Microorganisms are involved in all elemental cycles and therefore it is important to study their metabolism in the natural environment. A recent technique to investigate this is the hydrogen isotopic composition of microbial fatty acids, i.e., heterotrophic microorganisms produce fatty acids enriched in deuterium (D) while photoautotrophic and chemoautotrophic microorganisms produce fatty acids depleted in D compared to the water in the culture medium (growth water). However, the impact of factors other than metabolism have not been investigated. Here, we evaluate the impact of growth phase compared to metabolism on the hydrogen isotopic composition of fatty acids of different environmentally relevant microorganisms with heterotrophic, photoautotrophic and chemoautotrophic metabolisms. Fatty acids produced by heterotrophs are enriched in D compared to growth water with εlipid/water between 82 and 359‰ when grown on glucose or acetate, respectively. Photoautotrophs (εlipid/water between −149 and −264‰) and chemoautotrophs (εlipid/water between −217 and −275‰) produce fatty acids depleted in D. Fatty acids become, in general, enriched by between 4 and 46‰ with growth phase which is minor compared to the influence of metabolisms. Therefore, the D/H ratio of fatty acids is a promising tool to investigate community metabolisms in nature.

The core metabolism of microorganisms has a major influence on the hydrogen isotopic composition of their fatty acids. Heterotrophic microorganisms produce fatty acids with a deuterium to hydrogen (D/H) ratio either slightly depleted or enriched in D compared to the growth water, while photo- and chemoautotrophic microorganisms produce fatty acids which are heavily depleted in D. However, besides metabolism other biochemical and environmental factors (i.e. biosynthetic pathways, growth phase and temperature) have been shown to affect the D/H ratio of fatty acids, and it is necessary to evaluate the magnitude of these effects compared to that of metabolism. Here, we show that the effect of salinity on the D/H ratio of fatty acids depends on the core metabolism of the microorganism. While fatty acids of the photoautotroph Isochrysis galbana become more enriched in D with increasing salinity (enrichment of 30-40‰ over a range of 25 salinity units), no effect of salinity on the D/H ratio of fatty acids of the heterotrophic Pseudomonas str. LFY10 was observed ((ε)lipid/water of the C16:0 fatty acid of ~120‰ over a range of 10 salinity units). This can likely be explained by the relative contributions of different H and nicotinamide adenine dinucleotide phosphate sources during fatty acid biosynthesis.

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Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography-mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.

Abstract. Culture studies of microorganisms have shown that the hydrogen isotopic composition of fatty acids depends on their metabolism, but there are only few environmental studies available to confirm this observation. Here we studied the seasonal variability of the deuterium-to-hydrogen (D / H) ratio of fatty acids in the coastal Dutch North Sea and compared this with the diversity of the phyto- and bacterioplankton. Over the year, the stable hydrogen isotopic fractionation factor ε between fatty acids and water (εlipid/water) ranged between −172 and −237 ‰, the algal-derived polyunsaturated fatty acid nC20:5 generally being the most D-depleted (−177 to −235 ‰) and nC18:0 the least D-depleted fatty acid (−172 to −210 ‰). The in general highly D-depleted nC20:5 is in agreement with culture studies, which indicates that photoautotrophic microorganisms produce fatty acids which are significantly depleted in D relative to water. The εlipid/water of all fatty acids showed a transient shift towards increased fractionation during the spring phytoplankton bloom, indicated by increasing chlorophyll a concentrations and relative abundance of the nC20:5 polyunsaturated fatty acids, suggesting increased contributions of photoautotrophy. Time periods with decreased fractionation (less negative εlipid/water values) can potentially be explained by an increased contribution of heterotrophy to the fatty acid pool. Our results show that the hydrogen isotopic composition of fatty acids is a promising tool to assess the community metabolism of coastal plankton potentially in combination with the isotopic analysis of more specific biomarker lipids.