Back flux during anaerobic oxidation of butane support archaea-mediated alkanogenesis

Abstract

Microbial formation and oxidation of volatile alkanes in anoxic environments significantly impacts biogeochemical cycles on Earth. The discovery of archaea oxidizing volatile alkanes via deeply branching methyl-coenzyme M reductase variants, dubbed alkyl-CoM reductases (ACR), prompted the hypothesis of archaea-catalysed alkane formation in nature (alkanogenesis). A combination of metabolic modelling, anaerobic physiology assays, and isotope labeling of Candidatus Syntrophoarchaeum archaea catalyzing the anaerobic oxidation of butane (AOB) show a back flux of CO₂ to butane, demonstrating reversibility of the entire AOB pathway. Back fluxes correlate with thermodynamics and kinetics of the archaeal catabolic system. AOB reversibility supports a biological formation of butane, and generally of higher volatile alkanes, helping to explain the presence of isotopically light alkanes and deeply branching ACR genes in sedimentary basins isolated from gas reservoirs.

Fig. 1. Microscopy characterization of the Butane50 culture.

A Native aggregate of Ca. Syntrophoarchaeum butanivorans, Ca. S. caldarius (red-purple signal) and Ca. Desulfofervidus auxilii (blue-green signal) after double hybridization with general probes for Archaea and Bacteria and DAPI counterstaining. The Archaea appear magenta, and the Bacteria appear cyan due to overlay of probe and DAPI signals. Representative of n = 3 images. B Double hybridization of dispersed aggregates with probes specific for Archaea (green signal), and for Ca. S. butanivorans (orange signal; Ca. S. butanivorans cells were hybridized with both probes). C Double hybridization of dispersed aggregates with probes specific for Ca. S. butanivorans (red-purple signal) and for Ca. S. caldarius (green signal). Each of the (B, C) are representative of n > 60 images from n = 3 independent cultures. Insets in (B, C) show the average abundance (n > 5000 cells counted) of Ca. S. butanivorans (orange), other Archaea (green, B), Ca. S. caldarius (green, C), and Bacteria (blue, both panels). Scale bars = 5 μm, applicable to all panels. Details of the oligonucleotide probes used are summarized in Supplementary Table 1.

Fig. 2. Metabolic thermodynamic model of the anaerobic oxidation of butane (AOB) and reverse AOB.

A Refined central carbon and energy metabolism during AOB (black arrows) or reverse AOB (light blue arrows). Numbers in the figure correspond to reactions in Table 1. B The reducing equivalents released during AOB/consumed during rAOB include 2 XH₂, 5 NADH, 4 F₄₂₀H₂, and 4 Fd_red, which in total account for 26 electrons. C, D Quasi-equilibrium concentrations (mol L⁻¹) of AOB/rAOB metabolites considering a standard redox potential of XH₂/X of −0.220 V. C The AOB model considers energy recovery at F₄₂₀H₂ and Fd_red oxidation, energy investment at the butyl-CoM/butyryl-CoA conversion and at electron transport between MQ and X, and energy dissipation (∆G_dis) at oxidation of methylene-tetrahydromethanopterin. Metabolite concentrations were calculated under XH₂/X ratios of 2, 4, and 8, corresponding to AOB free energy change (∆G_cat) of −147.23, −124.91, and −102.58 kJ mol⁻¹ butane, respectively. D The rAOB model considers a switch in energy recovery and energy investment, with energy dissipation occurring at acetyl-CoA synthesis. Metabolite concentrations were calculated for XH₂/X ratios of 1000, 2000, 4000, and 8000, corresponding to rAOB ∆G_cat of −66.54, −88.87, −111.19, and −133.52 kJ mol⁻¹ butane, respectively. Δµ_H+ denotes the free energy required to translocate one proton across the membrane (−20 kJ mol⁻¹). Source data are provided as a Source Data file.

Fig. 3. Experimental data for the AOB cultures incubated with increasing fractions of ¹³C-labeled DIC.

A Consumption of butane (circles) and formation of sulfide (diamonds) in cultures supplied with ¹³C-DIC ranging from 1.12 to 98%. B Development of δ¹³C values (in ‰) in the residual butane pools (circles); color shades represent the same ¹³C-DIC labeling ratios as in panel A; δ¹³C of butane in sterile controls showed no variations during the same incubation time (squares). C Correlation of δ¹³C of residual butane pools with the ¹³C abundance in the DIC pool during stationary phases. Source data are provided as a Source Data file.

Fig. 4. Model output for the dynamics of δ¹³C_butane, back flux and free energy change during AOB under variable ¹³C/¹²C of CO₂.

The upper row shows the time-dependent model fitting (lines) of experimental data (circles) showing the evolution of δ¹³C in residual butane pools. The middle row shows that the contribution of the modelled back flux (f₋) to the net AOB process (f_net) followed similar trends under variable labeling conditions. The bottom row shows that the total free energy change of AOB (∆G) shifts along the depletion of butane and accumulation of CO₂ throughout the incubation. The shading shows the 95% confidence interval of the estimated values. Source data are provided as a Source Data file.

Fig. 5. Back flux under sulfate limitation or with variable starting butane concentrations.

A, D Temporal evolution of measured concentrations of butane (C₄H₁₀, circles) and sulfide (HS^–, diamonds), along with trend lines fitted using the first-order dynamic model. Gray shades indicate measurements from low-sulfate incubations (5 mmol/L sulfate) with three different starting ¹³C-bicarbonate concentrations (A; 1.12%, 50%, and 98%), or from incubations with two different initial butane concentrations (D; 3 mmol l⁻¹ vs. 6 mmol l⁻¹). B, E Development of isotope compositions of butane (${\mathrm{\delta }}^{13}{\mathrm{C}}_{{\mathrm{C}}_4{\mathrm{H}}_{10}}$). Color shades represent the same incubation conditions as in A; Error bars in B, E, are standard deviations of technical replicates (n = 3). C, F Ratio of back flux relative to the net AOB rate (f₋/f_net) and Gibbs free energy change of the overall AOB reaction (∆G) estimated from measured concentrations and/or isotope compositions. Black lines show calculated values for low-sulfate assays with 50% ¹³C-bicarbonate (C) and for low-butane assays (F). The f₋/f_net and ∆G calculated for the assay with 6 mmol l⁻¹ C₄H₁₀ 98% ¹³C-bicarbonate, and 28 mmol l⁻¹ sulfate are shown for comparison (grey lines in C, F). The shaded envelopes in (C, F) indicate the 95% confidence interval of estimated values. Source data are provided as a Source Data file.

Fig. 6. Global biogeography of ACR-encoding archaea and of AcrA sequences.

A Maximum likelihood tree of AcrA protein sequences. AcrA’s from experimentally validated anaerobic multicarbon alkane oxidizing archaea are highlighted with red squares. AcrA’s from Ca. Syntrophoarchaeum and other ACR-encoding genera are marked with red and cyan squares. The transparent circles reflect the number of AcrA fragments from IMG/M database that are placed to the corresponding branches by RAxML EPA. Clades of methanogenic archaea are collapsed for visualization purposes. Scale bar = substitutions per site. B Boxplot of relative abundance of Ca. Syntrophoarchaeum (red) and other ACR-encoding genera (cyan) shown in the AcrA phylogeny. C Biogeographic distribution of Ca. Syntrophoarchaeum (circles) and other ACR-encoding genera (triangles) across diverse biomes. D Biogeographic distribution of AcrA sequences retrieved from environmental samples. Sequences were retrieved from assembled metagenomes in the JGI IMG/M database. The phylogenetic placement of fragments is shown in A. The fragments that are annotated with C-terminus (PF02249) and N-terminus (PF02745) domains of McrA are shown with circles and triangles, respectively. Source data are provided as a Source Data file.