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Abstract

Determining the biogeochemical pathways utilized by microbes living in groundwater is
essential for understanding the subsurface C cycle and the fate of organic compounds, including
pollutants. The radiocarbon signature (Δ14C) of fatty acid methyl esters derived from microbial
phospholipids (PLFA) provides useful information for differentiating microbial C sources and infering
microbial metabolism. However, in subsurface environments, those analyses remain challenging. Here we
present a method combining large volume groundwater filtration (up to 10,000 L) and PLFA purification for
subsequent compound‐specific radiocarbon analyses. The analytical method involves conventional
chemical extraction of PLFA followed by purification of individual compounds by semipreparative
high‐performance liquid chromatography. Different saturated PLFA in amounts of up to 10 μg each can be
simultaneously separated on a C18 high‐load column using a mixture of MeOH/water and acetonitrile as the
mobile phase. Our procedure introduced dead‐Cext contaminations of 0.57 ± 0.29 and 0.35 ± 0.18 μg for
the high‐performance liquid chromatography and combustion/graphitization steps of the sample
preparation, respectively. However, tests on different high‐performance liquid chromatography C18 columns
revealed a large difference in dead Cext associated with column bleed. Modern Cext in the amount of
0.40 ± 0.20 μg was introduced by the combustion/graphitization step of the sample preparation, but other
steps did not add modern Cext. The entire method recovered ∼50% of the purified compounds on average,
but this did not affect their 14C content. This method will allow routine analysis of the Δ14C of PLFA
isolated from groundwaters or other sample types, revealing the relationships between microbial and soil‐derived C, sedimentary or dissolved C sources.