Thompson, Karen A

Acid mine drainage (AMD) and metal-contaminated tailings represent some of the most inhospitable aquatic environments on Earth, characterized by low pH, elevated metal concentrations, and chronic carbon limitation. Yet these systems support microbial consortia with remarkable resilience. Among the most conspicuous inhabitants is Euglena mutabilis, an acidophilic protist traditionally regarded as an indicator species of AMD but seldom thoroughly investigated. This thesis reframes E. mutabilis at the center of a fungal-algal-bacterial (FAB) consortium, demonstrating that its cadmium tolerance and persistence are emergent properties of the consortium.

Culture-based experiments revealed that E. mutabilis survival under cadmium stress declined when fungal and bacterial partners were disrupted, underscoring their indispensability. Glucose supplementation revealed the consortium's capacity for structural and metabolic reorganization: fungal hyphae bound algal cells into flocs, bacterial associates proliferated, and hormone production shifted. Hormone profiling suggested a distributed signaling system in which fungi contributed cytokinins (CKs) and gibberellins while algae produced methyl-thiolated CKs, jasmonic acid, and salicylic acid. Transmission electron microscopy revealed bacterial-like inclusions within algal vacuoles, suggesting facultative endosymbiosis or phagotrophic retention. Transcriptomic analyses revealed that cadmium stress suppresses light-harvesting complexes and growth-promoting hormone biosynthesis while activating metal transporters and chloroplast sequestration mechanisms.

Beyond stress physiology, the FAB consortium unlocked chemical diversity inaccessible to axenic cultures. Molecular networking revealed that environmental consortia consistently produced unique metabolite families, often linked to silent biosynthetic pathways. Metagenomic sequencing linked these products to bacterial gene clusters further supporting the view that metabolic innovation is an emergent property of the collective.

Together, these findings suggest that the FAB consortium should be understood not as a loose association but as a microbial superorganism. This framing extends beyond the holobiont concept by dissolving the hierarchy between host and symbiont: E. mutabilis, fungi, and bacteria are all indispensable, and the identity of the host itself becomes blurred.

By reframing E. mutabilis as the nucleus of a microbial superorganism, this work highlights both theoretical and applied significance. It advances ecological understanding of how life persists under geochemical extremes, while pointing to new opportunities for sustainable bioremediation and natural product discovery through the deliberate cultivation of naturally evolved microbial consortia.

Author Keywords: Algal symbiosis, Bioremediation, Co-culture, Hormones, Microscopy, Transcrioptomics

In Ontario, the dominant cash crop rotations consist of soybean (SB), which is a leguminous crop grown in rotation with maize (MZ) and winter wheat (WW). In addition to these crops, some farmers integrate cover crops (CC) into crop rotation, especially during the fallow period and winter seasons, to reduce nitrogen (N) losses via nitrate (NO3-) leaching and emission of N2 and the greenhouse gas nitrous oxide (N2O). This thesis focused on understanding the impact of crop phases in a MZ-(SB-WW)-CC rotation on the abundance of N-cycling bacterial communities that mediate nitrification and denitrification pathways. In addition, the influence of CCs on soil cytokinin (CK) profiles, which are plant growth-promoting hormones, were studied in a greenhouse trial to assess their potential impacts when integrating CCs into crop rotations. In particular, the relationship between traditional soil health parameters and the soil CK profiles was studied to understand how CKs might reflect biotic interactions and soil vitality. Results indicate N fertilizer application mono ammonium phosphate (MAP) and starter N:P: K (24:6:24) during WW planting in fall largely supported nitrifying bacterial communities (amoA) and potentially contributed to NO3- leaching. Management of MZ, which included spring-applied MAP resulted in larger denitrifying (nirK) bacterial communities, increasing the potential risk of N-loss via emission of dinitrogen gas (N2) and greenhouse gas N2O. However, CC soils had significantly lower nirK than MZ, reflecting the importance of strong and deep root systems of CCs, which have a higher ability to scavenge the substrates for denitrifying communities (NO3-). This highlights the importance of growing CCs in reducing the potential risk for N-loss via leaching and denitrification. Additionally, in the greenhouse trial, the ability of CCs to affect CK was detected, highlighting the importance of integrating CC in crop rotations. This is particularly noteworthy, given that total CK profiles showed strong associations with traditional soil health parameters such as labile or active carbon and soil microbial community diversity. It was concluded that total soil CK can be used as a novel and dynamic soil health measure. Future research on quantifying N2O fluxes and levels of NO3- in leachates would provide a more precise understanding of the impact of different crop rotation phases on N-dynamics in these fields. Further studies on single or combined measures of soil CKs are warranted to develop its potential as a practical and effective soil health parameter.

Author Keywords: Cover crops, Crop rotations, Cytokinin hormone, Nitrogen Cycle, qPCR, Soil health