Combined stable isotope, proteomic, metabolomics, and spatial specific analysis to track carbon flow through a hypersaline phototrophic microbial mat

Abstract:

Tracking labeled substrates through microbial mat systems can help elucidate carbon dynamics, species interactions, and niche partitioning, but the inherent microbial complexity of these systems makes them difficult to probe with single analytical techniques. Here we use a combination of different tools to track three labeled substrates through a benthic phototrophic mat from Hot Lake. Hot Lake is a hypersaline, meromictic lake located in an endorheic basin in north-central Washington which, despite extreme salinity and seasonal water temperatures (> 55 ˚C), hosts dense, phototrophic benthic microbial mats. Cyanobacteria are the dominant CO₂-fixing organisms in the system and we seek to understand the spatial and metabolic controls on how the carbon initially fixed by mat cyanobacteria is transferred to associated heterotrophic populations spread throughout the mat strata.

We performed ex situ incubations over a complete diel cycle with ¹³C labeled bicarbonate, acetate, and glucose. Traditional elemental analysis IRMS provided an estimate of bulk label uptake to total biomass and showed that both bicarbonate and acetate were incorporated only during daylight while glucose uptake was nearly constant through the cycle. Spatially resolved isotope analysis using laser ablation IRMS showed distinctive patterns between the different substrates with bicarbonate having highest uptake in the top third of the mat, acetate uptake focused near the mat’s center, and glucose showing similar uptake at all mat depths. Proteomic analysis showed a longer lag in substrate conversion to protein than to biomass and a distinct spike in the number of labeled peptides in the bicarbonate incubation near the end of the diel cycle. Proteomic analysis confirmed that photosynthetic organisms showed the highest rates of label conversion to protein but heterotrophic organisms also incorporated label into their peptides. Metabolomic analysis demonstrated the high conversion of organic substrates to storage compounds within the biomass. Taken together, these orthogonal data combine to show distinct differences in the fate of the applied substrates. Using this combination of analytical methods can build a detailed picture of carbon dynamics in complex microbial systems.