A sequence-based approach to identifying N2O fixing organisms

• Main Author: Kinkel, D
• Format: Thesis
• Language: English
• Published: Queen Mary University of London 2024
• Subjects: Iceland
• Online Access: https://qmro.qmul.ac.uk/xmlui/handle/123456789/97267

The increasing concentration of the powerful climate gas N2O has focussed research on its sources. In this thesis, I looked at the microbial ecology of experimental freshwater ponds that have been found to be a sink for N2O through N2O fixation. It is hypothesised that N2O fixation could be an alternative reaction carried out by the nitrogenase in cold environments. nifH-like sequences from a shotgun-metagenome from the ponds were translated into amino acids and compared to known NifH sequences. In this comparison two groups of pond NifH did not sit with their most similar reference NifH sequences as expected. Assuming that these sequences represented an adaptation for N2O fixation, they were used as templates to design specialised primers in order to examine any potential correlation of the nifH community to N2O fixation. The nifH community targeted by the novel primer pair had a better correlation to N2O undersaturation in pristine freshwaters in Scotland and Iceland compared to other established primers. This showed that the novel primers were more specific at targeting nitrogenases that potentially fix N2O. They were therefore used to characterise N2O fixing organisms in an incubation in N2O enriched medium. After incubating the biomass from the experimental ponds, the nifH community in the biomass was sequenced using the new primers and established primers for comparison. Although the enrichment of N2O fixing organisms was not seen, the new specialised primers were more sensitive in capturing shifts in the overall nifH community. This was seen in the Proteobacteria due to the N2O enrichment. The specialised primers allowed an examination of the proteobacterial community at a higher resolution, that can be exploited further in future transcriptomics experiments on N2O fixation in pristine freshwaters functioning as sinks for N2O.