Stable Isotope Probing and Raman Spectroscopy for Monitoring Carbon Flow in a Food Chain and Revealing Metabolic Pathway

Abstract

Accurately measuring carbon flows is a challenge for understanding processes such as diverse intracellular metabolic pathways and predator-prey interactions. Combined with stable isotope probing (SIP), single-cell Raman spectroscopy was demonstrated for the first time to link the food chain from carbon substrate to bacterial prey up to predators at the single-cell level in a quantitative and nondestructive manner. Escherichia coli OP50 with different ¹³C content, which were grown in a mixture of ¹²C- and fully carbon-labeled ¹³C-glucose (99%) as a sole carbon source, were fed to the nematode. The ¹³C signal in Caenorhabditis elegans was proportional to the ¹³C content in E. coli. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm^–1 and thymine at 747 cm^–1 Raman bands), were used to quantify the ¹³C content in E. coli and C. elegans over a range of 1.1–99%. The phenylalanine Raman band was a suitable biomarker for prokaryotic cells and thymine Raman band for eukaryotic cells. A biochemical mechanism accounting for the Raman red shifts of phenylalanine and thymine in response to ¹³C-labeling is proposed in this study and is supported by quantum chemical calculation. This study offers new insights of carbon flow via the food chain and provides a research tool for microbial ecology and investigation of biochemical pathways.