Microbial mediated carbon and nitrogen feedbacks to elevated CO2 and switchgrass establishment in grassland ecosystems

Anthropogenic climate change is the greatest scientific and political challenge of the twenty-first century. Since the industrial revolution, global climate change has elevated greenhouse gas concentrations in the atmosphere, increased the global mean surface temperatures of the Earth, caused a rise in sea levels, enhanced the frequency of extreme weather events, and is having severe effects on the biosphere. Climate change has also accelerated in recent decades, and many uncertainties remain about the lasting effects on ecosystems and human welfare. Additionally, nitrogen deposition has increased dramatically and may alter terrestrial ecosystem functionality. Therefore, nitrogen deposition combined with climate change may accelerate soil carbon loss, creating more ‘marginal lands’ of low nutrient availability. To address these concerns, it is vital to understand the response of soil microbes to aspects of climate change, nitrogen additions, and changes in land usage. Microorganisms are essential in biogeochemical cycling and regulating carbon feedbacks between the biosphere, soil, and atmosphere. However, their responses to elevated CO2, N deposition, and changes in land usage (e.g., bioenergy crop production) are difficult to predict, limited by a lack of mechanistic understanding due to the complexities of microbial communities, their considerable ecosystem functionalities, and intricate interactions between plants and other microbes. Advancements in high throughput metagenomic sequencing and environmental microbiology have allowed for a deeper understanding of the critical role microbial diversity and functions have in maintaining ecosystem functionality. This dissertation focuses on grassland soil microbial communities and their responses to elevated CO2, nitrogen deposition, and bioenergy crop establishment. It goes on to further address more theoretical questions associated with fundamental microbial ecology as they relate to species-time-area relationships and seeks to quantify the effects the microbial community has on soil carbon stocks in marginal lands. The first research chapter focuses on answering questions related to how free-living diazotrophic microorganisms respond to elevated CO2 and nitrogen deposition treatment, both in terms of their functionality and diversity. Soils were collected from the Cedar Creek Ecosystem Science Reserve in Minnesota. Heavy 15N2 gas soil incubations were utilized to assess the nitrogen fixation rates and functionality of the diazotrophic community, while high-throughput amplicon sequencing of the bacterial, fungal, and diazotrophic communities allowed for quantification of the diversity changes due to treatment effects. Elevated CO2 and nitrogen deposition significantly stimulated nitrogen fixation rates, while the combined treatment reduced rates back down to ambient levels. The treatments had no significant effect on diazotrophic richness, but the community dissimilarity between treatments was significant, revealing an effect on the beta diversity of diazotrophs. Fungal communities under nitrogen deposition were marked by a drop in the relative abundance of fungi in the Glomeromycota class, containing arbuscular mycorrhizal fungi. The functional potential of the bacterial community assessed by GeoChip DNA microarray revealed a reduction in signal intensity for many nitrate reductase-related genes (i.e., narG, nirK, and nirS) under nitrogen addition. Additionally, recalcitrant carbon cycling genes (i.e., vanA and ligninase) functional potential was elevated under the combined treatment. This study reveals the implications that elevated CO2 and nitrogen deposition combined may reduce free-living diazotrophic functionality and could alter soil nutrient cycling, which may lead to a depletion of native carbon stocks in the soil. Next, switchgrass establishment was investigated and its ecological impacts on soil chemistry, soil biota, and trace greenhouse gas emissions in highly eroded marginal lands of low soil nutrient quality. We performed a comparative analysis using seasonal soil chemical profiling, high-throughput amplicon sequencing of the bacterial community, and in situ field measurements of trace gas fluxes over a seventeen-month timeline to monitor changes between two contrasting soil types with different plant cover (i.e., switchgrass or fallow with annual weeds/grasses). Our results revealed carbon accumulation in the topsoil at our sandy loam site with switchgrass but not at the clay loam site with switchgrass. Significantly elevated CO2 emissions were observed from the clay loam switchgrass plot, along with reductions in microbial diversity (both alpha and beta). Surprisingly, methane consumption was significantly reduced by an estimated 39 and 47% at the clay loam and sandy loam switchgrass plots. Together, the results of this study suggest that differences in soil carbon stocks and greenhouse gas fluxes are different at highly degraded sites with switchgrass. Then, the temporal and spatial microbial diversity was assessed to ask how switchgrass establishment alters prokaryotic species-time-area relationships in nutrient-poor soil and what biotic or abiotic factors regulate the changes in diversity over space and time. By using paired plots at each site, with and without switchgrass, and tracking the soil physicochemical properties and bacterial biodiversity using 16S rRNA high-throughput sequencing, it was found that taxa turnover was higher over time than with increasing area. Soil heterogeneity was also found to be higher at the clay loam site, and that this heterogeneity was related to and helped in explaining the species-time-area relationships observed from each of the plots. Seasonal turnover of soil phosphorus concentrations was positively related to the species-area relationship, perhaps reflecting the linkage changes in phosphorus have with plant growth and thus the effect plants have on the overall bacterial diversity. This study links the importance of soil nutrient condition and heterogeneity to regulating the soil bacterial biodiversity for different spatial scales and over time. Finally, the potential impact on deep soil carbon stocks is evaluated under deep-rooted switchgrass to establish if the long-term accumulation of root biomass in the deep soil layers influences heterotrophic respiration rates of microorganisms and has consequences on the soil carbon balance. We examine this using soil taken along a depth profile of cores from paired plots in two contrasting soil types that either have or do not have switchgrass growing for at least ten years prior to sampling. A heavy isotopic 13C priming soil incubation experiment was conducted in the lab to determine heterotrophic respiration rates along with a BioLog functional degradation assays, PLFA microbial biomass estimates, and high-throughput sequencing of the bacterial communities of the native soil, and both before and after isotopic priming. A differential response to plant residual carbon inputs was found between long-term selected microbial communities from different sites depending on the root system. The results suggest that soil type and microbial community structure play important roles in regulating the deep carbon pool and the potential for increasing the soil carbon sink capacity with deep-rooted plants in nutrient-poor soils. Overall, the work presented in this dissertation provides evidence of prokaryotic communities’ response to elevated CO2 and nitrogen deposition altering community functionality, reveals the ecological impacts of switchgrass establishment and consequences on microbial diversity at scale and over time, and evaluates the priming effect of the subsurface soils under deep-rooted switchgrass. The findings presented here represent novel insights into understanding microbial-mediated carbon and nitrogen cycling in grassland ecosystems and assess the sustainability of switchgrass cultivation from a microbial perspective on marginal lands. Keywords: climate change, soil microbial community, nitrogen deposition, heterotrophic respiration, metagenomics, alpha and beta diversity, species-time-area relationship, ecosystem function, priming effect, microarray, GeoChip, high-throughput sequencin