OPTIMISATION OF THE BIOMASS AND FATTY ACID PRODUCTION OF THE MICROALGA ISOCHRYSIS AFF. GALBANA (clone t-iso) for aquacultural and industrial purposes

PhD Thesis  by I.P. Tzovenis,

Faculty of Agricultural and Applied Biological Sciences, Ghent University, Belgium, 2001, 306 pp.

Summary:

Microalgae mass cultures constitute a potential tool to effectively use the vast re­sources of the sun and the ocean in the future. Today the most important use of the microalgae is their use as live-food in aquaculture and some sporadic uses as source of highly unsaturated fatty acids in specialty food products such as baby ­formulas. These uses are based in great part on the unique fatty acid profiles of es­pecially the marine microalgae comprising highly unsaturated fatty acids biosyn­thesised via unique pathways from de novo fatty acid products.

In Chapter I (Introduction) the basic components of this thesis were identified along with the research objectives. Hence the objective of this work was to opti­mise the growth of a microalgal strain with a desired fatty acid spectrum in use in the aquaculture industry, and to explore the possibility of its candidacy for the oleochemical industry. As test organism was selected the haptophyte T-ISO (Isochrysis aff. galbana, Tahitian strain). This organism is a unicellular phyto­plankter isolated recently (1977) from tropical waters with the advantage of good growth at high temperatures (25-30^°C) comprising a highly unsaturated fatty acid spectrum due to its high w3 PUFA content and in particular of DHA.

In chapter II (Microalgae mass cultures) all the current knowledge regarding the production and use of microalgae has been reviewed. In particular for aquaculture a special survey was included referring to the qualitative and quantitative production of microalgae in the Mediterranean basin during the year 1996. According to this report the "Milford" stepwise batch scale-up method was found to be the most adopted one. Therefore this approach was followed in this thesis as well.

In Chapter III (Fatty acids of microalgae) the literature concerning taxonomical distribution (including those of macroalgae for comparison), biochemistry, metabo­lism and regulation of fatty acids in microalgae has been thoroughly reviewed. In this part the effect of the culture (environmental) conditions on the fatty acid spec­tra was extensively documented. Furthermore it was identified that there are three different lines of microalgae response i.e. the prokaryotic (Cyanobacteria), the green (Chlorophyta), and the non-green ones, the latter being paraphyletic and much less understood. In this context we designed simple factorial experiments to explore the light energy requirements of the growth of T-ISO and consequently its impact on the strains' fatty acid profile. The most desired light regimes were then expanded in subsequent factorial experimental designs with different temperature, salinity and carbon dioxide regimes in order to come up with some recommenda­tions for producing high yields of cells or biomass, comprising the desired fatty acid profiles. Both, acclimated (exponential phase harvests obtained from cultures extensively adapted to the applied conditions) and transient response (post­exponential harvests at the end of the light-limiting phase prior to the nutrient lim­ited early stationery phase) were taken into consideration in order to simulate the metabolism under continuous and semi-continuous (or low dilution rate continu­ous) modes respectively. Three concurrent replicate cultures were tested under each regime.

Chapter IV (Determination of fatty acid content in microalgae: methods compari­son and development of a rapid and efficient micro-protocol) deals with the devel­opment of a rapid, reliable and cost-effective analysis protocol for the analysis of the microalgal fatty acids. This work was based on the comparison between differ­ent extraction and derivatisation methods on fresh and frozen samples.

In Chapter V (Effect of different light regimes on the DHA content of Isochrysis aff. galbana, clone T-ISO) are presented the results relating the content of DHA (the most important fatty acid of T-ISO) to the growth under different light re­gimes. It was demonstrated that all different light regimes discriminate from each other on the basis of the fatty acid profile. The culture variables affect the fatty acid composition and kinetics in a perplex synergistic/antagonistic way, hence the ef­fects of culture regimes should be considered rather than of a particular variable. The acclimated response was different from the transient response (highly variant). Furthermore it was suggested that for rations based on cell numbers or biomass targeting fatty acid enrichment of aquaculture animals the data for the particular fatty acids should be interpreted per cell or per biomass as the qualitative (propor­tions to TFA) content might be misleading and hardly offer any true stoichiometry. Finally it was suggested that the production protocols should be followed with utter consistence as the culture variables not only differentiate the fatty acid contents but also the clonal stability of the strains.

In Chapter VI (optimisation of T-ISO production rich in essential fatty acids. I: effect of different light regimes on growth and biomass production) the results showed that the specific growth rate of T-ISO maximised with an increase of the total photon flux supplied per day irrespective of the photoperiod. Under continu­ous light, the cell size of T-ISO (both CDW and cell volume) correlated positively to PFD with a further increase when the cells were transiently light-limited in the post-exponential phase. In contrast, CDW under discontinuous light increased only at subsaturating PFD with a significant decrease in the post-exponential phase. Cell size (volume) did not correlate to CDW under discontinuous light, revealing an intracellular density change particularly for the 16:08 h L:D regimes. As a result biomass yield and productivity displayed differences between continuous and dis­continuous light while cell yield and productivity were simply a function of total PFD per photocycle. Continuous light imposed a certain constraint on the biomass productivity, yet not restraining the biomass yield from being maximal amongst all photopenods, at the highest PFD.

In Chapter VII (optimisation of T-ISO production rich in essential fatty acids. II: effect of different light regimes on the production of fatty acids) results showed that for 12:12 and 24:0 h L:D, the fatty acid pattern could be summarised as PUFA>SAFA>MUFA while for 16:08 h L:D, as SAFA>PUFA> MUFA which reflects a differential acclimation of the strain under different light-dark cycles. For 12:12 h L:D the biomass content of PUFA was significantly higher than for the other light regimes with differences located in the ω3 fraction, the ω6 content being rather constant. ω3 HUFA (DHA mainly) increased both in absolute and relative terms under conditions providing adequate energy for the elongation/desaturation pathway and it was speculated that under not light-limiting conditions there is op­timal co-operation among energy influx and PUFA pathway that permits increase of DHA content with increasing photon flux density. Furthermore it was suggested that under short daylength conditions such as 12:12 h L:D or under continuous light, ω3 PUFA accumulate in order to optimise the photosynthetic process. The ω3/ω6 and DHA/EPA ratios within the tested range of conditions were optimal according to the literature for fish and shellfish nutrition requirements. The produc­tion of ω3 HUFA in T-ISO was influenced by the total photon flux available per photocycle in a similar manner as for growth. The capacity of the strain for storing lipids was limited under the conditions tested. Consequently the fatty acid content followed the biomass yield, and productivity pattern. In the context of aquaculture it was recommended that a light regime of 12:12 h L:D at PFD within the photo­limitation-photoinhibition range offers certain advantages for the culture of T-ISO. Alternatively, if a high investment could be substantiated, continuous cultures un­der 24:0 h L:D at the same PFD range could serve as an optimisation basis using advanced photobioreactors.

In Chapter VIII (optimisation of T-ISO biomass production rich in essential fatty acids. III: effect of different light, temperature, salinity and C02 regimes on the biomass production) it was demonstrated that specific growth rates maximise (ca. 1.6 d^-') under continuous light and high temperatures in carbon dioxide boosted cultures. Cell size minimises under continuous light at optimal temperature with little change in the post-exponential phase, while in the other culture regimes cell size maximises in the exponential and drops in the post-exponential phase. Cell and biomass volumetric productivity during the exponential phase follow the pattern of specific growth rate. Post-exponential yields maximise under continuous light re­gimes with carbon dioxide enriched aeration while during the exponential phase, harvests seem to be independent of the regimes tested. The great acclimation ca­pacity of the strain for non resource-constraints renders within the tested range the effect of temperature and salinity indifferent for maximal post-exponential harvest.

Finally in Chapter IX (optimisation of T-ISO biomass production rich in essential fatty acids. IV: effect of different light, temperature, salinity and C02 regimes on the fatty acid production) it was demonstrated that well-acclimated T-ISO cells under the different culture regimes tested contained a rather constant fraction of TFA per biomass. The unsaturation degree of TFA increased under growth non­favourable regimes including the light-limited post-exponential phase, except for 12:12 h L:D x 25°C x p-exp. In gross terms, fatty acid variation in T-ISO is dic­tated by the interaction of photoperiod, temperature and growth phase. Neverthe­less, temperature seems to play the most critical role while interactions with light and CO2 regimes dictate fluctuations that differentiate further the fatty acid profile. Salinity within the range tested did not affect significantly any of the lipid outputs considered. For the culture regime 24:0h L:D x 25°C X CO₂+, well acclimated T-­ISO maximised specific growth rate (µ_max), and biomass and ω3 HUFA (mostly DHA) exponential volumetric productivity. Under the same light and CO₂ regime, biomass post-exponential volumetric yields were highest and temperature inde­pendent. Hence, this regime could serve as a basis for further optimisation for con­tinuous production of T-ISO biomass rich in DHA. A worth to consider alternative could be the culture regime 12:12h L:D x 25°C x CO₂+, since, although the growth rate was lower, the DHA productivity was comparable to the highest one.

Targeting the production of DHA, the culture regime 24:0h L:D x 19°C x CO₂+, could be an adequate choice since both, DHA content of biomass, and DHA pro­ductivity were maximal while, both biomass and DHA content at the end of the post-exponential phase were also maximal. For DHA rich rations based on cell numbers, regimes such as the 12:12 h L:D x 25°C x CO₂+, and 24:0 h L:D x 19°C x CO₂+, could be used for exponential batch harvests or the 24.0h L:D x 19°C x CO2+, for post-exponential batch harvests.

It has been shown in this research that microalgae modulate their fatty acid compo­sition to optimise their growth. Under well executed acclimation protocols cells exhibit a remarkable capacity for adaptation to different culture conditions, with some variation attributed to both different strategies and putative clonal instability. Out of these results a production scheme for hatcheries could be selected to be fur­ther optimised with the best available or affordable technology.

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