Intact polar lipids of ammonia-oxidizing Archaea: structural diversity application in molecular ecology

Non-extremophilic Crenarchaeota are ubiquitous, and comprise a major component of the microbial assemblages in many modern-day systems. Several studies have analyzed glycerol dialkyl glycerol tetraether (GDGT) membrane lipids synthesized by non-extremophilic Crenarchaeota to interpret the presence, distribution, and activity of these microbes in various environments. The use of cellular membrane lipids in molecular ecology studies provides added value to conventional (meta)genomic approaches, largely in the form of independence from biases associated with the extraction and analysis of nucleic acids. However, disentangling biomarker lipid signals derived from living and dead cells has remained a challenge, as core lipids are recalcitrant to degradation and can persist as molecular fossils for significant periods of time. This thesis describes investigations aimed at developing the use of intact polar lipids (IPLs) in ecological studies of (ammonia-oxidizing) Crenarchaeota, as IPLs containing polar head groups bound to the core GDGT are assumed to best represent living Crenarchaeota. To this end, improvements to both indirect and direct IPL-GDGT analytical methods were made, with the latter based largely on information obtained from four novel enrichment cultures of ammonia-oxidizing Crenarchaeota (AOA). All of these AOA synthesized abundant crenarchaeol, providing support for the hypothesis that crenarchaeol is specific to archaeal ammonia-oxidizers. In addition, crenarchaeol-based hexose-phosphohexose was the only IPL common to all species, pointing to this as the best biomarker for tracing living AOA. A selected reaction monitoring method developed to detect crenarchaeol-based IPLs at low levels was applied to suspended particulate matter from the Arabian Sea and the North Sea. Abundant AOA-specific IPLs were recovered from the Arabian Sea, and corroboration of these data with rRNA and AOA-specific functional genes showed a specific preference of AOA for the oxygen minimum zone (OMZ) boundaries. A comparison between these findings and IPL and DNA-based data generated for anammox Bacteria, illustrated a unique distribution throughout the OMZ with their respective niches separated by > 400 m vertical distance. Coupling of archaeal ammonia oxidation and anammox is, therefore, unlikely here, despite theory predicting this possibility. Crenarchaeol-based IPLs were also used to track the seasonal occurrence and carbon-fixation activity of marine AOA in the coastal North Sea. Increases in AOA abundance were notable during the winter months between November and February. Incubations with 13C-bicarbonate resulted in label incorporation into the tricyclic biphytane derived from IPL-crenarchaeol, showing that the Crenarchaeota in the North Sea surface waters actively fix bicarbonate during their winter blooms. Lower 13C-incorporation was observed in incubations containing nitrification inhibitors (Nserve or chlorate), further indicating that these Crenarchaeota are predominantly ammonia-oxidizers. The present study demonstrates that intact polar GDGTs are excellent tools to study the ecology of Crenarchaeota, and adds to our knowledge on the role of AOA in both carbon and nitrogen cycling in two important marine settings