Combined carbon, hydrogen, and clumped isotope fractionations reveal
differential reversibility of hydrogenotrophic methanogenesis in
laboratory cultures
Abstract
Stable isotope analysis has been widely used to aid the source
identification of methane. However, the isotopic (13C/12C and D/H) and
isotopologue (13CH3D and 12CH2D2) signatures of microbial methane in
natural environments are often different from those in laboratory
cultures in which methanogens are typically grown under optimal
conditions. Growth phase and hydrogen (H2) concentration have been
proposed as factors controlling the isotopic compositions of methane,
but their effects on the relationship among carbon, hydrogen and
doubly-substituted “clumped” isotopologue systems have not been
assessed in a quantitative framework. Here we experimentally investigate
the bulk (δ13C and δD) and clumped (∆13CH3D) isotopologue compositions
of methane produced by hyperthermophilic hydrogenotrophic (CO2-reducing)
methanogens using batch and fed-batch systems at different growth phases
and H2 mixing ratios (Methanocaldococcus bathoardescens at 82 or 60 °C
and on 80 or 25% H2; Methanothermobacter thermautotrophicus ∆H at 65 °C
and on 20, 5 or 1.6% H2). We observed a large range (18 to 63‰) of
carbon isotope fractionations, with larger values observed during later
growth phase, consistent with previous observations. In contrast,
hydrogen isotope fractionations remained relatively constant at –317 ±
25‰. Linear growth was observed for experiments with M. bathoardescens,
suggesting that dissolution of gaseous H2 into liquid media became the
rate limit as cell density increased. Accordingly, the low (and
undersaturated) dissolved H2 concentrations can explain the increased
carbon isotope fractionations during the later growth phase. The δD and
Δ13CH3D values indicated departure from equilibrium throughout
experiments. As the cell density increased and dissolved H2 decreased,
Δ13CH3D decreased (further departure from equilibrium), contrary to
expectations from previous models. Our isotopologue flow network model
reproduced the observed trends when the last H-addition step is less
reversible relative to the first three H-addition steps (up to CH3-CoM).
In this differential reversibility model, carbon, hydrogen and clumped
isotopologue fractionations are largely controlled by the reversibility
of the first three H-addition steps under high H2 concentrations; the
last H-addition step becomes important under low H2. The magnitude of
depletion and decreasing trend in Δ13CH3D values were reproduced when a
large (≥6‰) secondary clumped kinetic isotope effect was considered in
the model. This study highlights the advantage of combined bulk and
clumped isotope analyses and the importance of physiological factors
(growth phase) and energy availability (dissolved H2 concentration) when
using isotope analyses to aid the source identification of methane.