Classification and Identification of Archaea Using Single-Cell Raman Ejection and Artificial Intelligence: Implications for Investigating Uncultivated Microorganisms

Archaea can produce special cellular components such as polyhydroxyalkanoates, carotenoids, rhodopsin, and ether lipids, which have valuable applications in medicine and green energy production. Most of the archaeal species are uncultivated, posing challenges to investigating their biomarker components and biochemical properties. In this study, we applied Raman spectroscopy to examine the biological characteristics of nine archaeal isolates, including halophilic archaea (Haloferax larsenii, Haloarcula argentinensis, Haloferax mediterranei, Halomicrobium mukohataei, Halomicrobium salinus, Halorussus sp., Natrinema gari), thermophilic archaea (Sulfolobus acidocaldarius), and marine group I (MGI) archaea (Nitrosopumilus maritimus). Linear discriminant analysis of the Raman spectra allowed visualization of significant separations among the nine archaeal isolates. Machine-learning classification models based on support vector machine achieved accuracies of 88-100% when classifying the nine archaeal species. The predicted results were validated by DNA sequencing analysis of cells isolated from the mixture by Raman-activated cell sorting. Raman spectra of uncultured archaea (MGII) were also obtained based on Raman spectroscopy and fluorescence in situ hybridization. The results combining multiple Raman-based techniques indicated that MGII may have the ability to produce lipids distinct from other archaeal species. Our study provides a valuable approach for investigating and classifying archaea, especially uncultured species, at the single-cell level.