaco Basin in Venezuela. Bulk P concentrations from both cores suggest that although phosphorite formation in the ETNP during interstadials is not as widespread as previously thought, the very high accumulation rates in the Gulf of Tehuantepec and Mazatlan Margin lead to total Holocene phosphorus burial rates that are up to 4-5 times higher than had been estimated in previous studies. These observations lead to the argument that the ETNP may play a more important role in regulating global P budgets than was previously thought and call for an improved appreciation of the benthic microbial communities that modulate biomes at tropical latitudes.

Nitrogen (N) generally limits primary production across large areas of the world's oceans. Allochthonous inputs of N (i.e., "new" N) via N2-fixing organisms (diazotrophs) are crucial for sustaining primary production and are often associated with net export of organic matter (OM) from surface waters. Diazotroph N (ND) contribution plays an integral role in supporting oceanic food webs and regulating the flux of OM into and through the oceans (e.g., the biological pump). Stable isotope techniques were used to trace the input and movement of new N through oceanic food webs. Laboratory experiments were performed to determine elemental and isotopic shifts of OM exposed to microbial and metazoan processing. δ15N of OM was typically higher when exposed to microbial communities, with no difference in δ15N of OM between experiments incubated at different temperatures (4°C and 25°C). In separate experiments, shrimp digestion did not alter the δ15N of OM through digestion, but the δ15N of macerated OM was enriched in 15N. Both of these experiments provide insight into the mechanisms driving variations in the δ15N of OM in the world's oceans. To assess the role of diazotrophs in oceanic food webs, we used the distribution of δ15N to quantify the relative ND contribution to suspended particle N (PN) and mesozooplankton N biomass (NZOOP) in the subtropical North Atlantic (STNA). Qualitatively, ND contribution was often high for both PN and NZOOP, with the highest contributions occurring in the mixed layer. Our results also indicate higher ND contribution to both PN and NZOOP in the western portion of the basin than in the east. ND contribution to larger mesozooplankton at depth further suggests that migrating mesozooplankton transport ND out of the mixed layer. Quantitatively, ND trophic transfer efficiency was lower than bulk N trophic transfer efficiency, suggesting low assimilation of ND by mesozooplankton. Overall, we estimated a ND pool turnover time on the order of weeks for our region of study. These findings demonstrate that ND is laterally and vertically variable in the STNA, and that the ND pool is sensitive to perturbations on short timescales. We discuss the global implications of our findings and their implications for the N cycle and elemental fluxes through oligotrophic oceans.

The overall aim of the study was to provide better insights to the ecological role and impact of cyanobacteria in Baltic Sea (BS) bay, coastal and open sea areas. Biomass and heterocyst development of diazotrophic, heterocystous cyanobacteria were monitored over several years simultaneously as physical parameters such as nutrients and temperature. Nitrogen fixation was estimated as well as its transfer in the BS food web. Even after decades of debate there is still controversy whether eutrophication of lakes and estuaries/coastal areas should be managed by reducing phosphorus only or also nitrogen. Central to this debate is whether nitrogen fixation by cyanobacteria can replace shortages of combined nitrogen quickly enough to make phosphorus the limiting nutrient and nitrogen removal pointless or even harmful. Also, it is not clear if available combined nitrogen inhibits heterocystous cyanobacterial nitrogen fixation and if it is used for their growth in situ. A large ecosystem-wide experiment started in Himmerfjärden bay in year 1997, where the N-loadings and release depth from a modern sewage treatment plant (STP), located in the inner part of the bay, were modulated. The STP creates a steep gradient of nutrients and stable nitrogen isotopes, which can be used to study uptake of combined nitrogen, as well as biomass development and primary productivity. A 35-year long data series was used to achieve good insights into phytoplankton development and primary productivity in the Baltic Sea over the last couple of decades. These in vivo long time series, based on monitoring data, in combination with shorter series (2-3 seasons, including measurements of colony stoichiometry and stable isotopes), have resulted in a unique meta-dataset, allowing for high-resolution observations into the role of the cyanobacteria in the Baltic Sea ecosystem. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Os objetivos deste estudo foram avaliar a concentração de Cd, Pb, Cr e Ni nos tecidos da cana-de-açúcar, constatar a presença de bactérias fixadoras de nitrogênio (BFN) na variedade IAC87-3396 cultivada em ambiente com altas concentrações de metais e avaliar a tolerância das bactérias metais pesados \"in situ\" e \"in vitro\". A cana-de-açúcar IAC87-3396 foi cultivada em área com altos teores de metais. Antes do plantio foram aplicados 5 mitigadores de metais pesados. Para detecção e quantificação de níquel, chumbo, cromo e cádmio foram realizadas análises em ICP-MS em amostras de tecidos da cana-de-açúcar (folha +1, raízes e tolete pré-germinado) obtidos na colheita da cana-planta e cana-soca. A quantificação das bactérias foram realizadas em amostras da parte aérea e das raízes, da cana-planta (colheita) e da cana-soca (6 meses e colheita), utilizando a metodologia do tolete pré-germinado. O ensaio \"in vitro\" consistiu em acrescentar ao meio de cultura 4 concentrações de Pb, Cd, Ni e Cr inseridos isoladamente e em combinações e proceder a inoculação em placa de petri de 4 bactérias (Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, Azospirillum amazonense e Burkholderia tropica). O comportamento nas diferentes concentrações foi avaliado atribuindo-se valores aos diferentes padrões de crescimento. Para os metais, a maior concentração foi encontrada nas raízes e a menor foi encontrado na folha +1. No campo os valores da folha +1 aumentaram da cana planta para a cana soca e os valores das raízes diminuíram. Comparando campo e tolete pré-germinado, na cana planta a concentração nas raízes foi menor do que o encontrado no campo e na parte aérea o tolete teve uma concentração maior para o Ni e Cd. Na cana soca o campo possui as maiores concentrações tanto para parte aérea como para raízes. Na quantificação das bactérias, a comparação entre os períodos mostrou que a quantificação no geral é maior na cana planta. Na cana planta a quantificação foi maior na parte aérea, no 6 meses da cana soca o predomínio é na raiz e na colheita da cana soca existe uma distribuição mais uniforme das bactérias no interior da planta. No ensaio \"in vitro\" a concentração máxima de metais tolerada foi a máxima estudada para Pb (248 mg L-1), Cd (8 mg L-1) e Ni (40 mg L-1). O Cr e a associação entre 2, 3 e 4 metais proporcionaram o não crescimento das bactérias. Para a maior parte das bactérias houve associação significativa entre os padrões de crescimento e as concentrações de metais no meio de cultura. Na variedade IAC87-3396 foram encontrados os 4 gêneros de BFN, mesmo nas raízes onde os teores de Pb, Ni, Cr e Cd foram maiores. A tolerância das BFN aos metais \"in situ\" diferiu da \"in vitro\". O cromo e a associação entre os metais limitaram o crescimento \"in vitro\" de todas as bactérias. A metodologia do tolete pré-germinado não refletiu as condições de concentração de metais em materiais coletados no campo. A cana-deaçúcar tem potencial para ser uma cultura fitoextratora e acumuladora de Pb, Ni, Cr e Cd / The objectives of this study were to evaluate the concentration of Cd, Pb, Cr and Ni in the tissues of sugarcane, noting the presence of the BFN in IAC87-3396 variety grown in contaminated environment and evaluate the BFN tolerance to heavy metals \"in situ\" and \"in vitro\". Sugarcane variety IAC87-3396 was grown in an soil contaminated by metals. Before planting, five mitigators of heavy metals were applied. Analysis were performed to detect nickel, lead, chromium and cadmium in tissues of sugarcane, including leaf +1, roots and pregerminated sett (shoot and root) at harvest of the plant cane and ratoon cane. To quantify the nitrogen-fixing bacteria, three tests were performed comprising the shoots and roots, one in the plant cane (at harvest) and two in ratoon cane (6 months and harvesting) using the methodology of pre-germinated sett. The test \"in vitro\" consisted of adding to the culture medium Pb, Cd, Ni, Cr, mixed metals and inoculated in a petri dish of 4 bacteria (Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, Azospirillum amazonense and Burkholderia tropica). The behavior in different concentrations was evaluated by assigning values to different growth patterns. For all metals, the highest concentration was found in the roots and the lowest values were found in leaf +1. This values in leaf +1 increased from plant cane to ratoon cane while in root it decreased. In relation to the pregerminated sett, metal concentration in root of plant cane was lower than that found in the field while the leaf had a higher concentration only for Ni and Cd. The field ratoon cane has the highest concentrations for both the aerial and root part. For the quantification of bacteria, the comparison between the measurement periods showed that in general it is greater in the plant cane. The location of bacteria inside the plant revealed that, in the plant cane, it was higher in the shoot. However, for the first six months of ratoon cane the predominance of bacterias was in the root. At harvest of the ratoon crop a more uniform distribution of bacteria was found inside the plant. In the \"in vitro\" essay the maximum concentration studied was the maximum tolerated for Pb (248 mg L-1), Cd (8 mg L-1) and Ni (40 mg L-1). The association between Cr and 2, 3 and 4 metals provided no growth of bacteria. For most of the bacteria a significant association was found between growth patterns and concentrations of metals in the culture medium. All the four kinds of NFB were found in IAC87-3396 even in roots where the levels of Pb, Ni, Cr and Cd were high. The tolerance of the NFB to these metals \"in situ\" is different from that \"in vitro\". The chromium and its association with other metals have limited growth \"in vitro\" of all bacteria. The methodology of pre-germinated sett did not reflect the conditions of metal concentration in material collected in the field. Sugarcane plant has the potential to be a culture of phytoextraction and accumulation of Pb, Ni, Cr and Cd

Os objetivos deste estudo foram avaliar a concentração de Cd, Pb, Cr e Ni nos tecidos da cana-de-açúcar, constatar a presença de bactérias fixadoras de nitrogênio (BFN) na variedade IAC87-3396 cultivada em ambiente com altas concentrações de metais e avaliar a tolerância das bactérias metais pesados \"in situ\" e \"in vitro\". A cana-de-açúcar IAC87-3396 foi cultivada em área com altos teores de metais. Antes do plantio foram aplicados 5 mitigadores de metais pesados. Para detecção e quantificação de níquel, chumbo, cromo e cádmio foram realizadas análises em ICP-MS em amostras de tecidos da cana-de-açúcar (folha +1, raízes e tolete pré-germinado) obtidos na colheita da cana-planta e cana-soca. A quantificação das bactérias foram realizadas em amostras da parte aérea e das raízes, da cana-planta (colheita) e da cana-soca (6 meses e colheita), utilizando a metodologia do tolete pré-germinado. O ensaio \"in vitro\" consistiu em acrescentar ao meio de cultura 4 concentrações de Pb, Cd, Ni e Cr inseridos isoladamente e em combinações e proceder a inoculação em placa de petri de 4 bactérias (Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, Azospirillum amazonense e Burkholderia tropica). O comportamento nas diferentes concentrações foi avaliado atribuindo-se valores aos diferentes padrões de crescimento. Para os metais, a maior concentração foi encontrada nas raízes e a menor foi encontrado na folha +1. No campo os valores da folha +1 aumentaram da cana planta para a cana soca e os valores das raízes diminuíram. Comparando campo e tolete pré-germinado, na cana planta a concentração nas raízes foi menor do que o encontrado no campo e na parte aérea o tolete teve uma concentração maior para o Ni e Cd. Na cana soca o campo possui as maiores concentrações tanto para parte aérea como para raízes. Na quantificação das bactérias, a comparação entre os períodos mostrou que a quantificação no geral é maior na cana planta. Na cana planta a quantificação foi maior na parte aérea, no 6 meses da cana soca o predomínio é na raiz e na colheita da cana soca existe uma distribuição mais uniforme das bactérias no interior da planta. No ensaio \"in vitro\" a concentração máxima de metais tolerada foi a máxima estudada para Pb (248 mg L-1), Cd (8 mg L-1) e Ni (40 mg L-1). O Cr e a associação entre 2, 3 e 4 metais proporcionaram o não crescimento das bactérias. Para a maior parte das bactérias houve associação significativa entre os padrões de crescimento e as concentrações de metais no meio de cultura. Na variedade IAC87-3396 foram encontrados os 4 gêneros de BFN, mesmo nas raízes onde os teores de Pb, Ni, Cr e Cd foram maiores. A tolerância das BFN aos metais \"in situ\" diferiu da \"in vitro\". O cromo e a associação entre os metais limitaram o crescimento \"in vitro\" de todas as bactérias. A metodologia do tolete pré-germinado não refletiu as condições de concentração de metais em materiais coletados no campo. A cana-deaçúcar tem potencial para ser uma cultura fitoextratora e acumuladora de Pb, Ni, Cr e Cd / The objectives of this study were to evaluate the concentration of Cd, Pb, Cr and Ni in the tissues of sugarcane, noting the presence of the BFN in IAC87-3396 variety grown in contaminated environment and evaluate the BFN tolerance to heavy metals \"in situ\" and \"in vitro\". Sugarcane variety IAC87-3396 was grown in an soil contaminated by metals. Before planting, five mitigators of heavy metals were applied. Analysis were performed to detect nickel, lead, chromium and cadmium in tissues of sugarcane, including leaf +1, roots and pregerminated sett (shoot and root) at harvest of the plant cane and ratoon cane. To quantify the nitrogen-fixing bacteria, three tests were performed comprising the shoots and roots, one in the plant cane (at harvest) and two in ratoon cane (6 months and harvesting) using the methodology of pre-germinated sett. The test \"in vitro\" consisted of adding to the culture medium Pb, Cd, Ni, Cr, mixed metals and inoculated in a petri dish of 4 bacteria (Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, Azospirillum amazonense and Burkholderia tropica). The behavior in different concentrations was evaluated by assigning values to different growth patterns. For all metals, the highest concentration was found in the roots and the lowest values were found in leaf +1. This values in leaf +1 increased from plant cane to ratoon cane while in root it decreased. In relation to the pregerminated sett, metal concentration in root of plant cane was lower than that found in the field while the leaf had a higher concentration only for Ni and Cd. The field ratoon cane has the highest concentrations for both the aerial and root part. For the quantification of bacteria, the comparison between the measurement periods showed that in general it is greater in the plant cane. The location of bacteria inside the plant revealed that, in the plant cane, it was higher in the shoot. However, for the first six months of ratoon cane the predominance of bacterias was in the root. At harvest of the ratoon crop a more uniform distribution of bacteria was found inside the plant. In the \"in vitro\" essay the maximum concentration studied was the maximum tolerated for Pb (248 mg L-1), Cd (8 mg L-1) and Ni (40 mg L-1). The association between Cr and 2, 3 and 4 metals provided no growth of bacteria. For most of the bacteria a significant association was found between growth patterns and concentrations of metals in the culture medium. All the four kinds of NFB were found in IAC87-3396 even in roots where the levels of Pb, Ni, Cr and Cd were high. The tolerance of the NFB to these metals \"in situ\" is different from that \"in vitro\". The chromium and its association with other metals have limited growth \"in vitro\" of all bacteria. The methodology of pre-germinated sett did not reflect the conditions of metal concentration in material collected in the field. Sugarcane plant has the potential to be a culture of phytoextraction and accumulation of Pb, Ni, Cr and Cd

Tropical seagrass ecosystems are highly productive and important for sustaining marine life and associated coastal societies. In this study, the diversity and role of nitrogen-fixing cyanobacteria associated with five common seagrass genera in coastal regions of the western Indian Ocean (WIO; Tanzania) were examined, as well as the impact of anthropogenic activities. Cyanobacteria were characterized morphologically and genetically (16S rRNA and nifH gene phylogeny), as were diel variations in nifH gene expression, NifH protein levels and nitrogenase activity. The results revealed that WIO seagrass beds supported rich cyanobacterial diversity and that these represented approx. 83% of total clones obtained (DNA and RNA nifH clone libraries). Non-heterocystous genera, such as Oscillatoria, Lyngbya, Leptolyngbya, Phormidium and Microcoleus dominated, while heterocystous morphotypes such as Calothrix were less frequent and unicellular morphotypes (e.g. Gloeocapsa, Chroococcus and Chroococcidiopsis) were few. Additionally, the phylogenetic analysis revealed several novel uncharacterized cyanobacterial clades. Cyanobacterial composition and nitrogenase activity varied over seasons and between the seagrass species. Day time nitrogenase activity originated primarily from heterocystous phylotypes, while non-heterocystous filamentous phylotypes fixed nitrogen at night. The highest activity in the diel cycle was 358 ± 232 nmol C2H4 g-1 h-1at 09.00 associated with epiphytes of the seagrass Cymodocea. Nitrogenase activity was consistently lower in anthropogenically disturbed (eutrophication) seagrass sites. Such data suggest that diazotrophic cyanobacteria may be a significant source of ‘new’ nitrogen in the often oligotrophic coastal regions of tropical oceans. It is also proposed that the rapid shifts in the cyanobacterial population and function found may also be used as early disturbance indicator in coastal management practices. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. / SIDA SAREC Bilateral Marine Sciences Project / The Swedish Foundation for International Cooperation in Research and Higher Education

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.

Seasonal dynamics of unicellular diazotrophs were investigated in the upstream Kuroshio and the northern South China Sea (SCS). Unicellular diazotrophs had been postulated as an important N2-fixing contributor for the phenomenon of N* in the SCS where abundances of filamentous Trichodesmium and Richelia were scarced. Samples were collected during four cruises between August 2008 and August 2009 in summer (CR1310 and CR910), winter (CR886), and late spring (CR899), respectively. Sampling stations located between 21¢XN-22¢XN and 116¢XE-122¢XE in the upstream Kuroshio off southeast Taiwan and covering the shelf and basin waters of the northern SCS. The abundance of the unicellular diazotrophs was determined using whole-cell immunocytochemical method in which antibody of nitrogenase was used as the probe. Cells containing nitrogenase can be visualized and counted after the antigen-antibody reaction under microscope. Unicellular diazotrophs were classified to four types according to their sizes and shapes. For diameters of those with 1-3 £gm and in coccoid shape are called 1-3 £gm C, diameters of 1-3 £gm and in rod shape are called 1-3 £gm R, diameters of >3-10 £gm and in coccoid shape are called >3 £gm C, and diameters of >3-10 £gm and in rod shape are called >3 £gm R. Surface abundance of the unicellular diazotrophs was highest in winter in both the Kuroshio and the SCS, followed by summer, and was least in late spring. Among four cell types, 1-3 £gm C usually was the most abundant group, followed by 1-3 £gm R and >3 £gm R, and was least for the group of >3 £gm C. The abundances between groups of 1-3 £gm C and 1-3 £gm R were positively correlated. Likewise, the abundances between >3 £gm C and >3 £gm R were positively correlated. However, the total abundance of small cells (1-3 £gm C+R) was not significantly related to the large cells (>3 £gm C+R). During summer and late spring, the abundance of unicellular diazotrophs in the SCS was 1.3-2 times of that in the Kuroshio. However, in winter the abundance in the Kuroshio was 1.2 times of that in the SCS. Surface water temperature was found negatively correlated to the abundance of 1-3 £gm C, >3 £gm C, >3 £gm R, and large cells (>3 £gm C+R), respectively. Significant correlations among surface water temperature and surface chlorophyll a, [NO2+NO3], SRP and N:P ratio implicated that the dynamics of cell abundances could be attributed to the correlated ecological variables of surface water temperature. The dynamics for the abundances of >3 £gm C, >3 £gm R, and large cells (>3 £gm C+R) were suggested to relate with the fluctuation of SRP concentration. Unicellular diazotrophs accounted for 60-90 % of total unicellular cells in terms of cell number. Vertical distributions of unicellular diazotrophs in the Kuroshio and the SCS were in similar trends, with maximum abundance in deep water during summer and late spring, and on surface water during winter.

In the subtropical oligotrophic ocean, nitrogen fixation is an important source of new nitrogen (N) for supporting biological production. Previous studies have found that nitrogen-fixing Diatom-Diazotroph Associations (DDAs) are in high abundance in the intermediate salinity zone of large river plumes such as the Amazon and Mekong rivers, while Trichodesmium spp. becomes more abundant at higher salinities. This recurring pattern in the Amazon River plume suggests that strong salinity and nutrient gradients within the river plumes may lead to a cascade in diazotroph communities. I hypothesized that the Mississippi River, a major source of freshwater, nutrients and sediments to the northern Gulf of Mexico, creates a similar distribution of diazotroph communities. The relationship between large diazotrophs and salinity was examined in samples collected in July 2011 during a flood outflow from the Mississippi River. The dominant DDA, Hemiaulus spp. – Richelia spp., was at greatest abundance (≈31,000 cells L-1) west of the birdfoot delta on the periphery of the plume (≈29 salinity) where bottom water hypoxia was also observed. Trichodesmium spp., a cyanobacterium genus that occurs in both colonial and free trichome morphologies, was abundant at both high (≈35) salinities east of the delta reaching 20+ colonies L-1, as well as in the fresher (≈28) waters of the plume where it reached 3,500 trichomes L-1. Diazotroph distributions were separated east and west of the Mississippi River outflow, with DDAs being most abundant over bottom water hypoxic regions to the west and Trichodesmium spp. in high abundance to the east. The diazotroph – salinity gradient relationships present within the Amazon River plume were not present within the Gulf of Mexico. This study suggests that environmental factors other than salinity, such as nutrients or hypoxia, are influencing the distribution of diazotrophs around the Mississippi River plume. The seasonal hypoxia seen in the Gulf of Mexico with the co-occurring DDA increase could appear in other river systems. / text