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 (13)C content, which were grown in a mixture of (12)C- and fully carbon-labeled (13)C-glucose (99%) as a sole carbon source, were fed to the nematode. The (13)C signal in Caenorhabditis elegans was proportional to the (13)C 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 (13)C 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 (13)C-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.

The application of Raman spectroscopy for the detection and identification of microorganisms

“…This technique was further exploited to distinguish 13 C‐labeled microorganisms within real seawater samples. The same group then made use of SIP‐Raman to track down 13 C signals from 13 C‐glucose, which was fed to E. coli cells, which then were easy prey for the nematode Caenorhabditis elegans . This observation of the carbon flow in a food chain from carbon substrate to bacterial prey to eukaryotic predator was made possible by following the red shift of the phenylalanine peak at 1001 cm −1 and the thymine peak at 747 cm −1 .…”

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

“…SCRS of MRCs were acquired to examine their 13 C incorporation based on the fact that some Raman bands of 13 C-labelled cells shift to lower wavenumbers upon the incorporation of 13 C from a growth substrate (Huang et al, 2004(Huang et al, , 2007(Huang et al, , 2009Li et al, 2013). SCRS of MRCs from 12 C-phenol treatments were used as controls.…”

Biological and Medical Applications of Multivariate Curve Resolution Assisted Raman Spectroscopy

“…Raman spectroscopy, when combined with a microscope, achieves sub-μm spatial resolution, which can provide detailed spaceresolved molecular information and visualize their intracellular distribution. Owing to its ability to detect biochemical changes at the molecular level, Raman spectroscopy has gained much attention as a bio-analytical tool in recent years, and has been successfully applied in myriad different problems in biological and medical sciences, which include, but not limited to, structural identification and label-free biomolecular imaging of living cells, [1][2][3][4][5][6][7] cellular metabolic pathways, 8,9 cellular discrimination, [10][11][12][13] tissue characterization, 14,15 disease diagnosis, prognosis and therapy. component analysis (PCA) and clustering analysis (CA) are two popular methods.…”