A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements. A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species. By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (-) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography. From a pool of 2.0 x 10(8)CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.