Probing Biological Nitrogen Fixation in Legumes Using Raman Spectroscopy

Abstract

Biological nitrogen fixation (BNF) by symbiotic bacteria plays a vital role in sustainable agriculture. However, current quantification methods are often expensive and impractical. This study explores the potential of Raman spectroscopy, a non-invasive technique, for rapid assessment of BNF activity in soybeans. Raman spectra were obtained from soybean plants grown with and without rhizobia bacteria to identify spectral signatures associated with BNF. δN15 isotope ratio mass spectrometry (IRMS) was used to determine actual BNF percentages. Partial least squares regression (PLSR) was employed to develop a model for BNF quantification based on Raman spectra. The model explained 80% of the variation in BNF activity. To enhance the model's specificity for BNF detection regardless of nitrogen availability, a subsequent elastic net (Enet) regularization strategy was implemented. This approach provided insights into key wavenumbers and biochemicals associated with BNF in soybeans.

Code and data availability

The authors state that the original data (Raman spectra and BNF/IRMS measurements) are openly available on FigShare under DOI 10.6084/m9.figshare.25909780. This is a paper-specific, publicly deposited dataset directly reproducing the study's phenotyping measurements. No author analysis code or trained model is reported