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Towards stable chemical fossils for anaerobic ammonium-oxidizing bacteria in palaeoenvironmental studies: novel cyclic aliphatic hydrocarbons as potential dia- and catagenetic products of ladderane lipids

Abstract

Hydrous-pyrolysis oils from anammox-enriched biomass (Jaeschke et al., 2008 conditions) are screened for thermally robust cyclic hydrocarbons beyond labile ladderane lipids; two-dimensional GC–MS, preparative isolation, 2D NMR, synthesis of model compounds, and δ¹³C signatures motivate tricyclic products possibly derived from C₂₀ ladderane ethers, while ReaxFF conformer screening plus B3LYP/6-311++G** in Jaguar refine stereochemistry and relative stabilities.

Summary

Anammox bacteria carry diagnostic ladderane lipids that do not survive deep thermal maturity, limiting paleoredox proxies. This study re-examines pyrolysate oils from prior hydrous pyrolysis of anammox-rich sludge (temperatures >200 °C) and identifies novel cyclic aliphatic hydrocarbons as candidate long-lived fingerprints. Analytical chemistry (2D GC–MS, GC–MS, preparative isolation, 2D NMR, δ¹³C) constrains structures; ReaxFF relaxations screen many stereoisomers before DFT refinements and Boltzmann-style mixture estimates.

Methods

Hydrous pyrolysis and fractionation (Jaeschke et al. protocol)

  • Feedstock: Dried wastewater sludge (~70% Candidatus Kuenenia stuttgartiensis biomass) from Dokhaven (Paques B.V.).
  • Reactors: 75 g solids + 500 g distilled water in 1 L Hastelloy-C276 reactors; 14 temperature steps (120–365 °C), 72 h each.
  • Oils: Recovered for 200–365 °C runs from water surface and reactor walls (benzene rinse). Al₂O₃ column fractions (hexane, hexane/DCM 9:1, DCM/MeOH 1:1); AgNO₃–silica cleanup of saturates; anteiso-C₂₂ internal standard for quantification (as described).

Organic analysis

  • Chromatography / MS: GC, GC–MS, GC-SMB-MS, and comprehensive 2D GC–MS per prior instrumental articles cited in the paper.
  • Isotopes: δ¹³C on appropriate fractions (instrumentation referenced).
  • Structure: Preparative GC isolation of a representative product (two co-eluting components), 2D NMR assignment, comparison to synthesized model compounds, mass spectral matching.

Computational chemistry (ReaxFF and DFT)

  • ReaxFF: Chenoweth et al. CHO parameter set for geometry optimizations (conjugate gradient) of 32 starting stereoisomers of a key intermediate (IV); gradient threshold 0.25 (kcal/mol)/Å; annealing 10–1000 K on 16 survivors within 10 kcal/mol of the lowest ReaxFF structure.
  • DFT: Jaguar 7.5, B3LYP, 6-311++G(d,p) on down-selected isomers; equilibrium mixture fractions from relative free energies (entropy neglected at 298 K in the stated approximation).

ReaxFF in this work is used for conjugate-gradient geometry optimization and short annealing temperature ramps (10–1000 K) in gas-phase cluster models (non-periodic boundary conditions; atom lists and stoichiometry for intermediate IV isomers are defined in the GCA Computational section rather than PBC slab supercells)—N/A for a Langevin/Nosé–Hoover thermostat-controlled NVT production run in the same sense as long molecular dynamics; full NVE/NVT Molecular dynamics with time step in fs is N/A as the headline result (see Jaguar B3LYP block for high-accuracy energies). N/A — ps/ns NPT melt trajectories; N/A — barostat-controlled GPa pressure; N/A — electric field; N/A — replica exchange-style sampling in ReaxFF (the DFT section instead reports formation energy-driven mixture estimates).

Findings

  • Pyrolysates at >200 °C contain alkyl-branched tricyclic hydrocarbons interpreted as 4-octyl-tricyclo[7.3.0.0²,⁸]dodecane-type frameworks (7-4-5 ring counts) from 2D NMR, with mass-spectral support via synthetic references.
  • δ¹³C of isolated material matches the strong ¹³C depletion of ladderane fatty acids from the parent biomass, supporting a biogenic ladderane origin.
  • A mechanistic proposal links products to ether cleavage and cyclobutane rearrangement from C₂₀ [5]- or [3]-ladderane glycerol ethers; ReaxFF/DFT rank stereoisomer stabilities and outline reaction pathway-style interconversion among congeners (see B3LYP barriers in the article for quantitative ranking). Comparisons to synthesized standards and literature retention times anchor the MS and 2D NMR identifications; δ¹³C match to source ladderane lipid signatures is the key biogenic line of evidence (authors note caveats where laboratory pyrolysis overestimates field maturity). Sensitivity to hydrous pyrolysis temperature steps (120–365 °C ladder in the reactor series) is central to which oil cuts are examined, although this summary does not reproduce every T-bin. For corpus use: treat the GCA pre-proof PDF in pdf_path as possibly non-final page layout; version-of-record is authoritative.

Limitations

Elsevier journal pre-proof PDF: layout and pagination may change. Hydrous pyrolysis is a laboratory maturation mimic, not a field diagenesis reactor.

Relevance to group

Adri C. T. van Duin co-authored the ReaxFF / computational chemistry thread on stereoisomer stability and pathways.

Citations and evidence anchors

  • DOI: 10.1016/j.gca.2025.12.040 — Geochimica et Cosmochimica Acta (pre-proof PDF in corpus).