Ratios of carbon isotopes in microbial lipids as an indicator of substrate usage

Abstract

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of delta13C values of palmitic acid (C16:0) with the delta13C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C16:0 with 13C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of 13C in C16:0 while incorporating the carbons of glucose, and most of them were enriched with 13C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in 13C compared to the phospholipid fractions. The delta13C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C14:0) was depleted of 13C compared to palmitic acid (C16:0) while octadecanoic acid (C18:0) was enriched. These results are important for the calibration of a new method in which delta13C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.

FIG. 1

Growth curves and isotope ratios of the palmitic acid (C_16:0) of Pseudomonas putida in defined medium with glucose (triangles) and glycerol (circles). Biomass was determined as the optical density at 600 nm (OD₆₀₀) and is shown as solid symbols, whereas the isotope ratios of C_16:0 are given as open symbols.

FIG. 2

Carbon isotopic fractionation, ɛ, of C_16:0 from four different carbon sources (for definitions, see Materials and Methods). The four diagrams have the same scale. Pseudomonas sp. strain 8001 was grown twice with all substrates and is shown as 8001a and 8001b to show biological reproducibility.

FIG. 3

Isotope ratios of biomass and C_16:0 from the different fractions of polar and bound lipids of Fusarium solani compared with the isotope ratio of the corresponding substrate. Abbreviations: PL, phospholipids; GL, glycolipids; BL, bound lipids. For clarity, the isotope ratio of the substrate and the biomass is shown three times for direct comparison with those of the fatty acid.

FIG. 4

Isotope ratios of different fatty acids of Pseudomonas putida in four different minimal media and one complex medium. Open triangles, C_16:0; solid squares, C_18:1ω7; crosses, C_18:1ω6. GL, glycolipids; PL, phospholipids; BL, bound lipids.