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Symbiotic nitrogen fixation is the greatest potential input of nitrogen into terrestrial ecosystems. As a result, nitrogen fixation is critical to the functioning of the land carbon sink and its capacity to offset anthropogenic CO2 emissions and climate change. However, our understanding of the controls over nitrogen fixation rates and nitrogen fixing tree abundance is limited, resulting in paradoxes such as the relative absence of nitrogen fixing trees at high latitudes (where nitrogen is most limiting and it seems that nitrogen fixation should be most beneficial) and tropical forest nitrogen saturation, a mechanistically poor representation of nitrogen fixation in terrestrial biosphere models, and incomplete theory for variation in the successional trajectories of nitrogen fixing trees. This dissertation consists of four chapters that examine the drivers of symbiotic nitrogen fixation rates and the abundance of nitrogen fixing trees as they pertain to latitude, climate, and nitrogen fixation strategies. In chapter 1, I develop a method to measure coupled nitrogen fixation and plant carbon exchange in real-time, non-destructively, continuously, and at the whole plant scale. This permits a study of the controls of nitrogen fixation rates over timescales that range from seconds to months. In chapter 2 and 3, I apply the method developed in chapter 1 to determine the temperature response of nitrogen fixation rates and the timescales over which nitrogen fixation is regulated. For chapter 2 and 3, I grew nitrogen fixing tree species of tropical and temperate origin and representing the two types of nitrogen fixing symbioses (rhizobial and actinorhizal) across a 10 °C gradient of growing temperatures. In chapter 2, I show that nitrogen fixation depends on growing temperature and geographic origin and peaks at 30-38 °C, which is 5-13 °C higher than previous estimates based on other nitrogen fixing symbioses and 3-7 °C higher than net photosynthesis. These findings have direct implications for how nitrogen fixation is represented in terrestrial biosphere models and are in direct contrast to terrestrial biosphere model predictions of a decline in tropical nitrogen fixation with warming associated with climate change. In chapter 3, I show that nitrogen fixation takes 1-3 weeks to be down-regulated by 50% following an alleviation of nitrogen limitation, 1-5 weeks to be up-regulated by 50% following the initiation of nitrogen fixation when nitrogen becomes limiting, and up to 4 months for nitrogen fixation to start following a drastic reduction in soil nitrogen supply. Theory says that time-lags in regulating nitrogen fixation start becoming important for plant competition and losses of available nitrogen from ecosystems if they are between 1 day and 1 week. Thus, time-lags on the order of multiple weeks are a significant cost of a facultative nitrogen fixation strategy and resolve the tropical nitrogen forest nitrogen paradox characterized by high losses of available nitrogen at the ecosystem scale in spite of down-regulation of nitrogen fixation at the individual scale. In chapter 4, I show that nitrogen fixing tree abundance is bimodal in all regions of the contiguous United States except the Northeast and that founder effects can explain this pattern and the persistence of nitrogen fixing trees in old forests. Using theory, I show that founder effects are most probable at intermediate soil nitrogen supply, when nitrogen fixers have a high relative capacity to uptake available nitrogen, and when nitrogen fixing trees are facultative in their nitrogen fixation strategy. These chapters provide a new tool for studying nitrogen fixation, critical data for improving terrestrial biosphere models and our understanding of how nitrogen fixation and nitrogen cycling varies across latitude and how it will change with climate change, and new theory for the successional trajectories of nitrogen fixers.

Mälaren är Sveriges tredje största sjö och även den dricksvattentäkt som försörjer flest människor i Sverige med vatten. Ett hot mot Mälarens vattenkvalité är algblomningar då vissa cyanobakterier producerar giftiga cyanotoxiner. Under 60-talet ledde övergödningen i Mälaren till kraftiga algblomningar och för att kunna råda bot på problemet infördes ett övervakningsprogram som nu har följt Mälarens utveckling i snart 60 år. I denna studie undersöktes med hjälp av detta övervakningsprogram vid vilka förhållanden cyanobakterier, framförallt de kvävefixerande Nostocales, frodas i Mälaren. Därefter önskades slutsatser kunna dras om vilka åtgärder som kan vara lämpliga för hålla nere populationen och därmed inte utgöra en risk för dricksvattenkvalitén.  Först genomfördes en långtidsstudie för att analysera vilka parametrar som historiskt har haft den tydligaste kopplingen till biovolymen växtplankton, cyanobakterier och Nostocales med hjälp av en multivariat regressionsmodell (PLS). Från detta framgick att de undersökta parametrarna kunde förklara 29 % av variationen i biovolymen för växtplankton, 41 % för cyanobakterier och 45 % för Nostocales. För växtplankton var pH och siktdjup de parametrar som kunde förklara störst del av variationen i biovolym och för cyanobakterier och Nostocales var istället vattentemperaturen, oorganiskt kväve/totalfosfor samt halten oorganiskt kväve de parametrar som hade tydligast koppling till biovolymen.  Därefter genomfördes en säsongsstudie för att kunna förklara nutida halter och säsongsvariationer för fem av Mälarens mätstationer (Ekoln, Galten, Granfjärden, Görväln och Södra Björkfjärden). Variationen av halten cyanobakterier kopplades närmast till temperaturen eftersom cyanobakterierna till stor del var begränsade till sommar och sensommar. Övriga växtplankton kunde däremot förekomma i höga halter även under våren. Att Ekoln hade betydligt högre halter oorganiskt kväve än Galten skulle kunna förklara skillnaden i andelen Nostocales mellan de två mätstationerna, där Galten hade en stor andel Nostocales och Ekoln en låg andel. Dock kunde kvävehalten inte förklara skillnader i fördelning mellan kvävefixerande och icke kvävefixerande cyanobakterier för de andra mätstationerna. Galtens stora tillrinning, grunda förhållanden och artrikedom skulle även kunna förklara bassängens höga halter av växtplankton under hela året.  Då temperaturen visat sig vara den parameter med tydligast koppling till cyanobakteriernas tillväxt är att begränsa den globala uppvärmningen den viktigaste åtgärden för att förhindra en ökad algblommning i framtiden. Kring resterande parametrar var slutsatser svåra att dra och i och med osäkerheten i frågan är en fortsatt noggrann miljöövervakning och handlingsplan när problemen väl uppstår av stor vikt. / Lake Mälaren is the third largest lake in Sweden and the source of drinking water for two million people. A threat to the water quality of Lake Mälaren is algal blooms since some cyanobacteria produce toxic cyanotoxins. In the 1960s, eutrophication in Lake Mälaren led to heavy algal blooms and in order to handle the problem, a monitoring program was introduced that has now followed Lake Mälaren's development for almost 60 years. In this study, the conditions under which cyanobacteria, especially the nitrogen-fixing Nostocales, thrive in Lake Mälaren was analysed using data from the monitoring program. Thereafter the goal was to draw conclusions regarding which measures may be needed to keep the population down so as not to pose a risk to drinking water quality. To begin with, a long-term study was conducted to analyse which parameters have historically had the clearest connection to the biovolume of phytoplankton, cyanobacteria and Nostocales using a multivariate regression model (PLS). This showed that the examined parameters could explain 29 % of the variation in phytoplankton biovolume, 41 % of the variation in the cyanobacteria biovolume and 45 % of the variation in Nostocales biovolume. For phytoplankton, pH and water transparency were the parameters that could explain most of the variation in biovolume, and for cyanobacteria and Nostocales, water temperature, the ratio between inorganic nitrogen and total phosphorus and the content of inorganic nitrogen were the parameters most clearly linked to the biovolume. Thereafter, a seasonal study was conducted to help explain current levels and seasonal variations for five of Lake Mälaren's measuring stations (Ekoln, Galten, Granfjärden, Görväln and Södra Björkfjärden). The variation of the cyanobacteria level was most closely linked to the temperature, as cyanobacteria was mostly limited to the summer. The ratio between inorganic nitrogen and total phosphorus and the content of inorganic nitrogen seemed to be an explanation for the difference between the high share of Nostocales in Galten where nitrogen was scarce, and the low proportions in Ekoln where nitrogen was abundant, but did not explain the distribution between nitrogen-fixing and non nitrogen-fixing cyanobacteria in the other basins. Galten's large inflow, shallow conditions and species richness could also explain the basin's high levels of phytoplankton throughout the year. With temperature as the parameter most clearly linked to the content of cyanobacteria, limiting global warming is the most important of measures to prevent increased algal blooms in the future. With regard to the remaining parameters, conclusions were difficult to draw and due to the uncertainty in the matter, continued close environmental monitoring and an action plan once the problems arise are of great importance.

In the Himmerfjärden Bay a large excess of nitrogen over phosphorus in the discharge from a large sewage treatment plant (STP) has suppressed growth of diazotrophic cyanobacteria in its inner parts. Implementation of nitrogen removal in the STP in 1997 drastically reduced nitrogen load and triggered growth of diazotrophs, mainly Aphanizomenon sp. This study is part of a long-term series of experiments with the overall aim to test how algal biomass and production in a receiving area can be reduced, without stimulating nitrogen fixation and biomass growth by diazotrophic cyanobacteria. Nitrogen removal was discontinued in the STP during two years (2007-8) and resumed in 2009, and the discharge shifted from 25 to 10 m depth, above the seasonal pycnocline. Cellular 15N showed that N2 was the most important N source for diazotrophic cyanobacteria, and that uptake of combined nitrogen was insignificant. As biomass was declining and at the end of the productive season, we could detect a small, but significant, increase in cellular δ15N at the inner bay stations (H3 and H4). However, this coincided with an increased proportion of Anabaena spp. of the total diazotrophic biomass. This may indicate that Anabaena spp. has a higher uptake of combined nitrogen compared with Aphanizomenon sp. or that declining populations of Aphanizomenon sp. take up combined nitrogen. We also found no evidence of uptake of combined nitrogen during the winter months when nitrogen supply is ample and Aphanizomenon sp. is devoid of heterocysts. During the first half of summer (week 21-27) heterocyst frequencies were higher at the outer stations B1 and H2, compared to the inner bay stations (H4 and H5). The lower frequencies at the inner bay stations are likely due to the reduced growth rate suffered by the Aphanizomenon sp. due to stronger competition for phosphorus by non-diazotrophs at these stations and hence lower need for heterocysts. Towards the end of summer conditions even out along the bay, as the surplus phosphorus from the spring bloom is used up at the outer stations and no heterocyst gradient is present. Heterocyst frequency varied significantly over the summer, with minimum values in the beginning of July, coinciding with the highest water temperatures, and higher frequencies in early and late summer. We suggest this is primarily due to a more efficiently functioning nitrogenase enzyme at high temperatures with a reduced need for “expensive” heterocysts. Spring heterocyst differentiation occurred around 4-6 weeks after depletion of dissolved inorganic nitrogen (DIN) and only when water temperature was 5-9 oC and a pycnocline established. It seems that temperature and light in concert will initiate growth, leading to an internal nitrogen deficiency which starts heterocyst differentiation. / Himmerfjärden eutrophication study

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

Thesis: Ph. D., Joint Program in Physical Oceanography (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 155-162). / This work concerns the nitrogen fixation and abundance of Trichodesmium colonies in the western subtropical-tropical North Atlantic and their connections with physical processes. Data were collected in fall 2010 and spring 2011, primarily using the Video Plankton Recorder (VPR). A data processing procedure for estimating the abundance of rare taxa was devised to take advantage of the accuracy of manual classification and the effort savings of automatic classification. The procedure entails selecting a subset of the original dataset, classifying it with automated software, and then manually correcting each classification. The method was validated through comparisons with fully classified VPR data and with abundance data based on microscopic enumeration on preserved samples. Correlations of Trichodesmium colony abundance with the eddy field emerged in two subsets of the VPR observations. In fall 2010, local maxima in abundance were observed in a series of cyclones. We hypothesized Ekman transport convergence/ divergence in cyclones/anticyclones as a driving mechanism. We investigated the process using idealized three-dimensional models of buoyant colonies in eddies. Elevated abundances in anticyclones in spring 2011 were correlated with anomalously fresh water, suggesting riverine input as a driver of the relationship. Finally, we evaluated the hypothesis of Davis and McGillicuddy (2006) that Trichodesmium nitrogen fixation in the North Atlantic may be underestimated by conventional sampling methods, based on their VPR observations of higher than expected colony abundances at depth in the subtropical North Atlantic. A bio-optical model was developed based on carbon-normalized nitrogen fixation rates measured in fall 2010 and spring 2011 and used to estimate nitrogen fixation over the VPR transects. Estimates of abundance and nitrogen fixation were similar in magnitude and vertical and geographical distribution to estimates compiled in a database by Luo et al. (2012). In the mean, VPR-based estimates of volume-specific nitrogen fixation rates at depth in the tropical North Atlantic were not inconsistent with estimates derived from conventional sampling methods. Based on this analysis, if Trichodesmium nitrogen fixation is underestimated, it is unlikely that it is attributable to underestimation of deep colony abundances due to mechanical disturbance during net-based sampling. / by Elise Marie Black Olson. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19778号 / 農博第2174号 / 新制||農||1041(附属図書館) / 学位論文||H28||N4994(農学部図書室) / 32814 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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