An integrative Raman microscopy-based workflow for rapid in situ analysis of microalgal lipid bodies

Sudhir Kumar Sharma, David R. Nelson, Rasha Abdrabu, Basel Khraiwesh, Kenan Jijakli, Marc Arnoux, Matthew J. O’Connor, Tayebeh Bahmani, Hong Cai, Sachin Khapli, Ramesh Jagannathan, Kourosh Salehi-Ashtiani

Oils and bioproducts extracted from cultivated algae can be used as sustainable feedstock for fuels, nutritional supplements, and other bio-based products. Discovery and isolation of new algal species and their subsequent optimization are needed to achieve economical feasibility for industrial applications. Here we describe and validate a workflow for in situ analysis of algal lipids through confocal Raman microscopy. We demonstrate its effectiveness to characterize lipid content of algal strains isolated from the environment as well as algal cells screened for increased lipid accumulation through UV mutagenesis combined with Fluorescence Activated Cell Sorting (FACS). To establish and validate our workflow, we refined an existing Raman platform to obtain better discrimination in chain length and saturation of lipids through ratiometric analyses of mixed fatty acid lipid standards. Raman experiments were performed using two different excitation lasers (λ = 532 and 785 nm), with close agreement observed between values obtained using each laser. Liquid chromatography coupled with mass spectrometry (LC-MS) experiments validated the obtained Raman spectroscopic results. To demonstrate the utility and effectiveness of the improved Raman platform, we carried out bioprospecting for algal species from soil and marine environments in both temperate and subtropical geographies to obtain algal isolates from varied environments. Further, we carried out two rounds of mutagenesis screens on the green algal model species, Chlamydomonas reinhardtii, to obtain cells with increased lipid content. Analyses on both environmental isolates and screened cells were conducted which determined their respective lipids. Different saturation states among the isolates as well as the screened C. reinhardtii strains were observed. The latter indicated the presence of cell-to cell variations among cells grown under identical condition. In contrast, non-mutagenized C. reinhardtii cells showed no significant heterogeneity in lipid content. We demonstrate the utility of confocal Raman microscopy for lipid analysis on novel aquatic and soil microalgal isolates and for characterization of lipid-expressing cells obtained in a mutagenesis screen. Raman microscopy enables quantitative determination of the unsaturation level and chain lengths of microalgal lipids, which are key parameters in selection and engineering of microalgae for optimal production of biofuels.