|The origin and fate of intact polar lipids in the marine environment|Brandsma, J. (2011). The origin and fate of intact polar lipids in the marine environment. PhD Thesis. [S.n.]: [s.l.]. ISBN 978-90-6266-286-9. 1-223 pp. hdl.handle.net/1874/213581
Microorganisms, such as bacteria, archaea and algae, are the most abundant organisms on Earth and they contain the bulk of the biosphere’s carbon, nitrogen and phosphor.They are also the main drivers of the biogeochemical cycles, and therefore the study of microbes in their environment (microbial ecology) is important for our understanding of Earth system functioning. Unfortunately, the very small size of microbes makes it difficult to study them in situ, and a range of methods is employed to determine their natural diversity, abundance and activity. One such approach is to measure molecular compounds present in a microbial cell. Rather than trying to obtain information about the organism through direct visual or morphological analysis, analysis of its pigments,proteins, DNA or lipids has been shown to contain a wealth of information about their source organism.A promising method for the identification, characterization and enumeration of microbial communities in the natural environment is the measurement of intact polar lipids (IPLs), the basic building blocks of biomembranes. These complex molecules areubiquitous in nature and have several characteristics that make them useful as proxies for living microbial cells. Within the large molecular diversity encountered in IPLs,certain types are uniquely synthesized by particular organisms, and these specific IPLs can consequently be used as biomarkers for those source organisms. Furthermore, IPLs are thought to degrade rapidly upon cell death, meaning they are indicative of livingcells and can thus be used for estimates of viable microbial cell numbers or biomass.Finally, microbes can actively remodel the IPL composition of their biomembranes in adaptation to their environment, meaning that information about environmental conditions can potentially be obtained from IPL measurements.However, the comparatively recent development of suitable analytical instrumentation for direct analysis of IPL molecules (i.e., the coupling of liquid chromatography to mass spectrometry through an electrospray ionization interface) means that only alimited number of studies have been done on IPLs in the marine realm, and in particular the water column. The aim of this thesis was therefore to investigate the origins, dynamics and fate of IPLs in the marine environment. First, the applicability of IPLs as a proxy for living microbial cells was re-assessed by determining IPL degradation rates in decaying diatom cultures, and in targeted study in which IPLs specific to anammoxbacteria were measured in marine sediments in parallel with other proxies for anammox bacterial abundance. Subsequently, a comprehensive analysis of IPLs in surface waters of the North Sea was performed, both on a spatial and a temporal scale. Theseresults were statistically compared with measurements of the environmental conditions and the microbial biogeography and their use as a tool in marine environmental microbiology was evaluatedA laboratory study of IPL degradation in decaying diatom cultures showed that, while degradation rates were initially high, they slowed progressively over time. Furthermore,a substantial fraction of the total IPL pool remained intact for several weeks after the cultures had reached full senescence. Overall, IPL concentrations correlatedwith total cell counts (including both living and dead cells) rather than with living cell counts, implying that they do not exclusively reflect living cells. IPL degradationrates were enhanced when bacteria were introduced into the culture, but also in this scenario the IPL pool did not disappear completely. These results show that IPL degradationis not as uniformly rapid and complete as is currently assumed. While circumstantial evidence suggests that in most marine environments the majority of the IPL pool is degraded fairly rapidly, and most IPLs can be used as proxies for living microbialcells, care should be taking in settings where IPL degradation may be impeded. For example, in anoxic sediments with a high organic matter content the possibility exists that part of the IPL pool escapes degradation and becomes fossilized.In marine sediments of the Gullmar Fjord in southwest Sweden, a specific biomarker IPL (C20--ladderane monoalkylether phosphatidylcholine) was used to trace abundancesof its source organisms: anaerobic ammonium oxidizing (anammox) bacteria.The good correlations found between ladderane IPL concentrations in the sediment and several other markers for ladderane bacterial abundances and activity show the applicability of this very specific biomarker. However, a discrepancy between concentrationsof the ladderane IPL and ladderane fatty acids was noted, which increased progressively with sediment depth. This shows that these fatty acids are readily fossilized and IPLs provide a better indicator for living microbial cells.To assess the value of IPLs as environmental and chemotaxonomic markers, a comprehensive analysis of the IPL composition, environmental conditions and microbial communitycomposition of marine surface waters was performed in the North Sea. Strong environmental gradients between the North Sea and its adjacent water masses of the eastern North Atlantic Ocean and Skagerrak/Baltic Sea result in a number of distincthydrogeographical regions. These in turn drive the spatial distribution of the diverse microbial community, resulting in biogeographic regions with distinct microbiologicalcompositions during summer.Comprehensive IPL analysis of the North Sea surface waters indicated the presence of a large structural variety, comprising thousands of different IPL species. The IPL poolwas dominated by seven IPL classes, with the sulphur-bearing glycerolipid sulfoquinovosyl- diacylglycerol (SQDG) being the most abundant. The glycerophospholipidsphosphatidylcholine (PC), -glycerol (PG) and -ethanolamine (PE), as well as the betaine lipids diacylglyceryl-trimethylhomoserine (DGTS), -trimethylalanine (DGTA) and -carboxyhydroxymethylcholine(DGCC) were present in smaller and roughly comparableamounts. The overall fatty acid compositions of the different IPL classes were quite distinct, with the SQDGs containing almost exclusively short-chain saturated fatty acids, while the PCs, DGTAs and DGCCs contained many long-chain polyunsaturatedfatty acids (PUFAs).Statistical comparisons were made between the environmental and microbial parameters and the IPL compositions measured throughout the North Sea (spatial distribution) and measured in the Marsdiep tidal inlet over a one-year time series (temporal variation). Little evidence was found for a direct influence of environmentalconditions on IPL composition, and it appears that in this area the microbial community composition is the dominant factor determining the IPL composition. Tentative microbial sources for the predominant IPL classes could be identified, but only withpoor taxonomic resolution (e.g., small algae, cyanobacteria, diatoms), and none of the statistical relationships were particularly strong. This was particularly striking in theMarsdiep time series, where the IPL composition of the surface water remained relatively constant throughout the year, despite major shifts in the phytoplankton community composition. Combined with the observation in other studies that a comparablesuite of IPLs predominates in marine waters at a variety of sites, it thus appears that the most abundant IPLs in the world’s oceans are non-specific. This lack of chemotaxonomic resolution of the majority of the IPL pool makes it difficult to target specific microbial groups by general IPL screening.To bring out the full potential of IPLs as biomarkers in (marine) environmental microbiology, it thus seems necessary to focus on specific biomarker IPLs, such as ladderanes for anammox bacteria. Unfortunately, general full scan IPL screening like thatperformed in the North Sea and at other marine sites, will detect the common, most abundant and least specific IPLs, which obscure the less abundant, but more specific IPLs. This deeper layer of information can be accessed by specifically targeting knownbiomarker IPLs, for example using selective reaction monitoring (SRM). Alternatively, it may be possible to detect these more relevant IPLs by applying techniques that yieldmore structural information, such as MS3, 2D-HPLC-MS or high mass accuracy MS, particularly in combination with bioinformatics approaches to deal with the very large datasets (meta-lipidomics). Regardless of the techniques chosen, more specific biomarker IPLs will need to be obtained from microbial enrichment cultures. If the remaining issues with IPL degradation can be resolved, and their chemotaxonomic specificity can be increased, IPLs will be a useful tool for detecting and quantifying microorganisms in the marine environment.