Production of polyunsaturated fatty acids from marine microorganisms

Abstract

Polyunsaturated Fatty Acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are increasingly attracting scientific attention due to their significant health promoting role in the human body. However, the human body lacks the ability to produce them in vivo. The limitations associated with the current sources of ω-3 fatty acids and ω-6 fatty acids from animal and plant sources have led to increased interest in microbial production. Marine bacteria provide a suitable alternative, although the isolation of production strains and the identification of operating conditions must be addressed before manufacturing processes become economically viable. Sea sediment samples were collected from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea. The isolates were screened for PUFA production using a fast colourimetric method and verified by gas chromatography/mass spectroscopy. The isolated PUFA producers were characterised and identified on the basis of 16S rRNA gene sequencing and analysis. Three different isolates were chosen for this study. These were labelled as 717, 66 and Hus-27. The chosen isolates were subjected to an optimisation study to maximise their productivity. This optimisation strategy included identifying a suitable production medium by applying a statistical design of experiment methodology (Plackett-Burman and Central Composite Design). A chemically defined media was identified for isolates 717 and 66 in order to determine the limiting media components and to study the effect of carbon/nitrogen ratio on the productivity of isolates. As an important step in the process development of the microbial PUFA production, the culture conditions at the bioreactor scale were optimised for isolate 717 using a Response Surface Methodology (RSM) revealing the significant effect of temperature, dissolved oxygen and the interaction between them on the EPA production. Two sets of continuous stirred-tank reactor (CSTR) experiments were also performed to test the effect of growth rates on EPA production and the effect of temperature at constant growth rate as this was identified as the most significant factor affecting EPA production. This optimisation strategy led to a significant increase in the amount of EPA produced by isolates under investigation, where the amount of EPA increased from 9 mg/g biomass, 33 mg/l representing 7.6% of the total fatty acids to 45 mg/g, 350 mg/l representing 25% of the total fatty acids using isolate 717. A significant increase was also achieved using isolate 66 with the amount of EPA increased from 5.5 mg/g, 14 mg/l representing 3.5% of the total fatty acids to 32 mg/g, 285 mg/l representing 15% of the total fatty acids. For isolate Hus-27 the amount of EPA increased from 0.6 mg/g, 3 mg/l representing 2.2% of the total fatty acids to 8 mg/g, 36 mg/l representing 8% of the total fatty acids. The stability of the produced oil and the complete absence of heavy metals in bacterial biomass are considered as an additional benefit of bacterial EPA compared to other sources of PUFA. To our knowledge this is the first report of a bacterial isolate producing EPA with such high yields making large scale manufacture much more economically viable.