Optimization Conditions For The Production Of Arachidonic Acid By The Microalga Parietochloris Incisa Under Indoors And Outdoors Conditions

ABSTRACT

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4ω6, AA). Accumulation of AA and docosahexaenoic acid (22:0, DHA) is necessary for brain and retina development in infants. While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. This is of practical value, since TAGs are the preferred chemicals to introduce AA into baby formulae. Studies using different cultures conditions were carried out to assess their effects on AA production.

Under different cell densities, low, medium and high (LD, MD and HD, respectively), the cells of P. incisa showed a different pattern of chlorophyll content. At LD, cells followed the process of nitrogen starvation, decrease in chlorophyll content, accumulation of carotenoids and showed signs of bleaching at the end of cultivation period. In the same way, cells grown at MD followed the same pattern though it was not more pronounced compared to the former. However, at HD, cells continued to increase the chlorophyll content, probably by continuing to build the photosynthetic membranes to maximize light harvesting. The AA content increased in all cultures irrespective of the initial cell density. Volumetric biomass accumulation was higher in HD cultures, eventually the volumetric AA yield was also higher.

The growth stage at which cultures were transferred to N-free medium had a pronounced effect on chlorophyll synthesis of P. incisa. The rate of chlorophyll synthesis was faster in cultures transferred in EXP (exponential) and TRANS (transition) phases.

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Similarly, the biomass accumulation was rapid in cultures transferred in the EXP and TRANS phases. The proportion of AA of total fatty acid increased faster in cultures transferred to N- starvation in the EXP and TRANS phases of growth. The proportion of AA almost doubled from 25.8% to 47.5% and from 25.8% to 49.8% of TFA, respectively at day 14. Cultures transferred in the E.STA (early stationary) and M.STA (middle stationary) phases of growth, had higher initial proportion of AA, amounting to 34.9% and 36.3%, respectively, at day 0. Their proportion of AA had increased slowly throughout the cultivation period, reaching 47.3% and 47.0% at the end of cultivation period.

Cultures growing on complete medium and supplemented with fatty acid precursors were able to incorporate and further modify both incorporated oleic acid and linoleic acid to AA. This was evident only with 3d cultures at the EXP stage. Older cultures did not demonstrate an enhancement in AA production, but rather a reduction. Supplementation of N-starved cultures did not enhance AA production.

Increasing the C/N ratio favors lipid accumulation in microalgae by inducing de novo lipid synthesis. Indeed, high CO2 significantly enhanced the TFA synthesis in P. incisa indicating that under enriched CO2 conditions the rate of de novo synthesis of fatty acid is higher. However, this phenomenon was only evident up to 10 days. Cultivation of P. incisa on BG11-N with high CO2 did not favor an increase in the proportion of AA, moreover, the proportion of 18:1 increased at the expense of AA. This might indicate either an inhibition of sequential desaturation or favored synthesis of shorter and less desaturated fatty acids. Increasing the CO2 level in HD cultures on BG11-N and tap water media did not cause any significant changes in the TFA and AA contents.

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In developing a large-scale cultivation for AA, it is necessary to utilize inexpensive medium component to lower the cost. Tap water and brackish water were used to induce starvation in P. incisa in HD cultures. Cells of P. incisa survived in tap water, which basically contains no nutrients for sustaining growth. More diluted cultures were not able to survive under those conditions. It is therefore clear that growing cells of P. incisa under HD can allow the cells to grow under nutrient-devoid medium. Cells of P. incisa were able to produce biomass rich in arachidonic acid on tap or brackish water. Not only is this media less expensive but also it saves labor in media preparation, reducing the final cost of the product.

By growing P. incisa under outdoors conditions, this study represented an ideal approach to making the realization of commercial production of AA possible. The volumetric productivity of AA and TFA, as well as the daily accumulation of AA were almost the same in outdoors and indoors cultures at day 8. On subsequent days, indoors culture attained higher productivities, probably such differences was due to contaminations that were microscopically observed in outdoors cultures at day 9.