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Abstract

Despite the global significance of the subsurface biosphere, the degree to which it depends
on surface organic carbon (OC) is still poorly understood. Here, we compare stable and radiogenic
carbon isotope compositions of microbial phospholipid fatty acids (PLFAs) with those of in situ potential
microbial C sources to assess the major C sources for subsurface microorganisms in biogeochemical
distinct shallow aquifers (Critical Zone Exploratory, Thuringia Germany). Despite the presence of
younger OC, the microbes assimilated 14C‐free OC to varying degrees; ~31% in groundwater within the
oxic zone, ~47% in an iron reduction zone, and ~70% in a sulfate reduction/anammox zone. The
persistence of trace amounts of mature and partially biodegraded hydrocarbons suggested that
autochthonous petroleum‐derived hydrocarbons were a potential 14C‐free C source for heterotrophs in the
oxic zone. In this zone, Δ14C values of dissolved inorganic carbon (−366 ± 18‰) and 11MeC16:0
(−283 ± 32‰), an important component in autotrophic nitrite oxidizers, were similar enough to indicate
that autotrophy is an important additional C fixation pathway. In anoxic zones, methane as an important
C source was unlikely since the 13C‐fractionations between the PLFAs and CH4 were inconsistent
with kinetic isotope effects associated with methanotrophy. In the sulfate reduction/anammox zone, the
strong 14C‐depletion of 10MeC16:0 (−942 ± 22‰), a PLFA common in sulfate reducers, indicated that
those bacteria were likely to play a critical part in 14C‐free sedimentary OC cycling. Results indicated
that the 14C‐content of microbial biomass in shallow sedimentary aquifers results from complex
interactions between abundance and bioavailability of naturally occurring OC, hydrogeology, and specific
microbial metabolisms.