title: Gas phase pyrolytic approach for stable isotope analysis of hopanoids lipids
Creators: name: Amariei, Anca Elena
Publisher: name: University of Glasgow
Year: 2024

description: Methanotrophs are known for their role in mitigating CH4 emissions, with aerobic methanotrophs adapted to a wide range of environments. In oxic settings, they are able to utilise this high global warming potential molecule as a source of energy, oxidising it to CO2, or as a source of carbon, incorporating the CH4-carbon’s (C-CH4) isotopic signature into the intramolecular structure of the membrane-located hopanoid lipids. Being decay-resistant, hopanoids can be used to study past CH4 cycles.
 
Compound-specific isotope analysis (CSIA) of diploptene, a hopanoid biomarker, has been initially employed in this work to identify the advantages and limitations of this technique to assess past CH4 consumption within three peatlands of Pastaza-Marañón Foreland Basin (PMFB). This study thus provides the first multi-biomarker, yet low-resolution, isotopic reconstruction of the three peatlands, with CSIA values of hopanoids, as well as of n-alkanes and fatty acids interpreted in conjunction with previously measured biomarker concentrations and a wide range of palaeoenvironmental data from the same peatlands or area. Isotopic values of n-alkanes and diploptene reflect the CH4-producing conditions of the area, fluctuating with past changes in each peatland’s history. It was however found that methanotrophs were not the sole producers of hopanoids and, due to source dilution and the fact that the three aerobic methanotroph types can also incorporate different amounts of CCO2, via distinctive pathways, diploptene’s methanotrophic isotopic signature can become muted in these (palaeo)environmental samples at the CSIA level.

 These limitations, together with low compound concentrations and co-elution, were also encountered in simplified, one-diploptene source methanotrophic lab cultures conducted in this work to study diploptene’s CSIA evolution in relationship to isotopically known CH4 and CO2 sources. Even when the bio-producer is known, diploptene’s CSIA was influenced by the varying quantities of C-CH4 and C-CO2 incorporated by type I and II methanotrophs. New tools are thus required to assess methanotrophic processes in natural environments and increase confidence in source attribution. This work develops theoretical and analytical methodologies for the gaseous pyrolysis of diploptene that could enable the detection of methanotrophy at the intramolecular (or position-specific) level through isotopic analyses. 

Analytical instruments capable of accessing this information are currently restricting these new isotopic applications through complex setups, molecule-specific uses and high costs. Gaseous pyrolysis is demonstrated in this thesis on a bespoke GC-FID instrument (prep-GC) adapted to overcome these limitations. The development of the prep-GC instrument and methodology took place in a step-like fashion, with experiments aimed at increasing and testing its capacities on compounds such as methanol, MTBE, 2,3 DMN, squalene and diploptene, and their multiple pyrolysates. The prep-GC is demonstrated to be able to analyse compounds with molecular masses between 32 - 410 g/mol and at least up to 850℃ pyrolytic temperatures, making it a universal analytical instrument for the pyrolysis of volatile and semi-volatile compounds. Experiments presented in this thesis demonstrate the ability of the prep-GC to heart-cut and concentrate, within a trap, compounds of interest, release, pyrolyse and transfer the pyrolysates to a second detector for quantification and identification. The analytical pyrolytic methodologies were further adapted and the results were confirmed through in-silico pyrolysis, using Reaction Mechanism Generator (RMG, Gao et al., 2016). 

Diploptene, and hopanoids in general, have never been subjected to pyrolysis to investigate if the C-CH4 moieties can be accessed and their relationship to the CH4 cycle. The pyrolytic breakdown mechanism of diploptene was assessed first on RMG and the results were experimentally studied. On RMG, for the simulated pyrolytic temperature of 750℃, isoprene was the main pyrolytic fragment, produced from squalene, which occurs as an unstable intermediate. During pyrolysis, the diploptene backbone therefore unfolded, undoing the action of the squalene-hopene cyclase, and was broken down into isoprene units. Theoretical and experimental evidence of diploptene, squalene and isoprene pyrolysis are presented to support isoprene as the main diploptene pyrolysis fragment between 550℃-625℃. Isoprene however is demonstrated to not provide new isotopic evidence when compared to diploptene, as it does not allow a direct isotopic investigation of the C-CH4 and C-CO2 incorporated through the different methanotroph bio-cycles. Isoprene ozonolysis is proposed as a future tool that could access this intramolecular information, post diploptene (and squalene) pyrolysis. Finally, although produced from the same predecessor (squalene), RMG simulations indicate that other hopanoids present different breakdown mechanisms, requiring individual breakdown pyrolytic assessments for future PSIA studies. 

The development of the prep-GC instrument for gaseous pyrolysis biomarkers at low environmental concentrations and the exploration of hopanoid pyrolysis mechanisms in this thesis contribute to advancing the understanding of methanotrophy and its detection via isotopic studies, paving the way for new intramolecular isotopic investigations.