UC Berkeley

Anthropogenic nitrogen pollution is a global phenomenon that facilitated unprecedented disruptions to the nitrogen cycle distinctly marked by human inputs of reactive nitrogen into the environment. The increased flux of reactive nitrogen has precipitated unchecked discharges into surface waters leading to eutrophication, severely disrupting existing ecological networks. Anaerobic ammonium oxidation (anammox) is a biogeochemical process that is commonly applied for the removal of reactive nitrogen from wastewater. These systems have been demonstrated to be highly efficient at removing nitrogen from wastewater side-streams with a lower carbon and energy footprint than conventional biological nutrient removal technologies. However, they have been plagued by long startup times and process instabilities which can cause reactor performance lapses that can require months of recovery. A critical element for discerning and responding to these performance lapses is a deep understanding of the microbial community within anammox reactors. Anammox reactors create a unique environment that promotes the selection of a distinct assemblage of bacteria that commonly co-occur with anammox bacteria. These bacteria form an assortment of symbiotic and antagonistic relationships with the anammox bacteria that are critical to their growth and activity as well as the performance of anammox based nitrogen removal systems. This dissertation seeks to understand which interactions are most important for community stability and maintenance of ecosystem function and how the circulation of metabolites influences microbial diversity. The results of this research contribute to a robust understanding of community dynamics that can be used to model and manipulate anammox microbial communities.Chapter 1 seeks to situate the phenomenon of nitrogen pollution and its complementary solutions by contextualizing anthropogenic pollution as a manifestation of settler colonial regimes of power and land relations. This is conducted through a historical grounding of the tenets of Western environmentalism within the broader scope of Western scientific ontology as a means to critically re-examine the motivations of environmental discourse. Historical and political conceptualizations of race, class, materialism, and power structures are utilized to elucidate the 2 foundations of the current global tenets of capitalism, imperialism, and settler colonialism. Paralleling this analysis is an investigation into the history of human relations with reactive nitrogen and how these relations have developed from Pre-History, to Antiquity, to the Middle Ages, to the modern era and how the conceptualizations of nitrogen have intersected with modes of production, commerce, and state power. This chapter culminates in a re-examination of the work of Haber and Bosch as a coalescence of nitrogen exploitation and imperial power. The implications of this historical moment are used to establish circumstances around modern relations to nitrogen, the utilization of science as a means to materialize state agendas, and the futility of technological solutions to environmental problems. Chapter 2 of this dissertation seeks to understand how competitive nitrogen dynamics affects the microbial ecology of the system. The widely accepted stoichiometric relationship of anammox dictates a NH4+:NO2- of 1:1.32, however the anammox bacteria also compete with bacteria performing denitrification and dissimilatory nitrate reduction to ammonia (DNRA) for this nitrite. To understand this dynamic, an analysis was conducted using 16S rRNA and shotgun metagenomic sequencing data from a reactor study where the influent NH4+:NO2- ratio was changed to 1:1.1. Sequencing data revealed significant decreases in the abundance of anammox bacteria and increased abundance of bacteria capable of utilizing DNRA. Despite significant changes in taxonomic composition, the reactor still maintained high levels of nitrogen removal efficiency. These results would suggest a dynamic relationship between the anammox and DNRA bacteria that can be symbiotic. Chapter 3 of this dissertation sought to evaluate the effects of a process disturbance on the microbial community in an anammox reactor. To understand these effects, an analysis was conducted using 16S rRNA and shotgun metagenomic sequencing data from a separate reactor study where the solids retention time (SRT) was decreased step wise from 50 days to 28.5 days. Metagenomic and 16S rRNA data revealed a strong deterministic effect of the disturbance that selected bacteria based on growth strategies where K-type strategists seemed to undergo negative selection and r-type strategists underwent positive selection. The results of the study also suggest a more complex relationship between the anammox bacteria and filamentous Chloroflexi bacteria that is critical to biomass aggregation and retention. These results also demonstrated a significantly different microbial community in the reactor recovery period compared to prior stable operation which would suggest high levels of functional redundancy. Chapter 4 seeks to examine the role of extracellular polymeric substance (EPS) degradation by heterotrophic bacteria in carbon cycling within anammox bioreactors. To investigate this phenomenon EPS was extracted from suspended anammox biomass through an alkaline and heat extraction method and then utilized as a carbon source in batch enrichments utilizing anammox biomass as an inoculum. Shotgun metagenomic and 16S rRNA sequencing were utilized to determine the response of the microbial community to enrichment on the different EPS solutions. TOC and spectroscopic analysis confirmed biological degradation of EPS by the enrichment culture and sequencing analysis demonstrated increased abundance of bacteria classified as Ignavibacteriae in alkaline EPS enrichments, Nitrosomonadaceae and Rhizobiaceae increased in 3 heat extracted EPS enrichments, and Ruminococcoceae, Commamonadaceae, and Acidimicrobiales insertiae increased in both enrichments. Metagenomic analysis also demonstrated that bacteria increasing in abundance were enriched in extracellular glycoside hydrolases and peptidases that could be utilized to depolymerize complex carbon molecules. These results would suggest the ability to utilize EPS as a carbon source in anammox reactors is broadly phylogenetically distributed across bacteria within the community. Chapter 5 aims to characterize the functional profiles of anammox bioreactor associated microbial communities through the framework of ecological strategies. Microbial trait-based analyses were conducted on publicly available metagenomes using microTrait, a suite of computational tools that annotates microbial functions and maps them to a dimensionally reduced trait space representative of diverse microbial growth strategies. Phylogenomic analysis of retrieved metagenomes demonstrated a strong recurrence of phylogenetically similar organisms in anammox bioreactor systems of various configurations and operational parameters. Results of the trait-based modeling analysis demonstrated that the trait profiles converged closely according to phylogeny for guilds containing specific lineages of Planctomycetes, Chloroflexi, Bacteroidetes, and Iganvibacteriae which had high occurrences of extracellular carbon depolymerization but contained phylogenetically divergent MAGs in guilds enriched in other resource acquisition and stress tolerance traits. Assessing these functional guilds with relation to the Y-A-S (yield, acquisition, stress) ecological framework indicates that some of these guilds possess traits that appear to align with a high yield growth strategy characterized by investments in aerobic respiration and glyoxylate metabolism while others favor specialization in resource acquisition and stress tolerance. Overall, these groupings would indicate that the conditions of anammox bioreactors create a distinct set of ecological niches characterized by various growth and competition strategies. Ultimately, the results of this research will support the development of a robust kinetic growth model that can accurately predict the substrate utilization, growth, and microbial interactions in anammox bioreactors. The creation and refinement of these models will promote the design, operation, and maintenance of more resilient anammox bioreactors for municipal and industrial wastewater nitrogen removal. Furthermore, these results will also contribute toward a deeper understanding of the application of ecological theory to complex microbiomes and offer insights into the understudied nuances of microbial metabolic networks and materials exchanges. The contextualization of this biological phenomenon with a sociopolitical analysis of the current state of nitrogen in global commerce is critical to a wholistic understanding of the utility of these technologies, their shortcomings, and opportunities to redirect political momentum towards structural changes to mitigate nitrogen pollution to the environment.