Hydrogen isotopes of lipids as a proxy for central metabolism and carbon use efficiency in soil microbes

Microbial carbon use efficiency (CUE) represents the proportion of carbon consumed by microbes that is accumulated in biomass instead of respired, and is important for understanding carbon cycling and storage in soils. Existing methods for quantifying CUE rely on additions of isotopically labeled material and/or incubations in laboratory settings, which may differ from in situ conditions. We propose an alternative strategy to study microbial growth and metabolism using hydrogen isotopes of microbial phospholipid fatty acids (δ²H_PLFA). 

In bacterial monocultures, δ²H_PLFA values are strongly influenced by central metabolism. In particular, the most ²H-depleted PLFAs produced by heterotrophic bacteria are those with precursors derived from the Embden-Meyerhof-Doudoroff (EMP) and pentose phosphate pathways. When glycolysis proceeds through the Entner-Doudoroff (ED) pathway, fatty acids are significantly enriched in ²H. However, the highest δ²H_PLFA values are from cultures grown on precursors from the tricarboxylic acid (TCA) cycle as the sole carbon source. These naturally occurring differences in δ²H_PLFA values in cultures are one to two orders of magnitude greater than spatial and temporal variability soil water δ²H values. Therefore, δ²H_PLFA values offer the opportunity to detect relative changes in TCA activity by soil microbes. As the TCA cycle is typically associated with higher respiration and lower CUE, and specific PLFAs are primarily derived from distinct microbial groups, δ²H_PLFA values have potential as indicators of CUE for different groups of soil microbes.

Soil communities are inherently more diverse and dynamic than monocultures. As a first step to assess the utility of δ²H_PLFAs as indicators of group-specific metabolism, we established a two-species system using a gram-negative bacteria (Pseudomonas sp.) and gram-positive bacteria (Bacillus sp.). These taxa produce distinct PLFAs from each other and utilize distinct pathways for glycolysis. We grew monocultures and co-cultures in a minimum media (M9) with either glucose or succinate as the sole carbon source. We harvested cultures at mid-exponential phase and measured δ²H values of extracted fatty acids.

First, we confirmed the results of previous monocultures with different strains of Pseudomonas and Bacillus. When grown on glucose, Bacillus, which uses the EMP pathway, produced fatty acids with an average δ²H value of -184 ± 12 ‰, while Pseudomonas, using the ED pathway, produced fatty acids with an average δ²H value of -20 ± 2 ‰. When they were grown on succinate and thereby forced to rely on the citric acid cycle, both bacteria produced fatty acids that were much more enriched in ²H (δ²H = +24 ± 5 ‰ for Bacillus and +192 ± 5 ‰ for Pseudomonas). When grown in co-cultures on glucose, δ²H values for PLFAs produced by Bacillus were similar to when it was grown alone on glucose (-189 ± 8 ‰), but δ²H values for PLFAs produced by Pseudomonas increased to +58 ± 18 ‰, indicating an increase in TCA cycle activity due to consumption of acetate secreted by Bacillus. These results demonstrate how metabolic changes driven by community interactions can be detected through δ²H_PLFA values and provide a foundation for applications in more complex systems.