Simultaneous, High-Precision Measurements of δ2H and δ13C in Nanomole Quantities of Acetate Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry.

Stable hydrogen isotope compositions (2H/1H ratios) have been an invaluable tool for studying biogeochemical processes in nature, but the diversity of molecular targets amenable to such analysis is limited. Here, we demonstrate a new technique for measuring δ2H of biomolecules via Orbitrap mass spectrometry (MS) using acetate as a model analyte. Acetate was chosen as a target molecule because its production and consumption are central to microbial carbon cycling, yet the mechanisms behind acetate turnover remain poorly understood. δ2H of acetate could provide a useful constraint on these processes; however, it remains uncharacterized in nature due to analytical challenges. Electrospray ionization (ESI)-Orbitrap MS circumvents these challenges and delivers methyl-specific H-isotope compositions of acetate with nanomole sensitivity, enough to enable analyses of environmental samples. This approach quantifies the methyl-specific δ2H and molecular-average δ13C of acetate simultaneously while achieving <3 and <0.5‰ uncertainty, respectively. Using optimized ionization and Orbitrap parameters, this level of precision is obtained within 15 min using only 15 nmol of acetate. As a demonstration of our analytical approach, we cultured three acetogenic bacteria and found a large 2H-fractionation between acetate and water (>310‰ depletion) associated with the Wood-Ljungdahl pathway, while fermentation expressed a muted (∼80‰) fractionation. With its high precision and sensitivity, Orbitrap MS is a promising tool for investigating these signals in nature after offline purification. Furthermore, the ESI-Orbitrap method presented here could be applied to other molecules amenable to ESI, including central metabolites and sugars, greatly expanding the molecular targets used in hydrogen isotope biogeochemistry.

Environmental controls on bacteriohopanepolyol profiles of benthic microbial mats from Lake Fryxell, Antarctica.

Bacteriohopanepolyols (BHPs) are pentacyclic triterpenoid lipids that contribute to the structural integrity and physiology of some bacteria. Because some BHPs originate from specific classes of bacteria, BHPs have potential as taxonomically and environmentally diagnostic biomarkers. For example, a stereoisomer of bacteriohopanetetrol (informally BHT II) has been associated with anaerobic ammonium oxidation (anammox) bacteria and suboxic to anoxic marine environments where anammox is active. As a result, the detection of BHT II in the sedimentary record and fluctuations in the relative abundance of BHT II may inform reconstructions of nitrogen cycling and ocean redox changes through the geological record. However, there are uncertainties concerning the sources of BHT II and whether or not BHT II is produced in abundance in non-marine environments, both of which are pertinent to interpretations of BHT II signatures in sediments. To address these questions, we investigate the BHP composition of benthic microbial mats from Lake Fryxell, Antarctica. Lake Fryxell is a perennially ice-covered lake with a sharp oxycline in a density-stabilized water column. We describe the diversity and abundance of BHPs in benthic microbial mats across a transect from oxic to anoxic conditions. Generally, BHP abundances and diversity vary with the morphologies of microbial mats, which were previously shown to reflect local environmental conditions, such as irradiance and oxygen and sulfide concentrations. BHT II was identified in mats that exist within oxic to anoxic portions of the lake. However, anammox bacteria have yet to be identified in Lake Fryxell. We examine our results in the context of BHPs as biomarkers in modern and ancient environments.

Bacteriohopanepolyols across environmental gradients in Lake Vanda, Antarctica.

Bacteriohopanepolyols (BHPs) are bacterial membrane lipids that may be used as biological or environmental biomarkers. Previous studies have described the diversity, distribution, and abundance of BHPs in a variety of modern environments. However, the regulation of BHP production in polar settings is not well understood. Benthic microbial mats from ice-covered lakes of the McMurdo Dry Valleys, Antarctica provide an opportunity to investigate the sources, physiological roles, and preservation of BHPs in high-latitude environments. Lake Vanda is one of the most stable lakes on Earth, with microbial communities occupying specific niches along environmental gradients. We describe the influence of mat morphology and local environmental conditions on the diversity and distribution of BHPs and their biological sources in benthic microbial mats from Lake Vanda. The abundance and diversity of C-2 methylated hopanoids (2-MeBHP) are of particular interest, given that their stable degradation products, 2-methylhopanes, are among the oldest and most prevalent taxonomically informative biomarkers preserved in sedimentary rocks. Furthermore, the interpretation of sedimentary 2-methylhopanes is of great interest to the geobiology community. We identify cyanobacteria as the sole source of 2-MeBHP in benthic microbial mats from Lake Vanda and assess the hypothesis that 2-MeBHP are regulated in response to a particular environmental variable, namely solar irradiance.