Abstract
The stable hydrogen isotope composition of persistent biomolecules is
used as a paleoenvironmental proxy. While much previous work has focused
on plant leaf wax-derived n-alkanes, the potential of bacterial and
archaeal lipid biomarkers as carriers of H isotope signatures remains
underexplored. Here we investigated H isotope distributions in the
membrane lipids of the ammonia-oxidizing chemoautotroph Nitrosopumilus
maritimus strain SCM1. Hydrogen isotope ratios were measured on the
biphytane chains of tetraether membrane lipids extracted from
steady-state continuous cultures cultivated at slow, medium, and fast
growth rates. In contrast to recent work on bacterial fatty acids, where
the direction and magnitude of isotopic fractionation varies widely (ca.
600 ‰ range) in response to the choice of substrate and pathways of
energy metabolism, archaeal biphytane data in the present work are
relatively invariant. The weighted average 2H/1H fractionation values
relative to growth water (2εL/W) only ranged from 272 to 260 ‰, despite
a three-fold difference in doubling times (30.8 hr to 92.5 hr), yielding
an average growth-rate effect of 0.2 ‰ hr-1. These 2εL/W values are more
depleted than all heterotrophic archaeal and bacterial lipid H isotope
measurements in the literature, and on par with those from other
autotrophic archaea, as well as isoproenoid-based lipids in
photoautotrophic algae. N. maritimus values of 2εL/W also varied
systematically with the number of internal rings (cyclopentyl +
cyclohexyl), increasing for each additional ring by 6.4 ± 2.7 ‰. Using
an isotope flux-balance model in tandem with a comprehensive analysis of
the sources of H in archaeal lipid biosynthesis, we use this observation
to estimate the kinetic isotope effects (KIEs) of H incorporation from
water; from reducing cofactors such as ferredoxin, and for the
transhydrogenation reaction(s) that convert the electron-donor derived
NADH into NADPH for anabolic reactions. Consistent with prior studies on
bacteria, our results indicate the KIEs of reducing cofactors and
transhydrogenation processes in archaea are highly fractionating, while
those involving exchange of water protons are less so. When combined
with the observation of minimal growth-rate sensitivity, our results
suggest biphytanes of autotrophic 3HP/4HB Thaumarchaeota may be offset
from source waters by a nearly constant 2εL/W value. Together with the
ring effect, this implies that all biphytanes originating from a common
source should have a predictable ordering of their isotope ratios with
respect to biphytane ring number, allowing precise reconstruction of the
original δ2H value of the growth water. Collectively, these patterns
indicate archaeal biphytanes have potential as paleo-hydrological
proxies, either as a complement or an alternative to leaf wax n-alkanes.