Determining bacterially mediated fate of carbon: A stable isotope approach with a selection of cultivated marine bacteria

Bacteria are global players in marine DOC cycling but measuring how their genomic potential dictates geochemical function remains a challenge that limits our ability to generate an accurate determination of C fate. Such information is needed to both refine our understanding of marine biogeochemistry and establish baseline values to assess physiological impacts of warming and acidification due to climate change. To account for the complexity of microbial communities and chemical compositions, our approach uses a simplified system to examine the abilities of a library of 16 phylogenetically diverse bacterial isolates to incorporate and metabolize glycolate, an important component of DOC, using genomic analyses and isotope tracing at the single-cell level. Nanoscale secondary ion mass spectrometry (NanoSIMS) performed on over 500 cells revealed that 11 of the isolates incorporated large quantities of ¹³C-glycolate – up to 16 atom percent excess (APE) compared to natural abundance – revealing high affinity for the compound. While these uptake patterns were reflected in the genomes as the presence or absence of glycolate permeases and dehydrogenases, the lack of obvious relationships between family-level identities and the ability to metabolize the compound highlight DOC-assimilation strategies operating within individual genera. Semi-quantitative metabolomic analyses of spent media using LC-MS confirmed NanoSIMS observations; glycolate was absent or low compared to blanks, indicating significant bacterial drawdown. However, we did not detect differential metabolite signatures produced by the isolates from glycolate, suggesting minimal excretion of extracellular metabolites in monocultures. Ultimately, the single-cell heterogeneity in glycolate assimilation illustrated by NanoSIMS suggests that the metabolisms of subpopulations within the clonal community may differentially shunt C into biomass generation versus metabolite production versus CO₂ release via respiration. Ongoing work is quantifying this partitioning using cavity ring down spectroscopy and nuclear magnetic resonance to meld laboratory observations of bacterial C shuttling with the more intricate dynamics of marine microbial biogeochemistry in natural systems.