Insights into the methodological perspectives for screening polyunsaturated fatty acids-containing bacteria

Polyunsaturated fatty acids (PUFA) are vital molecules in the pharmaceutical, medical, and nutritional industries. Exploration of bacterial strains capable of producing significant amounts of PUFAs offers a promising avenue for biotechnological applications and industrial-scale production. However, an extensive screening of several samples from diverse sources is highly needed to identify a potential strain. The present study provides the results of the evaluation of 15 different screening methodologies (including changes in existing protocols in terms of reagent concentration, incubation temperature and time) for identifying PUFA-producing bacteria in comparison to the gold standard method (Gas chromatography-mass spectrometry), for the first time. The results determined the most effective techniques for each critical PUFA, leading to an optimized screening process that saves time and resources. The H₂O₂ plate assay using 0.5% or 1% H₂O₂ for 72 & 96 h of incubation at 15 °C consistently outperformed others for finding bacteria containing total nutritionally important long chain-PUFA (LC-PUFA), linoleic acid, and arachidonic acid. Whereas the 2,3,5-triphenyl tetrazolium chloride broth assay at 10-15 °C was the most effective and semiquantitative screening methodology for eicosapentaenoic acid (EPA) and alpha-linolenic acid-containing bacteria. Apart from the methodological perspectives, the study also revealed certain potential strains to be targeted in the ongoing research on PUFA-containing bacteria. Further, the manuscript forms the first report on the presence of docosahexaenoic acid (DHA) in Shewanella decolorationis, EPA in Psychrobacter maritimus and Micrococcus aloeverae, and both EPA and DHA in Arthrobacter rhombi. Altogether, the paper generates several thought-provoking insights on the methodological perspectives and identifies potential PUFA-containing bacteria with practical applications in future bacteria-based PUFA research.