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Incorporating white clover into intensive dairy production systems has the potential to increase their sustainability through increased milk output and reduced nitrogen (N) input. To support intensive stocking rates (<2.75 cows/ha) some inorganic N will still be required to support herbage production, particularly in the spring. However, sward white clover content, and hence N fixation has been shown to be suppressed by applications of inorganic N. In this thesis, the effects of varying N application rates on grass-clover swards as well as the effect of those swards on overall herbage production, dairy cow production, dairy cow rumen function and overall farm gate N balance were investigated. White clover inclusion increased herbage production at fertiliser application rates of up to and including 180 kg N/ha/year in the plotbased study described in chapter 4 of this thesis. The results from this demonstrated the negative relationship between increasing N fertiliser application rates and sward white clover content with sward white clover content decreasing as N application rate increased from 0 kg N/ha/year to 240 kg N/ha/year. Clover inclusion was found to increase milk solids production per cow and per hectare. There was no difference in cumulative herbage production between grass-clover swards receiving 150 kg N/ha/year and grass-only and grass-white clover swards receiving 250 kg N/ha/year. This indicated that the N fixing ability of the grass-white clover swards was supporting sward productivity at the lower N regime. Clover inclusion in the diet significantly altered the rumen function of the dairy cows with increased rumen ammonia and total VFA concentrations in the rumen of the grass-clover fed cows over those of the grass only and total mixed ration (TMR) fed cows. The farm gate N balance outlined the environmental benefits of white clover inclusion in grazed swards resulting in improved overall N use efficiency (NUE) and reduced N surpluses in the grass-white clover swards compared to grass-only swards. Overall, this thesis provides evidence that white clover can be successfully incorporated into intensive dairy production systems with benefits in terms of farm output and improved on-farm sustainability, though with both positive and potentially some negative environmental implications. The overall outcome of the project was to provide evidence that it is possible to maintain and benefit from clover inclusion in grass swards that are intensively managed at high N fertilizer rates, if grazing management is of a high standard with good control of pre- and post-grazing heights. The project also identified some knowledge gaps, particularly around achieving good spring sward productivity, differing environmental implications and longer-term stability of the clover system, that provide clear objectives for future research in this area.

In the next half-century the world will face an enormous food deficit due to a raising birth rate and longer life-spans of the global population. By 2050 there will be 9.5 billion people in the world who will require a 69% increase from current total food calories to prevent global starvation. Widespread hunger and starvation are linked directly to the productivity of crop growth and productivity in agriculture, especially in the third world. By 2030, Areas such as sub-Saharan Africa and Australia are predicted to have a 37% and 48% decrease in crop productivity respectively due to global warming. The first 'Green Revolution' increased the world's crop output by an order of magnitude using synthetic nitrogen fertilisers derived from the Haber-Bosch process. A process which last year yielded 180 million tonnes of ammonia, 82% of which was used for the manufacture of nitrogenous fertilisers. Global usage of synthetic nitrogenous fertilisers is increasingly unsustainable and a new approach to increasing crop productivity is required to face the ever-increasing food demands over the next 30 years. In this project we have contributed to the engineering of biofertilisers and plant growth promoting rhizobacteria that could alleviate the use of inorganic fertilisers and chemicals and serve as an economically viable alternative in developing countries. Synthetic biology tools and techniques are employed to engineer two model rhizobacteria. Azorhizobium caulinodans ORS571 and Azospirillum brasilense FP2, two nitrogen-fixing and known plant-associative rhizobacteria, are used as chassis. A suite of synthetic biology tools are characterised for the two rhizobacteria species as exemplars of the toolkit being used for the engineering of other significant rhizobacteria. Advanced logic-gate based genetic circuitry is developed to yield functional genetic circuits that can be engineered with characterised plant growth promotion (PGP) effectors to yield agriculturally-relevant phenotypes. A suite of these PGP mechanisms including auxin biosynthesis, phosphate solubilisation and antifungal compound (DAPG) biosynthesis, are built and characterised in both rhizobacterial strains The novel implementation of a CRISPR interference technology (CRISPRi) is demonstrated in both chassis. Genomic targeting of dCas9 is implemented to control the ammonia assimilation pathway of A. brasilense FP2 with the effect of significant ammonia release into growth media. This work builds the foundations for the future engineering of both logical genetic circuitry and the CRISPRi system with an input from a unique associative crop plant signal. This will allow a rhizosphere-specific phenotypic switch of the rhizobacteria from a fast-growing competitive state to a plant beneficial ammonia secreting state.

Mariner-based insertion sequencing (INSeq) has been used to characterise symbiotic fitness determinants in Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) on a whole genome-scale. An INSeq database has been constructed that defines the mutational phenotypes for 7316 genes (99.7% of the genome) in Rlv3841 across nine in vitro and in planta INSeq conditions, including growth on glucose or succinate at both 21% and 1% O2, growth in the pea rhizosphere, attachment to pea roots and pea nodulation (collaboration). A total of 463 genes were identified to be required for the successful nodulation of pea, and a genetic map was generated identifying the specific stages of symbiosis these genes are required in. There was identification of 55 genes required specifically for growth in the rhizosphere and 101 genes required specifically for root attachment. Analysis of the central carbon metabolism pathways indicated presence of a methylglyoxal pathway in Rlv3841 and suggested that in planta bacteroids might synthesise glycogen for carbon storage via gluconeogenesis of the plant-provided TCA- cycle intermediates. Only one gene, RL2393 (glnB), was found to be uniquely essential for growth under 1% O2 with succinate. RL2393 (glnB) encodes the nitrogen regulatory protein (PII). Mutation of glnB effectively generates a constitutively activated glutamine synthesis response that increases the removal of 2-ketoglutarate from the TCA-cycle to combine with ammonium to form glutamine. This removal of 2-ketoglutarate from the TCA-cycle becomes problematic under low O2, replenishing 2-ketoglutarate requires an additional turn of the TCA-cycle and the generation of NADH, NADPH and FADH2 reductant molecules whose reoxidation is limited by O2-availability. Ammonium assimilation similarly relies on the removal of 2-ketoglutarate. A reoxidation block model was proposed, in which the provision of dicarboxylates under low O2 prevents removal of 2-ketoglutarate from the TCA-cycle and the assimilation of ammonium. This reoxidation block model provides a novel driving force behind ammonium export in symbioses, alongside explaining why legumes always provide TCA-cycle dicarboxylates to fuel N2-fixation in the low O2 environment of the nodules.

A mesocosm experiment was carried out in a Norwegian fjord near Bergen in May 2006, with the main objective being the study of the effects of increasing concentrations of atmospheric CO2 (and associated effects such as increased acidification) on blooms of natural marine coastal plankton. Three mesocosms were bubbled with CO2(g) to achieve a high (~700ppm) CO2 concentration (pH ~7.8) to simulate predicted future conditions as a result of rising atmospheric CO2 concentrations. Another three mesocosms were treated as controls and bubbled with ambient air to represent a near pre-industrial scenario (atmospheric CO2 concentration ~300ppm, surface seawater pH ~8.15). Blooms in the mesocosms were stimulated by the addition of nutrients at a near-Redfield ratio ([N:P] ≈ [16:1]), and scientific measurements and analyses were carried out over the course of the blooms for approximately one month. Of particular interest in this study were the autotrophic plankton. The diversity and activities of these microorganisms under the two treatments was therefore investigated. By designing and using new degenerate primers specifically targeting ‘Green-type’ (Form IA and IB), ‘Red-type’ (Form IC and ID) and Form II RuBisCO, analysis of primary producers was carried out using PCR and either gDNA or cDNA (mRNA) templates from key time points spanning the complete duration of the blooms throughout the mesocosm experiment. Over 1250 novel RuBisCO large subunit sequences have been fully annotated and deposited in the NCBI GenBank® database. These sequences revealed distinct changes in the diversity of primary producers both over the courses of the blooms and between treatments. Particularly striking was the effect of acidification on the community structure of the eukaryotic picoplankton, Prasinophytes. A clade of prasinophytes closely related to Micromonas pusilla showed a distinct preference for the high CO2 conditions; a laboratory-based experiment confirmed the high tolerance of Micromonas pusilla to lower pH. Conversely, a clade related to Bathycoccus prasinos was almost entirely excluded from the high CO2 treatments. Clades of form II RuBisCO-containing dinoflagellates were also abundant throughout the experiment in both treatments. The high similarity of some of these clades to the toxin-producing species Heterocapsa triquetra and Gonyaulax polyedra, and apparent high tolerance of some clades to high CO2 conditions, is perhaps cause for concern in a high CO2 world and demands further research. In parallel with the RubisCO work, new primers were designed that target the gene encoding the Fe protein of nitrogenase (NifH). 82 Bergen genomic nifH sequences have been annotated and submitted to GenBank®. These sequences include those from organisms related to Alpha, Beta, and Gammaproteobacteria, and Cluster II and Cluster III sequences that align most closely with anaerobic Bacteria, Gram positive, and/or sulphur-reducing Bacteria. The biggest surprise, however, was the apparent abundance and significance of a Rhodobacter sphaeroides-like microorganism throughout the duration of the experiment in both treatments. Whilst this clade was unsurprisingly absent in the RuBisCO cDNA libraries, all but two of 128 nifH cDNA clones analysed were identical to the gene from Rhodobacter sphaeroides. This shows that this clade was potentially fixing N2 throughout the entire experiment, even in the presence of combined N added to both sets of mesocosms at the start of the experiment. A group of Rhodobacter sphaeroides-like microorganisms present at Bergen may therefore have been an unexpected source of new N during the experiment and contributed to the maintenance of the mesocosm communities as nutrients became depleted. One organism dominated the autotrophic communities after the blooms in both treatments. Synechococcus spp. Form IA rbcL clones most closely related to the coastal strain Synechococcus sp. strain CC9902 were recovered throughout the experiment but were particularly numerous toward the end of the experiment and dominated the “Green-type” libraries at this time. Initially, rbcL clones from these cyanobacteria were mostly derived from the ambient CO2 mesocosms but were equally distributed between treatments by the end of the experiment. This suggests that cyanobacteria related to strain CC9902 may be less tolerant of elevated CO2 (which was greatest at the beginning rather than the end of the experiment). However, despite the mesocosms being Pi-limited at the end of the experiment, several Synechococcus species (including those related to strain CC9902 and another coastal strain, CC9311) thrived. Following on from this observation, Pi uptake and assimilation mechanisms in a Synechococcus species were investigated in the laboratory. This led to the sequencing and characterisation of a pstS gene from the marine cyanobacterium Synechococcus sp. WH 8103. Unlike conventional pstS, it was discovered that the pstS II gene in this organism is constitutively expressed and unresponsive to or only weakly regulated by Pi supply. The use of PstS/pstS as a marker for P-limitation in natural samples, therefore, should be interpreted with caution.

V. faba fixes nitrogen effectively (Richards & Soper, 1979), however nitrogen fixation is reportedly energetically expensive and water deficit sensitive. Research was designed to determine whether medium nitrogen applications would result in increased productivities in V. faba, particularly during water deficits. Non-nodulated and nodulated V. faba were subjected to gradual water deficit imposition, and were supplied with a variety of medium nitrogen nutrition. Nitrogen fixing V. faba exhibited greater productivities than V. faba which were supplied with low medium nitrate concentrations (0.8 roM N), even during water deficits. Plant performance parameters (growth; net photosynthesis; nitrogen assimilatory enzyme activities; osmotic adjustment) were greater in nodulated than in non-nodulated 'no nitrate' supplied V. faba throughout water deficits, inferring water deficit tolerance for nitrogen fixation. However significantly greater plant performance paramaters were exhibited in V. faba when supplied with increasingly concentrated medium nitrogen nutrition (> 0.8 roM N) than when reliant on nitrogen fixation. In contrast to the bulk of previous literature, NR activities were maintained in V. faba until water deficits became severe, inferring a role for nitrate assimilation in nitrogenous osmotica production. Medium ammonia additions resulted in the exhibition of significantly increased root biomasses; cumulative leaf areas (important for a green manure crop); heights; and nitrogen assimilation in V. faba throughout water deficits, and accordingly in increased osmotic adjustment (including compatible solute accumulation), protein concentrations and vegetative yields. Greater plant productivities in v. faba when supplied with medium ammonia additions were attributed in part to lower associated assimilatory costs for ammonia than nitrate nutrition (Raven, 1992). Results indicated increased metabolism as opposed to storage of medium ammonia, and therefore potentially alleviated 'sink size' feedback inhibition of photosynthesis and nitrogen metabolism in V. faba when supplied with medium ammonia additions. Furthermore ammonia supplied V. faba may have been predisposed towards water deficit tolerance. In summary V. faba exhibited significantly greater nitrogen assimilation; osmotic adjustment; net photosynthesis; and growth when supplied with increasingly concentrated medium nitrogen nutrition (and particularly with medium ammonia additions) than when reliant on nitrogen fixation, both during periods of adequate irrigation and during water deficits.

The plant hormone ethylene has been extensively studied for its role in many plant developmental processes. This has led to the discovery of five ethylene receptors that are all involved in a complex signaling pathway that overlaps with several other phytohormone pathways, as well as stress responses. The role that ethylene plays in stress responses makes it a central signaling pathway in plant-microbe interactions. This interface has been well studied from the plant perspective, but there is limited data on how bacteria respond directly to ethylene. This work shows that a plant growth-promoting bacteria, Azospirillum brasilense, has a functional ethylene receptor capable of binding ethylene and inducing a response, making it the first plant-associated bacteria to have a receptor identified. The application of ethylene to A. brasilense alters attachment behavior in both biofilm formation assays as well as root colonization of both Arabidopsis thaliana and tomato. These changes in attachment behavior are likely mediated by a large transcriptomic response to ethylene that leads to an upregulation of central carbon metabolism and a downregulation of genes involved in nitrogen fixation. In agreement with the changes in transcripts, ethylene treated cells also have a distinct metabolic profile when compared to untreated cells, and this change can be detected as early as 8 hours after exposure to ethylene. Together, this data show that ethylene perception by A. brasilense alters the nitrogen metabolism of the cell in a way that inhibits colonization of the root and upregulates carbon metabolism and storage.

Macroalgae are important primary producers in the ocean and form expansive underwater forests to support diverse organisms in the ecosystem. They also contribute to a large amount of carbon export below the euphotic zone and carbon sequestration in the deep ocean in the form of detritus. As a result, there is an increasing number of studies suggesting long-term storage of carbon in macroalgae as a carbon dioxide removal strategy to mitigate climate change. However, in contrast to the growing attention of macroalgae as one of the blue carbon ecosystems, the fate of macroalgal-derived carbon after entering the deep ocean remains largely unexplored. Polysaccharides are a major component of both living and detrital macroalgae, and the degradation of these complex polymers is an important process for the turnover of carbon in the ocean. Macroalgal polysaccharides have diverse structures varying significantly in their monomeric saccharides, glycosidic linkages, branching sites, and modifications by chemical groups like sulfate. Therefore, the degradation of macroalgal polysaccharides requires an extensive number of enzymes produced by microorganisms specialized in this process. Carbohydrate-active enzymes (CAZymes) and sulfatases are the key enzymes typically organized in polysaccharide utilization loci (PULs) that mediate a cascade of pathways to degrade polymers to oligosaccharides and eventually to monosaccharides. However, we still have knowledge gaps in the details of the degradation processes and the various enzymes involved. In this dissertation, approaches like cultivation, genomic, and transcriptomic sequencing were applied to study the microbial degradation of macroalgal polysaccharides from cultures grown in the laboratory to marine sediments collected in the deep Santa Barbara Basin. A novel anaerobic bacterial strain NLcol2 was isolated from microbial mats in sediments offshore Santa Barbara, California, USA, and the name Pontiella agarivorans sp. nov. was proposed. It is the fifth cultivated representative in the phylum Kiritimatiellota with the ability to degrade macroalgal polysaccharides including agar and iota-carrageenan and is the first strain reported to be capable of nitrogen fixation. More than 10% of its genome also encodes CAZymes, sulfatases and nitrogenases that facilitate these metabolisms. To further study the enzymes and pathways involved in macroalgae degradation, a transcriptomic study was performed on strain NLcol2 growing with macroalgal polymers of agar and dried seaweeds compared with the monosaccharide D-galactose as control condition. During the growth of strain NLcol2 on polymers, 55% of the genome was expressed differentially, among which many CAZymes and sulfatases involved in the degradation pathways were significantly up-regulated. 12 putative PULs were identified and a selfish mechanism for agar degradation was proposed. The highly expressed genes involved in flagella formation and capsule polysaccharide synthesis with growth on seaweeds could promote motility and biofilm formation for better access and attachment to seaweed particles. To explore the ecological role of P. agarivorans and its relatives in the natural marine environment and to investigate the microbial communities enriched in kelp detritus on the seafloor, marine sediments with and without kelp detritus were sampled in the deep Santa Barbara Basin. The 16S rRNA gene survey showed an enrichment of phyla (Bacteroidota, Spirochaetota, Desulfobacterota, Campylobacterota, Verrucomicrobiota etc.) in kelp-laden sediments with cultivated relatives specializing in polysaccharide degradation, dissimilatory sulfate reduction and nitrogen fixation. Close relatives of P. agarivorans are widely distributed across the basin in low abundance in general but exhibited a high relative abundance in carbon-rich sediments with kelp detritus and at shallower depths. These microbial communities together contribute to the coupled biogeochemical cycling of carbon, sulfur, and nitrogen in the Santa Barbara Basin sediments in low oxygen conditions.

Net primary productivity and carbon sequestration are dependent on an adequate source of nitrogen (N) in terrestrial ecosystems. Biological nitrogen fixation, the microbially-mediated conversion of atmospheric N2 to biochemically reactive ammonia (NH3) serves as an important source of new N to many natural ecosystems. Despite its crucial role in global biogeochemistry, controls over the rate of this microbially mediated process remain poorly understood. This is particularly true in the context of land use (LU) alteration, a major driver of climate change and ecological disruption. In the Amazon Basin, LU change from primary tropical forest to agricultural operations, particularly cattle pasture, has been prevalent and ongoing for decades. To better understand shifts in N inputs resulting from LU change, this dissertation investigates the activity and diversity of free-living soil diazotrophic communities across the LU dichotomy of forest and pasture in the Amazon Basin. First, to establish a broader perspective on the microbial response to LU change in the Amazon, we performed a comprehensive review of all studies investigating microbial communities or microbially mediated processes across variable LUs and successional states in the region, highlighting both generalized and repeated findings, as well as points of disagreement which may indicate regional heterogeneity or methodological biases. This meta-analysis revealed that although trends in the alpha-diversity of microbial communities vary somewhat across studies, spatial biotic homogenization in pastures compared to primary forests has been a near-universal finding among prokaryotes, fungi, and several other taxonomic or functional subgroups. Additionally, a large proportion of studies have identified soil Al3+ content and extractable acidity to be major factors shaping community structure, emphasizing the importance of tropical mineralogy. Significant shifts in microbially-driven biogeochemical cycling with LU change have been identified as well. Most studies have found that soil C stocks increase slightly with pasture conversion, simultaneous with elevated methane emissions and a decline in the community proportion and taxonomic richness of methanotrophs. However, the most drastic and consequential nutrient cycle shift has been to the N cycle. Studies have found that rates of net mineralization and nitrification decline sharply in maturing pastures, coincident with reductions in nitrous oxide emissions and inorganic N pools. Recent studies have indicated that free-living diazotrophs increase in abundance and diversity with pasture conversion, but measurements of asymbiotic nitrogen fixation (ANF) have not been made across LUs to bolster these community findings. Addressing this knowledge gap in conjunction with community profiling was a primary focus of the original research presented here.To fulfill this aim, we surveyed three primary forests and three pastures (converted in or around 1972) in the state of Rondônia, Brazil near the end of the wet season in April of 2017. At each site, we established a 100 m2 quadrat and collected four replicate soil cores (0-10 cm in depth) from seven locations each. After homogenization, fresh soil was utilized for 15N2 gas incubations to calculate soil N incorporation attributable to ANF. Additionally, preserved soil samples were used for nucleic acid extraction and analysis, quantifying a suite of physicochemical measurements, and profiling the bulk soil metabolome. Community nucleic acids were utilized for marker-gene targeted amplification to serve as a proxy of absolute abundance and functional community structure, as well as to obtain profiles of potential diazotrophs within the broader soil metagenome. From this analysis, we concluded that soil ANF is indeed stimulated (47x increase) in active cattle pastures coincident with an augmentation in soil nifH copy number (18x increase; in line with previous observations), but that the two are not directly related within LUs. Using soil physicochemical parameters (including various pools of C and N, natural isotopic abundance, soil texture, pH, P, cation exchange capacity, and enzyme metallocluster constituents including Mo and V) for variable selection in multiple linear regression, we were unable to identify variables strongly associated with nifH community augmentation from forest to pasture. Substantially lower soil NO3- concentrations provided significant, but modest value in explaining the stimulation of ANF in mature, active cattle pastures compared to primary forests. Additionally, when forests were considered separately from pastures, nifH copy number and ANF rates (which were consistently near zero) were poorly explained by physicochemical parameters. Together these findings indicate that factors such as high inorganic N concentration as well as alternative, more productive N input pathways (i.e., canopy lichens, nodules, or the surface litter layer), which were not measured in this study, could largely suppress the activity of potential diazotrophic bacteria in primary forests. Within pasture soils, however, we found that nifH copy number was primarily associated with C pools. We found a modest, but significant association between pasture soil ANF rate and the ratio of low molecular weight extractable organic C to N, as well as the fraction of total N in the dissolved organic form, suggesting that this energy-intensive reaction is stimulated by a limited availability of N combined with sufficient fuel in the form of low molecular weight C compounds. Additionally, using a multigene approach to profile soil diazotrophs within the broader microbial community revealed that the genetic potential for asymbiotic diazotrophy is one of the most (if not the most) enriched soil microbial functions accompanying pasture conversion. This realization speaks to the immense influence that large-scale LU change can have on microbial communities, and the strong pressure for N replenishment in grazed (but unfertilized) pastures of the Amazon Basin. After observing that the absolute abundance of potential diazotrophs (based on DNA copy number) did not scale with ANF measurements in either LU, we aimed to further explore any meaningful relationships between diazotrophic communities and this crucial biogeochemical process they mediate. A previous analysis of the potential diazotroph community in forest and pasture soils of Rondônia found significant shifts in its structure. However, given the phylogenetic and trophic breadth of potential diazotrophs (i.e., microorganisms bearing nitrogenase-encoding genes, but not necessarily contributing to ANF), it is not clear whether these shifts have occurred independent of the larger soil prokaryotic community. By comparing potential diazotroph community structure (using DNA-based amplification of the nitrogenase marker gene, nifH) with that of all prokaryotes (by amplifying the 16S rRNA gene), we found that potential diazotroph community alpha-diversity was significantly higher in pasture soils, while the overall prokaryotic community did not reflect an increase. However, both profiles reflected a similar degree of community compositional dissimilarity with respect to LU change. Additionally, both communities exhibited significantly lower groupwise compositional dispersion in pasture compared to forest soils, agreeing with several earlier studies observing biotic homogenization of soil microbial communities with LU change. The compositional dissimilarities of both communities were associated with a similar subset of physicochemical conditions including clay content, pH, and total sulfur content, as well as the proportion of nitrogen in inorganic forms. We further investigated how these potential diazotrophic communities relate to the subset of taxa that are both metabolically active, and actively transcribing the nitrogenase enzyme (via RNA-based sequencing of nifH), to determine if the latter community exhibits a community structural response to LU change differing from that of DNA-based communities, and whether active communities may better explain ANF activity. In contrast to our expectations, we found that active diazotrophs did not reflect an increase in alpha-diversity with LU change and exhibited only a modest compositional response with no environmental correlates. Surprisingly, active, RNA-based communities across LUs were more similar to each other than they were to their DNA-based counterparts, showing an opposite trend in community dispersion with respect to LU. This increase in dispersion was related to ANF rates; we identified 17 of 882 taxa whose relative abundance scaled linearly and significantly (Pearson r = 0.88) with ANF. Of these, two OTUs, annotated as Bradyrhizobium and Enterobacteriaceae, reflected strong correlations with ANF on individual bases and were found to have the third and twentieth highest relative abundances among active pasture taxa, respectively. While paired potential and active diazotrophic communities (i.e., derived from the same sample) reflected a high degree of compositional dissimilarity from each other overall, by far the most drastic shift observed was the more than 100x enrichment of the photosynthetic cyanobacterial family Aphanizomenonaceae in active diazotroph profiles, irrespective of LU. Therefore, soil surface diazotrophs may also play an important role in providing N to pastures and forests alike, suggesting further work is needed to capture measurements of ANF activity under lighted conditions.

Diazotrophic microbes reside in soybean nodules; however, other non-nitrogen fixing bacteria are a part of the interior nodule microbiome. Results from a previous greenhouse study show that a novel species of Pseudomonas associates with soybean nodules as a plant-growth promoting rhizobacteria (PGPR). This study observes the soybean growth promoting potential of Pseudomonas spp. in a field setting. Additionally, this study observed differences in soybean growth promotion based on amending the plant with isolated strains or a mixed culture of the species' strains. Two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were either amended with isolated strains of the novel Pseudomonas spp. (referred to as PAMW1 and BUMW2 in this study), a mix of the two strains, or an uninoculated control. The study recorded measurements to observe growth, yield, and nitrogen fixation differences. The study uses two-way factorial ANOVAs and non-parametric, multivariate analyses to determine differences in growth promotion among samples. Soybean amended with PAMW1 has greater shoot mass, biomass, and height than other treatments. Through nonmetric multidimensional scaling (NMS), samples amended with a mixed culture or PAMW1 may be different regarding growth promotion relative to the non-amended samples. Univariate results support the hypothesis that the novel Pseudomonas spp. benefit soybean in a field setting. However, it is inconclusive whether a mixed culture amendment of multiple strains alters the overall growth promotion of soybean compared to samples amended with isolated strains.

Natural nitrogen fixation is done primarily by prokaryotes that reduce nitrogen gas(N2) to ammonia (NH3) using the enzyme nitrogenase in a process termed biological nitrogen fixation (BNF). BNF is an alternative to industrial fertilizers that could supply crops with the nitrogen they need, either through symbiotic or free-living associations. Engineering these associations requires an understanding of BNF that extends beyond nitrogenase, to the dynamic suite of proteins that support it. Azotobacter vinelandii is a model organism for studying BNF and contains more than 80 suspected BNF genes, many of which have an unknown function or lack experimental data showing direct BNF association. A. vinelandii is known for being a free-living and aerobic nitrogen fixer, making it both convenient for laboratory studies and biotechnologically relevant. Since most research in this organism has focused on nitrogen fixation, there have been few studies on how various carbon sources are metabolized. Characterizing this metabolism in A. vinelandii in the context of nitrogen fixation would help in engineering viable alternatives to Haber-Bosch. Consequently, this research builds on the contextual knowledge of BNF in A. vinelandii by using transposon mutagenesis to identify genes important to growth and/or nitrogen producing phenotypes. First, we used transposon sequencing (Tn-seq) to determine the genome-wide fitness of genes under diazotrophic, non-diazotrophic, and differing carbon sources. We then used the Tn-seq data from growth on the carbon source galactose to identify two galactose dehydrogenases predicted to complete the pathway of galactose metabolism missed by routine genomic annotation algorithms. Lastly, we explored the gene redundancy of NifA in nitrogenase regulation by characterizing two transposon mutagenesis mutants able to support the growth of algae in co-culture. Overall, this thesis expands on the knowledge of BNF in A. vinelandii by providing genome-wide fitness that quantify individual gene contributions to BNF, carbon metabolism and, which also explores gene redundancy in BNF regulation.

Nitrogen pollution from intensified agricultural production is a major driver of ecosystem degradation, contributing to aquatic eutrophication and global greenhouse gas emissions. Incorporating nitrogen-fixing leguminous intercrops into agricultural systems is an increasingly popular management strategy to mitigate those negative impacts. Using a perennial grainlegume intercropped agricultural system, this study aims to evaluate the potential for leguminous nitrogen fixers to support biomass yield of a well-developed perennial wheat, intermediate wheatgrass (Thinopyrum intermedium). The results of this study identified trends in improved forage quality, land use efficiency, and nitrogen fixation rates within the intermediate wheatgrass- alfalfa intercrop system. Findings also show that both white clover and alfalfa fix sufficient nitrogen to balance intermediate wheatgrass nitrogen uptake.

Intercropping and other forms of mixed cropping are both widely used and beneficial in many agricultural systems. One of the best-known examples is the grass-clover production model in pastures, in which nitrogen-fixation by symbiotic bacteria (e.g. Rhizobium spp.) benefits the grass as well as the host species of clover (Trifolium spp.) or other legume species. This is one example of transgressive overyielding, which refers to situations when the yield of two species growing together exceeds the yield of either of the species growing individually on the same area of land. This doctoral study aimed to investigate the benefits of companion plants in terms of soil biogeochemistry, focusing particularly on species that are companion plants to grasses. Literature is reviewed of plant species competition and co-existence, and rhizosphere biogeochemistry, primarily in the context of pastoral grasslands in the New Zealand High Country. The research project includes an investigation of whether there are nutritional benefits to nitrogen (N) -fixing plants when they grow in combination with grasses, rather than this being simply a relationship that involves spillover of nitrogen from legumes to grasses. This is extended to studies of phosphorus (P) -mobilizing plants and grasses, to investigate whether grasses provide reciprocal nutrients to cluster-rooted plants in the Proteaceae. The hypothesis underlying this study was that rhizosphere processes in grasses may be more adept than clovers at exploiting key trace elements in soils, and that these trace elements can be exchanged for N or P, thus also providing an explanation for species coexistence. Practical investigations consisted of pot experiments, a transplanted-soil core mesocosm experiment and field sampling, primarily concerned with nutrient deficient soils. This experimental work analysed the effects of different species combinations on (i) yield and (ii) plant and soil nutrient status (macronutrients and trace elements). Plant species combinations included both exotic and native species of grasses and legumes. The work was partly carried out on the Lincoln University Campus and at Mt. Grand Station, a university-owned high country pastoral farm in Hawea, Central Otago, from 2019 – 2022, with funding support from the Miss E.L. Hellaby Indigenous Grasslands Research Trust. The results showed that grasses played a critical role in acquisition of soil nutrients when they were growing with companion legumes or proteas in degraded soil. I found that legumes contained more nutrients (e.g. P, potassium sulphur, molybdenum and boron) when they were growing with grasses. Both native tussock grasses and exotic pasture grasses had significant positive impacts on native and exotic legumes in terms of nutrient uptake of a range of elements including Nitrogen and Sulfur. Both legumes and grasses benefited from growing together. Cluster-rooted species of Proteacea and grasses were found to have higher foliar concentrations of nutrients when they were growing together. The findings of this research provide evidence of facilitation between plant species with contrasting root systems when they are growing together in terms of procurement of key plant nutrients. Combinations of species can better exploit soil nutrients under conditions where fertility is constrained. This work is interpreted in the context of pasture production systems, protection of native biodiversity and land sharing by exotic and native species in the New Zealand High Country. The thesis is presented as an introduction to the study, a broad literature review, the inclusion of three published papers 1-3, one recently-submitted manuscript 4, a chapter describing additional work carried out during the PhD study, and a chapter containing the interpretive discussion and conclusions.

Nitrogen is an essential component of many biomolecules such as DNA and proteins. Biological nitrogen fixation is carried out by the enzyme nitrogenase. This multi-electron reduction requires the precise timing of electron and proton flow from the reductase, Fe-protein, through the [8Fe-7S] P-cluster to the catalytic site, FeMo-cofactor, located in the catalytic protein, MoFe-protein. Electron flow through nitrogenase is a dynamic process controlled by a conformational “gate” in which structural changes in MoFe-protein promote the accumulation of electrons at the FeMo-cofactor. Elucidating the mechanism thus requires a detailed understanding of structural changes that occur during catalysis. I have investigated the dynamics involved in nitrogenase catalysis, focusing on (1) the P-cluster’s dynamic role in orchestrating electron transfer to FeMo-cofactor and (2) structural characterization of the nitrogenase complex during N2 reduction. The experimental results discussed in Chapters 2 and 3 of this dissertation describe how the P-cluster has evolved to rest on the brink of stability, and that mutations to the redox-switchable, O-based P-cluster ligand (serine or tyrosine) result in a cluster that can reversibly lose Fe atoms. Furthermore, the O-based ligand protects the P-cluster from oxidative stress in vivo, is required for efficient diazotrophic growth under Fe-limiting conditions, and protects the P-cluster from metal exchange in vitro. These results indicate the native flexibility of the P-cluster are vital to its function in timing electron transfer to the active site. In Chapter 4, I discuss structural characterization of the nitrogenase complex at high-resolution during catalytic N2 reduction with cryoEM. Many structures were determined: structures of the complex during turnover at 2.6Å and 2.7Å, free MoFeP at 1.9Å, and ADP.AlF4—-inhibited complexes at 2.4Å and 2.8Å. Taken together, these structures have provided valuable insights into the mechanism of biological nitrogen fixation. The complex formed during turnover has a 1:1 Fe-protein:MoFe-protein stoichiometry, implying that there is negative cooperativity between the two halves of the complex. Furthermore, conformational differences between the two halves of MoFe-protein reveal previously unobserved conformational changes, which may be a part of the conformational “gate”. These observations provide critical insights into the dynamics required for nitrogenase function.

Nitrogen fixation efficiency is a desired trait in agriculture. Here we study nitrogen fixation traits in chickpea (Cicer arietium), which has special relevance given the crop’s predominance as an important source of protein and nutrition in the developing world. This study seeks to understand the relevance of genetic and transcriptional diversity of both the crop (C. arietinum), its two wild relatives C. reticulatum and C. echinospermum, and their cognate symbiotic microbes Mesorhizobium mediterraneum and M. ciceri. The experiments test the hypothesis that local adaptation is an ecological mechanism operating in wild systems to optimize biological nitrogen fixation, by quantifying biomass gain and nodulation phenotypes in a network of naturally-occurring host and bacterial genotype combinations. Plant biomass gain was greater for native (homologous) compared to non-native (heterologous) plant-bacteria combinations, leading to the conclusion that nitrogen fixation per se is more effective in co-evolved plant-microbe pairs, providing strong evidence of local adaptation in natural systems. C. echinospermun has effective symbiosis with its native, homologous symbiont M. ciceri and it is incompatible (nod-) with the heterologous symbiont M. mediterraneum. Conversely, C. reticulatum has more effective symbiosis with its native, homologous symbiont M. mediterraneum compared to heterologous M. ciceri. Interestingly, C. reticulatum has similar nodulation phenotypes with both bacterial species, and thus increased biomass gain in the homologous interaction is interpreted as greater efficiency of nitrogen fixation. The analysis of recombinant inbred lines derived from C. echinospermum X C. arietinum inoculated with either M. mediterraneum or M. ciceri document genetic segregation of symbiotic specificity, providing the basis for molecular genetic studies. Transcriptional profiling analysis of compatible and incompatible symbiotic pairs revealed responses to nitrogen (symbiotic and inorganic) that are both common and different among plant species. In particular, enhanced transcription of genes related to carbon metabolism and plastid-related functions is characteristic of M. ciceri nodules, while interaction with M. mediterraneum is associated with upregulation a diversity of processes, including nitrogen related gene expression, but largely exclusive of carbon metabolism. Whole genome analyses were used to characterize bacterial strains associated with chickpea cultivation in Pakistan and strains from the wild’s center of origin in South-Eastern Turkey. The analysis focused, in particular, on the provenance of genes involved in Type III secretion and Nod factor synthesis, informing us about the evolution of bacterial symbiosis in chickpea and bacterial loci relevant in the host-microbe interaction. The understanding gained from these studies informs fundamental questions about Cicer-Mesorhizobium co-evolution, and also has two broad practical implications: (1) for breeding of modern cultivars with improved biological nitrogen fixation, and (2) for developing improved microbial inoculants.

The overall motivations of this work were to better understand anthropogenic and environmental factors that can both inhibit or destroy, as well as promote biological soil crusts (biocrusts) in the North American southwest. Biocrusts are living mats that occupy the first few millimeters of soils in arid environments around the globe. They contribute to many ecosystem services including soil stabilization and nitrogen fixation.The specific research questions of this dissertation aim to unpack to complexity of these unique, specialized organisms capable of inhancing arid environments. The research here was approached from manuipulative and observational frameworks to increase our understanding of the ecology of biocrusts. Chapter I is an introduction to biocrusts, and the broader ecological concepts covered as part of this dissertation. In Chapter II, I present the research that was completed within the Nutrient Network (NutNet) framework at the Sevilleta, New Mexico. Here we showed that vascular plants respond positively to nutrients and consequently outcompete biocrusts for space. Chapter III represents the potential negative effects that nutrient deposition can have on nitrogen fixation of biocrusts. This study was done at the Jornada Basin Long-Term Ecological Research site in southern New Mexico. We also determined that encroaching shrubs were responsible for contributing more fixed nitrogen to the landscape compared to biocrusts, at least prior to the monsoon season. Chapter IV demonstrates the negative effect that livestock trampling has on the cover and function of biocrusts. We did this study at the Santa Rita Experimental Range in southern Arizona. We found that biocrust cover increased with increasing distance from watering holes within a piosphere framework. Chapter V illustrates the effects of elevation and soil parent material on biocrust cover and enzyme activity. This study was done between Phoenix (low elevation) and Flagstaff (high elevation) in Arizona. We found that elevation and soil parent material have significant effects on biocrust cover. Lastly, Chapter VI highlights our conclusions from this work as a whole and suggests future research.

Nutrient reduction, particularly with respect to nitrogen (N) losses, is an important goal for sustainably managed agroecosystems. Soil N-cycling microbial populations that modulate these processes are affected by agricultural management regimes. This research focused on the controls and dynamics of the major N-cycling microbial populations in high-input cotton field under agricultural management regimes and low-input native C4 forage grass systems under pasture management practices to determine the effects of management regimes on in situ seasonal dynamics of the functional microbes responsible for N fixation, nitrification, and denitrification processes. Molecular microbial ecology methods were combined with soil physicochemical properties and biogeochemical process rates to address research questions. The results showed that for soils in both cropland and grassland, the variability of the activity of N-cycling microbial populations was consistently and significantly higher than for functional potential. Agricultural season was a key factor affecting the variability of both functional potential and activity of N-cycling microbial populations regardless of agroecosystem types. In cotton fields, leguminous cover crops like hairy vetch promoted functional activity of N-cycling populations through the growing season, equaling or exceeding the effects of inorganic N fertilizer, suggesting the potential of leguminous cover crops for replacing inorganic N fertilization from the perspective of microbial ecology. In native C4 grasslands, high N fertilization rate changed community composition of both diazotrophs and ammonia-oxidizing bacteria (AOB), inhibited diazotrophs functional activity and diversity of diazotrophs but promoted AOB abundance, activity, and diversity as well as N2O emission and nitrification potential, indicating the negative influence of excessive N fertilization on soil functional microbial community, N fixation and reservation, and soil and downstream environmental health. Therefore, the significance of reduced N fertilization may be substantial both economically and environmentally. Our findings highlighted the fact that both genetic and transcriptional assessments can provide important information about the microbial populations and functions. Together, our work revealed insights into important relationships between microbial functional capacity and activity and associated nitrogen pools and processes in the field, adding to our understanding of the effects of soil health management practices on soil N-cycling microbial ecology.

Climate change effects are driving crop yield decreases globally, and yield penalties are often associated with increases in frequency and intensity of heat waves and soil moisture deficits, particularly during reproductive development. Additionally, climate change is driving increases in vapor pressure deficit (VPD) in recent decades, suggesting global atmospheric drying. In a U.S. study, VPD during reproductive stages was identified as the most important predictor of soybean yield variation. Investigations into the effect of VPD on soybean yields often consider its effect on crop transpiration rate (TR) and gas exchange, and such efforts have led to development of drought-tolerant soybean cultivars, which take advantage of particular TR responses to VPD. Despite such progress, no studies have yet examined the effect of VPD on nitrogen fixation, likely due to difficulties of taking non-invasive, real-time measurements.A common technique for monitoring nitrogen fixation rates non-destructively is the acetylene reduction assay (ARA), which has received critiques due to hazards posed to researchers and potential effects on root nodules. The first goal of this investigation was to develop an alternative method for non-invasively measuring nitrogen fixation that minimizes the above difficulties. A system was designed that enabled us to quantify N2 fixation as a function of the rate of by-product hydrogen gas (H2) production by soybean nodules. A second goal was to examine rates of nitrogen fixation in relation to concurrent changes in environmental variables, namely VPD and temperature. The system was successfully tested on two soybean genotypes in both field and controlled environment conditions. A key result is the confirmation of H2 production being associated with root nodules, seen by a lack of H2 signal from plants without nodules. Additional key observations include increases in H2 production rate from the morning to afternoon in the field, driven at least partly by increases in VPD, consistent with H2 production rate increases observed when shifting from low to high VPD in a controlled environment. Consistent genotypic differences were observed in both settings, signifying potential for genotypic diversity, which could be exploited in breeding efforts. This study invites further research into the effect of VPD and temperature on N fixation, as soybean growing environments continue to undergo increases in atmospheric drying. A second focus of this research on soybean is centered on plant water use, which is largely driven by evaporative demand as VPD increases drive increases in TR. Increased TR drives processes beneficial to carbon fixation, so in an environment where available water is not limiting, such behavior should lead to higher yield, or at least be neutral. However, in a water-deficit prone environment, genotypes exhibiting lower TR at high VPD will outperform those exhibiting a ‘profligate’ TR, by reducing the amount of water lost during the time of day when evaporative demand is the highest. Despite progress in phenotyping soybean TR responses to VPD, so far no QTL have been detected for traits directly reflecting such response curves, due to methodological and logistical challenges. The QTL mapping performed here resulted in the detection, for the first time, of two QTL for the response of TR to both low and high VPD. If confirmed, these QTL could be leveraged to design soybean varieties that are optimized for specific water availability regimes in order to maximize productivity.

Reduction of the reliance upon synthetic fertilizers is critical for a more sustainable agricultural future. Plant endophytes, especially those that engage in biological nitrogen fixation, provide a potential route towards this end. In this work, a deeper understanding of the plant endophyte and nitrogen fixing bacterium Gluconacetobacter diazotrophicus is presented through the following four chapters. The first chapter provides contextual background on this important plant endophyte and its potential for use in agriculture. Following this introduction, a large-scale transposon library of G. diazotrophicus is explored in the second and third chapters through highthroughput sequencing in a transposon insertion sequencing (Tn-seq) study. This Tn-seq study explores gene essentiality relating to nitrogen fixation as well as nutrient metabolism in G. diazotrophicus through rapid characterization across the entire genome. Lastly, the fourth chapter describes genetic manipulations of several key genes in G. diazotrophicus, performed to improve extracellular ammonium production to construct a strain for potential use as a biofertilizer. This work provides a deeper understanding of the genetic mechanisms essential to nitrogen fixation in G. diazotrophicus as well as those essential for growth under varied environmental conditions. As a nitrogen-fixing plant endophyte, further exploration of G. diazotrophicus as a potential alternative to synthetic fertilization brings us closer to a more sustainable agricultural future.

Iron-sulfur (Fe-S) clusters are among the oldest cofactors on the planet, used by proteins in almost all forms of life on Earth to carry out processes ranging from energy transfer to DNA replication. Among the organisms believed to use these Fe-S proteins more extensively than almost any others are the methanogens, an ancient lineage of archaeal microbes that produce methane as a required product of their metabolism. Methane, the primary component of commercial natural gas, is both a potent greenhouse gas and an important fossil fuel. It can also be renewably produced as a biofuel. Biogenic methane is almost entirely a product of archaea carrying out methanogenesis, a metabolic process with an absolute requirement for Fe-S clusters at multiple steps. They are also believed to be the lineage in which nitrogen fixation began, an activity that uses multiple Fe-S proteins to convert nitrogen gas into ammonia, which can be used by microbes and plants for growth. Despite this heavy reliance on Fe-S proteins, we know very little about how Fe-S clusters are generated in these methanogens. This process is well understood in bacteria and eukaryotes, but what little we know about archaeal Fe-S biogenesis suggests that the mechanisms in these organisms may not work the way they do in the other two domains. This dissertation presents several different but related projects undertaken to gain a deeper understanding of Fe-S cluster biogenesis in methanogenic archaea. Methanogens contain homologs of proteins known to function in Fe-S cluster biogenesis in bacteria and eukaryotes, including components of the ISC and SUF systems, as well as an ApbC Fe-S carrier protein. To test the hypothesis that these proteins have similar roles in methanogens, I employed biochemical and genetic approaches to assess the function of these homologs in the model methanogen Methanosarcina acetivorans. The primary avenue of investigation was a characterization of heterologously expressed putative ISC-type Fe-S cluster biogenesis proteins, and an assessment of the effects of deleting the genes encoding them, from M. acetivorans. Another subject of study was the putative Fe-S carrier protein ApbC, whose gene I also deleted. Finally, I demonstrated the feasibility of a CRISPR/Cas9 genome editing system that was recently developed for M. acetivorans by deleting the genes for a putative SUF-type Fe-S biogenesis system. My results demonstrate that M. acetivorans contains a functional ISC system but that the system is not essential. However, the results support the ISC system playing an important role in nitrogen fixation, potentially supplying Fe-S clusters to nitrogenase, but only when cysteine is the sulfur source. ApbC is also not essential but seems to be important for growth with certain sulfur sources, especially the thiosulfate ion. Deletion of SUF failed to produce any phenotype indicating SUF is not the primary Fe-S cluster biogenesis system in methanogens. Taken together, these results suggest that despite the evidence from bacteria and eukaryotes, and counter to the conclusions a bioinformatic approach would indicate, the ISC, ApbC, and perhaps even SUF cluster biogenesis systems individually appear surprisingly dispensable in methanogens.

Sustainable technologies are are often guided by our understanding of their natural equivalents. As such, our incomplete understanding of these natural processes limit our ability to design efficient and optimized synthetic schemes to address the challenges facing food and energy security. In particular, schemes using live organisms or purified proteins require that we either attempt to remove evolutionary features such as regulation or cope with unknown confounding factors resulting from the gaps in our knowledge. All three of the following chapters focus on furthering our understanding of the natural systems we wish to co-opt. The first chapter discusses producing biohydrogen via Azotobacter vinelandii, a soil bacterium that fixes nitrogen gas into ammonia aerobically using the enzyme, nitrogenase. Additionally, it shows how the produced biohydrogen can act as a proxy of nitrogenase activity such that we can quantify the in vivo inhibition that results from various nitrogenous compounds commonly found in the environment. The second chapter discusses growing green algae in turbidostat reactors as way to screen their compatibility with divergent growing technologies and commercial ventures. Lastly, the third chapter discusses how an oil-degrading marine bacterium, Marinobacter aquaeolei , produces wax esters via a partially defined wax ester biosynthesis pathway.

In Central Kentucky, rolling hill cropland presents a number of challenges related to soil sustainability. Increased topographic complexity can lead to increased erosion, inefficient crop nutrient use and increased nutrient loss. Further, grain crop yields can be variable across both space and time in rolling hill fields and are less resilient to changes in weather conditions than flatter, more homogeneous areas. More than 30% of cropland in Kentucky has a slope greater than 3⁰, which means a large swath of the row crop production land in the state is at increased risk of contribution to soil and water resource degradation. Managing these croplands in a way that secures the economic and environmental sustainability of Kentucky producers is critical to ensuring food system security on a local scale, and increase the viability of Kentucky ecosystems, both managed and natural. Cover crops, crops grown for ecosystem benefits rather than harvested for profit, may be a way of reducing the impact of complex topography on row crop production. Cover crops have been shown to reduce erosion losses, prevent nutrient loss to groundwater, and improve soil quality. Further when grown in a mixture, such as a cereal-legume biculture, cover crops have been shown to exhibit multifunctional traits, such as coupling N retention and release in synchrony with crop uptake. As such, they may present a management practice that can increase the sustainability of rolling hill agroecosystems, and possibly reduce the spatial and temporal variability in yield and biogeochemical cycling. There has been some research on the interaction between topography and cover crop implementation conducted up to this point. Across these studies however, to the author’s knowledge, there has not been a systems level analysis of the landscape position effect on cover crop growth and dynamics, decomposition, and the subsequent interactive effect of cover crop and topography on maize development and yield. Closing this knowledge gap provides an opportunity to increase our understanding of agroecosystem processes beyond individual crop growing seasons and determine how management of winter cover crops can be improved to enhance overall agronomic outcomes. Understanding how landscape topography impacts cover crop production, and in turn affects maize production, is critical to improving management of cover crops in Central Kentucky, and thus increasing agroecosystem sustainability. Here, we present the results of four experiments, which had the objectives of: 1.) Determining the effect of landscape position on cover crop biomass production and function, including species diversity and nitrogen fixation, 2.) Quantifying the effect of landscape position on the decomposition and N release rate of different cover crop treatments, 3.) Examining the scientific literature to determine how weather influences the relationship between grain production and topography, and 4.) Using field observations to calibrate a soil-crop system model for predictions of cover crop by topography interactions on maize yield and spatiotemporal yield stability under different climate scenarios. Our results, detailed in the subsequent chapters, indicate that cover crop growth and function respond strongly to topographic factors such as slope, and edaphic factors such as soil texture and inorganic nitrogen. Additionally, landscape position has a significant effect on the spatial distribution of soil water, and the surface soil temperature throughout the maize growing season, but this does not translate to spatial differences in the rate of cover crop decomposition. Our analysis of the relationships between topography and crop yield indicate that maize yield varies in its response to topographic factors based on growing season precipitation, such that at low precipitation, sloping areas yield poorly, but increase in yield as growing season precipitation increases. When we expanded our field data results to different climate scenarios using a soil-crop system model, we found that the presence of cover crop residue can decrease maize yield variability across time and space by raising yields in sloping positions via increased soil water storage, especially under dry conditions. The results presented here offer further evidence that the integration of cover crops into areas of sloping topography offer outsized benefits compared to areas of homogeneous terrain, including more stable yields, and increased N return. This work provides a means for maximizing the benefits of cover crop functionality for producers and increasing the overall sustainability of Kentucky agroecosystems.

Nutrient availability affects primary productivity, and nitrogen (N) is the limiting nutrient over large regions of the ocean. Biological N fixation (BNF) is the conversion of atmospheric dinitrogen (N2) to bioavailable forms of N (ammonium and amino acids). A select group of Bacteria and Archaea, known as diazotrophs, are the only organisms capable of this conversion. BNF is a critical source of N in nutrient-depleted (oligotrophic) marine waters. As such, BNF studies are a fundamental component for better understanding primary productivity and carbon cycling, as well as parameterizing predictive future global ocean models. Unicellular diazotrophs are widespread in marine systems and, in some cases, important contributors of BNF. This dissertation investigates unicellular diazotrophs at the single-cell level, including UCYN-A, a cyanobacterial diazotroph that lives in symbiosis with an algal host, non-cyanobacterial diazotrophs (NCDs), which are putative heterotrophs, and a specific NCD known as Gamma A, which is a commonly occurring organism throughout the tropics and subtropics that belongs to the class Gammaproteobacteria. The single-cell investigations used in these studies can provide high-resolution insights that are not possible from community-level analyses of BNF, such as the N2 fixation rate of individual cells and cell morphology. Single-cell nanoscale secondary ion mass spectrometry (nanoSIMS) analyses were used in chapters 2 and 3 to investigate the cell-specific N2 fixation rates of UCYN-A in the cold waters of the Bering and Arctic Seas where N2 fixation was not previously believed to be possible (chapter 2), and NCDs in the North Pacific Subtropical Gyre where we demonstrated the first direct evidence of NCD N2 fixation on open ocean particles (chapter 3). Chapter 4 focused on a specific uncultivated NCD, Gamma A, using geneFISH to visualize single cells in natural seawater samples. We successfully identified Gamma A cells by developing a fluorescent in situ hybridization (FISH) probe targeting the diazotrophic functional marker gene nifH, revealing Gamma A cells are most often attached to particles. Together these studies provide new insights into the distributions, activities, and physiologies of geographically widespread, unicellular diazotrophs, thus improving our understanding of their roles as sources of fixed N in the surface ocean.

With the surge of intermittent, renewable electricity, the storage of excessive electricity and reduction of CO2 or N2 into value-added chemicals is of great significance for a sustainable society. One viable route that fulfills such a target is to construct a hybrid inorganic-biological system that converts electricity into chemical energy and reduces CO2/N2 into commodity chemicals. In this general approach, water is split into H2 and O2 by renewable electricity and the yielded H2 is consumed by microbes as a reducing equivalent for CO2/N2 reduction. The throughput of this system is limited by the poor gas solubility in the aqueous environment. The control and design of the gas environment in these hybrid systems is crucial to achieving high throughput and sustainable reactions. To this end, my thesis has focused on combining perfluorocarbon (PFC) nanoemulsions with the hybrid system to enhance H2 or O2 gas solubility and optimize reaction performance. My first research project focused on combining PFC nanoemulsions with a hybrid biological inorganic CO2 fixation system to enhance the faradaic efficiency of acetic acid production (Chapter 2). PFC nanoemulsions are biologically inert and have reported high H2 gas solubilities. We determined that the nanoemulsions are compatible with the acetogenic microbe strain Sporomusa ovata and the electrochemical water-splitting catalysts used in the hybrid CO2 fixation system. When 2.5 % (v/v) PFC nanoemulsion was combined with the hybrid system we observed approximately 90 % or greater faradaic efficiency at all tested current densities. After 4 days, we were able to achieve an average acetic acid titer of 6.4 � 1.1 g�L−1 (107 mM), which equates to one of the highest reported productivities of 1.1 mM�h−1. With this observed enhancement, we decided to explore the change in the gas environment made by the addition of PFC nanoemulsions. We performed experiments to understand the mechanism of the observed enhancement when PFC nanoemulsions were added to the hybrid CO2 fixation system (Chapter 3). Experiments of flow cytometry were performed using fluorescently tagged PFC nanoemulsions to probe the association of the S. ovata with the nanoemulsions. We found a non-specific binding interaction between the nanoemulsion and the bacteria that could result in an increase in the local H2 concentration or an increase in the rate of H2 transfer to the microbes. Rotating disk electrode (RDE) experiments were performed as an electrochemical surrogate to understand the H2 transfer kinetics. Our experiments revealed that there was 3.5 times increase in the kinetic current density with the addition of the nanoemulsion. Additionally, we found that the local H2 concentration was 1.2 times the calculated bulk H2 concentration. This suggests that the addition of the PFC nanoemulsions resulted in a local increase in H2 concentration and an increase in the rate of transfer of the reducing equivalent to the microbe. We theorized that the addition of PFC nanoemulsions to other hybrid systems could result in similar enhancement and an ability to control the gas environment. With the success of the PFC nanoemulsion in the hybrid CO2 fixation system, my research shifted to focus on using the nanoemulsions with a hybrid N2 fixation system to enhance ammonia production and self-sufficiency (Chapter 4). The hybrid N2 fixation reaction utilizes Xanthobacter autotrophicus, a microaerobic bacterial strain that reduces N2 to NH3 through its nitrogenase enzyme. This has previously been accomplished through a precise supplied gas mixture, since the electrochemically generated O2 is poorly soluble in the aqueous solution. We added the PFC nanoemulsions to this system to utilize the electrochemically generated O2 and enhance the throughput of the N2 fixation system. Our experiments demonstrated that the use of PFC nanoemulsions drastically reduced the loss of cell viability in a supplied anaerobic atmosphere and led to a 3-fold increase in Faradaic efficiency of the system during 5-days of running. We performed mechanistic studies to understand the O2 gas-PFC nanoemulsion environment within the hybrid system (Chapter 5). Confocal microscopy images of the PFC nanoemulsions interacting with the microbes revealed binding and complete coverage of the microbe surface by the PFC nanoemulsions. Experiments of RDE were performed to ascertain the O2 transfer kinetics between the PFC nanoemulsions and the microbes. We found that there was a 20-fold increase in the kinetic O2 reduction rate when the PFC nanoemulsions were added. Assuming a Langmuir adsorption model of the PFC nanoemulsion binding to the microbe, we were able to use flow cytometry to determine the maximum number of nanoemulsions that could adsorb to the microbe surface and the equilibrium constant for the adsorption. Our results showed that the number of nanoemulsions that could bind to a given microbe or particle is dependent on the size of the particle, but the equilibrium constant is more influenced by the surface charge, with the -COOH functionalized microspheres having the largest binding affinity. In conclusion, my thesis research was able to integrate PFC nanoemulsions with hybrid biological inorganic systems to enhance throughput and alter the gas environment in the systems. The ability to understand and alter the gas environment using these PFC nanoemulsions can allow for other aqueous systems with poor gas transfer rates or gas restrictions to function with enhanced benefits. This dissertation also serves as groundwork for continued research in customizing the PFC nanoemulsion to achieve the optimal binding and gas environment.

Abstract: Alberta’s oil sands generate large volumes of tailings from bitumen ore processing. These tailings ponds produce biogenic methane, which can be measured across 60-80% of the tailings surface. Based on current surface area data and emissions studies, tailings ponds could account for 8% of Canada’s methane emissions. With a government mandate to reduce methane emissions by 45% of 2012 levels, understanding the dynamics of methanogenesis in tailings is highly important. Methane production from oil sands tailings is driven by hydrocarbon metabolism by the microbial community. For community growth and metabolism to occur, sufficient nutrients such as nitrogen (N) must be available in the environment. Several tailings ponds as well as reclaimed wet landscapes that incorporate mature fine tailings (MFT) are deficient in bioavailable N (NH4+, NO2-/NO3-) and yet continue to produce methane. Understanding this process with hydrocarbon amendments is important as it is generally assumed methane production will halt in the absence of nutrients. Additionally, literature suggests that methanogenesis is inhibited in the presence of amorphous Fe(III). This implies amendments such as amorphous Fe(III) oxides (rust) may be effective at reducing methane emissions in tailings ponds and potentially in reclaimed wet landscapes. In Chapter 2, we investigate methane production resulting from the degradation of n-alkanes and toluene in the presence and absence of fixed N in microcosms under an N2/CO2 headspace. Acetylene reduction assays indicated that N2-fixation was present in the absence of fixed N concurrent with n-alkane and toluene degradation, and methane production. Community gene analysis using 16S rRNA indicated that the bacterial community was dominated by fermentative bacteria and the archaeal community consisted primarily of Methanoseata in amended cultures. Functional gene analysis (DNA and mRNA) for the N2 fixing enzyme, nitrogenase (nifH) indicated genes can be expressed disproportionately to the coding DNA. In n-alkane cultures, nifH mRNA was predominantly expressed by Methanosaeta and in sequenced toluene cultures, nifH expression was detected in both Desulfovibrionales and Methanosaetaceae. These results suggested Archaea were responsible for most of the nitrogenase expression, and therefore N2-fixation in n-alkane amended N depleted treatments and in nearly equal proportions with Desulfovibrionales in toluene amended N depleted CNRL cultures. The presence of these genes in MFT suggest that anaerobic N transformations such as N2-fixation are possible in-situ. The results of this study support our hypothesis that N2-fixing microorganisms within the microbial communitycan support hydrocarbon degradation and methanogenesis in oil sands tailings communities. In Chapter 3, we investigated the use of amorphous Fe(III) as a methane inhibitor in conjunction with hydrocarbon degradation under N depleted conditions. As in chapter 2, we established cultures using tailings from three operators active at the time of this work, Albian, CNRL, and Syncrude. Cultures were established with and without fixed N and amended with toluene or n-alkanes in the presence of Fe(OH)3. 16S rRNA and functional genes were sequenced to define the microbial community. While no n-alkane degradation was observed, toluene degradation was recorded. In all cases, methanogenesis was inhibited. As observed in Chapter 2, N2-fixation as determined using an acetylene reduction assay was present in N deficient cultures alongside nifH expression. These data support our hypothesis that amorphous Fe(III) can be effectively used as a treatment to mitigate methane emissions by oil sands tailings communities while maintaining detectable rates of hydrocarbon degrading activity under N deficient conditions. In Chapter 4, we sought to determine if Fe(OH)3 could be used to inhibit methane production from the metabolism of citrate, a dispersant and labile methanogenic substrate found in some tailings ponds. Acetylene reduction assay revealed that N2-fixation was occurring in N deficient cultures, both with and without Fe(III). Methanogenesis was observed in the absence of Fe(III) as expected, however, methane was not inhibited in cultures treated with amorphous Fe(OH)3. We theorized this was due to abiotic reactions between citrate and Fe(OH)3 resulting in the formation of ferric citrate complexes. Unlike amorphous Fe(OH)3 ferric citrate complexes are not known to inhibit methanogenesis. These data suggest that Fe(OH)3 may not effectively treat methane emissions in ponds that contain a continuous source of citrate.

Nitrogen fixation (NFix) is an important yet understudied microbial process in aquatic ecosystems and especially in the Laurentian Great Lakes (LGL). Nitrogen fixation (NFix) or diazotrophy is a lynchpin in the nitrogen cycle that converts dinitrogen (N2) gas to ammonia (NH3) using the nitrogenase protein complex (Nif). Nitrogen fixation re-mobilizes atmospheric nitrogen and N lost to denitrification processes. Early work suggested the contribution of NFix in the LGL is minimal to the nitrogen budget. However, recent work has shown during bloom events, NFix can help alleviate nitrogen limitations. Evidence of nitrogen accumulation in the LGL, like in Superior, suggest that we do not have a firm grasp on the nitrogen cycle in large lakes. Furthermore, for most of the Great Lakes, there is a dearth of nitrogen fixation rates from near-shore and surface water environments. Thus, we sought to revisit NFix in the LGL and comprehensively sample from near and offshore stations and with depth to understand the spatial variability of NFix. In our quest to survey the Great Lakes, we carried out a series of preliminary studies to determine our methodology and understanding of nitrogen fixation is aqueous samples. Preliminary studies exploring seasonality and size fractionation showed detectable nitrogen fixation rates in Lake Superior, counter to previous expectations as a result of readily available dissolved inorganic nitrogen. Likewise, preliminary studies on nitrogen speciation and concentrations were found to inhibit nitrogen fixation rates in pure culture experiments. Inorganic nitrogen exemplified a significant threshold where nitrogen fixation rates decreased. In our Great Lakes survey, we found that each lake is significantly different in NFix rates from one another and that rates are depth dependent. Linear regression models show that chlorophyll-a and oxidized nitrogen species are correlated, directly and inversely, with NFix but with the caveat that lakes like Erie and Huron do not fully adhere to the model. Together this suggest that traditional controls on diazotrophy may not always adhere and that NFix is a significant source of N.

Drought, salinity, and low soil fertility have negative impacts on agricultural productivity, resulting in food scarcity and nutritional insecurity, particularly in Sub-Saharan Africa. Mungbean [Vigna radiata (L.) R. Wilczek] has seen increased interest as a short-duration and drought tolerant legume crop, capable of atmospheric N₂ fixation. Mungbean is a protein and iron-rich legume and can be used as vegetable or grain for human consumption or multipurpose crop. At present, few studies have simultaneously explored the best agronomic practices for mungbean cultivation and evaluated its potential for increasing crop yields via intercropping systems and improving soil fertility through biological N₂ fixation. To understand the agronomic practices and soil physical properties limiting mungbean production, the impacts of two mungbean cultivars (Berken and OK2000) with and without inoculation with Bradyrhizobium spp. grown in loamy sand and silt loam soils on mungbean growth and yield were investigated under glasshouse conditions. Promising results from this study led to the introduction of mungbean into pearl millet systems in Senegal and evaluation of the effects of intercropping on growth, yields, land equivalent ratio (LER), canopy cover estimates, and normalized difference vegetation index (NDVI). Finally, we evaluated plant growth and N₂ fixation of five mungbean genotypes grown in two soil textures using the ¹⁵N natural abundance technique leading to recommendations for those with the greatest overall benefit to the cropping system. The literature review shows mungbean often proposed as a strategic crop for increasing legume diversification within current cropping systems and providing increased food security as well as market diversification and economic sustainability. The greenhouse study revealed that OK2000 cultivar produced significantly higher yield when inoculated and planted on a silt loam soil than other treatments, indicating the importance of inoculation and soil texture in mungbean establishment. Intercropping mungbean and millet significantly (p≤ 0.05) increased combined yields (35% to 100% increase) and LER compared to sole millet cropping systems. Canopy cover estimates and NDVI values significantly increased up to 60% and 30%, respectively, in millet-mungbean intercropping over millet alone. The N2 fixation study showed that %Ndfa of mungbean was higher when grown in the loamy sand soil (27% increase). However, soil N uptake (235 mg plant⁻¹) and amount of N fixed (67 mg plant⁻¹) were greater in the silt loam soil. Among genotypes, IC 8972-1 significantly (p≤ 0.05) derived less N from the atmosphere (23%) but took more soil N (155 mg plant⁻¹) which yielded significantly greater dry biomass (7.85 g plant⁻¹) and shoot N content (200 mg plant⁻¹). The results from the N₂ fixation study indicated that choice of mungbean genotype can contribute to reducing N needs of agricultural systems. Overall, this research project demonstrated that mungbean has the potential for diversifying smallholder agriculture and adding biologically fixed N into soils, in line with transformative adaptation strategies being promoted for sustainable agriculture. Further research and development programs on good cultural practices, adaptation to cropping systems, and nutritional benefits for human consumption can promote mungbean cultivation in SSA.

White clover (Trifolium repens L.) is an integral component of mixed pastures in New Zealand temperate agriculture, providing quality feed and a sustainable source of fixed nitrogen (N) through its symbiosis with strains of Rhizobium leguminosarum bv. trifolii. While there has been much focus on identifying and applying Rhizobium strain inoculants for improved N-fixation, there has been less attention on identifying and exploiting plant genetic factors to develop cultivars that can consistently form effective Rhizobium symbioses. The complexity of the clover-Rhizobium interaction makes for a challenging but valuable breeding target which represents an important strategy for increasing the sustainability of New Zealand’s pasture-based agriculture. In this study, the phenotypic variation of N-fixation capacity across individuals from 17 white clover cultivars, including a wild ecotype (‘Tienshan’) and a T. repens × T. uniflorum interspecific hybrid cultivar (‘AberLasting’), released between 1920 and 2003 and representing three leaf size classes were investigated. N-fixation capacity was assessed by growing plants in vermiculite/McKnight’s solution, where N was provided either by an effective commercial strain (TA1) or partially effective (S12N10) N-fixing Rhizobium strain or supplied mineral N (positive control). Symbiotic traits recorded after 35 days of growth included Shoot and Root dry matter (DM), root to shoot ratio (RSR), and symbiotic potential (SP) which is the proportion of biomass generated through symbiosis relative to plants grown with mineral N. There were significant differences or interactions between and among cultivars and Rhizobium strains. Furthermore, there was a high degree of variability in N-fixation capacities of individuals within the cultivar populations. Multivariate analysis clustered cultivars across an N-availability gradient and identified some cultivars that performed equally well (‘S100’) or equally poorly (‘Barblanca’) or had marked differential performance (‘Dutch’ and ‘Tienshan’) across effective or partially effective strains. In order to derive key quantitative genetic parameters which could be utilised for improving N-fixation in white clover, a genetically structured F3 population of half-sibling (HS) families (n = 120) underwent the same screening assay as in the previous study, with N being provided either by the commerical strain TA1 or supplied mineral N (positive control). Shoot and Root DM, SP as well as RSR were measured and differed considerably among HS families, with 44% of families forming sub-optimal symbioses with TA1. Best linear unbiased predictor (BLUP) means for all traits were derived for each HS family and used to estimate significant additive variation and narrow-sense heritabilities, which ranged from 0.05 (RSR) to 0.33 (Shoot DM). This indicated there is additive genetic variability that can be exploited for improving the symbiotic interactions. These estimates also enabled the genetic gain for the examined traits to be predicted at 20%, 10% and 5% selection pressures while also identifying 24 elite HS families that could be utilised in future selection validation work. The feasibility of applying genomic selection (GS) for improving genetic gain in white clover-Rhizobium associations using a HS family structure was investigated. The phenotypes of 118 HS families and a genomic relationship matrix derived from ~155,000 genotyping-by-sequencing single nucleotide polymorphism markers of the HS families’ corresponding maternal parents were used for genomic prediction (GP) using five models. Cross-validation predictive ability (PA) of symbiotic traits ranged from 0.14 (Root SP) to 0.36 when two traits (Shoot DM and Shoot SP) were combined into a multi-trait Smith-Hazel selection index. The PAs of the five GP models were similar across all traits and therefore the computationally efficient GP model Kinship using GBS with depth adjustment-GBLUP (KGD-GBLUP) was selected. Different parameters influencing PA were assessed and identified a minimum of 94 HS families using a training: test combination of 80: 20%, respectively, over 100 iterations was sufficient for generating PAs in this population. This work provides an ideal foundation to assess genomic selection for breeding white clover germplasm for improved N-fixation and productivity by undertaking among-(using phenotype) and-within (using GS) HS family selection. To determine relationships between performance with pure strains and soil communities, a subset of cultivars (n = 4) and HS families (n = 4) screened previously for differential N-fixation capacities were assessed with two pure strains (TA1 and S12N10) and five soil rhizobia extracts. Differences in the N-fixation capacities with pure strains reflected those from the previous experiments. While there was considerable variation in symbiotic traits among populations with the five soil extracts, performance with the effective Rhizobium strain TA1 was often indicative of performance with the soil communities. Using multivariate pattern analysis, distinct clusters among the white clover populations were identified, which appeared to be driven by the number and appearance of nodules. In summary, the findings of this thesis will provide valuable information for facilitating the improvement of N-fixation in white clover in future breeding efforts.

Anabaena sp. PCC 7120 is a filamentous, nitrogen-fixing cyanobacterium that uses spatial separation to perform photosynthesis and nitrogen fixation simultaneously. Under fixed nitrogen limiting conditions, Anabaena 7120 forms specialized cells, called heterocysts, to fix nitrogen. In this research, I sought to add to the knowledge surrounding the process of heterocystous nitrogen fixation in Anabaena 7120. Cyanobacteria universally produce alkanes, which have been suggested to play a role in helping the organism adapt to abiotic stress, such as diazotrophic conditions. In my first study, I sought to identify the genes required for production of the hydrocarbon heptadecane (C17H36). Through using a double crossover approach and determining loss of function, I identified two genes (alr5283 and alr5284) responsible for heptadecane production in Anabaena 7120. In my second study, I employed the alkane knockout mutant to study the impact of alkanes in nitrogen-fixing conditions. My results showed that heptadecane production spikes immediately following nitrogen starvation. Correspondingly, the mutant, which is unable to produce heptadecane, exhibited a fragmented phenotype with a lower nitrogenase activity in the days immediately following nitrogen starvation. These results suggested that heptadecane may play an important role in maintaining membrane integrity for cell-to-cell connections, especially during the initial response to nitrogen starvation. In my last study, I knocked out 16 genes whose encoded proteins had been found only in heterocysts (Qiu 2018). Two of these gene mutants (for all3132 and alr0731) were unable to grow under aerobic nitrogen-fixing conditions. I was able to complement the alr0731 mutant, and it regained its ability to grow in diazotrophic conditions. Therefore, I categorized alr0731 as a Fox gene (unable to fix nitrogen in the presence of oxygen). This gene encodes an anaerobic ribocucleoside triphosphate reductase activating protein that is needed for conversion of ribonucleoside triphosphates (NTPs) to deoxyribonucleoside triphosphates (dNTPs). Because cell division (requiring DNA replication) and gene rearrangements have been linked to heterocyst differentiation, Alr0731 may be primarily important in the process of heterocyst differentiation. Future work will continue to add to the understanding of key regulators and contributors to the process of heterocyst differentiation and nitrogen fixation in cyanobacteria.

Meeting soybean demand on existing cropland area for a global population of 9.7 billion people by the year 2050 requires narrowing the existing gap between average producer yield and yield potential. Soybean relies on two sources on nitrogen (N): biological N2 fixation and indigenous soil N supply. As soybean yield continues to increase, it seems critical to know if there is a yield level at which potential contribution of indigenous nitrogen sources and fixation becomes insufficient to meet crop N requirements for high yields, while still maintaining or increasing protein and oil concentration. This study evaluated N limitation across 29 high-yield soybean environments in Argentina and Nebraska from 2015 to 2017. Each environment included a ‘zero-N’ treatment, which forced the crop to rely on biological N2 fixation and indigenous soil N, and a ‘full-N’ treatment, which provided an ample fertilizer N supply during the entire crop cycle based on novel protocol developed also in this study. Seed yield and protein concentration in full N were 11% and 3% higher than zero-N, respectively. The magnitude of the difference depended upon the yield level of the production environment, ranging from 0 kg ha-1 at 2.5 Mg ha-1 up to 900 kg ha-1 at 6 Mg ha-1. Seed yield responses were directly related with increases in accumulated N in aboveground biomass (70 kg N ha-1), without changes in nitrogen use efficiency. The N limitation was mitigated in environments with large contribution of indigenous soil N supply. The maximum rates of N limitation occurred before the seed filling and the plant mechanisms and processes underlying seed yield and protein concentrations were leaf area index, absorbed solar radiation, and N remobilization. Finally, there was a trade-off between biological N2 fixation and indigenous soil N supply with fixation reduced less than proportional per unit increase in indigenous N sources. There was a temporal asynchrony between biological N2 fixation and N demand, that is, biological N2 fixation was not sufficient to meet plant N demand as the latter increased and the contribution of indigenous soil N supply decreased. The peak of indigenous soil N supply was the most important factor explaining variation in the N limitation across environments. Findings from this study will help to narrow soybean yield gap to meet future food demand. Advisor: Patricio Grassini

Land managers across the Appalachian region have been dissatisfied with the progress of native temperate forest regeneration on surface mines reclaimed under Surface Mining Control and Reclamation Act of 1977 (SMCRA) protocols. These sites are commonly colonized by invasive plant species and have inadequate soils. Many of the nearly 50 former surface mines located within Cuyahoga Valley National Park (CVNP), Ohio, underwent reclamation following SMCRA protocols as an effort to restore native forest habitat, but park staff have also found succession to occur at a far slower rate than originally anticipated. While many studies have been conducted on acidic coal mines, reclaimed sites in CVNP were mined for non-coal resources and are alkaline. Here I investigated the plant communities and soil chemistry of reclaimed surface mines in CVNP to determine the implications of SMCRA protocols on the landscape on sites with alkaline soils. First, I analyzed how vegetative communities on reclaimed surface mines at CVNP changed over a 28-year chronosequence with particular regard to woody species and invasive species. Natural succession of sites was occurring slower than park staff anticipated, as the presence of woody species did not increase significantly over time. However, this did not appear to be a result of invasive plant colonization because exotic plant presence decreased over time, pointing toward inadequate soil conditions rather than interactions among the plant community withholding the growth of woody species. Results imply that SMCRA reclamation protocols do not improve site conditions sufficiently to facilitate natural succession as a tool to return native forests to mines within the timeframe envisioned by land managers.Second, I observed the effects of early colonization of woody nitrogen (N) fixing individuals on soil nutrient concentrations and vegetative communities at the mines. I compared an invasive N-fixing species, autumn olive (Elaeagnus umbellata), to several other shrub species, including the native N-fixer black locust (Robinia pseudoacacia) and other species that do not fix N. Both N-fixing species had considerably higher leaf tissue N content and surrounding soil N fixation potentials compared to other woody species, yet mineralized soil N and tissue N content of herbaceous species around N-fixers did not differ from non-fixers. This suggests that N-fixers release minimal fixed N through either secretion or tissue senescence during the early stages of colonization. Surprisingly, surrounding exotic and invasive herbaceous species were less commonly associated with N-fixers than non-N-fixers. These findings are contrary to what others have observed when studying denser patches of later-succession N-fixers, and provides evidence that woody, N-fixing species do not diminish native plant communities on reclaimed mine sites during early stages of colonization.